Monthly Archives: August 2020

EDMJ-4-3-410-g002

Metabolomic Analysis Unfolds Connection of Obesity and Endometrial Cancer

DOI: 10.31038/EDMJ.2020433

Abstract

In recent years, obesity has been identified as a risk factor for several cancers including endometrial cancer. This paper identifies molecular mechanisms that are potential causes or effects of the correlation between obesity and endometrial cancer. From public sources, metabolites involved in endometrial cancer were extracted and analyzed with pathway analyses. Several elucidated pathways and mechanisms were involved with both obesity and cancer and were studied to better understand the relationship between obesity and endometrial cancer. These pathways include the Adipocytokine signaling pathway, Insulin resistance pathway, and Glucagon signaling pathway. The Adipocytokine signaling pathway showed a relationship between leptin, a body-weight regulating hormone, and the JAK–STAT3 pathway known to promote cancer cell proliferation. The Insulin resistance pathway shows insulin, an obesity-related hormone, activating the STAT3 protein as well as the PI3k–Akt and the mTOR signaling pathways, all of which can lead to increased cancer risk. The Glucagon signaling pathway gave evidence that glucagon may have anticancer properties as it activates the FOXO signaling pathway, which suppresses cancer cell growth. Metabolic syndrome as well as foods with high glycemic loads may hinder the anticancer potential of glucagon and lead to an increase in endometrial cancer risk. Such results can provide targets for reducing the risk or progression of cancer and provides information on the effects of certain lifestyles on endometrial cancer risk.

Introduction

Endometrial cancer is the cancer of the innermost lining layer of the uterus. Endometrial cancer is the most common cancer of the female reproductive organs. In 2020, the American Cancer Society projections for cancer of the uterus in the United States are 65 620 new cases and 12 590 deathsincluding 59 060 and 11 330 for endometrial cancer accordingly (https://www.cancer.org/cancer/endometrial-cancer/about/key-statistics.html, accessed February 17, 2020). Endometrial cancer has a variety of risk factors, such as advanced age, hormonal imbalance, and diabetes. Additionally, one of the most prevalent risk factors of endometrial cancer is obesity. In the US, an estimated 57% of endometrial cancer cases are directly attributable to being overweight or obese [1]. This correlation has been proven by several meta-analyses, such as the American Institute for Cancer Research’s report that for every increase of five Body Mass Index (BMI) units (kg/m2), there was a 50% increase in the risk of developing endometrial cancer [1]. Among the reasons for this correlation is the fact that fat tissue can alter hormonal balance and increase estrogen levels and is related to insulin resistance, all of which have been directly linked to increased risk of endometrial cancer. Furthermore, several mechanisms involved in obesity have been identified as risk factors for endometrial cancer. The identification of obesity-related mechanisms in endometrial cancer networks has shown the oncogenic effects of various pathways and proteins. Such biological functions within the body can activate cancer-causing pathways and genes or inhibit anticancer processes. The Akt signaling pathway, activated by leptin and other obesity-related hormones, was shown to have a correlation with endometrial cancer when cancer cell proliferation decreased as Akt was inhibited in Ishikawa cells [2]. Obesity-caused activation of the STAT3 protein through leptin and insulin resistance, increases endometrial cancer risk as it promotes induction of oncogenic genes such as c-Myc [3]. Insulin resistance, commonly associated with obesity, is related to activation of TNF and mTOR signaling pathways that promote endometrial cancer [4,5]. Obesity-related proteins and genes, such as c-Myc, TNF-ɑ, S6K, and eIF4E, contribute to cell proliferation, protein synthesis, and oncogenesis [6,7]. Other proteins and genes, such as FOXO1, have tumor-suppression properties [8]. Similar studies demonstrated that cancers of the breast, colon, rectum, esophagus, kidney, and pancreas have also been linked to being overweight or obesity (Figure 1) [9,10].

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Figure 1. Relationship of Body Mass Index and risk of endometrial cancer. Graph shows a positive correlation between BMI and endometrial cancer risk from several studies. Adapted from reference [10].

Materials and Methods

Approach Overview

Collected from public sources metabolite data were analyzed by Ingenuity® Pathway Analysis (IPA®, Santa Clara, CA; Krämer et al. 2014) [11] program. Created by the IPA program metabolite–gene networks were elucidated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) software and analyzed withGeneFriends package (Figure 2).

EDMJ-4-3-410-g002

Figure 2. Approach overview. Data from public sources are inputted to Ingenuity Pathway Analysis (IPA®) program and results are analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Analysis Tool yielding significant KEGG pathways. Then results analyzed by GeneFriends program to find co-expressed genes and cluster them.

Metabolite Selection and Analysis

Metabolites specific to endometrial cancer were selected from public sources: articles from PubMed and Google Scholar libraries, Human Metabolite Database (https://hmdb.ca/) and BiGG Models (https://bigg.ucsd.edu). To use significant metabolites only they were cut off by p-value and fold change.

Ingenuity® Pathway Analysis

Ingenuity® Pathway Analysis (IPA®; QIAGEN Inc., Redwood City, Calif., USA; Krämer et al. 2014; https://www.qiagenbioinformatics.com/products/ingenuity-pathwayanalysis) [11] is a web-based analysis software that allow to understand complex ‘omics data. Using a list of genes or metabolites, it allows identifying signaling and metabolic pathways, interaction molecular networks, and their functions.

David

The Database for Annotation, Visualization and Integrated Discovery (DAVID) is an online server that consists of the DAVID Knowledgebase and five integrated functional annotation tools for bioinformatics data. It allows, among other functions, functionally classify and annotate genes, convert gene IDs, search for interesting and related genes, highlight them on signaling and metabolic pathways, and view these genes dynamically.

GeneFriends

GeneFriends is a tool that creates a large co-expression map from a wide variety number of datasets. There are two variants to this tool: a dataset consisting of microarray datasets [12] and a dataset consisting of RNA-seq samples [13]. The RNA-seq map currently consists of 46 475 human samples and 34 332 mouse samples while the microarray map currently consists of 4164 human microarray datasets and 3571 mouse datasets. This tool is used to elucidate clustered co-expressed genes since genes that are activated simultaneously tend to share similar transcriptional regulation. Novel genes probably related to the disease of study can be elucidated based on a guilt-by-association approach when observed to be co-expressed with known candidate genes. The microarray version of the program GeneFriends was used to elucidate the genes coexpressed with our genes of interest using over 1000 microarray datasets [12].

Results

Metabolites Selection

Selected metabolites underwent two cutoffs:

• byp-value (p). Metabolites with p<= 0.05 considered as significant.

• by fold change (FC). To reduce noise, the metabolites with 1.33 < FC < 0.67 were used.

The list of metabolites selected is presented in Table 1.

Table 1: List of metabolites from endometrial cancer patients [14].

Metabolite

p-value

Fold change

1-Methylhistidine

0.952

-1.01

2-Hydroxybutyrate

<0.001

1.51

Acetic acid

0.296

-1.19

Betaine

0.988

1.00

L-Carnitine

0.386

-1.31

Creatine

0.214

-1.19

Citric acid

0.612

-1.16

Choline

0.443

1.11

D-Glucose

0.386

1.09

Glycine

0.375

-1.13

Glycerol

0.620

1.09

Formic acid

0.911

-1.01

L-Glutamic acid

0.843

-1.03

L-Tyrosine

0.540

-1.06

L-Phenylalanine

0.779

1.03

L-Alanine

0.816

-1.02

L-Proline

0.320

-1.10

L-Threonine

0.772

-1.04

L-Isoleucine

0.695

-1.04

L-Histidine

0.832

-1.03

Lysine

0.850

1.02

L-Lactic acid

0.601

1.06

Pyruvic acid

0.419

1.08

3-Hydroxybutyric acid

<0.001

2.76

L-Arginine

0.314

-1.10

Creatinine

0.661

1.06

L-Glutamine

0.849

1.01

L-Leucine

0.731

-1.04

L-Methionine

0.002

-1.35

L-Valine

0.702

-1.04

Acetone

<0.001

1.79

Gene-metabolite Network Analysis

The metabolites were analyzed by the IPA® program, elucidating gene-metabolitenetworks including the selected metabolites, genes, and other entities (Figure 3).

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Figure 3. Ingenuity Pathway Analysis networks 1 (a) and 2 (b) including endometrial cancer metabolites. Metabolites from the public sources [14] inputted into IPA, colored in blue. Proteins, colored yellow, were extracted and inputted into DAVID Functional Annotation Tool.

Many genes in these networks have a dual function involved in obesity/insulin resistance/diabetes mechanisms along with clear involvement in cancers including endometrial cancer (Table 2).

Table 2: Genes extracted from IPA networks created on the basis of metabolites in endometrial cancer.

BLMH

PPT1

BDNF

BTK

PRPF38A

FOXO1

C8orf44-SGK3/SGK3

PTK2B

HIF1A

CSK

RPN1

HSPB2

IGF1R

SGK1

MB

JAK2

SLC36A2

PHB

PDHA1

SND1

PPARA

PDPK1

SOCS3

PPARGC1A

PKN2

TTK

STAT5B

PNN

ALB

TNF

TP53

Network 1

In this network, a number of genes have dual function and are involved in cancer and obesity mechanisms. We will describe some examples of such dual genes (Figure 3a).

PDPK1 (PDK1)

PDPK1 (PDK1) (3-Phosphoinositide Dependent Protein Kinase 1) as a gene phosphorylating AKT1 and involved in MYC1 pathway is very important in mechanisms of various cancer including endometrial cancer [14,15].PDPK1 gene is also a key player in the obesity. It phosphorylates AKT1, which is important in insulin signaling. Inhibiting of PDPK1 affects AKT1 function and consequently insulin signaling and weight gain [16].Intervention of a lifestyle decreasing insulin resistance led to significant degrease of PDPK1 (PDK1) gene expression confirming its dependence on obesity-related parameters [17].

IGFR1

IGFR1 (Insulin-like growth factor 1 receptor) is an important gene activating PI3K/ACT signaling pathway. Interesting to note that its expression growth proportionally to BMI and is leads tocancer prognosis [18]. Levels of activation of IGFR1 were found to be significantly higher in patients with endometrial cancer and insulin-resistant Diabetes II [19].

PPT1

Palmitoyl-protein thioesterase 1 (PPT1) is a lipoprotein;it has been shown that PPT1 gene is overexpressed in insulin-resistant patients [20]. PPT1 also significantly promotes tumor growth; PPT1 plays an important role in autophagy in cancer cells along with activation of mTORC1 [21].

SOCS3

As pointed by Sutherland and colleagues, silencing of the suppressor of cytokine signaling‑3 (SOCS3) by hypermethylationmay promote the oncogenic transformation of epithelial tissues and support growth of tumors [22]. It brings this gene in the set of tumor-suppressing genes. In obesity, SOCS3 plays a role of negative regulator of leptin-activation signaling actually increasing the leptin-resistance [23]. So, we can speculate that in both cases—cancer development and obesity methylation—silencing of SOCS3 would lead to disease enhancement.

BTK

BTK (Bruton’s tyrosine kinase) functions as a dual regulator of apoptosis and plays numerous other roles in cancer development [24]. BTK also plays a significant role in obesityleading to insulin resistance [25].

TTK

TTK protein kinase is a dual specificity protein kinase associatedwith cell proliferation. TTK has been found to be expressed in cancers such as pancreatic ductal adenocarcinoma, where cell proliferation was weakened when TTK was knocked down [26]. High expression levels of TTK was also found in clear cell renal cell carcinoma cells, and cell proliferation and invasion were suppressed with the knockdown of TTK [27].

SGK1

SGK1 (Serum/glucocorticoid regulated kinase 1) is involved in cellular stress response. SGK1 is known to be stimulated by insulin-related diseases as well as hyperglycemia [28]. SGK1 has also been found to have positive correlations with cancer progression and metastasis [29].

Network 2

Network 2 also contains dual-function genes. Several examples are presents below (Figure 3b).

TP53

Function of TP53 as a tumor suppressor is well known and does not need more explanation. In the same time not very obvious its role in obesity. Interesting findings demonstrated Morikawa and colleagues [30]. They found that TP53 expression changes to increase (positive) in patient with close to normal BMI. In the patients in obese category TP53 mostly has decreased expression (negative).Among the patients of invasive bladder cancer normal weight, overweight, and patients with obesity had a TP53 mutation frequency of 68.4%, 44.8%, and 25%, respectively (P < 0.05)” [31]. It means that mutational frequency of this gene is lower in obese patients. These fact that cancer rate is higher and TP53 expression is lowerin patients with lower BMI leads to a suggestion that obesity plays a significant role in cancer independently of the TP53 mutations. It can be related to higher probability of cancer proliferation in the adipose cells, more intensive development of blood vesicles in the tumors of obese patients, and other factors.Interesting to note that we found the same relations in some viral proteins impact to cancer development. We elucidated that presence of HPV viral proteins in patients with ovarian cancer leads to cancer development requiring less cancerogenic mutations than ovarian cancer without HPV (Shang, 2020 in press).

FOXO

FOXO (Forkhead box O3) phosphorylation leads to insulin resistance initiated by ER stress [32]. FOXO genes family has various roles in cancer development. Inactivation of FOXO function leads to uncontrolled cell proliferation and accumulation of DNA damage associated with cancer development [33].

PPARA

PPARA (Peroxisome proliferator-activated receptor-alpha) polymorphisms lead to cancer development [34]. It is also known that liver oncogenesis is significantly dependent on miRNAimpact on PPARA gene [35].

HIF1

HIF-1 (Hypoxia-inducible factor-1) regulates cellular response to hypoxia.As a transcription factors it increases concentration of genes involved in angiogenesis, cell survivaland other cancer-related activities [36]. In the same time HIF1 activation leads to white adipose tissue expansion in obese patients [37].

PHB

PHB (Prohibitin) is a “pleiotropic protein that has roles in both adipocytes and immune cells” [38]. PHB has been found to be upregulated in tumor cells from leukemia and lymphoma and it is associated with cancer progression [39]. PHB expression has been linked to both diabetes and cancer [38].

BDNF

BDNF (Brain Derived Neurotrophic Factor) has been found to play a role in cancer cell growth and progression through the stimulation of oncogenic pathways [40]. BDNF expression and mutation have also been associated with obesity [41].

Signaling Pathways Analysis

Genes elucidated the IPA networks as interacting with the endometrial cancer-related metabolites were analyzed by the DAVID program (Table 2). As expected, several KEGG pathways involving cancer were identified, including proteoglycans in cancer, prostate cancer, and pathways in cancer (Table 3). Moreover, several pathways that were identified were associated with obesity, which could reveal underlying mechanisms involving obesity and endometrial cancer.In order to understand the relationship between obesity and endometrial cancer, the adipocytokine signaling pathway, insulin resistance pathway, and glucagon signaling pathway from the list of these pathways were closely analyzed.

Table 3: KEGG pathways identified by the DAVID program.Count – number of inputted genes in the selected pathway.

Pathway Name

Count

p-Value

Insulin resistance

6

1.60E-05

Adipocytokine signaling pathway

5

6.30E-05

Proteoglycans in cancer

6

3.00E-04

Hepatitis C

5

5.90E-04

Prostate cancer

4

2.10E-03

Glucagon signaling pathway

4

2.90E-03

PI3K-Akt signaling pathway

6

3.20E-03

Thyroid hormone signaling pathway

4

4.50E-03

AMPK signaling pathway

4

5.50E-03

FoxO signaling pathway

4

7.00E-03

Insulin signaling pathway

4

7.60E-03

Hepatitis B

4

8.70E-03

Central carbon metabolism in cancer

3

1.50E-02

Fc epsilon RI signaling pathway

3

1.60E-02

Prolactin signaling pathway

3

2.50E-02

Pathways in cancer

5

2.70E-02

Herpes simplex infection

4

2.80E-02

HIF-1 signaling pathway

3

3.50E-02

Osteoclast differentiation

3

5E-02

Sphingolipid signaling pathway

3

5E-02

Adipocytokine Signaling Pathway

There is a high correlation between obesity and leptin concentration levels. Leptin is a hormone that regulates appetite, body mass composition, and energy expenditure. This hormone, while promoting lower food intake and fat loss, is often found in high concentrations alongside obesity when the hormone fails to induce weight loss. This effect is known as leptin resistance. Hyperleptinemia, where high levels of leptin are present in the bloodstream, is almost exclusively observed in obese animals. Leptin is primarily secreted in adipocytes and its expression is increased by obesity-related factors, such as overfeeding and insulin resistance [42,43]. The expression of leptin can be a risk factor for cancer. Leptin activates JAK, which then phosphorylates STAT3 (Figure 4). The activation of STAT3 is shown to significantly increase cell proliferation in endometrial cancer cells. When the phosphorylation of STAT3 was blocked with an inhibitor, the cell growth of the cancer cells decreased [2]. The activation of STAT3 leads to the induction of several oncogenic proteins, including c-Myc. In endometrial cancer cells, expression of the c-Myc gene increases cancer cell proliferation, and when the gene is knocked down, proliferation of the tumor cells dramatically decreases [6].

EDMJ-4-3-410-g004

Figure 4. KEGG pathway of adipocytokine signaling pathway selected by DAVID program. Leptin is a hormone that regulates obesity-related functions, such as body mass, appetite, and food intake. Leptin is shown to activate JAK–STAT3 pathway known to drive the proliferation of cancer tumor cells.

Insulin Resistance Pathway

Insulin resistance characterized by resistance to the effects of insulin on glucose uptake is a commonly known result of obesity and is a fundamental aspect of type 2 diabetes. In obesity, weight gain causes an increase in islet β cells, which in its turn increases insulin production. It has been shown that with increased insulin production, hyperinsulinemia occurs and induces insulin resistance [44]. Insulin resistance is related toactivation both the STAT3 and PI3k–Akt pathways (Figure 5). Through IL-6, obesity activates the STAT3 protein, which is known to increase cell proliferation in endometrial cancer through induction of the c-Mycprotein [6]. Additionally, insulin resistance is related to activation the PI3k–Akt and the mTOR signaling pathways (Figure 5). mTOR plays an important role in glucose metabolism and is activated with increased levels of insulin and glucose. Hyperactive mTOR occurs with obesity and nutrient overload, likely due to hyperglycemia or hyperinsulinemia—bothinvolved in the development of insulin resistance and obesity [45]. The activation of mTOR can lead to an increased risk of cancer. mTOR activates S6K and eIF4E (Figure 5), which are responsible for protein synthesis and have been shown to promote cancer cell survival [45]. Obesity leads to the expression of TNF-ɑ (Figure 5). TNF-ɑ can stimulate estrogen synthesis and has a direct impact on cell proliferation in the endometrium; overexpression of TNF-ɑ has been linked to endometrial cancer risk [7].

EDMJ-4-3-410-g005

Figure 5. KEGG pathway of insulin resistance from DAVID. Insulin resistance, which is commonly associated with obesity, activates both STAT3 and PI3k–Akt pathways. mTOR, activated by hyperinsulinemia and hyperactive in the obese population, can also lead to increased cancer risk.

Glucagon Signaling Pathway

Several meta-analyses have shown that a high glycemic load diet can increase the risk for endometrial cancer [3,46,47]. Glycemic load (GL) measures the effect a carbohydrate has on blood sugar levels as well as the amount consumed. Diets containing high GL with foods having high glycemic index are commonly associated with weight gain and obesity [48]. High GLs increases blood glucose and insulin levels, which leads to a decline in blood sugar levels within hours, ultimately creating a state of hunger that causes higher food intake and weight gain [49]. During the postprandial stage, a meal with the same nutrition but a higher GL can increase blood sugar levels two-fold, causing hyperglycemia. Hyperglycemia stimulates insulin production to lower blood sugar levels, while inhibiting glucagon release, which in turn would increase blood sugar levels [50]. Increased insulin production could potentially lead to hyperinsulinemia and insulin resistance, increasing the risk of cancer. Furthermore, glucagon has been shown to have anticancer properties on tumor growth as well as breast cancer [51,52]. Glucagon activates the FOXO signaling pathway and phosphorylates FOXO1 (Figure 6). FOXO1 is considered a tumor suppressor gene that is deactivated by insulin and the PI3k–Akt pathway. FOXO1 expression suppressed endometrial cancer cell proliferation in xenograft models and inhibited cell migration in Ishikawa endometrial cancer cells [8]. Diets with a high GL increase insulin levels while inhibiting glucagon release, reducing the activation of FOXO1 and its tumor suppressing properties, causing an increased risk of cancer.

EDMJ-4-3-410-g006

Figure 6. KEGG’s glucagon signaling pathway selected by DAVID program. Foods with high glycemic loads cause hyperinsulinemia and inhibit glucagon release, potentially leading to insulin resistance and obesity. Glucagon, having anticancer properties, can lead to an increased risk of cancer when inhibited.

Similarly Expressed Genes Analysis

To understand more comprehensively the common molecular mechanisms standing behind similarities in endometrial (among the others) cancer with obesity and related events, we performed analysis with the program GeneFriends [12,13]. We submitted to the program our list of genes elucidated by IPA program on the basis of endometrial cancer metabolites. The resulting co-expression networkconsists of four clusters having increased numbers of interconnections (Figure 7). Gene Ontology (GO)—analysis of existing clusters of similarly expressed genes brought the following results.

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Figure 7. Similarly expressed genes for the genes elucidated based on endometrial cancer metabolites. Green colored nodes represent genes from the original list.

Cluster 1

This contained mostly the genes related to immune response and related pathways activation (Table 4). These results support already pointed fact that immune response related processes are common for cancer and obesity. As noted by Pérez de Heredia and colleagues [53],“Obesity … is known to impair the immune function, altering leucocyte counts as well as cell-mediated immune responses. In addition, evidence has arisen that an altered immune function contributes to the pathogenesis of obesity”. Here, we see the obvious positive feedback when immune response alterations and obesity enhance each other leading to increase of both manifestations. In all cancers, inflammation is a part of the cancer development mechanism and immune response is common to tumor cells. Obesity is also related to inflammation. It is considered a condition causing chronic low-grade inflammation in patients [54]. Adipose tissue produced pro-inflammatory molecules including interleukins 1 and 6, TNFα, IFNγ, etc. and anti-inflammatory interleukins 3, 4, 10, and Ra [54,55].

Table 4: GO biological processes of the Cluster 1 of similarly expressed genes.

GO biological processes complete

P-value

Response to organic substance (GO:0010033)

2.66E-56

Immune system process (GO:0002376)

2.29E-53

Immune response (GO:0006955)

9.78E-52

Response to cytokine (GO:0034097)

4.18E-51

Cellular response to organic substance (GO:0071310)

1.92E-50

Cellular response to chemical stimulus (GO:0070887)

4.69E-50

Cellular response to cytokine stimulus (GO:0071345)

1.10E-49

Response to chemical (GO:0042221)

1.59E-44

Response to stimulus (GO:0050896)

1.55E-43

Cytokine-mediated signaling pathway (GO:0019221)

7.93E-43

Cell surface receptor signaling pathway (GO:0007166)

1.60E-40

Defense response (GO:0006952)

4.06E-39

Response to external stimulus (GO:0009605)

9.91E-39

Regulation of cell death (GO:0010941)

2.84E-38

Signal transduction (GO:0007165)

1.44E-37

Regulation of immune system process (GO:0002682)

1.08E-36

Positive regulation of immune system process (GO:0002684)

3.40E-36

Signaling (GO:0023052)

7.86E-36

Cell communication (GO:0007154)

1.17E-35

Regulation of cell population proliferation (GO:0042127)

1.21E-35

Immune effector process (GO:0002252)

1.76E-35

Cell activation (GO:0001775)

2.98E-35

Cellular response to stimulus (GO:0051716)

3.31E-35

Regulation of programmed cell death (GO:0043067)

1.07E-34

Leukocyte activation (GO:0045321)

4.09E-34

Regulation of apoptotic process (GO:0042981)

7.09E-34

Response to stress (GO:0006950)

8.63E-34

Cluster 2

This contains a number of biochemical reactions-related processes including interactions of various molecules within the cell environment, metabolic processes (Table 5). We will show in some examples that many ofthese processes are common for obesity and cancer.

Table 5: GO biological processes of the Cluster 2 of similarly expressed genes.

