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Dysregulation of Cell-Mediated Immunity in Patients with Long ICU Stay Affected of Multiple Organ Dysfunction Syndrome and Multifocal Candidiasis

DOI: 10.31038/IDT.2024523

Abstract

Introduction: The endogenous immunosuppression in critically ill patients with Multiple Organ Dysfunction Syndrome, requiring long-term ICU, can play an important role in the prognosis of Multifocal Candidiasis.

Method: Prospective study with control group (33 healthy volunteers) and study group (43 critically ill patients non neutropenic admitted to ICU with invasive mechanical ventilation), about cellular immunity in vitro, using Lymphoblastic Transformation Tests (LTT) stimulated with phytohemagglutinin (PHA). Simultaneously, the study group is followed up on appearance of Invasive Candidiasis and in vivo immunity study, using skin tests during the first week of ICU stay.

Results: There are significant statistical differences between the control group and the study group in relation to LTT PHA results. These differences are also found in the study group in relation to mortality and when skin tests are negative. When analysing the subgroup of patients with ICU stay longer than 12 days, there are statistically significant differences in LTT PHA when comparing the test performed in the 1st week in ICU in survivor patients with Invasive Candidiasis and the control group, but there are no differences with the 2nd determination. There are statistically significant differences when comparing the results of LTT PHA among patients with Invasive Candidiasis according to mortality in the 2nd determination, but these differences were not identified in the 1st week of ICU.

Conclusions: Both in vivo cellular immunity studies, using skin tests, and in vitro studies with the LTT PHA, help to establish a prognosis in critically ill patients with Multiple Organ Dysfunction Syndrome requiring long-term ICU care and in which a Multifocal Candidiasis has been identified. Monitoring of T-cell dysregulation and endogenous immunosuppression can help to identify patients who may benefit from new immunomodulatory therapeutic strategies.

Keywords

Aptamer, Crimean-Congo hemorrhagic fever, Glycoprotein, Plaque, Virus

Introduction

In order to make Multifocal Candidiasis equivalent to Invasive Candidiasis, and thus initiate an early antifungal treatment to prevent death attributable to Candida spp. in non-neutropenic critical ICU patients who are neither neutropenic nor affected by AIDS [1,2], we analysed the factors that could contribute to the transformation from colonization of Candida spp. [2] to Multifocal Candidiasis and subsequent disseminated candidiasis. It is universally accepted that Candida spp. infections are linked to the antibiotic era [3]. The prolonged use of broad-spectrum antibiotics leads to an intestinal microbiological imbalance, promoting an excessive growth of candida in the intestinal lumen and thus facilitating the translocation of these fungi into the interior of the organism [4]. As an example, in the observations that were made, it was very common that, after treating with antibiotics treatment with broad-spectrum antibiotics for a sepsis due to Pseudomonas spp., if the patient survived and remained in critical condition, it was very common to detect the appearance of an invasive candidiasis. However, in these ICU patients who remain in a critical condition for a prolonged period of time (more than 7 days), other factors related to ICU treatment and monitoring can be identified, which should be considered as facilitators of Multifocal Candidiasis [5]:

  • Gastrointestinal dysfunction: Malnutrition and/or absence of enteral nutrition due to intestinal paresis; breakdown of the intestinal physiological barrier; treatment with parenteral nutrition, gastric protectors and prolonged nasogastric tube
  • Prolonged vesical catheterization
  • Prolonged arterial or venous catheter
  • Mechanical ventilation more than 7 days
  • Hemofiltration techniques

In some cases, this list can also be added treatment with prolonged treatment or high doses of corticosteroids (30% in these patients) [6]. It is at this point where it is questioned whether there may be in these patients an immunodeficiency acquired in the ICU and that this may play a role as important as the prolonged antibiotic treatment.

Methods and Materials

Prospective Study with Control Group

Study Group (43 patients): Patients over 18 years old and middle ages of 61 ± 10,8 (37-86), admitted to ICU with invasive mechanical ventilation without history of neutropenia or AIDS, and need for mechanical ventilation for more than 48h. There was no informed consent from the patients because their level of consciousness on admission to the ICU does not allow it. Family members have been informed about participation in the study, its risks and that this participation will not modify any of the protocols required in patient care throughout their stay in the ICU. Only the patients with the consent of their relatives have been included. Mortality rate of this study population was 46%. In 12 cases the UCI stay was lesser than 12 days with 42% mortality rate, and in 31 cases UCI stay was more than 12 days with a 48% mortality rate. The diseases that were the reason for ICU admission, grouped as follows were:

  • Chronic Obstructive Pulmonary Disease with respiratory failure requiring invasive mechanical ventilation prior to ICU admission (15 cases)
  • Postoperatory complicated cardiac or abdominal surgery with more than 24 h. of mechanical ventilation (12 cases)
  • Patients with Tetanus disease with invasive mechanical ventilation (13 cases).

Control Group (33 healthy volunteers): Group formed by subjects who have been informed prior to the acceptance of being included in this study.

Immunity Cellular Evaluation

  1. In vivo immunity study using skin tests, only in the study group. The test is considered positive if they present, at least in one of the puncture sites, an induration greater than 5 mm in diameter against the selected antigens (Streptokinase- Streptodornase 1/4, Candidin 1/500, P.P.D. 1/1000, Toxoplasmin). The test is performed during the first week of ICU stay and is not repeated at any other time.
  2. In vitro immunity study using Lymphoblastic Transformation Tests (LTT), lymphocyte extraction is performed in both Technique: An adjustment of lymphocytes to 2×106 cells/ ml is performed. Three control cultures and three stimulated with phytohemagglutinin (PHA) are grown for 72 h. at 37°C, with aerobic culture and 5% CO2. After this time, the slide extension and May-Grünwald, Giemsa staining is performed. Once the staining is done, the percentage of blasts over the total population of lymphocytes is calculated. This test, in the study group, is performed during the first 6 days of stay in the ICU and then every 12-17 days until discharge from the ICU.

Diagnosis of Multifocal Candidiasis

From the inclusion of the patients in the study group, cultures to identify Candida spp. are carried out once a week, until the ICU is discharged, to identify Candida spp. in five foci:

  1. Blood cultures
  2. Respiratory secretions from tracheobronquial aspiration
  3. Urinary cultures from closed circuit bladder catheterization. Positive only with more than 5000 colonies/ml.
  4. Simultaneous presence of positive cultures in the pharyngeal swab and in the gastric aspirate obtained through the nasogastric tube
  5. Other foci. Positive cultures from drainages or wound exudates

Candida colonization is defined when this study demonstrates the presence of Candida spp. in only one of these foci and the blood culture is negative. Candida multifocality or Invasive Candidiasis is defined when positive cultures are identified simultaneously in two or more of these foci and/or positive blood cultures.

Variables and Statistical Analysis Methodology

The following variables were analysed: ICU stay, ICU outcome, presence or absence of Candida spp. multifocality, presence of positive skin tests and results of LTT stimulated with PHA. Data were analysed using the SPSS statistical program. Categorical variables were compared among groups with Chi-square or Fisher exact test as appropriate. Continuous variables were analysed with the Student’s t or Mann-Whitney U test when the distributions departed from normality and described as mean or median (standard deviation or variance). Statistical significance was established at p value < 0.05 on two-tailed testing. For statistical analysis, patients were grouped according to mortality (D: dead/S: survivors) or the presence of positive skin tests (P: positive/N: negative).

Results

No significant differences were detected between the number of days spent in the ICU and mortality (42% mortality in stays of less than 13 days vs. 48% in stays of more than 12 days, X2 = 0.16). Significant differences were detected between patients according to whether or not they had positive skin tests and mortality (mortality of 27.8% in patients with positive skin tests vs. 78.3% with negative skin tests, X2 = 10.45 (p = 0.001)) and between presence of multifocal candidiasis and mortality (30% mortality in patients with colonization vs. 73.9% in patients with multifocality, X2 = 8.29 (p = 0.004)). These differences are more significant if only the group with stays longer than 12 days is analysed (10% mortality in patients with colonization vs. 71.4% in patients with multifocalities, X2 = 10.24 (p = 0.001)). There were no significant differences between positive skin tests and the presence of Multifocal Candidiasis (50% of patients with colonization had negative skin tests vs. 60.9% of patients with multifocality, X2 = 0.48).

Related on the LTT PHA done:

  1. There are significant statistical differences between the control group and the study group, in relation to mortality (LTT PHA control group (group C) = 39.25% (6.79), LTT PHA study group survivors (group S) = 29.22% (11.67), LTT PHA study group dead (group D) = 22.89% (13.11). Statistical differences between group C and group S: t = 3.86 (65 d.f.), p = 4×10-4; between group C and group D: t = 6.29 (65 d.f.), p =6×10-8; between group S and group D: t = 2.45 (66 d.f.), p = 0.002). Statistically significant differences are not found when comparing the results of the LTT PHA of the first determination between group S and group D (LTT PHA group S = 27.29% (10.15), LTT PHA group D = 22.68% (12.33), t = 1.49 (34 d.f.), p = 0.131).
  2. There were significant statistical differences between the control group and the study groups, whether the skin tests were positive or negative (LTT PHA control group (group C) = 39, 25% (6.79), LTT PHA study group with positive skin tests (group P) = 30.03% (12.16), LTT PHA study group with negative skin tests (group N) = 21.52% (12.41). Statistical differences between group C and group P: t = 3.8 (66 d.f.), p = 0.001; between group C and group N: t = 6.98 (60 d.f.), p = 5×10-9; between group P and group N: t = 3.39 (62 d.f.), p = 0.002). There were significant statistical differences when comparing the results of LTT PHA of 2nd determination between group P and group N (LTT PHA group P = 30, 07% (10.73) and LTT PHA group N = 19,28% (10,59), t = 2,13 (18 d.f.), p = 0.025). Statistically significant differences are not found when comparing the results of the LTT PHA of the 1st determination between group P and group N (LTT PHA group P = 29.68% (8.27) and LTT PHA group N = 22.53% (13.05), t = 1.5 (34 d.f.), p = 0.129).
  3. When analysing the subgroup of patients with ICU stay of more than 12 days, there were significant statistical differences between the control group and the study group according to survivors with colonization (1st study LTT PHA: t = 3,8 (39 d.f.), p = 0,001; 2nd study LTT PHA: t = 3,461 (35 d.f.), p = 0.002) and dead with multifocality for Candida spp (1st study LTT PHA: t = 5,82 (41 d.f.), p = 1,3×10-6; 2nd study LTT PHA: t = 6,09 (43 d.f.), p = 4,5×10-7). There were only significant statistical differences between the control group and the study group according to survivors with multifocality for Candida spp in the 1st study LTT PHA (1st study LTT PHA: t = 3,957 (37 d.f.), p = 0,0005; 2nd study LTT PHA: t = 0,603 (35 d.f.), p = 0,329). There were only significant statistical differences between dead with multifocality for Candida spp. and survivors with multifocality for Candida spp. in the 2nd study LTT PHA (1st study LTT PHA: t = 0,751 (14 d.f.), p = 0,291; 2nd study LTT PHA: t = 3,764 (14 d.f.), p = 0.002) (Table 1). There were no deaths in patients with candida colonization of more than 12 days.

Table 1: LTT PHA results, 1st determination and 2nd determination, comparing control group with patients staying in the ICU for more than 12 days in the study group, collected according to mortality and presence or not of Multifocal Candidiasis.

Characteristics of the groups LTT PHA 1st determination

LTT PHA 2nd determination

Number of cases

Median Variance Number of tests Median

Variance

Control Group Healthy volunteers

33

39.25% 46 33 39.25% 46

Study Group with days of UCI stay > 12

Survivors and Candida spp colonization

8 27.69% 104.62 4 26.70%

30.99

Survivors and multifocal candidiasis

6

25.75% 111.35 4 37% 54.67

Dead and multifocal candidiasis

10 20.75% 166.17 12 20.78%

163.54

Discussion

Critical illness is defined by presence of altered organ function in acutely ill patients such that homeostasis cannot be maintained without medical intervention in ICU, such as mechanical ventilation, vasoactive support for hemodynamic, renal replacement therapy, and so on [7]. The Sequential Organ Failure Assessment (SOFA) score is a simple method of assessing and monitoring organ dysfunction in critically ill patients [8,9]. The prognostic value of skin tests in critically ill patients has been observed for years [10]. There is also evidence of the prognostic value of Multifocal Candidiasis in these non- neutropenic critically ill patients. The detection of Multifocal or Invasive Candidiasis [2] can be an indicator of acquired immunosuppression in ICU patients affected by a Multiple Organ Dysfunction Syndrome [10]. In the patients of the study, the need for mechanical ventilation for more than 24 h. together with continuous sedation, before identifying Multifocal Candidiasis, indicates that their SOFA score is higher than 5. This fact confirms that the selected patients are admitted to the ICU with a Multiple Organ Dysfunction Syndrome. The LTT PHA carried out in this study demonstrate that the cellular immunity of non- neutropenic patients requiring mechanical ventilation for more than 48 h. presented significantly lower values than in a control group of non-neutropenic subjects. These results confirm the existence of an immunological response known as endogenous immunosuppression [11] in non-neutropenic critically ill patients. At the same time, these statistically significant differences disappear in the 2nd determination of the LTT PHA in critically ill patients with an ICU stay of more than 12 days and who survive. Therefore, improvement of this T-cell dysregulation [12] will condition ICU outcome in patients who remain in critical condition for more than 12 days. This observation raises the possibility that quantitative and qualitative kinetic monitoring of T-lymphocytes in ICU patients may help to identify those who may benefit from new immunomodulatory therapeutic strategies [13]. A possibility of this monitoring can be found following the proposal of the REALIST score [14].

Conclusions

Both in vivo cellular immunity studies, using skin tests, and in vitro studies with the LTT PHA help to establish a prognosis in critically ill patients requiring long-term ICU care and in which a Multifocal Candidiasis has been identified. Monitoring of T-cell dysregulation and endogenous immunosuppression can help to identify patients who may benefit from new immunomodulatory therapeutic strategies.

Aknowledgements

Thank you to Torres Rodriguez JM. MD PhD, who served as scientific advisor.

References

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  2. Ibañez-Nolla J, Nolla-Salas M (2024) Multifocal Candidiasis can be considered a form of Invasive Candidiasis in critically non neutropenic patients. IJID 147: 107171.
  3. Monto Ho (1981) Non-bacterial infections in the Critical Care State of the Art (vol 2). Ed Shoemcker, UC y Thompson WL. Fullerton. California 1-12.
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  9. George C, Robin M, Carlet J, Rapin R, Landais C, et al. (1978) Cellular immunity skin testing and sepsis in intensive care patients: relationship between results and Nouv Presse Med 7: 2541-2544.
  10. León Regidor MA, Ayuso Gatell A, Díaz Boladeras R, Robusté Morell J, Soria Guerrero G, et al. (1993) Candidiasis in an intensive care unit. Rev Clin Esp 193:49-54.
  11. Munford RS, Pugin J (2001) Normal responses to injury prevent systemic inflammation and can be Am J Respir Crit Care Med 163: 316- 321.
  12. Luperto M, Zafrani L (2022) T-cell dysregulation in inflammatory diseases in Intensive Care Medicine Experimental 10: 43.
  13. Rol ML, Venet F, Rimmele T, Moucadel V, Cortez P, et (2017) Rheanimation Low Immune Status Markers (REALISM) project: A protocol for broad characterisation and follow-up of injury-induced immunosuppression in intensive care unit (ICU) critically ill patients. BMJ Open 7: e015734.
  14. Tremblay JA, Peron F, Kreitmann L, Textoris J, Brengel-Pesce K, et al. (2022) A stratification strategy to predict secondary infection in critical illness-induced immune dysfunction: the REALIST Ann Intensive Care 12: 76.

Assessing the Effectiveness of Financial Management Technical Assistance to Immunization Programs:A Case Study on the Use of Mobile Money Payment Mechanisms in the Expanded Program on Immunization (EPI) Liberia

DOI: 10.31038/IJNM.2024551

 
 

The availability and adoption of mobile money services have transformed financial inclusion and service delivery in many developing countries (Donovan, 2012). In Liberia, the immunization program post Ebola Virus Disease (EVD) has faced challenges in efficiently managing financial resources and making timely payments to vaccinators and other health workers, thereby hindering the effectiveness of immunization service delivery. Hence, this mixed- method study was conducted in November 2024 to assess the impact of integrating mobile money payment mechanisms into the financial management system of the Expanded Program on Immunization in Liberia. The review of existing literature suggests that mobile money has the potential to enhance financial inclusion and improve the efficiency of immunization service delivery and other health services in the health sector. Mobile money has been shown to facilitate risk-sharing and promote welfare, though the evidence on its direct economic impact is still limited. (Aron, 2018) The use of mobile money for immunization programs specifically has shown promising results in increasing vaccination coverage through SMS reminders and educational tools for health workers. (Oliver‐Williams et al., 2017). In the case of Liberia, the integration of mobile money into the immunization program’s financial management modalities has helped address the challenges of timely payments and better assurance that payments reach persons for which they are intended, thereby improving health worker motivation and the overall effectiveness of the program. Liberia is dealing with the aftermath of brutal civil wars, the last of which ended in 2003. The infrastructure challenges include poor road infrastructure and underfunded health sector. Gavi, the vaccine alliance is the main funder for routine immunization in the Liberia. An review by the funder’s auditors in 2018 recommended improvements in the financial management systems for the grants at the Ministry of Health. The actions put in place included assurances that payments that MOH/EPI made at community level reached their targeted beficiaries, with timely submission of accountability reports to the center in Monrovia. In 2019, in response to severe economic constraints, the government implemented a salary harmonization exercise which included a cap on personnel recruitments. Subsequently, the development partners provide funding for monthly stipends for recruited health workers.

Highlights from Liberia’s Routine Immunization Program

Despite being one of the world’s least developed countries with low health sector funding, having a health expenditure per capita of $112 in 2021, Liberia immunization coverage for DTP 3 / Penta 3 is 82% (WUENIC 2023 Report) of its targeted population between the ages of 0-23 months by December 2023, from 78% in 2022 thereby recording a percentage increase of 4%. The country relies on the support of international organizations like GAVI, The Vaccine Alliance, USAID, The World Bank, WHO and UNICEF to undertake activities which assure a high immunization coverage. The COVID-19 pandemic affected immunization seeking behaviours because of fear, hesitancy, and mistrust about vaccines, which resulted in a drop in immunization coverage rates for routine vaccines. However, the government of Liberia and its partners were strategic in making a substantial comeback through the development and implementation of an immunization recovery plan, which culminated in increased immunization coverage, as evidenced by the WUENIC 2023 report. It can be recorded that the use of a mobile money payment mechanism for timely and transparent payment of fit-for-purpose immunization workforce (vaccinators and other health workers) was one of the enablers for Liberia’s success in the attainment of 81% fully vaccinated coverage for COVID-19 vaccine. Based on the progress made in increasing the immunization coverage as measured by DTP3 and MCV1, Liberia has been consistent over the past two years with attaining the Millennium Challenge Corporation (MCC) indicator for immunization thereby setting the government on track for obtaining a new MCC Compact.

Trend in Routine Immunization Coverage Despite Low Spending

By 2023, Liberia achieved a notable increase in vaccination coverage. For instance, according to the WUENIC report, the DPT3 vaccine coverage will rise from 65% in 2020 to 82% by 2023. This improvement was largely due to intensified and targeted vaccination efforts (i.e., Outreach, Periodic Intensification of Routine Immunization-PIRI campaigns, etc.), timely payments of remunerations, supportive supervision and the strengthening of the immunization program and, by extension, the health system. Despite these gains, challenges remain, including reaching zero-dose children in remote and underserved areas and maintaining consistent vaccine supply chains. Continued efforts and support from global health partners will be crucial to sustain and further improve immunization coverage in Liberia (Figure 1).

Figure 1: Efforts and support from global health partners.

A Key Lesson on the Use of Mobile Money Payment Mechanism in Enhancing Transparency, Accountability, and Improving Program Performance within the Liberia Expanded Program on Immunization

Mobile money has significantly transformed financial transactions of the Expanded Program on Immunization at the Ministry of Health, Republic of Liberia since its introduction in 2021. The Expanded Program on Immunization entered into contract with the two main mobile telecommunications services providers, MTN Lonestar and Orange. With the contracts, the mobile providers set up payment platforms at the Ministry of Health Office of Finance Management (OFM) and trained users. The EPI and OFM teams conducted a nationwide registration of vaccinators and their particulars including, health facilities assigned, county, names, phone number, and supervisor names. With this information, a payee master list was created and submitted to the mobile providers who validated the names and phone number. With this validated list, the Ministry of Health was able to make periodic payments to health workers. Liberia’s implementation of mobile money and significant advancements in routine vaccination coverage rates exemplify the impact of innovative digital solutions in enhancing the speed and efficiency of financial transactions, as well as in developing customized strategies for expanding coverage and addressing outbreaks of vaccine-preventable diseases. This situation illustrates the substantial outcomes that can be achieved through appropriate innovation and tailored strategies, even in constrained fiscal conditions. The accomplishments of Liberia’s Expanded Program on Immunization can be distilled into seven essential lessons.

National and Subnational Leadership

Based on one of the findings from Gavi’s Audit and Investigation in 2018, Gavi contracted a financial management firm to provide technical support and capacity building to the Expanded Program on Immunization and the Office of Financial Management (OFM). Said recommendation was embraced by the honorable minister and the senior management team of the ministry of health which play a crucial role in the effective implementation of mobile money for immunization programs in Liberia. At the national level, the honourable minister of health, the senior management team of the ministry, the EPI manager, the Office of Financial Management, the internal audit unit, and the compliance unit play pivotal roles in creating policy frameworks that facilitate the adoption and use of mobile money systems by the Expanded Program on Immunization (EPI) and other programs subsequently. This includes providing regulatory support and ensuring that the mobile money payment is secure and accessible to all immunization service providers (e.g., vaccinators and other health workers). For instance, a mobile money steering committee was established and headed by the honourable deputy minister for administration. By promoting public-private partnerships, national leaders encourage investment in mobile money infrastructure and ensure that the technology reaches even the most remote areas, thereby ensuring that other projects and programs adopt said payment mechanisms. At the sub-national level, county health officers, officers-in-charge, and community structures were essential in bridging the gap between national policies and community practices. At the community and health facilities levels, the community development committee and the health facility development committee were charged with the responsibility during their monthly sitting to leverage their close ties with community members in advocating and explaining the use of mobile money in health services, including immunization programs. Working directly with health workers and subnational leaders has helped in the effective implementation of mobile money systems that align with the specific needs and contexts of health facilities and their communities. Moreover, the collaboration between national and subnational leadership is crucial to enhancing the overall effectiveness of mobile money in immunization programs. National leaders can provide the necessary resources and policy support, while subnational leaders ensure that these policies are effectively implemented on the ground. By fostering a coordinated approach, leaders at all levels can address challenges such as technological literacy, network connectivity, and financial inclusion. Strong leadership ensures that the benefits of mobile money are maximized, leading to improved vaccination coverage rates and better health outcomes for the population. A key lesson learned is that strong leadership at the national and sub-national levels through collaborative effort is essential in transforming health service delivery, including immunization, and achieving broader public health goals in Liberia. The successful implementation of mobile money payments within the Expanded Program on Immunization is a glaring demonstration.

