Monthly Archives: September 2021

fig 7a

A Serotonin 2A-Receptor Decoy Peptide Potently Lowers Blood Pressure in Male Zucker Diabetic, Fatty, Hypertensive Rats

DOI: 10.31038/EDMJ.2021523

Abstract

Aims: To test whether a novel 5-hydroxytryptamine 2A decoy receptor peptide, SN..8 (Sertuercept), administered via intraperitoneal injection, acutely lowers arterial blood pressure in obese, hypertensive male Zucker diabetic rats (ZDF). To examine the safety, tolerability and possible reno-protective effects following chronic alternate daily administration of Sertuercept (for 10 weeks) in the male ZDF rat.

Methods: Systolic and diastolic blood pressure were determined at baseline and regular intervals for up to 48 hours after a single IP administration of either Sertuercept (2 mg/kg), vehicle (saline) or an identical concentration of a scrambled sequence of the decoy receptor peptide, LN…8, in male ZDF and Zucker lean rats using tail cuff plethysmography. Plasma autoantibodies were obtained in thirteen male ZDF rats for determination of 5-hydroxytryptamine 2A receptor-mediated neurotoxicity using an acute neurite retraction assay in mouse neuroblastoma cells. Rats were sacrificed at 25-weeks of age, the kidneys were perfused, fixed and sections were stained using Masson’s trichrome for semi-quantitative determination of glomerular and interstitial fibrosis.

Results: Sertuercept (2 mg/kg IP) potently lowered systolic and diastolic blood pressure in both 11-week-old and 25-week-old male ZDF rats and in a subset of hypertensive Zucker lean rats. There was no significant blood pressure-lowering effect of vehicle (saline) or scrambled peptide sequence (LN.8). Blood pressure-lowering was rapid in onset (15-30 minutes following IP injection) and sustained for at least 24 hours. Alternate daily IP administration of 2 mg/kg dose of Sertuercept vs. scrambled peptide (for 10 weeks) was safe, well-tolerated and associated with a significant decrease in glomerulosclerosis in 25-week-old male ZDF rats. Plasma autoantibody-induced neurotoxicity correlated significantly with the global index of renal fibrosis severity in 25-week-old male ZDF rats.

Conclusions: These data indicate potent arterial blood pressure-lowering efficacy from a decoy receptor peptide comprised of a second extracellular loop region of the human 5-hydroxytryptamine receptor. Chronic administration of the decoy receptor peptide (10 weeks) was safe, well-tolerated and protected against renal glomerulosclerosis in the male ZDF rat.

Introduction

Obesity-associated hypertension is a major risk factor for adverse cardiovascular and renal outcomes in adult type 2 diabetic populations [1,2]. The underlying pathophysiologic mechanisms are complex and may include inflammation, lipotoxicity, and endothelial cell dysfunction as contributory factors [1]. Obesity is thought to drive sympathetic nervous system overactivation in the kidney [3] contributing to hypertension and the development of left ventricular hypertrophy [4]. Left ventricular hypertrophy is a risk factor for heart failure and myocardial infarction which both increase substantially in adult obese, hypertensive type 2 diabetes mellitus [5,6].

Volume and pressure overload cardiac hypertrophy is driven by catecholamines [7] and other hormones (e.g. angiotensin II) which (in the case of alpha 1β adrenergic R) activates Gq/phospholipase C-coupled signaling pathways in cardiac cells [8]. A hallmark feature of classical ligand-G-protein receptor interaction(s) is ligand-occupied receptor desensitization (via phosphorylation) mediated by G-protein coupled receptor kinases (GRKs) [8]. Receptor autoimmunity, on the other hand, is characterized by humoral IgG receptor-targeting autoantibodies (e.g. TSH receptor or beta-adrenergic receptor) that elicit longer-lasting receptor activation underlying various human pathologies such as Graves’ disease [9] or dilated cardiomyopathy, respectively [10]. Recently, we reported that subsets of human diabetic micro-vascular disease, stroke, refractory hypertension and/or chronic kidney disease harbored increased plasma IgG, 5-hydroxytryptamine 2A receptor (5-HT2AR)-targeting autoantibodies that caused long-lasting Gq11/phospholipase C/Ca2+ signaling activation in endothelial cells and in neurons [11-13].

The 5-HT2AR is expressed on arterial vascular smooth muscle cells where it mediates 5-HT induced arterial vasoconstriction [14]. We developed a novel decoy receptor peptide comprised of a subregion of the second extracellular loop of the 5-HT2A receptor involved in mediated long-lasting receptor activation [15]. The decoy receptor peptide, SCLLADDN (Sertuercept) prevented human 5-HT2AR-targeting IgG autoantibodies’ endothelial and neuronal cell toxicity in vitro [13]. The aim of the present study was to test whether the decoy (5HT2A) receptor peptide (Sertuercept) acutely lowers blood pressure in an animal model of obesity-associated hypertension harboring spontaneously-occurring plasma 5-HT2AR agonist autoantibodies [16].

The male Zucker diabetic fatty rat (ZDF) is a well-known genetic model of obese, hypertensive, dyslipidemic type 2 diabetes mellitus [17,18]. In the ZDF rat, agonist plasma 5-HT2AR targeting IgG autoantibodies appeared to develop around the same time as obesity and diabetes and caused persistent Gq11/PLC/Ca2+ signaling in cells [16]. We tested whether chronic administration of active decoy 5HT2A receptor peptide vs. scrambled peptide (for ~10 weeks) might protect against deleterious cardiac hypertrophy or glomerulosclerosis associated with chronic moderate-severe hypertension in the ZDF rat.

Methods

Synthetic Peptides

All synthetic peptides were synthesized at Lifetein Inc. (Hillborough, NJ). The lyophilized peptides were aliquoted and stored (in the presence of dessicant) at -40 degrees C prior to use. On the day of intraperitoneal (IP) administration, an aliquot of lyophilized peptide was reconstituted in sterile saline at the appropriate concentration of 2 mg/kg. Reconstituted peptide was prepared fresh before each injection. Lyophilized peptide was stored for up to 4 weeks (at – 40 deg C) prior to obtaining newly-synthesized peptide needed in chronic drug administration experiments.

Sertuercept (Decoy Receptor Peptide)

A linear synthetic peptide, SCLLADDN, having amino acid sequence identical to that of a fragment of the second extracellular loop region of the human 5-hydroxytryptamine 2A receptor was synthesized and had ≥95% purity.

Scrambled Peptide Sequence LD..8

The scrambled peptide had a sequence of LASNDCLD (LD.8) and a purity of 96.37%, MW 849.91.

Animals

All procedures were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Medical Center (East Orange, New Jersey). Male ZDF and lean (+/?) Zucker rats were obtained from Charles River Laboratories (Kingston, NY) at approximately 6-7 weeks of age. All rats were single housed upon arrival, without enrichment. Rats were acclimatized for two weeks prior to experimental procedures. Rats were provided ad libitum access to food and water and maintained in a 12 h light/dark cycle with lights on at 0630. All procedures occurred during the light phase of the cycle. Blood pressure testing and the effects of chronic peptide administration (for 10 weeks) on cardiac or renal endpoints was conducted in several cohorts of animals.

Cohort 1: Three 25-week-old male ZDF and three, age-matched, male Zucker lean rats(ZLR) treated acutely with IP Sertuercept (ZDF) vs. saline (vehicle) (ZLR).

Cohort 2: 11-week old male ZDF (n=5) and Zucker lean rats (n=4) treated acutely with IP Sertuercept.

Cohort 3: Two groups of 12-week-old male ZDF rats (n=6/group underwent chronic treatment with alternate daily IP, free Sertuercept (2 mg/kg) vs. IP scrambled LD..8 (2 mg/kg) continuously between 13.5-23.5 weeks of age, i.e for 10 weeks. The rats were sacrificed at 25 weeks of age. Body weight was determined immediately before sacrifice and after perfusion. The hearts were excised and weighed for determination of the heart-to-(perfused) body weight ratio.

Cohorts 1 & 3: 25 weeks of age: Rats were anesthetized with xylazine/ketamine prior to undergoing non-survival surgery during which they were perfused with 4% paraformaldehyde in PBS. The kidneys were post-fixed for 24-48 hours in 4% paraformaldehyde and then stored in 70% ethanol at 4 deg C prior to shipment to Histoserve, Inc. (Germantown, MD) where they were sectioned and stained with H&E and Masson’s trichrome. Blood was obtained by cardiac puncture immediately before non-survival surgery for isolation of plasma IgG autoantibodies.

Blood Pressure Monitoring

Tail cuff blood pressure measurement was performed using an automated CODA noninvasive blood pressure system (Kent Scientific, Torrington, CT.). Rats were placed on an insulated warming platform in a well-heated room to ensure proper body temperature. Cuff monitors were provided in different sizes to accommodate rats of different ages and having different body weights. After the animal was positioned in the clear plastic restraint holder, the appropriate-sized cuff was slid over the rat’s tail. The blood pressure system uses volume pressure recording tail-cuff technology and displays up to six blood pressure measurements per cycle.

Renal Histology

Comparisons between untreated male Zucker strain (lean vs. fatty) or male ZDF rats randomized to 10 week’s treatment with (Sertuercept vs. scrambled peptide) were made by Dr. Jerrold M. Ward (veterinary pathologist) using Masson’s trichrome fibrosis score. The fibrosis score ranged from 0-4, where 0 = no fibrosis, 1= minimal fibrosis, 2= mild fibrosis, 3= moderate fibrosis, and 4= severe fibrosis. An individual glomerular score and an interstitial score (for the one kidney examined from each individual rat) was determined separately. The glomerular score consisted of the number of glomeruli affected by fibrosis among 30-40 glomeruli examined per kidney. In addition, the overall glomerular score was divided into two sub-scores: one sub-score each was derived from glomeruli examined in regions unaffected or affected by interstitial fibrosis. Glomeruli affected by fibrosis represents an average of the two individual subscores obtained in 30-40, mean 37.5 glomeruli, examined per kidney. The veterinary pathologist examiner was unaware of the treatment assignment group during examination of histologic sections of rat kidneys. An ‘index of renal fibrosis’ global severity score was comprised of the interstitial fibrosis score (1-4) times the (percent affected glomeruli per kidney x 10).

Protein G Affinity Chromatography

Rat IgG autoantibodies were isolated from 25-week-old male ZDF plasma by protein G affinity chromatography as previously reported [16].

Acute Neurite Retraction Assay

A 1/100th dilution of the protein G eluate of ZDF rat plasma was added to 35 mm dishes containing mouse neuroblastoma (N2A) cells (ATCC, Rockville, MD) cultured in DMEM with 10% fetal calf serum. Acute neurite retraction was determined after 5-10 minutes incubation in the presence of rat IgG autoantibodies as previously reported [16].

Mouse Neuroblastoma (N2A) Cell Survival Assay

The assay was performed using an MTT assay as previously reported [16].

Urine Albumin/Creatinine Ratio

One day prior to sacrifice, rats were individually housed in metabolic cages for 3-4 hours for collection of urine samples. Thymol was added to urine samples to inhibit bacterial growth and the samples were kept frozen at -40 C prior to determination of albumin and creatinine concentrations. The rat albumin ELISA kit was obtained from Novus Biologics, Inc (Centennial, CO); the creatinine colorimetric assay kit was obtained from Cayman Chemicals, Inc (Ann Arbor, MI).

Statistical Analysis

Comparisons were made using Student’s unpaired t-test.

Results

Acute Blood Pressure-lowering Effect of SN..8 Peptide (2 mg/kg IP) in Male ZDF Rats

The male ZDF rat spontaneously develops hypertension by age 8-10 weeks, and manifests proteinuric nephropathy by approximately 18 weeks of age [19]. Sertuercept is a linear synthetic peptide having an amino acid sequence SCLLADDN identical to a region of the second extracellular loop of the 5-HT2A receptor involved in mediating long-lasting activation [15]. A single 2 mg/kg IP dose of Sertuercept caused highly significant (41-42%) acute decreases in systolic and diastolic blood pressure in three 25-week-old male ZDF rats tested, rats that were naïve to prior drug exposure (Table 1). The onset of acute blood pressure-lowering occurred between 15-40 minutes following IP drug injection. In two of three rats tested, IP Sertuercept administration caused sedation and borderline hypotension both of which resolved spontaneously after approximately ten minutes. Rechallenge (1 week later) with a single IP (2 mg/kg) dose of Sertuercept caused reproducibly large drops in systolic and diastolic blood pressure which were sustained for 4 hours or longer (Figure 1). There were no untoward acute or long-term side effects observed for up to 18 days following the repeat drug exposure. Taken together these data demonstrate that a single 2 mg/kg intraperitoneal injection of the peptide SCLLADDN (dissolved in sterile isotonic saline) causes acute substantial lowering of systolic, diastolic and mean arterial blood pressure in the older adult 25-week-old, hypertensive Zucker diabetic fatty rat.

Table 1: Change in systolic blood pressure in 25-week-old male ZDF rats before and after peptide SCLLADDN injection.

Systolic blood pressure

Animals Day-2. Day 0, pre-injection 15-40 mins post-injection.

P-value*

ZDF 1-3

166 + 24 161 + 21 95 + 13

0.018

DIASTOLIC BLOOD PRESSURE

ZDF 1-3 101 + 23 112 + 2 65 + 11

0.05

Percent lowering of systolic blood pressure after peptide (161-95)/161 = 41.
Percent lowering of diastolic blood pressure after peptide (112-65)/112 = 42.
Percent lowering of mean arterial pressure (125-75)/125 =40.
Results are mean mm Hg ± SD in three, obese male Zucker diabetic fatty rats (25-weeksold),
average weight approximately 500g who had blood pressure monitored two days before, 30 minutes before and then 15-40 minutes after receiving 2 mg/kg intraperitoneal injection of the linear synthetic peptide SCLLADDN in sterile saline.
*P-value is comparing mean systolic or diastolic blood pressure immediately before and
after the peptide injection.

fig 1a

fig 1b

Figure 1: A single intraperitoneal dose of Sertuercept (2 mg/kg) acutely lowered systolic (A) and diastolic blood pressure (B) in the Zucker diabetic fatty rat. Each point represents the mean ± SEM values in two-three, 25-week-old male ZDF rats.

Acute Effect of IP Administration of Saline in Older Male Zucker Lean Rats

Age-matched male Zucker lean rats do not manifest diabetes, obesity or hypertension when fed the same diet as ZDF rats. Baseline mean systolic and diastolic blood pressure was in the normal range in 25-week-old male ZLR rats (N=3), and it did not change significantly following IP administration of 0.5 mL vehicle (sterile saline) in each rat (Table 2).

Table 2: Effect of saline injection on systolic blood pressure in Zucker lean, non-obese rats (ZLR).

Systolic blood pressure

Animals Day-2. Day 0, pre-injection 15-40 mins post-injection.

P-value*

ZDF 1-3

166 + 24 161 + 21 95 + 13 0.018

DIASTOLIC BLOOD PRESSURE

ZDF 1-3

101 + 23 112 + 2 65 + 11

0.05

Intraperitoneal injection of 0.5 mL of sterile saline had no significant effect on diastolic blood pressure in three Zucker lean male rats, 25-weeks-old.
*P-value comparing mean systolic and diastolic blood pressure before and after saline injection.

Acute Effect of Sertuercept in Younger Male ZDF and Zucker Lean Rats

We next tested a 2 mg/kg IP dose of Sertuercept in 11-week-old male ZDF rats (N=5), and age-matched male Zucker lean rats (N=4). Sertuercept caused acute significant mean systolic and diastolic blood pressure-lowering (19-23%) in five of five 11-week-old male ZDF rats tested (Table 3). Acute blood pressure-lowering was well-tolerated; none of the five male ZDF rats experienced any untoward side effects including hypotension or acute sedation.

Table 3: Acute blood pressure- lowering effect of SN..8 in 11-week-old male ZDF rats.

Systolic blood pressure

 Animals Before 15-45 mins post-injection

P-value*

 ZDF (N=5)

167 ± 24 (N=10) 135 /-+ 18 (N=17) 0.0004

Diastolic blood pressure

 ZDF (N=5)

118 + 22 (N=10) 91 + 15 (N=17)

0.002

Results are mean ± SD.
Mean acute SBP-lowering (167-135)/167 = 19%.
Mean acute DBP-lowering (118-91)/118 = 23%
Mean acute MAP-lowering (134-106)/134 = 21%.
*P-value: comparing mean systolic or diastolic blood pressure before and after IP injection
of 2 mg/kg dose of Sertuercept (SN..8).

Three of four, 11-week-old Zucker lean rats tested had normal blood pressure at baseline and Sertuercept (2 mg/kg IP) did not significantly alter blood pressure acutely in normotensive rats (Table 4). In one of four Zucker lean rats that manifested baseline hypertension, a single dose of Sertuercept (2 mg/kg IP) acutely lowered systolic and diastolic blood pressure to statistically significantly lower levels compared to baseline (Table 4). These data suggest that 2 mg/kg IP Sertuercept effectively lowers blood pressure in both young and older male ZDF rats and in a subset of hypertensive Zucker lean rats.

Table 4: Acute effect of Sertuercept on blood pressure in 11-week-old male Zucker lean rats.

Blood pressure

Before injection

30 minutes after injection

P-value*

Zucker lean #1

130/72

120/78

NS

Zucker lean #2

124/80

122/83

NS

Zucker lean #3

130/85

132/98

NS

Zucker lean #4

149/102 (N=6)

130/91 (N=4)

<0.05

*P-value comparing mean blood pressure before and after IP injection of 2 mg/kg Sertuercept.

Sustained Blood Pressure Lowering Effect of Sertuercept in Adult Male ZDF Rat

The duration of Sertuercept’s blood pressure-lowering action was evaluated in 25-week-old male ZDF rats (N=3) administered a single IP 2 mg/kg dose. The control group were age matched male ZDF rats (N=3) who received 2 mg/kg IP administration of LD..8, a scrambled peptide sequence comprised of the same eight amino acids as in Sertuercept arranged in a random order. Sertuercept caused 25-30% significant systolic and diastolic blood pressure-lowering which was sustained for 24 hours or longer (Figure 2A). Blood pressure returned to baseline elevated levels 48 hours after Sertuercept administration (Figure 2A). The scrambled peptide LD.8 sequence (2 mg/kg IP) had no significant systolic or diastolic blood pressure-lowering effect at time points up to 24 hours (Figure 2B). These data suggest that a single 2 mg/kg Sertuercept dose (but not a scrambled peptide having the same amino acids) promotes relatively long-lasting significant systolic and diastolic blood pressure-lowering in adult hypertensive male ZDF rats.

fig 2a

fig 2b

Figure 2: Sertuercept (SN..8) (2 mg/kg) single intraperitoneal injection caused long-lasting systolic (A) and diastolic blood pressure-lowering (B) in 25-week- old male ZDF rats. Each point represents the mean ± SEM values as described in Materials and Methods.

Cardio-protective Effect of Chronic Administration of Sertuercept in Male ZDF Rats

Two groups of 13.5-week-old male Zucker fatty rats (n=6/group) having matching baseline mean capillary glucose concentration and body weight (Table 5), were randomly assigned to chronic 10 weeks’ treatment (between 13.5 and 23.5-weeks of age) with either alternative daily Sertuercept (2 mg/kg) or an identical 2 mg/kg concentration of LD..8 scrambled peptide. These animals were part of a neuroprotection experiment and at 14-weeks of age, half in each drug treatment group (n=3) experienced mild traumatic brain injury (via lateral fluid percussion) or sham injury. One rat in each drug assignment group (assigned to mild traumatic brain injury) failed to gain significant weight post-injury and was excluded from the analysis. In the remaining 10 rats (n=5/peptide drug group) 10 weeks’ IP treatment with Sertuercept was associated with a significantly lower mean heart-to-body weight ratio (3.3 mg/g vs. 4.0 mg/g; P =0.02, Figure 3) compared to scrambled peptide treatment. These data suggest that 10 weeks’ alternate daily IP administration of Sertuercept (2 mg/kg) may have reduced the development of cardiac hypertrophy – a serious long-term complication of moderate, uncontrolled hypertension.

Table 5: Baseline characteristics in 13-week-old ZDF rats before chronic peptide administration.

Scrambled peptide (N=6)

Sertuercept (N=6)

P-value

Body weight (g)

374.2 ± 24.7

 385.8 ± 34.2

0.55

Capillary glucose (mg/dL)

442 ± 54

 381 ± 120

0.32

Results are mean ± SD. Capillary glucose concentration was determined by tail nick method as previously reported [16].

fig 3

Ten weeks treatment of male ZDF rats with alternate daily SN..8 (2 mg/kg) (vs. scrambled peptide) was associated with significantly lower heart-to-body weight ratio.

Figure 3: Chronic administration of alternate daily intraperitoneal Sertuercept (2 mg/kg) was associated with significantly lower heart-to-body weight ratio than in baseline matched ZDF rats treated with IP scrambled LD.8 peptide (2 mg/kg). Results are mean ± SEM.

Reno-protective Effect of Chronic Administration of Sertuercept in Male ZDF Rats

Interstitial renal fibrosis occurs spontaneously in male Zucker diabetic fatty rats. In two groups of untreated male Zucker diabetic fatty or lean rats, Masson’s trichrome stain of kidney sections was used as an indicator of severity of interstitial renal fibrosis. There was a significantly greater degree of interstitial renal fibrosis in the twenty-five-week-old male ZDF rat compared to age-matched male Zucker lean rats (Figures 4, 5a and 5b). Interstitial fibrosis is the most important renal lesion in male ZDF rats. It can be present as early as 5 months of age in some male ZDF rats, but in other rats may take a considerably longer time (8-12 months) to develop. In twelve male ZDF rats who were randomized to 10 weeks’ treatment with either Sertuercept or scrambled peptide, one rat in each treatment group was excluded from the analysis because of inherent ‘fast progression’ to renal fibrosis. In the remaining ten rats, male ZDF rats treated for 10 weeks with Sertuercept (vs. scrambled peptide) manifested a significantly lower percentage of glomeruli affected by fibrosis (10% vs. 21.5%; P = 0.03; Figure 6). Representative images of 25-week-old male ZDF rat kidney sections in which glomeruli are affected by fibrosis are shown in Figure 7A and 7B. The pattern of fibrosis is consistent with focal segmental glomerulosclerosis. Glomerular changes associated with collagen deposition in the rat glomeruli included: loss of normal glomerular structure, obliteration of capillaries, increased mesangium, adhesion to Bowman’s capsule and periglomerular fibrosis (Figure 7). Vascular lesions were not easily seen, and there was only minimal interstitial inflammation (not shown in Figure 7).

fig 4

Untreated twenty-five- week old male Zucker diabetic fatty rats experience significantly greater interstitial renal fibrosis compared to age-matched, untreated male Zucker lean rats.

Figure 4: Male Zucker diabetes fatty rats (N=3) or Zucker lean rats (N=6) who did not receive chronic administration of blood-pressure lowering or control peptide(s) were sacrificed at 25-weeks of age for renal histopathologic examination. Interstitial renal fibrosis-the most important indicator of renal disease in the male ZDF rat was significantly increased compared to age matched male Zucker lean rat.

fig 5a

fig 5b

Figure 5: Representative 10X Massons trichrome stain of kidney from age-matched, 25-week-old Zucker lean (A) or Zucker diabetic fatty rat (B) showing lack of interstitial fibrosis in Zucker lean strain compared to mild interstitial fibrosis (blue staining, arrow) in ZDF rat kidney.

fig 6

Ten weeks continuous treatment with alternate daily SN..8 (mg/kg) (vs. scrambled Peptide) was associated with significantly less glomerular fibrosis (sclerosis) in 25-week old male ZDF rat.

Figure 6: Chronic administration of alternate daily intraperitoneal Sertuercept (2 mg/kg) was associated with significantly less glomerular sclerosis (fibrosis) than in baseline matched ZDF rats treated with IP scrambled LD.8 peptide (2 mg/kg). Results are mean ± SEM.

fig 7a

fig 7b

Figure 7: Representative 10X (A) and 20X (B) Massons trichrome stain of kidney from a 25-week-old ZDF rat affected by mild glomerular fibrosis (blue staining, arrows).

In the ZDF rat, plasma IgG autoantibodies were previously reported to cause dose-dependent acute neurite retraction in mouse N2A cells downstream of 5-HT2A receptor-mediated PLC/IP3/Ca2+ and RhoA/Rho kinase signaling pathways activation [16]. Here we tested whether plasma autoantibody neurotoxicity (a marker of long-lasting 5-HT2AR activation) was correlated with increased global renal fibrosis (interstitial and glomerular) in 13 male ZDF rats, 5 treated and 8 others not treated with the decoy receptor peptide. There was a statistically significant overall correlation between ZDF rat plasma autoantibody neurotoxicity and increased global renal fibrosis index in the male ZDF rats (Figure 8). Mean urine albumin/creatinine excretion ratio did not differ significantly in 25-week-old ZDF rats who were matched for baseline body weight and glucose concentration prior to assignment to 10 weeks’ alternate daily treatment with Sertuercept vs. scrambled peptide [0.826 ± 0.527 g/g (N=5) vs. 0.818 ± 0.391 g/g (N=6); P =0.98)].

fig 8

Index of renal fibrosis global severity in 25-week-old male ZDF rats is linearly significantly correlated with plasma autoantibody serotonin 2A receptor bioactivity.

Figure 8: Index of renal fibrosis global severity was linearly, significantly correlated with neurotoxicity in plasma (obtained at the time of sacrifice) autoantibodies isolated from the same rats. A 1/100th dilution of ZDF rat autoantibodies was incubated with mouse N2A cells for 5-10 minutes. Acute neurite retraction was determined as described in Material and Methods. Results on acute neurite retraction were compared to global renal fibrosis severity score obtained from histologic examination of the kidney from the same ZDF rats.

Taken together these data suggest that plasma autoantibodies which activate PLC/IP3/Ca2+ signaling leading to acute neurite retraction may contribute (in part) to renal fibrosis which is the most important renal lesion in the ZDF rat. Although the precise mechanism is unknown, Sertuercept may afford renoprotection in part by interfering with autoantibody-induced long-lasting 5-HT2A receptor activation coupled to PLC/IP3/Ca2+ signaling activation in renal glomerular cells.

Discussion

Chronic hypertension is a significant risk factor for the later occurrence of adverse cerebrovascular, cardiovascular and renal outcomes in humans [20-22]. Hypertension, diabetes, and obesity each increases the risk of heart failure occurrence in humans [23]. The global prevalence(s) of aging, obesity and diabetes is expected to increase over the next several decades driving further increase in the global prevalence of obesity-associated hypertension [24].

In the 2015 Global Burden of Disease Study, systolic blood pressure was the leading risk factor accounting for the largest number of global deaths and disability-adjusted life years [24]. In a systematic review and meta-analysis of the cardiovascular effect of blood pressure-lowering, every 10 mm Hg reduction in systolic blood pressure was associated with significant reductions in the risks for major cardiovascular events, coronary heart disease, stroke, heart failure and all-cause mortality [25]. In high risk patients without diabetes (SPRINT), intensive vs. standard blood pressure-lowering was associated with substantially lower rates of cardiovascular death and all-cause mortality [26].

Long-term adherence to anti-hypertensive medication regimen is critical in reducing cardiovascular event occurrence and mortality risk [27]. Yet few once-daily, long-acting anti-hypertensive medications achieved sustain substantial blood pressure-lowering over 24 hours.

For example, in a study comparing valsartan and long-acting amlodipine, four-fold escalation of the initial starting dose of each medication, i.e. valsartan (40 to 160 mg) and amlodipine (2.5 to 10 mg) was undertaken. Even then, once-daily long-acting amlodipine (10 mg) caused a 10% reduction in systolic blood pressure and once-daily valsartan (160 mg) had no significant systolic blood pressure-lowering effect after 24 hours [28]. Single IP administration of (2 mg/kg) Sertuercept caused 19-27% reductions in systolic and diastolic blood pressure after 24 hours.

Sustained, substantial blood pressure-lowering by Sertuercept could lead to improved medication adherence, and perhaps be useful in the treatment of acute severe hypertension (in the emergency room) by obviating the need for costly, hospitalization. Sertuercept was generally well-tolerated except at the highest doses tested (2.5 mg/kg or slightly higher) at which dose a few animals experienced brief somnolence likely secondary to spontaneously-reversible hypotension.

Our observation that ten weeks’ alternate daily chronic IP treatment with Sertuercept (2 mg/kg) vs. scrambled peptide (2 mg/kg) resulted in significantly lower heart-to-body weight ratio (in male ZDF rats matched for diabetes and obesity) suggests Sertuercept may be cardioprotective perhaps in part via a sustained blood pressure-lowering, i.e. hemodynamic, effect. Chronic administration (for 10 weeks) of Sertuercept (2 mg/kg) in the same cohort of rats was associated with significantly less glomerulosclerosis compared to age-matched male ZDF rats treated with an identical concentration of scrambled peptide sequence.

“It is of interest that the global severity of renal fibrosis index in the male ZDF rat was correlated significantly with plasma autoantibody neurotoxicity, neurotoxicity shown previously to be mediated via 5-HT2A receptor positively coupled to PLC/IP3/Ca2+ and RhoA/Rho kinase signaling pathways activation [16]. Plasma IgG from a male ZDF rat that experienced early progression to moderate renal fibrosis (Fig 9A) displayed five-seven- fold greater potency (in neurite retraction assay) compared to IgG in three age-matched male ZDF rats who experienced only mild- minimal renal fibrosis at 25-weeks of age (Fig 9B).  Rat IgG autoantibody-induced acute neurite retraction was dose-dependently inhibited (IC50 = 18 ug/mL) by co-incubation with Sertuercept (Fig 9C), a peptide comprising a sub-region of the second extracellular loop of the human 5-HT2AR located near the orthosteric binding pocket [15] and which normally functions to prevent constitutive receptor activation.

fig 9a,b

fig 9c

Figure 9: Marked difference in potency of acute neurite retraction in mouse N2A cells induced by autoantibodies from A) a 25-week-old male ZDF rat having moderate global renal fibrosis index or B) three 25-week old male ZDF rats, each having mild global renal fibrosis index C) dose-dependent inhibition of bioactivity by Sertuercept (SN..8).

In a prior report, ZDF rat plasma autoantibodies displayed increased binding (in an ELISA) to a linear synthetic peptide corresponding to the second extracellular loop region of the human 5-HT2A receptor [16].  The 5-HT2A receptor peptide binding autoantibodies first appear in the male ZDF rat plasma around 8.5 weeks of age and persist in the circulation until at least 30 weeks of age [16]. In addition to their presence in plasma during the period of development of renal fibrosis, we used protein G affinity chromatography to recover autoantibody from urine in a subset of male ZDF rats that also displayed increased binding in the 5-HT2A receptor ELISA.

Proteinuria is a leading risk factor for chronic kidney disease progression, however, residual risk for disease progression is substantial and may be mediated in part by additional factors associated with chronic inflammation and immune dysregulation.   One such factor may be autoantibodies which target receptors expressed on cells in the renal glomerulus. Identification of the relevant target receptors may lead to novel therapies to reduce residual risk for CKD progression.

In 15 of 20 older adults with chronic kidney disease previously tested (including diabetic and hypertensive nephropathy) plasma IgG displayed mean 3-fold increased binding (vs. background) to a linear synthetic peptide corresponding to the second extracellular loop of the human 5-HT2A receptor. [13].   In one such patient having biopsy-proven hypertensive glomerulosclerosis which progressed to end-stage-renal disease (Fig 10), Gq11/IP3-mediated neurotoxicity in the plasma autoantibodies was dose-dependently inhibited by co-incubation of N2A cells with low microgram per milliliter concentrations of the second extracellular loop region 5-HT2AR peptide (Fig 10).  Co-incubation of the patient’s autoantibodies (100 nM) with a 20 ug/mL concentration of Sertuercept nearly completely prevented (95%) IP3-mediated acute neurite retraction (in vitro) [13].  A 2 mg/kg IP dose of Sertuercept is expected to result in a plasma concentration of roughly 30 ug/mL in the ZDF rat, i.e. sufficient concentration to neutralize the bioactivity in moderately potent 5-HT2AR targeting autoantibodies.”

fig 10a,b

fig 10c

Figure 10: Human hypertensive glomerulosclerosis plasma IgG autoantibody caused A) dose-dependent inhibition of mouse neuroblastoma cell survival (MTT assay) and B) acute neurite retraction in mouse N2A cells which was prevented by co-incubation with low microgram per milliliter concentrations of a linear synthetic peptide having identical amino acid sequence as the second extracellular loop (ECL2) of the human 5-HT2AR C) 10X Masson’s trichrome stain of renal biopsy specimen showing global and segmental glomerulosclerosis (lines) and hyaline arteriolosclerosis (arrow).

The 5-HT2A receptor is expressed on renal mesangial cells where it promotes protein kinase C/MAPK/ERK signaling pathway activation and increased transforming growth factor-beta expression which promotes renal fibrosis and scarring [29]. Prior studies from a number of different laboratories reported reno-protective effects from various 5-HT2A receptor antagonists in animal models of CKD or in humans [30].

The mechanism of action of Sertuercept is not known, but it may differ from other 5-HT2A receptor antagonists. In a recent report, brief exposure of neuroblastoma cells to active, human diabetic 5-HT2AR targeting autoantibodies resulted in significant downregulation of mRNA expression in the G protein-coupled receptor kinase 3 gene (GRK3) [31].

G-protein receptor kinases are a family of serine-threonine kinases which are activated by certain ligand occupied G-protein coupled receptors causing phosphorylation of the GPCR and directing arrestin-mediated receptor desensitization [8]. G-protein receptor kinase 3 has an in vivo substrate specificity for ligand occupied-alpha-1B adrenergic receptor [8] which mediates norepinephrine-induced blood pressure elevation or the ionotropic effect of epinephrine and norepinephrine in cardiac muscle. In a recent study [8], transgenic mice harboring a cardiac specific, constitutively-active mutant alpha1b adrenergic receptor developed cardiac hypertrophy which could be prevented by simultaneous cardiac overexpression of GRK3.

One possible mechanism of action of Sertuercept may be preventing downregulation of GRK mRNA expression by agonist 5-HT2AR autoantibodies thereby restoring normal level of GRK3 mRNA expression in vascular tissues expressing both 5-HT2AR and alpha-1 adrenergic receptor.

Of interest, GRK3 mRNA expression in human lymphocytes was reported to be inversely associated with systolic and diastolic blood pressure [32] perhaps consistent with a regulatory role for GRK3 gene expression in the maintenance of normal blood pressure in humans.

In summary, the decoy 5-HT2A receptor peptide (SCLLADDN) which is identical to a second extracellular loop subregion implicating in promoting long-lasting receptor activation, caused acute and relatively long-lasting significant systolic and diastolic blood pressure-lowering in the Zucker hypertensive diabetic fatty rat. There were no untoward long-term side effects after chronic (10 weeks’) administration, and early data suggested chronic blood pressure-lowering may have been reno-protective and cardioprotective in a small cohort of rats matched for baseline diabetes severity and obesity.

Acknowledgments

Mihal Grinberg and Julia Burton for expert technical assistance. The study was supported by grants from the Department of Veterans Affairs Technology Transfer Program/BLRD (ORD, Washington, DC) and a grant from the New Jersey Commission on Brain Injury Research (Trenton, New Jersey). The views expressed in this article are solely those of the author and do not necessarily represent the position or policy of the Department of Veterans Affairs or the United States Government.