 

GO biological process complete

P-value

Lipid metabolic process (GO:0006629)

9.98E-12

Steroid metabolic process (GO:0008202)

7.41E-10

Small molecule metabolic process (GO:0044281)

1.66E-09

Regulation of hormone levels (GO:0010817)

1.48E-09

Organic hydroxy compound metabolic process (GO:1901615)

5.50E-09

Cellular response to xenobiotic stimulus (GO:0071466)

1.24E-08

Regulation of blood circulation (GO:1903522)

2.42E-08

Xenobiotic metabolic process (GO:0006805)

3.68E-08

Organic acid metabolic process (GO:0006082)

2.88E-08

Regulation of system process (GO:0044057)

3.87E-08

Monocarboxylic acid metabolic process (GO:0032787)

3.26E-08

Hormone metabolic process (GO:0042445)

3.62E-08

Negative regulation of endopeptidase activity (GO:0010951)

3.57E-08

Negative regulation of proteolysis (GO:0045861)

4.50E-08

Cellular lipid metabolic process (GO:0044255)

4.85E-08

Carboxylic acid metabolic process (GO:0019752)

5.82E-08

Negative regulation of peptidase activity (GO:0010466)

5.53E-08

Oxoacid(ketoacid) metabolic process (GO:0043436)

8.09E-08

Regulation of wound healing (GO:0061041)

1.05E-07

Response to oxygen-containing compound (GO:1901700)

1.61E-07

Negative regulation of hydrolase activity (GO:0051346)

1.73E-07

Response to stress (GO:0006950)

1.78E-07

Platelet degranulation (GO:0002576)

4.45E-07

Response to hormone (GO:0009725)

4.35E-07

Regulation of response to wounding (GO:1903034)

5.55E-07

Response to toxic substance (GO:0009636)

8.87E-07

Regulation of localization (GO:0032879)

8.76E-07

Response to inorganic substance (GO:0010035)

1.43E-06

Regulation of endopeptidase activity (GO:0052548)

1.96E-06

Fatty acid metabolic process (GO:0006631)

2.67E-06

Terpenoidmetabolic process (GO:0006721)

2.88E-06

Secretion (GO:0046903)

3.20E-06

Alcohol metabolic process (GO:0006066)

3.33E-06

Regulation of peptidase activity (GO:0052547)

4.13E-06

Regulation of transport (GO:0051049)

5.69E-06

Regulation of response to external stimulus (GO:0032101)

7.24E-06

Isoprenoidmetabolic process (GO:0006720)

8.05E-06

Tissue homeostasis (GO:0001894)

8.98E-06

Drug catabolic process (GO:0042737)

9.34E-06

Regulation of heart contraction (GO:0008016)

1.01E-05

Drug metabolic process (GO:0017144)

1.17E-05

Negative regulation of response to external stimulus (GO:0032102)

1.30E-05

Oxidation-reduction process (GO:0055114)

1.32E-05

Lipid Metabolic Process(GO:0006629)

Long and colleagues [56] pointed that “…energy metabolism, especially lipid metabolism, is significantly elevated during carcinogenesis.Furthermore, abnormality of lipid metabolism promotes cancer development, invasion and metastasis…”. In breast cancer the concentration of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) are significantly higher than in normal tissue. Moreover, concentration of TC and TG significantly higher in patients with metastases [56-58].

Oxoacid (Ketoacid) Metabolic Process

It was shown that key ketolytic enzymes BDH1 and OXCT1 can be biomarkers for possible ketogenic diet for cancer treatment [59]. Low expression level of these genes leads to sensitivity of cancer cells for low carbohydrates treatment.

Cluster 3

Thiscontains mostly processes-related to chromatin assembly, DNA processing, and epigenetic regulation (Supplemental Table S1). Experimental clinical studies of Ebot and colleagues [60] confirmed a link between obesity and prostate cancer through chromatin regulation. Their study demonstrated that in tumor tissues of very overweight/obese vs. healthy weight men, fifteen gene sets were enriched, while they did not change in normal tissues. They noted that patients with high tumor expression of chromatin-related genes had worse clinical characteristics and increased risk of lethal disease independent of grade and stage [60]. Wang with colleagues found that disruption of the bromodomain containing protein 2 (Bdr2), which plays a role in chromatin remodeling, leads to metabolically healthy obesity, with hyperinsulinemia, but enhanced glucose tolerance and low blood glucose, without T2D [61,62].Obesity causes changes in the acetylation distribution in specific cis-regulatory regions. This was supported by the fact of increased gene expression in the carcinogenic genes [63]. Authors also noted that premalignant lesions in obese patients’ colons have more probability to evolve to malignancy [63].

Cluster 4

.This contains mostly processes related to cell cycle (Supplemental Table S2). For obese patients, a number of cell-cycle-related genes were overexpressed including genes mostly involved in adipogenesis that most probably led to expansion of the adipose tissue during obesity [64]. Overexpressed genes promoting cell cycle were also found by Baranova and colleagues [65]. In cancers,defects of cell cycle is one of the most important molecular mechanisms of the disease: “Deregulation of the cell cycle engine underlies the uncontrolled cell proliferation that characterizes the malignant phenotype” [66]. Otto &Sicinski [67] pointed that cell cycle proteins are a promising target for anticancer drugs.

Discussion

Endometrial cancer is the cancer of the endometrium, contributing to an estimated to more than 10000 deaths in 2020. Obesity is recognized as one of the largest factors of increased risk and severity among different cancers, including endometrial cancer. There have been numerous population studies and meta-analyses supporting that an increase in BMI can significantly increase endometrial cancer risk. However, analysis of the molecular mechanisms involving both obesity and endometrial cancer can help elucidate their relationship and provide possibilities for new endometrial cancer therapies. Metabolites related to endometrial cancer obtained from the public sources were analyzed by the IPA program that created the gene–metabolites networks including the genes that interact with inputted metabolites. From the given list of endometrial cancer related genes and metabolites, DAVID Functional Annotation Tool identified several KEGG pathways involved in obesity, including insulin resistance, glucagon and adipocytokine signaling pathways.These pathways revealed potential targets for cancer treatment. For example, the STAT3 pathway, which was involved in both insulin resistance and adipocytokine signaling, could be targeted or inhibited which may reduce the effects of obesity on cancer risk. Lifestyle alterations, such as avoiding excessive food intake and high GL diets, can reduce metabolic syndrome, hyperinsulinemia, and weight gain, which can reduce endometrial cancer risk.In addition, further study on shared obesity and endometrial cancer biological processes via co-expression GO analysis may elucidate how their disease risks are positively correlated.

Data Availability

All data generated are presented in the article and supplemental materials.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Authors’ Contributions

VLK and IFT designed the study concept; KQW extracted the data; KQW created protein networks and extracted signaling pathways; BKP elucidated the genes with the similar expressions and did GO-analysis of the results; KQW, IFT, and VLK analyzed results; KQW, VLK, and IFT were involved in drafting the manuscript; IFT critically reviewed the manuscript for the intellectual quality of the study. The authors had full access to all data in the study. All authors have given their final approval to the final version of the manuscript.

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JDMR-3-2-305-g001

Are the Consequences of Gastrointestinal Infections of SARS-CoV-2 Underestimated?

DOI: 10.31038/IDT.2020121

 

With more than 7.8 million confirmed cases and over 430,000 deaths COVID-19 has had an impact on the whole world (16.06.2020). In many countries the peak of the first wave seems to be passing and strict lockdown measures are being eased gradually. It is important to implement risk mitigation procedures to ensure the safety of those people returning to the workplace and participating in “public life”. Despite mounting evidence that faeces are infectious there seems to be little discussion of this, no public awareness of risk, or implementation of potential mitigation strategies. In addition, lasting alterations in the gut microbiome of patients infected with SARS-CoV-2 have been observed in at least two pilot studies. Dysbiosis in the gut microbiome influences different biochemical functions and is associated with diverse diseases, effecting the immune system, metabolic states and mental health. In consequence, there could be unidentified long-term health risks of SARS-CoV-2 infections in the gut.

In a meta-analysis of 60 studies, comprising 4243 COVID-19 patients Cheung et al. showed that the pooled prevalence of any gastrointestinal symptoms was 17.6% (95% CI: 12.3-24.5%), with 11.8% occurring with non-severe COVID 19 symptoms and 17.1% with severe symptoms [1]. Not all studies reported all individual gastrointestinal symptoms, consequently the pooled prevalence of anorexia was 26.8% (95% CI: 16.2-40.8), diarrhoea 12.5% (95% CI: 9.6-16.0), nausea/vomiting 10.2% (95% CI: 6.6-15.3), and abdominal pain/discomfort 9.2% (95% CI: 5.7-14.5).

Using immunofluorescent staining, Xiao et al. showed expression of the ACE2 protein in the glandular cells of gastric, duodenal, and rectal epithelia [2]. They also demonstrated staining of viral nucleocapsid protein in the same epithelia cell populations. In addition, the authors showed that of 73 hospitalized patients in the study, 53% tested positive for SARS-CoV-2 RNA in stool. Wu et al. confirmed these findings and went a step further and followed the patients over time showing that respiratory samples remained positive for SARS-CoV-2 RNA for a mean of 16.7 days (SD 6.7) and faecal samples remained positive for a mean of 27.9 days (SD 10.7) after first symptom onset [3]. These findings have been repeated and verified in a number of additional studies, in adults and children, asymptomatic, mild and severe COVID-19 patients. In addition, Wu et al. showed that from 60 patients that were released from hospital after clinical recovery 10% were still positive for SARS-CoV-2 RNA in anal swab [4]. At least four independent studies have been published reporting the isolation of infectious SARS-CoV-2 from stool samples. For example, Wang et al. reported the isolation of live SARS-CoV-2 from stool samples (non-diarrhoea) from two patients [5]. This is of particular interest since various studies have shown the production of bioaerosols during toilet flushing [6]. That is significant since new data shows that airborne transmission plays a major role for the spread of SARS-CoV-2 [7]. Indeed, in a report Liu et al. studied the potential aerosol transmission of SARS-CoV-2 in two Wuhan hospitals [8]. The authors reported elevated levels of viral RNA in the patient toilet areas and recommended proper disinfection and ventilation. In addition, Van Doremalen et al. showed that viable SARS-CoV-2 could be detected in aerosols up to 3 hours after aerosolization, and up to 2-3 days on plastic and stainless steel [9].

First studies of the gut microbiome of COVID-19 patients showed a significant decrease in diversity and abundance compared to healthy control (HC) [10]. This was combined with the increased relative abundance of opportunistic pathogens like Streptococcus and Rothia. The study showed significant disease-specific shifts in the overall microbiota composition between COVID-19 and H1N1 patients and HC. Changes in the gut microbiome have been shown to associate with various diseases, for example IBS, IBD, type 2 diabetes, cardiovascular condition and depression. Further studies are needed to evaluate the long term effects on SARS-CoV-2 infections in the gut and possibly a restorative treatment regime needs to be developed.

Taken together these results strongly indicate that SARS-CoV-2 infections in the gut have short- and long-term effects. The short- term effects are gastrointestinal symptoms and in about 50% of the patients viral shedding in stool occurs. Toilet flushing can create infectious bioaerosols that are a potential risk factor for oral/nasal- faecal transmission of SARS-CoV-2. Cleaning procedures of toilets in hospitals, care facilities, schools, nurseries, work places, restaurants, trains, busses, airplanes and public spaces will need to be looked at and perhaps improved.

Conflict of Interest

I am a founder, director and shareholder of TiKa Diagnostics Ldt., however the company is specialized in diagnostics of Mycobacteria (for example TB) in humans and animals, and has not provided funding or any other support/influence for this review.

Acknowledgement

I thank Martin Cranage, Mark Bodman-Smith, Deborah Baines, Kathleen Kirmer and Ralf Mikut for their feedback.

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JPPR-3-2-314-g004

Food Supplements in the Treatment of Ophthalmic Diseases: Preclinical and Clinical Studies

DOI: 10.31038/JPPR.2020323

Abstract

At least 2.2 billion people in the world suffer by vision impairment or blindness (World Health Organization, 2019). Apart from infective diseases amenable to antibiotic treatment, cataract and corneal diseases resolvable by surgery, or refractive defects treatable with spectacles, most of the remaining pathologies do not have a cure. They can be treated with drugs or by surgery to alleviate symptoms and to slow down progression, but the underlying cause of the disease is usually not fully resolvable. Therefore, the best strategy to limit the burden of these ophthalmic diseases is prevention. In fact, almost all ophthalmic pathologies have a known etiology and pathway of development (although often not all the elements in the different steps have been elucidated), so that they are amenable to possible preventive treatments. Aim of this review is to illustrate how food supplements might help in the prevention and treatment of these ophthalmic diseases.

Introduction

Among the 2.2 billion people in the world affected by any type of eye condition, the World Health Organization estimates 196 million with Age-related Macular Degeneration (AMD), 146 million with diabetic retinopathy (DR) and 76 million with glaucoma [1]. No clear estimate exists on the global presence of dry eye, though the English Agency known as Global-Data UK Ltd. in a document published on June 2018, considering the 8 more industrialised Countries (US, France, Germany, Italy, Spain, UK, Japan, and China), reports a prevalence of 267,680,785 cases in 2016, destined to raise to 286,308,974 cases in 2026, therefore with an annual growth rate of 0.70% [2]. Approximately 200 million eyes globally are affected by cataracts causing a visual acuity less than 6/60, which become 3-4 times more including cataracts causing an acuity below 6/18 [3], accounting for one third of the worldwide blindness [4]. Globally, over 300 millions adolescents (less than 19 years old) are affected by progressive myopia [1], which in older ages may bring serious complications, like glaucoma or retinal detachment. All these eye pathologies pose a serious threat to vision, and have a strong social and economic impact. It has been estimated in 2014 by the National Eye Institute that the annual economic burden of vision loss, eye diseases and vision disorders in the U.S. amounted to 139 billion $ [5]. Now, what all these pathologies have in common is that there is no efficient pharmacological treatment for any of them. Resolutive surgery is possible in the case of cataract, but it is not devoid of – although minimal – side effects [6]. Surgical approaches are routinary applied also to the other pathologies, but most often they are not curative, and can at best attenuate their progression [7,8]. However, for all of them there is a link with lifestyle and alimentary habits, which becomes more evident in older ages [9,10]. This opens a space for prevention, early treatment and association of dietary habits or use of food supplements with pharmacological or surgical treatments. Aim of this review is to illustrate the scientific evidence supporting the use of food supplements in the management of eye pathologies.

GLAUCOMA

Glaucoma is an optic neuropathy, including a group of similar pathologies affecting the retinal ganglion cells (RGC), the optic nerve (ON) and the connected visual structures, causing vision loss and eventually blindness [11]. Primary open angle glaucoma (POAG) is the most common form of glaucoma (around 90% of the cases). It may occur in association with elevated intraocular pressure (IOP), in which case it is described as hypertensive glaucoma, or with normal IOP, in which case it is designed as normal tension glaucoma (NTG) [12]. In either case, the common pathological marker is the death of RGC by apoptosis, which eventually leads to the degeneration of the whole visual pathway, including the lateral geniculate nuclei and the visual brain cortex through the mechanism known as trans-synaptic degeneration [13,14]. This mechanism is present in other neurological diseases such as Alzheimer, Parkinson and Amyotrophic Lateral Sclerosis [13]. Therefore, also POAG is considered a slow progressing neurological disease [13]. Both extrinsic and intrinsic mechanisms contribute to RGC apoptotic death. Among the first, there are mechanical and metabolic factors. The compression of the ON at the lamina cribrosa (the structure present at the optic disk through which the ON leaves the eye) due to IOP elevation, leads to a decreased axoplasm flow and neurotrophins circulation between the periphery and the nucleus of the cell, necessary for RGC survival [15]. Endothelial dysfunction and decreased blood perfusion of the ON head (ONH) is a common finding in POAG [16], and may become a dominant trigger in NTG [17]. These events may activate or aggravate the intrinsic mechanisms, like glial cell activation and neuroinflammation, glutamate excitotoxicity and calcium release from dying cells, oxidative stress, starting a domino effect of RGC death, and further aggravating the pathology [18-20]. More recently, mitochondrial dysfunction has been added as a main mechanism among the different causes that can possibly lead to RGC death and glaucoma [21]. However, from the clinical perspective, the only pharmacologic therapeutic approach is based on the reduction of the IOP, both for hypertensive POAG and for NTG [22]. However, several clinical studies have shown that IOP reduction is a necessary, but not sufficient condition to arrest POAG progression [23]. Moreover, due to the structure of the retina, and the redundance of the cell network system carrying light signals from the eye to the brain, usually more than 50% of RGCs are dead when the first visual field abnormalities are detectable [24]. Hence, a neuroprotective strategy is becoming more often associated with the classical IOP-lowering treatment to try and improve the therapeutic outcome of POAG patients [25]. We have already reported that neurodegeneration is caused by several different types of insult to RGC, therefore an effective neuroprotective treatment should be endowed with multiple effects on different targets [25]. To this purpose, a single molecule with pleiotropic effects, or an association of molecules joining together their therapeutic mechanisms, should be considered (Figure 1).

JPPR-3-2-314-g001

Figure 1. Neurodegeneration in PoAG. The main recognized mechanisms of neurodegeneration in POAG are the excessive pressure at the level of the lamina cribrosa, where the optic nerve leaves the eye globe, and/or the decreased blood perfusion at the optic nerve head (ONH) (upper panel). These events may lead to injury and apoptosis of retinal ganglion cells (RGC), and the progressive loss of sight. Indirect neuroprotective agents are those mainly acting on the IOP decrease and the improvement of blood perfusion (bottom left). Direct neuroprotection works by blunting those events directly leading to cell injury and apoptosis (bottom right).

Brimonidine is a drug and it is an α-2A adrenergic receptor agonist, present in eye drops medications either alone, or in association with other hypotonizing drugs (timolol or dorzolamide). It is a paradigm of a multipurpose molecule, being active both on the hypotonizing and the neuroprotective side, and has clearly demonstrated that neuroprotection and hypotonizing effects can be dissociated. In fact, brimonidine, beside the hypotonizing effect on IOP [26], has also shown several neuroprotective effects, such as improved perfusion pressure at the ONH [27]; rescue of RGC from apoptosis by increasing the ratio bcl2/BAX [28]; prevention of the release of pro-apoptotic intracellular calcium [29]; blunting of the excitotoxicity due to excessive glutammate release [30] and upregulation of the neurotrophin BDNF expression by RGC [31]. Brimonidine neuroprotective effects in POAG have  been demonstrated both in preclinical animal model  systems [32] and in a seminal clinical trial of several years, showing that the neuroprotective effects achieved by brimonidine were indipendent from the hypotonizing effects that could be equally achieved by timolol [33].

Food Supplements

Epigallo-catechin-gallate (EGCG) is the main catechin of green tea, and also displays several activities with neuroprotective value. It has no effect on IOP, but it improves vascular perfusion by stimulating the expression of the endothelial form of nitric oxide synthase [34]; it is an iron chelator and a potent antioxidant, reducing oxidative stress and lipoperoxidation of cell membranes, thus shielding RGC from oxidative damage [35], and it is easily absorbed and distributed throughout the body [36]; it has anti-apoptotic activities, contrasting the activation of proapoptotic genes and favoring the expression of antiapoptotic ones, thus defending RGC from traumatic apoptotic death [37,38]. Its antioxidant and neuroprotective effects on RGC have been demonstrated in a mouse model in which oxidative damage was induced by intravitreal injection of sodium nitroprusside. When EGCG was given together with SNP, apoptotic cell death of RGC was significantly blunted [39]. Accordingly, in a mouse model of retinal ischemia/reperfusion, resembling acute closed angle glaucoma, oral

administration of EGCG could rescue RGC from apoptotic death, as seen by the conserved electroretinogram (ERG) response and thickness of the ganglion cell layer, and by the decreased expression of apoptotic caspases [40]. The antioxidant activity of EGCG has been proposed as a possible remedy to blunt the effects of mytochondrial dysfunction on glaucoma progression [41]. A clinical trial on 18 patients with POAG established that EGCG oral supplementation could improve – although slightly, because of the short treatment time – their pattern electroretinogram (PERG), which is an early alteration detectable in glaucoma subjects [42].

Citicoline is the current name of cytidine-5’-diphosphocholine. Citicoline is naturally present in all cells, serving as an intermediate in the biosynthesis of phosphatidylcholine, a component of biological membranes [43]. Citicoline has been one of the first neuroprotectors proposed [44], and has shown neuroprotective effects in Alzheimer disease, stroke, and Parkinson’s disease, as well as in glaucoma and amblyopia [45]. Some citicoline neuroprotective effects are mediated by the restoration of phosphatidylcholine levels and maintainance of sphingomyelin and cardiolipin levels (constituent of the inner mitochondrial membrane, involved in the cell energetic metabolism); by anti-oxidant effects, due to the increase of glutathione reductase (GR) activity and glutathione (GSH) synthesis and the decrease of PLA2 activity and lipid peroxidation; by the restoration of Na+/ K+ ATPase activity, essential for the proper functioning of neurons and glial cells. The choline provided by citicoline is used for the biosynthesis of the neurotransmitter acetylcholine, the stimulation of tyrosine hydroxylase activity and dopamine release [46]. Moreover, citicoline showed antiapoptotic effects by enhancing antiapoptotic bcl2 expression in a rat model of partial ON crush [47], and by decreasing apoptotic caspases expression in explanted rat retinas cultivated in toxic high glucose cell culture medium [48]. Over the years, citicoline has been studied in several glaucoma clinical trials. Initially, it was given by intramuscular injection, with a notable improvement on both PERG (RGC function) and VEP (Visual Evoked Potentials: gives the function of the whole visual axis) response [49,50]. In a further study, it was shown that oral administration of citicoline to glaucoma patients gave the same protection as the intramuscular injection, thus improving patients’ compliance [51]. Finally, also topical citicoline formulations as eye drops were shown to be effective in improving PERG and VEP of glaucoma patients over a period of 4 months [52].

Coenzyme Q (ubiquinone) neuroprotective activity also appears to be linked with its effects on mitochondria. In fact, coenzyme Q10 (CoQ10: the prevalent form found in humans) is a potent antioxidant, a membrane stabilizer and an important cofactor of the mitochondrial electron transport chain for the production of adenosine triphosphate (ATP) [53]. In the DBA/2J mouse model of spontaneous glaucoma, diet supplementation with CoQ10 significantly decreased the ratio between pro-apoptotic and anti-apoptocic genes in the retina, thus preventing apoptotic cell death. Moreover, mitochondrial DNA and the Tfam transcription factor of the oxydative phosphorylation system in the retina were functionally preserved. This resulted in increased RGC survival and preservation of ON axons, and blunting of astroglial activation in the retina and ONH [54]. Massive RGC death typically results in the release of an excess of glutamate, and a paroxystic activation of glutamate receptors. This leads to the opening of the mitochondrial permeability transition pore (MPTP) through which cytochrome C is released, signaling for the activation of caspase 9 and the apoptotic cascade. CoQ10 showed a further specific antiapoptotic effect by maintaining MPTP in the closed conformation thus preventing the triggering of apoptotic cell death [55]. In a rat model of mechanic ON injury a topical formulation of CoQ10 + vitamin E was administered as eye drops for 4 weeks. Decreased gliosis, increased anti-apoptotic gene expression, and preserved Tfam were observed in the retina of CoQ10 treated mice, which resulted in increased RGC survival [56]. Most recently, a multicenter, controlled clinical trial on 612 POAG patients has started, having visual field progression as the primary outcome, with the aim to evaluate the neuroprotective effects of an ophthalmic solution of CoQ10 and Vitamin E [57].

Saffron (crocus sativus) is a traditional medicinal plant containing in its stigmas many metabolites, among which the carotenoids crocin, crocetin and lycopene and the xantophyl zeaxanthin [58]. Crocin and crocetin are the main bioactive components [59], which can promptly be absorbed through the gastrointestinal tract, diffuse into organs and easily traverse the blood brain barrier (BBB) [60]. They have shown a protective effect on the integrity of the BBB during cerebral ischemia [61], which effect could likely be transposed also on the protection of the blood retinal barrier (BRB) during degenerative retinopathies, such as in AMD or DR [62]. The neuroprotective properties of saffron have been demonstrated in several ophthalmic pathologies and attributed to the important anti-inflammatory, antioxidant, and anti- apoptotic properties of its bioactive components [63]. In glaucoma, retinal ischemia triggers neuronal cell death. Crocin and crocetin blunt oxidative stress and inhibit RGC death in mouse and rat models of ischemia/reperfusion [64-66]. Excitotoxicity by N-methyl-D- aspartate (NMDA) released by dying RGC is a common event during glaucoma progression [19]. Oral administration of crocetin prevented the inner retinal damage induced by intravitreal injection of NMDA in mice, by inhibiting the caspase pathway [67]. Most recently, a mouse model of laser-induced ocular hypertension (OHT) has been used to show the anti-inflammatory and neuroprotective effects of a saffron extract titrated to 3% in crocin. Both gliosis and RGC death were efficiently prevented by crocin oral supplementation in OHT eyes [68]. Clinical studies with saffron used so far IOP as the sole endpoint. Daily oral administration of 30 mg of a saffron infusion for three weeks produced an IOP decrease in POAG patients [69], whilst a discontinuous (twice per week) oral treatment with an even higher amount of 1 gr for a month gave no variation on the IOP [70], suggesting that continuity of treatment rather than dose could be the relevant strategy. The toxic effects of saffron become evident for daily doses higher than 5 g [71]. Therefore, since its human therapeutic use is in the range of milligrams, it has virtually no toxicity [72].

Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a natural phenol, a phytoalexin naturally produced by several plants (it can be found in foods such as grapes, peanuts, blueberries, and dark chocolate) in response to microbiological attacks [73]. It is endowed with potent anti-oxidant and anti-inflammatory properties, which are likely responsible for its cardioprotective [74], neuroprotective [75], and anti- aging effects [76]. In fact, resveratrol has shown neuroprotective effects in neuroinflammatory and neurodegenerative diseases like stroke and Alzheimer’s disease [77,78]. Most interestingly, resveratrol has been shown to blunt the damage of ischemic brain injuries [79], likely by preserving brain mitochondria function after ischemic/reperfusion damage [80], and extend the lifespan of organisms from yeast to mammals [81]. All these beneficial activities of resveratrol appear to be mainly mediated by its stimulating effect on AMPK [82], which in turn stimulates the activity of SIRT1 (the mammal homologue of Sir2), the longevity-associated gene [83]. Several preclinical evidence support the efficacy of resveratrol in controlling the glaucomatous disease. In primary trabecular meshwork (TM) cells subjected in vitro to chronic oxidative stress (40% O2) resveratrol treatment blunted the production of free radicals and inflammatory markers (IL1α, IL6, IL8, and ELAM-1), also exerting antiapoptotic effects [84]. In a mouse model of glaucoma induced by clogging of the TM,

intraperitoneal injections of resveratrol partially protected RGC from apoptotic cell death [85]. RGC dendrites protection by resveratrol was demonstrated in mice fed for one year with a resveratrol supplement, and then subjected to ON crush, which simulates the compression at the ONH further to acute IOP elevation. In this case the protective effect appeared to be associated with increased expression of the nuclear C/EBP homologous protein and the nuclear X-box-binding protein-1 [86]. Intravitreal injection of resveratrol could mitigate the retinal ischemic injury in a rat model system of ischemia/reperfusion obtained by a sudden elevation of the IOP, simulating an angle closure glaucoma. In this case the effect was related to the containment of the upregulation of matrix metalloproteinase-9, heme oxygenase-1 (HO-1), and inducible nitric oxide synthase [87]. Mitochondrial protection by resveratrol treatment was shown in hypertensive model systems in vitro with RGC5 neuronal cells, and in vivo in rats fed with a resveratrol supplement before artificial clogging of the TM. In both systems neuronal cells improved mitochondrial function and survival was dependent on the activation of the AMPK/SIRT1/ PGC-1α signaling pathway, which is a critical pathway regulating mitochondrial biogenesis and function [88,89]. Finally, a critical process in the development of glaucoma is neuroinflammation, which is sustained by activated astrocytes producing cytotoxic mediators triggering RGC apoptosis. Resveratrol given in vitro to astrocytes under oxidative stress blunted their activation and improved their survival by decreasing caspases activation, toxic Tau processing, and neurofibrillar tangles formation [90]. Beside its direct neuroprotective activity on RGC, resveratrol may also work to decrease IOP. Topical administration of resveratrol eye drops in a rat model system of steroid-induced hypertensive glaucoma [91] could decrease by 25% the IOP via a mechanism involving adenosine receptors [92]. This hypotonizing effect was correlated with higher levels of metalloproteinase-2 in the aqueous humour (AH) and a decreased TM thickness beside a restoration of the retinal morphology and its redox status [92]. In the clinics, it has shown some degree of efficacy in neurologic pathologies such as Alzheimer disease and Friedreich ataxia [93], but no clinical data are available for glaucoma patients.

Melatonin is a very ancient molecule widely distributed in nature, in the vegetal and the animal world [94]. Melatonin is best known as a circadian regulator of sleep and other cyclical bodily functions [95]. Accordingly, melatonin is not produced only by the pineal gland, but also by several other organs, thus influencing through paracrine signaling the physiological functions of the whole organism [94] by differential binding to its three receptors MT1-3 [96]. In the eye, it is made by the retina [97], the ciliary body [98] and the lachrymal gland [99]. Melatonin and its metabolites have antioxidant and free radical scavenger activities including both reactive oxygen and nitrogen species [100,101], thus protecting from oxidative damage photoreceptors and other ocular tissues [102]. In preclinical studies melatonin showed clear neuroprotective effects. In a mouse model of ischemia/reperfusion of the retina after sudden IOP elevation, intraperitoneally injected melatonin prevented the toxicity due to the increased expression of the hypoxia inducible factor (HIF-1α) and Muller cells gliosis, thus improving RGC survival [103]. In a rat model of hypertensive glaucoma obtained by clogging of the TM, subcutaneous implantation of a pellet slowly releasing melatonin improved the antioxidant potential in the retina, decreased glutamate excitotoxicity with the final results of improving RGC survival and rescuing the retinal function as evidenced by the ERG [104]. In a rabbit model of intravitreal glutamate toxicity, simultaneous melatonin injection could blunt the oxidative stress and improve survival of RGC by decreasing their apoptotic cell death [105]. In a recent work with a rat model of hypertensive glaucoma obtained by episcleral injection of hypersaline solution, the intravitreal administration of Poly lactic-co-glycolic acid microspheres loaded with melatonin, CoQ10 and dexamethasone releasing the drugs for over 30 days, could preserve RGC from apoptotic cell death [106]. Interestingly, not only melatonin, but also agomelatine, the novel MT1/MT2 receptor agonist antidepressant, has shown significant neuroprotective features [107]. Melatonin not only shields RGC from apoptotic death, but also shows a direct effect on the IOP. Circadian changes of the IOP appear to be linked to melatonin [108], which has shown hypotensive effects on IOP [109], apparently mediated by the MT3 receptor [110,111]. Most recently, it has been shown that a nanomicellar formulation of melatonin and agomelatin given topically as eye drops is able to decrease by over 30% the IOP of normotensive rats, and by over 50% the IOP of hypertensive rats, after clogging of the TM [112]. Clinically, it has been reported that oral treatment with agomelatine could further decrease by 30% on average the IOP of 10 glaucomatous patients under maximum tolerated medical therapy (MTMT) with different hypotonizing drugs [113].

Cannabinoids are naturally present in our organism – including eye tissues and the retina – as endocannabinoids: anandamide, 2-arachidonoylglycerol and palmitoylethanolamide [114,115]. Phytocannabinoids (pCBs: agonists of the endocannabinoid receptors CB1 and CB2) are the active molecules found in cannabis (Cannabis sativa), and are mainly represented by Δ-9-tetrahydrocannabinol (THC), cannabinol and cannabidiol. THC is the molecule mainly responsible for the psychoactive effect of marijuana, while cannabinol is much less a psychotropic agent, and cannabidiol is not psychoactive at all [116]. Phytocannabinoids (much of all THC) have shown efficacy both as modulators of the IOP [117,118] and as neuroprotective agents [119]. It is likely the interaction of pCBs with the CB1 receptor that can lower the IOP by modulating production and outflow of AH [120,121]. The neuroprotective activity possibly depends on the antioxidant and antinflammatory effects of pCBs [122]. However, their psychotropic activity did not allow their free systemic use in ophthalmic diseases [123]. A valid alternative could be a topical formulation given as eye drops. Indeed, THC in nanotechnological formulations topically administered to normotensive rabbit’s eyes could reduce the IOP by 30% of the baseline value [124]. In a case report observation, oily based galenic ophthalmic formulations of pCBs with different proportions of THC and cannabidiol were given to patients unresponsive to drug treatments, however with little effect on the IOP. Only one patient with open angle glaucoma secondary to Fuchs heterochromic iridocyclitis had a partial response, probably due to the antinflammatory effect of the formulation with a higher content of THC [125].

Forskolin is a labdane diterpene extracted from the roots of the plant Indian Plectranthus barbatum (Coleus forskohlii). It is a lipid- soluble molecule that can cross cell membranes, thus working as a receptor independent activator of adenylate cyclase, finally resulting in the intracellular increase of cyclic adenosine monophosphate (cAMP) and cAMP-mediated functions [126]. Forskolin lowers IOP [127] by two independent and original mechanisms, both triggered by cAMP elevation: it decreases net secretion of AH by the ciliary body by a mechanism that involves the chloride channels and a reabsorption of AH from the posterior chamber into the stroma [128,129]; and by enhancing AH outflow through the TM by a mechanism that involves cell actin cytoskeleton disassembly through PKA activation and Rho kinase inhibition [130]. None of the existing glaucoma drugs (carbonic anhydrase inhibitors, beta-blockers, alfa-agonists, prostaglandin analogs) has similar mechanisms of action, therefore it might be expected that forskolin effects add upon those of such drugs. In fact, a clinical study in which POAG patients under MTMT were treated with a food supplement containing forskolin and rutin – an antioxidant and vasoactive ingredient [131] – showed that the food supplement resulted in a further decrease of the IOP, independent of the drug that the patients were taking [132,133]. Moreover, forskolin is also endowed with direct neuroprotective activities, mediated by the activation of paracrine signaling, due to the induction of the neurotrophin BDNF (brain derived neurotrophic factor) expression by astrocytes and vascular endothelial cells [134,135] and to the translocation of its receptor TrkB to the neuron cell membrane [136]. Preclinical data with a rat model system of hypertensive glaucoma obtained by clogging of the TM confirmed both the IOP control and the neuroprotective efficacy of oral forskolin [137]. Clinical data further corroborated the double efficacy of forskolin-based oral supplements. In a randomized, controlled clinical trial on 45 patients affected by POAG compensated by topical drugs, the 30 patients taking the food supplement with forskolin and rutin experienced during the 6 months of the trial a progressive decrease of the IOP and a concomitant increase of the PERG amplitude, suggesting an improvement of RGC survival [138]. Forskolin is also the basis of another association, with homotaurine – a neuroprotective compound used in Alzheimer [139] and Parkinson’s [140] disease – and carnosine, a wide spectrum antioxidant neuroprotector [141,142]. Preclinical evidence on a rat model of retinal ischemia/reperfusion obtained by sudden barometric increase of the IOP has shown a clear neuroprotective synergistic effect of the association of the three compounds intravitreally injected [143]. Improved RGC survival was reported, correlating with the induction of PI3K/Akt, the inhibition of GSK-3b and the reduction of calpain activity, all known to be linked to neurodegenerative events [143]. Results from a randomized, controlled clinical trial on 22 patients affected by POAG with IOP compensated by topical drugs, showed that patients taking the oral supplement containing the three components had a significant further decrease of their IOP, an improvement of PERG amplitude at 6, 9, and 12 months, and of foveal sensitivity at 12 months, whilst all the above values remained stable in untreated control patients [144]. Most recently, a new association of forskolin with homotaurine and spearmint extract – rich in polyphenols and rosmarinic acid with antinflammatory and anti-oxidative effects, likely preventing neuron degeneration [145] – has been tested in a mouse model of ON crush, simulating the sudden compression at the ONH during acute glaucoma. The oral supplementation of forskolin, homotaurine, spearmint extract, and vitamins of the B group resulted in a significant improvement of RGC survival, correlating with a preservation of the ERG photopic negative response, which depends on the RGC health status, and with a decrease of inflammatory cytokines and pro-apoptotic markers [146]. Similar results were also obtained in a mouse model in which RGC degeneration depends on progressive IOP elevation, obtained by intracameral injection of polyetylen-glycol, which clogs the TM and decreases AH outflow [147].

Vitamins have been suggested as potential neuroprotective agents, and a recent metanalysis found relevant associations with vitamins A and C [148]. Deficiency of vitamins of the B complex has been reported in patients with glaucoma [148,149], which specifically showed significant lower serum levels of vitamins B1 and B12 [150,151]. Insufficient amounts of vitamins B6, B9 and B12 may lead to increased levels of homocysteine [152,153], known to be toxic to RGC [154], and found in relevant amounts in the AH and plasma of POAG patients [155]. A recent meta-analysis showed a high prevalence of hyperhomocysteinemia in patients with pseudoexfoliative glaucoma [156], which another study associated with decreased levels of vitamin B6, B9 and B12 [157]. Accordingly, increasing vitamin B9 intake appeared to lower the risk of pseudoexfoliative glaucoma probably by reducing homocysteine levels [158]. Vitamin C is an antioxidant, and an important factor involved in tissue repair [159]. Biochemical analyses have shown that the glycosaminoglycan content of TM in healthy subject is different from that of POAG patients, which contains less hyaluronic acid (HA) and more chondroitin sulfate [160]. Vitamin C stimulates the synthesis and secretion of HA in TM cells, more in those cells derived from a glaucomatous eye than from a normal eye, thus re-equilibrating the production of extracellular matrix (ECM) glycosaminoglycans [161]. Addition of vitamin C supplements was found to be associated in a dose-dependent way with decreased prevalence of glaucoma in a population sample in the United States [162]. In a large cross-sectional study on Afro-American women, a correlation has been described between higher consumption of food rich in vitamins A and C and carotenoids and a decreased incidence of glaucoma [163]. In a different epidemiologic evaluation, NTG patients were found to have lower blood levels of vitamin C and increased levels of uric acid [164]. Vitamin C has a role in maintaining lysosomal function and correct protein turnover in TM cells; therefore, an insufficient concentration of vitamin C in plasma and AH may alter the physiological function of TM cells, resulting in a decreased outflow capability and an increase of the IOP [165]. Vitamin E is a strong antioxidant [166]. Several studies on glaucoma patients have reported low levels of vitamin E in their blood [162,167] or AH [168]. A low intake of Vitamin A as retinol equivalents appears to be associated with an increased risk of POAG [150]. Vitamin D plays a role in the signaling pathways related to bone and mineral metabolism, cellular proliferation, immune modulation, and oxidative stress [169,170]. A high prevalence of vitamin D deficiency is observed in patients with glaucoma, confirmed by low circulating amounts of vitamin D in these subjects [171,172]. Metabolomic analysis on plasma samples from 72 POAG patients compared to 72 healthy controls revealed alterations of vitamin D metabolic pathways correlating with vitamin D insufficiency in POAG patients [173]; moreover, the presence of specific vitamin D receptor polymorphisms were found to be relevant risk factors in the development of glaucoma [174]. Accordingly, other studies have reported low serum levels of vitamin D in POAG patients [175].

OCULAR SURFACE DYSFUNCTION(Dry Eye)

The most recent definition of the dry eye syndrome by “The Tear Film and Ocular Surface Society Dry Eye Workshop Report”, defines the key points and possible therapeutic targets of the disease. Dry eye is “a multifactorial disease of the ocular surface” leading to tear film instability in which “hyperosmolarity, ocular surface inflammation, and neurosensory abnormalities play etiological roles” [176]. The etiology of dry eye is complex, and no cure for it is available. Artificial tears provide a way to blunt signs and symptoms of the disease, but with limited efficacy, both in terms of intensity and duration. Therefore, there is an increasing interest in the use of nutritional supplements as a complementary approach for the prevention and management of the dry eye syndrome [177]. Many scientific reports have highlighted an association between incidence and severity of dry eye disease and nutritional deficiencies [178-183]. The identification and appropriate use of nutraceuticals able to work on dry eye may provide insights into dry eye pathogenesis and increase the efficacy of conventional therapies.

Polyunsaturated Fatty Acids

Among dietary supplements, there is a growing interest in the use of polyunsaturated fatty acids (PUFAs) to prevent or alleviate signs and symptoms of dry eye [184,185]. PUFAs are involved in the synthesis of photoreceptor outer segment disks, and in ocular surface physiology; therefore, they are essential for proper visual development and retinal function [186]. PUFAs can be grouped into two main families: omega-3 and omega-6 [187]. The omega-3 include docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and alpha-linolenic acid (ALA), while omega-6 include linoleic acid (LA), gamma-linolenic acid (GLA) and arachidonic acid (AA). Multiple clinical trials looked at the effect of omega-3 and omega-6 supplements in the treatment of dry eye syndrome. Miljanović et al., evaluated the association between the dietary intake of different ratios of omega-3 and omega-6 and dry eye syndrome occurrence in a large cross sectional study on women. Results showed that women with a higher dietary intake of omega-3 had a lower prevalence of dry eye symptoms and that a high ratio of omega-6/omega-3 (>15:1) was associated with a greater prevalence (more than 2-folds) of dry eye syndrome [188]. A meta-analysis of 9 randomized controlled trials (collected between 2002 and 2011) on PUFAs dietary supplements reported an improvement of the burning sensation and reflex lacrimation in dry eye patients assuming PUFAs [189]. A similar meta-analysis of other 7 randomized controlled trials collected from 2007 to 2013 reported not only the improvement of the above symptoms, but also an improvement of signs like Schirmer Test and Tear Break Up Time [190].

Meibomian gland dysfunction (MGD) is a chronic disease of the homonymous glands [191-193], leading to altered secretion of the tear film lipid layer, finally resulting in increased tear evaporation and tear osmolarity, a trigger for dry eye signs and symptoms [191-193]. One key factor of the potential benefits of systemic supplementation with PUFAs is the evidence that dietary fatty acids can be incorporated in lacrimal gland phospholipids [194-196]. Wojtowicz et al., explored the potential effect of dietary supplementation with omega-3 on lipid composition in a case-control trial, where treated patients received for 90 days fish oil, containing 450 mg of EPA, 300 mg of DHA, and 1000 mg of flaxseed oil (rich in ALA). At the end of the study, most of the patients (about 70%) became asymptomatic with an increment of tear secretion [197]. Another study recruiting a large sample of postmenopausal women, showed that high omega-3 consumption and moderate omega-6 consumption were protective against MGD [198]. Tear film hyperosmolarity is a focal event in dry eye syndrome [199]. In a double-blind, randomized, parallel trial, consumption of sea buckthorn oil (high in omega-3 and omega-6 fatty acids) for 3 months attenuated the increase in tear film osmolarity during the cold season and positively affected symptoms in dry eye patients [200]. Furthermore, a moderate daily dose of long-chain omega-3 PUFAs given over three months reduced tear osmolarity and increased tear stability in subjects with dry-eye disease [201]. In a different clinical study on patients with contact lens associated dry eye, 6 months of oral treatment with evening primrose oil rich in the omega-6 GLA resulted in improvements in dryness symptoms at 3 and 6 months and in overall lens wearing comfort at 6 months [202]. Oleñik et al., evaluated efficacy and tolerability of a food supplement containing a combination of omega-3 and antioxidants (a mixture of DHA 1050 mg, EPA 127.5 mg, DPA 90 mg + vitamins C/E and GSH + trace elements) on dry eye related symptoms in patients under treatment with artificial tears [203]. After 12 weeks, treated patients showed a significant improvement of symptoms with a lesser need of using artificial tears and a better tolerance to contact lens wearing. Comparable outcomes were achieved with treatment administered for shorter periods of time [204] or with lower concentrations of omega-3 PUFAs [205]. In another recent study, dietary omega-3 fatty acids administration showed efficacy in alleviating computer work- related dry-eye symptoms, associated with altered tear evaporation rate (Figure 2) [206].

JPPR-3-2-314-g002

Figure 2. Eye dryness and ocular surface dysfuntion. The malfunctioning of one ore more components of the ocular surface functional unit may lead to pathologic alterations of the ocular surface eventually causing eye dryness, discomfort and pain (upper panel). Some food supplements have been shown to prevent and/or alleviate symptoms and signs of dry eye (bottom panel).

Chronic inflammatory processes associated to immunologic activation have a pivotal role in dry eye pathogenesis [207,208] and PUFAs have been shown to have significant anti-inflammatory activity [209], which might turn beneficial in avoiding the excessive damage caused by chronic inflammation. In a mouse dry eye model induced by scopolamine injection in a dry environment, treatment with topical ALA for 9 days resulted in a significant decrease of inflammatory cytokines like TNF-α, and a reduction of corneal fluorescein staining in comparison to untreated controls [210]. Barabino and colleagues investigated the efficacy and anti-inflammatory activity of oral supplements of systemic ω-6 (LA and GLA) given twice per day for 45 days, on chronic ocular inflammation in patients with dry eye. They observed an improvement in symptoms and ocular surface inflammation in the study group patients when compared to controls [211]. A recent prospective case-control study showed that patients receiving an oral combination of antioxidants and omega-3 PUFAs for 3 months displayed reduced tear levels of inflammatory mediators such as the interleukins: IL-1b, IL-6, and IL-10 [212]. Brignole-Baudouin et al., in a multicentre trial, demonstrated that supplementation with omega-3 and omega-6 fatty acids can reduce the ectopic expression of the HLA-DR inflammatory marker and may help to improve dry eye symptoms [213]. Prostaglandins are well-known inflammatory mediators, playing a key role in the modulation of the inflammatory response in dry eye disease [214]. More specifically, tear levels of prostaglandin-E2 are known to be positively correlated with dry eye symptoms [215,216]. Black currant seed oil, rich in GLA and ALA, has been shown to modulate cell membrane lipid composition and eicosanoid production, finally reducing prostaglandin-E2 production [217]. Cermak et al. highlighted that in women with primary or secondary Sjögren’s syndrome (a systemic autoimmune inflammatory disease of the exocrine glands, including the lacrimal gland, thus resulting in a serious dry eye syndrome [218]), the intake of LA, and other specific PUFAs was significantly lower with respect to gender matched controls [219]. In another clinical trial involving 20 patients with primary Sjögren’s syndrome, an oral mixture of omega-6 induced a significant increase in anti-inflammatory PGE-1 content in tears, improving the symptoms of ocular discomfort and the signs derived from corneal epithelial defects [220]. The drug cyclosporine, present in eye drops approved for the treatment of dry eye disease [221], prevents T-cell activation and production of inflammatory cytokines, therefore breaking the inflammatory cycle of autoimmune dry eye disease and increasing the production of tears and mucin-producing Goblet cells in the conjunctiva [222]. However, the use of cyclosporine is not free from side effects which depend on the dose used; therefore it is important to maximise its effects in order to use lower doses. To this purpose, the effects of topical cyclosporine on dry eye signs and symptoms has been evaluated either alone, or in association with a prescription-only medical food supplement containing omega-3 and omega-6 PUFAs acids. It has been reported that a proper balance of omega-3 and omega-6 essential fatty acids could indeed improve the efficacy of cyclosporine on tear break up time and patients’ symptoms [223]. PUFAs have also proven to be effective in dry eye syndrome further to autoimmune rosacea disease. In fact, in rosacea patients which develop dry eye, diet supplementation with omega-3 for 6 months ameliorated dry eye parameters such as tear breakup time and Schirmer score [224].