Vaccinators’ Satisfaction with Mobile Money Payment

Bringing payment directly to the payees is a transformative digital solution that has been a game changer within the immunization landscape because of its availability and the reduction of bureaucratic bottlenecks and logistics. Out of 252 fit-for-purpose vaccinators interviewed from 252 health facilities across nine counties from northwest, north-central, south-central, and south-eastern Liberia, 63.1% (n=159) said that they were satisfied with the use of the mobile money for immunization transaction which demonstrates increased financial inclusion, especially for those who previously had limited access to traditional banking services due to geographical location and struggle to receive their just remuneration after conducting immunization services. Additionally, they indicated that it has brought a sense of respect and dignity to them within their communities. Table one provides a detailed breakdown of respondents’ reactions to the use of mobile money payment mechanisms for immunization operations. Several key lessons have been learned from mobile money platform use as an innovation in financial management, particularly in Liberia. These lessons include but are not limited to the following (Table 1):

  • Challenges associated with traditional banking: The issue of limited banking facilities and long distances due to its geographical location has been eliminated.
  • Improve access and convenience: Immunization services have been significantly enhanced, thereby eliminating the need for physical cash transactions and reducing logistical barriers for vaccinators and other health workers working in remote and underserved areas due to the absence of bank access and direct access to cash on the mobile phone.
  • Enhanced transparency and accountability in the financial aspects of the immunization program through the provision of digital records for all financial transactions.
  • Strengthening trust and engagement between the vaccinators and the This ensures the continuous access and utilization of immunization services by the communities, which is crucial to improving immunization coverage and the larger public health goal of Liberia.

Table 1: How satisfied are you with receiving your payments through mobile money?

Frequency Per cent

Valid Percent

 

 

Valid

Very satisfied

89

35.3 35.3

Satisfied

159 63.1

63.1

Neutral

4

1.6 1.6

Total

252 100.0

100.0

Transactional Time: How Long Does it Usually Take for Vaccinators to Receive Payment after the Outreach Vaccination Campaign?

Payment of vaccinators for an outreach and/or vaccination campaign can vary widely based on several factors. Ideally, a well- structured mobile money system should be able to effect payment within 24 to 48 hours after the conduct of a health facility outreach and/ or vaccination campaign has ended. However, out of 252 vaccinators interviewed, 54.4% (n=137) stated that they received payments for health facilities outreach within one to two days upon completion. At the same time, 45.6% (n=115) indicated that it takes more than five days. A key lesson learned is that the system is effective to an extent because more than fifty per cent of the participants attest to receiving their payment within one to two days. However, it is noteworthy that there are some situations in which payment may not come so quickly. For instance, administrative inefficiencies, verification processes, loss of network and/or technical issues related to the mobile money platform can further delay the payment period. In some instances, delays for vaccinators might last for days or even weeks to receive the requirements for payment due. Indefinite delays could lead to considerable inconvenience and dissatisfaction among vaccinators, which will negatively impact their morale and the effectiveness of the program. Hence, having a well-structured system for resolving issues in a timely manner will also help to enhance mobile money payments’ reliability and efficiency for this fit-for-purpose immunization workforce and other health workers.

What is Your Overall Experience with Mobile Money Payments Compared to Traditional Payment Methods for Vaccinators?

The use of mobile money platforms as cashless payments has greatly improved the payment process for vaccinators when compared to the traditional system (e.g., hauling cash around) as payments are sent directly to their mobile phones, vaccinators no longer must spend long hours travelling to collect their salary. Out of 252 vaccinators interviewed about their experience with the use of mobile money platforms compared to the traditional system, 59.9% (n=151) indicated that they had a better experience with the use of mobile money as opposed to the traditional system, citing bureaucratic bottlenecks and logistics as major challenges associated with the traditional system as shown in table 2. It was highlighted that mobile money saves time and minimizes the risk involved with transporting cash. Finally, the instantaneous nature of mobile money transactions means that vaccinators are paid quickly, boosting their finances and morale in an immediate fashion. That said, mobile money payments come with their challenges. However, remote areas may experience network issues that could result in delayed transactions, while some vaccinators may not be digitally inclined and thus could face challenges in using mobile money platforms. While mobile money is deemed efficient, some vaccinators expressed concern about abuse and/or other lingering concerns, as the threat of fraud or unauthorized access to accounts is ever-present. Nonetheless, mobile money has generally been a positive change, providing both greater efficiency and greater access over traditional banking and cash-based systems (Table 2).

Table 2: What is the overall experience with mobile money payments compared to traditional payment methods for vaccinators?

 

Frequency

Per cent Valid Percent
 

 

 

Valid

Much better

70 27.8

27.8

Better

151

59.9 59.9

About the same

26 10.3

10.3

Worse

5

2.0 2.0

Total

252 100.0

100.0

How Would You Rate Challenges Associated with Accessing Payments through Mobile Money Platforms for Immunization Activities?

Out of 252 vaccinators interviewed, the majority (48%, n=121) indicated that accessing mobile money is easy when compared to the traditional system because their incentives and/or salary are paid directly on their mobile phone, meaning that they (e.g., vaccinators) no longer need to journey long distances to collect their wages. This payment method has enhanced the time spent on financial transactions while minimizing the risk of carrying cash. The real- time characteristic of mobile money payments is that vaccinators receive their payments immediately, allowing them greater financial stability – and, with that, motivation. However, there are still issues being faced when using mobile money payments. For instance, network challenges are one of the key challenges highlighted by vaccinators working in rural and underserved areas. Transforming immunization financial landscape using mobile money platforms, however, has been a journey that while positive overall, has not been without some bumps along the way, such as sub-optimal network coverage, therefore it is imperative to have a cross-sectoral approach to improving telecommunications access and digitial payments in the more remote areas of the country (Figure 2).

Figure 2: Cross-sectoral approach to improving telecommunications access and digitial payments in the more remote areas of the country.

Leveraging Data, Tracking Performance and Payments, and Course Correction

To ensure efficient immunization spending, data-driven strategies like health facility microplanning, mobile money payment reconciliation, and community profiling have been essential in identifying, monitoring, and improving vaccination efforts. By mapping and profiling communities, the immunization program can develop tailored service delivery interventions to reach zero-dose children, missed children, and underserved communities. This targeted approach allows districts and counties to use health microplanning to pinpoint areas with low vaccination coverage rates effectively. By doing so, their efforts were concentrated on specific areas, enabling them to apply targeted interventions and adjust strategies in real- time. The use of data ensured that no community was overlooked and that corrective measures were both timely and effective. A key lesson learned is that understanding the target population and their locations is crucial to achieving successful immunization outcomes.

Hence, it is imperative to underscore the need for high-quality data to implement targeted interventions effectively. Therefore, investing in data quality is therefore critical for both routine immunization programs, financial decisions, and outbreak response efforts.

Immunization Supply Chain and Logistics Management Information System

In Liberia, the use of mobile money services has significantly enhanced communication on the immunization supply chain and logistics management information system by ensuring the conduct of monthly health facilities outreach and averting potential and/or reducing stockouts of vaccines and vaccine supplies. 70% (n=176) of respondents indicated that with the timely payment of outreach support and monthly incentives, they could transport their vaccines to avoid stockouts and maintain the health facility’s integrity with the communities. A key lesson learned is the importance of local leadership at the health facility level to avert stockouts of vaccines and supplies, especially in hard-to-reach areas, where delivering multiple vaccines is challenging due to geographical accessibility.

Association Between Outreach Payment and Increased DTP3/Penta3 Immunization Coverage

A study was conducted to determine the association between outreach payment and increased DTP3 / Penta3 immunization coverage. Out of 252 vaccinators interviewed, a chi-square test analysis was run to determine the likelihood of an association. However, the findings revealed that a value of 0.282 with 1 degree of freedom (df) and an asymptotic significance (p-value) of 0.595 were observed. This indicates that the observed data are not significantly different from what would be expected under the null hypothesis. Hence, the observed data reveals that there is no association between outreach payment and an increase in DTP3/ Penta 3 coverage (Table 3).

Table 3: Chi-Square Tests

 

Value

df Asymptotic Significance (2-sided) Exact Sig. (2-sided)

Exact Sig. (1-sided)

Pearson Chi-Square

.282a

1 .595    

Continuity Correction

.141 1

.708

   
Likelihood Ratio

.286

1 .593    

Fisher’s Exact Test

      .642

.357

Linear-by-Linear Association

.281

1

.596

   
N of Valid Cases

252

       

The Psychological and Social Impact of Immigration Policies: A Minority Stress Perspective

DOI: 10.31038/PSYJ.2024654

 
 

The incoming Trump administration has outlined plans to implement a series of immigration policies emphasizing stricter enforcement measures, prioritizing mass deportations, enhanced border security, and the rollback of humanitarian programs [1]. Key components are expected to include the reinstatement of the “Remain in Mexico” program, requiring asylum seekers to await their court proceedings outside the United States; rolling back Temporary Protected Status (TPS) for individuals from certain countries; targeting the Deferred Action for Childhood Arrivals (DACA) program; reducing pathways for family-based immigration; re-introducing the Social Security Administration (SSA) no-match letters program to identify employees who are unable to establish continued authorization to work in the U.S. [2]; and deploying the U.S. military to aid in deportation efforts [3]. While framed as mechanisms to enhance national security and uphold legal frameworks, these policies are likely to foster a hostile social environment that disproportionately affects immigrant populations in the country. When analyzed through the lens of social determinants of health and minority stress theories, the potentially far-reaching implications of these policies on mental health become evident.

Social Determinants of Health and Hostile Environments

Dynamic systems theory asserts that humans are susceptible to their social environments, and challenging conditions can profoundly affect physical and mental well-being. Aligning with this perspective, the World Health Organization recognizes that social and community contexts- where individuals are born, grow, live, work, and age – significantly shape health outcomes [4]. Hostile social environments shaped by stigma, prejudice, and racism can act as substantial sources of chronic stress, with detrimental effects on targeted individuals’ well-being [5].

The incoming administration’s proposed policies will likely intensify these challenges by fostering environments steeped in fear and uncertainty. The plan for mass deportations with the potential use of the military would exacerbate the sense of insecurity among irregular immigrants, increasing their anxiety and stress levels. Programs like “Remain in Mexico” could disrupt the social and community contexts central to health, forcing asylum seekers into precarious living conditions that lack access to stable housing, healthcare, and safety. These policies are likely to serve as structural mechanisms that amplify the chronic stressors already affecting marginalized populations.

Minority Stress and Stigma Consciousness

The chronic stress experienced by individuals belonging to marginalized social groups is referred to as minority stress. The sources of minority stress are categorized into external (distal) and internal (proximal) stressors. External stressors include observable experiences such as rejection, discrimination, and acts of violence (Meyer, 1995) -all of which are likely to escalate under the incoming administration’s stricter enforcement measures.

Internal stressors, on the other hand, involve internalized perceptions of stigma and societal prejudices, such as internalized racism or homophobia (Meyer, 1995). This process, also referred to as stigma consciousness [6], reflects an individual’s heightened awareness of sociocultural stereotypes and biases targeting their group. For immigrants in the U.S., stigma consciousness is further intensified by public narratives that dehumanize them and portray them as “threats” to social order and public security (E.g., Haitian immigrants eating pets).

Political leaders and public figures significantly shape societal perceptions and influence inter-group dynamics [7]. Provocative rhetoric that labels irregular immigrants as “illegal,” “criminal,” or even “pet-eaters” harms both communities. It creates immigraphobia, an irrational fear of incoming migrants and immigration, by fostering perceptions of threat to national identity, public safety, or resource availability amongst the American public, creating divisions and mistrust. Also, it simultaneously fuels stigma consciousness, fear, and anxiety within immigrant populations. Furthermore, the psychological strain of concealing one’s vulnerable identity, such as an immigrant with irregular status hiding their identity due to the fear of deportation, will add to the cumulative burden of minority stress (Meyer, 1995).

Research shows that stigma consciousness and vicarious experiences of racism – such as witnessing or hearing about discriminatory policies or deportations- foster pervasive fear and anxiety (Yip et al., 2022). Even in the absence of direct personal attacks, the anticipation of rejection, hostility, discrimination, or expulsion can place individuals in a chronic state of stress and hyper-vigilance, with long-lasting consequences for their mental health and well-being. The psychological harm resulting from prolonged personal, collective, and vicarious experiences of racism and discrimination can culminate in racial trauma [8] with symptoms of hyper-vigilance to threats, flashbacks, avoidance of others, heightened suspiciousness, and somatic symptoms such as headaches, heart palpitations (Comas-Díaz et al., 2019). Furthermore, research has consistently correlated perceived racial discrimination with increased severity of anxiety, depression, and psychological distress [9-13]. These findings underscore the profound and multifaceted potential impacts of planned exclusionary policies on the mental health of immigrants, highlighting the urgent need for systemic changes to mitigate these stressors.

Broader Impacts on Immigrant Communities

The ripple effects of the above-mentioned hostile migration policies may extend beyond individuals, disrupting the social fabric of immigrant communities. Deportations and fear-based environments can erode collective resilience, weaken informal support systems, and instill mistrust in institutions such as healthcare and education. This mistrust may discourage immigrants from accessing essential services, including mental health support, further compounding the psychological toll. Fear of exposure or legal repercussions can exacerbate this avoidance, leaving individuals without critical resources to mitigate the stressors they face.

Conclusion

The incoming Trump administration’s planned immigration policies are likely to contribute to a hostile sociopolitical environment, intensifying stigma consciousness and minority stress among immigrant populations. Based on research and theory, these policies are expected to exacerbate mental health disparities while undermining community cohesion and individual resilience. The resulting cycle of fear and exclusion could perpetuate systemic inequalities, highlighting the urgent need for more inclusive and equitable approaches to immigration and public health. By addressing the structural and systemic factors that contribute to chronic stress and trauma, policymakers can foster environments that promote mental health and well-being for all individuals, regardless of their immigration status.

References

  1. Alvarez P, Mattingly P (2024) Donald Trump’s plans on immigration are coming into focus | CNN Politics. CNN Politics.
  2. Benesch Attorneys at Law (2024) Sneak Peek into the Trump 2.0 Administration Immigration Landscape. JD Supra.
  3. Glebova D, Taer J (2024) Trump confirms plans to use military to deport migrants after declaring national emergency. New York Post.
  4. Cokley K, Krueger N, Cunningham SR, Burlew K, Hall S, et al. (2021) The COVID-19/racial injustice syndemic and mental health among Black Americans: The roles of general and race-related COVID worry, cultural mistrust, and perceived discrimination. Journal of Community Psychology. [crossref]
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  6. Pinel EC (1999) Stigma consciousness: The psychological legacy of social stereotypes. Journal of Personality and Social Psychology, 76: 114-128. [crossref]
  7. Newman B, Merolla JL, Shah S, Lemi DC, Collingwood L. et al. (2021) The Trump Effect: An Experimental Investigation of the Emboldening Effect of Racially Inflammatory Elite Communication. British Journal of Political Science, 51: 1138–1159.
  8. Comas-Díaz L, Hall GN, Neville HA (2019) Racial trauma: Theory, research, and healing: Introduction to the special issue. The American Psychologist, 74: 1-5.
  9. Cheah CSL, Wang C, Ren H, Zong X, Cho HS, et al. (2020) COVID-19 Racism and Mental Health in Chinese American Families. Pediatrics, 146: e2020021816. [crossref]
  10. Ertorer SE (2024) Racism and Mental Health: Examining the Psychological Toll of Anti-Asian Racism during the COVID-19 Pandemic. Genealogy, 8: 3.
  11. Haft SL, Zhou Q (2021) An outbreak of xenophobia: Perceived discrimination and anxiety in Chinese American college students before and during the COVID-19 pandemic. International Journal of Psychology, 56: 522-531. [crossref]
  12. Huynh MP, Yellow Horse AJ, Mai NM, Mantuhac J, Saw A (2023) Discrimination and psychological distress among Asian Americans during COVID-19: Gender differences in the moderating role of social support. American Journal of Orthopsychiatry.[crossref]
  13. Wenger MR, Lantz B, Gallardo G (2022) The Role of Hate Crime Victimization, Fear of Victimization, and Vicarious Victimization in COVID-19-Related Depression. Criminal Justice and Behavior, 49: 1746-1762.

Substance Abuse and Poor Sleep Among Adolescents: A Mind Genomics Cartography

DOI: 10.31038/PSYJ.2024653

Abstract

Ninety-nine young respondents evaluated different combinations of messages about the effects of substance abuse and sleep issues. The test stimuli were messages developed by generative AI (ChatGPT 3.5). The research followed the process established by the emerging science of Mind Genomics. The study investigated four elements (messages) each about four topics related to drug use: dangers and negative consequences, health alternatives to drug use, in depth consequences when one uses drugs, how poor sleep affects mood, energy levels, and ability to focus. The 16 elements were combined by experimental design, the specific combinations of elements (messages) differing for all respondents. The rating was “Describes Me.” Analysis by ordinary least squares regression showed that no single message strongly applied to the entire panel of 99 respondents. Clustering to generate three mind-sets showed three distinct groups, easy-to-interpret response patterns. These are Leisure Enthusiasts who do not respond to the negative elements (n=44), Serious Behavioral Problems (n=31), and Emotion Issues with Impaired Judgment (n=24). AI summarized the mind-sets clearly at two levels, first in terms of strong messages and suggestions for new ideas, and then at a more global level. The paper shows the feasibility of creating databases of the mind at low cost, within a day, with powerful insights and direction for communication emerging from the process.

Keywords

Adolescent sleep issues, Adolescent substance abuse, Generative AI, Mind genomics

Introduction

The growing problem of drug use leading to sleep disorders among adolescents is becoming increasingly relevant to medicine specifically, but to society in general. According to current research, misuse of drugs made for children and adults may, in some cases, negatively affect sleep patterns, leading to a variety of health problems and cognitive deficits [1-3]. A conventional approach by practitioners provides targeted treatments and therapies to address both the drug misuse problem and the sleep disorders, considered as two different indications. Problems frequently occur when teenagers resist therapy or do not cooperate with prescribed measures, either or both making it difficult for healthcare professionals to effectively address the problem. The result is that adolescents suffering from drug use and sleep issues may express themselves in a number of ways, using language indicative of bewilderment, frustration, and pessimism. Emerging societal difficulties associated with drug use that cause sleep problems in teens include increasing rates of scholastic underachievement, social isolation, and mental health illnesses. In turn, parents of these adolescents may report feelings of shame, powerlessness, and concern for their child’s well-being, exacerbating the issue [4-6].

Parents may go to considerable measures to address this issue— these include obtaining help from mental health specialists, attending therapy sessions with their child, and adopting tougher regulations and monitoring to handle substance addiction and sleep problems. However, the complexities of drug use and sleep issues in teens coupled with other behavioral issues involved in maturation make long-term remedies hard to achieve [7,8]. When addressing the growing problem of drug use resulting in sleep disorders among teenagers, a key issue is how drug use affects the brain and interrupts young people’s normal sleep habits. Adolescent sleep patterns may also be influenced by external variables such as drug usage, which can cause increased anxiety, restlessness, and interrupted sleep cycles, as well as the social determinant of drug use. Certain medicines such as stimulants and depressants have been reported to interfere with the synthesis of neurotransmitters which govern sleep, making it harder to fall and remain asleep. Other factors to consider which lead to teenage drug use are social and environmental situations. For example, peer pressure, stress, and a lack of parental monitoring may all contribute to young people commencing and continuing drug misuse habits [9].

Helping the Young Medical Professional to Understand the Adolescent Mind

Medical professionals play a vital role in understanding the minds of their adolescent patients, especially when it comes to identifying and addressing substance abuse issues. Experience can be a valuable asset in this regard. Seasoned doctors may have encountered a wider range of cases and developed a deeper understanding of adolescent behavior and mental health. However, not all doctors have extensive experience with adolescent patients [10,11]. One standard way to learn about a patient’s mind is to use questionnaires. At the time of this writing, fall of 2024, the practice of surveys is well-established in many domains, whether medical, commercial, and even recreational. The interested professional focuses on the topic and attempts to generate a set of questions about that topic, with these questions requiring simple answers. The common practice is to present these surveys, either or person, or more frequently on the internet. The survey-taker, known as the respondent, completes the questionnaire [12].

Questionnaires are powerful tools to gather information about adolescent patients, their experiences with substance abuse, as well as their sleep problems. By asking targeted questions about drug use, sleep patterns, and related behaviors, medical professionals can gain a better understanding of the factors contributing to these issues. Various questionnaires have been developed for use with teens, providing valuable data that can inform treatment plans and interventions aimed at addressing substance abuse and sleep disturbances in young people.One of the interesting sidebars of the “survey business” is the biases that the survey creator, viz., the “researcher” encounters. Biases range from non-response (refused to participate) to indifferent (assigned random responses), and all too often attempt to “outsmart” the researcher by guessing what are appropriate, socially acceptable answers, rather than real answers [13].

Creating a system that allows medical professionals to understand the minds of adolescents poses a significant challenge when the doctor lacks experience or the patient is non-communicative. Traditional methods of patient assessment may not always be effective with younger populations, who may struggle to express their thoughts and feelings verbally. In such cases, innovative approaches—such as using targeted questionnaires or assessments specifically designed for teens—could offer valuable insights into substance abuse and sleep-related issues. One metaphor for understanding the minds of adolescent patients is the concept of taking a patient’s blood at the start of the patient-doctor interaction. Just as a blood test can provide valuable insights into a patient’s physical health, a metaphorical “phlebotomy of the mind” could help medical professionals assess the mental and emotional well-being of their adolescent patients. By developing a systematic approach to understanding the minds of young people, doctors may be better equipped to address issues such as drug use and sleep problems [2,14,15].