Disclosure

Dr. Zimering is the Inventor on a pending patent assigned to the United States Government pertaining to results presented herein.

References

  1. Landsberg L, Aronne LJ, Beilin LJ, Burke V, Igel LI, Lloyd-Jones D, Sowers J (2013) Obesity-related hypertension: pathogenesis, cardiovascular risk, and treatment–a position paper of the Obesity Society and The American Society of Hypertension. Obesity (Silver Spring) 21(1): 8-24. [crossref]
  2. Hall J, Kuo J, daSilva A, DePaula R, Liu J, Tallam L (2003) Obesity associated hypertension and kidney disease. Curr Opin Nephrol Hypertens 12: 195-200. [crossref]
  3. Esler M, Rumantir M, Wiesner G, Kaye D, Hastings J, Lambert G (2001) Sympathetic nervous system and insulin resistance: from obesity to diabetes. Am J Hypertens 14(11 Pt 2): 304S-309S. [crossref]
  4. Woodiwiss A.J. Norton G.R. (2015) Obesity and left ventricular hypertrophy: The hypertension connection. Hypertens Rep 17: 539. [crossref]
  5. Kenny HC, Abel ED (2019) Heart Failure in Type 2 Diabetes Mellitus. Circ Res 124(1): 121-141. [crossref]
  6. Adlerberth AM, Rosengren A, Wilhelmsen L (1998) Diabetes and long-term risk of mortality from coronary and other causes in middle-aged Swedish men: a general population study. Diabetes Care 21: 539-545. [crossref]
  7. Rossi MA, Carillo SV (1991) Cardiac hypertrophy due to pressure and volume overload: distinctly different biological phenomena? Int J Cardiol 31(2): 133-141. [crossref]
  8. Eckhart AD, Duncan SJ, Penn RB, Benovic JL, Lefkowitz RJ, Koch WJ (2000) Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart. Circ Res 86(1): 43-50. [crossref]
  9. Adams DD (1988) Long-acting thyroid stimulator: how receptor autoimmunity was discovered. Autoimmunity 1(1): 3-9. [crossref]
  10. Jahns R, Boivin V, Hein L, Triebel S, Angermann CE, Ertl G, Lohse MJ (2004) Direct evidence for a beta 1-adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy. J Clin Invest 113(10): 1419-1429. [crossref]
  11. Zimering MB (2017) Diabetes Autoantibodies Mediate Neural- and Endothelial Cell- Inhibitory Effects Via 5-Hydroxytryptamine- 2 Receptor Coupled to Phospholipase C/Inositol Triphosphate/Ca2+ Pathway. J Endocrinol Diabetes 4(4): 10.15226/2374-6890/4/4/00184. [crossref]
  12. Zimering MB (2018) Circulating Neurotoxic 5-HT2A Receptor Agonist Autoantibodies in Adult Type 2 Diabetes with Parkinson’s Disease. J Endocrinol Diabetes 5(2): 10.15226/2374-6890/5/2/01102. [crossref]
  13. Zimering MB (2019) Autoantibodies in Type-2 Diabetes having Neurovascular Complications Bind to the Second Extracellular Loop of the 5-Hydroxytryptamine 2A Receptor. Endocrinol Diabetes Metab J 3(4): 118. [crossref]
  14. Watts SW, Morrison SF, Davis RP, Barman SM (2012) Serotonin and blood pressure regulation. Pharmacol Rev 64(2): 359-88. [crossref]
  15. Wacker D, Wang S, McCorvy JD, Betz RM, Venkatakrishnan AJ, et al. (2017) Crystal Structure of an LSD-Bound Human Serotonin Receptor. Cell 168: 377-389. [crossref]
  16. Zimering MB, Grinberg M, Burton J, Pang K. (2020) Circulating Agonist Autoantibody to 5-Hydroxytryptamine 2A Receptor in Lean and Diabetic Fatty Zucker Rat Strains. Endocrinol Diabetes Metab J 4(3): 413. [crossref]
  17. Kurtz TW, Morris RC (1989) Pershadsingh HA The Zucker fatty rat as a genetic model of obesity and hypertension. Hypertension 13: 896-901. [crossref]
  18. Clark JB, Palmer CJ, Shaw WN (1983) The diabetic Zucker fatty rat. Proc Soc Exp Biol Med 173(1): 68-75. [crossref]
  19. Coimbra T, Janssen U, Grone H, Ostendorf T, Kunter U, Schmidt H, Brabant G, Floege J (2000) Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int 57: 167-182. [crossref]
  20. Werle M, Bernkop-Schnürch A (2006) Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids 30(4): 351-67. [crossref]
  21. Fredersdorf S, Thumann C, Ulucan C, Griese DP, Luchner A, Riegger GA, Kromer EP, Weil J (2004) Myocardial hypertrophy and enhanced left ventricular contractility in Zucker diabetic fatty rats. Cardiovasc Pathol 13(1): 11-19. [crossref]
  22. Mazzaglia, G, Ambrosioni, E, Alacqua, M, Filippi, A, Sessa, E, Immordino, V, Borghi, C, Brignoli, O, Caputi, AP, Cricelli, C, Mantovani, LG (2009) Adherence to antihypertensive medications and cardiovascular morbidity among newly diagnosed hypertensive patients. Circulation 120: 1598-1605.
  23. Bui AL, Horwich TB, Fonarow GC (2011) Epidemiology and risk profile of heart failure. Nat Rev Cardiol 8(1): 30-41. [crossref]
  24. GBD 2015 Risk Factors Collaborators (2016) Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet 388(10053): 1659-1724. [crossref]
  25. Ettehad D, Emdin CA, Kiran A, Anderson SG, Callender T, Emberson J, Chalmers J, Rodgers A, Rahimi K (2016) Blood pressure lowering for prevention of cardiovascular disease and death: a systematic review and meta-analysis. Lancet 387(10022): 957-967. [crossref]
  26. SPRINT Research Group. Wright JT, Williamson JD (2015) A randomized trial of intensive versus standard blood-pressure control. N Engl J Med 373: 2103-2116.
  27. The Japanese Society of Hypertension Guidelines for the Management of Hypertension (JSH 2009). Hypertens Res 32: 3-107. [crossref]
  28. Eguchi K, Kario K, Hoshide Y, Hoshide S, Ishikawa J, Morinari M, Ishikawa S, Shimada K (2004) Comparison of valsartan and amlodipine on ambulatory and morning blood pressure in hypertensive patients. Am J Hypertens 17(2): 112. [crossref]
  29. Kasho M, Sakai M, Sasahara T, Anami Y, Matsumura T, Takemura T, Matsuda H, Kobori S, Shichiri M (1998) Serotonin enhances the production of type IV collagen by human mesangial cells. Kidney Int 54(4): 1083-92.
  30. Kaur, G. Krishan, P. (2020) Understanding Serotonin 5-HT2A Receptors-regulated cellular and molecular Mechanisms of Chronic Kidney Diseases. Ren Replace Ther 6, 25.  https: //doi.org/10.1186/s41100-020-00268
  31. Zimering MB, Delic V, Citron BA (2021) Gene Expression Changes in a Model Neuron Cell Line Exposed to Autoantibodies from Patients with Traumatic Brain Injury and/or Type 2 Diabetes. Mol Neurobiol. doi: 10.1007/s12035-021-02428-4. [crossref]
  32. Oliver E, Rovira E, Montó F, Valldecabres C, Julve R, Muedra V, Ruiz N, Barettino D, D’Ocon P (2010) beta-Adrenoceptor and GRK3 expression in human lymphocytes is related to blood pressure and urinary albumin excretion. J Hypertens 28(6): 1281-9. [crossref]
fig 5

Mitigating Risks from Negative Press through Rapid, Affordable, and Iterated Discovery of Effective, Targetable Messages

DOI: 10.31038/PSYJ.2021342

Abstract

The paper presents a novel, cost-effective, data-driven, rapid and scalable way to deal with crises in any industry where the crisis is the result of easily swayed consumer opinion. We introduce here a system which allows anyone to understand the complexities of a problem by dimensionalizing the problem into four relevant questions, and four answers to each question. This dimensionalization is accomplished through an interactive ‘consulting chat’ which guides anyone through to the deeper understanding by the foregoing deconstruction. The deconstructed elements (answers to the questions), are combined into vignettes, combinations of the answers, presented to a small, cost-effective group of consumer respondents through the web, who rate the combination in terms an aspect deemed relevant (here, the feeling towards the food industry, either negative or positively, respectively). The results are automatically and immediately analyzed, to reveal the contribution of each of the 16 answers, sending the researcher a user-friendly, presentation-ready data in an Excel format. The presentation provides a full analysis of the data, showing the contribution of each answer both to the rating (cognitive), and to the response time to the vignette (non-conscious physiological measure). The presentation further shows the contributions of each answer to the ratings, first by total panel, then by gender, then age, and finally uncovers both two and three new ‘mind-set’ segments, possibly unknown previously, with these segments defined on the basis of patterns of responses to the 16 answers. The approach is illustrated by simple, easy-to-implement study on the controversial topic of the ‘food industry’s responsibilities in the obesity crisis.’

Introduction – lies and today

Proliferating media, and today’s increasingly simple ways to send messages, are mixed blessings. Certainly, information gets passed around. And, in the case of truly helpful information, an increasing number of people have an opportunity to get to that helpful information. The result is an increasingly interconnected world, with the blessing of communications that can be shared to help people.

Today’s communication environment is not, however, an unmixed blessing, as the topic of ‘fake news’ continues to reveal, day after day, local crisis after local crisis. An aphorism often attributed to the pundit and writer Mark Twain, but probably more likely originating with that arch cynic, Jonathan Swift, is ‘a lie can travel halfway around the world while the truth is putting on its shoes.’ In Swift’s original statement, more than three centuries ago, written in the Examiner of 1710.

Besides, as the vilest Writer has his Readers, so the greatest Liar has his Believers; and it often happens, that if a Lie be believ’d only for an Hour, it has done its Work, and there is no farther occasion for it. Falsehood flies, and the Truth comes limping after it; so that when Men come to be undeceiv’d, it is too late; the Jest is over, and the Tale has had its Effect…

(Wikipedia, https://quoteinvestigator.com/2014/07/13/truth/)

How does one combat lies, disinformation, or today’s popular phrase, Fake News? One cannot stop the issue at the source. Yet, it might be possible to fight using information derived from the mind of the citizen, uncovering just what messages are relevant, and just what messages are believable. That is, knowing the mind of the audience ahead of time or during the time of crisis may provide the necessary arms by which to defeat false information, not by power, but by enhanced, knowledge-driven persuasion.

Experiments instead of opinions

The thesis of this paper is that professionals in an industry can prepare for the often-unexpected onslaughts of bad news, fake news, and outright lies by doing their homework ahead of time. The homework begins by identifying the topics which could become the center of controversy. When the time is appropriate, and the situation warrant, all that is needed is an iterative set of small, affordable, focused ‘experiments’, studies on the response to messaging. The messaging deals with the topic. The experiments produce solid knowledge, insights, but beyond the general insight, specific phrases to use, and specific phrases to avoid.

The goal of the experiments is to identify simply ‘what really works’, what produces the ‘right persuasion,’ not just nice to know facts. The approach presented here comes not from today, but from an aphorism of the great doctor, Louis Pasteur, who opined ‘chance favors the prepared mind.’ A modern corollary of this aphorism might be ‘Knowledge casts a light, cauterizing the ghosts of opinions which infect the shadows.’ (Source author HRM)

The notion of being prepared for negative situations is not a new one. It lies at the heart of every expertise. “Practice makes perfect” and other aphorisms, or perhaps platitudes, too numerous to count, are taken as truth, and they are. What is new in this paper is the use of experimental designs of ideas, to ‘dimensionalize’ issues and situations, identify problems, and identify different statements and counterstatements that could be made, all within an empirical, defensible, cost-effective framework. The notion is iterative experiments as tactics, not experiments as sources of grand knowledge. In other words, experiments to answer the issues of the ‘here and now’, in the same frame, ‘here and now.’

The origin of the ideas comes from two sources. In the mid 1990’s, author Jeffrey Ewald began to work with IdeaMap®, the precursor of today’s Mind Genomics. An ‘Early Adopter,’ Ewald explored the use of systematic study of arguments and refutations, first in business with the objective of the client selling something, and then with the objective of the client having a ready-made set of tested communications to use in a crisis.

The second source was legal research done by author Moskowitz and colleagues, at the behest of author (Professor) James Wren of the Baylor State University School of Law. With several colleagues, including R Rex Parris, a noted trial lawyer, Wren encouraged Moskowitz to work with law cases, reducing them to message components, and then test combinations of these messages as arguments in a law case, in order to identify which argument drove the response of the jury in the desired direction. The efforts eventuated in a recently published book on Mind Genomics and the law [1]. Further, Parris has used the approach to win in law cases, including one substantial award of several hundred million dollars, which he attributed to this use of experimental design of ideas [2].

Demonstrating the approach in a vertical – the world of food

We all eat. To the average citizen, food is good, food companies are bad, news about food is available everywhere, often simply interesting content such as favorite flavors of the season or cooking shows with notable personalities. And then there is the not-so-good side. The food companies are under siege for presumably making us obese, for hooking us on salt, sugar, and fat, for destroying the quality of tomato, especially the tomato flavor for the tomato to last longer and shipper better.

The list is endless. The food industry has many issues to face, more coming almost each day. We have to eat. Food is, for the most part, perishable. Little animals and plants like to attack food, ruining it during their effort to survive by poaching on what should be the domain of human beings. And, since food is necessary, and is really for all living creatures, the adaptations we have made to get and consume food manifest themselves in a society where food plays many important roles, some of which are grist for the mill of social and health issues. And so, the never-ending, swirling controversies around food, new ones cropping up every day.

Introducing the steps

Our focus here is on affordable yet ‘industrial-grade’ production of knowledge about the mind of the citizen, specifically what messages convince the citizen about a topic, and what messages do not convince the citizen. It is vital that we create a system to generate the necessary information easily, rapidly, at a low cost, and in a format that can be used by industry sectors and their associated PR firms, not to mention professionals and students alike. Most important, however, is that the information be valid from a rigorous, scientific perspective, and that the approach must be, like Caesar’s wife, above reproach.

To accomplish our goals, we turn to the experimental design of ideas or messages, known by the rubric of Mind Genomics. Mind Genomics has been vetted by peer review in a variety of different areas [3]. The science of Mind Genomics furthermore traces back to well accepted methods in experimental psychology, specifically the pioneering work of the late R Duncan Luce in mathematical psychology [4], and in the ongoing work of Norman Anderson [5]. A Google Scholar® search of the terms ‘conjoint measurement’ and ‘conjoint analysis,’ will bring up many papers of a refereed nature, reaffirming the acceptance by both the academic and business communities.

At its inception, conjoint measurement was a labor-intensive approach. It would soon be simplified and expanded by Wharton Professor, the late Paul Green, in the 1970’s through the late 1990’s [6, 7]. The vision of creating a fast system, self-authoring, inexpensive, and powerful, was introduced by Moskowitz and colleagues at the start of the 21st Century, and the rapid adoption of the Internet [8].

The actual steps have been developed to fit into the form of a smartphone APP, a symbol of today’s focus on fast, easy, connection, and democratic. Thus, as we go through the first example (with results) in detail, the reader will be able to see how the thinking behind the problem immediately transfers into steps that one follows to bring the project to life. We illustrate the approach using an example from the issue of ‘obesity,’ motivated by the oft-heard canard that somehow the food industry is causing obesity by its practices. The fact that obesity is increasing, is clear from statistics, and well-accepted. That is a ‘fact on the ground’. What is not true, however, is the canard. How then do we fight it with a strategy and with knowledge and data from consumers?

Introducing the process and illustrating how it is tailored for everyday problems

Step 1 – Rethink the notion of the scientific ‘project,’ and look at the effort in service of an issue

We all eat. Many are fanatic in their pursuit of healthier lifestyles. As the world uses machinery, cutting down on the expenditure of calories, the natural course of people is to consume more than they need, leading to food-based problems, such as obesity. The food industry, people, orientations about lifestyle, and an increasingly contentious, better-informed, information-swamped, less critical thinking public all combine for a perfect storm. The storm, to mix metaphors, generates a lot of heat, controversy, sometimes light. And all too often a mentality of jousting, fighting, and perhaps most distressing, ‘grandstanding’ in the name of something, whether that something has value or not.

We deal with one topic in depth, obesity, showing what can be done in a matter of a few hours, at low cost. We show how the scientific process need not be long and ponderous, need not be a process which requires months and years of expertise, but rather can be a disciplined way to create the necessary knowledge. The rationale is that we want to show how very straightforward it is to run a relatively simple experiment (or several iterative experiments) in just a few hours per experiment, in order to prepare for onslaughts in the media, whether that is broadcast media, text media, or social media.

There is a subtext to the choice. We are writing this paper as a demonstration that almost anyone can use knowledge to properly address issues raised by adversaries, whether interviewers looking for sensationalism, or simply groups of people putting in potentially incorrect, even inflammatory material. Furthermore, we take our ‘own medicine,’ here, setting up a study easily (in 20 minutes), and running the study. We also show how the study can be set up as a ‘chat,’ further making the science available to those who don’t even need to realize that they are ‘doing science.’

We also show the type of information one can obtain from so-called convenience sample, small groups of individuals, easy-to-find on the internet, or to recruit using a so-called consumer panel supplier. We show that the knowledge obtained comes not from the proper sampling of people (confusing people with the ideas that they have), but rather from identifying clusters of ideas (mind-sets), using the people almost as simply the carriers of mind-sts. In other words, using the language and metaphor of genomics, we are interested in the mental genomes, not in the body which happens for the moment to be carrying the mental genomes.

Finally, we are not presenting the well thought out, magnificently produced set of brochures in the way well-established, richer corporations do. Rather, we are appealing to those in a cash-starved environment, with no resources, no magic ‘white knight’ riding in at the last minute to save the day.

Step 2 – Describe the tool which allows us to create knowledge quickly

The tool to be used is BimiLeap (www.BimiLeap.com). BimilLeap is a reduced version of a larger technology known as Mind Genomics. Mind Genomics, in turn, is a statistics-based research tool, used to conduct experiments in which respondents are presented with systematically varied vignettes (descriptions of ideas of situations), and rate the vignette on a defined scale [9].

Although this instantiation of Mind Genomics appears to be nothing more than a plain-vanilla survey, the reality is that the Mind Genomics interview is really an experiment. The respondent is presented with a set of systematically varied vignettes, the variation being the selection of what individual messages are combined in the vignette to create the totality. By systematically varying the composition of the vignettes, and having respondents rate each vignette as a single test concepts, the subsequent statistical analysis (regression analysis) reveals the contribution of every element or independent variable to the response.

The experiment design embodied in a Mind Genomics study dovetails well with the topics being explored above. The topic is made explicit and concrete by asking four relevant questions, which tell a story. The relevant questions, in turn, are answered by four separate statements for each question or a total of 16 answers or ‘elements.’ The selection of the topic, the four questions, and the four answers to each question remains in the purview of the researcher. Although one is forever plagued by the nay-sayer’s aphorism ‘garbage in, garbage out,’ the reality of the exercise is that one is forced to think. Not everything is garbage, and indeed, with practice, one learns to think critically, with the data upon repeated practice, suggesting ‘less garbage, more substance.’

Step 3 – Example: Addressing the contention that ‘The Food Industry Causes Obesity’

An instructive way to appreciate the approach instantiated by the APP is through a case history of an actual problem. The study that we discuss here was presented to a large audience at the Chicago 2018 Annual Meeting of the Institute of Food Technologists (IFT). The session topic was how to communicate as professionals.

The rationale for the topic came from the ongoing attacks faced by the food industry. The food industry is often accused as active participants in the growth of obesity world-wide. The rationale for such accusations ranges from anger at the R&D efforts which are believed to use any ingredients which make economic sense, to marketing which is accused in the sensationalist press of communicating misleading information to consumers. And then there are those who argue that there is, in some unwritten way, an implicit contract between the company and its consumers for the company to take an active role in the consumer’s health.

How then does the BimiLeap, work in such a situation, especially in the hands of novices, who do not have the resources of a corporation behind them, a corporation with trained lawyers, a well-paid public relations firm, an advertising (or several) advertising agencies, and a legion of consultants? What happens when the research effort to build the requisite knowledge must be accomplished, from start to finish, in two hours or less. How can this be done, with a reasonable number of respondents (30 or more), each participating in an experiment lasting 4-5 minutes?

Observation from dozens of different studies suggest that the selection of the topic is easy. What becomes difficult is the following set of steps, after the topic is introduced.

It is important to keep in mind that the study that we report was set up in less than ½ hour, was easy to implement on the web, could have taken as little as one hour to complete had we used an easy source of respondents rather than asking people to volunteer for free. The study, when completed, generated a full report Excel, within one minute of the end of the study. The Excel file was emailed to the researcher (author Zemel), within two minutes after the end of the study.

Step 1 – Set up the ‘contents’ of the study (Table 1, Figures 1-5).

The set up comprises selecting a name for the study (viz., the topic), creating four questions, and for each question providing four answers. Table 1 shows the questions as one might fully ask them. Figure 1 shows the way they are recorded in BimiLeap (www.BimiLeap.com).

Table 1: The four questions and their answers. The table shows the four questions in actual question format.

table 1

fig 1

Figure 1: Define the name of the study, create four questions, and for each question provide four answers. The figure comes from the report, automatically generated at the end of the study, with all of the relevant set up slides captured as part of the research documentation.

fig 2

Figure 2: Create a third classification question (the first two are age, gender), create an open-ended question for the respondents to complete, and create an orientation page.

fig 3

Figure 3: The rating scale (left panel). Final thoughts of the researcher (middle panel), and number of respondents desired for the study (right panel).

fig 4

Figure 4: The respondent experience showing the classification question (left panel), and then one of the 24 vignettes shown as it would appear on a smartphone (right panel).

fig 5

fig 5 and 6

Figure 5 and 6: The Tino Space Chat. The figure shows the interchange between the researcher setting up the study and the chatbot. The objective is to make the set-up an interactive experience which simulates coaching.

It is as this stage that one is forced to think in a creative way. The selection of the topic, obesity and food, is straightforward. It is not hard to think of a topic, or even to refine our topic. The difficulty begins when it becomes time to ‘structure’ our inquiry, by asking four questions, and providing four answers to each question.

The first ‘stumbling block’ is the decreasing ability to think in a ‘systematic fashion’ We are not programmed in our education to think by asking a set of structured questions, although in reality that’s the easiest way to learn. A child asks questions. Despite the difficulties encountered by the researcher, it is vital that the researcher ‘stick with the script,’ and provide four questions. We simply require that the sequence of questions tell a story.

Figure 1 (middle panel) might have an actual question posted, such as ‘what is the advertising policy of the company?’ rather than just the word ‘advertising.’ Framing the question as a question versus framing the question as a simple statement makes no difference to the respondent, who will only see combinations of answers, and never the see the questions. The role of the questions is simply to facilitate the creation of answers.

From the point of view of science and education, it is becoming increasingly clear that we think easily in general terms, concepts, but have a hard time putting concrete ideas against those general terms. This step, questions, requires thought about STRUCTURE. We found formulating the four cogent question, to be modestly difficult for the researcher. Once the researcher formulates the four questions, a lot of the hard work has been done.

The four questions in Figure 1 are shown as one word each (ingredient advertising, prevention, responsibility, respectively.) The questions are short because they were selected by author Zemel, who had extensive previous experience. Instead of asking ‘what are the ingredients being discussed?’ Zemel simply put down the word ‘ingredients,’ knowing full well that the question is only used to generate the answers. The respondent never sees the underlying questions, but rather only sees the answers. The questions are selected to drive the production of the answers.

For each question, create four phrases which answer the question in different ways. The four answers pose a different challenge. Now, the task is not to think about structure, but rather to think about CONTENT. During the development of the BimiLep APP, it was first unclear how to generate this content. Putting the exercise into the form of question and answer made sense. The next issue was to determine the nature of the answers, how to make them readable, because it would be the answers, more correctly it would be the combinations of answers that would constitute the test stimuli.

The early efforts with BimiLeap ended up with researchers producing long-winded answers, complicated phrases involving different twists and turns of thinking. Others were simple one-word or two-word statements, i.e., very terse responses with little content. Neither type of ‘answer’ worked well. It became obvious that we need to specify how to write an answer. We came up with the suggestion that the answer be single-minded, and no longer than (approximately) 12 words. Formulating one’s thought in this structured manner called into play what might be called one’s ‘mental editor.’ No longer was the researcher grasping for structure in thinking, but rather now forced to provide answers selected with a focus on clarity of expression.

Advertising executives will recognize this step as one of the ways to create good ‘copy,’ i.e., good advertisements. Often, this creative step is described by the phrase ’problem-solution,’ namely pose a problem and then present a solution to that problem.

This step moves beyond the creation of a single, well-executed advertisement, or more correctly, a single, tested, vetted ‘concept,’ from which the advertising is to be created. Instead, the objective of this step is to create a bank of messages to be presented to the respondent in small, easy-to-read combinations. This first step, before creating the combinations, is to create the messages themselves, in a way which allows virtually ANYONE to do the basic creating. The question-answer format has been found by the authors to make the task easier. The question-answer format does not necessarily create brilliant ideas that will convince, but rather the format democratizes the process of coming up with material

Typically, consumer research studies using these approaches allow the researcher to ask many questions about who the respondent is, what the respondent believes, how the respondent has behaved with respect to the topic. The opportunity to acquire so much information about the respondent from the interview often ‘backfires,’ leading to data paralysis. The prevailing, typical attitude is ‘let’s not rush in and get the wrong information.’ Faced with the plethora of choice which potentially might produce analysis paralysis, the speed of the process is jettisoned in favor of ‘getting everything we can.’

BimiLeap was designed to be fast, modestly flexible, and inexpensive. The objective is to identify the ‘answers’, also known as element, which perform best, rather than acquire as much information about the respondent as possible. As of this writing (summer, 2021), BimiLeap acquires only three pieces of information about the respondent (age, gender, and a third question left to the researcher to select). The focus is clearly on the answers, the elements, not on the acquisition of extraneous information which ‘might somehow be useful.’

Figure 2 shows this third classification question. It is a sacrifice, of course, to leave information un-gathered, but it’s more important to have the study run quickly, and to recognize that deep knowledge will emerge when the researcher can do several of these small-scale studies, rapidly and affordably, building knowledge in a sequential, empirical, structured way. And, of course, create a library of responses to ideas, a library that can be searched easily to create even new constructs based upon the pattern of data from a set of somewhat related studies, dealing with one topic, or with a set of related topics.

Figure 1 shows how the app forces the researcher to ‘think through’ the issue by defining the topic, creating four questions, and then as an example, for the question regarding responsibility, provide four answers. BimiLeap forces the researcher into a structured way of thinking, beginning therefore with an undifferentiated issue, then a differentiation forced by the questions, and finally a further differentiation forced by the four answers to each question.

Once the questions are asked and the elements generated, the next steps create a rating scale (Figure 3, left panel), write instructions to have the researcher give some private, archival information about why the study and experiment were created (Figure 3, middle panel), and finally choose whether the study will be run free with data available to the world, or whether the study will be privatized, so that only the researcher knows the study and the data (see Figure 3, right panel).

Rating Scale: It is the rating question which allows the respondent to share his or her feelings. The objective of our project is to obtain a quick, almost intuitive response to the situation or the prospective client, which in this case is the world of food companies. A simple, rating question is:

 Based upon what you just read (the vignette). Please rate your feeling on the scale below

1=Feel very negative about the food industry … 9=Feel very positive about the food industry

Privatization: The privatization option was created to make the approach attractive to researchers who wanted to keep their efforts private, yet still wanted to use BimiLeap to create the data. There are always several constituencies when it comes to research. Everyone would like the research to be free, and instantly available, such as source material from Google about all sorts of information, as well as so-called Big Data. No one necessarily realizes that data ‘cost.’ The plaint is that ‘after all, the data are out there and freely available. How can you charge me for what I can get for free?’ The foregoing plaint is very real, altogether too common, and destined to produce stillborn, meager results if followed. Privatization addresses the need to satisfy the constituency which wants to do the research for their own interests. The default position of BimiLeap is that the data are public, when not ‘privatized.

The policy of BimiLeap is to be free for anyone to use, with the stipulation that the study is ported to the web, and parts available for anyone to see. One can purchase the study results in their entirety for a nominal sum. If the researcher wants to keep the results entirely private, the researcher need only select the ‘privatize option.’

Experimental design, test vignettes, and the respondent experience

We are accustomed to survey research, which asks a question, gets one of several possible answers, and analyzes the frequency with which each answer is provided. The results of this exercise give us a sense of what the respondent thinks about a topic, when asked a single-focus question.

Our experience here was also instructive. Many of us who have come from traditional backgrounds in testing products, but also in testing concepts and advertisements, are accustomed to the conventional practice of presenting one test stimulus, after which the respondent rates that test stimulus on a dozen or more scales, or rating attributes. What happens when one has many stimuli, but only one rating scale? The reason for the many stimuli is obvious; it is in the pattern of responses to the stimuli where the real knowledge lies. And the reason for only one rating scale is that with 24 test vignettes and a limited time, with unwilling or uninterested respondents, it is better to have a short interview than a long one.

Mind Genomics and BimiLeap follow a different path because they emerge from the heritage of experimentation, not the heritage of surveys. The respondent is presented with a set of elements, in our case a set of answers to the questions. The questions do not appear. The task of the respondent is to inspect this seeming random combination of messages (viz.,, answers, elements), and assign a single rating to the combination. The task is a bit jarring at first, because the respondent tries to be ‘accurate,’ carefully reading each of the combinations, the so-called vignettes. The combinations comprise different types of information, often information which may be somewhat contradictory, and certainly not put together in the most felicitous prose. Nonetheless, the respondent’s task is to read the test combination, preferably quickly, and assign a ‘gut-level’ reaction, an unintellectualized response.

The effort to force respondents to evaluate these disparate combinations of answers, our test elements, often produces a nervous respondent. Some complain that they don’t want to participate in a study where the ‘test stimuli do not make sense.’ They drop out. The remainder, most participants from a paid research panel, do not drop out, and end up responding in the desired ‘gut-level’ or intuitive fashion.

The aforementioned experimental design dictates the 24 combinations of elements, with the property that the 24 combinations comprise 2-tuples of elements 3-tuples, and 4-tuples, so that each element (i.e. answer) appears equally often, and that no vignette ever comprises more than one answer from the same question The 16 elements, embedded in the 24 vignettes, are combined in a way which makes the 16 elements statistically independent of each other, permitting regression analysis to be performed on the ratings, to relate the rating to the presence/absence of each element.

Finally, each respondent evaluates a different set of vignettes. The underlying experimental design is the same, but the combinations, the structure of the vignette is such that each respondent evaluates a unique set of combinations. This is called a ‘permuted design’ [10, 11]. The permuted design allows the research to cover a wide array of possible combinations, so one need not know anything about the topic, and yet the experiment will quickly reveal the important versus the unimportant elements.

Before the respondent begins to evaluate the vignettes, it is important to tell the respondent a little bit about the study, but not much. The introduction is the orientation page which tells the respondent about the study. The orientation page should be short, to the point, without giving information which could lead the respondent to answer one way or another. The orientation simply tells the respondent the least amount of information. Figure 4 (right panel) shows the orientation, at the top of the screen, and a specific vignette, or combination of elements below, with the rating scale at the bottom.

In terms of the actual user experience, today’s panel participants have moved beyond paper and pencil, and have even moved beyond the traditional computer screen, and onto a smartphone. Smartphones are ubiquitous. In light of the use of smartphones as both communication devices by phone, and as browsers with which to text information and to traverse the net, we created BimiLeap so that it would perform well when the respondent was browsing on a smartphone. Indeed, in today’s world, it’s not even clear whether consumers have computers. It’s best to allow the interview to occur on a smartphone, on the screen. Figure 4 shows the respondent screens, set up for a smart phone.

Improving the set-up experience by means of Chat

initial experiences with BimiLeap suggested that the actual process would be fairly simple. That suggestion turned out to be more optimistic than was the case. The early uses of the APP were made by individuals with a great deal of experience in setting up studies for Mind Genomics. When others began to use the APP it soon became obvious that their experience was far less smooth than was thought at the start of the process. It became obvious that a novice researcher, doing the Mind Genomics process for the first time was facing a host of problems, most of which were more complex than frustrating than had been the case before.

The problems encountered by those setting up both the obesity study and others ranged from hard-to-understand instructions about what it means to ‘ask a question,’ to ‘provide an answer,’ and even to create rating scales and open-ended questions. Quite simply, what appeared simple to experienced researchers required ‘coaching,’ and perhaps even more.

Author Savicevic suggested that the process move in a direction made possible by a chat bot. We are all becoming increasingly with chats, which engage in a simulated conversation. The chat was designed by the staff at Savicevic company, Tino Space, to act as a more intuitive, chat-based acquisition. Figures 5 and 6 show the set-up process, this time by a chat. The chat set-up, now in refinement, appears to be a substantial improvement in the user experience, at least for the researcher who has to do the set-up work.

The researcher experience – automatically-analyzed results in presentation-ready format (Figures 7-8)

fig 7

Figure 7: Full report in PowerPoint format.

fig 8

Figure 8: Information from the Excel file.

Today’s world operates on bites, small bits of information, presented in an entertaining fashion, or at least in a fashion which allows the information to be assimilated quickly. Rather than reading papers, even in areas which are very relevant, many people prefer to have the information presented in a manner that is exemplified by PowerPoint®. That is, rather than digesting the information for themselves from detailed text requiring thinking, many people prefer to have information ‘spoon-fed’ to them in a way which allows them to grasp the most important information.

In light of the emerging desire for fast, easy-to-understand information, called ‘the bottom-line’ or the ‘top-line,’ respectively, we have arranged BimiLeap to generate its own pair of reports, the first being a PowerPoint comprising all the relevant information (see Figure 7), and then an extensive, formatted Excel file with the relevant information, the summarized tables, and the raw data prepared for additional analysis (see Figure 8).

The report is a presentation-ready PowerPoint. The PowerPoint report is emailed to the researcher immediately after the close of the project, with the typical receipt of approximately one minute (by email). All the information in the report is either boilerplate or dynamically generated from the particular study being analyzed.

Accompanying the PowerPoint report is an excel file, also formatted, and presentation ready. The information page in Figure 8 is only one tab of a multi-tab Excel file, allowing the researcher to do further analysis of the data with other statistical analysis programs.

Part II – Explicating the resulting data – The food Industry and the issue of obesity

Illustrating how a study becomes one block in a scalable knowledge warehouse

An instructive way to appreciate the approach instantiated by the APP is through the data from our history. The study explicated here was presented to a large audience at the 2018 Annual Meeting of the Institute of Food Technologists (IFT), July 2018, in Chicago. The issue of the session was how to communicate as professionals.