Vitamins D and A

A nutritional deficiency, often associated to dry eye, is the lack of vitamin D [177,178,180,182,183]. Patients with vitamin D deficiency have a higher risk to develop dry eye disease [225]. The correlation of Vitamin D levels and inadequate sunlight exposure with severity of dry eye syndrome was highlighted in a recent study on Korean adults, concluding that sufficient sunlight exposure or vitamin D supplementation may attenuate signs and symptoms of dry eye [183]. Furthermore, an interesting correlation has been shown between single nucleotide polymorphisms in the vitamin D receptor gene and the incidence of dry eye disease in the Chinese Han population [226]. Vitamin D deficiency is relatively frequent in patients with primary Sjögren syndrome and also in this case its serum levels correlate with the severity of dry eye parameters [182,227,228]. In a retrospective observational study, in which subjects were divided into 3 groups according to serum Vitamin D levels (sufficient, inadequate or deficient group), tear break-up time and tear secretion were lower in the vitamin D-deficient group compared to the sufficient group [229]. In a recent work Dikci et al. observed that vitamin D deficiency may cause conjunctival squamous metaplasia and loss of goblet cells on the ocular surface in the eyes of 36 patients with mild to severe dry eye [230]. Inflammatory reactions and oxidative damage are common findings in dry eye, and in fact a study run on 217 patients (397 eyes) either normal or with various severity of dry eye has shown a correlation between its severity and the amount of oxidative tissue damage and polymorphonuclear leucocytes infiltration [231]. Within this frame, Eksioglu et al. evaluated the antioxidant effects of active vitamin D against high-dose radioiodine therapy-associated oxidative damage of the lacrimal gland in Wistar albino rats. Radioiodine caused significant oxidative stress and inflammation in lacrimal glands and vitamin D could blunt these effects, thus showing anti-inflammatory, antioxidant and radio-protective effects [232]. As mentioned before, chronic inflammatory events further to immunologic activation have a pivotal role in dry eye pathogenesis, and vitamin D is known to have immunomodulatory properties [179]. Further research on experimental animal models has provided evidence about the antinflammatory and immunomodulatory roles of vitamin D on the ocular surface, particularly in the cornea [233,234], and clinical data confirmed that a low serum vitamin D level is associated with different pathological states, such as autoimmune diseases, lymphoma, or neuropathy [218]. Reins et al. highlighted that vitamin D modulates the expression of inflammatory cytokines and protects corneal epithelial barrier function [180,235,236]. Antigen presenting Langerhans cells resident in the ocular surface epithelia have a role in aqueous tear-deficient dry eye pathogenesis [237], and topical administration of vitamin D can suppress ocular surface inflammation via inhibition of the increased Langerhans cells migration into mouse corneas [234]. The activation of inflammatory ROS-NLRP3-IL1β signaling axis induced by hyperosmotic stress has been recognized as a key priming stage of epithelial inflammation in dry eye pathogenesis [238,239]. Vitamin D has shown the ability to inhibit this signaling axis in human corneal epithelial cells under hyperosmotic stress via the activation of a competitive antioxidant signaling cascade dependent on the activation of Nrf2, a transcription factor regulating the expression of genes coding for antioxidant proteins [240]. Shetty et al. investigated the correlation between levels of serum vitamin D and tear-inflammatory proteins. They found that decreased serum vitamin D was significantly associated with higher levels of inflammatory interleukin IL-17A/F, interferon-γ, monocyte chemotactic protein 1, intercellular adhesion molecule 1, IL-4, IL-10, and decreased levels of antinflammatory IL-2 in tears of dry eye patients in comparison to healthy controls [241]. Altered tear fluid soluble factors with potential to modulate nociception exhibited a distinct association with the ocular surface discomfort status [242]. Vitamin D deficiency was found to be associated with neuralgia and chronic pain. In a case report it was observed that a vitamin D-deficient patient with corneal neuralgia had relief from burning sensation and pain by vitamin D supplementation, 1,000 IU/day, while topical therapies and lubricants were not effective [243]. It is thus probable that low vitamin D could contribute to the severity of ocular surface symptoms either by directly influencing nociceptive mechanisms or by affecting the presence of inflammatory cytokines [235,236,241,242]. Khamar et al., reported the dysregulation of tear fluid nociception-associated factors, corneal dendritic cell density, and vitamin D levels in dry eye patients. The use of lubricants (artificial tears or biological tear substitutes) is one of the strategies to manage dry eye symptoms [244,245], since tear film instability can lead to ocular surface damage [246]. Hwang et al., showed that Vitamin D enhanced the efficacy of topical carbomer- based lipid-containing artificial tears and Hyaluronic Acid (HA) in patients with dry eye and it may be a useful adjuvant therapy for patients with dry eye syndrome refractory to topical lubricants [247].

Vitamin A deficiency has also been suggested to be a contributory factor in the development of dry eye disease [248-250]. Vitamin A is essential for multiple functions in mammals including vision [251]; in fact it is a precursor in the formation of the rod photopigment rhodopsin [252]. An adequate consumption of vitamin A avoids night blindness, exophthalmia [253,254] and its deficiency is the main cause for blindness among children in third world Countries [255]. Apoptotic death of corneal epithelial cells is part of the pathological process of dry eye [256,257]. Vitamin A has been found to be able to prevent this event in a mice model of dry eye induced by benzalkonium chloride by decreasing the ratio between the pro-apoptotic gene Bax and the anti-apoptotic gene Bcl-2 [258]. In a recent clinical study on dry eye patients, tear ferning grades and tear osmolarity values were improved after vitamin A supplementation at a daily dose of 1,500 mg for 3 days [259].

lactoferrin

Lactoferrin deficiency in tears is a frequent finding in dry eye disease, especially in the elderlies [260]. Lactoferrin is a glycoprotein with several functions, including anti-inflammatory and antimicrobial, as well as promotion of cell growth, antiangiogenic and antitumoral [261]. Lactoferrin represents around 25% of tear proteins, with an average concentration in healthy subjects of 1.42 mg/ mL [262]. Its concentration decreases with prolonged closure of the eyelids, as it happens during sleep [263]. Lactoferrin concentration is also decreasing with age [264], and the decrease of its concentration in tear fluid correlates with the severity of dry eye disease [265-268]. Shimmura et al. reported a protective effect of lactoferrin against oxidative cellular damage in cultured corneal epithelial cells [269]. Accordingly, Fujihara et al. showed that lactoferrin eye drops may suppress the loss of corneal epithelial integrity produced in rabbits’ eyes by an ocular speculum blocking blinking [270]. Patients affected by Sjogren’s syndrome with dry eye symptoms and receiving 270 mg/day oral lactoferrin supplementation, showed improvements of both symptoms and tear film stability. This improvement reverted on cessation of treatment [271]. Moreover, oral lactoferrin administration preserves lacrimal gland function in aged mice by attenuating oxidative damage and suppressing subsequent gland inflammation [272]. Small incisions during cataract surgery may induce post-surgical dry eye, in which case oral lactoferrin may improve such condition [273]. Higuchi et al. showed the efficacy of Selenium-containing lactoferrin eye drops in a tobacco smoke exposure rat dry eye model and a short- term rabbit dry eye model of blinking block [274]. Rusciano et al. described the potential use of lactobionic acid, a functional mimetic of lactoferrin, in the management of age-related dry eye [260], and its efficacy is supported by the results of a recent study showing that lactobionic acid —with or without hyaluronic acid—favored wound healing both in vitro and in vivo, through an improvement of re- epithelization and the reduction of inflammatory markers [275].

Amino Acids

Amino acids are the building blocks of proteins and are essential for the construction and the functioning of the entire organism [276,277]. Amino acids are naturally present in human tears and their imbalance or deficiency could contribute to the development of the pathologies affecting the ocular surface [278]. Two different studies have reported that the concentration of amino acids is different in human tears and in serum [279,280] and that tear concentration changes in patients affected by ocular dryness [280,281]. As mentioned before, we know that chronic inflammatory events have a pivotal role in the pathogenesis of dry eye [207,208], and several studies have indicated that amino acids are involved in antinflammatory processes [282-284]. Dietary supplementation of arginine can enhance wound healing, regulate endocrine activity and potentiate immunity [285]. Glutamine may improve immunocompetence, thus reducing the susceptibility to infections and favoring the recovery of the seriously ill, therefore minimizing their hospital stay [286-292]. Branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, play critical roles in the regulation of energy homeostasis, nutrition metabolism, gut health, immunity and disease in humans and animals [293]. BCAAs have shown anti-inflammatory and anti-genotoxic activity in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages [294]. In a randomized, double-blind, placebo-controlled study, Dunn- Lewis et al. evaluated the antinflammatory properties and the capacity of improving physical performance of a multi-nutrient supplement containing leucine, isoleucine, valine, taurine plus anti-inflammatory plant extracts, and B vitamins. They reported that the multi-nutrient supplement was effective in improving the inflammatory status in both men and women, ameliorating the markers of pain, joints’ pain, strength, and power in men only, and both anxiety and balance (a risk factor for hip fracture) in women [295]. Refractive surgery may alter ocular surface homeostasis and Meibomian gland function, thus leading to the development of dry eye signs and symptoms [296-299]. Two different studies reported that oral administration of an amino acids mixture promotes the recovery and maintenance of corneal stroma health after refractive surgery [300,301]. In the first, Vinciguerra et al., demonstrated that oral supplementation of amino acids improved the healing rate of patients subjected to photorefractive keratectomy (PRK) and had shown re-epithelization defects, by favoring keratocyte growth and repopulation of the corneal stroma [300]. In the second study, Torres Munoz et al. reported that oral supplementation with a mixture of 13 different amino acids favored an active healing process and the remodelling of the stroma in patients operated of cataract surgery, by inducing growth and migration of stromal cells [301]. The identical oral supplement with 13 amino acids, used in the previous study, was also used by Roszkowska (Roszkowska, 2006, personal communication). The analyses of the corneal epithelium removed from patients during the procedure of PRK showed that amino acid treatment resulted in decreased EGF expression, suggesting a low proliferation rate of epithelial cells and keratocytes (hence, a decreased risk of scar formation); an increased expression of TGFβ, correlating again with decreased keratocyte proliferation and increased motility of epithelial cells (hence, faster re-epithelization); and a significant increase in the expression of polyamines, suggesting regulated differentiation and proliferation. Meduri et al. gave to patients undergoing PRK a cysteine oral supplement at a daily dose of 200 mg, then reporting shorter times for re-epithelization in comparison with eyes of untreated controls [302]. Observations at the confocal microscopy of the cornea of dry eye patients treated with eye drops containing HA and amino acids showed less hyper- reflecting cells in the epithelial layers (a sign of metabolic damage) in comparison to those receiving HA alone; nerve tortuosity in the subepithelial layer was also improving faster and better in the presence of topic amino acids [303]. The administration as eye drops of the amino acid taurine changed the tear proteome of contact lens wearers and dry eye patients shifting it towards the profile of healthy control subjects [304]. Taurine has then been included in a contact lens cleaning solution because of the regenerative effect on the tear film of contact lens wearers [305]. Taurine in combination with sodium hyaluronate has shown antioxidant and osmoprotective activity in in vitro and in vivo models of dry eye [306]. In albino rabbits, taurine effectively protected ocular surface tissues from chemical damage induced by hypochlorous acid, and arrested the progression of tissue damage (as measured by the level of lactate dehydrogenase activity) that had already been initiated by hypochlorous acid [307]. A mixture of taurine, alpha-ketoglutarate, pyruvate and pantothenate prevented corneal damage induced by 2-chloroethyl-ethyl sulfide [308]. Taurine could protect corneal stroma and epithelium from lactic acidosis caused by contact lens wearing [309], because of its acid buffering ability against lactic acidosis [310].

DIABETIC RETINOPATHY

Diabetic retinopathy (DR) is a multifactorial microvascular complication of diabetes mellitus caused by damage to the blood vessels of the retina, the light-sensitive tissue located at the back of the eye. DR has been included by the World Health Organization in the priority list of eye diseases which can be partly prevented, but not cured yet. In its early stages, DR may cause only mild vision problems but, over time, persistent high blood sugar levels can lead to the obstruction of the tiny blood vessels that nourish the retina, cutting off its blood supply. As a result, the eye reacts by triggering an abnormal growth of new retinal vessels, causing micro-hemorrhages and edemas in the macular region, thus leading to severe visual impairment and eventually blindness. These intra-retinal microvascular changes are used to classify DR into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). NPDR is characterized by a complex array of vasodegenerative lesions within the retinal microvascular bed, such as thickening of capillary basement membranes (BMs), loss of pericytes and vascular smooth muscle cells, capillary occlusion and microaneurysms [311]. PDR is caused by an abnormal growth of new blood vessels (retinal neovascularization) in response to inflammation and/or ischemic damage and hypoxia, eventually giving rise to vitreous hemorrhages and tractional retinal detachment. A direct consequence of inner blood–retinal barrier (iBRB) breakdown is the development of macular edema. Retinal neovascularization and macular edema are the result of increasing secretion of pro-inflammatory cytokines, and pro-angiogenic growth factors, among which predominates the vascular endothelial growth factor (VEGF) [312]. The retina is a highly metabolic active tissue, and high-glucose concentrations are particularly detrimental to its functioning. As reported by Sayin and collegues, chronic hyperglycemia is the main risk factor for DR because of its toxic effects on pericytes, to which vascular endothelial dysfunction may follow, also involving the retina [313]. A chronic hyperglycemic status will activate alternative energetic and metabolic pathways besides glycolysis, generating a toxic oxidative stress leading pericytes to apoptotic death, and causing vascular degeneration in the retina [314-316]. The physiopathology of DR could be represented by a pyramidal scheme in which the hyperglycemic state is at the bottom of the pyramid, immediately topped by oxidative stress, lipid peroxidation, apoptosis, mytochondrial alterations, leading to endothelial dysfunction and finally to retinal damage and neurodegeneration [317-319]. Under normal conditions, a certain amount of reactive oxygen species (ROS) can be tolerated, because they derive from the metabolic processes necessary to sustain cellular proliferation, differentiation and their physiological activities; however, if the production of free radicals overwhelms the capacity of the antioxidant defenses, the normal cellular metabolism will be disrupted by oxidative stress, degrading nucleic acids, proteins and enzymes, eventually leading to organ and tissue pathologies, such as DR [320]. Kowluru et al. [321] and Kaštelan et al. [322] have demonstrated that oxidative stress not only is involved in the physiopathology of DR, but also hinders its remission when glycemic values return within the normal interval. Typically, metabolic pathways altered in DR (polyol, hexosamine and protein kinase C pathways) lead to ROS imbalance, or result from oxidative stress, giving rise to advanced glycation end products (AGE). It is well known that visual impairment is already manifest in the early stages of DR, but currently no efficient treatment has been found to arrest its progression. Treatments have been developed for the late stages, and PDR can be treated or at least contained by pan-retinal laser photocoagulation, vitreoretinal surgery, or intravitreal injections of anti-inflammatory and anti-angiogenic drugs, even though retinal and visual function integrity cannot be preserved. Therefore, effective treatments for all stages of DR are needed to help in preventing or delaying the development and progression of this diabetes-induced visual dysfunction [323]. Most recently [324], it has been suggested to implement the use of nutraceuticals, which may act upstream the disease, reducing neuronal stress and favoring neuroprotection, in order to slow down its progression. Considering that antioxidants can act as free radical scavengers providing protection against oxidative stress, a good strategy could be to use nutrients with antioxidant and/ or anti-inflammatory activity and a good safety profile. In fact, in the last years the optimization of the glycemic and lipidemic control by a correct dietetic regimen has been shown to be of great relevance in controlling diabetes progression and its complications [325,326]. Thanks to their low toxicity in comparison to drugs, the use of food- derived bioactive molecules can be considered a good strategy to contrast the risk factors contributing to DR. In this paragraph, we will sum up the effects of nutritional strategies used as adjuvant therapies, on the different pathways involved in the physiopathology of DR (Figure 3).

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Figure 3. Diabetic retinopathy. Diabetic retinopathy is a frequent complication of diabetes, and mainly results from the oxidative stress and the inflammatory events triggered by the continuous hyperglycemic state of the organism and the retinal vasculature. Initially, local injuries are visible (NPDR), eventually leading to the proliferation of new vessels in the macular area (PDR) and the loss of sight (upper panel). The food supplements that have been shown some efficacy in slowing down the progression of DR have antioxidant, antinflammatory and vasoprotective activities (bottom panel).

Polyunsaturated Fatty Acids (PUFAs)

A healthy diet widely known and appreciated is the Mediterranean diet, rich in vegetables, fish and extra virgin olive oil containing antioxidant and anti-inflammatory factors. Among these, we can find phenolic antioxidants; gamma- and delta-tocopherols and tocotrienols; long chain PUFAs: omega-6 and mostly omega-3, from which DHA and EPA are derived; several carotenoids of which lycopene may be the most active; isothiocyanates from cruciferous vegetables; sulfur compounds from allium vegetables; terpenoids. Interestingly, most of these health-promoting nutrients function, at least in part, by raising the nuclear factor Nrf2 (nuclear erythroid factor 2-related factor 2), responsible for the transcription of over 500 genes in the human genome, most of which have cytoprotective functions [327]. The role of PUFAs in the retina has been intensely studied for their involvement in regulating vascular function and angiogenesis. Tikhonenko et al. have demonstrated, by transcriptomic analysis in diabetic rats, that diabetes induced a downregulation of retinal enzymes known as elongases, necessary to metabolize PUFAs. This event resulted in a decreased content of retinal DHA, as well as decreased incorporation of very long-chain poly unsaturated fatty acids, particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with the presence of an inflammatory status in diabetic retina, reflected by an increase in gene expression of the proinflammatory markers IL6, VEGF, and ICAM-1 [328]. Similar results were obtained by a lipidomic analysis showing that the omega-3 long-chain poly unsaturated fatty acids metabolites derived from the activity of cyclooxygenases and lipoxygenases, inhibit inflammation and angiogenesis [329]. Connor and colleagues showed that in mouse models of oxygen-induced retinopathy the dietary intake of omega-3- and omega-6 PUFAs decreased the avascular area of the retina and increased vessel regrowth after injury, thereby reducing the hypoxia- induced pathological neovascularization. Moreover, while the intake of omega-6 PUFAs increased the microglial production of the pro- inflammatory and pro-angiogenic cytokine TNF-α, such increase was prevented by the intake of omega-3 PUFAs, which are the precursors of the mediators neuroprotectin-D1, resolvin-D1 and resolvin-E1, known to be potent inhibitors of neovascularization [330]. This anti- angiogenic effect induced by omega-3 PUFAs on retinopathy in the mouse eye is comparable in magnitude to treatment with an inhibitor of VEGF [331]. Therefore, increased dietary intake of omega-3 PUFAs could be a useful addition to anti-VEGF therapy to control pathological retinal angiogenesis. Another important function of omega-3 PUFAs is correlated with the attenuation of apoptotic events induced by mytochondrial and endoplasmic reticulum oxidative stress by a reduction of caspase-3 activity in adipocytes via AMPK activation, and increased production of resolvins, a family of neuroprotector molecules derived from EPA and DHA [332]. Overall, omega-3 fatty acids act on different pathophysiological pathways of DR such as inflammation, oxidative stress and neoangiogenesis, showing a remarkable potential in the prevention of retinal disease progression.

Polyphenols

Polyphenols belong to a family of natural compounds characterized by one or more hydroxyl groups (OH) linked to a benzene ring. These natural substances are present in a variety of foods including grapes, berries, dark chocolate, coffee and tea to mention a few, and are characterized by strong antioxidant and anti-inflammatory power, so that they are widely used in the prevention and/or treatment of different pathologies [316,324]. Several studies have shown that dietary polyphenols exert a protective effect against progression of age-related ocular abnormalities such as cataract, glaucoma, DR and macular degeneration [333]. Based on their structure, polyphenols are classified into non-flavonoid and flavonoid compounds. Flavonoids consist of two phenolic rings linked to a pyranosic ring and are highly represented in fruits, vegetables, roots, and wine.

Anthocyanins (anthos = flower and kyanos = blue) are a subgroup of flavonoids, water-soluble pigments very common in the plant kingdom and in many typical foods of the Mediterranean diet such as blueberries, aubergines, peaches, oranges, figs, cherries and olives. Consumption of foods rich in anthocyanins has been associated with a reduced risk of cardiovascular diseases. As reported by Fang and colleagues [334] anthocyanins can be absorbed as such through the gastrointestinal wall, where they undergo an extensive first- pass metabolism and the metabolites so formed enter the systemic circulation. In fact, anthocyanin metabolites were detected in the blood stream in much higher concentrations than their parent compounds. Several studies reported that dietary intake of anthocyanins may confer benefits in different brain functions, including vision. For example, in blueberry-fed pigs, which are a suitable model to mimic human digestive absorption, anthocyanins were detected in all examined tissues, including brain and eyes, thus indicating that anthocyanins can accumulate also in tissues beyond the blood-brain barrier [335]. Other studies have shown the role of anthocyanins as antioxidants in the retinal pigmented epithelium (RPE) where they can neutralize the ROS formed by the metabolic activity of photoreceptors thus playing a role in neuroprotection as also shown by their protective effect in a model system of rat retinal neurons damaged by N-methyl- N-nitrosourea [336]. Song and colleagues [337] addressed the effects of blueberry anthocyanins on retinas of diabetic rats subjected to oxidative stress and inflammation, demonstrating their antioxidant potency resulting in increased expression of HO-1 and nuclear localization of the transcription factor Nrf2 (which controls the expression of antioxidant response element-dependent genes) finally resulting in increased expression of GSH and glutathione peroxidase, and reduced levels of malondialdehyde (MDA) and ROS. Moreover, cyanidins and the orthodihydroxy group of anthocyanins can inhibit lipid peroxidation by chelation of metal ions [338].

Another flavonoid with antioxidant and anti-inflammatory properties in the retina is quercetin. It has been reported that in mouse retinal photoreceptor cells, quercetin inactivated the pro-inflammatory transcription factor NF-κB through inhibition of both mitogen- activated protein kinases (MAPK) and Akt, reducing in this way VEGF-induced inflammation [339]. Kumar et al. [340] demonstrated the retinal neuroprotective effects of quercetin. In streptozotocin- induced diabetic rats, quercetin blunted inflammation and glyosis decreasing the retinal expression level of glial fibrillary acidic protein (GFAP) and NF-κB in specific retinal layers, such as the nerve fiber layer, the inner plexiform layer, and the inner nuclear layer. The effect of quercetin on NF-κB was also associated with decreased levels of TNFα and IL-1β. Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a non-flavonoid polyphenol, and a phytoalexin found in red wine and grape skin. Many studies demonstrated its antioxidative and anti- inflammatory properties, so that it has been often used to mitigate those eye diseases with a prevalence of oxidative stress, such as DR. In vitro studies have shown that resveratrol prevents ROS-induced apoptosis in retinal capillary endothelial cells stressed by high glucose, through the activation of the AMPK/Sirt1/PGC-1α pathway [341]. Other in vitro studies on the human pigmented epithelium ARPE-19 cell line put under chemically mimicked hypoxia by cobalt chloride have demonstrated that resveratrol could reduce oxidative stress and the production of pro-angiogenic and pro-fibrotic factors [342]. In the same model system, hower subjected to high glucose oxidative stress, resveratrol inhibited the stimulated secretion of inflammatory cytokines such as IL-6 and IL-8, and prevented the downregulation of gap junction associated connexin 43 while inducing the activation of TGFβ, PKCβ, and COX-2 [343]. In vivo studies by Chen and colleagues with streptozotocin (STZ)-induced diabetic rats, have revealed the effects of paraoxonase-1 (PON1) as an intermediary in the protective effects exerted by resveratrol. In fact, oral resveratrol treatment – via induction of PON1 in the retina – blunted the increased retinal vascular permeability, pro-apoptotic caspase-3 activity, retinal damage, as well as clearly showed an inhibitory effect on inflammatory markers, such as IL-1β, IL-6, TNFα, VEGF, IFN-γ and monocyte chemoattractant protein-1. Therefore, these data suggest that PON1 induction by resveratrol in the retina may be a promising therapeutic strategy to prevent the progression of diabetes-related retinopathy [344]. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)- 1,6-heptadiene-3,5-dione) is another non-flavonoid polyphenol extracted from the root of Curcuma longa and exhibits a wide range of pharmacological properties, which include antioxidant, anti- inflammatory, antimutagenic, antimicrobial and anticancer activities. For its anti-inflammatory and antioxidant properties, curcumin is exploited as oral supplementation therapy for retinal degenerative diseases, including DR [345]. The World Health Organization stated that an acceptable daily intake of curcumin can be up to 3mg/kg as a food additive [346]. However, this “golden spice” despite a good efficacy has a limited bioavailability due to poor absorption and rapid metabolism and elimination. Clinical trials have shown that curcumin is safe in humans and well tolerated up to doses of 12g/day [347]. The antioxidant and anti-inflammatory activities of curcumin (10 µM) have been tested on human retinal pigmented epithelial cells, human retinal endothelial cells and human retinal pericytes exposed to oxidative stress with high glucose. Results demonstrated a significant decrease of ROS concentration in retinal pigmented epithelial cells and of TNF-α release in retinal endothelial cells, while protecting retinal pericytes from high-glucose damage [348-349]. Human retinal endothelial cells exposed to high glucose and treated with curcumin (10 µM) showed an increase of HO-1 expression [350], a stress response protein that is highly inducible under various conditions, such as oxidative or heat stress. This observation suggests that curcumin may have both a direct and indirect antioxidant activity, due to the activation of Nrf2. Once activated, Nrf2 translocates into the nucleus and promotes the transcription of genes that encode antioxidant enzymes including HO-1 [351,352]. In vivo studies have been done in rats with STZ-induced diabetes and treated with curcumin given by different routes. Intraperitoneal injections of curcumin (80mg/ kg, once a day), prevented the retinal increase of malondialdehyde (MDA: a marker of oxidative stress) and the decrease of antioxidant GSH levels [353]. In the same model, Gupta et al. [354] have shown that oral curcumin (1 gr/Kg) showed hypoglycemic activity, decreased significantly the superoxide dismutase and catalase activities and prevented the increase of the proinflammatory cytokines TNF-α and VEGF in diabetic retinae. Moreover, they observed increased antioxidant levels of superoxide dismutase (SOD), catalase, and GSH and a lesser increase of retinal nitrotyrosine, a marker of oxidative protein damage, and of 8-hydroxy-20-deoxyguanosine, a marker of oxidative DNA damage [355].