The Mind Genomics Set up Process — AI Generated/ Human Edited Questions and Answers, Human Generated Classification and Rating Scale

An alternative approach to asking single, disconnected questions about a topic is to present people with combinations of ideas or messages, and instruct these respondents (viz., our survey-takers) to read the combination and to rate the entire combination. Furthermore, these combinations are comprised of single messages, simple phrases, seemingly thrown together in a haphazard manner, although later we will show that this seemingly haphazard manner is far from the case. The combinations are structured according to an underlying experimental design [16]. The task is rather easy, although boring. The respondent sits in front of a computer screen. The respondent sees combinations of messages emerge. The respondent simply rates the combination. The average person simply goes through these combinations in almost an indifferent fashion, feeling like some how they are guessing. The reality is that there is an underlying structure, the ratings make sense, and the results show how people think.

The process presents people with ordinary “slices of life” created in a way that the respondent cannot “game the system.” Rather, after a moment of surprise, most respondents sit down and do the task. The respondent ends up “grazing”, looking at the vignette and then assign a rating. The system invokes what the late Nobel economist called System 1 thinking, where the responses are virtually automatic. The process works very well with motivated as well as unmotivated respondents. There is no requirement that the respondent think about the topic. Rather, it suffices that the respondent pays some attention to the material, and not just type the same answer again and again [17]. Mind Genomics requires four questions and four answers to each question. If that is done, the rest of the exercise is simple. But how does the researcher come up with these questions? The early history of Mind Genomics revealed a major block to the successful use of the approach. Many people, including professionals, ended up ‘freezing’ when instructed to provide four questions. It was simple enough to name the study, but the creation of questions posed problems— generally emotional ones. As an example, consider Figure 1: Panel A shows the screen as presented to the user. Figure 1, Panel B shows a completed screen. Often the requirement to create a set of four questions which “tells a story” becomes a daunting task, an obstacle to be overcome in the research process [18,19].

Figure 1: Panel A shows the screen as presented to the user. Panel B shows a completed screen

Recent enhancement of the Mind Genomics platform has incorporated AI in the form of LLM, large language models (here ChatGPT 3.5). The access to the AI is by means of Idea Coach, a small rectangle in which the user can type the request. Table 1 (top) shows the query as provided to the AI, and Table 1B shows the 15 questions generated from this first iteration. The query is simple, stressing simplicity and understandability. Once the user is satisfied with a question, it is a simple matter to select that question. The selection of a question results in the insertion of the question into the study. When the question has been selected, it is straightforward for the researcher to edit the question, polishing and formatting it so that the subsequent AI effort to “answer” this question will generate meaningful answers, rather than just questions which end up with yes/no answers. The same process is used to select four answers for each question. Idea Coach maintains the selected questions, allowing the user to iterate to find answers and then polish them before inserting the answers into the template [20,21].

In the use of the LLM, the questions and answers usually require some editing. The questions should be edited to encourage expansive answers, using words like “explain in detail”. This embellishment is important because the AI will use the questions themselves to drive the creation of answers. The research requires answers which paint a word picture. The only way to get those answers painting a word picture is to instruct the AI to “explain” or “embellish,” or “describe how”. These are words which generate meaningful phrases painting the word picture.

The next step creates the self-profiling classification questionnaire which enables the researcher to gain more information about the respondent. Two questions are automatically asked—age and gender. The researcher can ask up to eight additional self-profiling questions, each with a possible eight answers. Table 2 shows the self-profiling classification created specifically for this study by the researcher.

Table 1: The instruction to AI to create the questions (top), and the 15 questions created in this iteration.

Table 2: The self-profiling questions and answers, completed at the start of the evaluation, before any vignettes are rated.

Creating Vignettes by Combining “Elements” (Answers)

Table 3 shows the four questions generated by the combination of the human researchers and AI. Under each question are the four answers. Each edited/polished question generated 15 answers. The researcher selected four answers, inserting the answers into the study after polishing them. As a consequence, the questions and answers in Table 4 are usually better than what would be generated even by an experienced professional. The user can run many iterations for questions and for answers, as well as polishing them to make them more precise.

Table 3: The “raw” material, comprising questions and answers

If this study was run as a typical study, then each of the 16 elements (A1-D4) would be presented as a single question, and the respondent (survey-taker) would evaluate each element independently. Of course, the researcher would randomize the order of the elements to reduce order bias. The biggest problem of these one-at-a-time evaluations is that they have no context—or at least the context may change depending upon the element.

The Experimental Design Underlying the Construction of the Vignettes

A better way might be to create combinations of these elements, doing so in a structured manner, so that the combinations, the vignettes, tell a story, albeit a story which has few connectives. Figure 2 shows an example of what the respondent might see.

Figure 2: Example of what the respondent might see

Figure 2 looks disconnected but the respondent evaluating the vignette ends up with a sense of what is being communicated. After evaluating the first two or so vignettes, most respondents stop fidgeting and simply look at the vignette, and rate it on the scale shown in Table 4. It is important to note that Figure 1 does not attempt to present a polished paragraph to the respondent, one which reads well, with all the connectives. Although one might be tempted to “pretty up” the vignette, the reality is that the respondent has an easier time “grazing through” the sparse structure presented by the vignette. Less effort is required to identify the information in the vignette, and consequently the respondent can quickly evaluate each vignette. The happy consequence is that the respondent can go through the 24 vignettes quite quickly without incurring much fatigue.

The vignette is rated by the respondent on a simple 5-point scale shown in Table 4. The scale has two sides. The left side, the first part read, has the respondent decide whether the description in the vignette describes or does not describe them. The right-hand side has the respondent decide whether the description is typical or not typical. The rating question captures two aspects of the vignette, fit to the person, and typicality of the statements. These are not explained to the respondents. Most respondents end up having an intuitive sense of what the rating scale means. Their answers suggest that this intuitive sense of its meaning operates in the interview, as we will see below.

Table 4: Five-point rating scale

Experimental Design to Create Vignettes Which Represent “Slices of Life”

The Mind Genomics effort allows exploratory research rather than requiring confirmatory research. As such, the studies need not be based on theory, with the goal of confirming or disconfirming a hypothesis. Rather, the Mind Genomics studies end up encouraging exploration, intuition, and iteration. It is easy to react to combinations of messages, vignettes. We do it all day long, as we react at an almost automatic level to the world around us. Rather than asking ourselves “what is important about this vignette” or this particular situation, a task requiring thought, we simply react to what is around us. In colloquial terms we “go with the flow”.

By presenting respondents with combinations of messages, i.e., mixture of messages such as the answers (elements) shown in Table 3, we put the respondent into a more natural situation, one which resembles daily experience. All the respondent has to do is react. The structure of the vignette allows the researcher to present slices of life to the respondent, have the respondent rate the combinations of these slices of life, and when done, properly enables the researcher to numerically estimate the driving power of each of the 16 elements, even when the respondent themselves cannot do so. The experimental design will enable us to determine the degree to which each of the 16 elements fits the respondent. The key to success here is to present the “right” combinations of elements, the “right” slices of life.

The experimental design for Mind Genomics comprises one basic specified set of combinations, which is permitted into several hundred variations. Each variation differs in the specific combinations, but the mathematical structure is maintained, and the design is tested to ensure that it runs in the statistical analysis.

  1. The design requires four questions (aka categories, silos), each question associated with four answers (aka messages, elements).
  2. The underlying experimental design creates 24 combinations or vignettes.
  3. Each vignette has a minimum of two elements and a maximum of four elements.
  4. Each element appears equally often, meaning that it appears five times in the set of 24 questions and is absent 19 times.
  5. The experimental design lays out the structure of each of the 24 vignettes. Some vignettes will have only two elements. Some vignettes will have three Most vignettes will have four elements.
  6. The absence of elements in a vignette means that the combinations of these vignettes are incomplete. That incompleteness is deliberate. It allows the researcher to estimate the contribution of each of the 16 elements to the rating because there are situations where the element is missing. This is important for regression analysis, specifically dummy variable regression analysis,
  7. Up to now, we have created a set of 24 combinations for one respondent with the property that each question can contribute at most one element (answer) to a vignette, sometimes contributing no answer to the vignette, but never contributing two answers to the This is important for statistics and also ensures that the vignette will not present mutually contradictory messages.
  8. The 16 elements are statistically independent of each other.
  9. The final benefit or the final piece of information is that all of these vignettes, these combinations form one experimental design.
  10. The final task is to permute the design. We keep the basic structure of the design, but we change the specific combinations by changing the element numbers. For instance, A1 may become A3, A2 may become A1, A3 may become A2, and A4 may remain as This permutation must be checked to make sure that the elements remain statistically independent of each other. The result is several hundred permuted designs.
  11. Every respondent is presented with a different set of 24 combinations, although the mathematical structure remains the The practical benefit is that the researcher need not know anything coming into the study. The researcher need not know the correct combinations because the system itself will take care of it.
  12. To sum up, the underlying experimental design ensures that the researcher can understand how people respond to ideas, by forcing them to respondent to combinations, the vignettes, the aforementioned “slices of life.” Through statistics, specifically OLS (ordinary least squares) regression used by Mind Genomics, the driving power of each element emerges immediately in a way that cannot be “gamed.”

Fielding the Study with a Panel Provider (Or with One’s Own Patients)

Once those bookkeeping steps are done, the user contacts the panel provider—in this case Lucid Inc., a panel aggregator with access to tens of millions of people around the world. The actual panelists are the appropriate group, adolescents, who have agreed to participate in these studies. They receive rewards. To the authors, these respondents, these survey-takers, are totally anonymized. We have no idea who they are, no idea the reward that they get. Typically, to run 99 people as respondents requires approximately an hour or two from the time the email invitation is mailed out until the respondents complete the 3–4-minute survey There is a tremendous benefit of having a panel of paid, motivated people. Otherwise, it might take weeks and months to get the same number of respondents.

The panel provider is contacted, and the request is made for a specific age group, market, etc. The age range requested was 15 to 21 years old. No two respondents evaluated the same set of combinations. The result is that the study allowed us to explore a wide variety of combinations. As noted above, a key benefit is that there is no need to know the topic at the start of the project.

Transforming the Data and Creating Models Relating Elements to the Newly Transformed Binary Variable

Each rating generated a rating on the 5-point scale. A rating of 1, 2 or 3 was converted to 0. A rating of 4 or 5 was converted to 100. A vanishingly small random number was added to each newly created binary variable, the aforementioned 0 or 100. The reason for that is purely prophylactic. With the vanishingly small random number (<10- 2), one does not influence the analysis through ordinary least squares regression, but one guarantees that every one of the respondents will have variability in their newly created binary variables. The analysis is straightforward, whether we do it at the level of a group such as the total panel of 99 respondents, by groups defined by who the respondent claims to be based on the self-profiling classification, or even by individuals.

Recall that the dependent variable, the newly created binary variable, takes on 0 or 100, and each of the 16 elements has the value 0 when absent from the vignette, and 1 when present in the vignette. We create a simple equation of the form listed below:

Dependent variable= k1A1 + k2A2 + k3A3… k16D4. The magnitudes of the coefficients, k1 – k16, tells us the degree to which the appearance of the element in a vignette drive the respondent to say, “that is me.”

Recall that our respondents were just sitting there being exposed to a variety of messages embedded or combined in these vignettes. They had no idea what was going on. It was a “blooming, buzzing confusion” to them, in the words of Harvard psychologist William James. But throughout the effort, the respondents just simply sat there, grazed, as we said, through the vignettes, and assigned a number. Most of the adolescents, had we asked them, would have said they were guessing and would shrug their shoulders.

Table 5 shows the coefficients. The coefficients show the degree to which the element is perceived as saying “this is me.” We can consider the coefficients as conditional percentages. Thus, a coefficient of 10 means that 10% of the answers would be “that describes me”, were the element to be put into the vignette. From many of these studies, it would appear that a coefficient around 20 would be considered statistically significant. The rationale for this number is that the coefficients estimated with an additive constant show that a coefficient around 10 is statistically significant based upon a simple T test of coefficients. A model without an additive constant would show that same value of 10 to be 20. Thus, we create a simple operational rule that we should look for high coefficients of 21 or higher in models estimated without an additive constant, viz., models that are said to go through the origin.

With the following in mind, Table 5 suggests that no elements can be said to read our operational criterion of 21 or higher.

Table 5: Coefficients for the total panel for each of the 16 elements. Coefficients of 21 or higher denote very strong performing elements. The elements are presented in descending order of magnitude for each question.

Mind-Sets: Moving to ‘Deducing how a Person Thinks’ by the Pattern of the Coefficients

Individuals vary in their preferences, coping mechanisms, and interactions with medical professionals. We are also aware that individuals may or may not be capable of identifying what they consider to be essential. With the aid of Mind Genomics, researchers are able to comprehend emergent groups of individuals whose decision-making processes adhere to distinct sets of criteria. Mind Genomics enables the researcher to identify different mind-sets. Mind-sets are defined as people thinking the same way about a topic. The important contribution of Mind Genomics is its ability to create these mind-sets at the level of the granular, at the level of the problem and its specificities. By having a bottom-up approach, one can create mind-sets for any specific problem, such as the one we are dealing with right now. One does not need mental gymnastics to translate macro mind-sets to specific topics, an issue often calling for creative re-thinking, with the mind-set data reworked and analyzed to produce an answer for a specific granular problem.

To create the mind-sets, one uses k-means clustering, a well- recognized statistical approach [22]. In our specific case of 16 elements, each respondent has 16 coefficients. We compute the distance between all pairs of the 99 respondents—a simple statistic. That statistic is called D, for distance, and is defined as (1 – Pearson correlation). The Pearson correlation quantifies the strength of the linear relation between two sets of data. A Pearson correlation of +1 means a perfect linear relation, whereas a Pearson correlation of -1 means a perfect inverse relation. Respondents with high values of D, near 2 are always in the same mind-set or cluster. Respondents with low values of D near 0 are generally in different mind-sets or clusters.

Once the respondents are assigned to either two clusters or mind- sets or separately three clusters or mind-sets, it is simple to create new groups for OLS regression, which we saw above for the Total Panel in Table 6. This time we run five regressions, first for the two mind-sets, and then for the three mind-sets.

Table 6 shows us the coefficients for the two mind-sets and then for the three mind-sets. The sum of the number of respondents is always 99. Each respondent fits into only one of the three mind- sets. There are empty cells in Table 6, corresponding to the elements whose coefficients are 5 or lower. Finally, Table 6 is sorted by the mind-sets, with all elements failing to score strongly in at least of the mind-sets put at the bottom of the Table. The mind-sets for the three- cluster segmentation by k-means make intuitive sense. The mind- sets are coherent, even though the entire analysis was done strictly by mathematical principles without any appeal whatsoever to the meaning of the elements. The mind-sets emerge quite clearly.

Table 6: How the elements performed when the 99 respondents were separately divided into two mind-sets, and then into three mind-sets respectively. Strong performing coefficients of 21 or higher are shaded. The table is sorted by the coefficients for the three mind-sets to highlight the differences among the mind-sets.

Table 7 shows the distribution of the selected answers for the three mind-sets, and for total panel. The distribution of the self-profiling answers by mind-set is unclear, in contrast to the clarity emerging from strong performing elements for each mind-set. There are some differences among the mind-sets, but the patterns are hard to discern, even though one might have expected to see more pronounced differences among the segments. It is this interpretability of mind-sets based upon very strong performing elements which enables Mind Genomics to create easy-to-understand “new knowledge.”

Table 7: The distribution of answers by mind-set and total for the self-profiling classification

Putting AI to the Task of Adding Insights to the Mind-Sets

The last part of our analytics from the study itself is the interpretation of the findings through artificial intelligence. The automated re-analysis looks at the results from each mind-set, considering only those elements in the mind-set which generated a coefficient of 21 or higher. Through generative AI (ChatGPT 3.5) the AI answers a fixed set of questions as shown in Table 8. The results for each mind-set and to answer a variety of prompts. AI looks only at the strong performing elements, previously defined as elements which have coefficients of 21 or higher. Therefore, if a mind-set does not have any elements of 21 or higher, it does not appear in this AI analysis.

Table 8 shows the analysis. The prompts shown in Table 8 give a sense of some of the deeper information and insights that might emerge from the data. We might characterize the material in Table 8 as preliminary material for additional insights. Or, to take a phrase from the late Professor of Computer Science at Havard University, Anthony Gervin Oettinger, the material in Table 6 could be considered TACT, Technical Aids to Creative Thought [23].

Table 8: AI summarization and deeper analysis of the strong performing elements for each of the three mind-sets

Instructing AI to Provide a Simple Overview

Final analysis is based on the need to simplify the results. We can take all of the information provided by AI detailed analysis in Table 8 and summarize it through one simple query as shown in Table 9. The artificial intelligence does a very good job of taking the material that it itself has generated and summarizing it. The bottom of Table 9 shows the summarization in terms of what the mind-sets are, in what aspects they differ, and what innovations can AI suggest. The important thing here is that we can have artificial intelligence summarize and summarize more. Here is a situation where less is more.

Table 9: AI summarization of the results previously presented in Table 8

Discussion and Conclusions

Mind Genomics studies offer a unique approach to understanding human behavior and decision-making, unlike traditional questionnaires. By presenting respondents with descriptions and scenarios, researchers can tap into their instinctual responses and emotions, providing a more accurate representation of their feelings about a topic or product. This method often involves experiments where participants are presented with varying descriptions or messages, making it difficult for them to guess the “right” answer. This allows researchers to capture genuine gut-level reactions, revealing hidden insights that may not be apparent through traditional questioning methods.

Mind Genomics studies offer a practical and efficient way to gather data, as they focus on individuals’ reactions to descriptions or messages, allowing researchers to quickly compile large databases of knowledge. Dividing people into groups based on their responses to different messages and descriptions allows for a deeper understanding of how individuals process information and make decisions. By identifying patterns in how different groups respond, researchers can tailor messaging and communication strategies to better reach and engage specific audiences.

The study presented here is an example of the effort put in versus the output emerging. The time to create the questionnaires can be measured in hours, certainly less than half a day. The time to set up the study itself and launch was another hour or two. The time to obtain the fully analyzed data was an hour or two, with the fully analyzed data emerging in the form of a user-friendly Excel file. Finally, the time to summarize the data a second time through AI was less than an hour. Altogether, the project could have been completed within 24 hours. The time to write the paper is, of course, longer, at least for the current iteration of Mind Genomics, but that time will “collapse” in future iterations.

Acknowledgment

The authors would like to thank Vanessa Marie B. Arcenas, Angela Louise C. Aton, and Isabelle Porat for helping to produce this manuscript.

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Crimean-Congo Hemorrhagic Fever DNA Aptamers Inhibit Plaque Formation In Vitro

DOI: 10.31038/IDT.2024522

Abstract

The top five candidate DNA aptamer sequences developed and published previously by Bruno et al. (BMC Research Notes 5: 633, 2012) against Crimean- Congo Hemorrhagic Fever virus envelope glycoprotein precursor peptides were screened for plaque reduction activity against viable CCHF Oman virus in a BSL-4 laboratory. Statistically significant reductions in plaque forming unit (pfu) counts for CCHF using SW-13 cell cultures were obtained when pretreated with these aptamer DNA sequences which demonstrated much greater efficacy versus a polyadenine 49mer control oligonucleotide at the same concentrations.

Keywords

Aptamer, Crimean-Congo hemorrhagic fever, Glycoprotein, Plaque, Virus

Introduction

Passive immunity for lethal hemorrhagic viruses such as Ebola has been demonstrated using convalescent antiserum [1-4]. Similarly, humanized monoclonal antibodies produced by Eli Lilly and Regeneron proved effective as passive immunity agents to protect patients suffering severe COVID during the pandemic [5].

Unfortunately, convalescent human antisera and humanized monoclonal antibodies are difficult and rather expensive to develop and mass produce. Nucleic acid aptamers present a much less expensive and more facile development alternative to humanized antibodies for industrial scale up of biologics to bind and block or inhibit progression of deadly viruses. A number of successful in vitro experiments exist reporting reductions in the number plaque forming units (pfu) or blockage of infections when viruses are pretreated with specific cognate aptamers and then added to cultures of mammalian host cells [6-9].

Crimean-Congo Hemorrhagic Fever (CCHF) virus is a widespread tick-borne virus member of the Nairoviridae with mortality reported as high as 30% in affected humans primarily across Europe and Africa [10]. As such, this virus is the subject of intense research [10-13]. While small molecule viral inhibitors have demonstrated significant efficacy against CCHF [12], humanized monoclonal antibodies are a prime target for the development of therapeutic medical countermeasures as well [10,13]. Human antibodies are known to synergistically neutralize the virus when bound to the envelope glycoprotein precursor [10]. In this work, one of the top 5 aptamers was shown to cause up to a 92.2% reduction in plaque forming units (pfu) and the other four aptamer DNA sequences also appear to exhibit significant efficacy as passive immunity pretreatment agents at least thus far in vitro.

Materials and Methods

Aptamer Development and DNA Sequencing

The SELEX aptamer development process against synthetic peptides derived from the CCHF envelope glycoprotein precursor (GenBank AHL45281.1) attached to magnetic microbeads was previously described by Bruno et al. [14]. All DNA aptamers or control oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA).

SW-13 Plaque Reduction Assay

The traditional pfu reduction assay did not work well with Vero cells in initial experiments for unknown reasons. Therefore, the method of Paragas et al. [11] was used with human SW-13 adrenal cortex epithelial cells (ATCC CCL-105) except that crystal violet dye was used instead of neutral red. SW-13 cells were cultured in the BSL- 4 4 laboratory of the Texas Biomedical Research Institute (TBRI; San Antonio, TX, USA) to a confluent monolayer in Dulbecco’s minimum essential medium (DMEM) plus 10% fetal calf serum (FCS) in 6-well plates at 37˚C with 5% CO2. The culture medium was replaced with a 1:2,500 dilution of stock CCHF Oman strain in 0.5 ml of DMEM plus 2% FCS with or without the aptamer levels indicated in the figures (0 to 1,900 nanomoles of aptamers) for 60 min at 37˚C with gentle rocking. Culture medium was removed from the wells and 5 ml of DMEM plus 1% penicillin-streptomycin-L-glutamine mixture and 16% methylcellulose overlay was added to each well. Thereafter, 6-well plates were incubated in a humidified incubator at 37° with 5% CO2 for 7-8 days. At the end of this week-long incubation period, the DMEM and methylcellulose overlays were removed and 5 ml of room temperature 10% neutral buffered formalin was added to each well as a fixative. Plates were refrigerated between 2°C to 8°C overnight (12 h minimum). The following day, the formalin was removed and wells were washed with Phosphate Buffered Saline (PBS). Plaque counts were performed manually by two technicians in agreement about the numbers of pfus following staining of each well in 1 ml of crystal violet, followed by removal of the dye, a gentle rinse with fresh tap water and air drying. Plaque counts were subjected to ANOVA statistical testing and found to show statistically significant differences for all of the aptamer-pretreated groups versus the blanks and ploy-A controls with p values <0.01.