The specific topic selected addresses the issue of combating ongoing negative press from activists and others who present their points of view in a variety of formats. The food industry is often accused as active participants in the growth of obesity world-wide. The rationale for such accusations ranges from anger at the R&D efforts which are believed to use any ingredients which make economic sense, to anger at marketing which is accused in the sensationalist press of communicating misleading information to consumers. And then there are those who argue that there is, in some unwritten way, an implicit contract between the company and its consumers for the company to take an active role in the consumer’s health.

How then does the, BimiLeap, work in such a situation, especially in the hands of novices, who do not have the resources of a corporation behind them, a corporation with trained lawyers, a well-paid public relations firm, an advertising (or several) advertising agencies, and a legion of consultants? What happens when the effort must be done expeditiously, to address a problem, and find the necessary messaging? The word ‘expeditiously’ means that there is relatively little time, perhaps a day or two at most, allowing perhaps one to six affordable, rapid iteration, each iteration totally complete, from start to finish in at most two hours. The challenge is to develop this approach, so that in 12 hours one can be fairly certain what to say and do, at least in terms of messaging.

This study, executed rapidly, shows what can be now done (2021), in as short a time as 1-2 hours, using a base of 30 respondents. A base of 100-200 respondents, easy to find on the Internet with online panel providers would take about the same time, approximately 1-2 hours at most. Note that the experiment from the point of view of the respondent takes about 4-5 minutes.

The foregoing sections have shown the set-up of this particular study on obesity and the perceived role of the food industry. We now move to the results, which emerged from the execution of the study with this small number of respondents. Just what emerged? And what conclusions can be made? The reader should keep in mind that the 32 respondents need not be the total number. There could be 132, or more respondents, just as easily. The question is really whether 32 respondents provide enough basic information, and whether one would be better off with the same study comprising 100 respondents, or say three studies, building upon each other, with say 32 respondents each. The answer to the foregoing question is not within the purview of this paper, other than to say that the opinion of the authors is that more studies, not more people for one study, provide a better strategy, assuming of course that there is a requisite minimum number of respondents for any one study.

Data analysis by building a model

The benefit of using the experimental design strategy emerges from the more powerful analytics which deconstruct the combinations, the vignettes, to the contribution of the individual elements, the messages or answers in Table 1. A paragraph is often more realistic than a single element, with the single element providing very little context. The respondent cannot ‘game’ the system, because too many different messages are present. This ‘blooming, buzzing confusion’ forces the respondent to assume a much less intellectualized role, and in turn to respond at almost a ‘gut level,’ the ‘System 1’ response so popular now in research thinking [12].

The actual ratings assigned by the respondent are transformed to a binary scale. The rationale for the binary scale, and thus the rationale for the transformation, is that managers and indeed almost everyone, more readily understands the meaning of a binary no/yes response, versus understanding the ‘meaning’ of a rating scale. Thus, when one is asked to rate ‘feeling about the food industry,’ the answers ‘feel positive’ versus ‘feel negative’ are easier to understand that the graded rating of ‘positive to ‘negative.’ We naturally gravitate to binary response, to no versus yes. For this study, ratings of 1-6 were transformed to 0, ratings of 7-9 were transformed to 100. Afterwards, the program added a very small random number to each transformed value, a value around 10-5. The random number ensures that the OLS (Ordinary Least-Squares) regression modeling incorporated in BimiLeap system does not ‘crash, when creating the model for the group, or the 32 individual respondent models, even when a respondent limits all the ratings of the 24 vignettes either to the low range of 1-6 (transformed to 0), to the high range (transformed to 100).

BimiLeap also measured the number of seconds from the moment that the test stimulus (combination of elements) appeared on the screen to the time that the response was made. BimiLeap then deconstructed the response time to the contributions of the individual elements, revealing what elements were processed more quickly (shorter response times), and what elements were processed more slowly (longer response times). The response time data warrants a totally separate paper by itself, and thus will not be reported here.

The experimental design and its permutations ensure that OLS regression can build an equation either for each person separately (individual-level model), and a model incorporating the data from many individuals, ranging from the total panel to a group incorporating, for example, only males versus only females (grand models). The specifics of experimental design and regression are well known and accepted in the academic and scientific communities [13].

At either the individual respondent level (so-called individual-level model), or at the group level (so-called grand model, BimiLeap computes the coefficient of a simple regression model, written as: Transformed Rating (Binary + random number) = k0 +k1(A1) …. k16D4). The small random number added to the transformed rating ensures that the OLS, ordinary least squares regression, will not crash, even though a particular respondent may have confined all 24 ratings to the range 1-6, or to the range 7-9. Confining the ratings to that range will result in all 24 cases for regression having the same value, 0 or 100, respectively. The small random number makes sure that such a situation will not a happen, for when that situation happens, the OLS regression ‘crashes.

Table 2, specifically the first set of numbers, shows the results from the total panel of 32 respondents. The data in Table 2 are sorted by the coefficient for the total panel. Despite what one might call a ‘thin sample,’ i.e., rather few people, even this small sample shows dramatic differences in the degree to which a specific element can drive a positive versus a negative feeling about the food industry.

Table 2: Parameters of the model relating the presence/absence of the elements to the binary variable ‘Feel positively about the food industry.’ Only positive coefficients are shown for the element.

table 2

Table 2 shows the data from total panel, gender, age, and then mind-set segments, to be discussed in the next section. The specific data are not the focus, as much as the structure of the data, and what the data provide.

  1. The additive constant tells us the basic likelihood that in the absence of elements, the respondent will have a positive feeling towards the food industry (rating of 7-9 on a 9-point scale, in the absence of any elements). The additive constant is a purely estimate parameter from regression analysis but does have this meaning of a ‘baseline.’
  2. Each coefficient tells us the incremental percent of respondents who would be positive (rate the combination 7-9) if the specific phrase, the element or answer, were to be incorporated into the combination. The important coefficients are those 10 or higher. Conventional practice from thousands of these experiments suggests that coefficients around 8 or higher correspond to relevant, meaningful elements in terms of driving external behaviors. The cut-off is not ‘fixed in stone,’ however.
  3. Negative coefficients mean that when the element is incorporated into a combination, we would observe a reduction in the number of respondents who would assign a combination the rating of 7-9. The negative coefficients are not relevant to disclose the pattern and are not shown in Table 2. Rather, the cell is left blank.
  4. The practical implication here is that one rapid study provides a great deal of specific information need to make a corporation ‘smarter’ in its dealings. The aggregation of these studies creates a database with structured form, linking together the corporation, the topic, the response of different people, all in a coherent fashion with numbers that can be compared to each other.
  5. To summarize, the binary variable used as the dependent variable is created by transforming the ratings 7-9 to 100, and the ratings of 1-6 to 0. The rating 7-9 means that the respondent is pro-food industry when reading the vignette. The coefficients and the additive constant can be combined with the sum showing how likely it would be to obtain a rating of 7-9 when the respondent reads the combination comprising the elements whose coefficients are being additive.

Different mind-sets and the opportunity provided through targeted, effective messaging

During the past sixty years, since approximately 1960, marketers and then others have realized that the world does not comprise individuals who think alike. Whereas this statement seems obvious now, it was not always so, especially during the early part of the 20th century and before, when people were classified by ‘who they were,’ not by ‘what and how they thought.’ Marketers have successfully used the notion of ‘segmentation’ to divide consumers into groups, and have struggled, occasionally successfully, to message to these segments in the appropriate ways [14].

Dividing people into like-groups, segmentation, is often done at first by what people are, and by what they do, and occasionally by how they ‘think’ about a topic. With respect to our project here on the food industry and obesity, the researcher might divide the respondents by who they are (age, gender, body type, where they live, etc.), by what they do (e.g., the types of foods they say they eat, the types of restaurants they frequent, etc.), and by what they think (e.g., how do they respond to topics such as who is responsible for behavior, what is important – health vs pleasure, when it comes to food).

Segmentation of the above type, based on general measures about a person, may or may not help us understand the nature of what is important when it comes to communicating about the food industry and its role (or non-role) in the obesity epidemic. One would hope that dividing the population into traditional subgroups, based either on WHO A PERSON IS, or HOW A PERSON THINKS would reveal clearly different ways of thinking about a topic. Table 2 suggests that dividing people by gender or by age may show differences, not necessarily clear patterns which can form the basis of deeper knowledge and thus more effective actions in the world of messaging. Traditional ways of dividing people assume, without proof, that WHO a person IS determines WHAT a person THINKS. It may not be the case, and in general, it is not the case.

A better way to address the problem of mind-set segments comes from creating these segments from the materials closest to the topic. Beyond the conventional breakout of respondents into self-defined groups, BimiLeap performs a ‘k-means clustering’ [15] of the regression coefficients, first constructing two mind-set segments, then constructing three mind-set segments. These segments are called ‘mind-sets’ because they divide the respondents in the study by the pattern of the coefficients, i.e., by the pattern emerging when the respondent is confronted with a specific, targeted, defined, and quite limited situation.

In our case, we create or perhaps really discover underlying mind-set segments of a specific nature, relating to the issue of how one feels about the food industry, based upon the pattern of responses to the 16 messages. Since each respondent generated a set of 17 numbers (additive constant, 16 coefficients, one per answer), it is straightforward to cluster the respondents based on the pattern of the 16 coefficients.

For these data the k-means clustering, done on the 16 coefficients for the 32 respondents, suggested either two or three different clusters or mind-sets. Selecting the appropriate number of clusters is a subjective matter. The authors’ criteria are parsimony (fewer clusters are better), and interpretability (the clusters should tell a meaningful story).

The two-mind-set solution suggested one group interested in the food industry as a purveyor of ingredients, and the second group interested in the food industry as responsible for the well-being of its customers. Here are the strongest performing elements for the two-cluster solution, i.e., the solution which suggests two mind-sets.

For the purposes of this chapter, it is important to note that the performance of these elements is dramatic, +15 or higher, meaning that dividing people into mind-sets, viz., how they think, produces more knowledge and possibly better actions than simply dividing people by who they are. Such high coefficients almost never occur when these studies divide respondents by who they ARE, for the simple reason that a typical subgroup of individuals who LOOK SIMILAR from the outside often think in many different ways, ways, never be fathomed by simply knowing who a person IS.

Mind-Set 1 of 2 – Focuses on corporate moral responsibility to SELL better ingredients.

B2 Works to improve labels which educate consumers. Shows just what they are buying 23

A1 Tries sincerely to include the best/healthiest ingredients 22

Mind-Set 2 of 2 – Focuses on the corporate responsibility to EDUCATE consumers in healthy eating

D1 Take active responsibility for its customer’s health, and encourages with education and offerings 41

D2 Takes some responsibility for customer’s health, does some reformulation to make offerings more healthful 34

B2 Works to improve labels which educate consumers. Shows just what they are buying 25

D3 Operates fairly, but talks about the need for the customers to take charge..  outreach of education only to customers, schools 25

C1 Supports and funds health-related efforts which educate consumers to eat healthfully 23

The three-mind-set solution suggests that the original Mind-Set 2 of 2 (the food industry is ‘responsible to EDUCATE,’) really comprises two smaller groups, Mind-Set 2 of 3 feeling that the food industry is only partly responsible for the customer’s health/well-being for food, and Mind-Set 3 of 3feeling that the food industry and the customer are partners in the customer’s health-well-being.

Mind-Set 1 of 3 – Focuses on corporate moral responsibility to SELL better ingredients.

A1 Tries sincerely to include the best/healthiest ingredients 29

A3 Uses ingredients with a focus on taste, nutrition, targeting and business 20

B2 Works to improve labels which educate consumers. Shows just what they are buying 19

Mind-Set 2 of 3- Focuses on corporate moral responsibility to EDUCATE consumer.

B2 Works to improve labels which educate consumers. Shows just what they are buying 50

C1 Supports and funds health-related efforts which educate consumers to eat healthfully 32

B1 Since efforts to PREVENT member companies from targeting susceptible populations. Children, obese, low-income 31

D1 Take active responsibility for its customer’s health, and encourages with education  and offerings 25

C2 Partners with government, and cooperates in programs to fight obesity 23

Mind-Set 3 of – Focuses on corporate moral responsibility to SELL better ingredients AND EDUCATE.

D1 Take active responsibility for its customer’s health, and encourages with education  and offerings 44

D2 Takes some responsibility for customer’s health, does some reformulation to make offerings more healthful 38

D3 Operates fairly, but talks about the need for the customers to take charge … outreach of education only to customers, schools 35

The three-cluster solution may or may be useful. Certainly the two-cluster solution helps us a great deal to understand deep differences in the attitudes of the population. Cluster analysis, recall, is simply a heuristic to divide objects, here people, into homogeneous groups, homogeneity defined by mathematical criteria. It is the job of the research to choose the most parsimonious number of mind-sets. The goal is interpretability (the segments must be dramatically different in terms of that to which they respond), and parsimony (the ideal is as few mind-sets as possible.

We have focused on uncovering potentially new-to-the-world mind-sets, simply by running the Mind Genomics experiment. How, then, can the practitioner make use of this discovery? Before we finish up with the last topic, another new discovery, response times (engagement time), let us remain with the discovery of the two mind-sets. The PR strategist working for the food industry, or even a scientist interested in the minds of consumers, might wish to move beyond simply discovering the mind-sets. The practical application of the knowledge tells the food professional in the industry what to emphasize. But what should the professional say to a new person, a person not known beyond basic information about WHO and PURCHASE BEHAVIOR?

Recently, authors Moskowitz and Gere have created a system called the Personal Viewpoint Identifier, or PVI. The objective of the PVI is to create a small questionnaire, six questions long, the pattern of responses to which assign a person to the correct mind-set, with a probability substantially greater than change, although not perfect.

Figure 9 shows the PVI as seen by a respondent, after the PVI is set up. The actual PVI program can be accessed at www.PVI360.com. The research need only put in specified information emerging from the Mind Genomics study, information readily available from the Excel results (see Figure 8). Figure 9 shows three panels.

The left panel acquires relevant information about WHO the respondent IS

The middle panel allows the respondent to answer up to four relevant questions

The right panel presents the six questions in randomized order, these elements selected automatically by the PVI to maximize the chances of a correct assignment.

The PVI is sent out by link, the PVI is done by a person, and the feedback (mind-set membership, feedback information etc.) is sent both to the person and to a database under the researcher’s control. In this fashion, the mind-sets emerging from the study move from ‘nice to know’ facts to a way to optimize messaging to a given individual even immediately after the PVI have been completed.

Discussion & Conclusion

The project of science, and the process empowered by Mind Genomics and BimiLeap

Science, as commonly taught in schools, relies on the isolation and study of variables. There is an often- unstated belief and practice that careful, meticulous, painstaking work is the preferred approach to science. That which we know, according to such worldviews, must be obtained with care, and analyzed with precision. It may be acceptable in the everyday real world to do things ‘in the moment,’ but real science must be done with agonizing precision in order to be perceived as relevant and meaningful.

The research approach promoted in this article moves in a different direction. The notion is that with a reasonably powerful process to acquire knowledge and to understand the world, it may not be necessary to be as meticulous. In fact, the reality of the everyday is that we function quite well by grazing information, rather than sitting down to comprehend the information.

Corporate (and other) learning gained in short, easy-to-do, scalable arrays of studies

The most important outcome of this study is ‘what did we learn,’ and ‘was it worth the effort?’ We can answer those two questions easily. In additional to answering the question for the total panel we discovered new-to-the-world mind-sets with respect to an issue of obesity as a social issue linked with the food industry, Discovering mind-sets in the way we did it, with very few respondents (32), means that we are using the research to uncover sets of ideas which ‘travel together.’ We are not studying the entire world to discover these mind-sets, but rather using a convenience sample to discover the existence of the mind-sets. The distribution of such mind-sets in the population remains an issue to be addressed by larger-scale studies, powered by the PVI, the personal viewpoint identifier.

What is important to keep in mind is that we have established of mind-sets, done with a small sample of randomly chosen individuals, differentiating that discovery from the equal important but quite different question of these segments distribute in the population, another question that we can address, given a parallel piece of easy-to-use software.

There is a second outcome. That is the possibility learning a ‘lot more by simply doing a lot more.’ The evolution of science has been to increasingly larger, more expensive, more ponderous studies. That may be the case for the natural sciences and the physical sciences. As yet, it is not the case for applied science dealing with those topics where ‘the mind of man about external topics is king.’ Where topics can be broken up into aspects, dimensionalized, and studied with small, convenience samples, the Mind Genomics process will flourish, powered by BimiLeap and PV3I60.

Finally, anyone can become a researcher, not just those with advanced degrees, funding, and the permission of an IRB, Internal Review Board. We can envision an entire world of young and not so young researcher, in school, in companies, in play groups, all adding to the sum of knowledge through these experiments.

Envisioning one possible future of knowledge-building made possible by Mind Genomics

The efforts made in this study are, in actuality, quite simple, and the execution expedient. The objective of the effort was not to study the problem in detail, producing an archival document which investigates the problem from many different aspects. This latter approach characterizes much of today’s science, namely the attempt to define the parameters of the problem, and proffer a solution through experimentation, with one solution for one problem added to one solution to another problem, until the general issue is somewhat solved, or at least illuminated by the concatenation of these often-disparate solutions to a set of connected problems.

The approach presented here moves in a different direction, presenting the approach as a standardized method for create knowledge about a topic. There is already the ingoing assumption that the solution will not be perfect, that the range of the aspects studied will be limited, and that the data will be obtained through a small sample of respondents, not a large sample. We can liken this approach to an MRI of the mind, as the mind deals with various topics. Each study, comprising four questions, sixteen answers, perhaps 30-50 respondents, can be likened to one MRI photograph. The brain, or in our case, the mind, does not come into view and cannot be understood by one snapshot, one study alone. Rather, it is the buildup of the snapshots, the dozens, hundreds, thousands, tens of thousands of studies, and perhaps more, which begin to produce a general outline of what’s happening in the mind. Each snapshot alone is a minor effort, but the array of snapshots put together becomes the MRI of the mind, the picture of the mind, taken through this simple, scalable, affordable approach.

Acknowledgment

AG thanks the support of the Premium Postdoctoral Researcher Program

References

  1. Moskowitz H, Wren J, Papajorgji P (2020) Mind Genomics and the Law. 1st Edition. LAP LAMBERT Academic Publishing.
  2. Parris RR (2009 ) Jury Hands Down Stunning $370 Million Verdict Against Georges Marciano, GUESS? Inc Founder and Art Dealer, Announces R. Rex Parris. GES| 7/28/2009 5:21:29 PM LOS ANGELES, Jul 28, 2009 (Globe Newswire via COMTEX News Network).
  3. Moskowitz HR, Porretta S, Silcher M (2005) Concept Research in Food Product Design & Development. Ames, IA: Blackwell Publishing Professional.
  4. Luce RD, Tukey JW (1964) Simultaneous conjoint measurement: A new type of fundamental measurement. Journal of Mathematical Psychology 1: 1-27.
  5. Anderson NH (2001) Empirical Direction in Design and Analysis. Scientific Psychology Series. Routledge. Taylor & Francis Group New York.
  6. Green PE, Krieger AM (1991) Segmenting markets with conjoint measurement. Journal of Marketing 55: 20-31.
  7. Green PE, Srinivasan V (1990) Conjoint analysis in marketing: new developments with implications for research and practice. Journal of Marketing 54: 3-19.
  8. Moskowitz HR, Gofman A, Itty B, Katz R, Manchaiah M, et al. (2001) Rapid, inexpensive, actionable concept generation and optimization – the use and promise of self-authoring conjoint analysis for the foodservice industry. Foodservice Technology 1: 149-116.
  9. Moskowitz HR (2012) ‘Mind Genomics’: The experimental, inductive science of the ordinary, and its application to aspects of food and feeding. Physiology & behavior107: 606-613. [Crossref]
  10. Gofman A, Moskowitz HR (2010) Application of isomorphic permuted experimental designs in conjoint measurement. Journal of Sensory Studies 25: 127-145.
  11. Moskowitz HR, Gofman A (2004) System and method for performing conjoint measurement. Provisional patent application, 60/538,787, filed.
  12. Kahneman D (2011) Thinking, fast and slow. New York: Farrar, Straus and Giroux.
  13. Box GEP, Hunter J, Hunter S (1978) Statistics for Experimenters. New York, John Wiley.
  14. Claritas (1999) “PRIZM Cluster Snapshots: Getting to Know the 62 Clusters”.
  15. Jain AK, (2010) Data clustering: 50 years beyond K-means. Pattern recognition letters 31: 651-666.
fig 7

Choosing a Banquet Hall: A Mind Genomics Cartography of the Ordinary

DOI: 10.31038/MGSPE.2021112

Abstract

Respondents each evaluated 24 unique combinations of messages (vignettes) pertaining to the choice of a banquet hall, rating each vignette on a Likert scale for likelihood to choose the banquet hall for their next event, and then selecting one of five emotions to show their feeling about the banquet hall as described. The approach, Mind Genomics, revealed the part-worth contribution of each of 36 messages to both choice (question #1) and to feeling/emotion (question #2). Respondents fell into one of three different mind-sets, based upon their patterns of choice, MS1 – Practical aspects of hiring a banquet hall; MS2 – Focus on cost; MS3 – Focus on experience. Membership in the mind-sets did not correlate with any aspects of the respondent as determined by a self-profiling classification. The paper presents a PVI, personal viewpoint identifier, six questions on a 2-point scale, developed by the mind-set segmentation, which assign a new person to one of the three mind-sets.

Introduction

Anthropology and Sociology

The study of everyday events by psychologists, sociologists, anthropologists and even commercially- oriented market researchers, creates a foundation of knowledge about a society. Researchers observe from the outside, often talk to those in the society, and then describe what they see, often in very readable papers and books, occasionally in charts and statistics. Those who observe people in their own environment, talk to them, record these interactions, and perhaps even substantiate some of the information with charts and tables, provide a very readable account of the people as those people go about their daily lives.

The focus of this paper in on a very simple aspect of daily life, specifically hiring a banquet hall. Rather than presenting this topic from the point of view of statistics or economics such as how many halls are rented, whom, for what reasons, and so forth, we approach it from the perspective of the inner, personal aspect, the thinking which may go on in the mind of the prospect who is about to hire the hall.

Banquets and banquet halls present a plethora of interesting aspects to study. Banquets are ceremonial meals. A number of papers present to us the nature of the banquet in ancient times, as well as the role of the banquet and banquet hall today. This paper is not a review of the extensive literature about banquets and banquet halls other than to recognize the rich academic history that the public feasting has enjoyed, whether the academic work focuses on what happened across history [1,2], on the position of banqueting and banquet halls in society [3-5], and even on the nature of banqueting in different cultures [6-8].

There is a practical aspect as well, today’s economy. High-priced banquet activities at wedding receptions have significantly contributed to the growth in the overall profits of the food and beverage (F&B) departments of hotels [9]. Restaurants are, by their very frequency, more important economically than are banquets. Yet, banquets are critical for the hospitality world [10-14]. Almost 70% of the food and beverage revenue of hotels in the U.S. is generated by banquets. Fifty percent of these profits come from weddings in the United States.

When we turn to the topic of ‘banquet’ in the academic literature, searching through Google Scholar we have many more hits, about 51,000, but many of these are relevant to issues of service quality and customer satisfaction on the one hand, and historical aspects of banqueting on the other. Indeed, banqueting as a topic in and of itself is of less interest than banqueting as a social institution, and how it may fit into the complex of daily institutions in one’s culture, whether that culture be current around the world, or part of the daily events of historical times, such as banqueting during the days of the Roman empire, and how the behavior changed with history. There is very little about the topic of ‘banquet halls,’ however.

Despite the paucity of information about banquet halls (not banquets themselves!) in the academic literature, such as the importance of the different reasons for choosing a banquet hall, there are some relevant papers, such as Ling Guan’s master’s thesis in Iowa State University [15] on ‘Push and pull factors in determining the consumer’s motivations for choose wedding banquet venues: A case study in Chongging, China. Guan introduces the notion of two types of factors for the choice of venue; push and pull. four push factors (“seeking relaxation and knowledge”, “fulfilling prestige”, “escaping from daily routine”, and “social networking”) derived from the extracted 10 push items and six pull factors (“budget”, “atmosphere”, “facilities”, “wedding services”, “transportation”, and “service and quality”). Peng & Wang [16] also talk about the wedding banquet as a set of decision with different strategies for decision making.

The alternative to a rigorous academic study of the topic of banquet halls is a study of the commercial banquet hall, how the hall is used, how the hall is chosen, the position of the banquet hall in everyday life. One need only look at the local newspapers of a community, at the websites of business in that community, or just drive around the main streets to get a sense of the nature of the banquet facilities that are offered, how these are presented to the prospective customers. In the same spirit, one can request a market research study about attitudes and practices with regard to banquet halls, or even commission a focus group of banquet hall customers or prospects to discuss their experiences with, and their attitudes towards banquet halls. No doubt such commercially relevant studies have been done, not so much for publication as for business use, to know, for example, how to present one’s banquet hall to customers to increase business.

The Internet is an increasingly popular source of knowledge about daily people, especially because the content of the Internet matches the interests of those who search for information. Popular topics like health have hundreds of millions of sites. When we look for the phrase “banquet halls’ in Google, we end up with approximately 46,000 hits as of July 30, 2020. People also search for the practical aspects of banquet halls such as ‘what are the different types of banquets?, ‘In a banquet hall a good business, what is a banquet hall?’, How do banquet halls charge?’, What happens at a banquet?’, ‘What is a banquet menu?’, and so forth. These are the relevant questions. The websites which address these questions are usually commercial sites, providing an answer to the question, and then offering a particular banquet-related service to the person who is doing the searching.

A good idea of what can be found for in a search for a banquet is the following:

Banquet halls are among the most popular types of venues for events, particularly wedding. The popularity stems from the benefits these halls bring such as stress-free planning for families and couples. These banquet facilities are typically all-inclusive which means a lot of the little details are covered such as catering, seating, decorating and so much more.

https://www.uniquevenues.com/banquet-halls

Applying Mind Genomics to study what appeals to a person asked to choose a banquet facility

Banquets provide a topic which can contribute to the understanding of choice for celebratory events, events that are affordable and which move beyond the everyday dining behavior that one does at a restaurant. Studying the choice of options for a banquet brings into play the recognition of clearly different factors influencing that choice. The different aspects range across topics such as the nature of the information to which people pay attention when choosing a banquet, the influence of the person who is presenting the information, the income of the person giving the banquet, the choice of banquet amenities, and so forth. The reality is that the academic literature on both hospitality and choice, respectively, recognizes the importance of such information, but there is little to be found which treats the options of choice in a banquet hall in a rigorous fashion.

As the 21st century progresses, and with the plethora of available methods to measure and to analyze, what is missing is a deep psychological understanding of the everyday. We can study the economics and sociology of banqueting, either by reading papers in the scientific literature, by immersing oneself in the veritable flood of advertisements in the popular press and Internet, or from going through the verbatims of in-depth interviews with consumers or the mountains of survey results run by market researchers. Yet, unless there is a sharp focus, one will obtain a great deal of ‘data’ but not understand the ‘mind’ of the customer of a banquet hall. The topic is simply too limited for today’s scientist.

What is needed is a fast, inexpensive, knowledge-creating system to understand any topic where judgment is key, where the data are scarce and where the experience is widespread. Mind Genomics is that key. Mind Genomics was borne out of the desire to understand the mind of the person as that person lives life, doing so in the spirit of Weber to study the ‘whole’ but also in the spirit of experimenting science. Mind Genomics is a branch of experimental psychology, one informed by consumer research in the world of the applied, informed by the statistics of experimental design, and inspired by the images of the MRI (magnetic resonance imagery) in medicine which takes pictures of tissues from different angles, and inspired by genomics which focuses what drives individuation [17-19].

Mind Genomics begins with notion that people may not know what they are thinking but will know It when they see it. The strategy of a Mind Genomics experiment is to combine elements, messages, descriptions of various aspects of the topic (banquet halls), create small vignettes whose composition is known, present the combinations to respondents, obtain a response to the combination, and then estimate the degree to which each element in the combination drives the response. The experiments are quick, affordable, informative, and archival. Most of all, the experiments are targeted to answer question in a direct fashion.

We show the use of Mind Genomics to study banquet facilities, beginning with the topic (Banquet facilities), moving through the selection of questions and answers, and then on to the actual study, the data and analysis, the discovery of mind-sets even within this limited world, and finally the creation of a tool to discover these mind-sets in the population. The demonstration study, which can be developed and run in less than a day, provides the potential to generate a ‘Wiki of the Mind’ for topics relevant to society, in different cultures, different situations.

Explicating the process through the study of choosing a banquet hall

Step 1 – Choose the topic, questions, and answers

The topic is banquet halls. In this particular version of Mind Genomics, we deal with the 6×6 design, comprising six questions, and six answers to each question. Table 1 shows the six different questions, and for each question six different answers. The questions and answers are left to the researcher(s). The questions and answers emerge after about 20-30 minutes of brainstorming among a group of four researchers who have never experienced Mind Genomics research before. Mind Genomics has been developed to promote rapid iterations, so one need not spend a great deal of time thinking about the questions and answers, and perhaps even overthink.

Table 1: The raw material for the Mind Genomics study of the banquet hall; six questions and the six answers to each question

table 1(1)

table 1(2)

The objective of Mind Genomics, the creation of the aforementioned ‘wiki of the Mind’ requires that the test stimuli be relevant for everyday life, and not simplistic statements of the type that one would not typically encounter. That is, the stimuli in Mind Genomics are of the type that would be appropriate for the quotidian, commercial and social aspects, rather than artificially created stimuli, manipulated so that the responses to these artificial stimuli would be able to support a hypothesis about behavior, or disprove it. Thus, one can look at the material in Table 1 in the light of an ethnographic report of the types of messages one might encounter in every-day life.

A key aspect of Mind Genomics is the reality that one need not be ‘right’ at the start of the Mind Genomics experiments. Whereas in most research the effort requires a great deal of planning, the selection of the ‘correct’ test stimuli, the appropriate scales, and the appropriate respondents, the science of Mind Genomics was created to more realistically simulate the exploration of the everyday, where one does not weigh the alternatives in a carefully considered manner, in the fashion called System 2 by Nobel Laureate Daniel Kahneman [20]. Rather, the typical approach is the more automatic approach, called System 1, or in the thought of Harvard psycholinguist George Miller, TOTE, Test, Operate, Test, Exit [21]. One need not be exact to study inexact behavior. One need only be systematic, consistent, affordable, fast, and scalable to create archivable, solid knowledge.

Step 2 – Combine the elements into short, easy to read vignettes, or test concepts, each vignette comprising a maximum of four elements, and a minimum of two elements

Figures 1 and 2 show the same vignette, comprising four elements. The elements are placed one atop the other, left justified, set up to be easy to read. No effort is expended to tie the elements together.

fig 1

Figure 1: Example of a 4-element vignette, showing the first rating scale (likely to choose)

fig 2

Figure 2: Example of the same 4-element vignette, showing the second rating scale (select the emotion)

The vignettes are created by an underlying statistical plan called an experiment design [22]. The design for the 6×6 (six questions, each with six answers), comprises 48 vignettes, each element appearing five times, and absent 43 times. A vignette can have at most ONE element or answer from a question. This particular property of incompleteness is necessary to ensure that the 36 elements are statistically independent of each other.

Each respondent evaluates a unique set of 48 vignettes, different from the set evaluated by the other respondents. This strategy is known as a permuted design [23], ensuring that the combinations different across respondents. The strategy is to uncover the underlying pattern in the data (viz., how the elements drive the responses) by testing many combinations only once and putting together the pattern from these many combinations. The strategy is similar to the manner of the MRI (magnetic resonance imagery), which takes many pictures of an object from different angles and combines these by computer to create a 3-dimensional image. The Mind Genomics strategy differs dramatically from the conventional approach of selecting a limited set of vignettes (e.g., 48), to represent all combinations, and then testing the same 48 vignettes with many respondents to average out random error.

Step 3 – Invite the respondents to participate, and collect the ratings

Each respondent is invited through an on-line panel company, specializing in these types of studies. It is important to work with a panel provider because otherwise the response rate is low, and the study may take a week to complete, rather than the more normal hour or two. Panel companies have lists of respondents who have agreed to participate. Most panel companies can tailor the list of respondents by a variety of criteria ranging from simple geo-demographics to self-stated attitudes and behaviors. For this study, the criteria were general, since the focus was on ideas appealing to the general public. Figure 3 shows the introduction to the vignettes, including the scales on which the vignettes will be rated.

fig 3

Figure 3: Orientation screen, showing instructions to the respondents, the two scales, and the expected time that will be needed to complete the Mind Genomics experiment. The experiment is positioned as a ‘survey’.

Step 4 – Create the database, to prepare it for regression analysis, and for cluster analysis, respectively

The database for the Mind Genomics study comprises one row for each vignette, for each respondent, respectively. Each of the 57 respondents evaluated 48 unique vignettes, generating a set of 2,736 rows of data. The first 36 columns of data corresponded to the 36 elements, with the database showing the number ‘1’ for those elements appearing in the particular vignette, and the number ‘0’ for those elements absent from the particular vignette. Since a vignette could comprise 2-4 elements, only 2-4 cells contained the value ‘1’, and the remainder contained the value ‘0.’ The 37th column contained the rating 1-9, and the 38th column contained the choice of the emotion.

The rating scale, values shown in the 37th column, was transformed to a binary scale, TOP3, following the practice in consumer research and public opinion studies. It is not easy to explain what is meant by any rating scale, unless each point is labelled. Researchers argue over the labelling of each point. An easier way is to bifurcate the scale into a binary scale, with the lower part of the scale (1-6) transformed to 0, and the upper part of the scale (7-9) transformed to 100. The ‘cut-point’ (6,7) is defined arbitrarily. The effect creates a ‘No/Yes’ variable, one easier explain. In order to ensure that it would be possible to run an OLS (ordinary least-squares) regression analysis on the 48 vignettes for each respondent, the Mind Genomics program added a very small random number (<10-5), to ensure some variation in the dependent variable. Otherwise, the transformed ratings from a respondent might all end up 0 or 100, were the respondent to have confined the rating to the low end of the scale, 1-6, or to the high end of the scale.

The ratings in the 38th column, the selection of emotions, were treated in a different fashion. There were five emotions. The rating scale is known as a ‘nominal scale,’ with the values 1-5 standing for different emotions. To prepare the data for analysis by OLS regression requires the expansion of the one scale into five new scales, one scale for each emotion, respectively. The expansion added five new columns of data. The respondent had to select one emotion for each vignette. The newly created variable corresponding to that emotion was coded as ‘100’ plus a very small random number. The remaining newly created variables corresponding to the four emotions not chosen were coded as 0 plus a very small random number. Thus, the five emotions were coded to immediately reveal which feeling/emotion was selected.

At this point the data matrix is ready for analysis by OLS (ordinary least-squares) regression, a well-accepted statistical technique, also colloquially known as curve-fitting. The OLS regression is executed in two steps, first to create 57 individual level models in preparation for clustering to reveal mind-sets, and then run with groups of respondents, defined by who they are (e.g., Total Panel, Gender, Age), and by how they think about the topic of banquets, here specifically three mind-sets. This paper presents only the data from the Total Panel, and from three mind-sets, respectively.

Step 5 – Create an individual-level model for each of the 57 respondents, using OLS regression, and then cluster the 57 respondents into two, and then three groups (mind-sets)

One of the continuing themes of Mind Genomics is that people differ from each other in the way they think about a topic. These different ways are called mind-set. Step 5 discovers these mind-sets.