Carotenoids

Carotenoids are naturally occurring pigments found in fruits and vegetables, but also in plants, algae, and photosynthetic bacteria. Carotenoids can be divided into two main classes: carotenes (precursors of vitamin A) and xanthophylls (non precursors  of vitamin A). Carotenes are non-polar molecules, containing only carbon and hydrogen atoms, while xanthophylls are polar carotenoids, containing at least one oxygen atom [356]. Several studies demonstrated the beneficial power of carotenoids on human health and more specifically on eye health, exerting primarily antioxidant effects against AMD, age-related cataract (ARC), uveitis and DR [357]. Humans cannot synthesize carotenoids which must be supplemented by foods in order to be distributed to various tissues, especially to the retina. Lutein and zeaxanthin are non-provitamin A carotenoids from the xanthophyll family. These carotenoids are characterized by OH groups on each end. Their amphipathic character allows them to be inserted into the lipid bilayer of cell membranes and in the outer monolayer of lipoproteins. These xanthophylls have been found in the human retina, particularly in the macular region [358]. The highest concentration is found in the fovea, and in primates they are responsible for the characteristic central yellow coloration known as the macula lutea. The fovea contains approximately 13ng/ mm2 of carotenoids, compared with approximately 0.05ng/mm2 in the peripheral retina [359]. The main mechanism by which lutein and zeaxanthin are involved in the prevention of retinal diseases appears to be due to their local antioxidant activity and their capacity to filter the blue light, responsible of photo-oxidative damage [360]. Elimination of lutein from the diet of experimental animals resulted in early degenerative signs in the retina, and patients with an acquired condition of macular pigment loss (Macular Telangiectasia) showed a serious visual handicap, highlighting the importance of macular pigment. Accordingly, a recent clinical study reported that thanks to lutein capacity of filtering short-wavelenght light, its administration to patients with NPDR led to some improvement in glare sensitivity [361]. Another study on diabetic rats treated with zeaxanthin has shown its ability to bring the retinal concentration levels of VEGF and intercellular adhesion molecule-1 (ICAM-1: related to the inflammatory state of the tissue) to values comparable to healthy controls. Moreover, zeaxanthin supplementation prevented diabetes-associated retinal damage by decreasing nitrotyrosine levels, DNA oxidative damage, lipid peroxidation and the decrease of retinal SOD [362]. Similarly, lutein administration to diabetic mice resulted to prevent retinal oxidative stress and restored normal retinal ROS levels [363]. Hu et al. addressed the potential benefits of supplementation with lutein and zeaxanthin in type 2 diabetic patients with diagnosed NPDR [364]. In this study, plasma concentration of these carotenoids was substantially lower in the diabetic group than in normal subjects at baseline. After 3 months treated patients presented higher plasma concentrations in comparison to untreated controls, correlating with reduction of the macular edema and improvement of visual acuity. Overall, these data indicate that lutein and zeaxanthin are main actors on the photo-oxidative stage, by quenching oxygen singlets, scavenging free radicals and protecting retinal cells from oxidative damage. Their dietary intake may be used to ameliorate or even to reverse vision loss in DR patients. Astaxanthin is a third member of the xanthophyll family, a ketocarotenoid generated by the oxidation of β-carotene, and is a common pigment in algae, fishes, and birds. One of the most important properties of astaxanthin is a potent antioxidant activity, almost 10 times higher than other known compounds such as polyphenols or β-carotene [365]. Because of this property, astaxanthin has been studied, mainly in animal and cell models, for its possible role in the treatment of chronic diseases involving oxidative stress, such as in diabetes and its complications. Yeh et al. [366] addressed the effect of orally administered astaxanthin on STZ-induced diabetic rats. Ocular tissues from astaxanthin and lutein treated rats showed a significant reduction of oxidative stress and inflammatory mediators depending on NF-κB transcription activity, and increased levels of antioxidant enzymes, thus preserving retinal architecture and function. Zhou and colleagues further demonstrated that astaxanthin can regulate the glycemic state and reduce insulin resistance, and can also exert an anti-inflammatory and anti-angiogenic effect by decreasing the expression of NF-κβ and TNF-α, thus inhibiting the expression of proinflammatory molecules such as ICAM-1, the monocyte chemoattractant protein-1 and VEGF [367]. In conclusion, several studies indicate that astaxanthin, zeaxanthin and lutein may blunt the generation of oxidation products in the retina, so that their oral uptake could be a good strategy to control the development of DR by reducing the oxidative damage to DNA, proteins, and lipids.

Vitamin c

Vitamin C is a water-soluble vitamin that exists in two main forms, ascorbic and dehydroascorbic acid. It is an important nutraceutical used as a support to therapy to treat oxidative-stress-induced diseases. Vitamin C scavenges ROS such as superoxide and peroxynitrite in plasma and cells (thus avoiding damage to proteins, lipids and DNA), prevents occludin dephosphorylation and the loosening of tight junctions. Several studies have reported that serum vitamin C levels were lower in diabetic patients in comparison to healthy subjects [368], and these levels were much lower in diabetic patients with DR [369]. The vitreous level of vitamin C is responsible for the regulation of oxygen tension and oxidative stress in the eye, which are implicated in retinal ischemia and the development of PDR [370]. Duarte et al. have demonstrated that in healthy individuals the concentration of vitamin C in vitreous humor is remarkably higher as compared to that in serum [371]. In patients with PDR, the vitreous level of vitamin C was decreased up to tenfold compared to healthy controls and was associated with the degree of macular ischemia, suggesting that vitreous vitamin C depletion may contribute to macular ischemia in PDR patients [370]. Ascorbic acid (AsA) has anti-inflammatory, immunostimulant, and antibacterial properties, and is used as adjuvant therapy in many inflammatory disorders [372]. In a double- blind cross-over trial [373], 8 diabetic patients were treated with AsA tablets showing that the supplementation of 1000 mg AsA/day for 2 weeks reduced the activity of the enzyme aldose reductase thus inhibiting the polyol pathway, which is activated in DR. Diabetic hyperglycemia triggers apoptosis of vascular pericytes thus impairing vascular regulation and weakening vessels, especially in brain and retina. May et al. demonstrated that vitamin C prevented high glucose- induced perycytes apoptosis, that was largely due to the activation of the receptor for advanced glycation end products (RAGE) [374]. AsA may also have a role in angiogenesis. A recent study conducted on human umbilical vein endothelial cells (HUVECs) has shown that AsA prevented the increase of endothelial barrier permeability induced by VEGF. This effect may contribute to reduce the macular edema in DR [375].

Alpha-lipoic Acid

Alpha-Lipoic Acid (1,2 dithiolane-3-pentanoic acid) (aLA), also known as thioctic acid, is a natural thiol antioxidant. After oral supplementation, it is quickly transported to intracellular compartments and reduced by enzymes to dihydrolipoic acid. This reduced form has many biochemical functions. It acts as biological antioxidant, metal chelator, and regenerates by reducing their oxidized forms other antioxidant agents such as vitamin C, E and GSH. It also functions as modulator of the signaling transduction of pathways involving insulin or NF-kB [376]. aLA plays an essential role in mitochondrial bioenergetic reactions, which makes it useful in managing diabetic vascular complications such as retinopathy and peripheral neuropathy. In an in vivo study [377], the mitochondrial functional integrity, biogenesis and DNA copy number were analyzed in the retina of STZ-diabetic rats. Animals under good glycemic control and fed with aLA have shown a lesser dysregulation of retinal mitochondrial biogenesis as measured by citrase synthase (a marker of mitochondria functional integrity) and the presence of fewer acellular capillaries (a marker of DR). In another experimental study [378], Wistar rats were treated with aLA (60 mg/kg bodyweight) i.p. daily for 30 weeks. Results demonstrated that aLA treatment reduced oxidative stress, NF-kB activation and angiopoietin-2 expression, and reduced the amount of VEGF in the diabetic retina by 43%. In a randomized, double-blind placebo-controlled study, 32 patients with preretinopathic diabetes were treated with aLA (400 mg/daily) in combination with other antioxidants (genistein and vitamins). Oxidative stress levels in plasma and changes in the full field ERG were evaluated. Results revealed that the oral treatment with antioxidants in preretinopathic diabetes subjects may have a protective effect on retinal cells, as detected by ERG analyses observed after 30 days of treatment [379]. In conclusion, several studies show that nutritional supplementation of natural molecules, as discussed above, protects neuronal and vascular cells from inflammatory damage and mitochondrial degeneration, thus slowing down DR progression and helping diabetic patients to spare their vision for longer times [380].

AGE-RELATED-MACULAR DEGENERATION

AMD is a progressive degenerative disease of the retina principally affecting the macula, a highly specialized region of the central retina responsible for fine and colour vision. It is currently considered the leading cause of visual impairment and blindness among patients over 60 years [381]. AMD can occur in two distinct forms: the most frequent (roughly 90% of the cases) is the dry (atrophic, non- exudative) form, characterized by diffuse insoluble debris (known as drusen) causing the death of RPE cells and photoreceptors [381]; the wet (neovascular, exudative) form is less frequent (roughly 10% of the cases) but still responsible of the majority of blindness cases, and is characterized by the development of abnormal new vessels inside the macular region (choroidal neovascularisation) [382]. Oxidative stress and inflammation along with genetic risk factors are all involved in AMD pathogenesis [383]. An important and characteristic clinical hallmark of AMD is the presence of drusen – small yellow or whitish accumulations of extracellular debris (made of proteins and lipids) located between the basal lamina of the RPE and the inner collagenous layer of Bruch’s membrane [382,384]. In particular, these protein/ lipid deposits are associated with an inefficient metabolism of RPE cells, which cannot get rid of catabolic products which then tend to accumulate in the extracellular spaces giving rise to drusen bodies [385]. Another early molecular event of AMD is the accumulation within RPE cells of lipofuscin, a remnant of the recycling of photoreceptor outer segments, that contributes to RPE degeneration [386]. Oxidative stress is a leading mechanism in the generation and progression of AMD, therefore antioxidant self-defense mechanisms are crucial for the mainteinance of retinal health. Two main targets of oxidative stress in RPE cells are the elements involved in autophagy and the transcription factor Nrf2. Dysregulation of both autophagy and the induction of antioxidant enzymes regulated by Nrf2 may trigger the cascade of events finally leading to lipofuscin and drusen generation, and start the inflammatory process causing AMD [387]. Inflammation processes thus induced occur through recruitment of macrophages and microglia, and complement activation [388]. Allelic variations of genes coding for complement factors, beside those participating in lipid metabolism or angiogenesis, constitute risk factors for AMD development [389]. Other risk factors include age, genetic predisposition, exposure to light, race, smoking habit, altered body/mass index, and diet [390-392]. Currently, the only therapy approved for the treatment of the dangerous wet form of AMD involves the inhibition of the pro-angiogenic factor VEGF in the retina. However, the search for more effective treatments of dry AMD is actively ongoing (Figure 4) [387].

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Figure 4. Age related macular degeneration. Oxidative stress and inflammation further to ageing, a wrong lifestyle and hyper reactivity of the natural immunity (mainly the complement system) may lead to the accumulation of catabolic debris (drusen) between the RPE and the Bruch’s membrane (dry or atrophic MD), and eventually to the rupture of the Bruch’s membrane and the invasion of new blood vessels into the macular area (wet or neovascular MD) (upper panel). Food supplements that may be useful to blunt these events have antioxidant, antinflammatory, antiapoptotic and antiangiogenic effects (bottom panel).

Food Supplements

In recent years, epidemiological and clinical studies have suggested the importance of nutrients and food supplements to prevent or slow down the progression of AMD in the elderly. In particular, since free radicals and ROS are known to be the principal factors behind this pathology, most of the nutrients studied have antioxidant and free radical scavenger characteristics [382,393]. They belong to several classes: polyphenols (anthocyanins, tannic acid, quercetin, curcumin, EGCG, resveratrol and herb rosemary), carotenoids (lutein, zeaxanthin, and β-carotene), vitamins (A, C, D and E) and minerals (zinc, selenium and copper). Other molecules with relevant antiaging and antioxidant properties used for AMD are aLA, omega-3 fatty acids and Lactobacillus paracasei KW3110 [382,394-396]. The first important trial evaluating food supplements for AMD prevention has been the Age-Related Eye Disease Study (AREDS) started in 1992. Results have shown that a formulation with vitamin C (500 mg), vitamin E (400 IU), β-carotene (15 mg) and zinc (zinc oxide 80 mg and cupric oxide 2 mg) showed a 25% risk reduction of the progression to advanced AMD over 5 years in patients with intermediate or advanced AMD [397]. A second trial, designated AREDS2, conducted between 2006 and 2012, was carried out to explore the added benefits of lutein, zeaxanthin and omega-3 fatty acids to the AREDS1 diet [398]. This study provided an opportunity to further refine the original AREDS formulation by eliminating ß-carotene (because of increasing risk of lung cancer in smokers) and lowering the dose of zinc (because of the potential health risks of taking large quantities of zinc). AREDS2 found that lutein and zeaxanthin reduced the risk of developing advanced AMD by a further 10 per cent. For participants who had a poor dietary intake of lutein and zeaxanthin a further decrease of 20% of the risk of progression to advanced AMD was observed; omega-3 fatty acids did not appear to further improve the efficacy of the AREDS1 diet, and the reduced amount of zinc conserved the same effects of the original higher amount [398,399].

Polyphenols

Anthocyanins (a subgroup of flavonoids, with cyanidin and delphinidin among the most common) are red–purple pigments found in plants [400]. Berries, such as blueberry, blackcurrant, strawberry, and wolfberry (better known as ‘goji berry’), are all rich in anthocyanins [401]. They have shown favorable effects on AMD for their antioxidant and anti-inflammatory properties, as demonstrated by both in vitro and in vivo models. Anthocyanins such as cyanidin 3-glucoside (C3G) promote regeneration and new synthesis of rhodopsin (the photosensible pigment of rod photoreceptor cells) and protect the retina from overexposure to visible and UV light [402-404]. A clinical study on 72 post-menopausal women showed that combining anthocyanin supplements with lutein and zeaxanthin did not influence their uptake in serum and macular pigment, so that an additive protection effect could be expected [405]. Tannic acid (a tannin derivative) contained in the polyphenolic extract of some plants, has been shown to protect human RPE cells from UVB damage, by inhibiting the activation of the transcription factor STAT3 and the downstream induction of the pro-inflammatory cytokine IL-6 and downregulating the expression of the complement factor B, also involved in the inflammatory cascade leading to AMD [406]. Quercetin is an another natural polyphenol with known antioxidant and anti-apoptotic properties, the protective efficacy of which has been demonstrated both in vitro and in vivo models. RPE exposed to cytotoxic and inflammatory effects induced by 4-  hydroxynonenal [407] or by blue light [408] were prevented by quercetin. In a different model of peroxide-induced oxidative stress model in RPE cells [409] quercetin treatment influenced the transcription of genes involved in apoptosis regulation, including the anti-apoptotic Bcl-2 and the pro- apoptotic BAX and FADD, and the effectors of apoptosis Caspase-3 and Caspase-9. Intraperitoneal administration of quercetin blunted the photo-oxidative damage of rat retinas’ photoreceptors exposed to intense light [410]. Curcumin, a polyphenol from Curcuma longa (turmeric plant) belonging to the ginger family, is also able to modulate apoptosis, inflammation and oxidative stress defense proteins [411]; these effects follow the upregulation of the anti-apoptotic protein Bcl- 2, the downregulation of the pro-inflammatory transcription factor NF-kB, and the upregulation of the antioxidant proteins SOD, GR and HO-1, as shown in human retinal cell models exposed to oxidative damage [350,412]. An active phenolic component of green tea, EGCG, protected human ARPE19 cells from UVA-induced damage by decreasing both the production of hydrogen peroxide and the activation of the pro-inflammatory MAPK and COX-2 [413]; moreover, it has shown a protective effect in RPE cells against oxidative stress-induced damage by modulating the expression of the transcription factor Nrf2 [414]. Resveratrol (3,4,5-trihydroxystilbene), a polyphenolic antioxidant belonging to the stilbene family and commonly found in grape skin and seeds, has been shown to be protective against UVA-induced damage in ARPE19 cells [415] and to prevent the development of choroidal neovascularization by modulating protein kinase A (PKA) in macrophages [416]. A recent study has suggested that resveratrol could mitigate the adverse effects induced by repeated injections of bevacizumab (anti-VEGF antibody) in wet-AMD therapy [417]. One of the major limitations of anti-VEGF therapy is an excessive reduction of the available secreted VEGF, which is also important to maintain the homeostatic function of the RPE. In ARPE-19 cells, various combinations of resveratrol and bevacizumab prevented VEGF reduction observed after bevacizumab alone treatment [417]. A prospective, randomized clinical trial (NCT02625376) started on August 2015, the results of which have not been released yet, aimed to evaluate the safety and efficacy of resveratrol to reduce the progression of exudative AMD between two groups of patients, either receiving 250mg of resveratrol or placebo, after 24 months of follow-up. The common herb rosemary (Rosmarinus officinalis) contains a variety of polyphenolic compounds which, combined with zinc, decreased the amount of CEP-proteins (carboxyethyl-pyrrole modified proteins, found in drusen and considered a significant risk factor for the development of AMD), and improved photoreceptor cell survival in rat retinas exposed to photo-oxidative damage [418]. Chronic supplementation with rosemary antioxidants has been suggested to be a useful addition to the therapeutic benefits of AREDS supplements in slowing down the progression of AMD [419,420].

carotenoids

Lutein, zeaxanthin and meso-zeaxanthin are carotenoids present in high concentration in the macula where they form the macular pigment protecting the retina from the oxidative stress constantly induced by light [421]. They are not synthesized by mammalian cells but found in egg yolk and vegetables such as fruits, spices, lettuce, broccoli, and spinach [422]. In an oxidative stress model obtained in manganese superoxide dismutase knockout mice, zeaxanthin supplementation blunted the oxidative stress and preserved the structure of the RPE [423]. Human clinical studies demonstrated that lutein and zeaxanthin, as well as βcarotene supplementations have protective effects on AMD, improving visual performance measured as contrast sensitivity, glare tolerance, and photo-stress recovery [396,424]. Several other studies have shown that high blood levels of lutein and zeaxanthin correlate with a significant lower risk of AMD [425,426].

Vitamins

Vitamins A, C and E appeared to reduce the risk of macular degeneration according to the AREDS studies I and II [397,398]. Positive outcomes concerning high dietary intakes of fruits and vegetables rich in pro vitamin A and vitamin C have been reported [394,427]. Vitamin C is known to be a potent antioxidant protecting proteins, lipids, carbohydrates and nucleic acids from free radicals and ROS damage [428]. Moreover, the AREDS Study Group has revealed that high vitamin C intake is associated with a reduced likelihood of neo-vascular AMD [429]. Vitamin E, similarly to vitamin C, is a powerful antioxidant. Its deprivation leads to lipofuscin accumulation [430], retinal damage [431] and loss of photoreceptors [432]. High dietary intakes of vitamin E have been clinically correlated with a slower progression of AMD [433]. Vitamin D may exert beneficial effects on AMD prevention by modulating the immune system and inhibiting inflammation and angiogenesis. The role of vitamin D in the progression of AMD has been addressed recently, because the cognate receptor is expressed by mammalian RPE [434]. However, among the many clinical observations and studies run on the efficacy of vitamins A, C, E, not all the results could confirm their efficacy on the decourse of AMD [396]. As to vitamin D, its role in the prevention of AMD seems to be ascertained, though the amount to be given as food supplement is still in need of better clarification [435].

Minerals

Zinc, copper and selenium play important roles in the normal functioning of antioxidant enzymes and in retinal cell survival [436,437]. Supplementation of zinc and copper decreases the risk of AMD progression suggesting that these metals play an important role in the homeostasis of AMD and in retinal health [438]. In particular, zinc supplementation effectively blunts the risk of AMD and visual loss by its antioxidant properties, and by intervening in the regulation of the light-rhodopsin reaction, in the modulation of synaptic transmission and of complement activation [438]. Selenium is a well-known anti-oxidant agent, and may reduce the risk of AMD by blunting the oxidative damage of membrane lipids and by participating in the activity of GSH antioxidant defense [437]. Moreover, a diet deficient in vitamin E and selenium in laboratory animals resulted in a consistent decrease of total PUFAs in RPE and in retinal rod outer segments, which could be recovered by an adequate diet supplementation [439]. However, more clinical evidence has to be raised in order to better understand the role, the efficacy and the dietary doses of these minerals in the prevention and treatment of AMD.

Alpha-Lipoic Acid

aLA is a naturally occurring fatty acid found in some foods such as yeast, spinach, broccoli and potatoes [376]. It works as a cofactor of mitochondrial dehydrogenase and as a free radical scavenger, regenerating endogenous antioxidant systems like vitamins C and E and the enzyme SOD [440]. aLA dietary supplementation has been shown to improve vision-related quality of life in dry AMD patients most likely by improving the antioxidant defense [441]. aLA entered a phase 2 clinical trial in May of 2016 sponsored by the University of Pennsylvania (NCT02613572), with the aim of assessing safety and tolerability of the 800 and 1200 mg daily doses in patients affected by geographic atrophy [417].

Omega-3 Fatty Acids

The deficiency of omega-3 fatty acids DHA and EPA in photoreceptor membrane lipids play an important role in the pathogenesis of AMD because their imbalance contribute to drusen formation in the RPE and sub-RPE layers [442]. Oral treatment with a complex mixture of fatty acids (commercially available as Macular- FAG®) has been used in a mouse model system of AMD induced by subretinal injection of polyethylene glycol (PEG-400). Results have shown a blunting of complement-mediated inflammation, a decrease of macrophage recruitment and modulation of the production of pro-inflammatory and angiogenic cytokines, finally resulting in a functional rescue of retinal electrophysiology [443].

Lactobacillus Paracasei KW3110

Anti-inflammatory agents appear to be optimal candidates for AMD prevention or slowing of its progression. Most recently, Morita et al. have demonstrated that Lactobaciullus paracasei KW3110 (a lactic acid bacterium) may activate M2 macrophages (macrophages associated with anti-inflammatory reactions), thus suppressing blue light-induced retinal inflammation both in vitro and in vivo. These results suggest that L. paracasei KW3110 may have a preventive effect against degenerative retinal diseases, including AMD [395].

MYOPIA

Aristotle is considered to be the first person who described myopia. The term myopia derives from the Greek’s verb μύειν, which means ‘to close’ and ωψ which means ‘eye’, and together describe a typical action of myopic individuals who squeeze their eyes to improve farsight vision. This action reduces the opening of the iris diaphragm, thus increasing the focal depth. Scientifically, myopia (near-sightdness) is a spherical refractive error that occurs when rays of light running parallel to the optic axis focus the image anterior to the retinal plane [444]. This disorder is mostly due to an over-growth of the eyeball, which brings the retina behind the focal plan [445]. According to the recent definition by the IMI (International Myopia Institute), myopia can be divided into low myopia and high myopia, in which the spherical equivalent refractive error is ≤ −0.50 diopters (D) and ≤ −6.00 D, respectively [445]. Myopia is among the most common ocular disorders causing visual loss. In 2010 a systematic analysis estimated that 1.9 billion of people in the world were myopic (about 27% of the world’s population), among which 70 million had high myopia [446]. Holden et al. estimated that in 2050 there will be almost 5 billion myopic people (50% of world’s population) among which almost 1 billion with high myopia [447]. However, myopia is not equally distributed worldwide; 90% of the youngsters are myopic in East Asian countries, while in the United States and Europe their proportion remains around 50% [448]. The prevalence of myopia also varies with age. In the Beaver Dam Eye Study, data suggested that the prevalence of myopics decreased from 42.9% among adults aged 43-54 years to 14% in individuals older than 75 years [449]. To date, hundreds of genes and around twenty chromosomal loci have been identified to be associated with the occurrence of refractive error and myopia, although they cannot fully explain it [450-452]. A seminal study published in 1969 concerning Inuit people living in North Alaska, showed that only 2 out of 131 adults, grown up in an isolated group, were myopic. On the contrary, about 50% of the next generation individuals were shortsighted [453], demonstrating that genes alone cannot explain such increase in just one generation, because genetic changes require longer times. However, genes can determine the individual susceptibility to environmental factors, and familiarity is evident within families and within populations [454-456]. Environmental factors including socio-economic and the time spent in outdoor activities have also been correlated with myopia development. While the time spent indoor represents an environmental risk, the concomitant outdoor time may be an important protective factor [457-458]. High level of outdoor activity is positively correlated with lower odds of myopia in different epidemiological studies [459-461]. The idea that outdoor activities reduce the onset of myopia, introduces the concept that the amount of exposition to bright light has a pivotal role in this prevention. In fact, light stimulates the release of dopamine and its accumulation in the retina, which starts a signaling cascade involving the retinal pigmented epithelium, finally slowing down the growth of the eye globe [462-465]. The molecular etiology of myopia is not clear yet. In a recent study, we observed that atropine, a molecule used to slow down myopia progression in children and teenagers, increased extra-cellular matrix production from scleral fibroblasts in vitro, thus contrasting the scleral thinning often observed during myopia progression; on the contrary, atropine effect on choroidal fibroblasts resulted in a decrease of ECM components production, suggesting an alternative mechanism, by which atropine might improve choroidal blood perfusion, known to be reduced in myopic eyes [466]. Further investigations are needed to better clarify the physio-pathological events leading to myopia. Current anti-myopic treatments include: use of eye-glasses or soft contact lenses, orthokeratology, topical administration of low dose atropine or pirenzepine, oral administration of 7-methylxanthine, corrective surgery. However, in many cases a “rebound effect” can be observed after interruption of treatment (Figure 5) [467-470].