Results

Figure 1 shows the results of initial plaque reduction studies in which the means of triplicate well readings with 2X standard deviation bars appeared to produce a >50% reduction in the mean number of pfu per well from pretreatment of the Oman CCHF virus with 47 nmoles of aptamer 2 versus the >120 pfu for the blank control without any aptamer pretreatment additive. The blank in this initial experiment was comparable in number of pfu versus the poly-A 49mer control wells. The other aptamers in this first experiment did not reduce plaque formation >50%, but also demonstrated statistically significant reductions in plaque formation.

Figure 1: Initial plaque reduction screening experimental results for the top 5 aptamers and a poly-adenine 49mer all at 47 nmole of DNA per well except for the zero aptamer added blank. The averages and 2X standard deviation bars for 3 measurements are shown for each group.

In the second set of higher dose studies (Figure 2) with only aptamers 1 and 2, the aptamers exhibited a >90% reduction (up to 92.2% for aptamer 1 at 1,900 nmoles of aptamer) which was dose- dependent. Although the poly-A control exhibited some dose- dependent reduction in plaque formation as well in Figure 2, it was not nearly as effective at plaque reduction versus the more specific CCHF aptamers (~50% reduction versus >90%) at the 1,900 nmole concentration level.

Figure 2: Dose-dependence plaque reduction results for aptamers 1 and 2 versus the poly-A 49mer and the blank group at the higher DNA concentrations indicated. The averages and 2X standard deviation bars for 3 measurements are shown for each group.

Discussion

Jalali et al. developed numerous aptamers against the conserved internal nucleoprotein (NP) of CCHF which produced excellent reagents for sensitive detection [15] of many different variants of lysed CCHF viruses. However, anti-NP aptamers are not very useful as therapeutics or prophylactics for passive immunity since they do not bind the viral surface or prevent viral fusion with the host cell [6]. Here the author describes the first in vitro testing of aptamers developed against the CCHF envelope glycoprotein precursor complex [14] which could inhibit or prevent CCHF viral fusion and host cell entry as demonstrated by the present data (Figures 1 and 2). The top 5 aptamer DNA sequences reported here (Table 1) were mostly generated against synthetic peptides in the Gc region of the CCHF envelope glycoprotein precursor [14] (amino acids 1041 through 1684) which form spikes on the viral surface involved in viral host cell entry [16]. The author has previously reported 3-D molecular models of the top 5 aptamers docked with the CCHF envelope glycoprotein precursor [17] which bear some resemblance to the precursor domain II regions bound by the antibodies Mishra et al. have reported [10] making the results reported here quite plausible.

Table 1: Aptamer DNA sequences.

Aptamer No.

DNA Sequence 5’-3’

1

ACA GTT AGA GCT TGC CGT ATG CCT TTG TTA ACA TAA

2

ACT AAC CGA ATG GCA GTT TCC CCC TTA TCC ATC TAT

3

GGG ATA GGG TCT CGT GCT AGA TG

4

CGC TGA AGC AAG ACA TTA TCG GGA CAT TGC CGT GA

5

TGA CAC GCG TAC GGG TCC GGA CAT GTC ATA ACG GAC

Conclusions

In this preliminary in vitro study, the top 5 aptamer candidates from Bruno et al.’s previous publication [14] demonstrated solid in vitro potential to act as passive immunity biologics. CCHF aptamers 1 and 2 in particular demonstrated strong dose dependence in vitro with >90% efficacy at the 1,900 nmole dose and deserve further research attention as possible alternatives to antibodies for the passive therapy of CCHF. The biggest issues with using aptamers in vivo is their small size making them subject to rapid kidney clearance and susceptibility to serum nucleases. However, aptamer pharmacokinetics can be greatly enhanced by covalent addition of heavier inert blocking agents such as polyethylene glycol or some proteins to the 3’ end [18-20].

Acknowledgments

Funding was provided by a U.S. Defense Department SBIR Contract No. W911SR22P0007. The author thanks Dr. Ricardo Carrion and Dr. Michal Gazi of the Texas Biomedical Research Institute (TBRI) in San Antonio, TX for maintaining the SW13 host cell cultures and conducting plaque assays in the BSL-4 laboratory.

References

  1. PJ Lachmann (2014) Traditional passive immune therapy for emerging Ebola Emerg Microbes Infect 3: e81. [crossref]
  2. S Lu (2014) Using convalescent whole blood or plasma as passive immune therapy for the global war against Emerg Microbes Infect 3: e80. [crossref]
  3. Casadevall, LA Pirofski (2015) The Ebola epidemic crystallizes the potential of passive antibody therapy for infectious PLoS Pathog 11: e1004717. [crossref]
  4. E Mire, JB Geisbert, KN Agans, EP Thi, AC Lee, et al. (2016) Passive Immunotherapy: Assessment of Convalescent Serum Against Ebola Virus Makona Infection in Nonhuman Primates. J Infect Dis 214: S367-S374. [crossref]
  5. N Cimolai (2021) Passive Immunity Should and Will Work for COVID-19 for Some Clin Hematol Int 3: 47-68.
  6. SH Jeon, B Kayhan, T Ben-Yedidia, R Arnon (2004) A DNA aptamer prevents influenza infection by blocking the receptor binding region of the viral hemagglutinin. J Biol Chem 279: 48410-48419. [crossref
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  8. M Wongphatcharachai, P Wang, S Enomoto, RJ Webby, MR Gramer, et al. (2013) Neutralizing DNA aptamers against swine influenza H3N2 J Clin Microbiol 51: 46-54. [crossref]
  9. M Wang, MC Hao, Y Huangfu, KZ Yang, XQ Zhang, et al. (2024) A Universal Aptamer for Influenza A Viruses: Selection, Recognition, and Infection Inhibition, 7: 249-258.
  10. K Mishra, J Hellert, N Freitas, P Guardado-Calvo, A Haouz, et (2022) Structural basis of synergistic neutralization of Crimean-Congo hemorrhagic fever virus by human antibodies. Science 375: 104-109. [crossref]
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  12. O Ferraris, M Moroso, O Pernet, S Emonet, A Ferrier Rembert, et (2015) Evaluation of Crimean-Congo hemorrhagic fever virus in vitro inhibition by chloroquine and chlorpromazine, two FDA approved molecules. Antiviral Res 118: 75-81. [crossref]
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  14. JG Bruno, MP Carrillo, AM Richarte, T Phillips, C Andrews, et al. (2012) Development, screening, and analysis of DNA aptamer libraries potentially useful for diagnosis and passive immunity of BMC Res Notes 5: 633. [crossref]
  15. T Jalali, M Salehi-Vaziri, MH Pouriayevali, SLM Gargari (2021) Aptamer based diagnosis of crimean-congo hemorrhagic fever from clinical Sci Rep 11: 12639. [crossref]
  16. K Mishra, CL Moyer, DM Abelson, DJ Deer, KEl Omari, et (2020) Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38. Journal of virology 94: e02005-19. [crossref]
  17. JG Bruno (2022) Successes and Failures of Static Aptamer-Target 3D Docking Int J Mol Sci 23. [crossref]
  18. JG Bruno, R Crowell (2008) Selective glutaraldehyde-mediated coupling of proteins to the 3’-adenine terminus of polymerase chain reaction J Biomol Tech 19: 177-183. [crossref]
  19. H Dougan, DM Lyster, CV Vo, A Stafford, JI Weitz, et al. (2000) Extending the lifetime of anticoagulant oligodeoxynucleotide aptamers in blood. Nucl Med Biol 27: 289-297.
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Reflections Upon Political and Phamaceutical Deceits Perpetuated During the Covid Years – An Epitaph to Humanitarianism in Modern Times

DOI: 10.31038/PSYJ.2024652

 
 

A almost laughable to ponder upon where it all started and nearly grim and shameful when we realise as humans that we are reckless, careless, and foolish (Santosh Kalwar).

Looking back upon Covid’s entry into our lives, in the following narative we remove the wrapping of fraudulent news, to reveal how the spectre of a pandemic was used to coerce society into submission. To thank for all this we note the co-ordinated forces of a greedy pharmaceutical industry, shadowy governmental control plus an over-trusting population compliant in their own ruination. Not to mention the related pandemics of bankruptcies, depressions and suicides subsequently fermented. Ongoing are the results of ineffective vaccinations [1] causing more hospitalisations than the original virus (dones 2022); growing cases of autoimmunity [2]; nor is this yet over for the consequences are with us still [3,4]. Many vaccine effects are being covered-up by the term long-covid, where the guilty blame the disease rather than the longer term effects of vaccination. If this weren’t enough, and especially pertinent to modern times, are long-lasting effects steming from destruction of human rights and desecration of the human soul. As psychotherapists we see it in our clients plus those upon the streets. At the last, through complience, we all played a part in this all too real-life horror story. Many who previously believed in a more humane, just, compassionate and democratic society, now realise they were hood-winked into allowing all that’s best in us be destroyed. It’s not entirely our fault we fell for the prevailing chronicle, were we not primed to believe that politicians and medical experts, for all their personal flaws were trying to do the best they can? But to enhance compliance, governments used mass psychology and orchestrated fear [5], plus censored news [6] to keep us malleable. These ploys worked excellently and on far too many are working still. But now, finally, we perceive a wind of change blowing in the air (Mandavilli New York Times 202@. Indeed, the public’s belief in vaccination, since Covid, remains still at an all time low [7].

From the jig-saw of information arrayed below, drawn from multiple research studies, social and statistical surveys, news leaks and the accounts of ex-insiders at tbe time, we invite you to build your own picture, very different from the one officially broadcast by government, news media and big pharma – all guilty agencies who had a vested interest to supress the truth. Personally, although we would love to believe otherwise, reading between the lines it appears likely a group of powerful people regard the ordinary members of the public as surplus to their requirements. We, the public, don’t see through all this because evil makes us feel bad, sad, desturbed and deeply uncomfortable; so we resist accepting the world could work this way, for denial of unpleasant reality is part of our everyday coping mechanism. Nevertheless, uncomfortable facts must be faced if we are to awake fully to what’s before us. With Covid, even if we accept pharmaceutical bribes, commercial corruption and medical incompetence, there has to be another over-arching agenda for conspiracy of this proportion to thrive? How could blunders of such magnitude have been tolerated, condoned and succesful without large scale subterfuge sanctioned by our appointed guardians and rulers? There are few nurturing parents in our story, but many controlling and power-hungry ones. Are we then ‘Lost Children’? Regards the criminal reasons behind the Covid pandemic, the following are most widely circulated in public fokelore:

  1. Culling of the world population via vaccination;
  2. Experimental Implementation of mass control;
  3. Pathing the way for an economic power-driven global financial re-set.

Sadly, there is some confirmation these were designed to work hand-in-hand.

Everyhing ofcourse remains unproven to the uninformed public eye, but disturbing enough data nevertheless keeps pumping out via litigation within law courts, whistle blowers jumping ship and courageous individuals like J. F. Kennedy Jr [8] for us to consider some pretty shocking accusations, which we survey under the following headings:

Suspicions of a Plan-demic…

Bribes and inflated figures of pandemic deaths…

Formally denied side-effects of vaccinations now emerging…. The ineffectiveness of vaccination…

Culpability of Big Pharma…

Draconian intentions of governments and WHO… Press and News Supression…

Over-estimation of vaccine take-up…

Under the above we share factual reportage prior to a brief synopsis, and at the close share a summary of our findings and personal impressions.

We hope the information herein will bring you upto speed and initiate you into the ranks of the ‘informed public’.

Looking back at our earlier reflective studies listing lethal side-effects and the ineffectiveness of vaccines, bribery in high places and corruption plus propoganderisation of the news [9,10] are all too sadly proving true. After reading this article you will have to hand sufficient data to make up your own mind.

What a story to tell the world how Coronavirus has become impotent and Triendless to reward ultra rich with more rights (Qamar RaTiq).

Suspicions ofa PlanQemic

“Each wave of terrorizing creates its effects more easily — añer a breathing spell — than the one that preceded it because people are still disturbed by their previous experience. Morality becomes lower and lower, and the psychological effects of each new propaganda campaign become strongep it reaches a public already soñened up” (doost Meerloo).

The Research and Evidential base:

In 2017 and 2018 every nation on earth started buying and selling millions upon millions of virus test kits labelled ‘Covid19 Test Kits’, you can still see this on the website of the World Integrated Trade Solution! How could so much be prepared and so many nations be in ‘the know’ so early on? What is more, only five months before the coronavirus outbreak WHO instructed all the nations to immediately prepare for an imminent World influenza pandemic. Similar foresight caused the Australian government to publish a manual for a coronavirus pandemic, a few months before it even surfaced.

  • If the above weren’t enough to raise suspicions, why do American Government records show that the S. Department of Defense (DOD) awarded a contract on the 12th November 2019, to Labyrinth Global Health Inc, for ‘COVlD-Research’, interestingly in the Ukraine, one month before the alledged emergence of novel coronavirus and 3 months before Covid was officially dubbed Covid-19 or even known to exist? Further more, in relation to this spectre of bacterial warfare, Peter Daszak appears as a bridge, in that he was previously involved with weaponising drugs and subsequently employed by the US government as a Covid Health Advisor. And his health advice? In a minuted public meeting: he advised “We must create public h)/pe to get the public to accept an international virus vaccine”[11]. What are we to make of this? In relation to the link of germ warfare to Covid, although Corona virus was declared a dead issue and supposeably written off by WHO in 2007, why did the US government continue to spend millions of dollars upon it? Could it be Corona was still seen as a highly malleable bio-weapon so its research went underground in military laboratories? More disturbingly, it appears we all eventually became the enemy it was directed towards!
  • The person who technically owns the World Health Organization through his financing of it, is Bill Gates, the number 1 vaccine dealer in the world, who predicted: ‘There is no doubt in an)/one’s mind that we will experience a surprise outbreak of an infectious disease during the first term of the Trump administration’ (Kennedy 2021). Such foresight! He even went on public record threatening “We’ve Not Seen the Worst of Covid”. Clearly a continuing pandemic mind-set was in certain people’s interests. It is common knowledge that The World Health Organization had an official plan primed for 10 years of ongoing infectious diseases in waiting, from 2020 to 2030, which they entitled ‘A Decade of Pandemics’ – all ready and willing to go when Covid arrived! 2030 is also the year when ‘Agenda 2030’, a formally recognised United Nations, WHO and Bill Gates initiative geared to a worldwide centralization of eco-political power is envisaged as coming into Again, it appears someone knows something we don’t and is merrily planning a take-over!
  • To iron out any further doubts consider again Dr David Martin’s testimony to the Corona Investigative Committee [12], in which he states: ”The National Institute of Health’s grant Al23946-08, issued to Ralph Baric at the University of North Carolina at Chapel Hill (officially classified as affiliated with Dr. Anthony Fauci’s NlAlD by at least 2003), shows they began work on synthetically altering the Coronaviridae (the coronavirus family for the express purpose of general research, pathogenic enhancement, detection, manipulation, and potential therapeutic interventions targeting Covid, as early as May 21, 2000, at which time Dr. Baric and UNC sought to patent critical sections of the coronavirus family for their commercial benefit”(Ibid). In one of several papers derived from work sponsored by this grant, Dr. Baric published what he reported to be the full length cDNA of SARS CoV in which clearly stated that SAR CoV was based on a composite of DNA segments: “Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoVUrbani strain, and have rescued molecularl)/ cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones”(Ibid). In line with this, on April 19, 2002, the Spring before the first SARS outbreak in Asia, Christopher M. Curtis, Boyd Yount, and Ralph Baric also filed an application for U.S. (Patent 7,279,372) for a method of producing recombinant coronavirus. In the first public record of these claims they sought to patent a means of producing: “an infectious, replication defective, coronavirus”. In short, the U.S. Department of Health and Human Services was involved in the funding of amplifying the infectious nature of coronavirus between 1999 and 2002, way before SARS was detected in humans. Following evidence of the Patents Office how can we ever doubt that Coronavirus came from a lab? With great difficulty we respectfully suggest. Many portants of the Covid have reached the public since 2000, which caused some to suggest we were being softened up for the kill! In 2003 the TV series Dead Zone aired an episode entitled ‘The Plague’, showing a coronavirus pandemic. Everything we have experienced was shown: Chinese origin of the virus, masks, lockdowns, quarantines, test swabs! Even the cure was mentioned: chloroquine! In like vein, a musician in 2013 composed a song about a coronavirus pandemic, that would occur in 2020! He said he could predict this because he had been investigating so called ‘conspiracy theories’. Supporting the notion that some people knew before-hand about the pandemic, in 2006 a movie was also released called V for Vendetta, which depicts a coronavirus pandemic that results in worldwide tyranny. The theme repeated throughout the movie is ‘This is for your safety”! So clairvoyance or something else more sinister? One has to ask were we being subject to subconscious psychic preparation and subliminal preparation? Even the UN got into the act of prophesying the pandemic when in 2012 it published a comic book titled ‘INFECTED’, which tells the story of a global pandemic that starts in a market in China, leads up to worldwide medical tyranny which is praised as the best solution. You can download this comic from the UN website cited in the bibliography of this article {U/V 2012). Taken together, all these tend to suggest, as in mass psychology, we were possibly being primed and softened-up (an old KBG ploy) to expect a pandemic?
  • Dr. Reiner Fuellmich, is an international trial lawyer who has successfully sued large fraudulent corporations like Volkswagen and Deutsche Bank, has a worldwide network of lawyers who have listened to over a hundred experts from every field of science. Reiner claims to have collected undeniable evidence that the Covid pandemic was in fact a planned criminal operation. According to him, a second Nuremberg trial may be needed to prosecute all who are complicit in this unprecedented crime against humanity. Besides enrolling international lawyers and a judge, Dr. Fuellmich has organized a Grand Jury proceeding, where experts from the WHO, UN, CDC, Pfizer, Military, Secret Services are all presenting evidence that Covid was a criminal planed operation [13]. Either Dr Fuellmich has gone mad and his many witnesses likewise, or mass conspiracy is well nigh proven! Note that a criminal court in Peru has already ruled that this pandemic is a criminal operation run by Bill Gates, the Rockefellers, George Soros and other billionaires! But don’t worry, it may only be another conspiracy theory for us to dismiss!
  • The notion of a plandemic is further credited by the testimony of Marion Koopmans, a WHO virologist from the Netherlands working at the Wuhan bio-lab, who confirmed on Dutch television that the World Health Organization had been working on the Covid agenda for many years, while at the same time developing a Pandemic Treaty, which gives them complete totalitarian control over all the nations in the world, so that whenever they declare a pandemic, something they can do whenever they want based on any test they choose – governments had to obey! Luckily WHO’s megalomaniac macinations of a Pandemic Treaty was derailed by a consortium of 3rd World nations and Russia, who refused to sign their consent, though it had been already signed by governments in Europe and the USA.
  • As widely predicted, another “pandemic”, namely Monkeypox (dones 2022), stands currently waiting in the wings poised to take Covids This infection mimics many of the symptoms of COVID jab- induced shingles — so much so, the Department of Health in Queensland, Australia, used the same photo to illustrate both infections (the photos have since been removed from the website). Please note: “Unless there has been some genetic alteration, either through evolution or intentional genetic manipulation, fmonkeypozj is not a significant biothreat, and has never been considered a high threat pathogen in the past. So, stop the Tear mongering, misinformation and disinTormation”Dr. Robert Malone (Fearless Speech). Monkeypox is a virus endemic in Africa which emerges sporadically after transmission into humans from animal hosts; is typically spread by close human contact and is readily controlled by classical public health measures. It does not have a high mortality rate. So please please remove from your mind the necessity of standing in line for your Monkey-pox vaccination. Ask also what happened to Omicron, Delta and Alpha? Did they just go out of fashion as fear mongering moved on?

Authors Comments: The conspiratorial nature of Covid was picked up early [14,15] and its man-made origins confirmed in records from The Patents Office January 2000 to June 2008, firstly addressing investigation of canine coronavirus gene uses on Jan 28th 2000) by Pfizer Inc (https.fff patents.justia.comfpatentf6372224);in the subsequent isolation of Corona virus from humans on April 12th 2004, also by Pfizer (https.fffpatents.justia.comfpatentf7220852);plus in the ongoing investigation into antiviral agents for the treatment, control and prevention of infections by coronaviruses on Apr 28th 2004 by Sequoia Pharmaceuticals Inc (https.fffpatents.justia.comfpatentf7151 163); finally, and most incriminating of all, by the investigation of amino acid sequences directed against envelope proteins of a virus and polypeptides for the treatment of viral diseases upon Jun Sth 2009 by Ablynx N.V (https.fff patents.justia.comfpatentf919378). It all becomes very difficult to believe that Coronavirus came from any other than a lab or that it was ever a new or novel strain. Sceptics amongst you please click any one of the above patent references to see for yourself. WHO, appear to have very powerful political ambitions on a world wide scale, not forgetting a principle funder in vaccine multimillionaire Bill Gates. Personal and comercial interests, we suggest, may have corrupted WHO’s stated purpose. We all tend to forget how large commercial enterprises have more wealth than many countries and wield much more power than most nation states, enabling their wealthy owners to force ‘their version of reality’ on us. Add to all this Patent Office records extending back to 2000, military involvement, leakage from public meetings and overlap of warnings from whistle-blowers, plus preparation of the public mind-set by pandemic films and dramas, and the notion of a Plan-demic, to our minds, is much more than merely circumstantial What is more, WHO in trying to push through Monkeypox as an International Emergency, even though US Government data agrees it is impossible to distinguish between Monkeypox, chickenpox and shingles, all appear highly invested in repeating their earlier Covid success! More tellingly, as the Covid-1 9 vaccination increases the risk of singles by a shocking 4925Oó (The Ezpose 2022Ç, they would appear to have set the conditions for a further pandemic? Unsurprisingly the main clusters of Monkeypox (vaccine induced shingles) are emerging in areas where Pfizer vaccinations predominated! Intriguingly, when I clicked on Dr. Reiner Fuellmich’s website and sites relating to his legal legal actions against Covid, most were rapidly blocked or otherwise made impossible to access! A host of attacks on the validity of his inquiry also litter the net. This is common practice I find when ‘the authorities’ and Google do not want discomforting facts to energe!

“Menticide (brainwashing) is an old crime against the human mind and spirit but systematized anew. It is an organized system of psychological intervention andjudicial perversion through which a fruling class can imprint their own opportunistic thoughts upon the minds of those they plan to use and destroy” (doost Meerloo).