The experimental design ensures that the 36 elements arrayed in the 48 vignettes are set up to allow for OLS regression, relating the presence/absence of the 36 elements to the binary response (TOP3), which was either 0 (rating 1-6) or 100 (rating 7-9). The OLS model was estimated without an additive constant, called forcing the regression through the origin. Estimating the coefficients without an additive constant will make it easier to cluster the respondents into either two mind-sets or into three mind-sets, respectively. The equation for the individual respondent is written as: Top3 = k1(A1) + k2(A2) … k36(F6).

The OLS regression is run 57 times, one for each respondent. The preparation stage using OLS regression generates the necessary data matrix comprising 57 rows, one per respondent, and 36 columns, one per element. The matrix is then subject to cluster analysis [24]. The cluster analysis puts the respondents into two groups and then into three groups. Respondents in a cluster show similar patterns of coefficients. Cluster analysis simply provides the solutions but does not decide the number of clusters (mind-sets). It is the researcher who selects the number of mind-sets based upon two criteria. Criterion #1 is interpretability. The strongest performing elements must tell a coherent story. Criterion #2 is parsimony. It is better to have fewer clusters than more clusters, given equal interpretability.

Step 6 – Create the model for the Total Panel and for each of three cluster which emerge

The clustering in Step 5 suggested three groups, also known as mind-sets. Two groups or mind-sets were hard to interpret. The final analysis to understand what drives selection of the banquet hall comes from putting all of the RELEVANT data together into one file and running one grand OLS regression on the data. This time the regression equation has an additive constant whose purpose will be explained below.

Right after the OLS regression on the Total Panel come three separate OLS regressions, first on the data from all respondents assigned by cluster program to Cluster 1 (Mind=Set1), second on the data from all respondents assigned to Cluster 2 (Mind-Set 2), and third and finally, on the data from all respondents repsondents assigned to Cluster 3 (Mind-Set 3). The one equation for each OLS regression is expressed as: Top3 = k0 + k1(A1) + k2(A2) … k36(F6). The analysis below will reveal just how radically different are the mind-sets of these groups. The rationale for the additive constant is that in the absence of specific information about the banquet hall there is still a proclivity to choose the banquet hall. The additive constant measures that proclivity.

Step 7 – Create a model for the Total Panel showing the linkage of each element to every one of the five emotions (scale 2)

Recall that Step 4 above, preparing the data for OLS regression, mapped the second rating scale, selection of emotions, to five new binary scales, one per emotion. The data are ready for OLS regression. This time the dependent variables are the five newly created variables, one per emotion, with the values 0 or 100, depending upon the selection of the emotion. The independent variables are the 36 elements. The model is run without the additive constant, this time because in the absence of elements there is no proclivity to choose an emotion, and therefore the additive constant is by definition 0. The reality is that whether we include an additive constant or not, the coefficients of the elements will show the truly strong performing elements.

Step 8 – Create a PVI (personal viewpoint identifier) to assign new people to one of the three mind-sets

Study after study in the world of Mind Genomics reveals that the respondents in the same mind-set fall into different geo-demographic, behavioral, and attitudinal groups. The Mind Genomics study for banquet halls is such a narrow topic that we would not expect the mind-sets to distribute by gender, age, and so forth. Thus, there needs to be a way to assign a new person to one of the mind-sets which emerge, if only to improve the nature of the interaction between sales/manager of the banquet hall and customer hiring the services of the banquet hall for a particular event. The topic of banquet halls is very narrow, and it is quite unlikely that the scientific literature can provide much guidance about how to identify the mind-sets, if even the scientific (or business) literature recognizes the existence. The PVI will be used to create a tool comprising six question which will assign a new person to one of the three mind-sets.

Results

Total panel versus emergent mind-sets

Respondents cannot easily tell the researcher what is important versus what is not important. Yet, the coefficients from the model reveal immediately how strongly each element drives the TOP3, the rating ‘I would choose’.

We begin with the additive constant. The additive constant tells us the conditional probability of a person responding 7-9 on the 9-point scale, viz., I would likely choose this banquet hall from my next event. Keep in mind that most respondents are not in the immediate situation of choosing a banquet hall. We would expect, therefore, that the respondents have not thought about the banquet hall. What is remarkable, but not surprising, is the very low additive constant, 6, one of the lowest for total panel in the many Mind Genomics studies conducted by author HRM. At a basic level the respondents are simply not interested in the banquet hall. The respondents are being honest. They are reading the vignettes and, not being interested in a banquet hall, they respond that they are not interested.

Beyond the additive constant is the contributory power of the different elements. The typical standard errors are around 3-4 for the coefficients of the elements. Table 2 shows the strong performing coefficients for the 36 elements by Total Panel, and by the three mind-sets which emerge, and discussed below. The elements are shown in descending order based upon the three mind-sets. To allow patterns to emerge, we will remove any coefficients lower than +8. We lose some of the fine-grained information, but the patterns more clearly emerge.

Table 2: Performance of the elements by Total Panel and by the three mind-sets. Only strong performing elements are shown, with coefficient of 8 or higher.

table 2(1)

table 2(2)

table 2(3)

table 2(4)

We can sort the elements based upon the Total Panel, and pull out the very strong performing elements, those elements with coefficients of +10 or higher. The coefficient tells us the increased (or decreased) percent of responses of magnitude 7-9 beyond the percent shown by the additive constant when the element is inserted into the vignette. For example, the additive constant is 6 for the Total Panel, meaning that in the absence of elements we expect to see 6% of the ratings be 7-9. Incorporate the element D4, Open Bar included at no extra cost, and we can expect an additional 16% of the responses (viz., 22%) to be 7-9. Choose D3 instead, Only $150 per person for closed event, and we can expect to lose 9% of the respondents, viz., end up with a sum of -3, viz., virtually none of the rating being 7-9. Put both D4 and D4 in together, and we can expect the percent of ratings 7-9 to be 6% (additive constant) + 16% (Open Bar …) – 9% ($150 per person) or 13%.

There are seven very strong elements. They make sense. These are the elements with coefficients of +10 or higher. There is no single ‘theme’ appropriate to these seven elements. They range from open bar to photographer, to flexibility, and so forth. It should be kept in mind that without the Mind Genomics experiment it would be highly unlikely for anyone to be able to predict that each of the seven elements would be perform well. It takes an experiment to reveal the winners.

Open Bar included at no extra cost

Professional photographers upon request

Want to have a fun night out with your friends… Host a theme party at our fascinating hall

Want an unforgettable prom night… Host it here and it will be a night you will never forget

Need a last-minute party? We are fast, fun and friendly

All the information is on our website…only a click away

All rooms are customizable to fit your party size

There are elements with coefficients of 0 or lower. These are not necessarily ‘bad’ but may be either ‘bad’ (ratings of 1-3) or ‘irrelevant’ (ratings of 4-6). We would have to analyze the data from the opposite side of the scale (1-3 transformed to 100; 4-9 transformed to 0). That analysis in the reverse direction would tell us whether the negatives are truly ‘bad’ (1-3) or merely irrelevant (4-6.)

The more revealing results emerge when we consider the nature of the mind-sets, what attracts these mind-sets, and the existence of an underlying theme for each mind-set. Respondents may or may not know about mind-sets. Indeed, unless the topic of banquet halls comes up as a focus of one’s conversation and plans, it is unlikely that one would even pay attention to what’s important about a banquet hall unless challenged to answer. The Mind Genomics experiment will, however, reveal these mind-sets in about 5-10 minutes.

The clustering program (Step 6 above) suggested three groups of respondents, based upon the pattern of coefficients. The three right-most columns of data show the strong performing elements for each of the mind-sets. An element performing well in two mind-sets appears, twice, once for each mid-set in which is performs well. The name to be given to the mind-set comes from the nature of the elements which perform best.

Mind-Set 1, with 33 respondents, has an additive constant of 13. The respondents are not particularly interested in the banquet hall but respond positively to elements of a very general nature. One gets a sense that Mind-Set 1 comprises individual who respond to general, practical information, but are not thinking of the specifics of an event.

All the information is on our website…only a click away

Want an unforgettable prom night… Host it here and it will be a night you will never forget

Want to have a fun night out with your friends… Host a theme party at our fascinating hall

Need a last-minute party? We are fast, fun and friendly

Easy to find parking near the hall

Mind-Set 2, with 10 respondents, has an additive constant of -48, but shows 29 of the 36 elements scoring very well. Mind-Set 2 is definitely interested in the particulars of the banquet. The very low additive constant is deceptive and is a statistical aberration to correct for the exceptionally high-scoring elements appropriate for a person who actively contemplates hiring a banquet hall. We can get a sense of their seriousness by looking at the elements which generate the exceptionally high coefficients of 30 or above. These are the respondents who are in the target audience.

Open Bar included at no extra cost

Most affordable rates per hour …4 hours per night

All rooms are customizable to fit your party size

Our professional event planner takes care of interior decorations

Keep More in your wallet with our flexible offers

All fees and taxes included in final price

Only $120 every additional hour

Take a step outside onto our one of a kind designed balcony

Professional photographers upon request

Mind-Set 3 with 14 respondents shows the highest additive constant, 27. They are modestly interested in the notion of a banquet hall, but the highest scoring elements give a sense of a casual shopper looking at the offer of a banquet hall. The only element which strongly interests them are ‘All rooms are customizable to fit your party size.’ One gets a sense that this is information which is ‘nice to know’ but the respondents are not excited by the idea of a banquet hall in the same way that respondents in Mind-

Set 2 are.

All rooms are customizable to fit your party size

Lost your valuables? We keep them safe in our safe

Open Bar included at no extra cost

Have a nice view of the great scenery

Linking elements to feelings/emotions

In the world of consumer research, the recognition of emotion as a key driver of purchase has a long history. Indeed, a great deal of the consumer research is on the emotions evoked by products, services, and advertisements. When emotions are invoked the typical approach instructs the respondents to select the one or two emotions which ‘fit’ the vignette. The standard analysis measures the percent of respondents assigning one or another emotion to the test stimulus, either for the entire test stimulus, or during the period that the test stimulus is being evaluated, e.g., for a video.

The Mind Genomics approach differs. The goal is to link each individual element to every one of the emotions. It is impossible to do that in a simple way because there are several different emotions, and the respondent’s job is extremely difficult to divide up the emotional feeling into the different parts. One might have the respondents select the most dominant emotion experience, the approach used here, or instruct the respondent to check ‘all which apply,’ not used here, but also feasible.

The mapping of the one rating (select the single emotion which best describes the feeling) into the five alternative emotions, allows the researcher to link the 36 elements to emotions. As noted in Step 7above, the analysis uses OLS regression, without incorporating the additive constant. The coefficients in the OLS equation can be considered to be the linkage between the element and the emotion. The linkages for the total panel appear in Table 3. Again we focus only on the strong linkages, this time defined as +8 or higher, based upon previous experiences with linkages in Mind Genomics.

Table 3: Linkages between the elements and the five feelings/emotions. Only strong linkages are show (>= 6)

table 3(1)

table 3(2)

table 3(3)

Most of the elements link strongly to feeling/emotion, as shown by the shaded cells. A few elements link strongly to two emotions. For example, element F3, Have the wedding of her dreams, a phrase not particularly interesting to anyone not contemplating marriage, links both to disinterested and to interested, presumably because of the very specific nature of the phrase, viz., a wedding.

Assigning a new person to a mind-set at any time in the knowledge development process

Knowing the nature of the mind-sets for topics of the everyday builds up the ‘Wikipedia of the Mind’. Beyond knowledge, however, is the opportunity to use the information for further research, or for applications such as sales. Rapidly and affordably uncovering the nature of the underlying mind-sets of people on any topic that can be dimensionalized is one distinct contribution of Mind Genomics. The second contribution is the ability to assign a new person to one of the mind-sets.

In the world of consumer research, sociology, and the like, there is a pervasive belief that ‘birds of a feather flock together,’ or more specifically, people who ‘look alike think alike.’ This notion underlies some of the strategies of companies such as Facebook, which observe a specific behavior of different people, and find commonalities among these people in terms of “who they are.” One can market to this newly created group by marketing to people which look like them, on a variety of dimensions, so-called look-alikes.

It is attractive to search for assignment rules which put new people into newly developed mind-sets. Those who work with so-called ‘Big Data’ believe that it is possible to do so, if only the algorithms and the computing power is sufficient, only if the data to be processed is sufficient, and only there are enough cases so that one is not working with random noise. The attraction of such ‘hope’ is enormous, despite the fact that the use of Big Data is to assign individuals to specific, granular segments, of a type that may not even be suspected.

Such marketing strategies make sense when one looks at behavior from the ‘outside-in’, searching for common properties of a group of people which are of interest. Mind Genomics works from the opposite direction, knowing that there are certain ways of ‘thinking’ about a topic, and then determining how a new person thinks about the topic. The approach is not to find lookalikes, but rather to create a simple test which assigns a person to a mind-set. The test, called a PVI, personal viewpoint identifier, is applied to the data of the original group of respondents which defined the mind-set, and then applied to new individuals.

Table 4 shows the distribution of the respondents by total panel and by the three mind-sets. The respondents completed a short classification questionnaire at the start of the study. The questionnaire provided the respondent’s gender, age, and the reason(s) that the respondent might be interested in hiring a banquet hall. The respondents could select any number of the different reasons for hiring a banquet hall. It is clear from Table 4 that the mind-sets are found in all of the groups into which the respondent can define herself or himself. It is not easy, if even possible, to develop a simple assignment rule to mind-set based simply upon the pattern of who the respondent says she or he IS, or what she or he would hire a banquet hall.

Table 4: Distribution of the respondents into groups (gender, age, reason for hiring a banquet hall.)

table 4

Mind Genomics provides an entirely different approach to assigning people to groups, one working from the bottom up, from the simple granularity of an experiment that can be conducted in an hour or two, or several iterative experiments that can be conducted in a day or so. The approach used by Mind Genomics is called the PVI, the personal viewpoint identifier.

The PVI uses the basic data from the mind-sets (see Table 2), perturbing the data with random numbers added to the coefficients. Through a Monte-Carlo simulation the PVI identifies six elements which best differentiate among the mind-sets, based on a two-point scale. The PVI then generates 64 patterns. Each pattern maps to one of the two or three mind-sets.

Figure 4 shows the introductory page to the PVI, which obtains relevant information about the respondent. Each question can be suppressed. Some of the information is appropriate to marketing, other information, e.g., about time, relevant to scientific studies about decision making and diurnal rhythms and so forth. Figure 5 shows the actual PVI itself, comprising background questions about attitudes and usage, as well as the six question, which appear in randomized order, different for each respondent. The six questions appear after the background questions. Finally, Figure 6 shows the feedback to the respondent regarding the mind-set to which the respondent is assigned. The information from Figures 4-6 are maintained in a user-accessible database. Thus, the PVI provides both ‘gamification’ to interest the respondent, and archival information for subsequent work. The respondent can also be immediately led to a video or to a ‘landing page’ on a website, depending upon the mind-set to which the respondent has been just assigned.

fig 4

Figure 4: Introductory page to the PVI

fig 5

Figure 5: The background questions and the six PVI questions

fig 6

Figure 6: Results from the PVI, sent to the respondent. The mind-set to which the respondent is assigned is shown by the shaded cells.

Discussion and conclusion

The Mind Genomics experiment detailed here represents a divergent way of thinking in social science. As noted in the introduction, researchers in the social science usually forego the experimental method in favor of observation when the topic is everyday behavior. The psychologists and economists might study everyday behavior, but with the exception of researchers such as Robert Shiller [25], few economists and psychologists study the simple, every-day experience, which constitutes the warp and woof of social life.

The story changes a little when we move beyond anthropology, sociology and classical economics to some aspects of experimental social psychology, behavioral economics, and occasionally consumer research, respectively. The experimentation focuses on what might be considered unusual perturbations of the everyday, such as varying prices or prices expressed in different ways. The objective is, through the experiment, to express some generality of behavior, such as the fact that people like prices specified by text rather than by numbers When it comes to consumer research the effort is no so much on experimentation at all as on the role of the test stimulus (the nature of the banquet hall, etc.) in driving choice.

Mind Genomics provides the researcher with a new set of tools, systematic experimentation that can be done using simple to vary stimuli (viz., combinations of messages, or combinations of visuals [26], and a simple respondent task (e.g., read/look, and then rate the combination on the basis of criteria laid out in a rating scale provide to the respondent.) In doing so, Mind Genomics may open up our understanding of ‘typical’ human behavior of the everyday, a rich source to understand the ‘mind of man.’

Statistical appendix – establishing consistency of response

The impetus for this statistical appendix comes from the continuing question by those introduced to the method, name ‘can people really do this task?’ We are accustomed to many people in the applied profession of consumer research who believe that ordinary people simply cannot be consistent when evaluating mixtures of messages. The ingoing assumption is that unless the messages are simple, the respondent becomes totally confused. This assumption is reinforced by exit interviews in which respondents ‘complain’ that they did not do the study correctly because they did not ‘know’ what the researcher wanted to hear, and thus they feel that their answers were random.

In order to address these ongoing issues, one can point to the data and show that the pattern of responses is meaningful, at least at an intuitive level. An example of this is element D4, ‘Open Bar included at no extra cost’ with a coefficient of +16 for the Total Panel. Such suggestions of valid results appear in study after study, but are not considered sufficiently rigorous. Some critics want statistical evidence of reliability, such as split half reliability.

One way to show consistency of responses, which may be a form of validity, is to demonstrate that the ratings accurately ‘track’ the stimuli, at the level of the individual respondent. That is, within the confines of the Mind Genomics experiment, one can create a model for each respondent relating the numerical ratings on the 9-point scale to the presence/absence of the 36 elements. The degree to which the ratings ‘track’ the elements is a measure of the consistency of the ratings, and in some respects to the validity of the respondent’s ratings within the confines of the Mind Genomics experiment.

The approach to establish consistency is to compute the Pearson multiple correlation for each respondent, showing the strength of a linear relation between the elements and the ratings. The Pearson multiple linear correlation goes from a perfect +1 (perfect linear relation), down to 0 (no apparent relation).

Figure 7 shows the distribution of multiple linear correlations for the respondents, based on 48 observations, and 36 predictors. The R values were computed by relating the presence/absence of the 36 elements to the 9-point rating, using OLS (ordinary least-squares) regression .More than half of the multiple linear correlations are 0.6 or higher, suggesting that despite the apparent difficulty of choosing the relevant information from the vignette, respondents act in a consistent manner, and in a seeming interpretable, rational manner as well.

fig 7

Figure 7: Distribution of the multiple Pearson R values across the 57 respondents.

Acknowledgement

Attila Gere wishes to acknowledge and thank the Premium Postdoctoral Research Program of The Hungarian Academy of Sciences

References

  1. Dunbabin KM (2003) The Roman Banquet: Images of Conviviality. Cambridge University Press.
  2. Graham JW (1967) A banquet hall at Mycenaean Pylos. American Journal of Archaeology 71: 353-360.
  3. Anglo S (2017) Overcrowding at court: A Renaissance problem and its solution: temporary theatres and banquet halls. In Architectures of Festival in Early Modern Europe 201-212.
  4. Chau AY (2008) The sensorial production of the social. Ethnos 73: 485-504.
  5. Meads C (2001) Banquets Set Forth: Banqueting in English Renaissance Drama. Manchester University Press.
  6. Edwards W (1987) The commercialized wedding as ritual: A window on social values. Journal of Japanese Studies 13: 51-78.
  7. Lau CK, Hui SH (2010) Selection attributes of wedding banquet venues: An exploratory study of Hong Kong prospective wedding couples. International Journal of Hospitality Management 29: 268-276.
  8. Schoenfeld S (1987) Folk Judaism, elite Judaism and the role of bar mitzvah in the development of the synagogue and Jewish school in America. Contemporary Jewry 9: 67.
  9. Adler H, Chienm TC (2005) The wedding business: a method to boost food and beverage revenues in hotels. Journal of Foodservice Business Research 7: 117-125.
  10. Choi JH, Choi YJ (2017) The effect of service scape of banquet hall at special class hotels on customer satisfaction and positive word-of-mouth intention. Korea Tourism Research Association 32: 125-145.
  11. Kim YK (2016) A study on the relationship among service quality and customer satisfaction of wedding hall restaurants, and recommendation intensity-focus on the moderating effect of wedding hall and hotel image. Culinary Science and Hospitality Research22: 252-266.
  12. Kim GC, Lee YJ (2009) A study on the importance and performance of the display of dining space for Hotel banquets. Culinary science and hospitality research 15: 173-187.
  13. LaFleur T, Hyten C (1995) Improving the quality of hotel banquet staff performance. Journal of Organizational Behavior Management 15: 69-93.
  14. Noone BM, Kimes SE, Renaghan LM (2003) Integrating customer relationship management and revenue management: A hotel perspective. Journal of Revenue and Pricing Management 2: 7-21.
  15. Guan L (2014) “Push and Pull Factors in Determining the Consumer’s Motivations for Choosing Wedding Banquet Venues: A Case Study in Chongqing, China”. Graduate Theses & Dissertations. 13851.
  16. Peng MC, Wang RY (2017) Evaluating wedding banquet halls using a novel multiple-criteria decision-making model. Advances in Management and Applied Economics 7: 13-28.
  17. Moskowitz HR (2012) ‘Mind genomics’: The experimental, inductive science of the ordinary, and its application to aspects of food and feeding. Physiology & behavior 107: 606-613.
  18. Moskowitz HR, Gofman A, Beckley J, Ashman H (2006) Founding a new science: Mind genomics. Journal of sensory studies 21: 266-307.
  19. Moskowitz HR, Gofman A (2007) Selling Blue Elephants: How to Make Great Products that People Want before they even Know they Want Them Pearson Education.
  20. Kahneman D (2011) Thinking, Fast and Slow. Macmillan.
  21. Miller GA, Galanter E, Pribram KH (1960) Plans and the Structure of Behavior. Henry Holt and Co.
  22. Box GE, Hunter WH, Hunter S (1978)Statistics for Experimenters (Vol. 664). New York: John Wiley & sons.
  23. Gofman A, Moskowitz H (2010) Isomorphic permuted experimental designs and their application in conjoint analysis. Journal of Sensory Studies 25: 127-145.
  24. Jain AK, Dubes RC (1988) Algorithms for Clustering Data. Prentice-Hall, Inc.
  25. Shiller RJ (2017) Narrative economics. American Economic Review 107: 967-1004.
  26. Gofman A, Moskowitz HR, Fyrbjork J, Moskowitz D,  Mets T (2009) Extending rule developing experimentation to perception of food packages with eye tracking. The Open Food Science Journal 3: 66-78.
  27. Moskowitz MR, Ashman H, Minkus-McKenna D, Rabino S, Beckley JH (2006) Databasing the shopper’s mind: Approaches to a ‘mind genomics’. Journal of Database Marketing & Customer Strategy Management 13: 144-155.
fig 1

Family Stress, Responses, and Mind-Sets: An Exploratory Mind Genomics Cartography

DOI: 10.31038/PSYJ.2021341

Abstract

We introduce the emerging science of Mind Genomics to understand how ordinary people feel when they are presented with different vignettes about a couple’s behavior in tough economic times. Respondents each rated 24 unique vignettes describing the economic situation, the time of year, what the couple does in light of coping with the economic situation, and situation at home resulting from the coping efforts. The Mind Genomics method allows the respondent to predict what might happen to the couple. The approach introduces a projective approach to understanding social problems.

Introduction

During a discussion between authors Peer and Moskowitz, the issue arose as to whether there would be a better way to understand the feelings underlying family violence, especially between the adult couple. There are quite a number of papers and books devoted to the topic, so the contribution of this paper is methodological, rather than substantive[1-3].

The literature is filled with different reports about family violence co-varying with economically hard times [4], with the woman taking a job outside the home and the conflicts about the sizes of the salary [5-6], as well as issues such as external factors which would seem unrelated, such as the time of year [7-8] and even external cues such as football games on television [9]. The recent and ongoing Covid-19 epidemic, world-wide, and its seemingly never-end demand on family life is also now an excellent source for family discord, violence, and simply the normal reactions to a drawn-out social stressor.

For the most part, the literature of the family and family issues during time of difficulty approaches the data from the outside in, from observations of behaviors, and attempts to find general patterns. There is a rich world of knowledge from the ‘inside out’, from the point of view of the people in the family, but for the most part this knowledge is confidential, the outcome of private therapy sessions between therapist and family. The topics and issues can be discussed by the therapist in professional meetings and in written form for journals and the like, as long as the relevant identifying information is disguised to accord with privacy laws.

Advancing our understanding by using new tools to quantify, but go from the ‘inside out’

During the past four decades, researchers studying consumer behavior have been interested in questions which move beyond ‘what happened’ or ‘how does the consumer think,’ and into issues that might be called ‘what if thinking.’ The term ‘what if’ refers to the effort to create a model showing what the person might do under different situations. The value of ‘what if’ modeling is patently clear when the issue comes to identifying the decision rules of a person, especially when the objective is to sell the person a product or a service. The objective of all these research techniques is to ‘understand’ the problem [10].

The notion of a model of decision making can move beyond issues of economics, where one might naturally think of the usefulness of the model. What might happen when the modeling is used to create a structure to understand the alternatives possible in everyday behavior, behavior that does not involve a choice among alternatives, but simply a yes/no. For example, what might happen to our knowledge of social issues if we can understand what a person would do in various circumstances?.

In the past decade there has been a concerted effort to understand the mind of people who are presented with description of social situations, instructed to predict of what might happen, using a scale whose numbers are later analyzed to create mathematical models. The research ranges from studies of decision making in courtrooms[11], to studies of social distancing during the time of the Covid-19 epidemic [12], and on to issues involving corruption in the world of education [13]. The approach, Mind Genomics, described below, presents a new approach to understanding how people make decisions, doing so in a way which prevent the respondent from ‘gaming’ the study, giving the answer that the research is expected to hear.

How Mind Genomics works to understand the problem, yet prevent politically correct answers

This paper focuses on a limited topic of home behavior during a period of external economic stress. The approach uses the emerging science of Mind Genomics, a science whose origins can be traced to psychophysics (a branch of experimental psychology), to statistics (specifically experimental design and so-called functional measurement), and finally to consumer research (focus on common, everyday issues, expressed in specifics, rather than in general, and vague language).

Mind Genomics as a science began with the effort to understand how we react to ‘signals,’ or ‘messages’ in the environment. Typically, researchers focusing on the perception and understanding of the external stimuli would identify the test stimuli, and isolate the stimulus and the subject, so that the subject could focus on the stimulus. In this way the researcher could try to eliminate other factors, noise or random variability, which would confound the results. Occasionally, the researcher might wish to introduce distractions as part of the research task, in which case the experiment would be crafted to introduce both a known stimulus and known ‘noise,’ the aforementioned extraneous variability. This approach can be used both for qualitative research (e.g., anthropological research about shopping)[14], or for standard questionnaires.

Mind Genomics went a different direction, deliberating creating combinations of stimuli of known composition (mixtures of messages), presenting these to the respondent and getting an answer, such as a rating. The objective of Mind Genomics is to measure the intuitive response of the subject to the test stimuli, doing so in a noisy environment, but noise which can be factored out during the analysis. In that way, the Mind Genomics effort identifies the subject’s response to the test stimulus, understands the role of the distractor variables, and produces a quantitative measure of the subject’s response. At the same time, it becomes impossible for the subject to ‘game’ the system, viz., to provide so-called politically correct answers of the type that would be socially acceptable, even though misleading.

The Process of Mind Genomics applied to the projection of emotions onto a situation

The study here exemplifies the approach taken by Mind Genomics. In the interests of description, understanding, and discovery, we explain the approach with a case history, one dealing with expected responses in one’s home during a stressful situation. The study was developed from discussions with author Christine Peer. The process follows a set of choreographed steps which move on to a defined experiment generating data that can be immediately analyzed to reveal patterns. In the vernacular of science, one the effort can be defined as a ;cartography,’ to study a social situation, rather than an effort to prove or in contrast to falsify a hypothesis. Mind Genomics, viewed in this context, can be thought of as more ‘description’ of a situation, at least in the mind of a person presented with alternative ideas.

The Mind Genomics process proceeds in a systematic fashion, from the choice of topic and test materials to the creation the test stimuli (vignettes or combinations of messages), the evaluation of the test stimuli, the creation of ‘equations’ or ‘models’ showing how the test stimuli ‘drive’ the responses, and then the extraction of meaning and implications from the data. Over the past six years the process has been templated, allowing anyone to become a researcher (see www.bimileap.com). The templated system, doable even in a demonstration model, sets up the experiment, runs the experiment on the internet, acquires the necessary data, and automatically analyzes the results to generate results usually each to interpret. The statistics are standard ones (experimental design, regression analysis, clustering). The rapid, virtually automatically executed study allows the researcher, even a novice, to spend the valuable time interpreting data, generally data that most people find easy to understand. Patterns emerge clearly, as we will see from these data. With this emerging reality of rapid experimentation, the vision of a science of the mind, a science of the everyday experience, becomes feasible with low cost and little effort, available to all.

Step 1: Select a topic and create the raw materials. The topic sets the focus of the study. Typically, the topic constitutes a circumscribed set of experiences described in words. The topic could be described by a word, or a phrase, portraying a situation. For this study, we were inspired by the opening line of Tolstoy’s novel, Anna Karenina: Happy families are all alike; every unhappy family is unhappy in its own way”. Our topic was the ‘unhappy family.’

Following the choice of topic, create four questions which ‘tell a story.’ The questions are selected to move the ’story along’, but never appear in the test material (viz., the vignette described below). The hardest part of the Mind Genomics exercise often is the selection of the ‘appropriate’ questions because the questions will constitute the backbone of the vignettes, even though the questions never appear. As we see below, the four questions sketch out a reasonable, logical outline. Question B might have preceded Question A, but this decision was to follow the order below.

Question A: What is the current situation of the person?

Question B: What are the local constraints?

Question C: What is the situation of the wife or the husband?

Question D: What happens afterward?

The final part of the first step creates four answers to each question. The answers should be descriptive phrases which ‘tell a story,’ rather than simple yes/no terms which would not be found in a story. The objective is to create small stories, albeit stories without the necessary connectives. The stories or vignettes will comprise 2-4 phrases, presented in centered, stacked format, on a screen.

Table 1 shows the 16 answers, with the answer attached to one of the four questions. It is clear from Table 1 that each of the 16 elements is a simple description, with no hint of the emotional response of the members of the family to each other, although the element can describe the emotional condition of an individual with respect to the circumstances at large, such as elements C3 and C4 about the husband’s emotions in general. It is also clear that the elements paint short ‘word pictures’ and move beyond simply noticeably short and non-evocative phrases. The one element which is noticeably short is B4 (It’s summertime), which is meant to elicit the feelings about summertime.

At this point, it is important to note that the study appears to be simply notions, ideas thought up as convenience stimuli. That is true. The underlying objective of Mind Genomics is to make research easy, quick, iterative, and affordable. Unlike a great number of other research approaches, Mind Genomics encourages guessing about the correct test stimuli to use. Being able to iterate quickly, to pivot in an hour or two, means that the 16 answers or elements shown in Table 1, and indeed even the questions, can be tested in a study, the promising ideas kept and refined, the less than promising ideas discarded, and in their place new ideas introduced.

Table 1: The 16 elements (answers to the four questions)

table 1

2. Field execution. The field execution comprises a short, 3-4 minute ‘interview’ with a respondent. The respondent remains totally anonymous, both in terms of disclosure of identity by the panel provider (Luc.id), and by the researcher setting up the experiment. For both Luc.id and the Mind Genomics technology, BimiLeap®, the requirement is for everything to be anonymized, unless specifically requested by the researcher, and accepted by the respondent.

The online panel company, Luc.id, Inc., invites the respondents to participate. The respondents are compensated, but the details of the compensation are not relevant to the researcher. The respondents click on a link embedded in the invitation and are led first to a classification page, which thanks the respondents for participating, and which asks them to record their age, gender, and their marital status (question #3). This third classification question is in the purview of the researcher to write. For this study, the classification question was phrases as: What is your marital situation 1=single2=married3=living together4=divorced5=Not applicable

After the respondent completed the classification question, the respondent next read the orientation page, and rated 24 systematically varied vignettes, using the same rating scale. The BimiLeap program recorded the rating, the response time (time between appearance of the vignette on the screen and the rating), and then recorded the data in a database for almost-immediate analysis.

The respondents are introduced to the study by an orientation and a rating question. By design, the orientation is short. In this way, it is left to the specific elements to specify the situation. It will be the elements which will become the key to understanding the mind of the respondent. The less information in the orientation the more that the respondent will use the elements to drive the rating.

Here is a set of snapshots of families. Please read the full snapshot and tell us what will happen within the foreseeable future. Read the whole snapshot. Is it going to be peaceful, or do you sense some family violence brewing?

What will happen in the foreseeable future with this family?

1=peace and love …9=some violence(Bot=Peace, Top =Violence)

fig 1

Figure 1: The appearance of a test vignette on the smartphone

Figure 1 shows an example of the vignette as it would appear on a smart phone. The format is set up to make it easy to scan the vignette, pick out the relevant material (an almost automatic behavior), and then rate the combination.

To the unaided eye, the vignettes appear to be haphazard combinations of elements, thrown together at random. The presentation of these types of combinations ‘frustrates’ the respondent who is trying to answer ‘correctly,’ viz., to ‘get it right.’The impression of a haphazard combination is very far from the reality, however, it is important to keep in mind that the 24 vignettes are created according to a strict plan which ensures that each element appears equally, that all elements are statistically independent of each other, and that the data for each respondent suffices to allow for a regression model to be created for that one respondent. The latter feature ensures the ability to estimate the necessary parameters (coefficients), allowing for clustering or segmentation according to the pattern of coefficients.

Each respondent evaluated a specific, and unique set of 24 vignettes, some vignettes comprising two elements (answers from two different questions), some comprising three elements (answers from three different questions), and the remaining comprising four elements (answers from all four questions). Each element appears five times across the 24 vignettes designed for an individual respondent. By unique is meant that the vignettes tested by one respondent were mathematically identical to the vignettes test by other respondents, but the actual combinations were different for each respondent. This approach allows the Mind Genomics effort to cover a great deal of the so-called ‘design space,’ the combinations that could be created. The approach, called the permuted design method[15], makes the Mind Genomics approach a good tool to learn, even when absolutely nothing is known about the topic. One need not know the most ‘promising region’ to test, something which frees the researcher from losing out when the initial guess is incorrect. Each experiment covers a lot of the design space, with as few as 20-30 respondents.

One final point is important to reiterate. This point is the strategy of experimentation which sacrifices precision of measurement (averaging out the variability), replacing it precision by identify the pattern, even though the individual points are variable. The analogy in medicine is the the MRI, magnetic resonance imaging. The pattern emerges from the many different combinations tested. With 50 respondents, for example, the study here covers 1200 combinations. The underlying strategy is to permute these combinations, keeping the mathematical structure the same, but changing the specific combinations [15]. With the 1200 combinations tested in this study (minus a few possible duplicates across respondents), and with measurement at each point, each combination, we have the opportunity to evaluate many different regions of the ‘design space,’ see what works, and then redo the study, focusing on that part of the space. We have the benefit of evaluating many different combinations, and not having to know anything at the start. A few iterations, and a researcher can ‘home in’ to promising areas, viz., topics driving violence.