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Figure 5. Myopia. Myopia mostly results from the elongation of the eye globe, which brings the macular region of the retina out of focus, and puts a mechanical stress on this delicate tissue, increasing the risk of a retinal detachment and/or laceration (upper panel). Food supplements that might be useful are those reinforcing the matrix of the sclera, thus retarding its abnormal growth and elongation (bottom panel).

Nutrition and Food Supplements

The use of food supplements to correct nutrition deficiencies is a strategy widely accepted and used to improve the general health and try to prevent many different pathologies, also including eye diseases. Eating brightly colored foods like green vegetables and fresh fruits, loaded with vitamins, is highly recommended also to intervene on eye growth. Moreover, the consumption of fish like salmon, tuna and mackerel is very important for a sufficient intake of omega-3 fatty acids, needful to the brain and the eye to modulate neuronal cell membrane activity and their kinetics of transport systems [471]. In the study KNHANES VII (Korean National Health and Nutrition Examination Survey 2016-2017) the prevalence of high and low grade myopia among South Korean children 5-18 years of age and the associated risk factors were analyzed. In this study, a correlation was noted between high body mass index and high myopia [472]. In another study on Asian children a high dietary intake of cholesterol and fat was found to be associated with myopia progression [473]. Glycemic index can be regarded as a risk factor for myopia and refractive error is more frequent in young patients with newly diagnosed type I diabetes mellitus [474,475].

Microelements: Microelements are important during the entire life, since they are essential co-factors for many enzymes, and more specifically they have a critical role to support a normal growth in children. Fedor and coworkers [476] found an association between low serum concentration of zinc and selenium, and a high Cu/Zn ratio in a cohort of 83 Polish myopic children aged 7-17 years, which might suggest that a dietary supplement of Zinc and Selenium could be indicated in myopic children. However, the finding was not confirmed by another study on American children [477], and more clinical data are necessary to consider microelements as a necessary supplement in the control of myopia progression in children.

Vitamin D: Although there is no direct evidence on the effect of vitamins on myopia progression, some studies have shown a correlation between vitamin D and myopia. The biosynthesis of vitamin D is promoted by sunlight exposure, and might be related to the time that individuals spend outdoor. Intake of folate and calcium also correlate with the amount of circulating vitamin D. In a study on 22 young American students (13 to 25 years old) the 14 myopes showed lower blood levels of vitamin D (average of 3.4 ng/ml) compared with non-myopes when adjusted for age and dietary intakes, despite the fact that both groups spent similar amounts of time outdoor [478]. In a Western Australian Pregnancy Cohort (Raine) Study, Yzar and co-workers [479] analyzed 946 children during a follow-up of 20 years. Among these, 23.4% were myopic and had lower serum vitamin D3 concentrations compared to non-myopic individuals. Univariate analysis showed that lower serum vitamin D3 concentration was associated with higher risk of progressive myopia (with a cut-off value of 50 nmol/L the odds ratio for developing myopia with low vitamin D3 was 2.63). Two more studies investigated the correlation between low serum levels of vitamin D3 and myopia. A first study analyzed 2,666 European children aged 6 years [480], and the second study enrolled more than 15,000 Korean young adults (aged 20 years or more) [481]. Both studies found an association between low serum levels of vitamin D3 and presence of myopia, independently from the time spent outdoor.

7-Methylxanthine: 7-Methylxanthine (7-MX) is an intermediate metabolite in the synthesis of caffeine in plants, and a product of the metabolism of caffeine in humans [482]. Unpublished evidence by K. Trier had shown an effect of caffeine on the production of ECM components in the sclera of treated rabbits. Further evidence by the same author has shown that 7-MX – a non-selective adenosine receptor antagonist – may have a role in the retardation of myopia progression. 7-MX effects resemble those of atropine (a muscarinic receptor antagonist), both increasing ECM production and collagen fibril diameter, thus increasing thickness of the sclera [483,484]. A thicker sclera should be less prone to axial elongation, which is the main cause of myopia development. In a recent study, infant macaques with myopia induced by the use of corrective lenses were treated with 100 mg/kg of oral 7-MX twice daily, demonstrating the ability of 7-MX to retard myopia progression in primate puppets [483]. 7-MX is a common human dietary ingredient for coffee drinkers, which has shown no significant side effects during long term treatment. In a clinical study, it has been shown to limit eye elongation and myopia progression in childhood myopia [485].

crocetin: Another promising natural compound is crocetin, a carotenoid compound consisting of a C-20 carbon chain contained in saffron and used in traditional herbal medicine [486]. This powerful compound is able to delay the growth of cancer cells by inhibiting the synthesis of nucleic acids and the activation of defense anti-oxidative systems, interfering with growth factor signaling pathways and finally inducing cancer cells apoptosis [487]. In a murine model of lens-induced myopia, a dietary crocetin supplement delayed myopia progression. Compared to controls (n = 14), crocetin administration resulted in a significant smaller change of refractive errors and axial elongation [488]. In a randomized clinical trial, 69 myopic children aged 6 to 12 years received either a placebo or oral crocetin for 24 weeks. The spherical equivalent refractive error progressed during the 6 months of the study by -0.41± 0.05 Diopters in placebo controls, and by −0.33 ± 0.05 Diopters in crocetin treated (p < 0.05); the axial length increased by 0.21 ± 0.02 mm in placebo controls, and by 0.18 ± 0.02 mm in crocetin treated (p < 0.05), thus showing the potential effects of crocetin in slowing down myopia progression [489].

CATARACT

Cataract is the opacification of the normally transparent lens of the eye, which may occur centrally (nuclear and posterior subcapsular) thus impairing vision, or in periphery (cortical cataract) [490]. Oxidative stress in the lens, occurring as consequence of normal aging, or accelerated by genetic and environmental causes, plays an important role in the onset and progression of cataract [491,492]. Currently, the only treatment for cataract is surgical removal of the cloudy lens, which typically is then replaced with an intraocular lens (IOL) during cataract surgery (Figure 6).

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Figure 6. Cataract. Oxidative damage is the main and only recognized factor for lens opacification (upper panel). Antioxidants like vitamin C, lutein and zeaxanthin are the only food supplements that have shown some efficacy in retarding the insurgence of cataract (bottom panel).

Food Supplements

Despite the evidence indicating oxidative damage to lens epithelial cells as a likely common cause for cataractogenesis, the efficacy of nutritional supplements and antioxidants in the management of age- related cataracts is still under debate. Several studies reported that nutritional supplements showed a long-term inhibitory effect on the development of sight threatening cataract. A randomized clinical trial enrolling 14,000 U.S. male physicians older than 50 years with a follow-up of 11 years indicated that long-term daily multivitamin use had a slight, though significant effect in decreasing the risk of cataract [493]. A nutrition survey including more than 30,000 women older than 49 years followed for over 7 years detected a 13% lesser risk of developing cataract among those indicating a higher dietary intake of antioxidants. The main contributors to dietary total antioxidant capacity in the study population were fruit and vegetables (44.3%), whole grains (17.0%) and coffee (15.1%) [494]. Another large study of adult women in the United States correlated the eating of foods rich in a variety of vitamins and minerals with the delay of cataracts development [495]. A 10-year study of more than 2,400 older adults in Australia found that participants with the highest quintile of total intake (diet + supplements) of vitamin C had a reduced risk of incident nuclear cataract. An above-median intake of combined antioxidants (vitamins C and E, beta-carotene, and zinc) was associated with a reduced risk of incident nuclear cataract. Antioxidant intake was not associated with incident cortical or posterior sub-capsular cataract [496]. A large study of female health professionals in the United States enrolled 1802 women aged 50 to 79 years with intakes of lutein and zeaxanthin above the 78th (high) and below the 28th (low) percentiles. Four to seven years later, it was found that diets rich in lutein and zeaxanthin are moderately associated with a decreased prevalence of nuclear cataract in older women [497]. However, a Cochrane metanalysis including 9 clinical trials involving more than 117,000 patients concluded that dietary supplements of beta-carotene, vitamin E, and vitamin C had no clear beneficial effects on the inhibition of ARC development [498]. It has to be considered though that clinical studies on cataract development and progression require several years of observation, during which time the compliance of patients to treatment indications can be less tight, and that the endpoint parameters of such studies can be highly subjective. Most recently, a randomized, prospective, double-blind, placebo controlled human clinical trial has been launched to test the efficacy of a 6 months treatment with a nutritional supplement in inhibiting nuclear cataract progression on 20 patients subjected to Pars Plana Vitrectomy surgery. The nutritional supplement contained 11 compounds with reported cataract inhibitory properties: Riboflavin, L-GSH, C-Phycocyanin (as Spirulina Algae Extract), LA, Pyruvate (as Calcium Pyruvate), aLA, Quercetin, Turmeric, Silybin (as Milk Thistle Extract), Lutein, Zeaxanthin, and Astaxanthin [499]. In this study, cataract formation and progression were monitored by the serial Pentacam Nuclear Staging measurements (Oculus, Arlington, Wash.), supposed to be an objective, quantitative, and reproducible measurement of nuclear cataract density. Results showed a marginal borderline effect of the nutritional supplement, which however might suggest some efficacy, given the short observation time and the paucity of enrolled patients [499]. All nutrients associated with cataract prevention in clinical studies can be found in eye vitamins and vision supplements. Many experts believe these substances should be acquired from a healthy diet rather than from nutritional supplements. However, very often it happens that common dietary habits may lack key nutrients because not containing enough fruits and vegetables. In such occurrence, it could be wise to consider taking one or more daily nutritional supplements to assure the organism the intake of all the nutrients needed for optimum eye health. Antioxidant vitamins and phytochemicals that are supposed to reduce the risk of cataracts include vitamins A, C and E, lutein and zeaxanthin; consumption of fish high in omega-3 fatty acids has also been linked to a potential reduction of the risk of cataracts or their progression [500]. Here follows a sample of recent research suggesting the role of such nutrients in the prevention of cataracts.

Antioxidants and Vitamins

Seminal preclinical studies performed in the 80’s of the past century on cataract induced in mice by hyperbaric oxygen exposure [501] or by photo-oxidative stress in isolated rat lenses [502] have highlighted the potential protective activity of antioxidants, also including vitamin C, against cataract. The level of AsA is known to be low or absent in cataractous patients, with a concomitant increase of di-hydro-AsA (the oxidized form of ascorbic acid) [503]. However, the concentration of vitamin C in the aqueous humor and lens of human subjects could be increased after supplementation of 2 gr per day for 2 to 4 weeks prior to lens removal [504]. Along the same line, the Second National Health and Nutrition Examination Survey reported an inverse association between serum AsA and self-reported cataract [505], confirmed by the findings of the Nutrition and Vision Project, in which the inverse association between cortical and posterior subcapsular cataract with the use of long-term vitamin C supplement was evidenced [506]. These results are also consistent with those from the Beaver Dam Eye study where the 5-year risk to develop nuclear or cortical cataract was 40% and 60% lower among individuals who reported the abitual use of multivitamins or any other supplement containing vitamin C [507]. Daily oral use for 3 years of a mixture of antioxidant micronutrients including vitamin C produced a small deceleration in the progression of ARC [508]. In a population-based cross-sectional analytic study, which included 5638 people (aged ≥60 years), a strong inverse association between vitamin C and cataract was described [509]. Finally, a recent prospective cohort study, has shown that vitamin C protected against nuclear cataract progression as evaluated approximately 10 years after the baseline [510].

lutein and Zeaxanthin

Lutein and zeaxanthin are the main carotenoids found in the eye and look like promising nutrients in the fight against cataracts. Recent preclinical and clinical works have addressed the risk of developing cataracts in relation to dietary assumption of these nutrients.

Diabetic cataract is a frequent eye complication in diabetes. Dietary lutein effects on the prevention of diabetic cataract have been addressed in a rat model of diabetes induced by streptozotocin [511]. An oral lutein supplement (0.5 mg/kg) did not significantly affect blood glycemic values or body weights in diabetic rats. Most (81%) of diabetic rats’ eyes developed lens opacity and 43% developed mature cataract. Diabetic rats treated with lutein, developed lens opacity in 38% of the eyes and no mature cataracts were observed. Diabetic rats’ lenses showed a parallel increase of MDA and a decrease of GSH levels. Lutein treatment blunted MDA levels, though not raising GSH, hence indicating that lutein effects on the prevention of diabetic cataract may work through the inhibition of lipid peroxidation, finally suggesting that a lutein dietary supplement, combined with glycemic control, might work in the prevention of diabetic cataract [511]. The Nurses’ Health Study enrolled over 77,000 US female nurses aged 45- 71 y, and observed for 12 years. Results showed that those subjects with a dietary intake of high amounts of lutein+zeaxanthin (more than the average amount of 6 mg/day registered in the study) had a reduced need for cataract surgery [511]. The POLA (Pathologies Oculaires Liées à l’Age) study evaluated the associations of plasma lutein and zeaxanthin and other carotenoids with the risk of age- related maculopathy and cataract in 899 residents in the south of France and aged > 60 years. Results were strongly suggestive of a protective role of the xanthophylls, in particular zeaxanthin, for the development and progression of age-related maculopathy and nuclear cataract [512]. The Melbourne Visual Impairment Project recruited 2322 permanent residents aged > 40 years, with a follow-up of 2 years. This study confirmed the inverse association between high dietary zeaxanthin intake and prevalence of nuclear cataract [513]. On the contrary, the AREDS2 study concluded that daily supplementation with lutein/zeaxanthin had no detectable effects on rates of cataract surgery or vision [514]. However, these results are biased by the fact that the enrolled population was relatively well nourished and given at baseline a multivitamin supplement which could already have a protective effect on cataract progression, thus masking the effects of lutein and zeaxanthin [515].

Conclusion

Most often pathologies result from the loss of homeostasis of the whole organism, and may involve one or more organs at a time. Pache and Flammer defined POAG as “a sick eye in a sick body” [516], and indeed this definition might be extended to more ophthalmic pathologies, which can be derived or aggravated by imbalances of the homeostatic equilibrium of the body. Food supplements aim at re-establishing such equilibrium, both at the organism level, and sometimes specifically at the organ level, in case an organ is in major need of some specific nutrients or defense molecules that can be delivered by the food supplement. In fact, medical treatments are specifically aimed at the diseased organ, and their effect might be potentiated by food supplements cooperating with the drug(s) both at the specific site, and more generally at the organism level. Several decades of preclinical and clinical research have substantiated this hypothesis, telling us that nutrition is the most important element in our lifestyle influencing our health and disease status. Food supplements can thus be seen as nutritional aids to prevent disease, but also to cooperate with the treatment of diseases, of the eyes, but of other organs as well.

Abbreviations

JPPR-3-2-314-g007

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Severe Spinal Column Deformity from Scoliosis with Harrington Rods Implant

DOI: 10.31038/AWHC.2020344

 

Severe scoliotic deformity of the thoracolumbar spine imposes a significant anesthesia challenge for non-spine surgery. Patients with severe scoliosis are at increased risk for perioperative morbidity and mortality due to underlying pulmonary and cardiac dysfunctions [1-3]. Stress, pain, mechanical ventilation, and surgery-induced inflammation can further increase the risk of postoperative cardiopulmonary failure. We present a preoperative chest radiograph demonstrating extensive thoracolumbar scoliosis with Harrington rods implant, anatomic distortion, and bony dysmorphism (Panel A, white arrow). The patient underwent a living donor kidney transplant under general anesthesia. Preoperative anesthesia and surgical planning is crucial and should focus on airway difficulty, ventilation management, positioning, new kidney location, and postoperative pain management.

The kidney transplant is a heterotopic transplant surgery meaning the kidney is placed in a different location than existing kidneys. The new kidney is on the right or left side of the abdomen to allow the donor kidney to be easily anastomosed surgically to blood vessels and the bladder of the recipient. Due to the extensive deformity of the spinal column and right chest wall (Panel B, black arrow), the operation was performed in the left lateral decubitus position. Moreover, the donor kidney was placed to the right iliac fossa to decrease the risk of left lung atelectasis, restricted breathing, and sprinting from pain.

Ultrasound-guided quadratus lumborum was difficult in this patient due to atrophy of trunk muscles, chronic scarring, and artifacts from the implant, which required careful assessment of anatomical landmarks to perform a successful nerve block.

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References

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  3. Kulkarni Anand H, Ambareesha M (2007) Scoliosis and anaesthetic considerations. Indian Journal of Anaesthesia. 51: 486-495
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Treatment of Vaginal Atrophy with the Aqueous Extract of Triticum vulgare: Comparison between Different Pharmaceutical Forms

DOI: 10.31038/AWHC.2020335

Abstract

Decreased estrogenization in post-menopausal women causes changes in the lower urinary tract. Vulvovaginal atrophy (VVA) is a pathological condition resulting from those changes. VVA has a negative effect on the quality of life therefore prompting a search for well tolerated therapeutic options. This article was aimed to evaluate whether the participation of the patient in the choice of the therapy may improve the adherence to the treatment. In particular, there are two distinct pharmaceutical forms in vaginal cream and pessaries based on the Triticum vulgare extract (TVE) for the treatment of post-menopausal VVA.  We examined the clinical reports of48 women with VVA who chose to be treated with Fitostimoline® 20% vaginal cream (based on TVE, 2-phenoxyethanol; glycerine; white vaseline; sodium cetyl stearyl sulfate; cetyl stearyl alcohol; decyl oiled; methyl parahydroxybenzoate; propyl parahydroxybenzoate; purified water. Produced by Farmaceutici Damor, Naples, Italy) and 43 who preferred Fitostimoline®600 mg pessaries (based on TVE,2-phenoxyethanol; macrogol 400; macrogol 1500; macrogol 12000.Produced by Farmaceutici Damor, Naples, Italy) for a 3-monthtreatment. Signs and symptoms of VVA were available during the baseline and after 12 and 90-day treatment visits.

Adhering to the patient’s choice allowed for a very high compliance to the prescribed therapy in the whole study population, and we recorded a significant reduction of the total symptom score (TSS) inboth women treated with the vaginal cream and in those who underwent pessary therapy, without any differencein TTS and in its single components between thetwo pharmaceutical formulation groups.

Thus, the availability of two distinct formulations of Fitostimoline® pessaries and vaginal cream seems to be particularly useful for the treatment of VVA.

Keywords

Vulvovaginal atrophy, Fitostimoline® pessaries, Fitostimoline® vaginal cream

Introduction

Vulvovaginal atrophy (VVA) is a common condition associated with decreased estrogenization of the vaginal tissue. It can occur at any time in a woman’s life cycle, but it is more frequent in the postmenopausal phase, since it affects around 90% of postmenopausal women [1]. Other than menopause, VVA may appear during hypoestrogenic conditions, such as lactation, various breast cancer treatments, and use of certain medications. In these cases, VVA may resolve spontaneously when estrogen levels are restored [2]. VVA symptoms are all dependent from decreased estrogenization and include: Dryness, soreness, irritation and dyspareunia with increased urinary frequency, urgency, and urge incontinence [3]. Clinical findings include the presence of pale and dry vulvovaginal mucosa with petechiae. Vaginal rugae disappear, and the cervix may become flush with the vaginal wall. A vaginal pH of 4.6 or more supports the diagnosis of VVA because the low level of estrogen may decrease the number of lactobacilli thus causing increased pH of the vagina [4].

Only a quarter of women with VVA seek for treatment [5]. The

first- line treatment of VVA is a continuous sexual relationship, using non-hormonal lubricant over the counter vaginal [6]. The systemic and local estrogen administration is recommended for the treatment of VVA in postmenopausal women [7]. Although hormone therapy may alleviate the short and long term complications of menopause, such as hot flashes, night sweats, and VVA, it may increase the risk of breast and endometrial cancer [8]. Because of the adverse effects of estrogen therapy, some women prefer not to use this method for alleviating VVA, and they tend to use non-hormonal medication for this matter[9]. In this regard, it is noteworthy the evidence that a local treatment based on hyaluronic acid is more effective than hormonal therapy in improving VVA symptoms [10]. More recently, it has been showed that the aqueous extract of Triticum vulgare (TVE) in cream [11] and pessaries [12] formulation could reduce sign and symptoms of VVA in postmenopausal women. TVE containing mainly poly/ oligosaccharides’ components has been extensively used in different pharmaceutical forms. The Damor Farmaceutici TVE, because of its global patent based on its production process, has unique characteristics that lead to its application in VVA treatment. In fact, as recently demonstrated, Damor TVE has a regenerating [13,14], antinflammatory [15] and anti-MMP9 activity [16], and antioxidant activity [17], which play a crucial role in the treatment of VVA. Furthermore, these recent studies suggest that Damor TVE is at least as efficacious as hyaluronic acid in favoring tissue re-epithelization and in contrasting inflammation. For this reason, Damor TVE cream and pessaries formulations (Fitostimoline® vaginal cream and pessaries) are used in the gynecological clinical practice as an alternative to hyaluronic acid in the treatment of VVA.

Since in a long-term treatment it may be useful to involve the patient in the choice of the pharmaceutical formulation to use, in order to improve adherence to the treatment, the availability of two different pharmaceutical formulations based on Damor TVE, may be particularly useful to improve the adherence to the treatment. Thus, we used a large population of women with VVA treated with the vaginal cream or the pessaries of Damor TVE, according to their preference, to evaluate in real word clinical practice whether this “therapeutic agreement” may improve the compliance in a long-term treatment.

Patients and Treatments

In an Outpatient Gynecological Clinic database we selected 91 women, aging between 18 and 70 years, with evidence of VVA (physiological, pharmacologically induced or surgery-dependent) who had symptoms and objective signs of vaginal atrophy plus amenorrhea for at least 12 months, who chose to receive one of following treatments for a 3 months’ period:

• Fitostimoline 20% vaginal cream (based on TVE, 2-phenoxyethanol; glycerine; white vaseline; sodium cetyl stearyl sulfate; cetyl stearyl alcohol; decyl oiled; methyl parahydroxybenzoate; propyl parahydroxybenzoate; purified water. Produced by Farmaceutici Damor, Naples, Italy).

• Fitostimoline 600 mg pessaries (based on TVE, 2-phenoxyethanol; macrogol 400; macrogol 1500; macrogol 12000. Produced by Farmaceutici Damor, Naples, Italy).

The treatments were locally applied every evening for 3 months. Patients were asked to avoid vaginal sexual intercourse during the entire study period.

We excluded women with other gynecological diseases (in addition to VVA), like the presence of metabolic or endocrine diseases (e.g. uncontrolled diabetes mellitus) or of other local/systemic diseases that could potentially interfere with the study parameters (concomitant treatment with antibiotics/antiseptic agents, steroidal and non-steroidal anti-inflammatory drugs, analgesics (except paracetamol as pain killer)). All patients gave their informed written consent to the use of their clinical data for scientific purposes.

Outcome Measures

Signs and symptoms of VVA were available during the baseline visit and after 12 and 90 days of treatment. Six subjective symptoms (burning, pain, itching, vaginal dryness, dyspareunia and dysuria) and 5 objective signs (mucous dryness, pale mucosa, thin vaginal folds, mucous fragility, petechiae) of vaginal atrophy were evaluated by a semi-quantitative scale (0 = absence of the sign/symptom; 1 = mild sign/symptom; 2 = moderate sign/symptom; 3 = severe sign/ symptom) expressed for both single signs and symptoms and for their sum (total symptoms score, TSS). Adverse events occurrence and vital signs (blood pressure, heart rate, body temperature) were reported as safety parameters. At the end of the treatment period, a satisfaction opinion was expressed by the patient in terms of excellent, good, to be improved, negative. For the statistical analysis, post hoc simultaneous multiple comparisons were done by Bonferroni’s analysis.