Bribes and Inflated Figures

“We’ve seen repeated cases of corruption, and that is the second pandemic in many ways” (dohnathan Cushing Transparany International Global Health Team)..

The Research and Evidential base:

  • Dr Scott Jensen, Senator of Minnesota, put on public record via Fox News, that “Hospitals receive as much as 13,900 dollars for every patient they register as Covid-19. For every death resulting from Covid-19, that amount is tripled to. 30,900 USD”[16]. On a far lesser scale some Romanian GPs offered a hundred pounds to poor peasant folk in the provinces to take the vaccine! If that much was offered to patients how much were GP’s themselves getting? We personally have already uncovered, locally in Romania, monetary incentives for re-classification of those otherwise dead, to Covid deaths.

One of the informants for our earlier articles was an Orthodox Priest who was asked to confirm a Covid death for a road traffic accident victim. He refused and went public on TV! Also consider, death from any cause within 28 days of a positive test for SARS-CoV-2 was recorded in several countries as a “Covid death”. With case-rates and Covid deaths highest among the Triple Vaccinated many governments are now asking where did the ‘Science’ of the pandemic go wrong? The hype and the statistical facts don’t add up!

There are literally scores of viruses, even common cold viruses, which infect the human airway in elderly and infirm people to give rise to severe illness. But these appear to have been intentionally or accidently re-coded as Covid-19 – how else can we account for the disappearance of the common cold and seasonal influenzer during our Covid years? Least we forget about prior conditions contributing to Covid deaths, of 29,135 Covid deaths registered in Romania until April 2021 (Statistica, 2021), only 227 were admitted by the Minister of Health to have died of Covid alone, without severe aggravating prior conditions! Thus proving a massive exaggeration of figures to the tune of 28,908 excess deaths! A similar over estimation of deaths was similarly reported in the UK and USA. Please ask yourself why was it so important to lie to us by official bodies?

Authors Comments. Obviously, there was a concerted effort from many quarters to inflate and escalate Covid figures to glorify the pandemics potency. Bribes were also paid to this end and health policies modified. Why was it important for us to be deceived? We mentioned in an earlier article that if you once test positive in the UK, you can end up being registered ‘Covid positive for life’, to later be attributed ‘a Covid death’ statistically. In this way viral deaths soar frighteningly and swell existing figures. Not surprisingly, in this fear inducing context, no figures for recovery from Covid were ever circulated thus magnifying its potential as an imagined death sentence in public eyes. As we have evidence of the UK weaponising fear [17], we shouldn’t be too surprised by this petty dramatisation. More peturbingly, is money the main reason for all this? Or is there something more sinister afoot? De- populatuon perhaps? So does Covid and its vaccination shade into this? The argument politicians like Kissenger [18] and philosophers such as Yuval Noah Harari [19] espouse, is “we need to depopulate the planet out of necessity or we won’t survive as a species”. It’s an old cover story. Evil always believes itself to be doing good. Over-population and the burden of an aging populance have been live issues for many decades. The older we get the more pressure we put on state pensions, health services and public spending. Should a man of my age of 76 in light of such logic then be let live?

°The masses have never thirsted añer truth. They turn aside from evidence that is not to their taste, preferring to deity error, it error seduce them. Whoever can supply them with illusions is easily their master; whoever attempts to destroy their illusions is always their victim”(Gustav Le Bon)

Formally Denied Side-effects of Vaccinations Now Emerging

“Maybe it is not COVID-19, but the government should come out and explain what’s causing these deaths. We don’t have the freedom or the Tacts to say iT it is COVID-19” (AdoIT Mkono).

The Research and Evidential base:

  • Official UK Government data shows a 73Oó increase in the number of Young Adults and Teens suffering Heart Attack, Myocarditis and Stroke since the Covid-19 Vaccine roll-out. Another study found Covid-19 Vaccination increased risk of suffering a Stroke by 11,361Oó [20]. Is this just the tip of the iceberg? When they discovered there was a spate of heart attacks in children post vaccination, ‘Pfiser added an extra anti heart-attack ingredient (The Ezpose 2021), as if to acknowledge their culpability around vascular side-effects, ‘Sudden Death Syndrome’ appears to be a catch all diagnosis, invented by medics to cover vaccine induced heart attacks, such as the worrying number of Professional Athletes (890) who suffered cardiac arrests and deaths (579) post vaccination, and FIFA’s increase in Football Deaths to the tune of 300Oó over the past year (The Ezpose 2022e). So much evidence yet authorites are still loath to link it formally to But its not just strokes, why are so many post vaccination also going blind? And how come Covid-19 vaccination increases the risk of suffering miscarriage by 1517Oó? And the poison cherry upon the vaccination cake, a recent study found Covid-19 Vaccination increases risk of Death by whatever cause by at least 4,800Oó{//tid}/ Just let that sink in! Do you remember queing for your booster shot? Lucky you – not!
    Data recently published by the UK’s Office for National Statistics indicates that it only took 4 to 5 months after a Covid-19 vaccination for so much damage to be done to our immune system that some suffered death (The Ezpose 2022c), many are still suffering now (Dutcher et al 2024; & Lam et all 2024). In light of this, whilst we were all distracted by the Russia-Ukraine war, the UK Gov. confirmed that the Triple Vaccinated seem to be more rapidly developing Acquired Immuno-deficiency Syndrome (AIDS) than others, a finding also confirmed by a follow-up Canadian study. The damage has sadly been done, and we, the guinea-pigs,p are still bearing the consequences.
  • A New Modelling Study, no doubt funded by Big Pharma, goes against all others to conclude ‘Unvaccinated are a danger to the Vaccinated’; but Real-World Data and a growing number of authenticated studies prove COVID Vaccines increase risk of Covid infection by around a whopping 400Oó! There was an obvous war of disinformation going on out there. Andreas Schíifbeck, a director of a large German medical insurer, reported his company’s data indicated serious adverse effects were running at approximately 1 0 times the official rate reported by the Paul Ehrlich Institute (PEI), the official German vaccine regulator. Mr Schíifbeck got summarily fired for his troubles, despite only calling for further anaIysis. Two months on, an extensive study at the Charité Universitãtsmedizin Berlin (a large medical research university owned by the Federal State of Berlin in Germany), came up with data from long-term observational research sustaining his concerns. They reported: “The number of serious complications a€er vaccinations against Sars-CoV-2 is 40 times higher than previously recorded by the Paul Ehrlich Institute” (HART 2022). They further observed that suspected cases are not officially reported, and so the numbers of serious vaccination reactions at the Paul Ehrlich Institute remain significantly lower than in the Charité study (Ibid). As under-reporting is standard, it is estimated that only 1 0Oó of serious reactions and between 2 and 4Oó of non-serious reactions were ever If only government advisors and others had read the British Medical Journal’s warning and hesitated longer before adopting experimental and untested vaccinations [21]. Sadly, the BMJ’s caution fell on deaf ears.
  • Damage to our immunity by vaccination just won’t go away, a CDC (Centre for Disease Control & Provention) study found Covid-1 9 Vaccination increases risk of suffering Autoimmune Disease Myocarditis by 13,200Oó; another demonstrated that children’s risk of death due to lowered immunity increases by 51 00Oó following Covid-1 9 Vaccination compared to Unvaccinated Children (Office of National Statistics data). Add to this a more recent peer-reviewed study by Scandinavian researchers who looked at 1 million people in Denmark, Finland, Norway, and Sweden, that concluded almost two years ago, that the mRNA-based CVD ’gene vaccines’ caused myocarditis and pericarditis, and we can’t say we weren’t fore-warned. Heart and vascular conditions, plus autoimmunity, shout out in all statistical returns
  • but where is investigative journalism on all this? Nowhere! The National Health Service has also confirmed, in response to a freedom of information request, that ambulance call-outs relating to immediate care required for heart conditions doubled in the whole of 2021 and are still on the rise in 2024. But the most concerning figures, published by The Office for National Statistics, reveal that between January 2021 and March 2022 a total of 69,466 people died within 28 days of Covid-1 9 vaccination, and 1 09,408 people died within 60 days of vaccination in England [22]. Where were the news services? Why were we not informed of the facts? Doctors were also reporting, somewhat paniced, that a sudden upsurge in ’Sudden Adult Death Syndrome’ had emerged amongst vacvinated adults under the age of 40. Consequently, GP’s are still urging all under 40’s to go for a heart check-up. Covid-1 9 Vaccination is also being blamed for the UK Office for National Statistics data showing vaccinated adults aged 18 to 39 having a 92Oó higher mortality rate (per 1 00,000) than unvaccinated adults. Public Health Scotland data similarly reveals there has been a 67Oó increase compared to the historical average in 15 to 44-year-oIds suffering cardiac arrest, myocarditis, stroke, and other cardiovascular diseases since this age group was offered Covid-1 9 injections. One can understand full well why the pharmaceutical companies negotiated a no sue no liability clause in their supply of public vaccinations with side effects running rampant even today! Again, we have to accept the fact that untested vaccinations and profits came before responsibility for maimed lives!
  • Talking to grave diggers in our local cemeteries, we were personally informed, that since Covid the death rate amongst the young and old has tripled!

Authors Comments: Panic appears to be growing in many governments as the Fully Vaccinated have a higher Covid Hospitalisation-Rate than the Unvaccinated. Growing figures also testify to the inefficiency of masks, lockdowns, vaccinations, Covid passports, thus opening the door for litigation! The more so as AIDS; as viral authorities predicted is still showing-up as a fact of full vaccination. Is it not time we stopped calling the pandemic of heart attacks in the young ‘Sudden Death Syndrome’ and give them their real name, ‘Death by Covid Vaccination’? This is after all the true cause, as most governments well know. But why should governments support the truth when it means political suicide? Understandably, medical doctors who supported vaccination and advised their community accordingly, are also reluctant to share statistics which make clear that Vaccinated Young Adults have a 92Oó higher mortality rate (per 1 00,000) than Unvaccinated Young Adults. Combine all this with ambulance call-outs for heart illness having doubled since the beginning of the Covid-1 9 Vaccination Campaign, and orthodox medicine, it would appear, has hung itself by its own petard! But the wall is crumbling, for even a World Health Organization study [23] concludes risk of suffering Serious Injury due to COVID Vaccination is 339Oó higher than the risk was of being hospitalised with COVID-1 9! It is interesting timing for WHO to now expose the inefficiency of vaccines, the very things they pushed – are they changing sides in order to survive the incoming tide turning against them? When WHO and the BMJ agree on a point, it can’t be ignored. Will Big Pharma do the same and fess up we wonder? Insurance companies, who have no reason to lie, in reports from 201 9 (the last normal year before the pandemic) to 2020 (the year of the Covid-1 9 virus) report an increase in Group Death Benefits of only 9 percent; but group death benefits in 2021 when vaccine was introduced, increased 164 percent. Indeed the precise numbers for Group Death Benefits taken from Lincoln National’s annual statements for these years are 201 9: 8500,888,808; 2020: 8547,940,260; 2021: 81,445,350,949 [24]. Please read these figures again. When commerce is God, all else, morals and Christain virtues it seems become expendable. There is also another area we need consider, assisted deaths. It is now being suggested in several quarters, somewhat cynically, that we gave up two years of our life because Midazolam was used prematurely to end the lives of thousands of over 70’s in care homes (Telegraph 2021), though relatives were told their next of kin had died of Covid-1 9. Legalised murder no less! Looking at the bigger picture, we were conned by health and government alike into believing Covid was so very very lethal! So the alternative motive? Euthanasia or an experiment in mass population control? Such notions as these we deemed unbelievable prior to Covid, but many now believe they are are worth considering – so low is civic trust! Lastly, regarding the prospect of vaccine induced AIDS, this was prophesied by an official German Government study in January 2022 [25], it came right on cue explaining many of the associated peaks of illness earlier described and is with us still. The amazing thing is, some people thrive following triple vaccination, so research is acutely needed to identify catalysts that counter the effects of vaccination; but here’s the catch, grants for remedial research can’t be allocated until vaccine damage is openly accepted – and there remain forces at work hell bent on preventing this!

“Logic can be met with logic, while illogic cannot – it confuses those who think straight. The Big Lie and monotonously repeated nonsense have more emotional appeal… than logic and reason. While the fpeople are still searching for a reasonable counter-argument to the Tirst lie, the totalitarians can assault fthemj with another” (doost Meerloo).

The Ineffectiveness of Vaccination

“Success belongs to those who accept mistakes as stepping stones” (Hermann d Steinherr)

The Research and Evidential base:

  • At the height of the Covid pandemic a report noted 89Oó New Covid Cases were mostly within the fully vaxxed, another that the fully Vaccinated accounted for a shocking 73Oó of all Covid-19 Deaths across New Zealand since the its beginning in March Indeed, triple/double Vaccinated accounted for 81Oó of the record breaking numbers of Covid Deaths in New Zealand as late as March/April 2022. Simply, in terms of Covid and its variations, we are currently in the midst of a pandemic of the vaccinated! There is also another factor worth considering: “Those who had received a second dose over siz months ago had higher monthly ASMRs {Autonomous Sensory Meridian Response) for deaths involving COVID-19 than those who had received a second dose less than siz months ago, indicating possible waning protection from vaccination over time” (Office for National Statistics 2022). With the risks of side effects and waning effectiveness, vaccinations appear to exacerbate the problem rather than resolve it! But if immunity is being compromised by vaccination is it any surprise more vaccinated folk are getting infected?
  • Pfizer research data of 80,000 pages, ordered recently to be delivered to the law courts by a High Court Judge, whom they had approached for the opposite, namely 50 years legal restriction from public access under the Data Protection Act, proves beyond doubt they knew that vaccinations harmed pregnant women and that the vaccine was only 12Oó Though they claimed it to be 95Oó successful! So what can we trust about them? It would appear nothing! As I write this the Federal aurhorities are still investigating executives of Pfizer to possibly charge them with multiple counts of fraud relating to Covid vaccine. Truth will eventually out, but far too late for too many.
  • A comparison of official Government reports from so many countries are confirming that Covid-19 vaccines are not only ineffective, but causing as earlier cited, large scale Antibody-Dependent Enhancement (The Ezpose 2022c). If this weren’t enough official figures from the United Kingdom’s PHE Vaccine Surveillance Report UK (Gov 2021) suggest ‘fully vaccinated’ people are losing on average of about 5Oó percent of their immune systems function per week! It is further reported that doubly injected “People aged 40-69 have already lost 40% of their immune system capability and are losing it progressively 3% to 6.4% per week” (Ibid). Current figures remain unavailable! Still governments continues to push vaccination! We pray the above figures and predictions are wrong, or that our bodies will be able to manufacture counter-measures to stop this deterioration, for everything points to fully vaccinated people suffering degrees of an ‘acquired immunodeficiency syndrome’ (AIDS) at a galloping rate.

Authors Comments: We wish to appologise to our readers that some of our quotes cannot be fully referenced, especially in this section, as they were taken off the website before we had time to formally record them! When we returned to many sites challenging vaccination, we also found these had also mysteriously disappeared, and in their place we found a plethora of pro-vaccination messages rubbishing earlier claims. Having said this, enough sites remain to support that the fully Vaccinated are nearly 3 times more likely to die of Covid-1 9 than the Unvaccinated. How do you feel about having been coned into considering, if not indeed having a proven useless Covid injection with life threatening side-effects? Not forgetting ever more follow up boosters where you are asked to willingly accept an even greater risk to your health? We personally know of too many local cases where vaccination has heralded cerebral changes, cardio-vascular problems and autoimmune reactions, for us to doubt the above statistics. Small blood vessels in the eye, brain and heart seem especially to suffer. As we speak ever more variants are being conjured up and we”re being offered further vaccinations for vaccination induced diseases! A brilliant commercial plan for Big Pharma yet again! It is no small wonder that statistics are causing government concern, with the fully vaccinated, on the international scene still having a higher Covid hospitalisation rate than the unvaccinated, and the triple vaccinated now up to 5 times more likely to be infected with Covid-1 9 than the unvaccinated. But this is small wonder when vaccine effectiveness has fallen as low as minus 391 Oó! A recent scientific study confirmed COVID vaccines were causing severe Autoimmune-Hepatitis days after WHO issued a ‘Global Alert’ about new Severe Hepatitis strain among Children! Was WHO offering a cover story for vaccine side effect? And why when we Google for ‘Covid vaccination side effects’ do we get sore arms and headaches but nothing about serious side effects?

Governments, through mis-information fostered by Big Pharma have damaged those they were appointed to protect, yet so many of us still turn a blind eye? An extensive wide ranging study by several universites, plus the editor of the BMJ, report via the Social Science Research Network (SSRN), have reported that Covid vaccines are more likely to put you in hospital than keep you out [26]. We rest our case.

“Totalitarianism is man’s escape from the fearful realities of Iife into the virtual womb of the leaders. The individual’s actions are directed from this womb — from the inner sanctum man need no longer assume responsibility for his own Iite. The order and logic of the prenatal world reign. There is peace and silence, the peace of utter submission” (doost Meerloo,).

Culpability oF Big Pharma…

“Medicine being a compendium of the successive and contradictory mistakes of medical practitioners, when we summon the wisest of them to our aid, the chances are that we may be relying on a scientific truth the error of which will be recognized in a few years time” (Marcel Proust).

The Research and Evidential base:

  • Pfizer, from their earliest vaccine trial-runs kept ‘adverse reaction reports’ which testify they were already aware of 1,223 deaths and 42,000 complaints describing 158,893 side-effects! Obviously this is less a case of neglegence than For they knowingly released a dangerous drug upon the population. Keean Bexte, an independent journalist formerly employed by Rebel News, pointed out how 1,223 people died within the first 28 days after being inoculated with the BioNTech Pfizer vaccine during trials — and it was still approved for use (https.fft.cofBPzXvjUTsa)!. There are also reports that the vaccine killed all animals during an earlier trial. Many are justifiably angry the vaccine was allowed onto the open market despite all proven risks. Pfizer, yet again are demonstrating they put money before lives! Just 90 days after the release of Pfizer’s mRNA vaccine they knew and reported mounting deaths and side-effects under a heading of “general disorders” (note how death is subsumed here under general disorders); the most frequently reported disfunctions were nervous system ones – over 25,957. Still Pfizer went full-steam ahead without further investigation. This information comes as consequence, as earlier noted, of 1,000s of classified research documents from Pfizer being released by court order.
  • “The immuno-compromised SHOULD NOT Be Given Covid Injections”, so said the AstraZeneca CEO, this again tends to confirm that Big Pharma was alerted to the autoimmune side effects of its vaccines from the earliest trials! Even the key inventor of mRNA vaccine, Dr Robert Mason, is on record as challenging the lack of transparency regarding vaccine side-effects. He is pro-vaccination but considers the with- holding of side-effects He is especially concerrned re the true number of cardio-vascular complications, which he claims are 20 per cent higher than reported. As Public Health Officials, are forced to make policy on a best-guess way forward rather than factual basis, so damage continues to rise.
  • Kennedy’s book ‘The Real Anthony Fauci’, reveals how Fauci launched his career during the early AIDS It is claimed Fauci partnered with pharmaceutical companies to sabotage safe and effective off- patent treatments for AIDS and orchestrated a series of fraudulent studies, before pressurising US Food and Drug Administration (FDA) regulators into approving a deadly chemotherapy treatment he had good reason to know was worthless against AIDS, but made him money! Shades of his role in Covid times?
    Kennedy further alleges Fauci repeatedly violated federal laws to allow his Pharma partners to use impoverished and dark-skinned children as lab rats in deadly experiments with toxic AIDS and cancer chemotherapies. Strangely, Kennedy’s account has not been legally contested by Fauci and hence he’s not been sued! Why?
  • Pfizer is on public record since 2000 as paying over $4,660,896,333 (four thousand six hundred and sixty billion, eight hundred and ninety six thousand three hundred and thirty three dollars) in 71 criminal fines due to corrupt practices, inclusive of false claims; flaws in drug and medical safety; unapporoved off-label promotion of medical products; missing research and healthcare records; improper government contracting and competition; plus environmental Pfizer has been found repeatedly guilty of Negligence, Fraud and Bribery over the past two decades and fined accordingly, but was aeemingly too established and powerful to be brought down! Why should we ever trust their plea for more preventative vaccines? Especially in light of a Bio-distribution Study of Pfizer Covid-19 Injections, which suggested use of mRNA Vaccines should be suspended immediately! How could a company like this be let continue to practice? I guess we must never under-estimate the power of bribes and the willingness of officials to accept them.
  • Ivermectin emmerged early in the pandemic as effective in Covid, yet big pharma, abetted by government and WHO reatedly quashed any and every alternative treatment. For example, we mentioned in earlier articles that India went against the instructions of WHO and mandated the prophylactic usage of Ivermectin, effectively eradicating COVID-1 9 in areas where it was used. The Indian Bar Association of Mumbai, subsequently brought criminal charges against WHO Chief Scientist Dr. Soumya Swaminathan for recommending against the use of Ivermectin. Similar repressive scenarios have been reported internationally and further court cases are pending.

Authors Comments : Big Pharma got itself quite a business plan! Working first with WHO to dilute the definition of ‘pandemic’; spending billions cosying-up medico-political support via free lunches and cash incentives; pouring money into viral research and when a suitable candidate came along declaring it with WHO’S blessing a ’pandemic’. They even committed the worst virological sin of all – vaccinating at the height of a pandemic forcing the virus to mutate to survive! Even first year student nurses know this is dangerous and eeckless! How did they bring in previously prepared vaccinations? By coercing goverments into signing-off their culpability for side-effects, thus allowing them to offer lame experimental vaccinations over-and-above existing legal safeguards and law. And when these untested vaccines produced varients such as Alpha, Amicron, Delta, and Monkeypox they sought to vaccinate againt these also! Grand plan don’t you think? Commercially sound, ethically barren. We must also note the concerted effort to suppress the following alternative treatments: HYDROXYCHLOROQUINE – Used by tens of thousands of physicians worldwide, FDA approved for over 65 years, hydroxychloroquine is considered a safe and virtually 100Oó effective medicine for Covid-1 9. BUDESONIDE – Discovered as a 100Oó effective cure for Covid-1 9 by Dr. Richard Bartlett, who saw over 500 covid patients recover after treating them with this asthma medicine. As his randomised control study proved. IVERMECTIN – Dr Pierre Kory treated numerous corona patients to discover that Ivermectin is a true miracle drug for Covid1 9. A more recent study by ten medical experts reviewed the evidence and concluded that Ivermectin both prevents and cures Covid-1 9. CHLORINE DIOXIDE – Biophysicist Andreas Kalcker was reported as bringing down the covid daily death rate in Bolívia from 1 00 deaths a day to almost zero, with this substance he has researched for over 13 years. Even now alternative treatments to vaccination are being rubbished upon the net. So why were alternative treatments not investigated more? Rather than quashed so quickly – follow the money line and ask who has most to gain? It all boils down, yet again, to money before human lives! And when ‘ mass formation’ cum collective hysteria was cited to explain the populations sheep-like behaviour, Gates and big pharma likewise rubbished the work of all scholars associated with mass hysteria from Gustave le Bon to Mattias Desmet. Bill Gates even put out media message to say it was a made up term with no value! From Nazies to Covid and back again! Lies from the powerful masquerade as shining truths.