A total of 50 respondents participated, all from the United States. New subgroups defined as the three mind-sets, will be discussed below. For now it is simply relevant to think of these mind-sets as individuals with different patterns of response to the vignettes.

3. Relating elements to responses. The Mind Genomics point of view is that the valuable information is in the parameters of the model relating the presence/absence of the 16 elements to the ratings. The first step to create the model defines two new dependent variables, both from the rating. Recall that the rating scale is anchored on both side, with 1 being a response of ‘love’ and a 9 being a response of ‘some violence.’ We are interested in the ability of the elements to drive love or violence, respectively. In order to address the issue of two opposite objectives, we create two new binary variables, Love and Violence, respectively. These two newly created binary variables make the interpretation much easier when we look at the tabulated results.

The actual transformation is:

                  Rating of 1-3 transformed to 100 (Love), ratings of 4-9 transformed to 0 (Not love)

                  Ratings 7-9 transformed to 100 (Violence), ratings of 1-6 transformed to 0 (Not violence)

To complete the transformation, a small random number (<10-4) is added to all of the transformed ratings, in order to add artificial but miniscule variation in the newly created binary variables, Love, Violence. The addition of this small random number ensures that the dependent variable will have some minimal level of variability, required for the OLS (ordinary least-squares regression)o work, and not to crash.

In the presentation of the results, we will presently only the positive coefficients, AND NOT REPORT coefficients which are either 0 or negative, respectively. The underlying themes emerge more clearly when we focus only on the positive coefficients. The negative coefficient simply means ‘absence of’.

4. Relating elements to responses – extending the analysis to individual level models to create new to the world mind-sets. One of the hallmark benefits of the Mind Genomics approach is its ability to uncover mind-sets, defined operationally as different patterns of coefficients for the same set of elements and the same rating attribute. We will create one group of mind-sets, defined a separate, non-overlapping groups of responses who show similar patterns of coefficients, both for Violence and for Love, respectively. That is, we have two types of behaviors, violence (ratings of 7-9) and love (ratings of 1-3). We will create a separate pair of models for each of the 50 respondents, one model or equation for violence vs the 16 elements, and the second for love vs the 16 elements. The independent variables for each respondent were set up by the aforementioned ‘permuted design’, producing a valid experimental design for each respondent. As a consequence, OLS regression allows us to create a valid pairs of equations or models for each respondent.

To create the individual-level equation, we fit a simple linear equation, without an additive constant, doing so for each respondent, once for the 16 elements vs the response ‘violence,’ and for the same 16 elements vs the response ‘love,’ respectively. The calculation generates 32 coefficients, 16 coefficients for the equation for violence, and a parallel 16 coefficients for the equation for love. There are no additive constants in either model.

The clustering which follows is a purely mathematical effort. There is no effort to interpret the data at the tie of clustering, although such an effort might be viable For Mind Genomics studies the clustering produces easy-to-label clusters called mind-sets, easy perhaps because the test stimuli on which the clustering is based, coefficients of elements, use cognitively rich stimuli.

5. Patterns emerging from models. Once the respondents have been identified according to the relevant criteria (total, age, gender, relationship status, membership in the mind-set from clustering) the relevant data for a group are analyzed twice, once creating an equation for Violence (ratings 7-9 converted to 100, otherwise converted to 0), and once creating an equation for Love (ratings 1-3 converted to 100, otherwise converted to 0). This time the equation does have an additive constant.

Binary Variable (Violence or Love) = k0 + k1(A1)+k2(A2)…k16(D4)

The additive constant is the expected proportion of the responses to be 100 (viz., rating 7-9) when there are no elements. The experimental design introduced above ensures that all vignettes comprised 2-4 elements, meaning that the additive constant is a purely estimated parameter.

Patterns for ‘violence’: The additive constant can be interpreted as the expected percent of the responses the respondent will rate a vignette 7-9 in the absence of elements. Of course, that is not possible since by design all vignettes comprised 2-4 elements. Nonetheless, the additive constant is a valid measure, one that plays the role of a baseline feeling. The top of Table 2 shows the summary table for the response ‘violence’.

  1. The total panel is 27 – violence will be the outcome for one out of every four responses
  2. Males judge the outcome as violence far more frequently than do females(39 vs 16)
  3. Young people judge the outcome as violence more than do older people (31 vs 21)
  4. Single people judge the outcome as violence more than people with partners (married, in a relationship)

We now proceed to the individual elements, and the patterns emerging from the groups. As noted above, in the interest of clarity we do not present coefficients which are 0 or negative, but rather present only coefficients equal to or higher than 2. We also shade coefficients of +8 or higher, because it is around +8 that a coefficient reaches statistical significance (T value around 1.5 or higher).

Table 2 (Top; Violence) suggests no clear pattern by key subgroup, but some elements which drive expected violence, at least among some respondent groups. These trigger elements leading to expected violence are:

B1 – Companies are firing employees, as perceived by females and respondents aged 50+. This means that when these respondents read a vignette, the element B1 is likely to trigger the expectation of some violence occurring.

C4 – The husband is sad and depressed, as perceived by females, older, and those with current with partners.

Patterns emerging for ‘love’: Table 2 (Bottom; Love) reveals very low additive constants, most around 12 or lower, except for the younger respondent (age21-49) showing a still-low additive constant of 20.

The two elements bringing almost universal love are descriptions of the season: (B3 – Middle of the winter Christmas) and B4 (It’s summertime)

We conclude from this first analysis that there are few strong differences among the groups. Only a few elements emerge to drive either violence or love.

Table 2: Models relating the presence/absence of the 16 elements to either violence or to love. The data come from the groups as they specified themselves in the up-front classification step.

table 2(1)

table 2(2)

6. How one element influences another (scenario analysis). The permuted experimental design brings with it an unexpected capability to uncover interactions among elements. The underlying experimental design is set up to make all the 16 elements statistically independent of each other. If every respondent simply evaluated the predesignated 16 combinations, it would be impossible to discover synergies between elements, where the presence of a pair of elements in the same vignette ‘turbocharges’ the rating, so the rating is much higher than one would predict from the simple sum of the coefficients.

The strategy for creating the scenarios follows a set of simple, based upon the notion that each of the elements in the study appears five times for every respondent, and is absent 19 for every respondent. Let us now select one of the four questions, for example question B. Question B comprises four elements presenting information about the time of year, and what companies are doing, respectively. We consider our four elements to be strata, and sort all of the vignettes into the four strata defined by the elements, as well as into the fifth stratum defined by all the vignettes which, by design, lack an element.

The previous exercise creates five strata. In each stratum, the element B is held constant, or does not appear. We now have five new data sets, each with elements from Questions A, C and D present. We simply run two sets of five equations, using as the dependent variables Top3 (Violence) and Bot3 (Love), respectively. The 12 independent variables are A1-A4, C1-C4, and D1-D4.

Table 3 show the five regression models. Each column corresponds to one of the five strata, defined by B1-B4, as well as B0 (B absent from the vignette). Each row corresponds to one of the 12 remaining elements. The top of Table 3 shows the coefficients for Top3 (violence), sorted by ascending order of additive constant. The bottom of Table 3 shows the coefficient of Bot3 (Love), sorted once again by ascending order of the additive constant.

Table 3 shows us much great performance of the elements as drivers of violence and love, respectively. Once the elements are constrained to be fixed in a vignette, they set the ‘stage’ for the ideas. We can see various new patterns emerge, allowing a deeper insight into the topic. For example, when we look at the model for B3 (it’s in the middle of winter Christmas), we see a low proclivity for violence (additive constant is 10, the lowest basic proclivity). ON the other hand, there are specific events which occur which substantially increase the likelihood of violence. Examples are A1 (The local economy is stressed and in recession), and A3 (the children are having problems).

Let us compare the violence expected in winter to the violence expected in summer. We now turn to the last column, for element B4 held constant. The additive constant is much large, an extraordinary 44.Yet, there are no other elements which drive expected violence.

We now move to the bottom of Table 3.We see that the same element, B3 (it’s in the middle of winter. Christmas) brings happiness, viz., synergizes with A2 (the local economy is growing).And, when it is summertime, rather than wintertime, element A3 (the children are having problems) bring love to the family, not violence. That is, the same element (A3)can drive violence (winter) or drive love (summer).

It is patterns like these which are the ‘value add’ to a Mind Genomics cartography. We are able to get a sense of new patterns, some of which make intuitive sense, and some which may spur an ‘aha’ moment.

Table 3: Scenario analyses, holding constant each of the four elements (and the no-element) from Question B, and estimating the model using the remaining 12 elements.

table 3(1)

table 3(2)

7. The allure of mind=sets as organizing principles. Our previous analyses of the data suggested some effects, such as love expected to emerge during two special times, Christmas in the winter and during summer, respectively. One can investigate the literature of the social and psychological sciences, and in doing so discover these disconnected nuggets which intuitively feel as if they are ‘weak signals’ emerging from a deeper, more coherent reality. The problem is that these signals emerge unexpectedly, and do not allow for a deeper investigation without first requiring a hypothesis of just ‘what is happening’.

Mind Genomics circumvents these problems, first by providing a method of clustering based upon a small, tightly defined topic, and then allowing the research to be done efficiently, inexpensively, and in a manner which moves stepwise through the problem in simple and illustrative steps. The clustering method is totally a theoretical, in terms of the meanings of the clusters. The clusters are labelled by which elements score highest and tell a ‘coherent’ story. The method of clustering is known as k-means clustering. The ‘distance’ between people in k-means clustering is known as ‘D’ defined as (1-Pearson R), where the Pearson R is the Pearson linear correlation between each pair of respondents, computed on the 32 coefficients [16].

The clustering performed on the data did not make any assumptions, because none needed to be made. The models were created for each respondent. The two decisions were to combine the models for one individual (violence and love together to extract people similar in both), and then to extract three mind-sets. Two, three, four, and even more mind-sets could have been extracted. The ideal is to work with as few mind-sets as possible (parsimony), but have each mind-set tell its own coherent story (interpretability). The data suggested that two mind-sets may have been the more parsimonious, but the patterns of the coefficients were not clear. Too much information seemed to cross the mind-sets, suggesting the need for a third mind-sets.

The results from the clustering to generate three mind-sets appear in Table 4 Again, we show only the additive constant, and the elements with positive coefficients for each mind-set. From these, we might name the mind-set. The Top of Table 3 shows the results for the response ‘violence’, the bottom of Table 3 shows the results for the response ‘love’. We will present the additive constant, and then piece together a story from the strong performing elements for that mind-sets.

Mind-Set 1 = High violence constant (42), very low love constant (12). Mind-Set 1 is prone to violence when the economy is stressed and in recession, but that is all. Mind-Set 1 is prone to love when things are better, when its summer and winter and when things are going well. Mind-Set 1 is also, however, just as prone to love when people are getting fired. Mind-Set 1 might be called reactive to the outside world, to when, and to what’s happening’

Mind-Set 2 = lower violence constant (24) and very low love constant (15). Mind-Set 2 is probably a person who is depressive but can be cheered up by the season.’

Mind-Set 3 = lowest violence constant (14), lowest love constant (6), strong reactor to the family situation. Mind-Set 3 is most likely to shut off from the family, miss the time, and feel anxious until the wife begins to clearly help out.

The important thing about Table 3 is that the elements which are strongest appear to paint a picture, which makes intuitive sense. Not everything ‘hangs together’ but we are dealing with a small sample of individuals, and the first effort, done in the period two days. The elements can be refined to expand the focus.

Table 4: Models relating the presence/absence of the 16 elements to either violence or to love. The data come from the three mind-sets which emerged from clustering. Only the positive coefficients are shown.

table 4(1)

table 4(2)

Discussion and conclusion

As we see from the cursory data from 50 respondents, the data provided by Mind Genomics is rich, indeed far richer than one might expect from a method emerging out of consumer research. One of the reasons for the rich information comes from the effort of Mind Genomics to provide a context for each stimulus. Rather than responding to a set of disconnected questions, the respondent evaluates a unique set of 24 vignettes, each of the vignettes more likely to tell a story than a single question would be.

In our study we take many pictures of the family and ask what might be happening for that particular picture or vignette. It is only later that we put together the individual snapshots (responses to the vignettes) into a coherent whole, an action made straightforward by the use of individual-level experimental designs, and permuted experimental designs. Mind Genomics capitalizes on both, identifying pictures from disparate combinations, and covering a lot of the ‘design space’ of possibilities, using the strategy of permuted experimental design.

The study reported here can be considered to be a cartography, an exploration of the ‘territory’ of the topic, rather than an attempt to confirm or falsify hypotheses. Mind Genomics gives us an opportunity to move in a variety directions, in the spirit of exploratory research, mapping the mind of people as they think about stressful situations, or even as they live through the stressful situation. The objective is not to accept or reject a hypothesis about ‘how behavior works’ or ‘how the mind works.’ Rather, the objective is to find repeating patterns of behavior, or stated patterns of thinking, either separate from the situation, or even in the middle of the situation. A good example of the approach can be found in [17], which deals with the types of behavior that teens want from doctors. That type of information is gathered in the same spirit as these data, namely understanding behavior in stressful situations.

The data lend themselves to the systemized creation of knowledge, literally at an industrial scale, across topics, countries, people, and external situations. For example, we might run this same experiment during several seasons of the year, and in several venues with varying economic conditions, as well as with people who are known to be prone to family violence versus people without that history. All of these approaches will end up creating, in rapid pace, an affordable database of the mind of family violence and family affection, a database that can be extended world-wide with very little effort. The patterns and the increased knowledge, and perhaps even many more ‘ah ha’ moments await the research. The approaches were laid down more than two decades ago, but the methodological advance is fresh, and the data continuing to pile up, in well-managed databases which maintain their value year after year because they reveal the nature of the ‘mind’ and ‘mind-sets’ confronted with situations inevitable emerging from the daily life of people world-wide[18-20].

References

  1. Ammerman RT, Hersen M (eds) 2000 Case studies in family violence. Springer Science & Business Media.
  2. Denzin NK (1984) Toward a phenomenology of domestic, family violence. American Journal of Sociology. 90 : 483-513.
  3. Momirov J, Duffy A (2011) Family violence: A Canadian introduction. James Lorimer & Company.
  4. Anderberg D, Rainer H, Wadsworth J, Wilson T (2016) Unemployment and domestic violence: Theory and evidence. The Economic Journal126 : 1947-1979.
  5. Aizer A (2010) The gender wage gap and domestic violence. American Economic Review100 : 1847-1859.
  6. Showalter K (2016) Women’s employment and domestic violence: A review of the literature. Aggression and Violent behavior31 : 37-47.
  7. Rotton, J., & Cohn, E. G. 2002. Climate, weather, and crime. In : RB Bechtel, A Churchman (Eds.), Handbook of Environmental Psychology. John Wiley & Sons, Inc. pg : 481-498.
  8. Shortlands ND. https://www.shortlands.co.uk/crisis-at-christmas/
  9. Card D, Dahl GB (2011) Family violence and football: The effect of unexpected emotional cues on violent behavior. The Quarterly Journal of Economics126 : 103-143.
  10. Moskowitz HR, Gofman A (2007) Selling Blue Elephants: How to Make Great Products that People Want Before They Even Know They Want Them. Pearson Education.
  11. Moskowitz, Howard, James Wren, Petraq Papajorgji (2020) Mind Genomics and the Law. 1st Edition. LAP LAMBERT Academic Publishing.
  12. Moskowitz H, Prendi V, Gere A, Harizi A, Papajorgji P (2021) Mind-sets of worried citizens and the ‘real-world experiment’ of Covid-19: A mind genomics cartography. Edelweiss Applied Science and Technology 41-49.
  13. Gere A, Papajorgji P, Moskowitz HR, Milutinovic V (2019) Using a Rule Developing Experimentation Approach to Study Social Problems: The Case of Corruption in Education. International Journal of Political Activism and Engagement6 : 23-48.
  14. Mariampolski H (2006) Ethnography for Marketers: A guide to Consumer Immersion. Sage.
  15. Gofman A, Moskowitz H (2010) Isomorphic permuted experimental designs and their application in conjoint analysis. Journal of sensory studies25 :127-145.
  16. Likas A, Vlassis N, Verbeek JJ (2003) The global k-means clustering algorithm. Pattern Recognition, 36 : 451-461.
  17. Gabay G, Moskowitz HR (2015) Mind Genomics: What Professional Conduct Enhances the Emotional Wellbeing of Teens at the Hospital?. Journal of Psychological Abnormalities.
  18. Moskowitz HR (2012) ‘Mind Genomics’: The experimental, inductive science of the ordinary, and its application to aspects of food and feeding. Physiology & behavior107 : 606-613.
  19. Moskowitz HR, Gofman A, Beckley, J. & Ashman, H., (2006) Founding a new science: Mind genomics. Journal of sensory studies21 : 266-307.
  20. Boserup B, McKenney M, Elkbuli A (2020) Alarming trends in US domestic violence during the COVID-19 pandemic. The American Journal of Emergency Medicine38 : 2753-2755.

Status of Menstrual Dignity during the COVID-19 Pandemics

DOI: 10.31038/AWHC.2021444

Abstract

Background: Violence against women takes different forms, often reflecting cultural patterns. Forced segregation and other dangerous or at least discriminatory practices during menstruation can be observed in a number of cultures, such as Nepal, but also in other regions. The present pandemic with its special risks and lockdown measures must be expected to potentially cause additional problems for women in the critical time of menstruation.

Aims and methodology: The aim of our study was to collect information on the experience of women in different regions and identify risk factors for such practices, such as education, health belief systems and bias in the communities together with the impact of the COVID pandemic on these factors. The survey was conducted online to keep safety protocols necessary during the SARS 2 pandemic. To identify possible key factors we conducted a qualitative/mixed method survey resulting in categories and vivid descriptions relating to violence and discrimination. 139 participants, (age range 13-48 years, 85.8 percent female, else LGBT) from different countries, including both low-economy countries with high rates of reported discrimination such as Nepal and India, but also from the US participated in the survey.

Results: Patients reported experience of bias and insufficient or incorrect information by parents, and later in the communities. Lockdown measures impacted in some cases, but in general to a lesser degree on access to dignified hygienic measures required during menstruation, as compared to before the pandemics, but was reported to increase the social stress and reduce social support. Shame, insecurity and distress during menstruation continued and were described as main adverse factors influencing well-being and psychological health.

Keywords

Women, Gender, Menstruation, Discrimination, Dignity, Pandemics, COVID-19

Background

The World Health Organization (WHO) confirmed the coronavirus outbreak as a pandemic on 11 March 2020. From the outset of the pandemic, the United  Nations and various countries  are working towards a large-scale, coordinated, and comprehensive health response. Women are disproportionately affected by the COVID-19 crisis because of the gender and social norms combined with the disruption of services and the special challenges of factors such as menstruation or pregnancy [1]. The impact must be seen     as multifactorial, including the impact of infection, long COVID, vaccination, and the different COVID prevention and lock-down measures. They have been documented to affect some population groups such as women and migrants in different ways [1-3].

A number of publications have  also  reported  the  impact  on the physical health of women such as sex hormones, fertility and spontaneous abortion [4-6] and on mental health [7]. Several authors, such as Abuhammad et al. have further observed increased problems related to domestic violence in the Pandemics [8,9]. Abdelbadee et al. have further drawn attention to the specific problems to be observed in low-income countries [10].

The pandemic has been exacerbating existing inequalities between women and men in almost all areas of life [3]. However, menstrual discrimination (taboos, stigma, abuses, restrictions, discriminations) has not been studied in the context of the pandemic at least in Asia  in spite of the well published earlier concerns in regard to the politics of menstruation during the pandemic by Jahan [11], by the first data published by Aolymat on women in Jordan [12] and several other authors [13-15].

Therefore, this study was conducted to fill this gap mainly by contributing to a better understanding of menstrual dignity and the experiences of women and LGBTQI during menstruation and observed changes during the COVID-19 pandemic.

Methodology

The study adopted a mixed-method qualitative method integrating qualitative components based on open questions with a short structured questionnaire to elicit general social data and basic information on menstruation related health literacy developed for  the study. We also decided to use a multi/transcultural approach by recruiting participants from different countries and cultures as culture has been identified as a major factor in menstrual practices and related health belief systems [16]. The survey modules separated questions regarding the situation in general and before the pandemic and those regarding the changes observed during or due to the pandemics.

A survey of 139 participants from different countries was conducted using an open online platform developed for the project.

Not all respondents filled out all items in the survey, though all participants answered to at least 90% of the questions. The respondents had been recruited through a public platform delivering the survey. All participants gave informed consent during registration, data were stored anonymised on a safe server and will be erased in due time following common data safety protocols.

The age range of participants was between 13-48 years, 85.8 percent were female and 11.2 percent and the rest identified themselves as LGBTQI (Lesbian, Gay, BiSexual, TranSex, Queer and Intersex). The participants were from ten countries: Bangladesh, Bhutan, India, Israel, Madagascar, Namibia, Nepal, Philippines, Uganda, and USA. All respondents had education backgrounds ranging from higher education to master’s degree; 22.1 percent finished high school;

58.8 percent held bachelor degrees and 19.1 percent master’s degree as highest level of education. Only nine percent of the respondents revealed that they were people with physical or mental disabilities at the time of responding to the online survey.

Findings

General Findings

Knowledge about Menstruation

The respondents reported to have learned about menstruation from different sources, which included mainly their mothers, other family members, school, internet, or from friends. This happened in some participants when they were between age 5-13 years, and in the majority only when they were 10-12 years old.

In the qualitative part of the survey, using the open questions based on an interview guideline, respondents reported:

‘My grandmother gave me a “menstruation talk” starting at age eight up till age 13 and gave me information that was appropriate at each age as I come from a family that has a high risk of menstrual problems. Post age 13, I have received informed from friends & gynecologists’.

‘Although school syllabuses contain reproductive and sexual education, students are not properly provided with knowledge. I learnt most of it from my mother and later by studying myself ’.

‘When my mother was menstruating, my father asked for help from me while cooking.’

Initial Reaction

Respondents stated that their first reactions after having the menstruation were as follows.  Sixty-six  percent  of  respondents  felt scared, seven percent felt sick, and seven percent considered menstruation normal and reacted accordingly. Ten percent of responses on this question noted that the question did not apply to them (all members of the LGBTQI group), and another 10 percent had an ambivalent experience.

‘When I looked into my pants, I was devastated, thinking I was about to die. Nobody likes looking at blood, in whatever sense’.

‘Did I eat something wrong, or did I had an accident that makes my internal parts bleed’

‘Damn, will I survive with this massive loss of blood?

‘During menarche, I thought I was exposed to some deadly disease and angrily went to my mother and claimed that I feel I would die soon as I am bleeding. Then, my mother explained that you are menstruating and it’s a natural process which a woman has to undergo every month’.

Discrimination during Menstruation

Even in regular menstruation, many of the 77 respondents on this items experienced discrimination during menstruation directly and indirectly, as described in the open responses. Mostly, respondents described discrimination as verbal, emotional, as denial of menstrual products, and as impaired mobility. Out of 77 respondents, 23 participants stated that they remained silent while experiencing discrimination. They felt hurt, traumatized and pained though they did not do anything inadequate in their own perception. This group simply tried to ignore discrimination and most of them stated they had no energy to “fight back”. Some of them also said that they felt like “avoiding people”, “running away” or it her acts of social withdrawal.

‘I would not want to be a part of the community where people do not understand and respect a natural phenomenon as such’.

‘I have left an office internship because my supervisor wasn’t sensitive enough. So instead of working from the office, I chose to work in a different department in the same organization. I have also complained about this insensitive behaviour of the supervisor’.

‘I feel inferior to the persons who perpetuate violence towards me, and I feel pressured by societal expectations to abide by the greater publics expectations, so therefore I do not feel empowered to speak up even though I am aware I should do so.’

Out of 77, 49 respondents tried fighting back. They educated  and empowered themselves by sharing facts and then started raising questions about the experienced acts of discrimination. Some reported to have argued with community and family members, and to have used different forms of direct confrontation, including screaming. Further they reported engaging in teaching about natural phenomena and human rights in the community, and involving community members in a dialogue on menstruation.

‘I tried understanding why this is happening and why was it considered a taboo. I usually have these conversations with my mother because she is usually the one who perpetuated the discrimination and had herself not experienced any problems with my family. Still, while visiting the village, I hide the fact that I’m menstruating.’

‘When it comes to PMS comments and menstrual stigma from peers, I educate them politely. However, for elders, I have given up on explaining because they won’t budge’.

However, even while fighting back, they experienced feeling tortured, weak, sad, annoyed, oppressed, helpless, depressed, embarrassed, frustrated, worthless, tired, ashamed, guilty, disappointed, and awful (using the verbs in the qualitative part of the online review). Only few positive emotions were reported, such as feeling proud, or courageous.

‘Once, while I was visiting Lonavala, there was a temple I wanted to see, and I couldn’t because I was during the period. So I asked my mom, “Why can’t I go inside the temple just coz I’m bleeding” she told me, “you shouldn’t question faith”!!! And I felt very bad. I wanted to see the temple from the inside. But, I was so damn eager to see it, and I couldn’t’.

‘Very bad. Life on this planet exists due to the occurrence of menstruation, and they say we are weak for having it. Really!’

‘I feel disappointed at our education system and anger towards those conditioned to perpetuate discrimination by patriarchal mind sets’

‘I would surely feel shame, that’s awful, but it’s always the first emotion I feel when someone perpetuates some sort of violence on me. But then, I’ll call back and feel courageous. Then, last, I’ll be sad about how people can be so rude and uneducated’.

Openness Regarding Menstruation, Disclosure

Only 29 respondents stated that they freely talk with family members, in most cases because they are health workers (menstruator or non-menstruators) and asked for medicine or pads without any hesitation.The rest of the respondents (n-59) talked only with female members of the family such as mother, elder sister, grandmothers, and aunts and their discussions were more focused on ‘do’s and ‘do not’ including restrictions associated with menstruation, menstrual products, and menstrual symptoms or illness.

‘Yes, I talk about menstruation with my family. Talks would be something about how to maintain good hygiene during periods and techniques to ease cramps’.

‘With my mother only. Not my sister or father. It is very awkward. I only ever ask her for tampons when I run out. I spoke to her when  I was younger about what age she got her first period and how to use tampons and pads.’

‘As my father does not menstruate, and we were taught not to speak of “these things” with anyone other than mother, my conversation is mostly limited with her’.

80 respondents remained silent in public about menstruation, thinking for example that “it was disrespectful towards elder members of the family”. They stated they did not receive support from the family and considered it a matter of “hush and girl thing” (personal or private). Reasons mentioned included “Men should not know anything”, “conservative family”, “they hate the topic”, “would be avoided by men”.

“Maybe in African cultures, it is regarded as a taboo. However, we are fighting for fundamental rights, and everyone could be a part of this”.

“Because my parents believe menstruation should be kept a secret and that it is not conversation meant for the living room”.

However, respondents knew that their friends had been menstruating through observation of  their  various  behaviors,  which included blood stained clothes, their experiences of severe cramps, their being more emotional than usual, being hesitant to walk or to walk with precaution, the increased frequency of toilet breaks, avoidance of regular activities, from whispering among girls friends, asking for a favour like a pad, and by their having “pimples”. Segregation was common in their experience.

‘In Islam, a person is unable to perform “ablution” because it needs proper hygiene, so the person cannot perform religious duties’

‘In our culture, girls stay away and are considered untouchable. They are restricted from staying anywhere and from touching anything casually. They shouldn’t touch anybody. In the case of males, they shouldn’t even go nearby.’

‘Yaa. In our society, they stay separately during periods and wear old clothes. So it can be known easily.’

Anxiety during Menstruation in the Bathroom

Only 23 responses registered that they worried about using the toilets at home due to fear  of staining the blood, lack of water, fear of getting scolded, and running out of the menstrual hygienic aids. Respondents reported that they “avoided anybody who knows about the state of menstruating”, its being “like a murder scene”. Some reported also fears including “fear of contamination from the toilet”, “the sound of the opening of the pad”, and “fear of some household members”.

‘As you can touch nothing, you should beg for being handed everything. If you use your bathroom, it should be cleansed.’

‘Fear of getting scolded if the floor gets blood on it, lack of water supply-inability to flush the blood out, fear of getting scolded for forgetting used menstrual products wrapped in the bathroom after taking a shower.’

Understanding of Dignified Menstruation

A total of 62 percent of respondents mentioned that they would perceive freedom during menstruation as dignified menstruation. They considered that hygienic products (access to menstrual pads at home and workplace), freedom of individual behaviour, and family support as being the elements to make menstruation dignified.

Menstruation during the Pandemic

Out of 104 responses, fifty participants expressed that they experienced increased direct and indirect discrimination as compared to before the pandemic. They expressed varieties of violence in this context: physical (once), verbal, emotional, and denial of services. They reported not being allowed to go to the temple, not allowed to cook food, not allowed to consort with their husbands, prohibition to touch some plants (Tulasi), etc.

In the part of the questionnaire asking for experiences first observed during the pandemics, they reported:

‘”seems like you’re ALWAYS on your period,” “stop using it as an excuse,” “don’t you dare bleed on my bed,” not much.’

“One of my friends was beaten because she touched a water purifier.”

‘My brother couldn’t really understand when I first menstruated. People wouldn’t talk about this subject. He even complained about the smell of the blood. He would also force me to do my chores, and I really felt bad because he found it was unfair that I would go to sleep because of some “random illness once a month.”

Respondents experienced difficulties due to travel restrictions to purchase medicine for pain management and menstrual products, as online orders popular in high-income countries during the pandemics are not always available or affordable. On the other hand, when for example the government in India announced a COVID relief package, the menstrual pads were not considered essential by the authorities.

Still, only few of the participants (n= 10) reported that they run out of menstrual products, experienced scarcities, or experienced increased expenses, especially after the second and third months     of the lockdown . Several participants reported that they were not aware that the frontline health (pathologists, nurse, doctors etc.) workers supporting menstruation practices struggled so  much during menstruation due to lack of extra PPE (Personal Protection Equipment’s), menstrual products, and hygiene facilities at their workplace, while others commented on the situation especially in India, as mentioned before. Even the media highlighted the scarcity of toilet paper but did not care about menstrual products to cover in their media, as did some governments in their support emergency plans described above.

‘When the Indian government was making a list of industries that need to operate during the lockdown to provide necessary items, menstrual products were not included in the initial stage…”

Such restrictions during menstruation were accented during COVID-19 [11], and the Pandemic can be seen as discrimination and inequality in health care as in the above example. Women cannot speak about the discomforts and problems openly, cannot move freely, have to work with discomfort and pain, have difficulties in getting a shower and wash their belongings, feel ashamed when buying pads, feel pressure all the time even when they are at home with family, and expressed that they felt isolated or were marginalised even before the pandemic.

‘Girls are treated like as male COVID-19 patients (without special consideration)’

‘I always have, pandemic or no, been flabbergasted by the rules of not entering temples during periods, a duality of our society where you are fascinated by the femininity of goddesses but are filled with stigma when it comes to menstruation.’

‘Women should not talk about it to anyone, especially boys, because it makes you lose dignity. To “control the flow of my blood” and not to make a mess on my bed’.

‘We have to work very hard though we are agonizing in pain while the men of the house just sit and relax with no cramps or anything’.

‘Not able to use the kitchen. I need to ask for someone else to provide something like hot water. The situation is the same during the pandemic as before’.

‘This time, I had gone to my husband’s home in lockdown. I was asked not to enter the kitchen for four days and avoided drinking milk tea too. I have to use all utensils separately. I feel so bad, but I follow it because I was tired of cooking every morning to evening for about four months. Otherwise, I wouldn’t tell anybody. But secretly, I entered the kitchen to get water and food.’

Many menstruating women with a disability, transmen, immigrants, and refugees had suffered more discrimination during menstruation. They were compelled to use old clothes due to the lack of availability of menstrual products. However, only few reported feeling ashamed in managing menstrual products in front of the men’s family members, including children.

‘During the COVID buying PAD was the main issue trans-man. However, they can’t buy the PAD at the time of menstruation because shop owners ask many more questions and they can’t answer, so they use a single PAD for up to three days too’.

Only few of the members of this group thought that the menstruating persons were affected more by the pandemic due to their bleeding status.

Earlier Existing Practices of Segregation can be Accentuated

‘My uncles and aunts in the village think that menstruating women are more likely to develop COVID 19, so they isolate them for 14 days when menstruating. However, it’s not a very logical conclusion.’

In addition to these practices, some respondents also reported increased difficulties like menorrhagia, uneasiness, or the need to work more due to the absence of domestic help during the pandemic. Similarly, participants reported that they could not buy pain killers to ease the cramps, and their period to be irregular due to lack of exercise or change in lifestyle in this situation. Likewise, some participants used the pad sparingly to minimize the waste and to better hide from non-menstruating sisters and other family members,

‘Have to work more at home as domestic helpers are not available because of COVID-19 so I have no rest and have to work through pain’.

‘Since I am not at school, during my  periods, I am just in bed   all day crying until the pain passes. Because I can’t go out to buy painkillers, as they are more expensive than ever, and I consequently am in dire need to distract myself from the pain and focus on studying or something. Sometimes, I exercised heavily to dull the pain’.

Other women reported “I used few products to avoid the waste in our bins. I am worried that my non-menstruating sister or father will see the products in the bin and be embarrassed.”

Use and Management of Menstrual Products during the COVID-19 Pandemic

Regarding use of menstrual products, from the 106 participants who responded to this part of the survey, 82 participants used menstrual pads. Three used menstrual cups, and one used tampons. Respondents considered themselves privileged more than others in many ways; such as “living in urban areas”, by an “online supply”, and because of “access to money”. Participants bought menstrual pads from the grocery shop, pharmacy, and online, as they were open even during the lockdown. Few of them purchased in bulk before the imposing of lockdown. Therefore, most respondents, but depending on the local situation, continued the use of menstrual products as before.

‘All pharmacies are open since the number of COVID-19 cases is not very high in my area.’

‘I always bought a big pack of sanitary pads that is sufficient for 3 4 months, sometimes more … so I didn’t feel any shortening.’

‘I’m fortunate enough to have easy access to products. Medicine shops are open. They are available there’.

‘I use a menstrual cup. It’s pretty hygienic and reusable, plus it’s so comfortable I almost forget that I’m on my period’.

‘I had to “stock-pile” tampons before the lockdown, as we are not going to the shops often enough. I am managing okay, but we would be in a difficult situation if we hadn’t had money to buy extra tampons.

A total of ten respondents applied alternatives to manage the menstrual blood during lockdown like sleeping, or cloth pads because of financial limitations as the work and salary of their parents were interrupted.

‘It’s been quite challenging mainly to the girls in my community, lack of money to buy sanitary pads since also parents are no longer working’.

The transmen suffered much when they had to get out and buy the pads.

‘It’s pretty tough because it’s not like I can run to shops when I’m out of pads’.

Regarding waste management of such menstrual products, 75 respondents said that they were practicing as usual by throwing them in a in garbage tank either at home or in the municipality, seven burned, five re-used the products (cups and cloth), and one buried them in their field. Thus, they did not have significant problems managing the waste due to COVID-19. However, they struggled to wrap them up in newspapers, plastics, by hiding them underneath of bed, etc. due to already pre-pandemic taboo and stigma around menstruation.