Results

Out of 91 women recruited, 48 were treated with Damor TVE vaginal cream and 43 with the pessaries. The two groups of women were comparable by demographics and clinical characteristics (Table 1). The menopause origin for the majority of women was physiological (Table 2). Only a few women had a pharmacological or surgical menopause (Table 2). However, the origins of the menopause were comparable between the two groups of women. All the patients were completely adherent to the treatment since all completed the 90-day treatment period with a compliance, percentage of the prescribed therapy assumed, above 95%: 98% for Fitostimoline® vaginal cream and 95% for Fitostimoline® pessaries (Figure 1).

Table 1: Patient demographic characteristics.

Pessaries
(N = 34)

Cream
(N = 38)

p Value

Age (years)

51.4 ± 11.0

53.2 ± 10.3

NS

Menopause (months)

32.0 ± 34.5

37.5 ± 31.7

NS

Table 2: Menopause origin.

Pessaries
(N = 34)

Cream
(N = 38)

p Value

Physiological (%)

93.1

89.6

NS

Surgical (%)

4.7

6.3

NS

Pharmacological (%)

2.2

4.1

NS

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Figure 1. Patients adherence to the treatment with Fitostimoline® vaginal cream and Fitostimoline® pessaries.

By evaluating together patients treated with the vaginal cream and the pessaries, a significant reduction of the TSS was detected both at visit 2 and at visit 3 (Figure 2). When the TSS score was evaluated separately for women treated with Fitostimoline® vaginal cream and those treated with Fitostimoline® pessaries no significant difference in the TSS was detected between the treatment groups (Figure 3). As shown in Figure 3, a significant reduction in the TSS occurred from visit 1 to visit 2 in the two study groups (TSS = 20,1 at visit 1 and 14,4 at visit 2 to for Fitostimoline® pessaries; TSS = 19,1 at visit 1 and 10,9 at visit 2 for Fitostimoline® vaginal cream). A further significant reduction of the TSS score was observed from visit 2 to visit 3 either in patients treated with the vaginal cream (TSS = 10,9 at visit 2 and 5,7 at visit 3) than in those treated with the pessaries (TSS = 14,4 at visit 2 and 8,8 at visit 3) (Figure 3).

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Figure 2. TSS evaluation for treatment with both Fitostimoline® pessaries and Fitostimoline® vaginal cream.

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Figure 3. TSS variation in patients treated with Fitostimoline® pessaries and Fitostimoline® vaginal cream.

Also the single components of the TSS score of VVA were evaluated for both groups of women (Figures 4 and 5). A significant reduction of the main VVA symptoms of pain, burn, itching, dryness, dyspareunia, dysuria, petechiae was observed both in patients treated with the vaginal cream and those receiving the pessaries at visit 2 and visit 3 versus the basal visit (Figures 4 and 5). Furthermore, a statistically significant progressive improvement of all these symptoms evaluated was observed from visit 2 to visit 3 (Figures 4 and 5) without any difference between the groups of treatment (Figures 4 and 5).

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Figure 4. Symptoms variation after treatment with Fitostimoline® pessaries.

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Figure 5. Symptoms variation after treatment with Fitostimoline® vaginal cream.

Finally, in order to assess whether the women were satisfied by the chosen treatment, vaginal cream or the pessaries, the patient’s satisfaction was evaluated. As indicated by Figure 6, 65,1% of women treated with Fitostimoline® pessaries and 68,8% of women treated with Fitostimoline® vaginal cream reported an “excellent satisfaction”, 32,6% and 31,2%, respectively had a “good satisfaction”, while only 2.3% of women treated with Fitostimoline® pessaries referred a “negative satisfaction’’.

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Figure 6. Evaluation of the satisfaction to the treatment with Fitostimoline® pessaries and vaginal cream.

Discussion

The transition from evidence medicine, “one size fits for all”, to precision medicine, “tailored treatment”, requires not only the demonstration of the efficacy and tolerability of a given treatment obtained in randomized trials but also its detailed characterization which can be obtained exclusively in daily clinical practice using large groups of patients. Furthermore, in order to solve the problem of the low adherence and compliance to the prescribed therapy observed in all the long-term treatments, it has been suggested that the patient should be adequately informed and involved in the choice of the therapy.

Thus, the availability of large database of Outpatient Clinics seems particularly useful to satisfy these needs for very common diseases which require long-term treatments. In order to verify this assumption for VVA we obtained from the data base of an Outpatient Gynecological Clinic the clinical records of 91 women with VVA who had been involved in the choice of the treatment and had undergone a 90-day therapy with Fitostimoline® vaginal cream or with Fitostimoline® pessaries. The results of the present study confirm that the involvement of the patients in the choice of the treatment markedly improves the adherence and the compliance to the therapy prescription since at our knowledge there are no reports in the real world clinical practice describing a 100% adherence in a 3 month treatment with the assumption of more than 95% of the drug prescribed.

Our results do not allow any speculation of the efficacy of TVE pharmacological formulations in the treatment of VVA since we have no comparison with placebo or active drug. However, experimental studies suggest that there is a rationale for the use of TVE for the treatment of VVA [13-16] and evidence of their efficacy in the treatment of VVA obtained in clinical studies are available in the literature [11,12]. An indirect support to these data seems to be corroborated by the comparison between the percentage of success achieved in our population, as expressed by the reduction in TSS score, and that reported with the use of hyaluronic acid in the treatment of VVA (Figure 2) [10].

Furthermore, the observation that there are no differences between TVE pessaries and vaginal cream either in the evaluation of the TSS or in the evaluation of the single symptoms, support the possibility to allow a personal choice between the two pharmacological formulations. Finally, the time course of the therapeutic effect that appears to consolidate over time seems to suggest that the mechanism of action of the Fitostimoline® formulations is actually pathogenic rather than symptomatic. The limitations of this study are that this is not a clinical trial but an observational study on a wide range of cases. For this reason, we do not have a validation of efficacy which however already exists for this products and it was not in the objective of the study which consisted in assessing whether the availability of different pharmaceutical forms by favoring the “therapeutic concordance” can be useful in improving compliance.

Conclusion

For the purposes of therapeutic agreements between physicians and patients, which is the mechanism currently suggested to improve adherence to therapy, the availability of different pharmaceutical forms represents an issue of great relevance. In order to manage the choice by the physician, however, it is essential to have full knowledge of the potential of the different pharmaceutical forms. For this reason, we can conclude that the two distinct formulations of TVE, pessaries and vaginal cream, didn’t show any significant difference in the treatment of the VVA, both in terms of efficacy and of compliance, and could be alternatively used for the treatment of VVA.

References

  1. Palacios S, Nappi RE, Bruyniks N, Particco M, Panay N (2018) The European Vulvovaginal Epidemiological Survey (EVES): Prevalence, symptoms and impact of vulvovaginal atrophy of menopause. Climacteric 21: 286-291. [crossref]
  2. Mac Bride MB, Rhodes DJ, Shuster LT (2010) Vulvovaginal atrophy. Mayo Clin Proc 85: 87-94. [crossref]
  3. Castelo-Branco C, Cancelo MJ (2008) Compounds for the treatment of atropic vaginitis. Expert Opin Ther Pat 18: 1385-1394.
  4. Sturdee DW, Panay N (2010) Recommendations for the management of postmenopausal vaginal atrophy. Climacteric 13: 509-522. [crossref]
  5. Castelo-Branco C, Rostro F (2007) Treatment of atrophic vaginitis. Therapy 4: 349- 353.
  6. Noumova I, Castelo-Branco C (2018) Current treatment options for postmenopausal vaginal atrophy. Int J Women’s Health 10: 387-395 [crossref]
  7. North American Menopause Society (2007) The role of local vaginal estrogen for treatment of vaginal atrophy in postmenopausal women: 2007 position statement of the North American Menopause Society. Menopause 14: 355-369. [crossref]
  8. Wentzensen N, Trabert B (2015) Hormone therapy: Short-term relief, long-term consequences. Lancet 385.
  9. Ibe C, Simon JA (2010) Vulvovaginal atrophy: Current and future therapies. J Sex Med 7: 1042-1050. [crossref]
  10. Jokar A, Davari T, Asadi N, Ahmadi F, Foruhari S (2016) Comparison of the hyaluronic acid vaginal cream and conjugated estrogen used in treatment of vaginal atrophy of menopause women: a randomized controlled clinical trial. Int J Community Based Nurs Midwifery 4: 69-78. [crossref]
  11. Mollica G, Bonaccorsi G, Martinello R (2008) Evaluation of efficacy and tolerability of Fitostimoline vaginal cream (Damor Farmaceutici) in the treatment of vaginal inflammation and vulvar dystrophy: A double-blind randomized controlled trial Gazzetta Medica Italiana Archivio per le Scienze Mediche 167: 87-95.
  12. Mollica G, Bonaccorsi G, Martinello R (2008) Valutazione di efficacia e tollerabilità di Fitostimoline Ovuli Vaginali (Damor Farmaceutici) nel trattamento delle affezioni flogistico-distrofiche della vagina. Studio controllato in doppio cieco. Gazzetta Medica Italiana Archivio per le Scienze Mediche 167: 97-103
  13. Sanguigno L, Minale M, Vannini E, Arato G, Riccio R, et al. (2015) Oligosaccharidic fractions derived from Triticum vulgare extract accelerate tissue repairing processes in in vitro and in vivo models of skin lesions. J Ethnopharmacol 159: 198-208. [crossref]
  14. D’Agostino A, Pirozzi AVA, Finamore R, Grieco F, Minale M, et al. (2020) Molecular mechanisms at the basis of pharmaceutical grade triticum vulgare extract efficacy in prompting keratinocytes healing. Molecules 25: 431. [crossref]
  15. Sanguigno L, Casamassa A, Funel N, Minale M, Riccio R, et al. (2018)Triticum vulgare extract exerts an anti-inflammatory action in two in vitro models of inflammation in microglial cells. PLoS One. 13: 197493. [crossref]
  16. Funel N, Dini V, Janowska A, Loggini V, Minale M, et al. (2020) Triticum vulgare extract modulates protein-kinase b and matrix metalloproteinases 9 protein expression in bv-2 cells: Bioactivity on inflammatory pathway associated with molecular mechanism wound healing. Mediators of Inflammation 1-13.
  17. Antonucci I, Fiorentino G, Contursi P, Minale M, Riccio R, et al. (2018)Antioxidant capacity of rigenase®, a specific aqueous extract of triticum vulgare. Antioxidants (Basel) 7: 67. [crossref]

The Effect of COVID-19 Pandemic on Urolithiasis Management and Urologists in the Gulf Countries: A Survey of Urologists in GCC

DOI: 10.31038/IJNUS.2020211

Abstract

Objective: To explore the effects of COVID-19 on urolithiasis management and on the medical practice of urologists in Gulf countries.

Methods: A web-based survey was sent to urologists in the six countries in the Gulf Cooperation Council (GCC). The survey consisted of 23 questions about their working environment, urolithiasis management experience, and the policies of their facilities during the COVID-19 pandemic.

Results: During the one-week survey period, responses were received from 191 urologists working in the six GCC countries. Responses were received from urologists in all six countries but the numbers differed markedly. Of the 191 urologists who responded, 160 (83.8%) were experienced urologists and 31 (16.2%) were urology residents. Eighty-four (44.0%) volunteered for COVID-19 management rather than urology, 22 (11.5%) were infected with COVID-19 and 38 (19.9%) sought mental health support. Clinical duties related to urolithiasis management were reduced for most of the urologists, with elective procedures postponed and urolithiasis management confined to emergency conditions. In the absence of COVID-19 infection, 67 (35.1%) preferred to actively manage ureteral stones, whereas the remaining urologists preferred less invasive methods, such as conservative or instant drainage.

Conclusion: COVID-19 is hazardous to urologists in GCC countries, with 11.5% being infected and most reducing their clinical duties related to urolithiasis management. Although urolithiasis management in GCC countries during the COVID-19 pandemic was generally consistent with worldwide guidelines, some differences were observed, with further studies warranted to compare different management strategies.

Introduction

COVID-19 is a species of coronavirus that causes severe acute respiratory syndrome (SARS-COV-2) [1]. The first documented cases were in Wuhan, China, in September 2019. Infection subsequently spread worldwide, with the World Health Organization announcing an epidemic in March 2020 [2]. Since then, all countries worldwide have started to lockdown, with many activities being restricted, including flying, attending schools, participating in and watching sports activities, shopping at malls, and public gatherings. The health system has also been affected, with elective procedures and clinics being restricted. These restrictions have affected urologic practice and patient management [3]. Although the populations of the six countries in the Gulf Cooperation Council (GCC) have similar demographic characteristics, each country has devised its own lockdown strategy to deal with the pandemic. The present study explored the effects of the pandemic on the management of urolithiasis patients across the Gulf countries, as well as the impact of the pandemic on urologists practicing in these countries.

Methodology

A web-based survey was developed by a group of urologists from the six GCC countries. The survey, written in English, included 23 general and specific questions about COVID-19, including ten questions with free-response prompts. Upon developing the survey, it was sent to another few urologists as a pilot for validation.

The survey was sent to many urologists in the GCC countries who were easily approachable by the study investigators during the week of 10-16 May 2020. The 23 questions on the survey are detailed in Figure 1.

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Figure 1. Questions of the survey.

Results

Responses were received from 191 urologists, representing the six GCC countries. Of these respondents, 72 (37.7%) were from Qatar, 39 (20.4%) from Kuwait, 30 (15.7%) from the United Arab Emirates (UAE), 19 (9.9%) each from Oman and the Kingdom of Saudi Arabia, and 12 (6.3%) from Bahrain (Figure 2).

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Figure 2. The effect of COVID-19 on urologists in each country.

Of the 191 urologists, 145 (75.6%) worked in governmental hospitals, including 27 (14.1%) at academic centers, and 46 (24.4%) worked in private practice. The respondents differed in working experience, with 82 (42.9%) being consultants, 54 (28.3%) being specialists, nine (4.7%) being senior registrars, ten (5.2%) being registrars, four (2.1%) being assistant registrars, and 31 (16.2%) being urology residents. Of the respondents, 124 (64.9%) were general urologists, 42 (22.0%) were endo-urologists, and 25 (13.1%) were primarily interested in subspecialties in addition to urolithiasis.

Interestingly, 128 (67%) respondents changed their regular urology practice in response to COVID-19, 48 (25.1%) continued their routine practice, and 15 (7.9%) stopped practicing medicine following the lockdown. The lockdown reduced duty hours for 118 (61.8%) of the respondents and increased duty hours for 22 (11.5%), whereas 49 (25.7%) had almost the same number of duty hours, and two (1.0%) stopped practicing medicine.

Outpatient clinics and elective surgeries were closed or limited to minimize contact with patients. Only 26 (13.6%) reported no changes in the management of their clinics, whereas 37 (19.4%) closed their clinics, 91 (47.6%) changed to telemedicine phone consultations, four (2.1%) are consulting by video conference, and one (0.5%) is communicating with patients through the facility website.

Of the 191 respondents, 118 (61.8%) are not operating on patients infected with COVID-19, and 139 (72.8%) have stopped performing elective urolithiasis procedures. In contrast, 44 (23%) have reduced the number of elective urolithiasis procedures, whereas eight (4.2%) have not changed the number of procedures. Ninety-two respondents (48.2%) continue to perform the same number of emergency urolithiasis procedures, whereas 66 (34.5%) have reduced and 27 (14.1%) have increased the number of procedures. The remaining five (2.6%) urologists have stopped performing any operations, even emergency operations.

ESWL management was stopped by 98 (51.3%) of the urologists and reduced by 64 (33.5%) but remained unchanged by 18 (9.4%).Most of these respondents have changed the mode of anaesthesia to spinal anaesthesia. For example, 76 (39.7%) reported using spinal anaesthesia for all their patients and 16 (8.5%) for patients with proven COVID-19 infection. In contrast, 67 (35.1%) reported no change in anaesthesia preference in response to the pandemic.

To prevent infection, 112 (58.6%) urologists are screening their patients preoperatively, and 32 (16.5%) have worn full PPE routinely since the start of the pandemic.

In managing ureteral stones in patients lacking urinary tract infection and with unknown COVID-19 status, 67 (35.1%) urologists prefer more conservative management, 53 (27.7%) favour instant drainage followed by ureteroscopy after the easing of lockdown measures, 40 (20.9%) reported no change in management and 27 (14.1%) favour ureteroscopy for stone clearance. This is illustrated in Figure 3. These physicians were highly susceptible to COVID-19 infection, with 22 (11.5%) urologists found to be infected by positive swab results. Hospitals have been advised to support their employees psychologically, as evidenced by the 38 (19.9%) who received mental health support. Assessment of the impact of COVID-19 on these urologists showed that 107 (56%) were not affected, 55 (28.8%) had mild salary reduction, 25 (13.1%) experienced an almost 50% reduction in salary, and three (1.6%) stopped working because of the lockdown. Urologists are actively involved in COVID-19 management, with 84 (44%) shifting their medical practice from urology to volunteer in the treatment of COVID-19 patients. The impact of COVID-19 pandemic on urologists is illustrated in Figure2.

Discussion

About one-third of the respondents were from the state of Qatar. Response rates do not reflect the relative number of urologists in the six countries but may reflect the short time provided for the return of the survey. A large country like KSA requires more than one focal urologist, as well as additional time to reach urologists throughout the entire country. In 2016, approximately 257 urologists were estimated to be working in governmental hospitals (2), suggesting that our survey included only a small percentage of practicing urologists-most of the urologists who participated in the survey work in the governmental sector. In contrast, more than 50% of practicing urologists in the United States work in private practice [3].

The outbreak of COVID19 led most of the GCC to adopt lockdown measures, including preventing people from entering these countries during the outbreak period. Furthermore, medical services to treat COVID-19 patients were increased in most of these countries, including an increase in staff to provide these services. This led to an increase in staff exposure to COVID-19, increasing the incidence of COVID-19 among the staff. This, in turn, reduced the number of staff members available to treat COVID-19, requiring the deployment of urologists to work in COVID-19 facilities and reducing the ability to provide non COVID-19 related services.

Similar to other countries, most countries in the GCC have utilized telephone communications or telemedicine to manage outpatients, restricting surgery to emergency and oncology procedures. Also, the duty hours for physicians increased from 8 to 12 hours per day, making more staff members available to work in COVID-19 facilities. This was reflected in the survey results, as nearly two-thirds of the urologists were treating COVID-19 infected patients, whereas only about 25% continued practicing urology, which likely consists of emergency and oncology services. Similar changes were applied during the outbreak of COVID19 in Singapore [4].

EAU guidelines advise following local recommendations to test staff and patients for COVID-19 if resources are available [5]. Nearly two-thirds of the respondents are testing their patients for COVID-19 preoperatively, and only 16% reported wearing full PPE. This does not conform to EAU guidelines, perhaps because of a relative lack of availability of the test kits or the equipment for full PPE. The EAU guidelines advise full PPE, irrespective of the COVID status of the patient [5].

Testing patients for COVID-19 is crucial because asymptomatic patients from Wuhan, China, who tested positive for COVID-19 before surgery, had a postoperative mortality rate of 20% [6].

Since the EAU guideline considered that surgery is harmful to the patients if he is tested COVID positive, this should result in a change in the practice of management of urolithiasis. Our survey showed inconsistencies in the management of urolithiasis during the COVID-19 pandemic. For example, 20% of urologists surveyed did not alter their management of ureteral stones in the absence of infection, with only 38% selecting conservative management (Figure3).

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Figure 3. Management of ureteral stones in patients lacking urinary tract infection and with unknown COVID-19 status.

Studies have advised that, whenever possible, patients should be treated non-surgically or surgery should be deferred until the demands for ventilators and inpatient beds are reduced [7]. The EAU guidelines recommend treating intermediate priority patients if the capacity is available but not during COVID-19 surge [5].

Healthcare workers are members of the frontline response team and are therefore highly susceptible to infection. The WHO–China Joint Mission on COVID-19 reported that by 20 February 2020, there were 2,055 laboratory-confirmed cases of COVID-19 among healthcare workers, with 22 (1.1%) deaths [8].In Italy, at least 2,629 health workers, or 8.3% of all infected individuals, have been infected with coronavirus since the pandemic broke out in February [9]. In our survey, 11.5% of respondents were laboratory confirmed as having COVID-19. In addition to the infection itself, COVID-19 may be an independent risk factor for mental health problems in health care workers, including depression, anxiety, insomnia, and distress [10,11]. The WHO recommendations for health workers mention various hazards, including exposure to the pathogen, long working hours, psychological distress, fatigue, occupational burnout, stigma, and physical and psychological violence [12]. The WHO recommendations include providing access to mental health and counselling resources. This is supported by our survey, which found that 20% of the respondents had been provided mental health support by their facilities. The lockdown has also had an economic impact on the urologists working in this region. For example, our survey found that 75% of the urologists working in government hospitals, as well as some in private hospitals, were mildly affected economically, whereas around 15% experienced a greater economic impact, which may worsen the psychological impact of the pandemic on health care workers. The effect of COVID-19 on urologists in each country is illustrated in Figure 2.

Despite surveying a relatively large number of respondents, the results of this survey were limited by its narrow distribution among urologists in the region, which may have led to uneven distribution and collection bias. This may have been avoided had the survey been kept open for a longer period. However, this survey explored the common and variable practices among the health care organizations in the Gulf countries and emphasized the need for further studies to compare various practice strategies. The survey also showed the relatively high risk of COVID-19 infection to urologists, which warrants further evaluation by well-designed observational studies.

Conclusion

COVID-19 is hazardous to urologists in GCC countries, with 11.5% being infected and most reducing their clinical duties related to urolithiasis management. Although urolithiasis management in GCC countries during the COVID-19 pandemic was generally consistent with worldwide guidelines, some differences were observed, with further studies warranted to compare different management strategies.

References

  1. Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, et al. (2020) The species Severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2. Nature Microbiology 5: 536-544.
  2. Otaibi KE (2016) Challenge facing the urologist in Saudi Arabia in the future. Urol Ann 184-188.
  3. The State of the Urology Workforce and Practice in the United State (2018).
  4. Chan MC, Yeo SEK, Chong YL, Lee YM (2020) Stepping Forward: urologists’ Efforts during the COVID-19 outbreak in Singapore. EurUrol 78: 38-39. [crossref]
  5. European Association of Urology. https://uroweb.org/wp-content/uploads/EAU-Guidelines-Office-Rapid-Reaction-Group-An-organisation-wide-collaborative-effort-to-adapt-the-EAU-guidelines-recommendations-to-the-COVID-19-era.pdf
  6. Lei S, Jiang F, Su W, Chen C, Chen J, Wei W, et al. (2020) Clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of COVID-19 infection. EClinicalMedicine 21: 100331. [crossref]
  7. Stensland KD, Morgan TM, Moinzadeh A, Cheryl T, Briganti A, et al. (2020) Consideration in the triage of urologic surgeries During the COVID-19 pandemic. EurUrolEpub 77: 663-666.
  8. World Health Organization. 2020. Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19)
  9. Psychiatry Res (2020) Jun; 288: 112972. Published online 2020 of Apr 13. doi: 10.1016/j.psychres.2020.112972. PMCID: PMC7152886
  10. SaiSpoorthy M, KarthikPratapa S, Supriya M (2020) Mental health problems faced by healthcare workers due to the COVID-19 pandemic- A review. Asian J Psychiatr 51: 102119. [crossref]
  11. Jianbo Lai, Simeng Ma, Ying Wang, ZhongxiangCai, Jianbo Hu, et al. (2020) Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019. JAMA Netw Open 3: 203976.
  12. Coronavirus disease (COVID-19) outbreak: rights,roles and responsibilities of health workers, including key considerations for occupational safety and health. 18 March 2020. COVID-19: Schools, businesses and institutions. WHO reference no.: WHO/2019-nCov/HCW_advice/2020. https://apps.who.int/iris/rest/bitstreams/1272583/retrieve.

Facemasks are Not Effective for Preventing Transmission of the Coronavirus

DOI: 10.31038/JNNC.2020321

 

On February 29, 2020, Jerome Adams, the Surgeon  General of  the United States, tweeted that, “They [facemasks] are NOT effective in preventing general public from catching coronavirus.” Within a few months, he and the Centers for Disease Control and Prevention (CDC) were stating that facemasks are effective for preventing transmission of viral illnesses in the community, and within a short time, governments in the United States and elsewhere were mandating the wearing of facemasks in public. The explanation for this reversal in policy focused on asymptomatic carriers in public. However, the evidence from Randomized Controlled Trials (RTCs) indicates that surgical facemasks have no effect on the transmission of viral illnesses in the community. Based on their physical characteristics, one would not expect surgical facemasks to reduce viral transmission by asymptomatic carriers. The size of a coronavirus is about 0.1 microns and the pore size of surgical masks is in the range of 50-100 microns: the pores are 500-1000 times the size of the virus. Aerosol droplets are about 3 microns in diameter, so surgical masks would not be expected to block them either.