The world is in the grips of mass formation—a dangerous, collective type of hypnosis—as we bear witness to loneliness, free-floating anxiety, and fear giving way to censorship, loss of privacy, and surrendered freedoms (Mattias Desmet).

Draconian Intentions of Governments and WHO

“The worrying issue is that all levels of government — not only Federal — are abusing the emergency decree to continue with direct awards without any restrictions” (Eduardo Bohârquez director of Transparency International).

The Research and Evidential Base

  • Research undermining the effectiveness of face masks suddenly became political when Covid arrived! Whar are we to believe? The facts or political message? The accepted research findings in total confirm the efficacy of face masks is lacking, while adverse physiological, psychological and health effects of masks are clearly Note Dennis Rancourt’s exhaustive review of all existing scientific literature on face masks which concluded that masks offer no protection against viruses [27]! Quote: ”No RCT study with verified outcome shows a benefit for HCW or community members in households to wearing a mask or respirator. There is no such stud)/. There are no exceptions. Likewise, no stud)/ exists that shows a benefit from a broad policy to wear masks in public”(Ibid). Two final points, firstly during the Spanish Flu in 1918-1919 most people died of bacterial pneumonia caused by extensive wearing of masks. Secondly, as any bright school-boy will tell you, at the molecular level viruses are too small to be deterred by paper face masks – they fly right through the molecular structure of paper masks! Like trying to repel mosquitos with chicken wire! Yet governments went ahead enforcing masks and lockdowns though both were proven to be scientifically indefensible! Forcing the population into masks would therefore seem more an issue of control than one of preserving health!
  • As director of the National Institute of Allergy and Infectious Diseases (NIAID), Anthony Fauci, dispenses $6.1 billion in annual taxpayer-provided funding for chosen scientific research, allowing him to dictate the subject, content, and outcome of scientific health research across the globe. Fauci uses his financial clout to wield extraordinary influence over hospitals, universities, journals and thousands of influential doctors and scientists – whose careers and institutions he has the power to ruin, advance, or reward in an authoritarian manner. As an influential force within WHO Fauci has played a major role in the mess we are in now. J. F. Kennedy (Jr) has gone on public record stating that in early 2000, Fauci shook hands with Bill Gates in the library of Gates’ $147 million mansion, cementing a partnership that would aim to control an increasingly profitable $60 billion global vaccine enterprise with unlimited growth potential. Through funding leverage and carefully cultivated personal relationships with heads of state and leading media and social media institutions, the Pharma-Fauci-Gates alliance exercises dominion over much global health policy. But not for much longer as this truth is now out in Kennedy’s best selling book.
  • An investigative 229 page report, early on in the Covid game, came out in 2020 by a consortium of Reserve French Army Officers which drew links between The Gates Foundation and WHO, 5G, governmentally controlled vaccination and It identified that Covid-19 pandemic as troublingly being ‘foreseen’ by representatives of the military, industrial complexes, governments, pharmaceutical giants and shadowy “philanthropic” organisations. As for the reasons for Covid: ”The management of the ‘health crisis’seems to be a pretext for a totalitarian global take-over, inclusive of an intention to impose a global cryptocurrency, a vaccine with nano-chips and a subcutaneous electronic chip”(AFRAO 2020). The report identified massive corruption at the heart of WHO; saw Covid-19 as a biological and electromagnetic war supported by a vast “smoke-and-mirrors” operation designed to sow confusion among the ranks of medical and hospital personnel; it speculated that “Covid-19” could be the preparation for a much larger-scale joint operation, combined with a smokescreen to conceal large-scale tests of the 5G weapon for criminal ends that remain to be clarified. Links were also made between: 5G installations, both terrestrial and aerial (Elon Musk’s satellites in low-Earth orbit), as all part of a “total war project”. This independent inquiry came out in 2020 and guess what, was labelled as a conspiracy theory and left unreported by the main media. It may read in part as somewhat over-stated but… could it be nearer the truth than we care to realise if ‘The Great Reset’ is set to occur this next year or two? Time will no doubt tell.
  • 160,000 COVID Deaths were claimed by the UK government, yet the NHS confirms just 5,115 people have died of COVID-19 in England since beginning of Pandemic! As earlier stated, we can’t reference this because the UK Government has taken anti-Covid stats down from its statistical and health service public view websites! A similar governmental falsified over-estimation of deaths was also discovered in the US, where Centers for Disease Control and Prevention (CDC) were directed to not count anybody as vaccinated within the first 14 days, until their second dose, so those who suffered death or side effects within this period would enter the records as unvaccinated! Thus blatently falsifying statistical returns. So why was it so important to panic the country? To magnify death rates? What answer would you give? Some suggest this was a ploy of government to weaponise fear {Dodsi+’oxh 202f}/ But to what purpose? An experiment in population control? Certainly the CIA has been implicated in the production and refinement of Digital Vaccine Passports (Daily Veracity. 2022), and many other countries are exploring tracking systems for their population, so the issue remains live.
  • Governments worldwide are, it appears, working in lock-step fashion to bring in Digital I.D. and a Social Credit The EU has already agreed to expand online censorship with a ‘Digital Services Act’. In similar vein, Vienna is becoming a testing ground for a Chinese-style social credit system, and Canada is moving forward with plans to implement digital coding of population, while Italy has announced the roll- out of dystopian Social Credit System where compliant citizens will be rewarded for “Good Behaviour”. All much in keeping with the digital Identity introduced by Nigeria’s federal government, which has ordered telecommunication companies to bar calls from phone numbers not yet linked to a National Identity Number. Today Nigeria tomorrow the world! But you see the patten – control and monitor the masses, irrespective of human dignity or civil rights or individual privacy?
  • As eluded to earlier, the UK Health and Security Agency has been forbidden to publish any further statistical data on Covid-1 9 cases, hospitalisations and deaths by vaccination status, because its figures embarrass government by showing the triple vaccinated population are on the verge of developing Acquired Immunodeficiency Syndrome and the double vaccinated are suffering Antibody-Dependent Enhancement. In this way the truth is formally buried, for fear of challenging their own double So the UK Gov. uses hardworking taxpayer’s money to advance their agenda to control an official version of ‘the truth’. The Ministry of Truth is patently invested in mistruth in the UK!

Authors Comments. The handling of Covid 19 has been cited as preparation for ‘Agenda 2030’, a UN initiative whose shadow intentions, if not its press hand-out, have been said to support a system of total, worldwide centralization of power, influencing everything from governments to free market economy, with the goal of full centralization to allow a handful of people to micro-manage every aspect of trade and business. WHO ardently backs Agenda 2030 and seems to be working towards a like goal. Covid is seen by some as an initial experiment serving the creation of this economic empire, where a concept called the “sharing economy” is created in which people own less property and have less privacy. Sounds far fetched, but a lot of material is on the net re banking reset geared towards ensuring the security of existing power holders! Worryingly, Obama, Zuckerberg and Gates have acknowledged adopting the philosophy of Yuval Noah Harari, a Jewish philosopher who states the role of culture is to forbid (Harari 2018) He acknowledges that if governments and corporations succeed in hacking the human animal the easiest people to manipulate will be those “who believe in free will”. Harari observes: “We now have the technology to hack humanity and let everyone think and feel what we want. Tyrants always wanted to do thai but now for the first time we have the ability to do this. We will eradicate faith in God, end all free will, and make sure that humans think exactly what we want them to think”. His book,”21 Thoughts for the 21 st Century,” which supports the political lobby for population reduction was promoted extensively by Bill Gates, who wrote a foreword upon the cover. More desturbing is that Harari is being promoted massively by the United Nations, the World Economic Forum, the World Health Organization – hence Agenda 2030 cited above. The spectre of de-population seemingly won’t go away. 5G, was cited earlier as linked to depopulation, was referred to by the president of Chile who threatened on national TV: “5G will not only read your thoughts, it will inseW thoughts and feelings. And we will make sure it reaches every home in the country.”’What a charming fellow – not! The Jury is out whether this is rhetoric or fact. The military, Big Tech and various governments have all been implemented as playing their part in 2030, now said to be in its final stages. Ten years ago we would have laughed at such notions – now we seriously consider them. Lastly we must with great sadness note that many sources, both governmental and pharmaceutical, are now suspecting that the Covid-19 vaccination may lead to mass depopulation. These suspicions, further backed up by mounting evidence from research studies and confidential Pfizer documents the U.S. Food & Drug Administration has released by court order, confirm a similar scenario.

One can but remember a Kessinger memo leaked out from the US Government in 2014 seriously considering depopulation as a viable way forward! Something unimaginable was seemingly being held in mind prior to Covid.

“Modern technology teaches man to take for granted the world he is looking at; he takes no time to retreat and reflect. Technology lures him on, dropping him into its wheels and movements. No rest no meditation, no reflection, no conversation — the senses are continually overloaded with stimuli. Man doesn’t learn to question his world anymore; the screen of fears him answers-ready-made” (doost Meerloo).

Press and News Supression…

*‘It is a Turore of disinformation and attacks — one in which credible journalists are subjected to online violence with impunity; where Tacts wither and democracies teeter”(dulie Posetti).

The Research and Evidential base:

  • A volume entitled “Journalists for Hire” (Ulfkotte 2020), tells how perks are used to bribe writers and opinion makers to twist their reports. It illuminates first-hand how a tone of corruption is set from the top
  • ‘play along or quit’, plus to what degree the long arm of a NATO press office enrolls media to get Europe to support foreign Press as a political tool? Just like the old communist days is it not?
  • J.F. Kennedy’s book lays bare how Fauci and Gates control the media outlets, both conservative and liberal news services, as well as scientific journals, plus key government and quasi-governmental agencies, global intelligence agencies and influential scientists and physicians so as to flood the public with fearful propaganda about COVID-19’s virulence and pathogenesis. Power to muzzle debate and censor dissent. As noted earlier, as Kennedy has not been sued for these accusations they would appear true?
  • The afforementioned press whistle-blower Ulfkotte 2020), a respected journalist in his own right, reported how government inspired under-cover agents wrote articles in editorial offices that were subsequently published later under the names of well-known He also pointed out which journalists received bribes for their warped reporting, plus how prestigious “journalism prizes” were a reward system for cooperation, going so far as to name who received them. It was further evidenced, by the author, who died later under suspicious circumtances [28], that respected journalist names and outwardly respectable organisations as the Trilateral Commission and German Marshall Fund were behind-the-scene players that were regularly paid to influence German media with one-sided propaganda.
  • For evidence of how the bought press turns upon the free press we need look no further than the case of Dr Rancourt, who we heard earlier dared speak against the official narrative of face Like many other scientists whose findings had a potential to rock government policy and pharmaceutical desires, Dr Rancourt was censored, had his academic reputation tarnished and his profile removed from ‘ResearchGate.net’. A host of character assasinations and de-buncking of his findings rapidly followed, flooding the internet! John Hardie’s world renknown study which proved masks increase infection [29], likewise was dug up from te past to suffer a similar debunking fate when the pandemic arrived.

The new black is obvously white! And truth is as ever decreed by those in power – are they not the ones to re-write history?

  • “The COVID-19 pandemic has demonstrated how vital it is for people to be able to speak out and share And yet medical professionals, journalists, activists and others who have informed the public or questioned the way the crisis has been handled have faced harassment and attacks” [30]. So speaks a professional body representing journalists. In the UK a pincer movement by the Treasury and Ofcom – the UK’s communications regulator, gagged our once proud news media through threats of fines and sanctions should they dare go against government guidelines. Not that the UK’s the only culprit. In its 2021 Index, Reporters Without Boarders [31] reported a ’dramatic deterioration in people’s access to information and an increase in obstacles to news coverage.” The RSF goes on to state that the COVID-1 9 pandemic has been used to prevent journalists from accessing other wide- ranging information so as to restrict critical reporting. According to RSF’s international survey, ” journalism is currently completely blocked or seriously impeded in 73 countries and constrained in 59 countries. Taken together, these figures represent 73% of the 180 countries assessed. Only 12 out of the countries ranked are deemed to have a Tree and favourable environment for journalism (one fewer than last year). Norway, Finland, Sweden, Denmark, Costa Rica, Netherlands, Jamaica, New Zealand, Portugal, Switzerland, Belgium and Ireland” [32]. Look at the glaring absence of the US and UK, France and Germany from this list! While the UK merely took down the National Statistics website relating to Covid deaths, in Iran, the authorities imposed measures to prevent news media from scrutinising the pandemic-related death toll, and “In Hungary and elsewhere, anti-Take news laws effectively criminalise legitimate journalistic reporting on the pandemic… Cases abound of how the ‘COVID-19 ezcuse’has led to the inability of journalists to do their job of reporting medically endorsed effective public health measures, or to challenge lethal disinformation” (Ibid). So we have a ‘gagged press’ not a ‘free press’, and propaganda rarther than factual reportage peppers our news (HART 2021). Investigative journalists continue to have their hands tied behind their backs! Indeed many have left employment or been dismissed because censorship has made their job untenable. In this future free speech has also been silenced. Speak truth at your peril!

Authors Comments: The UK government, while professing to be democratic, uses public money to bribe a publically funded news media – the BBC, to fear bombard the public with government approved propaganda furthering its own political agenda (HART 2021)/. They are also reported as paying experts in mass psychological manipulation to preserve their standpoint as the one and only way (Dodsworth 2021). There really is no room left for us to doubt press censorship. We personally have lost all faith in politicians and governments and consider the national news services a sick joke. Journalists themselves are likewise very concerned [33]. In terms of the impact COVID-1 9 is having on media there has been a renewed closure of civil space, especially in more authoritarian countries. In Egypt, for instance, there are new laws to suppress reporting, and leading journalists have been arrested. In Myanmar, under the cloak of COVID-1 9/fake news, independent media has been shut down (Ibid). All of which is deeply worrying. Sadly, the UK can’t report its own governmental censorship or abuses of the home press for fear of governmental and Ofcom fines and reprisals! Hypocritical or what?

“The social transformation that unfolds under totalitarianism is built upon, and sustained by, delusions. For only deluded men and women regress to the childlike status of obedient and submissive subjects and hand over complete control of their lives to politicians and bureaucrats” (doost Meerloo).

Over-estimation of Vaccine Take-up

Mis-information is a virus unto itselT (Brianna Keilar).

The Research and Evidential base:

  • Contrary to what the press and TV say fewer than half of people living in parts of London, Birmingham and Leeds have had the first Covid jab, and statistics show below the 50 per cent vaccination mark in 13 other wards dotted across the country, including Interestingly, the lowest uptake for vaccinations was amongst Ph.D’s! Practiced, well read and informed researchers like ourselves no doubt.
  • Even the UK Gov report admits 19.2 million people in England have not had a single dose of a Covid-19 “Vaccine” and another 12 million have refused a 2nd or 3rd Probing a little deeper on top of the 19.2 million unvaccinated, a further 2.6 million who had the 1st dose refused the 2nd dose, and a further 9.1 million who had the 2nd dose refused the 3rd dose (Fearless-speach 2022). Bringing the possible number of people who have now woken-up to the lies and propaganda spouted by the Government and mainstream media over the past two years up to 30.1 million.
  • A similar case to the above is reported for the USA. In light of Mr and Mrs average Joe seeing the ineffectiveness of vaccination to prevent repeated Covid infection, plus damaging side effects on their family, neighbours and friends, is it really surprising CDC (Centers for Disease Control and Prevention) reports 74.2 million people in the USA have not had a single dose of a Covid-19 Vaccine and another 157 million have refused a 2nd or 3rd dose? Are you dear reader aware of friends or acquaintances of yours who have suffered post vaccine effects? Many are seeing through the earlier hype. Are sheep now finally changing into rams?

Authors Comments

In an earlier report we mentioned if you once test positive in the UK you can end up being registered ’Covid positive for life’, to sooner or later be attributed ’a Covid death’! Such ploys as this cause Covid deaths to soar frighteningly. One could easily be led to believe no one recovers from Covid! But almost all recover unless with serious medical pre-conditions, or if incubated or otherwise harmed by medical intervention. So much stistical subterfuge! Hopefully the recent refusal of vaccination is evidence of the masses waking up to the harm inflicted upon us? But sometimes it’s much harder to admit our mistakes than to make them – so this may take some time!

“But the order of a totalitarian world is a pathological order. By enforcing a strict conformity, and requiring a blind obedience from the citizenry, totalitarianism rids the world of the spontaneity that produces many of Iife’s joys and the creativity that drives society forward” (doost Meerloo).[34-50]

Concluding Summary

“Historically, pandemics have forced humans to break with the past and imagine their world anew. This one is no different. It is a portal, a gateway between one world and the next. We can choose to walk through i¿ dragging the carcasses of our prejudice and hatred, our avarice, our data banks and dead ideas, our dead rivers and smoky skies behind us. Or we can walk through lightly, with little luggage, ready to imagine another world. And ready to fight for it” (Arundhati Roy).

When you join up the dots of the information we have arrayed before you where does your reasoning take you?

For ourselves, reluctantly, we believe there is just too much data coming out to believe that Covid was a simple virus. Military path lab involvenent and patent office records, plus WHO macinations produce too much evidence of fore-knowledge and fore-preparation from such wide ranging sources, to suggest, Covid was manufactured and planned, and on a massive co-ordinated scale. Mass corruption from state governments and State suppliers of health, pharmaceuticals and WHO are all evidenced as keeping it going. Bribes of a very high calibre oiled the workings and governments silenced the press and TV. Though in the UK the public facing puppets kept it going almost seamlessly, here, in our home-base of Romania we saw through the cracks much sooner, as public facing systems were more flakey and politicians were thankfully less organised than elsewhere.

We wish there was more data that a straight forward virus was merely mis-handled and more proof that our conclusions are faulty. For there are no winners here! But such reliable sources as the British Medical Journal, Data Science Association, Health Advisory and Recovery Team, International Freedom of Expression Exchange, Social Science Research Network, UK Office for National Statistics, UK Statistics Authority and far too many university led research studies, plus records of The Patents Office back-up our findings. Deep down, we sincerely wish we were wrong, but we can’t find the verification for it. So we end up believing governments, spurred on by Covid, have ridden rough-shod over common good, human dignity and human rights, while exiziling humanitarianism, research and empirical observation to the wastelands. Consequently personal liberty has been restricted, justice and rationality suspended, and social responsibility all but forgot. The very things that make life the more enjoyable. In this light, as humanists, we feel morally compelled to write, as to sit idly by as moral cowards is not a self respecting option. Is silence during public atrocity not complience?

It took us some time to catch up with the Covid farce. Sinziana comes from a medical family and my first career was as a nurse and nurse teacher. Until Covid came along we both believed in vaccination. We were reluctant dissenters though ardent researchers, so research won us over in the end. In our earlier articles we were genuinely perplexed, now we have lost faith in government and medicine in general. At the last, the vaccinated are the ones paying the price. Prospectively, we fear they have a life vexed by possible auto-immunity and cardio-vacular problems ahead, not to mention a shortened life expectancy. This is a very hard reality to face, and we are past anger on the issue.

We know most people feed off the telly and news papers for their shot of truth, taking all other sources as suspect, so we will likely be enthroned further by writing this article as conspiracy theorists. But please, at least, survey the headlines of the references below to glean something of what propoganderised news is denying you. Feel free to shoot us down as the messangers but we beg of you – awake!

“It’s easier to fool people than to convince them that they have been Tooled” (Mark Twain).

References

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Digital Light Processing-Based 3D Printing Using Gelatin Methacryloyl as a Ink for Tissue Repair

DOI: 10.31038/NAMS.2024732

Abstract

Tissue engineering technology has demonstrated significant potential for tissue repair, enabling the restoration of damaged tissues or organs in terms of both structure and function. The utilization of digital light processing (DLP)-based 3D printing has gained significant traction in tissue repair due to its remarkable advantages in terms of printing resolution and efficiency, as well as mild printing conditions. Gelatin methacryloyl (GelMA) has emerged as a commonly utilized ink for DLP-based 3D printing due to its unique photoinduced crosslinking properties, controllable mechanical properties, degradation characteristics, and excellent biocompatibility. This paper primarily presents state-of-the-art technologies and applications involving DLP- based 3D printed GelMA constructs for bone repair, skin repair, and peripheral nerve repair. It aims to highlight the promising potential of DLP-based 3D printed GelMA constructs in regenerative medicine.

Keywords

Digital light processing, 3D printing, Gelatin methacryloyl, Tissue repair

Introduction

The repair and functional reconstruction of tissue and organ defects pose significant challenges in the field of regenerative medicine. Currently, autologous or heterologous tissue and organ transplantation are the most commonly used treatments; however, they are hindered by limitations such as traumatic trauma treatment, insufficient donor availability, and immune rejection. Tissue engineering research focuses on three core elements: cells, biomaterials, and tissue construction technology [1,2]. The realization of tissue and organ reconstruction relies on obtaining an adequate number of seed cells with specific biological activities, combining them with suitable biological scaffold materials, and employing precise construction techniques to create personalized physiological structures and functional organs or tissues in vitro. Therefore, personalized tissue engineering holds immense potential for advancements in the field of tissue repair and replacement. 3D printing technology offers rapid personalized manufacturing capabilities that can greatly contribute to tissue repair [3-5].Digital light processing (DLP)-based 3D printing utilizes a digital microscope device (DMD) to project images onto photosensitive liquid resin or bioink through surface exposure, enabling layer-by-layer solidification for printing purposes. DLP- based 3D printing technology enables the creation of personalized biological structures with precisely designable geometries, effectively bridging the structural disparities between engineered and natural tissues. It has found extensive applications in fabricating constructs such as artificial skin, bone implants, and nerve conduits. In comparison to other printing methods such as extruded and inkjet 3D printing, DLP-based 3D printing offers significant advantages in terms of resolution, efficiency, and working conditions. Its non-direct contact approach provides relatively gentle conditions for cell printing.