‘I wrap them in paper and throw them in the dustbin, which is collected by the municipality vehicle garbage.’

‘Hardest thing ever. I keep collecting used pads for 2-3 days under my bed. Then, later, when there’s no male member around, I cover myself and throw it in the bin downstairs.’

25 (total 76 responses) respondents reported hiding their menstrual pads and increased increased problems during the pandemic. They experienced irritation, sadness due to the unavailability of the dustbin, and a few experienced foul odors, unhygienic conditions and felt shy in front of others.

One respondent described her experience “I don’t like the smell of the product and my blood. And I don’t want my family to see and smell of that disposed stuff. I don’t want my family to see something that was near my private area. And I am also worried that the adhesives might give up, and the folded pad opens up, revealing a “bad” scene”.

Discussion

Pandemics or any other disaster can have a severe physical impact on menstruation and the health of women [17-20]. Menstruating women are experiencing increased discrimination during the COVID-19 pandemic due to silence  and  ignorance  about menstruation, but also in logistics and access to menstruation pads or cups during the pandemics as observed by our group. This can result in difficult situations for both the women and for their health care workers, as reflected in extreme measures – as example frontline health workers reportedly used birth control pills to stop menstruation at Wuhan, China. However, women have a dire need  for menstrual dignity through more freedom during menstruation. The impact of menstrual discrimination has a multi-level impact that affects mental, physical and social health and might lead to violation of the human rights of the women. The needs and priorities of women should be scrutinized and need to be addressed even in shifting priorities of service providers during quarantine, isolations, hospitals, travel restrictions and curfews, the ongoing financial crisis. Scarcity of menstrual products and other essentials in some countries might be affected to a different degree. Communities and families need education and improved health literacy to fight health belief models leading to discrimination or violent acts such as forced segregation.

The observations in our mixed method study are limited by the small sample size and by the methodology based on an online survey and can therefore not provide representative data on the countries of the participants, that can be generalized. Still, the results identify and highlight some of the problems pre-existing and those accentuated or created during the pandemic.

Conclusion and Recommendations

Menstrual discrimination prevails in many countries and the problem was neglected across all the countries during the COVID-19 pandemic in our study. Since menstrual discrimination is a form of gender-based violence, it is continuously manifested in various forms. Based on the findings, our recommendations are as following:

  1. Information should be available everywhere in numerous languages on the physiology of menstruation and all aspects of menstrual dignity to increase health literacy for all genders and decrease stigma and discrimination based on inadequate knowledge and traditional cultural practices or belief systems.
  2. The menstrual products should be included in the COVID-19 response packages, and other humanitarian response materials.
  3. The provision of water, menstruation-friendly toilet/bathroom, hand sanitizers, and a mechanism for safe, discrete and low barrier waste management needs to be ensured in all settings, including temporary shelters, as explored by some authors [21].
  4. Programs as described in 1, focusing on menstrual dignity need to be continued across all programs as a cross-cutting concern.

References

  1. Abate BB, Kassie AM, Kassaw MW, Aragie TG, Masresha SA (2020) Sex difference in coronavirus disease (COVID-19): a systematic review and meta-analysis. BMJ Open 10: e040129. [crossref]
  2. Türkan Akkaya-Kalayci ODK, Thomas Wenzel, Anthony Chen VC, Zeliha Özlü- Erkilic (2020) The impact of the COVID-19 pandemic on mental health and psychological well-being of young people living in Austria and Turkey: a multicenter study International Journal of Environmental Research and Public Health 17: 9111. [crossref]
  3. Abufaraj M, Eyadat Z, Al-Sabbagh MQ, Nimer A, Moonesar IA, et (2021) Gender- based disparities on health indices during COVID-19 crisis: a nationwide cross- sectional study in Jordan. Int J Equity Health 20: 91.
  4. Cosma S, Carosso AR, Cusato J, Borella F, Carosso M, et al. (2021) Coronavirus disease 2019 and first-trimester spontaneous abortion: a case-control study of 225 pregnant Am J Obstet Gynecol 224: 391 e1-e7. [crossref]
  5. Xiong Z, Li P, Lyu H, Luo J (2021) Observational Study of Working from Home during the COVID-19 Pandemic Using Social Media JMIR Med Inform.
  6. Bagli I, Ocal E, Yavuz M, Uzundere O, Bozkurt F (2021) Maternal deaths due to COVID-19 disease: The cases in a single center pandemic hospital in the south east of J Obstet Gynaecol Res.
  7. Ahorsu DK, Imani V, Lin CY, Timpka T, Brostrom A, et al. (2020) Associations Between Fear of COVID-19, Mental Health, and Preventive Behaviours Across Pregnant Women and Husbands: An Actor-Partner Interdependence Modelling. Int J Ment Health Addict 1-15. [crossref]
  8. Abuhammad S (2021) Violence against Jordanian Women during COVID-19 Outbreak. Int J Clin Pract 75: e13824. [crossref]
  9. Abujilban S, Mrayan L, Hamaideh S, Obeisat S, Damra J (2021) Intimate Partner Violence Against Pregnant Jordanian Women at the Time of COVID-19 Pandemic’s J Interpers Violence 886260520984259. [crossref]
  10. Abdelbadee AY, Abbas AM (2020) Impact of COVID-19 on reproductive health and maternity services in low resource countries. Eur J Contracept Reprod Health Care 25: 402-404. [crossref]
  11. Jahan N (2020) Bleeding during the pandemic: the politics of menstruation. Sex Reprod Health Matters 28: 1801001. [crossref]
  12. Aolymat I (2020) A Cross-Sectional Study of the Impact of COVID-19 on Domestic Violence, Menstruation, Genital Tract Health, and Contraception Use among Women in Am J Trop Med Hyg 104: 519-525. [crossref]
  13. Wilbur J, Kayastha S, Mahon T, Torondel B, Hameed S, et (2021) Qualitative study exploring the barriers to menstrual hygiene management faced by adolescents and young people with a disability, and their carers in the Kavrepalanchok district, Nepal. BMC Public Health 21: 476.
  14. Thapa S, Aro AR (2021) ‘Menstruation means impurity’: multilevel interventions are needed to break the menstrual taboo in BMC Womens Health 21: 84. [crossref]
  15. Levitt RB, Barnack-Tavlaris JL (2020) Addressing Menstruation in the Workplace: The Menstrual Leave In: Bobel C, Winkler IT, Fahs B, Hasson KA, Kissling EA, Roberts TA, editors. The Palgrave Handbook of Critical Menstruation Studies. Singapore 561-575. [crossref]
  16. Paudel Radha A, Mili, Kletecka-Pulker, Maria, Wenzel, Thomas (2019) The Construction of Power in Nepal: Menstrual Restriction and Rape Arch Women Health Care 2.
  17. Li F, Lu H, Zhang Q, Li X, Wang T, Liu Q, et (2021) Impact of COVID-19 on female fertility: a systematic review and meta-analysis protocol. BMJ Open 11: e045524.
  18. Li K, Chen G, Hou H, Liao Q, Chen J, Bai H, et (2021) Analysis of sex hormones and menstruation in COVID-19 women of child-bearing age. Reprod Biomed Online 42: 260-267. [crossref]
  19. Mishra N, Sharma R, Mishra P, Singh M, Seth S, Deori T, et al. (2020) COVID-19 and Menstrual Status: Is Menopause an Independent Risk Factor for SARS Cov-2? J Midlife Health 11: 240-249. [crossref]
  20. Phelan N, Behan LA, Owens L (2021) The Impact of the COVID-19 Pandemic on Women’s Reproductive Front Endocrinol (Lausanne) 12: 642755. [crossref]
  21. Hirai M, Nyamandi V, Siachema C, Shirihuru N, Dhoba L, et al. (2021) Using     the Water and Sanitation for Health Facility Improvement Tool (WASH FIT) in Zimbabwe: A Cross-Sectional Study of Water, Sanitation and Hygiene Services in 50 COVID-19 Isolation Int J Environ Res Public Health 18: 5641. [crossref]
fig 2

An Effective Technique for Simultaneous Indirect-Direct Teeth Traction Using Temporary Anchorage Device

DOI: 10.31038/JDMR.2021422

Abstract

Since the introduction of Temporary Anchorage Devices (TADs) in the orthodontic field, they have been proven to be versatile and multi-applicable in the management of various orthodontic situation. Here, we highlight the viability of simple technique using TADs for an effective and time-efficient space closure.

Text

Temporary anchorage devices (TADs) are widely incorporated in the orthodontic field. TADs can be utilized directly or indirectly for anchorage, intrusion, distalization and retraction. Traditionally, when TADs are used indirectly for teeth retraction, the anchored tooth is maintained in place by using a stainless-steel ligature wire from the tooth to the TAD, allowing the retracted tooth to slide along the arch wire using an active open coil spring against the anchored tooth, utilizing push mechanics for retraction. While in direct teeth retraction, TADs can be utilized for pull mechanics [1].

Introduced here is an effective approach for simultaneous indirect-direct retraction using TAD. In this technique; instead of ligating the anchored tooth to the TAD, the ligature wire is ligated on the arch wire instead while the open coil spring is active. This will allow the retracted tooth to slide along the arch wire without the need to keep an anchored tooth in place and delay its retraction. Moreover, the adjacent tooth can be directly retracted using an elastomeric power chain attached from the TAD to the tooth. Thus, providing a simultaneous indirect-direct tooth retraction by utilizing push-pull mechanics on different teeth at the same time (Figures 1 and 2).

fig 1

Figure 1: An illustration showing the application of TAD for simultaneous indirect-direct teeth retraction by utilizing push-pull mechanics on different teeth at the same time.

fig 2

Figure 2: Clinical application of the technique.

Additionally, the neighboring teeth can follow the retraction and exhibit lateral movement as a result of recoiling of the trans-septal fibers between the retracted and adjacent teeth, allowing driftodontics to take place. This technique can be modified for retraction or mesialization of the teeth (Figure 3).

fig 3

Figure 3: An illustration showing modification of the technique to be used for teeth protraction.

References

  1. Antoszewska-Smith J, Sarul M Łyczek J, Konopka T, Kawala B (2017) Effectiveness of orthodontic miniscrew implants in anchorage reinforcement during en-masse retraction: A systematic review and meta-analysis. Am J Orthod Dentofacial Orthop 151: 440-455. [crossref]
fig 1

Discovering the Pitfall of Using Horse Radish Peroxidase in Enzyme Linked Immunosorbent Assays for Detection of Pollen Specific IgE

DOI: 10.31038/MIP.2021224

Abstract

Enzyme Linked Immunosorbent assays incorporate a functional enzyme conjugate in at least one procedural step; horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most commonly used. Recent evaluations in our laboratory yielded disparaging results for pollen specific IgE between assays that incorporate a biotinylated anti-IgE primary tracer reagent and either streptavidin-horseradish peroxidase (SA-HRP) or streptavidin-alkaline phosphatase (SA-AP) as the enzyme secondary conjugate. A screen of 1008 randomly selected samples submitted by veterinarians for routine allergy testing identified 128 samples that yielded a response consistent with an expected classical profile (CP) of positive responses when evaluated with an anti-IgE-biotin primary tracer followed by SA-HRP as secondary tracer, and negative responses when tested without anti-IgE-Biotin. An additional 96 reactive samples yielded a non-classical profile (NP) of equal magnitude with or without the anti-IgE-biotin conjugated tracer. Adsorption of IgE from sera pools prepared from CP samples and heat inactivation of IgE reactivity in these pools readily reduced the signal evident in untreated pools. Similar treatment of NP pools had minimal effect on the signal generated. Inhibition evaluations using unconjugated biotin or streptavidin indicates that neither is involved in the aberrant reaction. However, inhibition evaluations with unconjugated heat inactivated HRP reduces the reactivity in the NP pool and that the reactivity evident in the CP pool is unaffected by free HRP inhibition. These results are consistent with the hypothesis that antibodies specific for an epitope on glycoproteins present in the allergen extracts cross reacts with a component of HRP and binds the secondary tracer molecule without interfering with its enzymatic reactivity. Collectively, the results provide evidence that warrants avoidance of ELISAs that incorporate HRP as the enzyme containing tracer reagent but confirms the functional utility of ELISAs that incorporate alkaline phosphatase as the report enzyme, for assays that are intended for detection of allergen specific antibodies.

Keywords

Allergen specific IgE, ELISA, Pollen allergy, Horse radish peroxidase, Alkaline phosphatase

Introduction

Horseradish peroxidase (HRP) is one of the most widely used enzymes in analytical applications. HRP readily combines with hydrogen peroxide (H2O2) and the resultant [HRP-H2O2] complex can oxidize a wide variety of hydrogen donors. Consequently, the enzyme is often used in biochemistry applications such as immunohistochemistry [1], western blots [2], and ELISA [3], where it is used to amplify a weak signal and increase detectability of a target molecule. In the diagnostic arena, HRP is widely used as an enzyme label in medical diagnostics and research applications. One such use is for identifying allergen specific IgE and multiple assays have been developed in both human and veterinary arenas [4-7].

It has been more than three decades since the first in vitro assay for detection of allergen specific IgE in dogs became commercially available and the functional characteristics of this assay were described [5]. The introduction of this enzyme-linked immunosorbent assay (ELISA) fostered a number of similar commercial assays, all of which rely upon the use of either polyclonal or monoclonal anti-IgE antibodies, or a recombinant human high-affinity IgE epsilon receptor fragment as primary tracer molecules [4-8]. Many of these assays incorporated HRP as the reporter enzyme while others used alkaline phosphatase conjugates. Comparative evaluations of these different tests demonstrated substantial variance of results for the various assays [9-14], but no efforts have been put forth to identify why such dramatic differences might exist. To address this issue more thoroughly, we opted to comparatively evaluate the responses of results evident in ELISA that incorporate either HRP or alkaline phosphatase (AP). We hypothesize that the differences in responses evident in the two assays resides in the binding differences of the reporter enzyme conjugates.

Materials and Methods

Sera

The serum samples used throughout were derived from dogs suspected of clinical allergy and had previously been submitted by veterinarians for evaluation using Stallergenes Greer macELISA for detection of allergen-specific IgE. The sole criterion for selecting individual samples for this study was the volume of sera remaining following the allergen testing exceeded 2.0 mL; but hemolyzed or lipemic sera were excluded. Samples were stored frozen (–20°C) for up to one year before being used in this study. A total of 1008 sera derived from individual dogs were each screened, in triplicate, on an allergen panel that contained extracts derived from ash, marsh elder, and ragweed pollens. All samples were screened using an HRP based ELISA, and each was evaluated with and with and without anti-IgE-biotin included in the assay. Following the initial evaluation, two separate sera pools were prepared based on the reactivity profiles evident when evaluated in the HRP ELISA. Subsequent evaluations of the sera pools were completed using both HRP and AP based ELISAs, and a portion of each pool was heated at 56°C to inactivate the functional binding of IgE in the assays.

Buffers

The buffers used throughout have been previously described, [6,15,16] and included: a) well coating buffer: 0.05 M sodium carbonate bicarbonate buffer, pH 9.6; b) wash buffer: phosphate buffered saline (PBS), pH 7.4, containing 0.05% Tween 20, and 0.05% sodium azide; c) serum and reagent diluent buffer: PBS, pH 7.4, containing 1% fish gelatin, 0.05% Tween 20 and 0.05% sodium azide.

Preparation of Coated Wells

Immulon 4HBH flat bottom strip assemblies (Thermo Electron Corporation, Waltham, MA) were used throughout and served as the solid phase for all ELISA evaluations. The twelve well strips were individually coated with the specified allergen extracts following a previously defined procedure [6]. Briefly, the individual extracts were diluted in bicarbonate buffer (pH 9.6) and 100 µL was added to each assigned well. Following overnight incubation at 4-8°C, the wells were washed with PBS, blocked with 1% monoethanolamine (pH 7.5) then air dried and stored at 4-8°C in resealable plastic bags until used. The allergen panel used for screening samples consisted of ash, marsh elder, and ragweed allergens. To compare HRP and AP based ELISA a panel that included 10 grasses, 10 weeds, and 4 trees was used.

IgE Detection Reagents

Monoclonal anti-IgE antibodies were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin ester (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s recommended procedure. Briefly, individual monoclonal anti-IgE solutions were dialyzed against 50 mM carbonate buffer pH 8.5. A 6 mM solution of biotin ester in bicarbonate buffer was added to each monoclonal solution to yield a molar ratio mixture of 12:1 (biotin: monoclonal). Following incubation at room temperature for 2 hours with constant agitation, excess biotin ester was removed by dialysis against Tris-Buffered Saline. The protein recovered from each reaction mixture was estimated by determining the optical density at 278 nm, assuming an extinction coefficient of 1.4 for each monoclonal IgG. A sufficient volume of glycerol was added to each monoclonal-biotin conjugate to yield a 50% solution before storage at –20°C. The IgE specificity of the monoclonal anti-IgE was confirmed by documenting the heat lability (56°C) of reactive serum components. A mixture of three monoclonal anti-canine IgE antibody preparations was optimized for use in the IgE specific ELISAs.

The substrate reagent used for the SA-HRP conjugate, o-phenylenediamine dihydrochloride (OPD), was purchased from Sigma-Aldrich (St. Louis, MO, USA). The SA-AP substrate reagent, p-nitrophenylphosphate (pNPP), was purchased from Moss Inc (Pasadena, MD, USA). Each enzyme conjugate was stored at 4-8° C in its respective stabilizing buffer, which was also purchased (Sigma-Aldrich, St. Louis, MO, USA).

Sample Evaluations – ELISA

The basic operational characteristics and procedures for the ELISAs have been previously described [5,6]. Briefly, serum samples were diluted 1 : 6 in diluent buffer. One hundred microlitres of the diluted samples was added to coated microwells and incubated overnight (14–16 h) at 4–8°C. The wells were washed twice with PBS-T, and 100 μL of biotinylated monoclonal anti-IgE-biotin mixture in diluent buffer was added to each well; when evaluated without anti-IgE-biotin 100 μL of diluent buffer was substituted for the biotinylated monoclonal anti-IgE-biotin mixture. After 2 h incubation at room temperature (22°C), the wells were washed thrice with PBS-T, and 100 μL of streptavidin-enzyme conjugate in diluent buffer was added to each well before incubation for 1 h at room temperature. Following a final washing (four cycles with PBS-T), 100 μL of appropriate substrate was added to each well. Following a 1 h incubation period, the HRP reactivity was stopped by adding 50 μL of 2M H2SO4 to each well while the AP reactivity was stopped by adding 50 μL of 20 mM cysteine to each well. Specific IgE reactivity to the allergens was then estimated by determining the absorbance of each well measured at 405 nM for the AP ELISA and 492 nm for HRP ELISA using an automated plate reader. All results are expressed as ELISA Absorbance Units (EAU) which are background-corrected observed responses expressed as milli absorbance [6].

Statistics

Statistical analysis was performed with a commercial software package (PRISM v9, GraphPad; La Jolla, CA, USA); P‐values ≤ 0.05 were considered statistically significant. The Student’s t-test was used to evaluate the significance of differences of observed responses.

Results

Definition of Classical and Non-Classical Responses

The first experiments undertaken evaluated the reactivity evident in serum samples that were evaluated on an allergen panel that included extracts of ash, marsh elder and ragweed; each sample was evaluated with and without an anti-IgE-biotin tracer conjugate. For those sera samples that were shown to possess allergen specific IgE for these allergen extracts when evaluated in an ELISA that incorporates HRP as the reporter enzyme, two representative reactivity profiles became readily apparent. The first, which is representative of what might be expected when a serum sample is evaluated with or without anti-IgE-biotin included in the assay. The signal generated with the inclusion of anti-IgE-biotin was not evident when the assay was completed using diluent only in place of anti-IgE-biotin. We have identified this reaction profile as a classical profile (CP). The second response profile that is observed with some serum samples were characterized as a non-classical profile (NP). For these samples the magnitude of signal that was yielded, for all allergens in the screen, without including anti-IgE-biotin in the assay were of the same order of magnitude (P<0.001) as the signals generated when this reagent was included. Approximately 22% (224/1008) of all the samples screened were reactive to the pollen allergens (Table 1). Of these reactive samples, 57.2% (128/224) yielded a reactivity profile consisted with a classical response, and 42.8% (96/224) responded in a non-classical manner. All subsequent evaluations presented in this document used pools of sera that were derived from these characterized samples. Only samples that exhibited a classical response were included in the CP sera pool; likewise, only samples that exhibited similar responses with and without anti-IgE-biotin in the evaluation were included in the NP sera pool.

Table 1: Incidence of pollen reactivity that behave in expected classical manner and non-classical manner (N=1008).

Samples

Number of Test Samples % of Screened Samples

% of Test Reactive Samples

Reactive to Test Antigensa

224

22.2

100

Reactive in Non-Classical Mannerb

96

9.5

42.8

Reactive in Classical Mannerb

128

12.7

57.2

aTall Ragweed, Marsh Elder, and White Ash.
bSamples reacting in non-classical manner are reactive with and without inclusion of the primary biotinylated tracer samples reacting in classical manner are reactive only when primary biotinylated tracer is included in the assay.

Allergen Reactivity Profile for Classical and Non-Classical Sera Pools

To characterize the reactivity profile for the CP and NP sera pools for a panel of grass, weed, and tree pollen allergens, each pool was evaluated using an HRP based ELISA as well as an AP based ELISA; each pool was evaluated with and without anti-IgE-biotin in the assay. The results demonstrate that the CP pool does, in fact, react to all pollens tested (Table 2) in a manner consistent with the definition of a classical reactivity profile. Signals of substantial magnitude were evident with all grasses, weeds, and trees, but the signals yielded without including anti-IgE-biotin were dramatically different (P<0.001) and were indistinguishable from the background responses (P<0.001). On the other hand, the signals evident with the NP pool were not significantly different (P=0.264) when evaluated with or without anti-IgE-biotin (Table 2). The average signal evident with the NP pool when evaluated on grass pollen allergens in the absence of anti-IgE-biotin was 76.7% of the signal that was generated in the presence of anti-IgE-biotin and ranged between 50.0-90.3% depending on the allergen tested. When evaluated in the absence of anti-IgE-biotin, the average signal evident with weed and tree pollen allergens was 93.3% (range 84.3-100%) and 87.8% (range 60.0-99.2%), respectively, of the signal evident when evaluated with the biotinylated reagent.

Table 2: Pollen reactivity of Classical and Non-Classical sera pools when evaluated with and without anti-IgE-biotin in an ELISA that incorporates HRP in the secondary tracer conjugate (mean ± SD of triplicate evaluations)

Allergens

  EAU    
  Classical Pool

Non Classical Pool

 

Biotin

NO Biotin Biotin

NO Biotin

Grasses
Bermuda (Cynodon dactylon)

3864 ± 29

6 ± 2 2266 ± 25

1871 ± 54

Brome (Bromus inermis)

3844 ± 50

39 ± 7 926 ± 25

463 ± 27

Johnson (Sorghum halepense)

3779 ± 36

0 ± 1 3246 ± 40

2931 ± 102

Kentucky Blue (Poa pratensis)

3839 ± 36

46 ± 10 773 ± 66

458 ± 69

Meadow fescue (Festuca pratensis)

3875 ± 29

123 ± 37 2200 ± 85

1818 ± 70

Orchard (Dactylis glomerata)

3868 ± 25

99 ± 32 2305 ± 53

1853 ± 94

Perennial Rye (Lolium perenne)

3854 ± 32

147 ± 49 1911 ± 212

1393 ± 46

Red Top (Agrostis alba)

3855 ± 62

32 ± 3 2111 ± 27

1801 ± 94

Sweet Vernal (Anthoxanthum odoratum)

3791 ± 75

101 ± 25 1585 ± 123

1317 ± 131

Timothy (Phleum pratense)

3861 ± 29

34 ± 10 1708 ± 38

1373 ± 89

Trees
Birch (Betula nigra)

2042 ± 172

33 ± 56 3031 ± 78

2937 ± 51

Box Elder (Acer negundo)

3819 ± 16

0 ± 6 1170 ± 37

702 ± 57

Quaking Aspen (Populus tremuloides)

3569 ± 105

0 ± 19 1683 ± 44

1670 ± 70

White Ash (Fraxinus Americana)

2357 ± 71

155 ± 15 3229 ± 53

3076 ± 41

Weeds
Cocklebur (Xanthium strumarium)

3493 ± 49

47 ± 54 3302 ± 17

2934 ± 49

English Plantain (Plantago lanceolata)

3649 ± 83

22 ± 16 1660 ± 28

1431 ± 113

Kochia (Bassia scoparia)

3809 ± 115

0 ± 3 3328 ± 44

3215 ± 69

Lambs Quarter (Chenopodium album)

3717 ± 55

0 ± 10 3381 ± 23

3218 ± 53

Marsh Elder (Cyclachaena xanthiifolia)

3781 ± 19

46 ± 5 3539 ± 26

3546 ± 35

Marsh Elder (Iva annua)

2633 ± 123

88 ± 13 3553 ± 48

3540 ± 24

Pigweed (Amaranthus palmeri)

3819 ± 50

52 ± 8 3271 ± 40

3032 ± 39

Ragweed (Ambrosia trifida)

3839 ± 10

68 ± 18 3474 ± 18

3418 ± 40

Sheep Sorrel (Rumex acetosella)

3696 ± 122

68 ± 73 3117 ± 3

2902 ± 148

Yellow Dock (Rumex crispus)

3663 ± 100

254 ± 27 2924 ± 82

2464 ± 149

All results were expressed as ELISA Absorbance Units (EAU; mean ± SD) which are background corrected observed responses (OD at 492 nm) expressed as milli absorbance.

Heat inactivation of IgE reactivity in sera

The effect of heating on the reactivity of the CP and NP sera pools to the various allergens demonstrated the that a significant portion (>90%; P<0.001) of the pollen reactivity evident in the CP pool (presumably IgE) was eliminated following heat treatment (Table 3).

Table 3: Pollen reactivity of Classical and Non-Classical sera pools following heat treatment (56°C, 4 h) when evaluated with and without anti-IgE-biotin in an ELISA that incorporates HRP in the secondary tracer conjugate.

Allergens

  EAU    
  Classical Pool Non Classical Pool
  Biotin NO Biotin Biotin

NO Biotin

Grasses
Bermuda (Cynodon dactylon)

264 ± 51

17 ± 11 895 ± 39

869 ± 42

Brome (Bromus inermis)

261 ± 47

71 ± 11 327 ± 12

333 ± 71

Johnson (Sorghum halepense)

174 ± 21

0 ± 55 1702 ± 56

1693 ± 48

Kentucky Blue (Poa pratensis)

246 ± 7

31 ± 9 258 ± 73

179 ± 55

Meadow fescue (Festuca pratensis)

282 ± 6

53 ± 4 943 ± 8

708 ± 8

Orchard (Dactylis glomerata)

241 ± 3

92 ± 15 1001 ± 4

806 ± 31

Perennial Rye (Lolium perenne)

282 ± 8

74 ± 18 686 ± 39

478 ± 29

Red Top (Agrostis alba)

236 ± 26

30 ± 6 924 ± 75

927 ± 82

Sweet Vernal (Anthoxanthum odoratum)

272 ± 25

295 ± 21 717 ± 62

706 ± 47

Timothy (Phleum pratense)

200 ± 6

24 ± 4 665 ± 13

435 ± 7

Trees
Birch (Betula nigra)

139 ± 43

37 ± 6 1879 ± 37

1818 ± 53

Box Elder (Acer negundo)

190 ± 43

7 ± 10 527 ± 45

374 ± 28

Quaking Aspen (Populus tremuloides)

103 ± 5

1 ± 41 687 ± 45

747 ± 39

White Ash (Fraxinus Americana)

246 ± 4

160 ± 11 2115 ± 26

2051 ± 9

Weeds
Cocklebur (Xanthium strumarium)

167 ± 22

52 ± 11 1560 ± 130

1217 ± 169

English Plantain (Plantago lanceolata)

198 ± 22

9 ± 7 631 ± 16

431 ± 17

Kochia (Bassia scoparia)

219 ± 8

28 ± 1 1967 ± 89

2016 ± 33

Lambs Quarter (Chenopodium album)

136 ± 25

0 ± 9 2302 ± 57

1977 ± 29

Marsh Elder (Cyclachaena xanthiifolia)

162 ± 1

24 ± 11 2730 ± 198

2867 ± 50

Marsh Elder (Iva annua)

157 ± 13

45 ± 6 2872 ± 64

2969 ± 19

Pigweed (Amaranthus palmeri)

258 ± 40

56 ± 6 1917 ± 15

1451 ± 41

Ragweed (Ambrosia trifida)

189 ± 7

55 ± 1 2518 ± 81

2556 ± 71

Sheep Sorrel (Rumex acetosella)

244 ± 28

25 ± 2 1757 ± 51

1449 ± 68

Yellow Dock (Rumex crispus)

396 ± 20

181 ± 6 1380 ± 80

1134 ± 51

A significant (P<0.001) portion of the pollen reactivity evident in the NP pool was also affected by heating; however, substantial reactivity (ca. 50%) remained following heat treatment and the reactivity evident with and without anti-IgE-biotin were not different (P=0.631). Similar profiles of reactivity were evident in the CP and NP pools when each pool was rendered deficient in IgE by immunoaffinity chromatography using solid phase bound anti-IgE (data not shown).

Reactivity of Classical and Non-Classical Sera Pools When Reacted with Free HRP

The blocking effects that free biotin, streptavidin, or HRP might have on the reactivity evident in the NP pool, but lacking in the CP pool, was evaluated by incubating each serum pool individually with an excess of each of these components. None of these treatments altered the reactivity profile of either of the CP or NP sera pools (data not shown). However, the results clearly demonstrated that evaluation of the NP sera pool with free HRP included in the assay, at varying concentrations, in place of the streptavidin-HRP conjugate, resulted in generation of a substantial signal that approximates one-half the magnitude of signal evident when the assay was completed including streptavidin-HRP (Figure 1).

fig 1

Figure 1: ELISA reactivity of Classical and Non-Classical sera pools to ragweed (mean ± SD of triplicate evaluations) following substitution of Streptavidin-HRP with varying concentrations of unconjugated horseradish peroxidase.

No significant differences (P<0.001) were noted for the responses evident with or without anti-IgE-biotin regardless of the concentration of free HRP that was included in the assay. However, the signals evident at 31.3 nG/mL or less of free HRP were significantly different (P,0.001) than the signals evident at concentrations of 250 ng/ml or greater which indicated a concentration dependent binding of the free HRP. A similar signal, presumably due to binding of the free HRP, was lacking in the CP sera pool. The addition of enzymatically inactive HRP had no dramatic effect on either the background responses (P, 0.001) or the signal generated with the CP pool when evaluated with or without anti-IgE-biotin (Figure 2). On the other hand, the magnitude of signal generated with the NP pool was dramatically diminished (P<0.001) when evaluated with or without anti-IgE-biotin.

fig 2

Figure 2: Effect of adding inactivated horseradish peroxidase on the reactivity of sera pools that exhibit Classical and Non-Classical reactivity profiles when evaluated using an ELISA (mean ± SD of triplicate evaluations) that incorporates horse radish peroxidase enzyme conjugate.

Reactivity of Classical Pool and Non-Classical Pool Using Alkaline phosphatase ELISA

Finally, both CP and NP sera pools were evaluated using an ELISA that incorporates a streptavidin-alkaline phosphate conjugate in place of the streptavidin-HRP conjugate. The results (Table 4) demonstrated that a substantial signal was yielded with the CP sera pool when evaluated with anti-IgE-biotin and that the signal was lacking in the assay without anti-IgE-biotin (P<0.001). Similarly, no signal was evident (P<0.001) with the NP sera pool when evaluated without anti-IgE-biotin. Yet, a substantial signal (P<0.001) indicative of allergen specific IgE was detected when using the alkaline phosphatase enzyme.

Table 4: Pollen reactivity of Classical and Non-Classical sera pools when evaluated with and without anti-IgE-biotin in an ELISA that incorporates alkaline phosphatase in the secondary.

Allergens

  EAU    
 

Classical Pool

Non Classical Pool

  Biotin NO Biotin Biotin

NO Biotin

Grasses
Bermuda (Cynodon dactylon)

3835 ± 58

6 ± 2 835 ± 10

3 ± 9

Brome (Bromus inermis)

3868 ± 23

39 ± 7 304 ± 10

22 ± 3

Johnson (Sorghum halepense)

3789 ± 28

0 ± 1 1190 ± 16

0 ± 10

Kentucky Blue (Poa pratensis)

3827 ± 53

46 ± 10 236 ± 26

39 ± 88

Meadow fescue (Festuca pratensis)

3858 ± 33

123 ± 37 815 ± 34

24 ± 9

Orchard (Dactylis glomerata)

3871 ± 19

99 ± 32 857 ± 21

39 ± 7

Perennial Rye(Lolium perenne)

3845 ± 51

147 ± 49 699 ± 85

0 ± 3

Red Top (Agrostis alba)

3821 ± 23

32 ± 3 782 ± 11

69 ± 14

Sweet Vernal (Anthoxanthum odoratum)

3783 ± 40

101 ± 25 556 ± 49

57 ± 9

Timothy (Phleum pratense)

3837 ± 43

34 ± 10 620 ± 15

25 ± 12

Trees
Birch (Betula nigra)

2042 ± 172

33 ± 56 1099 ± 31

0 ± 8

Box Elder (Acer negundo)

3819 ± 16

0 ± 6 369 ± 15

0 ± 1

Quaking Aspen (Populus tremuloides)

3569 ± 105

1 ± 19 560 ± 17

0± 4

White Ash (Fraxinus Americana)

2357 ± 71

155 ± 15 1162 ± 21

0 ± 5

Weeds
Cocklebur (Xanthium strumarium)

3493 ± 49

47 ± 54 1252 ± 7

12 ± 2

English Plantain (Plantago lanceolata)

3678 ± 65

22 ± 16 493 ± 11

0 ± 7

Kochia (Bassia scoparia)

3794 ± 71

0 ± 3 1257 ± 17

40 ± 1

Lambs Quarter (Chenopodium album)

3717 ± 55

0 ± 10 1218 ± 9

0 ± 5

Marsh Elder (Cyclachaena xanthiifolia)

3780 ± 19

46 ± 5 1290 ± 10

0 ±5

Marsh Elder (Iva annua)

2633 ± 123

88 ± 13 1356 ± 19

14 ± 1

Pigweed (Amaranthus palmeri)

3814 ± 49

52 ± 8 1228 ± 16

11 ± 14

Ragweed (Ambrosia trifida)

3754 ± 48

68 ± 18 1299 ± 7

0 ± 21

Sheep Sorrel (Rumex acetosella)

3751 ± 68

68 ± 73 1131 ± 10

0 ± 4

Yellow Dock (Rumex crispus)

3713 ± 57

287 ± 57 1027 ± 33

0 ± 2

All results were expressed as ELISA Absorbance Units (EAU) which are background corrected observed responses (OD at 405 nm) expressed as milli absorbance.