The development and application of printing ink is an important research connotation of dlp-based 3D printing technology. Conducting research and developing innovative bioinks that strike a balance between printability, biocompatibility, and mechanical performance is crucial for the advancement of bioprinting applications. GelMA, a photocrosslinkable hydrogel derived from gelatin modified with methacrylic anhydride, exhibits exceptional biocompatibility, visible light curing capabilities, and controllable mechanical properties, rendering it a versatile ink for printing purposes. During the printing process, GelMA is typically combined with cells, growth factors, and other bioactive substances to leverage 3D printing in fabricating tissue engineering models with specific structures and functionalities. This paper primarily presents state-of-the-art technologies and applications involving DLP-based 3D printed GelMA constructs for bone repair, skin repair, and peripheral nerve repair (Figure 1).

Figure 1: Application of DLP printing technology in tissue repair

DLP-Based 3D Printing

Based on the continuous application of DLP-based 3D printing in tissue engineering, higher printing accuracy and faster printing speed are required for practical implementation of DLP technology. Constant advancements in new printing technologies based on DLP technology are being made [6,7].

John et al. have designed a rapid continuous printing process based on DLP technology, which creates an oxygen-containing “dead zone” between the forming section and liquid precursors to enable continuous exposure printing and significantly enhance DLP printing speed [8]. Callum et al. have developed a dynamic interface printing technique based on DLP that utilizes sound modulation and restricted gas-liquid boundaries to generate centimeter-scale 3D structures within tens of seconds. This approach does not require complex feedback systems, specialized chemical reagents, or intricate optical elements while maintaining rapid printing speed and exhibiting immense potential for high-resolution and biocompatible applications {Vidler, 2024 #6}. Brett et al., on the other hand, have developed Computed Axial Lithographic (CAL) printing technology based on DLP where materials can achieve volumetric polymerization of arbitrary geometric shapes. Compared to traditional layer-by-layer printing methods, CAL’s polymerization approach eliminates the need for support structures, is suitable for high-viscosity fluids as well as solids, and significantly enhances print speed{Kelly, 2019 #4}. Regehly et al. employed dual-color photopolymerization in DLP-based 3D printing, wherein photoswitchable photoinitiators were developed. This technique utilizes two intersecting light beams of different wavelengths to achieve complete object fabrication by inducing localized polymerization within a confined monomer volume, resulting in enhanced speed and resolution for 3D printing. Recent advancements in DLP-based 3D printing have primarily focused on improving print speed, with various novel techniques offering additional technological approaches for bioprinting applications. In the realm of biological 3D printing technology, the choice of bioink plays a crucial role; currently, commonly utilized bioinks include alginate series bioinks, collagen bioinks, and GelMA materials [9]. CAL technologies introduce a printing paradigm shift because they are able to create entire objects at a time, rather than by adding basic building blocks in sequence. The study of Bernal et al. introduced the concept of volume bioprinting (VBP), which can manufacture an entire carrier cell structure of arbitrary size and structure in a time range of several seconds to tens of seconds. Using GelMA as printing material, bone scaffold and meniscus containing were constructed by volume printing technology. The rapid manufacturing advantage of CAL technology in bioprinting is verified [10].

GelMA

GelMA exhibits excellent biocompatibility and promotes cell adhesion and proliferation through its biological motifs, particularly the RGD sequences on gelatin molecules. The crosslinked network structure of GelMA not only serves as a scaffold for cells but also facilitates material exchange{Yue, 2015 #56} [11-13]. By adjusting the substitution degree of MA in GelMA, its mechanical properties such as viscosity, compressive strength, and tensile strength can be tailored to meet specific printing requirements. Additionally, for tissue repair applications, it is crucial that bioprinted structures are biodegradable. GelMA’s matrix metalloproteinase (MMP) site allows it to serve as an enzymatic degradation site recognized by injured organism cells [14]. As native cells fill the GelMA construct, they initiate its degradation while simultaneously repairing the area with their own cells to achieve tissue regeneration. With its exceptional biocompatibility, tunable mechanical properties, degradability, and photoinduced cross- linking capability, GelMA holds great potential for personalized and customized engineered living tissues or scaffolds for tissue repair [15- 18].

Applications of DLP-based 3D Printing Using GelMA

Tissue engineering aims to enhance or replace biological functions for the purpose of repairing damaged tissues and organs. DLP bioprinting enables precise distribution of cells, materials, and biological factors, offering a novel approach for fabricating artificial tissues. DLP-based 3D printing exhibits remarkable advantages in terms of resolution, efficiency, and operational conditions; moreover, its indirect contact formation method provides relatively mild conditions for cell printing. GelMA and its composite hydrogel system are exceptional biomaterials for tissue engineering due to their excellent biocompatibility, tunable mechanical properties, and degradability. The utilization of GelMA in DLP-based 3D printing holds great promise in various tissue repair applications including bone regeneration, skin reconstruction, as well as peripheral nerve restoration.

Bone Repair

The incidence of bone defects caused by trauma, infection, tumors, and congenital or metabolic diseases has significantly increased. These defects often result in functional disabilities and deformities, posing a clinical challenge for treatment. Traditional autologous and allogeneic bone transplantation methods have several limitations including inadequate tissue supply, donor site damage, immune rejection risks, and potential infections. Therefore, the development of new strategies for bone defect repair has become an urgent problem to be addressed [19]. On one hand, 3D printing enables personalized customization for individualized precision treatment; on the other hand, it reduces the cost associated with large-scale preparation [20]. The architecture of the bone repair scaffold can affect the effect of cell-induced regeneration of damaged bone. Song et al. combined bionic microporous GelMA/SilMA with hydroxyapatite (HAp) to prepare a bionic microporous GelMA/SilMA/HAp ink. By utilizing DLP-based 3D printing, they fabricated layered bionic microporous GelMA/SilMA/HAp (M-GSH) scaffolds. Animal experiments demonstrated that these bionic microporous scaffolds significantly enhanced tissue integration and bone regeneration after 12 weeks of implantation. Tissue engineering scaffolds for repair require not only adequate structural stability, but also good biocompatibility [21]. Gao et al. successfully developed GelMA/PEGDA/F127DA composite hydrogel scaffolds by DLP-based 3D printing, which not only facilitated cell adhesion and proliferation but also effectively promoted osteogenic differentiation of mesenchymal stem cells in osteogenic inductive environments. Intramembranous ossification and endochondral ossification are two ways of bone regeneration, in which hypoxia-inducing factor-1 α (HIF-1 α) signaling pathway can promote endochondral ossification and angiogenesis [22]. Gao et al. DLP-based 3D printing to fabricate structurally robust and biocompatible GelMA/PMAA hydrogel scaffolds, capable of chelating iron ions and continuously activating the hypoxia-inducible factor- 1α (HIF-1α) signaling pathway, thereby promoting endochondral ossification and angiogenesis processes as well as late-stage vascular formation and bone remodeling. The balance between cell survival environment and hydrogel cross-linking density is crucial for light sensitivity to achieve high intensity and good cell viability in cell- borne cartilage repair materials [23]. Shen et al., employing GelMA/ SG as bioink, created high-precision networked DLP printed scaffolds with superior shape retention compared to GelMA alone. In vitro experiments exhibited significant chondrocyte proliferation, while ectopic cartilage formation was evaluated by subcutaneously implanting the GelMA/SG scaffolds in nude mice. The utilization of these scaffold materials along with their manufacturing strategy offers potential solutions for future clinical challenges in cartilage repair. “Sr” is a potent anti-osteoporotic agent with anti-resorptive and anabolic properties, but with side effects when applying systemic administration. GelMA-SR doped nanosized hydroxyapatite (SrHA) composite hydrogel scaffold with controllable Sr delivery capability [24]. Cosmin et al. DLP-based 3D printing developed a novel GelMA- strontium-doped nanohydroxyapatite (SrHA) composite scaffold for bone tissue regeneration featuring controllable strontium (Sr) release capabilities. They investigated the biocompatibility of the composite hydrogel scaffold. In vitro cell culture demonstrated that osteoblasts could adhere and proliferate on the surface of the hydrogel. The DLP- prepared GelMA composite hydrogel tissue engineering scaffolds offer innovative avenues for research in bone loss repair [25].

Skin Repair

The prevalence of burn and skin ulcer patients is significant, with chronic skin ulcers having a severe impact on their quality of life. Therefore, there is a great need for the development of scaffolds or biomimetic skin through tissue engineering. By utilizing 3D printing techniques that combine human keratinocytes, fibroblasts, and endothelial cells, rapid preparation of vascularized microstructures and perfused skin grafts can be achieved. DLP-based 3D printing provides a rapid manufacturing method to curing human skin fibroblast (HSF) and human umbilical vein endothelial cell (HUVEC) hydrogel material to form biomimetic skin. Zhou et al. utilized a biomimetic bioink (GelMA/HA-NB/LAP) and DLP-based 3D printing to fabricate functional living skin (FLS). FLS possesses interconnected microchannels that facilitate cell migration, proliferation, and new tissue formation. By mimicking the physiological structure of natural skin, FLS promotes skin regeneration and neovascularization. The three-dimensional (3D) artificial skin model provides a variety of platforms for testing skin transplantation, disease mechanisms and skin tissue. However, achieving physiological complexes in such hierarchical structures, such as the neurovasculature with living cells, is extremely difficult [26]. Choi et al. DLP-based 3D printing printed a full-thickness skin model by employing methacryloyl-modified silk fibroin (SFMA) and GelMA, incorporating multiple cell types. They evaluated the printability, mechanical properties, and cell viability of SFMA/GelMA construct at various concentrations in order to determine the optimal printing concentration for artificial skin models. The simulation model confirmed that epidermal growth factor could enhance wound healing in both epidermal and dermal layers [27].

Peripheral Nerve Repair

Peripheral nerve injury is a significant cause of disability, often resulting in motor and sensory impairments. Treating peripheral nerve injuries poses a major technical challenge in the fields of reconstructive surgery and regenerative medicine. Utilizing DLP-based 3D printing, the rapid fabrication of personalized nerve conduit shows promise as an effective method to enhance peripheral nerve healing.The nerve conduit is a promising treatment for long-gap peripheral nerve injury, but with limited efficacy. Drug-releasable scaffolds may provide a reliable platform to construct a regenerative microenvironment for neural recovery. Tao et al. A functional nanoparticle-enhanced nerve conduit for promoting peripheral nerve regeneration was prepared by DLP-Based 3D Printing. The conduit consists of a gelatin-methylacryanyl (GelMA) hydrogel and drug-loaded poly (ethylene glycol) -poly (3-caprolactone) (MPEG-PCL) nanoparticles dispersed in the hydrogel matrix. Such nanoparticles in the conduit can release Hippo pathway inhibitors to promote nerve regeneration and functional recovery [28]. Zhang et al. fabricated a biodegradable self-adhesive bandage by utilizing a series of clickable functionalized monomers, including azide-modified gelatin methacryloyl and dibenzylcyclooctyne-modified GelMA. This bandage possesses the capability to envelop injured nerves and selectively release drugs for neural repair purposes. Through electrophysiological assessment and histological examination using a rat sciatic nerve transection model, it was confirmed that the drug-loaded self-adhesive bandage developed by Zhang et al. effectively promotes peripheral nerve regeneration and facilitates recovery [29]. Zhang et al. A double-branched GelMA neural conduit was constructed by DLP-based 3D printing .The efficacy of the double-branch nerve conduit was evaluated by the transfer of the rat tibial nerve to the peroneal nerve. The results of functional and histological evaluation showed that the double-branch nerve conduit could not only promote the regeneration and functional recovery of the peroneal nerve in the injury, but also retain the function of the donor nerve, demonstrating the potential application of this conduit in nerve transfer [30]. Wu et al. successfully employed continuous DLP printing to manufacture elastic hydrogel conduits encapsulating nanodrugs, utilizing GelMA/methacryloyl-modified silk fibroin (SFMA) composite hydrogels. This material exhibited favorable effects on cell adhesion, proliferation, and migration. Electrophysiological, morphological, and histological evaluations conducted through animal experiments demonstrated that the conduit effectively promoted axonal regeneration, myelin sheath regeneration, and functional recovery by providing an optimal microenvironment. Peripheral nerve injury is a common condition that often causes disability and poses challenges to the surgeon. Drug-releasable biomaterials provide a reliable tool for regulating the nerve healing-related neurorepair microenvironment. A self-adhesive bandage was designed to form parcels around the injured nerve to promote nerve regeneration and recovery [31].

Conclusion

DLP-based 3D printing technology offers significant advantages in resolution, efficiency, and working conditions. Its non-direct contact forming method provides relatively gentle conditions for cell printing, making it a promising construction technology for tissue engineering. GelMA composite hydrogels possess excellent biocompatibility, adjustable mechanical properties, and good formability, presenting enormous application prospects in tissue repair. By adjusting GelMA’s substitution rate and compositing with other materials, hydrogel systems can be prepared to meet different application requirements. However, prior to the clinical implementation of GelMA construct, it is imperative to address concerns such as standardizing GelMA synthesis methods and ensuring the safety of photoinitiators and hydrogel materials. Additionally, material properties should be expanded to meet more complex clinical demands such as controlled degradability of GelMA materials. For DLP printing technology and equipment to fully realize its personalized advantages in 3D printing applications like bedside printing technologies need further development.

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Superwetting Materials with Different Dimensions are Used in the Study of Oil-Water Separation

DOI: 10.31038/AFS.2024613

Introduction

With global warming, the shortage of water resources is aggravated, and a large amount of oily wastewater produced by the petroleum industry poses a threat to the ecological environment. The traditional oil-water separation methods have some problems, such as low efficiency, long cycle, complicated operation and secondary pollution. The appearance of ultra-wetting materials has brought new hope for solving these problems. This paper focuses on the application of ultra-wetting materials in the field of oil and water separation, introduces the limitations of traditional separation methods, expounds the research progress of ultra-wetting materials, wetting theory, and discusses various types of ultra-wetting materials in detail, and finally summarizes the shortcomings of existing materials and looks forward to the future.

Superwetting Oil-Water Separation Material

Super Hydrophobic – Super Oil Wet Material

Summarizing this type of material, compared with the previous two, its three-dimensional structure is equivalent to a random stacking of multi-level two-dimensional materials, which significantly enhances separation efficiency. Additionally, small oil droplets undergo demulsification, coalescence, and separation within the internal space, thereby achieving emulsion separation and efficient “dewatering”. However, for most non-metallic substrates, further research is needed to explore and improve their mechanical stability, reusability, and durability.

Super Hydrophilic – Underwater Super Oil Phobic Material

In summary, in the realm of superhydrophilic–underwater superoleophobic three-dimensional materials, there still exist a series of application issues such as structural instability, poor overall wear resistance, and susceptibility to contamination in complex environments. These issues serve as inspirations for targeted solutions in subsequent development efforts. Additionally, practical applications of the material are often limited by factors such as synthetic preparation methods. Nevertheless, overall, these “oil-removing” type three- dimensional wetting materials exhibit higher emulsion separation performance compared with two-dimensional materials.

Superhydrophobic – Superoleophobic Materials

According to the previous discussion, three-dimensional materials can utilize porous nickel foam substrates, directly forming micro-nano rough structures on the three-dimensional framework. Compared with two-dimensional materials where particles adhere to the surface and are combined with binders, this approach offers superior performance and a more efficient preparation process. For non-metallic substrate materials, the three-dimensional structure provides sufficient space to accommodate inorganic hybrid polymers, forming a unique porous structure and significantly enhancing their mechanical strength.

“Special” Ultra-Wetting Materials

Compared with two-dimensional materials, three-dimensional “special” superwetting materials exhibit enhanced water absorption capabilities. Additionally, the selection of these materials increasingly considers the application of green, biodegradable materials. To address common oil–water separation clogging issues, porous superamphiphilic materials offer a fundamental solution, with aerogel materials selectively capturing the water phase to improve separation performance.

Smart Switchable Superwetting Material

Two-dimensional switchable superwetting materials focus on reusability and recyclability while conserving energy. They achieve on-demand emulsion separation under external stimuli, effectively addressing the single-use issue of one dimensional materials. Additionally, the in situ growth method overcomes the stability problems common in most superwetting materials.

Summary and Outlook

In the future, green and biodegradable base materials have enormous development potential and prospects. The green recycling of materials for renewable use is a key direction for future research. However, current recycled materials still exhibit some apparent disadvantages: cumbersome and complex recycling processes, high energy input, poor durability, and a short lifespan during use. The future aims to combine repairability, self-cleaning, high corrosion resistance, and material recycling to form a completely new industrial chain and breakthrough direction in technology development.

Does Water Immersion Have a Role in Cord BDNF Levels and Neurological Development of the Baby: A Case Control Study

DOI: 10.31038/IGOJ.2024713

Abstract

Background: Brain-derived neurotrophic factor (BDNF) levels in maternal serum and umbilical cord blood serum samples from women who underwent water immersion during labor and those who gave birth without water immersion were compared.

Objectives: This study aimed to investigate the impact of water immersion on maternal and neonatal serum BDNF levels. A total of 57 pregnant women were included in the study, 32 in the non-water immersion group and 25 in the water immersion group. Serum BDNF levels were measured by ELISA an Enzyme-Linked ImmunoSorbent Assay (ELISA). For comparisons between groups, the independent samples t-test, Mann-Whitney U-test, and Spearman rho correlation test were used.

Results: No differences were observed in age, gravidity, parity, maternal BMI, infant weight, and infant sex (p=0.97, p=0.61, p=0.71, p=0.24, p=0.14, and p=0.88, respectively). There was no difference in maternal serum BDNF levels between the two groups were compared (p=0.152). Cord blood BDNF levels were found to be significantly different in the water immersion group compared to the conventional vaginal delivery group (p=0.03).

Conclusions: The high BDNF levels in the water immersion group suggest that this method may contribute to the neurological development of infants. We believe that water immersion can have a positive effect on the psychology of mothers and their babies.

Keywords

Hydrotherapy, Brain-derived neurotrophic factor, Immersion in water, Neurological development

Introduction

Hydrotherapy and water immersion are long-standing therapeutic techniques used in medicine. Thus, the popularity of this method has increased. Water immersion has become widely used worldwide as a non-pharmacological method for reducing the stress of labor pain [1-3].

Although water birth and hydrotherapy (water immersion or immersion in water) are thought to be similar techniques, they are different. While hydrotherapy is a non-pharmacological method to cope with labor pain, water birth is the use of hydrotherapy in the second stage of labor, and as the delivery takes place in water, it can be accepted as a birth method.

Studies have underscored the drawbacks of water birth, including the risks of maternal and neonatal infections as well as potential respiratory issues for newborns [3]. Water birth performed by skilled obstetric care providers in a hospital setting is a reasonable option for low-risk women and their newborns. In a recent study, the water immersion group demonstrated lower rates of neonatal intensive care, special care nursery admission, and perineal laceration than the control group. Furthermore, this review provides additional information on immersion water [4-6].

The benefits of water immersion for pregnant women are apparent. Owing to the buoyancy of water, hydrotherapy enables pregnant women to move their legs more easily. It has been proposed that water immersion during labor enhances maternal satisfaction and a sense of control [1].

Women who sense control during childbirth tend to have enhanced emotional well-being postnatally [1]. It is also known that immersion in water significantly reduces the anxiety of pregnant women [2]. Some professional associations, such as the Royal College of Obstetricians and Gynecologists and the American College of Nurse-Midwives, support uncomplicated healthy pregnant women having water births [7].

BDNF is a protein belonging to the neurotrophin subfamily that has various effects on the central nervous system. Neurotrophins is a crucial intracellular factor that contributes to the maintenance of neuronal function. BDNF forms neurotrophins, which are very important in protecting the nervous system and neuronal structure. BDNF plays a role in the differentiation of cells into neurons in the neural root during development by preventing neuronal death in cases such as trauma or ischemia in adult brain cells. It contributes significantly to the continuation of their vitality. In addition, BDNF has a significant impact on brain development in the prenatal and postnatal periods [8-10].

BDNF has been studied in mammals, mostly during the prenatal period and neurogenesis phase [8]. This stage is important because although neurogenesis is completed a few days before birth, most neurons in the hippocampus occur after birth [11].

We aimed to investigate the relaxing and pain-reducing effects of water immersion on maternal and infant cord serum BDNF levels.

Methods

Study Design

This prospective case-control observational study was carried out at the Zekai Tahir Burak Women’s Health Education and Research Hospital with the approval of the ethics committee (ethical approval statement: 58/2018) and in accordance with the Helsinki criteria. The women were divided into two groups: the control group consisted of 32 pregnant women who did not receive water immersion during labor and gave birth vaginally, and the case group consisted of 30 women who received water immersion during labor and gave birth vaginally.

Setting

We selected our study patients among full-term pregnant women between 37-40 weeks, hospitalized in the obstetrics clinic for delivery. Our study was conducted over a period of 6 months.

Immersion water was present in the special pools in the delivery room. Attention was paid to the cleaning of the pool. The bathtub was cleaned after each use as part of the precautions taken to prevent infection before immersion. First, the organic waste was removed and prewashed with running water. After preliminary cleaning with detergent and water, drainage pipes were cleaned and treated with chlorine tablets. The cleaning was completed after waiting for a certain period. Materials used in bathtubs, such as thermometers and hand dopplers, are also disinfected by surface disinfectants. After disinfection, cultures were collected from the bathtub surface, pool bottom, drain, and water flow areas. If the culture result is negative, the pregnant woman can be placed in a bathtub. All operations were performed by trained personnel.

Participants

A total of 62 pregnant women between the ages of 18 and 40 years, with pregnancies between 37 and 40 weeks, and who were in active labor during the examination, were included in our study. All pregnant women were in vertex presentation at the examination and had antenatal follow-ups in the maternity polyclinics of the same hospital. The amniotic membrane was observed to be intact during the examination. The exclusion criteria were as follows: pregnant women with a history of cesarean section, chronic disease, malpresentation, ruptured amniotic membrane, high-risk pregnancy, and medical and obstetric risks. Pregnant women with macrosomia fetuses and refugees on ultrasound who also had signs of active infection and fetal distress with bleeding were excluded from the study (Figure 1).

Figure 1: Inclusion and exclusion criterias.

The control group comprised of 32 patients with normal birth pain. No non-pharmacological or pharmacological pain relief methods were applied in this group. Labor was not induced.

Pregnant women in the case group (n=30) with cervical dilatation of 3 cm and 70% were taken to a pool, which had a temperature of 37–37.5⁰C and was wide enough for the woman to move freely. Fetal heart monitoring was performed at regular intervals using Doppler or non-stress tests (NST). The second stage of labor was carried out in a controlled manner outside the water. In both groups, as soon as the baby was born, it was placed on the mother’s womb, the cord was cut, and the delivery of the placenta and membranes was completed.

Venous blood samples were obtained from the mothers after birth. After the cord was clamped, blood samples were collected.

Measurements

Serum samples were separated by centrifugation at 5000 revolutions/min (2236 × g) for 10 min within 15–20 min of blood sampling. They were frozen immediately and stored at -80⁰C until the final analysis.

Serum BDNF levels were measured using an ELISA. The BDNF concentration was determined using the Elab Science Human BDNF ELISA kit (Elabscience Biotechnology Inc., Wuhan, China), which had a sensitivity of 18,75 pg/mL. The sandwich principle was used for the ELISA kit. The micro-ELISA plate provided in this kit was precoated with an antibody specific for human BDNF. Assays were performed according to the manufacturer’s instructions as follows: standards and samples were added to the micro-ELISA plate wells and combined with the specific antibody. Then, a biotinylated detection antibody specific for human BDNF and an avidin-horseradish peroxidase (HRP) conjugate were added to each microplate well and incubated. The free components were then washed away. The substrate solution was then added to each well. The enzyme-substrate reaction was terminated by the addition of a stop solution. The optical density (OD) was measured spectrophotometrically at a wavelength of 450 ± 2 nm. The OD value is proportional to the concentration of human BDNF. Maternal serum and infant cord blood BDNF levels (pg/ml) were recorded.

Bias

The case and control groups were selected from patients with the same characteristics. In contrast, only hydrotherapy was administered in the patient group. Five of the 30 patients in the case group were excluded from the study because their blood samples were damaged during transport. The number of cases had decreased to 25.

Statistical Analyses

Study Size

The mean standard deviation of BDNF in women giving birth in water was predicted to be 1200 ± 290 pg/ml. Thus, the effect size was calculated to be 0.741. With an alpha of 0.05 and power of 0.80, the sample size was determined to be 60 people in total, with at least 30 people in each group.

Variables

After birth, the weight and sex of the infants were recorded. In this study, age, gravidity, parity, BMI, baby weight, baby sex, and BDNF levels were compared between the two groups.

Statistical Methods

Whether the variables with numerical results in the study were normally distributed was examined using the Shapiro–Wilk test and graphs (histogram, boxplot, etc.). Normally distributed variables, such as age, infant weight, and BMI, were compared between the two groups using an independent sample t-test. While performing the independent samples t-test, Levene’s test was used for the equality of variances. The distribution of other numerical variables that did not show a normal distribution was compared between the two independent groups using the Mann-Whitney U test, which is a nonparametric test. Descriptive statistics are given as the mean standard deviation for numerical variables compared with parametric tests and median (min-max) for nonparametric tests. Sex, which is a categorical variable, was compared between the groups using the Pearson Chi-Square test. Yates’ correction was not used, and descriptive statistics for this variable are given as numbers and percentages. The relationships between numerical variables were analyzed using Spearman’s rho correlation coefficients. Statistical significance was set at p < 0.05. Analyses were performed using SPSS IBM Statistics 23.0 Program.

Of the 62 pregnant women included in the study, 32 were in the control group and all were considered suitable until the end of the study. As the serum samples of 30 pregnant women in the case group were collected during transportation, 25 were evaluated.

Results

Descriptive Data

When the demographic data of the two groups were examined, no differences were observed in age, gravidity, parity, maternal BMI, infant weight, and infant sex (p=0.97, p=0.61, p=0.71, p=0.24, p=0.14, p=0.88, respectively) (Table 1).

Table 1: Comparison of demographic characteristics, clinical features and BDNF levels of umbilical cord, and maternal serum between the control and case groups.

Variables

The control group (labor without immersion-no hydrotherapy) The case group (labor immersion in water-hydrotherapy)

P-value t/df/z/x²

Frequency

32

25  

Age (years) Mean ± SD

26 ± 5.5 26 ± 5.8

0.979

t: -0.027

df: 55

Gravida Mean ± SD (Median (Min-Max)

2.03 ± 0.7

2(1-4)

2.16 ± 0.8

2(1-4)

0.613

z: -0.505

Parity Mean ± SD (Median (Min-Max)

0.94 ± 0.6

1(0-2)

1 ± 0.6

1(0-2)

0.711

z: -0.370

BMI kg/m2 Mean ± SD

28.76 ± 4.7

27.49 ± 2.9 0.249

t: 1.166

df: 55

Baby weight kg Mean ± SD

3.304 ± 447 3.460 ± 329

0.149

t: -1.063

df: 55

Baby gender male/female n (%)

Male: 16 (50%)

Female: 16 (50%)

Male: 13 (52%)

Female: 12 (48%)

0.881

x²: 0.022

df: 1

Maternal serum BDNF levels pg/ml Mean ± SD

(Median (Min-Max)

110.07 ± 79.89

85.49(39.16-359.68)

130.83 ± 79.44

113.79(13.90-332.57)

0.152

z: -1.431

Umbilical cord serum BDNF levels pg/ml Mean ± SD

(Median (Min-Max)

160.47 ± 82.31

137.81(66.72-415.49)

226.48 ± 128.44

168.20(84.99-48.204)

0.033* z: -2.127

P-value <0.05 is considered as statistically significant.
BMI: Body mass index; BDNF: Brain derived neurotrophic factor; SD: Standard deviation.Min-Max: Minimal and maximal value.
An independent samples t-test (with t and df) was used to compare age, baby weight, and BMI. While performing the Independent samples t test, Levene’s test was used for equality of variances.
The Mann-Whitney U test (with p and z values) was used to compare gravida, parity, and BDNF.
The Pearson Chi-Square test (with value (x²) and df) was used to compare the gender distribution of babie. Yates’s correction was not us.

Outcome Data

When the maternal serum BDNF levels were analyzed, no statistically significant difference was observed between the two groups (p=0.152). However, cord blood BDNF levels were significantly different between the case group compared with the control group (p=0.03).

Neither maternal serum nor cord serum BDNF values differed according to sex (p=0.861 and p=0.718, respectively). A statistically weak but significant correlation was found between infant weight and maternal serum BDNF level (p=0.004; r=0.371). There was also no relationship between baby weight and cord BDNF level (p=0.642; r=0.063) (Table 2).

Table 2: Maternal and umbilical cord serum BDNF levels acccording to baby gender.

Variables

Female n=28 Male n=29 P-value

Maternal serum BDNF levels pg/ml Mean ± SD

(Median (Min-Max)

126.77 ± 92.05

87.03(39.16-359.68)

111.85 ± 66.38

96.95(13.90-312.64

0.861

z: -0.176

Umbilical cord serum BDNF levels pg/ml Mean ± SD

(Median (Min-Max)

189.94 ± 117.05

191.84 ± 104.02

0.718

z: -0.361

BDNF: Brain Derived Neurotrophic Factor; SD: Standard deviation; Min-Max: Minimal and maximal value.
The Mann-Whitney U test (with p and z values) was used to compare BDNF.

Discussion

Immersion in the waterbirth method provides many benefits in the form of maternal satisfaction, pain control, and easy movement in water. In this method, pain is reduced by hydrotherapy by taking pregnant women into the pool during labor, but birth takes place outside the pool. On the other hand, during a water birth, the pregnant woman is taken into the water during labor, and the birth takes place in the water. Both methods have been found to be beneficial for the emotional comfort of postpartum mothers [1]. However, studies on the neonatal benefits of these methods are limited. There are selected studies on immersion in water and the absence of fetal side effects during birth [4,12].

This study was designed to investigate the potential benefits of water immersion. The advantages of water immersion during labor or birth encompass reduced pain, expanded functional diameter of the true pelvis, improved quality of contractions, heightened release of endorphins, diminished reliance on opiates, increased mobility for the mother, and enhanced positioning during various stages of labor [13]. We aimed to investigate whether hydrotherapy has an increasing effect on maternal and infant cord BDNF values, and whether hydrotherapy has a positive neurohormonal effect. We compared BDNF, a neurotrophic factor in the serum of maternal and infant cord blood, in hydrotherapy and conventional vaginal delivery and found that maternal serum BDNF levels were not different. Although there was no statistical difference between maternal serum BDNFs levels, the mean values in the hydrotherapy group were significantly higher. This may be because of the small sample size. Despite this, cord serum BDNF levels were significantly higher in the hydrotherapy group.

Neurotrophins are important regulators of neural cell survival, development, function and plasticity. Mammals have four neurotrophins that are derived from the same ancestral gene [26]. Neurotrophins support neuron survival and prevent neuron apoptosis [14].

Neurotrophins play an important role in axon growth during development, higher neuronal function, morphologic differentiation, and neurotransmitter expression [15]. Thus, neurotrophins can play an important role in the development of the brain before and after birth. However, data on the presence and effects of neurotrophins in preterm infants are insufficient. BDNF and NT-3 are highly expressed in the cortical and hippocampal structures and have been linked to the survival and function of multiple neuronal populations [16].

BDNF was found to be related to hypoxic-ischemic encephalopathy, mental retardation, and autism in newborns. The importance of BDNF and NT3 in neurodevelopment in the intrauterine period has been emphasized. There is evidence that prenatal or maternal traumatic stress has a significant impact on neurodevelopment. In general, the earlier and more severe the trauma, the more impaired the neurodevelopment [17]. The better the mother’s comfort during delivery, the easier the mother’s adaptation to the mother’s puerperium, and the lower the rate of postpartum depression. Therefore, we believe that immersion in water may be beneficial to neurological development.

BDNF is important in neuronal plasticity [18]. BDNF has mostly been studied in mammals during the prenatal and neurogenesis stages, but relatively less in the postnatal period [8]. This phase is important because, although neurogenesis is completed several days before birth, most neurons in the hippocampus appear after birth [11]. In addition, since BDNF is very important in mammalian adults, our research goal was to investigate whether BDNF changes depending on the mode of delivery.

In the water immersion group, high BDNF levels in the cord blood, but not in the mother’s blood, may contribute positively to the neurological development of the newborn. Moreover, Kodomori et al. showed in their animal study that maternal BDNF contributes to the neurological development of the fetus through uteroplacental passage. In our study, high BDNF levels were detected in the cord blood of rats in the water immersion group. In the perinatal period, the blood-brain barrier is immature because circulating BDNF may reflect the level of BDNF in the central nervous system, and circulating cortical BDNF levels are correlated, as has been reported. Again, in previous studies, conditions such as surgery, stress, birth, and hypotension that cause stress in the central nervous system have been found to cause changes in BDNF release. Accordingly, since hydrotherapy is a less painful and emotional form of delivery, higher BDNF values were obtained in our study group [18-20].

We also investigated the relationship between infant weight and BDNF levels and found only a weak link between maternal serum BDNF levels and infant weight. All the infants included in this study were term. In a recent animal experimental study [21], the relationship between infant sex and BDNF was investigated, and it was shown that BDNF content increased in the brains of both male and female rat pups 0 h after hypoxia and 4 h in serum; however, only males had increased brain BDNF levels 4 h after hypoxia. When we investigated the relationship between baby sex and BDNF levels, we did not find any difference in BDNF levels between the sexes. This may be due to the small number of patients. As this subject has been extensively researched and the importance of BDNF and other neurohormones is increasing, more long-term studies are needed.

Recent studies have investigated the use of serum BDNF levels in Alzheimer’s disease and as a biomarker of schizophrenia and depression. We aimed to examine serum BDNF levels because we believe that hydrotherapy has positive effects on maternal psychology and protects against the development of postpartum depression. As hydrotherapy has positive effects on maternal psychology and postpartum depression, we examined serum values [21-23].

Neurotrophic factors play crucial roles in neuroprotection. Neurotrophins promote survival and reduce apoptosis in many populations of neurons [14].

Limitations of the Study

The small number of participants in our study and the fact that we did not follow mothers and babies in the long term may be a limitation of our study.

Conclusions

In light of these studies, the neuroprotective effects of neurotrophins, especially BDNF, including anti-apoptotic axonal development of neurons, neurodevelopmental effects that have healing effects in some neurodegenerative diseases, and their positive effects in diseases such as autism and mental retardation, have increased the importance of BDNF in recent years. The relationship between BDNF, other delivery modes, and hydrotherapy has not been previously studied. Although we have shown in our study that hydrotherapy delivery may have a positive effect on BDNF levels, we believe that immersion in water contributes to the development of neurons in newborns by increasing BDNF levels. We hope that our study will encourage future research on this very important subject and will shed light on future studies on this very important subject.

Declarations

  1. Conflict of interest: Not applicable
  2. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
  3. Ethical approval: Ethical approval was received from the Zekai Tahir Burak Women’s Health Education and Research Hospital with the approval of the ethics committee (ethical approval statement: 58/2018).
  4. Consent to Participate: All participants provided written informed consent prior to their participation in the study in accordance with the tenets of the Declaration of Helsinki.
  5. Authors’ Contributions:
    Conceptualization: Rahime Bedir Findik
    Investigation: Rahime Bedir Findik, Ozlem Uzunlar, Esin Merve Erol Koc
    Methodology: Rahime Bedir Findik, Ozlem Uzunlar
    Resources: Rahime Bedik Findik, Ozlem Uzunlar, Esin Merve Erol Koc
    Validation: Rahime Bedir Findik, Ozlem Uzunlar
    Supervision: Yaprak Ustun
    Writing – original draft: Rahime Bedir Findik, Ozlem Uzunlar
    Writing – review & editing: Rahime Bedir Findik, Ozlem Uzunlar
    Formal analysis: Jale Karakaya, Gulsen Yilmaz, Fatma Meric Yilmaz Mert
    All authors have read and agreed to the submitted version of the manuscript.
    1. Data availability: Data are however available from the authors upon reasonable request and with permission from [third party name].

    Abbreviations

    BDNF: Brain-Derived Neurotrophic Factor; ELISA: Enzyme- Linked ImmunoSorbent Assay; OD: The Optical Density

    References

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Evaluation of In Vitro Cell Viability and Cytokine Production by Mesenchymal Stem Cells Exposed to the Homeopathic Medicine Matricaria chamomilla D3

DOI: 10.31038/IJVB.2024823

Abstract

Chamomilla has long been recognized in traditional medicine for its established uses in herbal medicine and homeopathy. It is commonly recommended for treating respiratory, hepatic, gastrointestinal, and mental disorders. Additionally, it exhibits sedative, antiseptic, antiemetic, and anti-inflammatory properties and is frequently used to address issues related to teething in young patients. Despite its widespread use, scientific validation is essential to enhance the credibility of this medicine. In vitro studies offer a valuable approach for assessing the impact of homeopathic medicines on cellular functions, including cytotoxicity and cytokine secretion. Cell viability is typically evaluated through assays such as MTT, which measures cellular metabolic activity and provides insight into the proportion of viable cells following exposure to specific compounds. In the case of mesenchymal stem cells (MSCs) exposed to Matricaria chamomilla D3, the goal is to determine whether the homeopathic remedy affects cell survival or induces cytotoxicity. MSCs are known for secreting various cytokines that regulate inflammatory responses and promote tissue regeneration. Exposure to Matricaria chamomilla D3 may influence cytokine secretion, potentially altering the inflammatory response. This study evaluated the in vitro toxicity of injectable Chamomilla D3 in human mesenchymal stem cells, along with its potential anti-inflammatory effects, as evidenced by the reduction in the pro-inflammatory cytokine IL-8. The findings suggest that homeopathic Chamomilla D3 exhibits in vitro anti-inflammatory activity.

Keywords

Homeopathy, Vegetal, Complementary medicine

Introduction

Matricaria chamomilla, also known as chamomile, is a globally distributed plant [2]. It has a rich history in herbal medicine and homeopathy, with indications for treating various diseases [1]. Matricaria chamomilla is a versatile plant with multiple uses in folk medicine. It treats respiratory, hepatic, gastrointestinal, and mental alterations like stress and anxiety. It is also used as a sedative, antiseptic, and antiemetic, among others reported.

The phytochemistry, biological, and pharmacological properties of Matricaria chamomilla extracts are extensively characterized and systematically documented within herbal medicine. Its phytochemical composition encompasses over 120 bioactive compounds, including essential oils, terpenoids, and phenolic substances such as phenolic acids, flavonoids, and coumarins. These compounds impart a range of well-documented activities, including antioxidant, antibacterial, antifungal, antiparasitic, insecticidal, antidiabetic, anticancer, anti-inflammatory, antidepressant, antipyretic, anti-allergic, and analgesic effects [3].

According to the Homeopathic Medical Material, Matricaria chamomilla is indicated for various clinical manifestations such as irritability and hypersensitivity. Additionally, it may be relevant for treating otitis and diarrhea, especially in children, and for conditions associated with teething and gastrointestinal disturbances [4]. described effects such as anti-inflammatory and antispasmodic activities, among others, that support alleviating clinical symptoms. These effects may significantly improve the previously mentioned signs and symptoms [5].

The present study aimed to evaluate the anti-inflammatory activity of Matricaria chamomilla prepared according to the homeopathic pharmacopeia, specifically at a D3 potency and a concentration of 8 µL/mL. This evaluation focused on releasing the inflammatory cytokine Interleukin 8 (IL-8) and assessed the viability of healthy mesenchymal stem cells exposed to the medicine Matricaria chamomilla.

Materials and Methods

MTT Assay

The injectable homeopathic medicinal product Chamomilla D3 was tested on human mesenchymal stem cells (MSC) by the MTT test at a concentration of 8 µL/mL. Cell culture was performed in 75 cm2 flasks until reaching 80% confluence. Human mesenchymal stem cells in culture were trypsinized and distributed in a 96-well plate. After this process, the cells were incubated for 24 hours at 37°C in a 5% CO₂ environment. The test substance was prepared at 8 µL/mL and distributed into the designated wells. After 24-hour incubation, the culture medium was withdrawn and discarded. A volume of 50 μL of medium supplemented with 20% FBS was added, followed by 50 μL of medium containing the diluted test substance. The cells were incubated for an additional 48 hours in a CO₂ incubator, maintained at 5% CO₂ and a temperature of 37°C. After this period, the treatment medium was discarded, and 100 µL of the MTT solution was added to each well. The plate was covered with aluminum foil and incubated for 4 hours in an oven. Subsequently, MTT was removed, and 100 µL of DMSO was added to each well. The optical density was measured at 570 nm ± 10 nm using a plate reader. After this assay, the cytokine levels released upon exposure to the medicine were measured.

Cytokine Dosing

After reaching cellular confluence, cultured human mesenchymal stem cells were subjected to trypsinization and plated in 96-well plates. After 24 hours of incubation at 37°C with 5% CO₂, the culture medium was removed, and the wells were washed with PBS. Subsequently, 200 μg/mL of LPS (lipopolysaccharides from *Escherichia coli* O55: B5 – Sigma Aldrich), diluted in an antibiotic-free medium, was added in wells of positive control group and treatment group. Negative control group did not receive LPS, only culture medium. A subsequent incubation was performed for an additional 24 hours. Following the induction period of cellular inflammation with LPS, the medium was removed from the plate, and the wells were washed with PBS. The medicine was added at a final concentration of 8 μL/mL in treatment group. In negative and positive control groups, culture medium was added. The plate was then incubated for 24 hours under the previously described conditions.

After the treatment period, the supernatant was removed, and serum-free culture medium was added for 24 hours. The supernatant was collected for IL-8 analysis. IL-8 levels were measured using flow cytometry with the FACS VIA BD™ cytometer.

Results and Discussion

The present study assessed the cytotoxicity of the injectable homeopathic medicine Chamomilla D3 in human mesenchymal stem cells. The analysis revealed that, at the tested concentration, the material exhibited no cytotoxic potential (Table 1 and Figure 1).

Table 1: Cell viability obtained from the control and treated groups (Chamomilla 8 μl/mL) after MTT testing.

Cell viability(%)

Control

Chamomilla 8µl/mL
94

93

101

93
102

95

Figure 1: Cell viability (%) of the control and treated groups (Chamomilla 8 μl/mL): ns = no statistical difference.

Following the initial analysis, the release of the inflammation marker IL-8 by MSCs was evaluated both in response to LPS exposure and after treatment with the medicine. It was observed that LPS induced the release of IL-8, demonstrating its effectiveness in stimulating inflammation within the cell culture environment (see Figure 2). Additionally, cells previously “inflamed” by LPS were subsequently “treated” with the homeopathic medicine Chamomilla D3. The results demonstrated a significant reduction in the inflammation marker IL-8 following treatment. A similar pattern was observed in the control group, which consisted of cells maintained in a culture medium without LPS induction or Chamomilla D3 treatment (see Figure 2).

Figure 2: The results of the cytokine dosage test are presented in pg/mL for the following groups: the control group without LPS addition, the control group with LPS addition, and the treated group with Chamomilla at 8 μl/mL.

Homeopathy is frequently investigated due to its low likelihood of toxicity and minimal risk of causing side effects. According to Millstine [6], homeopathy can benefit the treatment of specific clinical conditions by potentially providing symptom relief.

Homeopathic Chamomilla is known for its anti-inflammatory effects, which can aid in treating various conditions [10]. Studies have demonstrated that the compounds in Chamomilla, such as flavonoids and terpenoids, possess properties that aid in reducing inflammation and alleviating related symptoms. As noted by Amsterdam [7], Chamomilla is also recognized for its calming and relaxing effects, which can contribute to alleviating stress and anxiety – factors often associated with inflammatory processes in the body.

Furthermore, regarding the anti-inflammatory properties of homeopathic Chamomilla, this article supports and validates the study by Scabello and Gardin [8], which examines injectable dynamized medicines available in Brazil. The authors noted that Chamomilla harmonizes the excessive action of the soul organization over the vital force, particularly within the digestive and menstrual spheres, and addresses general inflammation, per the principles of Anthroposophical Medicine.

In the homeopathic form, Chamomilla was identified as one of the ten most frequently used medicines for treating migraines, as highlighted and reviewed by Santos. Migraines are types of headaches that impair the patient’s quality of life.

Another property attributed to a medicine based on Matricaria chamomilla is its relaxation and analgesic effects. These effects were demonstrated in the study by Jyothis [9], which experimentally evaluated its impact on the central nervous system. As a result, a significant reduction in locomotor activity was observed, indicating muscle relaxation, analgesic effects, and anticonvulsant activity. Pinto [12] also reported relaxation effects in animals subjected to stress and depression.

The antibacterial and fungicidal actions of Matricaria chamomilla were emphasized in the study by, which explored its various aspects and properties. The study noted that its compounds impart sedative attributes, support digestion, and exhibit antimicrobial effects against bacteria and fungi [13].

Conclusion

The present study demonstrated the low in vitro toxicity of injectable Chamomilla D3 in human mesenchymal stem cells. Additionally, it suggested a potential anti-inflammatory action, as evidenced by a reduction in the levels of the pro-inflammatory cytokine IL-8. However, further studies are needed to confirm the homeopathic indications of its compounds in their homeopathic form and establish Chamomilla‘s in vivo anti-inflammatory activity.

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