Discussion

We characterized two different patterns of reactivity among dog serum samples that yield a positive reaction in an ELISA that incorporates HRP as the reporter enzyme. The classical profile of reactivity was evident in samples that yield a positive response when evaluated in ELISA that include all assay components but exhibited no response when tested in the absence of the anti-IgE-biotin test reagent; slightly less than 60% of the reactive samples tested yield this characteristic response. The non-classical profile of reactivity was characteristic of samples that yielded a response in ELISA when evaluated without anti-IgE-biotin tracer reagent; more than 40% of the pollen reactive dog serum samples yielded a response with this characteristic. The magnitude of response evident in these samples approximated the magnitude of response evident when the sample was evaluated in an ELISA including anti-IgE-biotin reagent.

Pools of these respective sera yielded responses that were consistent with the responses evident with the individual samples that comprise the pools. When evaluated with and without the anti-IgE-biotin reagent the character of the CP and NP pools were evident with all pollen allergens tested, which encompass 10 grasses, 10 weeds, and 4 trees. However, when evaluated against mite allergen extracts the responses evident with both the CP and NP pools were indistinguishable from background responses when evaluated in the absence of the anti-IgE-biotin reagent (data not shown). Thus, this serum dependent response appears to be restricted to pollen extracts. Neither the functional removal of IgE from the sera pools by heat treatment nor the physical removal of IgE using immunoaffinity chromatography substantially altered the response profile yielded with the NP pool. However, the magnitude of responses that were evident in the NP pool following heat treatment were reduced by 40% to 50% of the signal evident in the unheated sample, indicating that a portion of the response evident in the NP pool was likely the result of allergen specific IgE. Greater than 90% of the allergen specific IgE present in the CP pool was inactivated by heating and substantiates that the reactivity remaining in the NP pool following heat treatment was due to a serum component that is not IgE with specificity toward the pollen allergens. This conclusion is supported by the results observed following removal of IgE from the sera pools.

To define a plausible explanation for the results observed with this non-classical response we attempted to block the response using various unconjugated assay components that might be interacting with the responsible serum component, and in so doing allowed for the binding of the HRP-streptavidin enzyme conjugate component of the assay. Addition or substitution of any of the assay components with the unconjugated reactive motif (biotin, streptavidin, or HRP) did not alter the reactivity profile evident with either the CP of NP pools. However, substitution of Streptavidin-HRP with unconjugated, but enzymatically active, HRP yielded results consistent with the NP reactivity profile but had no effect on the CP pool. Such results were consistent with the hypothesis that a serum component evident in the NP pool was facilitating HRP (either conjugated or unconjugated) binding during the streptavidin-HRP incubation stage of the assay. To address this hypothesis, heat treated (90°C, 4 hrs.) HRP was simultaneously added, along with the streptavidin-HRP, at the appropriate stage of the assay. The results demonstrated that functionally inactive HRP does, in fact, substantially reduced the signal that was generated with the NP pool but did not alter the signal evident with the CP pool. These results, combined with the results yielded using a comparable ELISA that uses alkaline phosphate enzyme conjugate, provide conformation that a serum component in the NP pool was effectively binding the HRP component of the streptavidin-HRP reagent. The interaction between the streptavidin and the serum dependent component does not modify the enzymatic functionality of HRP.

The most obvious serum component that might allow for binding of HRP in an allergen specific ELISA without interaction of streptavidin-HRP conjugate with the anti-IgE biotinylated reagent is actually an allergen specific antibody. We propose that epitopes present on the pollen allergens that are cross-reactive with epitopes on HRP will specifically bind the various classes of antibodies, especially IgG, present in serum. During the subsequent incubation period with streptavidin-HRP the specific allergen bound antibody will also specifically bind to the cross-reactive epitope on the HRP molecule that is conjugated to the streptavidin but without interfering with the enzymatic function of HRP. With addition of substrate, a specific but non-related signal will result. Support for this hypothesis is the observation that antibodies specific for cross reactive carbohydrates (CCD) have been defined in a number of mammals including humans, dogs, and cats where the prevalence of anti-CCD IgE has been estimated to range from 20% to 70% [17-22]. Reaction with these molecules results in a false positive interpretation for many allergen extracts when evaluated using in vitro assays intended for detection of allergen specific IgE [23,24]. The relevant structure of the epitopes responsible for these false positive reactions has been characterized as a 1,3-fucose linked to the amide nitrogen of an asparagine residue of the protein [25,26]. Such carbohydrate containing structures have also been identified as specific carbohydrate epitopes of HRP. Furthermore, these specific N-glycans are widely distributed among pollens and invertebrate animals but are lacking in mammalian proteins [25,26] where they can be strongly antigenic. This being the case, we propose that epitopes of this sort that are present on the pollen allergens will specifically bind the various classes of antibodies present in sera which then concomitantly binds the HRP conjugate.

Collectively, the results presented herein document substantial differences in responses that were yielded in an ELISA that incorporated an HRP enzyme conjugate and one that incorporated an AP enzyme conjugate. The results provide supportive information that warrants avoidance of ELISAs that incorporate HRP as the enzyme containing tracer reagent that are intended for detection of pollen specific antibodies. Also included are results that support the functional utility of ELISAs that incorporate alkaline phosphatase as the report enzyme.

Acknowledgments

Funding for this study was provided by Stallergenes Greer. At the time of the study all authors were employees of Stallergenes Greer.

References

  1. Janardhan KS, Jensen H, Clayton NP, Herbert RA (2018) Immunohistochemistry in Investigative and Toxicologic Pathology. Toxicol Pathol 46: 488-510. [crossref]
  2. Ghosh R, Gilda JE, Gomes AV (2014) The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics 11: 549-560. [crossref]
  3. Aydin S (2015) A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 72: 4-15. [crossref]
  4. Ansotegui IJ, Melioli G, Canonica GW, Caraballo L, Villa E, et al. (2020) IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper. World Allergy Organ J 13: 100080. [crossref]
  5. Kleinbeck ML, Hites MJ, Loker JL, Halliwell RE, Lee KW (1989) Enzyme-linked immunosorbent assay for measurement of allergen-specific IgE antibodies in canine serum. Am J Vet Res 50: 1831-1839. [crossref]
  6. Lee KW, Blankenship KD, McCurry ZM, Esch RE, DeBoer DJ, et al. (2009) Performance characteristics of a monoclonal antibody cocktail-based ELISA for detection of allergen-specific IgE in dogs and comparison with a high affinity IgE receptor-based ELISA. Vet Dermatol 20: 157-164. [crossref]
  7. Stedman K, Lee K, Hunter S, Rivoire R, McCall K, Wassom D (2001) Measurement of canine IgE using the alpha chain of the human high affinity IgE receptor. Vet Immunol Immunopathol 78: 349-355. [crossref]
  8. Hensel P, Santoro D, Favrot C, Hill P, Griffin C (2015) Canine atopic dermatitis: detailed guidelines for diagnosis and allergen identification. BMC Vet Res 11: 196. [crossref]
  9. Wang J, Godbold JH, Sampson HA (2008) Correlation of serum allergy (IgE) tests performed by different assay systems. J Allergy Clin Immunol 121: 1219-1224. [crossref]
  10. Szeinbach, SL, Barnes, JH, Sullivan TJ, Williams PB (2001) Precision and accuracy of commercial laboratories’ ability to classify positive and/or negative allergen specific IgE results. Ann Allergy Asthma Immunol 86: 1-9. [crossref]
  11. Plant JD, Neradelik MB, Polissar NL, Fadok VA, Scott BA (2014) Agreement between allergen-specific IgE assays and ensuing immunotherapy recommendations from four commercial laboratories in the USA. Vet Dermatol 25: 15-16. [crossref]
  12. DeBoer DJ, Hillier A (2001) The ACVD task force on canine atopic dermatitis (XVI): laboratory evaluation of dogs with atopic dermatitis with serum-based “allergy” tests. Veterinary Immunology and Immunopathology 81: 277-87. [crossref]
  13. Codner EC, Lessard P (1993) Comparison of intradermal allergy test and enzyme-linked immunosorbent assay in dogs with allergic skin disease. Journal of the American Veterinary Medical Association 202: 739-43. [crossref]
  14. Hammerling R, De Weck AL (1998) Comparison of two diagnostic tests for canine atopy using monoclonal anti-IgE antibodies. Veterinary Dermatology 9: 191-199. [crossref]
  15. Lee KW, Blankenship KD, McCurry ZM, McKinney B, Ruffner R, et al. (2012) Intra and inter-laboratory reproducibility of a monoclonal antibody cocktail based ELISA for detection of allergen specific IgE in dogs: proficiency monitoring of macELISA in six laboratories. Vet Immunol Immunopathol 148: 267-275. [crossref]
  16. Enck K, Lee K, McKinney B, Lillard J, Globokar M, et al. (2020) Proficiency Monitoring of Allergen-Specific IgE macELISA – 2020. Integr J Vet Biosci 4: 1-7.
  17. Mari A (2002) IgE to cross-reactive carbohydrate determinants: analysis of the distribution and appraisal of the in vivo and in vitro reactivity. Int Arch Allergy Immunol 129: 286-295. [crossref]
  18. Holzweber F, Svehla E, Fellner W, Dalik T, Stubler S, et al. (2013) Inhibition of IgE binding to cross-reactive carbohydrate determinants enhances diagnostic selectivity. Allergy 68: 1269-1277. [crossref]
  19. Levy BJ, DeBoer DJ (2018) A preliminary study of serum IgE against cross-reactive carbohydrate determinants (CCD) in client-owned atopic dogs. Vet Dermatol 29: 243-290. [crossref]
  20. Lee KW, McKinney BH, Blankenship K, Morris, DO (2020) Detection and Inhibition of IgE for cross‐reactive carbohydrate determinants evident in an enzyme‐linked immunosorbent assay for detection of allergen‐specific IgE in the sera of dogs and cats. Vet Dermatol 31: 439-e116. [crossref]
  21. Piccione, ML, DeBoer DJ (2019) Serum IgE against cross-reactive carbohydrates determinants in healthy and atopic dogs. Vet Dermatol 30: 507-e153. [crossref]
  22. Gedon, NKY, Boehm TMSA, Klinger CJ, Udraite L, Mueller RS (2019) Agreement of serum allergen test results with unblocked and blocked IgE against cross-reactive carbohydrate determinants (CCD) and intradermal test results in atopic dogs. Vet Dermatol 30: 243-e90.
  23. Altmann F (2016) Coping with cross-reactive carbohydrate determinants in allergy diagnosis. Allergo J Int 25: 98-105. [crossref]
  24. Foetisch K, Westphal S, Lauer I, Retzek M, Altmann F, et al. (2003) Biological activity of IgE specific for cross-reactive carbohydrate determinants. J Allergy Clin Immunol 111: 889-896. [crossref]
  25. Jin C, Hantusch B, Hemmer W, Stadlmann J, Altmann F (2008) Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins. J Allergy Clin Immunol 121: 185-190. [crossref]
  26. Hemmer W, Altmann F, Holzweber F, Gruber C, Wantke F, et al. (2018) Immuno CAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels. J Allergy Clin Immunol 141: 372-381e3. [crossref]
fig 2

Kinetics and Spatial Distribution of Glial Derived Nerve Growth Factor during Experimental Peripheral Nerve Regeneration

DOI: 10.31038/IJOT.2021421

Abstract

Nerve growth factors have been used therapeutically to enhance nerve regeneration with variable results, suggesting that before its use as therapeutic agents is important to determine their timing and spatial distribution during peripheral nerve regeneration. One of the most important growth factors in nerve regeneration is glial derived nerve factor (GDNF). Thus, in this study was determined the kinetics of gene expression and cellular sources of GDNF in Wistar rats after transection of the sciatic nerve and resection of 5mm in its middle third.

Both proximal and distal nerve stumps were obtained at different time points with laser capture microdissection and used for RNA isolation for quantification of GDNF gene expression by Reverse Transcriptase Polymerase Chain Reaction. The cellular source was determined by immunohistochemistry. Our results showed that transcripts of GDNF were constantly expressed and exhibited two peaks, at 48 hrs being inflammatory macrophages the cells that showed the highest GDNF immunostaining. The second peak was seen at 17th to 26 th days, being the Schwann cells from the proximal nerve stump the highest GDNF immunostained cells. Thus, GDNF is constantly produced at the site of the injury during peripheral nerve regeneration with two maximal time points, during early and late regeneration, suggesting that its local administration could be used therapeutically to increase nerve regeneration.

Keywords

Growth Derived Nerve Factor, Nerve Injury, Peripheral Nerve Regeneration, Laser Microdisection

Introduction

Nerve growth factors are produced during embryo development, neurodegenerative diseases and after traumatic peripheral nerve injury [1]. During nerve regeneration diverse cells and its products participate, such as Schwann cells, macrophages and neurotrophic factors being one of the most important glial derived nerve factor (GDNF) [1].

Nerve growth factors have been administrated by various methods to enhance nerve regeneration [2]. These methods have had variable results in several experiments, perhaps because the timing and amount of the administrated factors have not been appropriate. Thus, it is important to know the kinetics of the neurotrophic factors production, in order to replicate the normal regeneration and in that way improve the reparative nerve process. The aim of the present study was to determine the kinetics and cellular sources of GDNF after section of the sciatic nerve in the rat.

Materials and Methods

Wistar rats six weeks old were anesthetized, the right sciatic nerve was exposed, dissected and transected in its middle third where 5mm were resected. Group of three animals were euthanized at 6, 12, 24, 36 hrs; and after 3, 4, 5, 6, 9, 10, 17, 22, 31, 43 days of nerve section. The surgical wound was opened, the sciatic nerve exposed and the distal part of the proximal stump was resected and tagged with silk suture in its proximal end. The same procedure was done in the proximal part of the distal nerve. Tissue samples were fixed and embedded in paraffin.  Animals work was performed according to the guidelines of the Mexican Institutional Animal Care and the local committee (permit 264).

Specific areas of nerve regeneration were obtained by laser capture microdissection (LCM) using an XTTM Microdissection System Arcturus XT, isolating the distal portion of the proximal nerve and the most proximal part of the distal stump.

To determine the gene kinetics of GDNF, RNA was isolated from the nerve tissue fragments obtained by LCM. Reverse transcription was performed using 5µg RNA, oligo-dt and Omniscript kit (Qiagen, Inc). Real-time PCR was done using the 7500 RT-PCR system (Applied Biosystems, USA) and Quantitect SYBER Green Kit (Qiagen). Specific primers for GDNF transcripts were designed (Primer Express, Applied Biosystems, USA), β-actin was used as housekeeping gene.

The same paraffin embedded tissue used for LMC was used for immunohistochemistry detection of GDNF, using a mouse monoclonal antibody (Santa Cruz Biotechnology, INC.) and anti-mouse rabbit immunodetector HRP/DAB (BIOSB,USA).

Results

Proximal and distal nerve stumps expressed GDNF transcripts in all the studied time points and showed two peaks, the highest was after two days of nerve injury in the distal nerve ending, while the second peak was at 17 and 26 days in the proximal nerve stump [Fig 1].

fig 1

Figure 1: Kinetics of GDNF gene expression during peripheral nerve regeneration. Sciatic nerve was dissected, sectioned and a small fragment was removed in a large group of Wistar rats. Three rats were euthanized at the indicated time points and the injured sciatic nerve and surrounded tissues were dissected, fixed by immersion and embedded in paraffin. The proximal and distal nerve stumps were isolated from these paraffin blocks by laser microdisection and total RNA was purified and used to quantify GDNF gene expression by RT-PCR. The number of GDNF mRNA copies related to 106 mRNA copies of actin as housekeeping gene are shown.

Histologically, after 6 hrs there was acute inflammation with numerous mast cells around perineurial blood vessels and epineurium [Fig 2A]. At 12 hrs, the epineurium and perineurium showed connective tissue hyalinization, dilated blood vessels surrounded by numerous lymphocytes, monocytes and neutrophils [Fig 2B]. The injured nerve showed vacuolization with cellular decrease. After two days, numerous lymphocytes and mainly macrophages were located around perineural vessels, on the epineurium and between nerve fascicles and muscle, the injured nerve showed detached Schwann cells. The epineurium show incipient granulation tissue, numerous mast cells, edema and focal rabdomyolisis [Fig 2C, 2D]. Many macrophages showed strong GDNF immunostaining, while weak staining was seen in some fibroblasts, endothelial and Schwann cells [Fig 3A].  At days 4 and 6, inflammation was mild with macrophages distributed between nerve fibers, in the epineurium and perineurium with some mast cells and fibroblasts. At day 7, the inflammatory infiltrate was slight and there were sprouts or nerve ramifications from the proximal nerve to the muscle tissue with some axons covered by Schwann cells [Fig 3E]. The nerve sprouts showed irregular shape, some were nodules constituted by Schwann cells from day 17 to 26 [Fig 3F, 3G], surrounded by mild fibrosis and chronic inflammation that progressively decreased [Fig 3H]. Numerous new formed nerve sprouts were constituted by Schwann cells with strong GDNF immunostaining, being the highest in the principal sectioned nerve, while macrophages showed lesser immunoreactivity [Fig 3].

fig 2

Figure 2: Representative micrographs of selected time points during peripheral nerve regeneration.
A) After 6 h of nerve injury there are acute inflammatory infiltrate in the epineurium with several mast cells (arrows).
B) Twelve hours after sciatic nerve section there are nerve vacuolization and hyalinization of epineural collagen (asterisk) with mild inflammatory infiltrate.
C) Two days after nerve injury there are intense inflammatory infiltrate into the nerve, its epineurium and neighbor adipose and muscular tissues.
D) High power micrograph of the lesion exhibited in C showed numerous macrophages dissecting the injured nerve, which showed extensive demielinization.
E) After one week of injury, there are slender prolongations or spouts (arrow) from the injured nerve.
F) After 17 days of injury there are numerous nerve sprouts with irregular shape and size (arrows), some of them have nodular morphology (asterisk).
G) High power micrograph of the same nerve showed in F exhibit numerous Schwann cells and few inflammatory cells.
H) After 43 days of nerve injury, some nerve sprouts show nodular organization resembling posttraumatic neuromas (asterisks).

fig 3

Figure 3: Representative micrographs of GDNF detection by immunohistochemistry during early and late sciatic nerve regeneration.
A) After 48 h of nerve injury there are numerous macrophages and fibroblasts that show strong GDNF immunostaining located in the perineurium, while few Schwann cells exhibit scarce immunereactivity (arrows).
B) Several perivascular inflammatory macrophages with strong GDNF immunostaining are seen around dilated blood vessels, which are revisted by strongly GDFN immunostained endothelium (arrows).
C) In the same early lesion, GDNF immunestained macrophages are seen into the injured nerve.
D) After 17 days of injury, regenerative sciatic nerve show strong GDNF immunostaining (asterisk), as well as its sprouts but in lesser intensity (arrows).
E) Collateral nerve ramifications or nerve sprouts are surrounded by thick fibrous epineurium and constituted by Schwann cells that show strong GDNF immunostaining.
F) High power micrograph show Schwann cells from regenerative nerve after 26 days of injury with strong GDNF immunostaining, while the inflammatory infiltrate is negative (arrow).

Discussion

The inflammatory response induced by peripheral nerve injury induces mast cells degranulation that release histamine and serotonin that enhance capillary permeability, facilitating macrophage migration [3]. Macrophages recruitment begins 2 to 3 days after nerve injury and peaks at about 7 or 14 days [4]. Macrophages and Schwann cells remove the injured tissue debris [5]. Macrophages secrete an enormous range of products, including cytokines and growth factors that are mitogenic for Schwann cells.  Our results showed that GDNF is constantly produced and its highest gene expression was very early, after two days of nerve injury in the distal stump, being macrophages its most important cellular source as suggested by immunohistochemistry. Other lesser numerous GDNF immunostained cells were the endothelium and fibroblasts. Few Schwann cells showed slight GDNF immunoreactivity in this early regenerative response.

GDNF was originally identified in astrocytes and later in other cell types [6]. GDNF is a potent trophic factor for embryonic motoneurons that enhance their cholinergic maturation and reduce cell death after axotomy. GDNF overexpression by muscle cells highly increases the number of axons in neuromuscular junctions. Administration of GDNF results in motor unit enlargement and continuous synaptic remodeling at the neuromuscular junction [7]. GDNF is also produced by macrophages and activated microglia in response to striatal and spinal cord injury, where these cells remain at the wound site producing increasing amounts of GDNF. In experimental autoimmune neuritis, GDNF is produced by macrophages and T cells [8]. GDNF is up regulated after several types of peripheral nerve injury and its administration to adult rats can change the phenotype of nerve fibers from unmyelinated to myelinated, where Schwann cells also aid axonal outgrowth and remyelinate the regenerating axon [9]. Previous reports showed that 48 hrs after peripheral nerve injury GDNF expression increased and Schwann cells are responsible for its expression [10]. These observations are in agreement with our results of time course gene expression, but in disagreement with our immunohistochemistry results, which indicated that macrophages are the most important cellular source of GDNF after early nerve injury, while Schwann cells are apparently the source of the later second peak production. At this later time there are numerous nerve sprouts that showed strong GDNF immunostaining in Schwann cells. Thus, it seems that GDNF is related to the nerve sprouts production that apparently try to reconnect nerve endings, and this growth factor could also participate in the production of post-traumatic neuromas, because strong GDNF immunostaining was seen in nodular structures located near to the sectioned nerve.

References

  1. Cattin AL and Lloyd AC (2016) The multicellular complexity of peripheral nerve regeneration. Curr Opin Neurobiol 39:38-46. DOI:10.1016/j.conb.2016.04.005.
  2. Zigmond RE and Echevarria FD (2019) Macrophage Biology in the Peripheral Nervous System after injury. Progress in Neurobiology 173:102-12. doi.org/10.1016/j.pneurobio.2018,12.001.
  3. Zochodne DW (2000) “The microenvironment of injured and regenerating peripheral nerves”. Muscle Nerve Suppl 9: 33-38. [crossref]
  4. Perry VH, Brown MC (1992) “Role of macrophages in peripheral nerve degeneration and repair”. Bioessays 14: 401-406. [crossref]
  5. Stoll G, Griffin JW (1989) “Wallerian degeneration in the peripheral nervous system participation of both Schwann cells and macrophages in myelin degradation”. J Neurocytol 18: 671-683. [crossref]
  6. Zurn AD, Baetge EE, Hammang JP, Tan SA, Aebischer P (1994) “Glial cell line-derived neurotrophic factor (GDNF), a new neurotrophic factor for motoneurones”. Neuroreport 30;6(1): 113-118. [crossref]
  7. Keller-Peck CR, Feng G, Sanes JR, Yan Q, Lichtman JW, et al. (2001) “Glial Cell Line-Derived Neurotrophic Factor Administration in postnatal life results in motor unit enlargement and continuous synaptic remodeling at the neuromuscular junction”. J Neuroscience 21: 6136-6146. [crossref]
  8. Ahn M, Jin JK, Moon C, Matsumoto Y, Koh C S, et al. (2010) “Glial cell line-derived neurotrophic factor is expressed by inflammatory cells in the sciatic nerves of Lewis rats with experimental autoimmune neuritis”. J Peripheral Nervous System 15: 104-112. [crossref]
  9. Höke A, Ho T, Craford TO, LeBel C, Hilt D, et al. (2003) “Glial Cell Line-Derived Neurotrophic Factor alters axon Schwann Cell units and promotes myelination in unmyelinated nerve fibers”. J Neuroscience 23: 561-567. [crossref]
  10. Fadia NB, Bliley JM, DiBernardo GA et al. (2020) Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in non human primates. Transl 12, eaav 7753. DOI: 10.1126/scitranslmed.aav.7753.
fig 6

Dual Arginine and Glutamic Amino Acids Delivery Effectiveness of Injectable Chitosan-Poloxamer P407 towards Wound Healing Application

DOI: 10.31038/JPPR.2021435

Abstract

The development of bioactive hydrogels has received much attention in the field of tissue regeneration. In the study, we utilized an injectable and biocompatible chitosan-Poloxamer P407 (CS-P407) hydrogel to deliver dual amino acids: glutamic and arginine. The amphiphilic CS-P407 copolymer structure was identified by 1H-NMR and FITR. The obtained copolymer solution shows the sol-gel transition point at body temperature (35-37°C), which is suitable for wound healing application. Through SEM imaging, this hydrogel presented a well-defined three-dimensional microporous network. In addition, CS-P407 exposed excellent bio-compatibility, with 90% fibroblast cell survival. The encapsulation of Arg and/or Glu did not induce any change to sol-gel transition behavior of CS-P407 as well as their biodegradation. The release of Arg and Glu from CS-P407 performed a sustainable profile following the non-Fickian kinetic model. The bioactive hydrogel may provide great potential for future clinical chronic wound management.

Keywords

Arginine, Chitosan, Glutamic, Hydrogel, Poloxamer P407, Wound healing

Introduction

Currently, the engineered bio-materials have significantly contributed to the field of tissue regeneration [1]. Specifically, engineered biomaterials, can control, regulate or mimic the natural regeneration process of tissue or organ. During this process, engineered biomaterials provide the framework structure to promote the migration and the proliferation of the target cells resulting in the promotion of the re-programming tissue [1,2]. In terms of mimicking extracellular matrix, the hydrogel is known as the best candidate in tissue engineering [2,3]. Hydrogels have a 3D network structure of hydrophilic polymeric with a controllable mechanical property as well as the ability to release growth factors sustainably; consequently, supporting the healing of damaged tissues [2-4]. More recently, hydrogel response to changing temperature through physical cross-linking has attracted extensive studies [5]. The outstanding advantages of the temperature-responsive hydrogel are of relative ease and do not require exogenous agents that may induce immune responses inside the body [5]. Additionally, the sol-gel transition behavior of thermal-sensitive hydrogel provides an injectable platform with minimal invasion compares to surgical delivery [4,5].

In the modern concept of tissue engineering, hydrogel-based natural materials such as chitosan, alginate, gelatin, or collagen have been fabricated [1-3]. Among them, chitosan is of great significance for tissue regeneration [5]. Chitosan has good biocompatibility, low toxicity, and rapid biodegradability. Various studies proposed the great pharmaceutical application of chitosan, including anti-bacteria, anti-inflammation, hemostasis, etc. [6,7]. Furthermore, chitosan contains abundant functional groups on its backbone; therefore, it is easy to modify or fabricate to optimize the hydrogel structure [6-8]. For example, chitosan could be co-polymerization with pluronic F127 to form the thermal sensitive hydrogel [6,8]. The system was considered an effective platform for drug delivery and tissue regeneration [5-7]. Poloxamer, an amphiphilic, thermo-sensitive, and FDA-approved Triblock copolymer of poly (ethylene oxide) and poly (propylene oxide), is one of the most studied platforms for the preparation of highly efficient hydrogels [5,6,8]. Chitosan-grafted poloxamer is widely exploited in cartilage regeneration, wound healing, burn healing, and anti-cancer drug delivery [7,9-13].

L-Arginine (Arg) is an essential amino acid that helps to prevent and treat circulatory diseases, alleviate fatigue, and stimulate the immune system. Arg is also known as endothelial nitric oxide synthase enzyme (eNOS) substrate [14], responsible for Nitric Oxide (NO) synthesis [15]. NO creates the signal for macrophage activation leading to the migration of fibroblast cells in wound healing process [14]. Along with Arg, L-Glutamic acid (Glu) is a necessary amino acid in the body, a precursor for collagen synthesis [15]. Collagen is a crucial protein for skin and tissue regeneration. It has been shown that the rate of collagen synthesis in tissues was completely dependent on the concentration of Arg and Glu in the micro-environment [16,17]. Therefore, Arg and Glu supplements into the hydrogel are necessary to ensure treatment outcomes of wounded areas. In this study, we develop a thermo-sensitive system of chitosan-poloxamer (CS-P407) hydrogel with Arg and Glu dual loading. The thermal-responsive behavior of the multifunctional hydrogel was evaluated by inverted tube method. The release profile with the kinetic model of Arg and Glu was also exanimated. Furthermore, the degradation of this system was investigated in the physiological medium. It is expected that CS-P407 in the combination with two amino acids could be used as a promising functional wound dressing in the future clinical treatment of chronic wounds.

Materials and Methods

Chemicals

Chitosan (CS) low molecular weight 85% deacetylated was supplied from Sigma. L-Arginine (Arg), L-Glutamic (Glu) and FMOC chloride (FMOC-Cl) were purchased from Sigma-Aldrich (USA). The cellulose dialysis membranes (molecular weight cut-off of 12-14 and 3.5 kDa) obtaining from Repligen were used to purify products. Mononitrophenyl formate-activated Poloxamer (NPC-P407-OH) was prepared in our previous studies at the Institute for Applied Materials Science as described [12,13]. All other chemicals were purchased from Thermo Fisher Scientific (Waltham, MA) or Fisher (USA) or of the analytical grade.

Synthesis of Poloxamer P407-Conjugated Chitosan Copolymers (CS-P407)

CS-P407 was prepared by the combination of Poloxamer P407 activated by mononitrophenyl formate with CS solution in acidic media (pH 4-5) followed by our previous report [9]. Briefly, NPC-P407-OH 10°C was added dropwise into CS solution (mass ratio of CS: NPC-P407-OH was 1:15). After 24 h, CS-P407 was dialyzed against distilled water using a cellulose membrane (MWCO = 12-14 kDa) in 5 days and then lyophilized to obtain the final product. 1H-NMR and Fourier Transform Infrared Spectroscopy (FTIR) measurements were used to identify the chemical structure of copolymers.

Characterizations of CS-P407 Hydrogels

Preparation of the CS-P407 Hydrogels and Bioactive Hydrogels

The grafted copolymer CS-P407 was dissolved in DI water (PBS, DMEM) contained vials (5, 8, 10, 12, 15, and 20% w/v) at 4°C. The temperature-responsive behavior of copolymer was investigated by sol-gel transition observation with a temperature range from 15 to 50°C, each measurement is spaced 5°C. The temperature of the incubator was stabilized in 5 min before dipping the test tube. At each investigated temperature, the test tube was kept in the incubator for 10 min to observe the gel-sol transition behavior [16,17]. The sol-gel transition of the bioactive hydrogels was conducted in the same method. The three-dimensional microporous network of the hydrogel was observed by SEM.

Adhesion Testing of the CS-P407 Hydrogel

The pigskin was cleaned of fat and cut into 2 squares with dimensions of 2.5 x 3 cm then soaked in 1X PBS solution (pH 7.4) for 2 h at 37°C. The pigskin was then fixed on the glass slide (25.4 x 76.2 cm) and left to stabilize for about 1 hour. The hydrogel (0.5 g) was drenched in cold water to evenly coat the pork skin. The remaining pigskin is then placed on top of the hydrogel-coated skin. The sample was stabilized at 37°C then pulled with a universal tester (Portable Tension Tester MTT 1500) at 10 mm/min until the piece of skin is separated. The value of the adhesive strength is calculated as the tensile strength at the point of the separated skin divided by the contact area of the skin. The unit of adhesive strength is measured in N/mm2, then converted to KPa (1 N/mm2 = 1000 KPa). The experiment was repeated thrice.

Cytocompatibility Test of the Hydrogels

Human fibroblasts cells (BJ (ATCC® CRL-2522™) were used in this study. The percentage of viable cells was determined by Sulforhodamine B (SRB) assay. The process was based on the guidance of Abcam. CS-P407 hydrogel was dissolved in distilled water then irradiated with a dose of 25 kJ for sterilization. The copolymer solution (100 µL) was evenly seeded in the culture disks with the dense of 104 cells/mL, then the culture media DMEM with the supplement of 10% FBS and 1% Penicillin Streptomycin was added to reach 0.5 mL. The untreated cells incubating with completed DMEM only was used as the control while the cell culture with 10 µL water was considered as the negative control and the cell culture with 10 µL Camptothecin (CPT) solution at the determined concentration (0.2 µg/mL) was considered as the positive control. The cells were incubated at 37°C, 90% humidity, 5% CO2 condition. At the designed time (4h, 24h, 72h, and 96h), the SRB kit was applied to each well. The results were recorded by ELISA at 570 nm. The percentage of viable cells was determined by the ratio of OD value of the treated cells to untreated cells. All the experiments were repeated thrice each case with 3 replications.

Release Profile and Release Kinetics of Amino Acid

CS-P407 Hydrogel Containing Amino Acid

The fabrication of Glu/Arg loaded CS-P407 hydrogel. CS-P407 (4.5 g) was dissolved in 20.5 mL DI containing Glu/Arg at 10°C. Copolymer solution with predetermined Glu/Arg concentration was lyophilized for further use in Glu/Arg content evaluation and in vitro release behavior. Since it is difficult to determine the content of Glu/Arg loaded CS-P407 hydrogels, a mediated-reagent (FMOC-Cl) was used to quantify different amino acids via reactions between amino acids and FMOC-Cl [18-20]. Pure Glu/Arg was dissolved in H2O with various concentrations (in the range from 1 to 10 ppm). The other stock solutions were prepared by dissolving 1.237 g H3BO3 in 100 mL H2O with pH was adjusted to 9 by NaOH 0.1 M and 100 ppm FMOC-Cl in acetonitrile. A mixture containing 3 stock solutions as follows Glu/Arg: H3BO3 : FMOC-Cl = 1:1:2 (v/v) was stirred at 30°C in 2h. The residual FMOC-Cl and FMOC-OH were eliminated by diethyl ether (5×5 mL). The remaining solution was diluted to 10 mL. UV-Vis spectra (Agilent 8453 UV-Vis Spectrophotometer) were used to determine each amino acid content at the absorb wavelength of 265 nm. The release profiles of loading agents from the hydrogels in vitro were characterized by self-diffusive method of Glu/Arg-loaded hydrogel contained in a cellulose membrane (MCWO = 3.5 kDa). Samples (2 mL hydrogel 18%) was immersed in the phosphate-buffered saline (PBS) pH 7.4 (20 mL) and shaken (100 rpm) at 37 ± 1°C. At predetermined intervals (0h, 1h, 3h, 5h, 7h, 9h, 12h, 24h, 36h, 48h), the released PBS was collected and replaced by fresh PBS. The number of released agents was measured using UV-vis spectrophotometer as mentioned. The experiments were repeated 3 times. The percentage of released Glu/Arg was calculated as follows:

formula 2

Where Cn is the concentration of Glu/Arg in the sample, Cn-1 is the concentration of Glu/Arg released at time t, Vs is the volume of incubation medium, and Vt is the volume of medium replaced at time t [21].

The release profiles of Glu/Arg were found to be suitable for zero and first-degree equations, Higuchi, and Korsmeyer [22,23]. The mean dissolving time (MDT) value was calculated from release kinetic data using equation (Mockel and Lippold) [24].

formula 3

Where n and k are the release exponent and the release rate constant from the Korsmeyer equation, respectively.

Degradation Profiles

Approximately 2 mL hydrogels (Mi) were fabricated in vials subsequently incubated at 37°C. The samples were prepared in distilled water and then diluted in 5 mL buffer PBS pH 7.4 or DMEM. After every 2 days incubation, the liquid was consecutively replaced by 5 mL culture media until the hydrogels had completely disintegrated. The weight of the remaining hydrogel is recorded by an electronic balance. The data profiles were expressed as the average of three measurements. The percentage weight loss is calculated as follows:

formula 1

Mi: initial gel mass (g); Mt: the remaining gel mass (g) and after the degraded time.

Results and Discussions

Characterizations of the Amphiphilic CS-P407 Copolymers

CS-P407 is synthesized based on the carbamate group synthesis reaction through a covalent bond between the carbonate group (NPC-P407-OH) and amino group (-NH2) of CS (Figure 1). The 1H-NMR spectroscopy of CS-P407 copolymer has been mentioned in our previous research [25,26]. Overall, the resonance signal of Poloxamer protons and methyl, methylene and methine group exist in 3.41 – 4.03 ppm and 1.10 ppm (-CH3 of PPO unit). The max resonance was achieved at δ = 1.97 ppm, δ = 2.90 ppm, and δ = 4.64 ppm for protons in glucosamine of CS backbone, which confirmed the structure of co-conjugated compound CS-P407. The FTIR result (Figure 2) describes the spectroscopic features of standard CS in 3368.02 cm-1 wavenumber due to oscillation of O-H bond, 1558.89cm-1 is the oscillation of N-H bond. The NPC-P407-OH spectroscopy shows that the peak in 2885.96cm-1 belongs to the C-H bond in the PEO fraction of Poloxamer P407. The peak at 1111.03cm-1 is caused by a specific C-O linkage of Poloxamer P407. These signals also occurred in CS-P407 spectroscopy at 2890.07cm-1 is a peak of C-H linkage. The stretched oscillation of the C-O bond shows a signal at 1111.88cm-1. The peak at 1571.05cm-1 indicates the -NH deforming signal of the amine group in CS, even though this signal doesn’t exist in CS-P407 spectroscopy due to the amine group has formed a bond with P407 and create an amide I group with a wavenumber at around 1650.04cm-1 [27]. To sum up, the FTIR spectroscopy data shows that the peaks observed are suitable with the expected functional groups in the compound structure of conjugated copolymer.

fig 1

Figure 1: Synthesis of thermosensitive copolymer CS-P407

fig 2

Figure 2: The FTIR spectra of P407, NPC-P407-OH, and CS-P407.

Characterizations of CS-P407 Hydrogels

The CS-P407 hydrogel was coated with a thin layer of approximately 2 mm on the glass slide and the samples were allowed to dry naturally. The sample was then measured by SEM to observe the hydrogel surface structure.

SEM imaging results of Figure 3 show that the CS-P407 hydrogel has an abundant-porous structure (1-2µm) formed by the overlapping network of CS-P407 copolymers. This is the most important characteristic of the hydrogel system (scaffold) since it is not only able to absorb the fluid exudate and maintain certain water content in the wound but also allows the fibroblasts to divide and migrate. The interconnected porous structure has an impact on the supply of nutrient and gas exchange in order to maintain cellular ingrowth and retain a high amount of water, and also it offers ideal material for the Glu/Arg delivery carrier system [10].

fig 3

Figure 3: C-P407 hydrogel surface structure (x1000 image on the left) and (x5000 image on the right)

Thermal Sensitivity of CS-P407 Solution

Obtained results of sol-gel transition investigation show that CS-P407 solution can create gel at a relatively low copolymer concentration (above 10% wt/v) at 32-37°C (Figure 4A). When replacing water with PBS buffer and DMEM cell culture media, the transition temperature is unaffected significantly. The thermosensitive hydrogel was formed via hydrophobic interaction of hydrophobic PPO domains in the CS-P407 as seen in Figure 4B. The phenomenon was reported in several studies [5,7,9,10].

fig 4

Figure 4: The phase diagram of the sol-gel transition (A); illustration of the sol-gel transition of the CS-P407 solution (B).

Adhesion of the Hydrogel Scaffolds

The cohesive ability of hydrogel material to the skin surface is a critical factor in creating material for wound healing. The cohesion process will suppress plasma leakage, prevent bacterial infection, maintain gaseous exchange and provide better access to bioactive compounds in the hydrogel. The tissue adhesive experiment was conducted in porcine skin. As shown in Figure 5, the adhesivity of CS-P407 hydrogels at 15%Wt, 18%Wt, and 20%Wt are 5.51 ± 0.88 KPa, 6.62 ± 0.7 KPa and 5.74 ± 0.74 KPa, respectively. This result was similar to Ji Hyun Ryu’s research in 2011 (The value in this study are about 5.3±2.6 KPa) [28]. At the same condition, P407 hydrogel exhibits a very low tissue adhesion, at which the value is 0.79 ± 0.26 KPa that is similar to a previous study (0.72±0.32 KPa) [29].

fig 5

Figure 5: Adhesion strength (KPa) of the material to the pigskin surface.

The experiment was repeated three times independently (n=3), and the errors are presented in S.E. Statistical significance of p < 0.001 is indicated by ***. Non-statistical insignificance of p ≥ 0.05 is indicated by ns.

This adhesive feature is formed by the positively charged amine groups in the CS backbone interact with the collagen matrix of porcine skin. It depends on chitosan, a polymer with strong adhesivity through its -NH2 groups. As a result, when attaching to the skin surface, CS-P407 can interact with the surface through hydrogen bonds, electrostatic bonds, hydrophobic interaction that make CS-P407 have better adhesive intensity.

Cytotoxicity of Hydrogel CS-P407

In biomedical material, biocompatibility, or the fitness and harmlessness of the material with the human body and related physiological activities, is the primary criteria to decide its possibility for application. Fibroblasts are vital for wound regeneration, especially in the division, differentiation, and migration stage of fibrocytes at the wound surface. These cells synthesize and secrete extracellular proteins, mainly collagen, to reconstruct the extracellular matrix of connective tissue. In this research, a preliminary cytotoxicity assessment was conducted on fibroblast cell lines from human skin. According to the results in Figure 6, CS-P407 hydrogel is non-toxic for fibroblast. CPT was used at 3 µg/mL, the fibroblast growing rate decreased 42.02 ± 8.05% after 4h of exposure and go down to 7.21 ± 5.38% after 24 hours with total cell death was observed after 48 hours. The number of cells on CS-P407 increase from 106.07 ± 3.52% after 4 hours to 136.8 ± 5.07% after 48 hours. This proves that besides its non-toxicity, CS-P407 can improve cell growth for fibroblasts.

fig 6

Figure 6: Percentage of BJ fibroblasts growth incubated in 0.1 mL of CS-P407, water (negative control) and CPT (positive controls).

In Vitro Amino Acid Release Profile and Release Kinetic Models

In this study, we investigated the proliferative capacity of human fibroblasts (ATCC® CRL-2522™) of free Arg and Glu. Tested concentrations of Arg and Glu from 0-250 µM and 0-500 µM respectively. Figure S.1 showed that the increasing concentration of active ingredients leads to cell proliferation, proving that both Arg and Glu support cell growth and increase wound healing. Arg’s support was significantly impactful on proliferation. Specially, 50 µM for Arg (number of cells = 9 x 104) and 250 µM for Glu (number of cells = 8 x 104) are the optimal concentration for cell proliferation. On the other hand, the excessive use of Arg and Glu also reduces cell growth. This inhibition of cell growth can be seen above 200 µM for Arg and above 450 µM. Based on obtained data, we selected the optimal carry concentrations of Arg and Glu are 50 µM and 250 µM, respectively, into CS-P407. These primary results are the premise to develop a hydrogel system CS-P407 to support treatment and wound healing, show great applications in medicine in general and tissue regeneration in particular.

The gelation temperature range was determined from the minimum temperature of gel formation Tgel to the temperature at which the gel begins to melt Tm. In Figure 7, from the measured results, it can be seen that the Tgel gelation temperature of CS-P407 carrying amino acids when mixed with distilled water is higher than that of PBS night medium and physiological DMEM medium at the same concentration. Gel forming temperature range in an aqueous medium is mostly narrower, the results are similar to that of CS-P407 hydrogel. The influence of the medium on the gelation of the hydrogel can be seen. It is understood that in the medium PBS and DMEM gel has higher mechanical properties, more stable. The above results show that the gel is effective in carrying amino acids, without losing the inherent mechanical properties of the system. The CS-P407 polymer system creates a gel at 30-35°C and the gel melting point is above 50°C, so it is very suitable for biomedical applications, especially wound treatment gels.

fig 7

Figure 7: The phase diagram of the sol-gel transition of CS-P407/Arg (A); CS-P407/Glu (B) và CS-P407/Glu/Arg (C)

Figure 8 represents the release rate of free Glu and Arg after 12 hours achieve 100% when CS-P407/Glu, CS-P407/Arg, and CS-P407/Glu/Arg rates are 32.53%, 47.28%, and 65.02%. After 48 hours, the release rate at CS-P407/Glu and CS-P407/Arg reach 43.97% and 96.83%, respectively. The Arg release profile of CS-P407 was faster compared to Glu. This difference is according to the carboxylated form of the 2 -COOH group in Glu at pH=7.4 has electrostatic interaction with the positively charged structure of CS-P407, which leads to a slower release rate. When both amino acids were captured in the hydrogel, their release rate reaches 80.59% after 48 hours. Thus, CS-P407 hydrogel helps decelerate the release rate of amino acids, increases their absorptivity at wounded tissue

fig 8

Figure 8: Release profile of amino acid (Glu/Arg) from the hydrogel CS-P407

Among all formulas investigated, the Korsmeyer-Peppas regression model has the greatest fitness (R2= 0.9155-9.8874) (Table 1 and Figure 9). With this model, the transport exponent (n) belongs to the (0.3078-0.5218) interval, indicating that the release mechanism of free amino acid and CS-P407/Glu are Fickian diffusions. With free amino acid, it is the passive diffusion, for CS-P407/Glu is due to the CS-P407 gel barrier interaction with Glu. On the other hand, with CS-P407/Arg and CS-P407/Glu/Arg, the release mechanism is non-Fickian, influenced by diffusion and swelling. The rate of diffusion and swelling are the same. The rearrangement of polymer chains happens in slow progress, while the diffusion triggers some abnormal effects over time [30,31].

Table 1: Amino acid release parameters of CS-P407/Glu, CS-P407/Arg, and CS-P407/Glu/Arg hydrogel were obtained to four different mathematical models of drug release kinetics.

Formulation

Mathematical models for drug-release kinetics
Zero order First order Higuchi

Power law

k0

R2 k1 R2 kH R2 K n

R2

Glu Free

0.0981

0.9836 0.5243 0.9459 0.2911 0.9980 0.3179 0.4122

0.9972

Arg Free

0.0935

0.9972 0.5182 0.9854 0.2771 0.9846 0.3339 0.3760

0.9760

CS-P407/Glu

0.0121

0.9231 0.1560 0.8463 0.0799 0.9790 0.1364 0.3078

0.9920

CS-P407/Arg

0.0238

0.9875 0.1805 0.9188 0.1369 0.9982 0.1422 0.4719

0.9974

CS-P407/Glu/Arg

0.0233

0.8275 0.1869 0.7269 0.1611 0.9195 0.2032 0.5218

0.9155

fig 9a

fig 9b

fig 9c

fig 9d

Figure 9: Release kinetics of Glu/Arg from CS-P407 fitted to four kinetic models: (A) zero-order kinetic model, (B) first-order kinetic model, (C) Higuchi model, and (D) Korsmeyer-Peppas model.

Table 2 shows that the MDT value of hydrogel is much higher than free amino acid, which means that amino acids have been entrapped inside the hydrogel matrix. This improves the usage efficiency of the bioactive compound with a short half-life. Thus, CS-P407 hydrogel has prominent potential in the drug distribution process in wound treatment.

Table 2: The fit kinetic model and the MDT value of the modulation formulas.

Formulation

Order of release t25% (hours) t50% (hours) t75% (hours) t90% (hours)

MDT (hours)

Glu Free

Fickian

0.5583 3.0000 8.0223 12.4849

4.7056

Arg Free

Fickian

0.4631 2.9265 8.6043 13.9741

5.0533

CS-P407/Glu

Fickian

7.1578 68.0652 254.1572 459.6082

152.3172

CS-P407/Arg

Non-Fickian

3.3073 14.3690 33.9318 49.9357

20.0140

CS-P407/Glu/Arg

Non-Fickian

1.4878 5.6165 12.2164 17.3257

7.2699

Degradation Profiles

The hydrogel degradability was evaluated by gravimetric analysis of CS-P407 in PBS (pH 7.4) or DMEM solution until the hydrogel was completely degraded. Figure 10 shows that CS-P407 hydrogel had a degradation time of 10 days in PBS buffer with pH 7.4, which was 1.7 times longer than the P407 hydrogel sample. This shows that the combination of Poloxamer P407 with CS (which is a bio-adhesive agent) has increased the stability of the hydrogel system. The results can be explained as the interweaving of the branched copolymer chains is increased by CS, leading to increase hydrogel stability during decomposition. Comparing degradation time in two physiological media, PBS medium pH 7.4 degraded more rapidly, when all samples have been downgraded after 12 days. DMEM medium has a longer degradation time, as the hydrogel sample remains after 16 days of investigation. Loading arginine and glutamic resulted in prolonging degradation of the bioactive hydrogels. This could be contributed by hydrogen bonding formation of amino acids and chitosan chains leading to increase the stability of the hydrogel matrix.

fig 10

Figure 10: Degradation profile of hydrogel samples in PBS pH 7.4(A) and DMEM (B).

Conclusion

The bioactive hydrogel loading glutamic and arginine amino acid was developed. CS-P407 18 wt/wt% copolymer solution strongly forms hydrogel at body temperature. The system exhibits a porous structure, high tissue adhesion, and cytocompatibility which is injectable for applying minimal invasion surgery. The optimum concentrations of Arg and Glu used in the CS-P407 system to increase cell proliferation are 50 µM and 250 µM, respectively. The CS-P407 hydrogel performed a suitable platform for controlling the delivery of these amino acids. The release behavior is affected by concentration diffusion and the swelling of the gel system. Arginine and glutamic encapsulation resulted in the prolonged degradation of bioactive hydrogels. The preliminary results could pave the way to apply the bioactive hydrogel in would healing.

Acknowledgements

This work was supported by the Ho Chi Minh Department of Science and Technology (number of contract 47/2019/HĐ-QPTKHCN)

References

  1. Pramanik S, Pingguan-Murphy B, and Osman NAA (2012) Progress of key strategies in development of electrospun scaffolds: bone tissue. Science and Technology of Advanced Materials 13: 043002.
  2. Wang K, Hao Y, Wang Y, Chen J, Mao L, et al. (2019) Functional hydrogels and their application in drug delivery, biosensors, and tissue engineering. International Journal of Polymer Science.
  3. Le PN, Huynh CK, Tran NQ (2018) Advances in thermosensitive polymer-grafted platforms for biomedical applications. Materials Science and Engineering: C 92: 1016-1030. [crossref]
  4. Bhattarai N, Gunn J, Zhang M (2010) Chitosan-based hydrogels for controlled, localized drug delivery. Advanced drug delivery reviews 62: 83-99.
  5. Choi JH, Joung YK, Bae JW, Choi JW, Quyen T, et al. (2011) Self-assembled nanogel of pluronic-conjugated heparin as a versatile drug nanocarrier. Macromolecular research 19: 180-188.
  6. Antoniraj MG, Kumar CS, Kandasamy R (2016) Synthesis and characterization of poly (N-isopropylacrylamide)-g-carboxymethyl chitosan copolymer-based doxorubicin-loaded polymeric nanoparticles for thermoresponsive drug release. Colloid and Polymer Science 294: 527-535.
  7. Huynh NT, Dang LH, Pham NO, Nguyen CT, Vu MT, et al. (2019) Injectable nanocurcumin-dispersed gelatin–pluronic nanocomposite hydrogel platform for burn wound treatment. Bulletin of Materials Science, 42: 1-10.
  8. Nguyen TTC, Nguyen CK, Nguyen TH, Tran NQ (2017) Highly lipophilic pluronics-conjugated polyamidoamine dendrimer nanocarriers as potential delivery system for hydrophobic drugs. Materials Science and Engineering: C 70: 992-999.
  9. Dang LH, Nguyen TH, Tran HLB, Doan VN, Tran NQ (2018) Injectable nanocurcumin-formulated chitosan-g-pluronic hydrogel exhibiting a great potential for burn treatment. Journal of healthcare engineering. [crossref]
  10. Pham L, Dang LH, Truong DM, Nguyen TH, Le L, et al. (2019) A dual synergistic of curcumin and gelatin on thermal-responsive hydrogel based on Chitosan-P123 in wound healing application. Biomedicine & Pharmacotherapy 117: 109183.
  11. Park KM, Lee SY, Joung YK, Na JS, Lee MC, et al. (2009) Thermosensitive chitosan–Pluronic hydrogel as an injectable cell delivery carrier for cartilage regeneration. Acta biomaterialia 5: 1956-1965. [crossref]
  12. Hosseinzadeh H, Atyabi F, Dinarvand R, Ostad SN (2012) Chitosan–Pluronic nanoparticles as oral delivery of anticancer gemcitabine: preparation and in vitro study. International journal of nanomedicine 7: 1851-63. [crossref]
  13. Nguyen VT, Nguyen TH, Dang LH, Vu-Quang H, Tran NQ (2019) Folate-conjugated chitosan-pluronic P123 nanogels: synthesis and characterizations towards dual drug delivery. Journal of Nanomaterials.
  14. Fujiwara T, Kanazawa S, Ichibori R, Tanigawa T, Magome T, et al. (2014) L-arginine stimulates fibroblast proliferation through the GPRC6A-ERK1/2 and PI3K/Akt pathway. Plos one 9: e92168.
  15. Aalto M, Lampiaho K, Pikkarainen J, Kulonen E (1973) Amino acid metabolism of experimental granulation tissue in vitro. Biochemical Journal 132: 663-671. [crossref]
  16. Szekalska M, Puciłowska A, Szymańska E, Ciosek P, Winnicka K (2016) Alginate: current use and future perspectives in pharmaceutical and biomedical applications. International Journal of Polymer Science.
  17. An D, Ji Y, Chiu A, Lu YC, Song W, et al. (2015) Developing robust, hydrogel-based, nanofiber-enabled encapsulation devices (NEEDs) for cell therapies. Biomaterials 37: 40-48. [crossref]
  18. Armstrong DW, Gasper M, Lee SH, Zukowski J, Ercal N (1993) D‐amino acid levels in human physiological fluids. Chirality 5: 375-378. [crossref]
  19. Nguyen CT, Dang LH, Nguyen DT, Tran KP, Giang BL, et al. (2019) Effect of GA3 and Gly plant growth regulators on productivity and sugar content of sugarcane. Agriculture 9: 136.
  20. Zhou W, Zhanga XY, Duana GL (2011) Liquid‐Chromatography Quantitative Analysis of 20 Amino Acids after Derivatization with FMOC‐Cl and Its Application to Different Origin Radix isatidis. Journal of the Chinese Chemical Society 58: 509-515.
  21. Nguyen TD, Nguyen TN, Nguyen TT, Ivanov IA, Nguyen KC, et al. (2019) Nanoencapsulation enhances anticoagulant activity of adenosine and dipeptide IleTrp. Nanomaterials 9: 1191.
  22. Kuksal A, Tiwary AK, Jain NK, Jain S (2006) Formulation and in vitro, in vivo evaluation of extended-release matrix tablet of zidovudine: influence of combination of hydrophilic and hydrophobic matrix formers. AAPS pharmscitech 7: E1-E9. [crossref]
  23. Sahu A, Kasoju N, Goswami P, Bora U (2011) Encapsulation of curcumin in Pluronic block copolymer micelles for drug delivery applications. Journal of biomaterials applications 25: 619-639. [crossref]
  24. Roni MA, Kibria G, Jalil RU (2009) In vitro studies of controlled release alfuzosin matrix tablets prepared with ethylcellulose and hydroxypropyl methylcellulose. Iranian Journal of Pharmaceutical Sciences.
  25. Dang LH, Vu MT, Chen J, Nguyen CK, Bach LG, et al. (2019) Effect of ultrasonication on self-assembled nanostructures formed by amphiphilic positive-charged copolymers and negative-charged drug. ACS Omega 4: 4540-4552.
  26. Nguyen DH, Nguyen VT, Nguyen CK, Nguyen TH (2016) Green processing of thermosensitive nanocurcumin-encapsulated chitosan hydrogel towards biomedical application.
  27. Nguyen TBT, Dang LH, Nguyen TT, Tran DL, Nguyen DH, et al. (2016) Green processing of thermosensitive nanocurcumin-encapsulated chitosan hydrogel towards biomedical application. Green Processing and synthesis 5: 511-520.
  28. Ryu JH, Lee Y, Kong WH, Kim TG, Park TG et al. (2011) Catechol-functionalized chitosan/pluronic hydrogels for tissue adhesives and hemostatic materials. Biomacromolecules 12: 2653-2659. [crossref]
  29. Lee Y, Chung HJ, Yeo S, Ahn CH, Lee H, et al. (2010) Thermo-sensitive, injectable, and tissue adhesive sol–gel transition hyaluronic acid/pluronic composite hydrogels prepared from bio-inspired catechol-thiol reaction. Soft Matter 6: 977-983.
  30. Mircioiu C, Voicu V, Anuta V, Tudose A, Celia C, et al. (2019) Mathematical modeling of release kinetics from supramolecular drug delivery systems. Pharmaceutics 11: 140. [crossref]
  31. Shaikh HK, Kshirsagar RV, Patil SG (2015) Mathematical models for drug release characterization: a review. World J. Pharm. Pharm. Sci 4: 324-338.
fig 1

Resource Optimization through Group, Pooling, Tests, Testing in the Detection of Asymptomatic People with COVID-19

DOI: 10.31038/MIP.2021221

Abstract

The current ongoing coronavirus disease 2019 (COVID-19 SARS2 and detection and isolation of infected people with the virus is crucial. Real-time polymerase chain reaction (RtPCR) test has proven to be most useful  in  viral  detection.  The  objective  of  this  work  is  to  determine  the  benefit  of  group testing for resource optimization and be able to expand the number of individuals who are screened to find healthy COVID-19 carriers. Groups from 2 to 27 of a total of 2,100 people were included. RT-PCR was done with Seegene kits for mRNA extraction and reagents in Biorad RT-PCR machine. Results: groups of between 4 to 6 people have been the ones with the best optimization results with 64 to 67%, this means that with 1 test it is possible to cover the same number of people and detect more quickly those who must be isolated and follow their contacts. Conclusion: Grouping tests can optimize resources up to 67%.

Background

The current ongoing coronavirus disease 2019 (COVID-19 SARS2) pandemic with mainly severe acute respiratory syndrome, is a serious global public health problem. The detection and  isolation  of infected people with the virus is crucial. Hitherto, the real-time polymerase chain reaction (RT-PCR) test has proven to be most useful in viral detection when performed using nasal and oropharyngeal exudate samples. Due to the global and rapidly progressive nature of the pandemic, the tests have been limited in supply with a high cost; therefore, their optimization is important. The Many millions of tests have been performed almost exclusively on an individual basis and are many millions of tests [1]. To date, the United Kingdom has performed the highest number of tests globally, with 486 billion tested inhabitants [2]. In the open population, the first positivity reported by South Korea, who had performed the greatest number of tests, the statistic of positives was 1.7% in the open population, and later in May 2020 they performed 10 million tests and only had 300 positives (positivity rate of 0.9% of positivity) between 1,553,5523.. In the United States of America, the positivity rate in the suspected sample population was 19.8% and 9.6% in the general population of 32,009,840 by July 25, 20204. Based on this percentage, the negativity rate in the general  population  is expected to be high. It is known that a person infected by COVID-19 (SARS- CoV-2) who is asymptomatic, has an approximate viral load of 6.76 x 106 viral copies in the first five days of infection [3-9]. Subsequently, the viral load decreases from day 6, to 3.44 x 10 viral copies. In addition, it has been described that the viral load is not detectable by day 28 in up to 39.93% of infected people. On the other hand, patients with symptoms can present a viral load of up to 7.11 x 108 viral copies [5-9]. Taking these findings into account, it was found feasible to do group tests (pooling), the first published antecedent for COVID-19 is from Yelin and collaborators [10] in which they make groups of 35 people. In later publications, theoretical calculations are made on the number of people to include in the groups and with automated equipment [11-13], finding savings and optimization of resources of 69% [13].

Objective

Determine the benefit of group testing for resource optimization and be able to expand the number of individuals who are screened  to find healthy COVID-19 carriers. In our laboratory to assess this observation at the beginning, we carried out a preliminary test with 19 people including technicians, doctors, administrative and cleaning staff, all asymptomatic, a sample was taken from each person and the pilot group was carried out, gathering aliquot of 19 people in a pool, performing a single RT-RCR test for COVID-19, obtaining a negative result. None developed any symptoms of COVID-19 in the next 2 months. Additionally, the same  procedure  was  performed  adding to that pool with an already known positive sample, obtaining the positive result in the pool sample.

Material and Methods

An observational, cross-sectional and descriptive study was carried out.

Selection and Inclusion Criteria

Open population with asymptomatic individuals who are considered healthy, including asymptomatic carriers, which is the objective of this study. Selection of people and groups: a call was made to individuals and companies of various types, who were named as suppliers: 1) Family group: people who live in the same home; 2) Work group: people who work in the same place; 3) Health group: health workers with or without direct contact with patients. The selection of the groups was initially proposed in groups selected by each supplier, without exceeding 30 people. People were recruited between May  21 and August 5, 2020. Exclusion criteria: People with respiratory symptoms of any type and degree and with fever were ruled out. Taking samples: It was carried out either in the laboratory or at the work sites. In both cases with the safety measures by the sampling personnel according to the WHO recommended. The transport was carried out with triple packaging. A nasopharyngeal sample was taken from each individual and placed in a tube with 2.0 ml of transport medium, from which an aliquot of 0.10 ml was taken from each individual to form the pool of that group, a single mRNA extraction was performed in each group. The samples were kept refrigerated for the first 24 hours and after that time they were kept frozen at -20°C until processing.

Sample Processing

Extraction of viral DNA/RNA, using Invisorb® Spin universal kit reagents Invitek Molecular Gmbh Berlin. In a 2 ml Safe lock tube, place 200 µl of the sample, with 200 µl HTL buffer, 20 µl of carrier DNA and 20 µl of Proteinase K, mix in vortex for 10 seconds, place the tube in thermomix and incubate with constant shaking x 10 minutes at 65°C and then 10 minutes at 95°C. Binding Add 260 µl of binding solution to the sample lysate and mix by pipetting up and down or vortex. The sample is incubated for 5 minutes at room temperature. The sample is transferred to the RTA Spin filter with the receiving RTA tube. The tube is closed and centrifuged for 1 minute at 11.1100 g. Discard the RTA Receiver with the filtrate and fit a new receiver tube. The samples are centrifuged at -20°C. Separately, the following reagents are prepared: 1) 5 µl of 2019-nCoV MOM reagent (Seegene®); 2) 5 µl of RNase-free water; 3) 5 μl of Buffer 5X Real-time One-Step (Seegene®), and 4) 2 μl of Real-time One-Step enzyme (Seegene®). The mixture is centrifuged briefly and 17 μl of One-step RT-PCR Mastermix (Seegene®) will be added.

8 μl of the group of samples, 2019-nCoV PC and RNAs-free distilled water will be added to the previous solution. Afterwards, the samples will be centrifuged and they will be placed in the equipment for RT-PCR. (2) The AllplexTM 2019-nCoV kit (Cat. No. RP10243X; Seegene®) will be used for the qualitative detection of SARS-CoV-2 (COVID-19). The kit evaluates the E, RdRP and N genes of SARS- CoV-2. (3) Polymerase chain reaction with reverse transcriptase. The samples will be placed in the equipment for RT-PCR (CFX96TM, BioRad®), with the following parameters: Cycle 1) Temperature 50°C, duration 20 minutes; Cycle 2) Temperature 95°C, duration 15 minutes; Cycles 3-44) Temperature 94°C, duration 15 seconds each; Cycle 45) Temperature 58°C, duration 30  seconds.  The  cycling  determined to evaluate the positivity of the sample is 45 cycles according to the manufacturer’s protocol.

Data Interpretation

The data obtained from the RT-PCR process will be automatically analyzed in the CFX96 ManagerTM program included in the BioRad® kit. The cut-off point to establish a sample as positive is the value of Ct ≤40.

Statistical Analysis

Frequencies and percentages were determined, Pearson’s correlation test was applied. The data analysis was carried out with the SPSSv23 program. A p value <0.05 was considered as statistical significance. Ethical and biosafety aspects: The protocol was approved by the research department of the General Hospital of Mexico Eduardo Liceaga of CDMX with number DI / 20/405103150.

Results

2,100 participants were included, 60% (1,260 people) were men and 40% (840 people) were women. The average age was 35 years, with a range of 18 to 65 years. The participants were gathered into 536 groups, which meant performing the same  number  of  tests. The groups consisted of a minimum of 2 people and a maximum     of 23 people and were selected by the suppliers (Table 1), 17.35% (93 groups) were positive with a total of 131 positive people, which means 6.2% of positive people out of the 2,100 included in the study. To detect positive people in each group, 372 additional tests were required, optimization of the test resource in total groups between    2 to 27 people was 56.76%, this figure was obtained in relation to the total number of people included that would be equivalent to the tests performed individually (n 2,100, 100%) and the number of tests actually performed, obtained from the sum of the number of groups (n 536) plus the number of additional tests, of each positive group individual tests were performed in groups. The additional tests where done in the positive groups as follows: from 2 to 7 people individually and in those from 8 to 27 in subgroups of 2 or 3 and of them the positive ones individually, obtaining a total of 372 additional tests, which added to the initial ones were 908, the % optimization in relation to the Initial theoretical of 2,100 tests and the performed test 908 equivalent to 56.76% (100-(908 * 100/2100). Table 1 shows the % optimization for each group, the small % was 2 people and the better between 4, 5 and 6 people. There were between 1 and 2 groups of 8, 9 and 11 to 23 people who were carried out very early in the study (May- June) that had a very high % optimization (87 to 94) and it should  be considered that at the beginning of the pandemic there were very few infected people. The number of members of each group had a low positive correlation with the number of additional tests performed (rho=0.13, p=0.009), while the number of additional tests performed had a high positive correlation with the number of tests that resulted positive for COVID-19 (rho=0.99, p=0.0001) (Figure 1) [14].

Table 1: Optimization% for each group.

People per group

Total no. of people in the groups n of groups Positive no. of groups no. of positive People Additional tests Total of Tests performed Theoretically individual tests % Optimization
2 482 241 48 49 96 337 482

30.08

3

132 44 8 8 24 68 132 48.48
4 220 55 6 11 24 79 220

64.09

5

580 116 15 32 75 191 580 67.07
6 294 49 8 12 48 97 294

67.01

7

63 9 4 8 28 37 63 41.27
8 16 2 0 0 0 2 16

87.50

9

9 1 0 0 0 1 9 88.89
10 50 5 0 0 0 5 50

90.00

11

22 2 0 0 0 2 22 90.91
12 24 2 1 1 12 14 24

41.67

17

17 1 0 0 0 1 17 94.12
19 19 1 0 0 0 1 19

94.74

20

40 2 1 2 20 22 40 45.00
21 42 2 0 0 0 2 42

95.24

22

44 2 1 7 22 24 44 45.45
23 46 2 1 1 23 25 46

45.65

Total

2100 536 93 131 372 908 2100

56.76

fig 1

Figure 1: From Left to Right % of optimization below the line. In top of line number of individuals in each group.

Discussion

The SARS COVID-19 pandemic persists till the end of 2020 and probably the first half of 2021, and in the absence of a specific drug and only treatments for the effects of the virus on the coagulation and inflammatory process, the isolation strategy and follow-up of contacts with real-time PCR tests should be followed. The Vaccines probably will modify the number of positive people, but still will be important, given the cost of the tests to be able to optimize resources and group or pool tests is an effective method to do it. Groups of between 4 to  6 people have been the ones with the best optimization results with 64 to 67%, this means that with 1 test it is possible to cover the same number of people and detect more quickly those who must be isolated and follow their contacts. The groups of two are not adequate since an initial test is required and if positive result requires 2 additional tests and this means to carry out for two people three tests. Positive groups of more than 10 people require performing individual tests   in subgroups of 2 or 3 and positive ones in individual tests, which is not practical since it takes at least two or three additional days to perform them and although the optimization obtained was of up to 94%, in time and at present time with the expected increase in the number of cases it would not be practical. We found groups of 4 to 6 people are optimal. 3 groups with false positives were detected, these show to start positive above cycle 33 of the 45 cycles of the Rt-PCR programming and the individual tests were always negative. In the groups with cycles less than 31, the positive person or persons were always detected. Two different brands of reagents were used to verify the results in the 3 false positive groups with the same results.

Conclusion

Carrying out group tests in asymptomatic people of open population for any reason, whether it is resuming activities or detecting cases of healthy carriers, is very effective and is optimal in groups 4, 5 and 6 people, this allows more tests to be carried out at a lower cost.

References

  1. Huipeng Ge, Xiufen Wang, Xiangning Yuan, Gong Xiao, Chengzhi Wang et (2020) The epidemiology and clinical information about COVID-19. European Journal of Clinical Microbiology & Infectious Diseases 39: 1011-1019. [crossref]
  2. Consultada 21 de octubre 2020
  3. http://ncov.mohw.go.kr/en/ Tests in Suth Corea updated 1 july 2020
  4. Ultimo acceso 1 de Agosto 2020
  5. Chen Y, Liu Q, Guo D (2020) Coronaviruses: genome structure, replication, and pathogenesis Deyin Guo ORCID.
  6. Zou L, Ruan F, Huang M, Liang L, Huang H et al (2020) SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients. N engl j med 382: 1171-1179. [crossref]
  7. Enfermedad por coronavirus, COVID-19 Ministerio de Sanidad España Centro de coordinación de alertas y emergencia sanitaria actualización 4 de abril 2020
  8. Tapiwa G, Cécile K, Dongxuan Ch, Andrea T (2020) Estimating the generation interval for COVID-19 based on symptom onset medRxiv
  9. Wölfel R, Corman VM, Guggemos W, Seilmaier M, Zange S (2020) Virological assessment of hospitalized patients with COVID-2019. Nature 581: 465-469. [crossref]
  10. Idan Yelin, Noga Aharony,  Einat Shaer Tamar,  Amir Argoetti, Esther Messer, et  (2020) Evaluation of COVID-19 RT-qPCR test in multi-sample pool. medRxiv preprint
  11. Roni Ben-Ami, Agnes Klochendler, Matan Seidel, Tal Sido, Ori Gurel-Gurevich, et (2020) Large-scale implementation of pooled RNA extraction and RT-PCR for SARS- CoV-2 detection. Clinical Microbiology and Infection PII: S1198-743X(20)30349-9.
  12. Michael G Becker, Tracy Taylor, Sandra Kiazyk, Dana R Cabiles, Adrienne FA Meyers, et al. (2020) Recommendations for sample pooling on the Cepheid 1 GeneXpert® system using the Cepheid Xpert® Xpress SARS-2 CoV-2 bioRxiv preprint
  13. Abdalhamid B, Bilder CR, McCutchen EL, Hinrichs SH, Koepsell SA, etal. (2020) Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Am J Clin Pathol 153: 715-718. [crossref]
  14. Ola Brynildsrud (2020) COVID-19 prevalence estimation  by  random  sampling in population – optimal sample pooling under varying assumptions about true prevalence BMC Med Res Methodol 20: 196.