A common rationale for wearing masks in public is that their purpose is not to protect you from others, but to protect others from you if you are an asymptomatic carrier: this is illogical. How can a mask protect someone else from you if it does not protect you from someone else? Also, asymptomatic carriers are, by definition, not coughing or sneezing in public, except very infrequently. Normal breathing and speaking emits primarily aerosols not droplets; droplets are the main concern for transmission of the coronavirus in public. Infectious symptomatic carriers who are emitting virus-laden droplets should be in quarantine, therefore it is not necessary to mandate facemasks to protect the public from them.

Four meta-analyses published to date report that no Randomized Controlled Trial (RCT) has ever shown a significant difference in viral transmission rate with and without facemasks in the general public. Brainard, Jones, Lake, Hopper and Hunter  [1] reviewed 3 RCTs   and found no difference in any of them; Cowling, Zhou, Ip, Leung, and Aiello [2] reviewed 4 RCTs and found no difference in any of them; Xiao, Shiu, Gao, Wong, Fong, Ryu and Cowling [3] reviewed 10 RCTs and found no difference in any of them; and Aggarwal, Dwarakananthan, Gautam and Ray [4] reviewed 9 RCTs and found no difference in any of them. These meta-analyses by four different groups from around the world have not found a single RCT that demonstrates a protective effect of facemasks for viral transmission in the general public. In discussing the Xiao et al. [3] Meta-analysis, Greenlagh, Schmid, Czypionka, Bassler and Gruer [5] said that, “…the authors conclude that there was no significant reduction in influenza transmission with the use of facemasks.” The situation, then, is not that there is a lack of evidence: there is replicated, controlled evidence that facemasks do not reduce viral transmission in the community.

For the sake of discussion, what if we made an assumption that facemasks reduce the rate of viral transmission in the community by 5%? For this discussion, we will bear in mind that there is no evidence that facemasks reduce transmission by even  this  much.  Further, let’s assume that the infection rate in the community is 5% and the mortality rate for infected healthy people under 60 is 0.1%. This would mean that wearing facemasks would reduce one’s risk of infection as a healthy person under 60 from 5% to 4.75%; the risk of death from COVID-19 would drop from 0.005% to 0.00475% due to wearing facemasks. This does not seem like a sufficient risk reduction to justify mandatory facemasks in public.

Wearing masks is not terribly inconvenient, but it does have  costs in terms of dollars, an energy burden from manufacturing and distribution, and pollution pressure on landfills, bodies of water and the environment in general. Given these considerations, and the evidence from RTCs, it would seem that mandates for wearing facemasks in public should be, at the least, reduced to recommendations, until there is definitive evidence that they are effective. For any other topic in medicine, one would expect a consensus to exist if all the available RTCs and meta-analyses found that a given intervention has no effect on the target problem. One would expect authorities to state that there is no need for the intervention. If the same standards were applied to public wearing of facemasks for COVID-19, there would be neither a mandate nor a recommendation for them. As discussed recently, facemask policies are only one of many problems with how medical authorities have handled the COVID-19 epidemic; many public health recommendations and statements have not been based on science [6].

References

  1. Brainard JS, Jones N, Lake I, Hooper L, Hunter P (2020) Face masks and similar barriers to prevent respiratory illness such as COVID-19: A rapid systematic review. Medrxiv doi:10.1101/2020.04.01.20049528.
  2. Cowling BJ, Zhou Y, Ip DK, Leung GM, Aiello AE (2010) Face masks to prevent transmission of influenza virus: a systematic review. Epidemiology of Infections 138: 449-456. [crossref]
  3. Xiao J, Shiv EYC, Gao H, Wong JY, Fong MW, et al. (2020) Nonpharmaceutical measures for pandemic influenza in non-healthcare settings – personal protective and environmental measures. Emerging Infectious Diseases 26: 967-975.
  4. Aggarwhal N, Dwarakananthan V, Gautham N, Ray A (2020) Facemasks for prevention of viral respiratory infections in community settings: A systematic review and meta-analysis. IndianJournal of Public Health 64: 192-200.
  5. Greenhalgh T, Schmid MB, Czypionka T, Bassler D,  Gruer L (2020) Facemasks  for the public during the COVID-19 crisis. British Medical Journal 369: m1435. [crossref]
  6. Ross CA (2020) Thoughts on COVID-19. Journal of Neurology and Neurocritical Care 3: 1-3.

Therapeutic Effects of Ozone Therapy that Justifies Its Use for the Treatment of COVID-19

DOI: 10.31038/JNNC.2020314

Abstract

SARS-Cov2, the virus causing COVID-19, is distributed globally since December 2019, causing a pandemic and there are currently no specific treatments available. Patients evolve differently and extreme cases have fatal outcomes after 10 days of being infected. The virus is known to cause Acute Respiratory Distress Syndrome (ARDS). Cytokine storm is considered to be one of the major causes of ARDS and multiple-organ failure. Due to the high lethality of SARS-CoV2 infections and its economic and social impact, it is necessary to seek new therapeutic procedures. It has been demonstrated that ozone therapy produces a significant improvement in blood flow and oxygenation of ischemic tissues. Also, ozone can achieve an equilibrium between Nrf2 and NF-κB factors, modulating the oxidative stress and the expression of pro-inflammatory cytokines. In clinical studies, ozone has a significant role in the treatment of pulmonary and vascular diseases. Today, ozone therapy represents the most practical approach for integrating standard therapies to achieve homeostasis. Therefore, due to the ozone therapeutical effects, it can be proposed as an adjunct therapy in SARS-CoV-2. Three randomized control trials (NCT04359303, NCT04370223 and NCT04444531) are pending classification and approval to start in Spain, one in Iran (IRCT20190618043923N4) and two more (NCT04366089 and NCT04388514) started in Italy one month ago.

Keywords

COVID-19, Immunomodulation, NF-κB, Nrf2, Oxidative stress, Ozone therapy

Introduction

According to the World Health Organization (WHO), viral diseases continue to emerge and represent a serious issue to public health. An epidemic of cases with unexplained low respiratory infections was first reported to the WHO Country Office in China, on December 31, 2019. The new virus was called SARS-CoV-2 and the disease cause was a “COVID-19” an acronym of “coronavirus disease 2019” [1]. Many of these patients deteriorated rapidly and required intubation and mechanical ventilation. Mortality rates are assumed to be around 3.7%. There is currently no effective treatment [2,3]. The therapeutic strategies to deal with the infection are only supportive. Prevention, aimed at reducing transmission rates within the community is our best weapon.

COVID-19 has characteristics of two known syndromes [4,5]:

• Macrophage activation syndrome [6]: a life-threatening complication characterized by hypercytokinemia (cytokine storm) with multi-organ failure. It is characterized by an uncontrolled activation and proliferation of T lymphocytes and macrophages, producing extensive tissue damage as endothelial lesions that lead to the production of microthrombi. Laboratory abnormalities include a decrease in white blood cells, platelet and hemoglobin. There is a production of a high level of transaminase, a marked increase in ferritin, and evidence for intravascular coagulation activation. The protagonist of this storm is mainly interleukin 6 (IL-6) which promotes the differentiation of B lymphocytes. The cytokine storm also stimulates the production of acute-phase proteins and further plays a role in thermoregulation, bone maintenance and the function of the central nervous system. During inflammatory diseases, infections, autoimmune disorders, cardiovascular diseases and some types of cancer, there is an increase in IL-6.

• Antiphospholipid syndrome [7]: it is an autoimmune system disorder that manifests clinically as recurrent venous or arterial thrombosis. This also alters the homeostatic regulation of blood coagulation. The D-dimer is elevated in most patients with pneumonia and other indicators of coagulation are abnormal. Thrombocytopenia is also observed, which seems to be associated with a poorer prognosis. Analytically, the presence of high levels of ferritin in the blood is striking. They appear to respond to an acute inflammatory process. Liver enzymes also tend to be elevated. The Fe2+ released into the blood, in the presence of hydrogen peroxide produces hydroxyl radicals (Fenton reaction). This is extremely toxic, causing oxidative damage, mainly pulmonary, but also systemic. The lung tissue damage stimulates the monocyte-macrophage system which contributes significantly to the inflammatory process. Taking into account all the ozone therapeutical properties, which will be explained below, it can be proposed as an adjunct therapy for patients with COVID-19.

Ozone Therapy and its Mechanism of Action

Ozone (O3) is an allotropic form of the element oxygen, containing one more atom than atmospheric oxygen. It is particularly unstable and decomposes spontaneously into diatomic oxygen, which, in practice, makes it very difficult to transport and store. Ozone therapy has been used for therapeutic purposes since the beginning of the last century and its use is increasingly demanded nowadays. It is characterized by the simplicity of its application, its great effectiveness and with good tolerance. International reports of adverse reactions to the application of ozone therapy place it among the lowest incidences with 0.0007% [8,9]. Ozone, at therapeutic doses, is capable of producing a small, transitory and controlled oxidative stress that stimulates a group of depressed biological functions without causing any adverse effect. This ozone’s preconditioning effect is capable of rebalancing the upset redox state in the organism [10]. Biochemically, when blood is exposed to ozone for several minutes, it reacts immediately with different molecules present in biological fluids, namely antioxidants, proteins, carbohydrates and, preferentially, polyunsaturated fatty acids (Criegee reaction), leading to the formation of alpha-hydroxy- hydroperoxides, hydrogen peroxide, ozonides and aldehydes such as 4-hydroxynonenal. These are important signaling molecules, with crucial roles modulating inflammation, cell proliferation, cell growth and cell death [11].

These alkenals can activate a nuclear transcriptional factor, called nuclear factor erythroid 2-related factor 2 (Nrf2) present in the cell cytoplasm bound to Keap-1 protein. Such a protein has -NH2 and, mainly, -SH groups (Cys273 and Cys288) which, by binding alkenals [for example 4-hydroxynonenal (4-HNE)] at picomolar levels, causes a conformational change favoring the dissociation of Nrf2. This is then imported into the nucleus where, after forming a heterodimer with Maf (musculoaponeurotic fibrosarcoma) protein, interacts with the Antioxidant Response Element (ARE) on DNA. Consequently, the synthesis of several antioxidative enzymes (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferases, NADPH- quinone oxidoreductase, heat shock protein 70, phase II enzymes and Heme-oxygenase-1) are upregulated in various organs [12]. Also, reduces iron overload, and subsequent oxidative stress that is induced by elevated ferritin [13]. The increase of antioxidant capacity is the crucial step to counteract the chronic inflammation typical of diseases aggravated by chronic oxidative stress. An improvement of the antioxidant response has been reported in patients with asthma and Chronic Obstructive Pulmonary Disease (COPD), as emphysema, treated with ozone therapy [14-16]. Specifically, improvements were seen in IgE levels, inflammatory response, respiratory tests and clinical status. Also, in patients with rheumatoid arthritis, ozone has exerted beneficial effects [17,18].

This ozone efficacy not only may be explained through its actions on cytokine control (diminished IL-1, IL-6 and tumor necrosis factor α-TNFα) but also can reestablish cellular redox balance. It is known that reactive oxygen species can function as a second messenger to activate the nuclear transcription factor NF-κB, which orchestrates the expression of a spectrum of genes involved in the inflammatory response. Nrf2 is able to modulate inflammation through multiple mechanisms, such as the regulation of redox homeostasis and the suppression of pro-inflammatory genes, either directly or through the interaction with NF-κB [19]. Inflammation increases local and systemic Reactive Oxygen Species (ROS) level while ROS enhance inflammation. The Nrf2-mediated ROS-homeostatic control can break this vicious cycle. Nrf2 reduces inflammation by preventing the recruitment of RNA polymerase II to start gene transcription of pro-inflammatory cytokines IL-6 and IL-1β [20]. The capability of Nrf2 to maintain redox homeostasis would prevent DNA damage, preserve proteostasis, and improve mitochondrial function while suppressing acute and chronic inflammation [20]. The antioxidant and anti-inflammatory effects of ozone involve activation of Nrf2, which is thus considered as a key factor for the efficacy of ozone treatments. A previous study reported that ozone preconditioning significantly reduced NF-κB expression and inhibited inflammatory responses in liver ischemia/reperfusion injury [21]. Ozone can achieve an equilibrium between Nrf2 and NF- κB, modulating the expression of pro-inflammatory cytokines with an important effect in cytoprotection (Figure 1) [20].

JNNC-3-1-304-g001

Figure 1. Ozone and its relation with Nrf2 and NF-κB.
Ozone, at therapeutic doses, is capable of producing a small, transitory and controlled oxidative stress. The nuclear transcription factor Nrf2 is usually present within the cytosol as a complex with Keap-1 protein. The 4-HNE (ozone active metabolite) binds to Cys 151 of Keap1 and suppresses the constitutive inhibition of Nrf2, which then translocates into the nucleus. After binding to Maf, Nrf2 binds to ARE and switches on the synthesis of highly cytoprotective enzymes (SOD, catalase, GSH, heme-oxygenase-1, HSP, etc) maintaining a redox balance. NF-κB is also a redox- regulated transcription factor, involved in inflammation, immune function, cellular growth and apoptosis. In resting, it exists in an inactive form complexed with the inhibitor IκB. In the presence of oxidative stress, H2O2 (ozone active metabolite) activates a tyrosine kinase that phosphorylates IκB and causes its detachment from the inactive complex. The heterodimer moves promptly from the cytosol into the nucleus, where it regulates gene expression forming new proteins such as cytokines (IL-1, IL-2, IL-6, IL-10, TNF-α), COX-2, iNOS, adhesion molecules (ICAM), tissue factor, immunoregulatory molecules. At the same time, these two pathways inhibit each other at their transcription level via protein-protein interactions or through secondary messenger effects [19]. Nrf2 opposes the transcriptional upregulation of proinflammatory cytokine genes. Nrf2 binds to the proximity of inflammatory cytokine genes, including IL-6 and IL-1β, and inhibits their transcription. Nrf2 pathway also inhibits NF-κB mediated transcription by preventing the degradation of IκB-α. At the same time, Nrf2 upregulates the expression of genes coding antioxidant proteins. Similarly, NF-κB mediated transcription reduces the Nrf2 activation by reducing the ARE gene transcription, among other factors. Therefore, it can be considered that ozone is involved in the balance between these two transcription factors.
Nrf2, nuclear factor erythroid 2-related factor 2;Keap1, Kelch-like erythroid cell-derived protein; Maf, musculoaponeuroticfibrosarcoma; ARE, antioxidant response element; HO-1, heme oxygenase-1; 4-HNE, 4-Hydroxynonenal; HSP, Heat shock protein; SOD, superoxide dismutase; GSH, reduced glutathione; H2O2, hydrogen peroxide; TNF, tumor necrosis factor; COX-2, cyclooxygenase-2; ICAM, intercellular adhesion molecule; iNOS, inducible nitric oxide synthase.

Besides, Nrf2-activator may attenuate the Toll-Like Receptor (TLR) mediated aberrant inflammation by activation of intrinsic cytoprotective proteins and suppression of pro-inflammatory mediators. Hence, these two major signaling pathways may interact differentially and their cross-talk can be manipulated to regulate inflammation [22]. TLR activation is critical in the initiation of an inflammatory response against pathogens by triggering the production of inflammatory cytokines, enhancing adaptive immunity [23]. Simultaneously, a negative feedback mechanism also exists that could prevent the over-activation of TLR signaling that may otherwise result in chronic inflammation or autoimmunity. Nrf2 activation interferes with the expression of pro-inflammatory proteins and suppresses inflammation. The interaction of TLR and Nrf2 helps in the regulation of the inflammation process. The linkage between TLR signaling and Nrf2-Keap1 pathway may serve as a bridge between immune regulation and oxidative stress responses through the regulation of inflammation [22]. It has been demonstrated that ozone preconditioning improved renal inflammation and damage by blocking the activation of TLR4-NF-κB pathway in renal ischemia/ reperfusion injury. Also, ozone significantly reduced the mRNA level of TNF-α, IL-1β, IL-6, ICAM-1 (Intercellular Adhesion Molecule 1) and MCP-1 (monocyte chemoattractant protein 1) [24]. On the other hand, medical ozone, in vitro, has proven to be effective against viruses, bacteria, fungi and spores, destroying cells membrane and viruses envelop [25].

Ozone Therapy and its Positive Effects in the Treatment of Patients with COVID-19

Among the therapeutic effects of ozone therapy that favors the positive evolution of patients with COVID-19 are:

– Ozone improves oxygen metabolism increasing cellular oxygenation. Improving the hexose-monophosphate shunt, due to the activation of 2,3-DPG which, by binding to the β–chain of hemoglobin (Hb), causes a shift to the right of the Hb dissociation curve. This enhances the release of oxygen in the hypoxic tissues. There is also an improvement of the glycolytic pathway on erythrocytes significantly increasing their ATP content [11,13], recovering the elasticity of the red cell membrane thus improving blood rheology and capillarity [26]. There is a significant improvement in blood flow and oxygenation of ischemic tissues due to ozone treatment [27-30]. This is due to Nitric Oxide (NO), S-nitrosothiols cooperating with Carbon Monoxide (CO) and released prostacyclin [31,32]. Different preclinical and clinical studies have demonstrated the effect of ozone in modulating the NO levels and its importance in the protection of the vascular endothelium cells [32-34].

– Ozone is capable of inducing the release and modulation of interferons and related cytokines. Also, stimulates antioxidant defense systems, counteracting the state of hyperinflammation, cytokine storm and oxidative stress, suffered by patients with COVID-19. This is achieved through the increase in Nrf2 factors and restoring cellular redox balance [35,36]. There is also the activation of heme oxygenase-1 (HO-1) by increasing the release of CO and bilirubin. This contributes to reducing inflammation [37]. Several preclinical and clinical studies report a decrease in proinflammatory cytokines as IL-1, IL-6, TNFα, as well as ICAM-1, MCP-1, among others [24,38-45]. Ozone was able to modulate the phagocytic cells in peripheral blood and the mechanisms on how messengers can activate immunological response leading to the therapeutic biological effects [46,47]. This is a very positive effect on COVID-19 infection. The inflammatory response is a hallmark of severe SARS-CoV-2 infection, cytokine storm can lead to the death of these patients. The protective effect of ozone therapy was achieved by its anti-inflammatory property through the modulation of nucleotide- binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome, enhancing the antioxidant activity of Nrf2 and inhibiting apoptosis [48,49]. The NLRP3 inflammasome is a critical component of the innate immune system that mediates caspase-1 activation and the secretion of proinflammatory cytokines IL-1β/IL-18 in response to microbial infection and cellular damage. On the other hand, activation of Toll-Like Receptor (for example TLR4) by SARS-CoV-2 causes a biochemical cascade that begins with the formation of pro-IL-1 cleaved by caspase-1 and followed by activation of the inflammasome. IL-1 is secreted outside the macrophage, mediating lung inflammation, fever and fibrosis, and provoking severe respiratory problems [50]. It has been demonstrated that ozone preconditioning protected the rat kidney from reperfusion injury via modulation of the TLR4-NF-κB pathway [24].

– COVID-19 patients suffer from microthrombi due to increased viscosity and erythrocyte aggregation, among other factors. Ozone has an antiplatelet effect, increases some prostacyclins (like PGI2) leading to vasodilatation, as well as modulates antithrombin III [31,51]. All these effects, in conjunction with better blood circulation, can help to decrease the hypercoagulation phenomena present in these patients.

– Ozone can block the virus’s ability to replicate by balancing the cellular redox state, through the control of Nrf2 [52,53]. SARS-CoV-2 cell entry depends on Angiotensin-Converting Enzyme 2 (ACE2) and Transmembrane protease, serine 2 (TMPRSS2). SARS spike protein S will attach to ACE2. Following attachment to ACE2, viral entry requires S protein priming, which is performed by TMPRSS2 cleaving S protein. TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host, and TMPRSS2 inhibitors have been investigated as a potential therapeutic target for SARS-CoV-2. Nrf2 activators have an important role in reducing viral pathogenesis via inhibiting virus entry through inhibit TRMPSS2 [54,55]. Nrf2 activators may offer multiple ways to regain control of important pathways to increase resistance and slow viral replication. Application of an NRF2 activating agent, ACE2 mRNA was down-regulated 3.5-fold and TMPRSS2 was down-regulated 2.8-fold in human liver-derived HepG2 cells [56]. Exacerbated lung injury in Nrf2−/− mice was associated with increased pulmonary expression of inflammatory cytokines (TNF-α, IL-1β, IL-6) and with decreased pulmonary antioxidant and detoxifying enzymes relative to Nrf2+/+ mice [57]. Furthermore, pretreatment with the Nrf2-ARE inducer sulforaphane significantly attenuated Respiratory Syncytial Virus (RSV)-induced bronchopulmonary inflammation, epithelial injury and pulmonary viral expression in Nrf2+/+ mice [58]. Results from the study confirmed an association of oxidative stress in RSV pathogenesis and provide compelling evidence for an important regulatory role of Nrf2-ARE as a host defense mechanism against RSV disease. Another study found an inverse relationship between the levels of Nrf2 expression and influenza A viral entry and replication within nasal epithelial cells [59]. In response to experimentally applied mechanical ventilation, greater levels of lung alveolar and vascular permeability and inflammatory responses were found in Nrf2−/− compared to Nrf2+/+ mice [60]. In mice, Nrf2 deficiency caused augmented ovalbumin-driven airway inflammation and hyperresponsiveness. In this study, the enhanced allergic response in Nrf2−/− mice was associated with more pronounced lung mucus cell hyperplasia, eosinophilic infiltration, increased Th2 cytokines IL-4 and IL-13 and suppressed multiple antioxidants relative to Nrf2+/+ mice [61]. In an experimental sepsis model, Nrf2 deficiency increased the inflammation and mortality of mice against bacterial endotoxin (LPS)- and cecal ligation and puncture-induced septic shock [62]. This indicates that Nrf2 is a novel modifier of sepsis that determines survival by mounting an appropriate innate immune response. Data, therefore, suggest that Nrf2-ARE activators exert protective effects on LPS-induced inflammation, and suggested their potential therapeutic role for intervening sepsis syndrome. Taking into account that ozone stimulates Nrf2 [28,36,37,63], this could be an important physiological mechanism to block endogenous COVID-19 reduplication by preventing contact with receptors of SARS-CoV-19 through downregulation of ACE2 and TMPRSS2, inactivating the ability of the virus to enter cells [55]. The re-equilibration of the cellular REDOX state achieved with the ozone therapy is also important in the induction of cytokines synthesis in monocytes and lymphocytes and in the release of HO-1 and heat shock proteins which are potent activators of the immune system [12,64].

Conclusion

In summary, the positive aspect of ozone therapy is the ability to activate several defense mechanisms that cooperate to regain a normal redox system and a modulation of the NFκB/Nrf2 pathway. Today, ozone therapy represents the most practical approach for integrating standard therapies to achieve homeostasis. Therefore, due to the ozone therapeutical effects, it can be proposed as an adjunct therapy in SARS- CoV-2. Three randomized control trials (NCT04359303, NCT04370223 and NCT04444531) are pending classification and approval to start in Spain, one in Iran (IRCT20190618043923N4) and two more (NCT04366089 and NCT04388514) started in Italy one month ago.

Highlights

Ozone Therapy can be used for the treatment of COVID-19.

Ozone can achieve an equilibrium between Nrf2 and NF-κB,
modulating oxidative stress and pro-inflammatory cytokines.

Ozone counteracts hyperinflammation, cytokine storm and oxidative stress.

Ozone improves oxygen metabolism, blood flow and oxygenation of ischemic tissues.

Author Contributions

SMC – works on the conceptualization, drafting, editing and revision. JAMM, AHM, FJHT and JBN – work on the conceptualization and critical review of the manuscript. All authors have approved the final version of the manuscript.

Conflict of Interest

The authors declare they have no conflict of interest.

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Farmers Market Fruit & Vegetable Rx Program Shows Clinically Significant Quality of Life Outcomes Using Standardized SF-36 Health Survey

DOI: 10.31038/PEP.2020121

Abstract

Introduction: Fruit and vegetable prescription programs at farmers markets have shown some statistically significant changes in health markers. To date, little is known about whether these programs also improve Quality of Life (QoL). The current study examined if QoL improvements occurred after participation in a fruit and vegetable prescription program.

Methods: Our team of volunteer clinicians enrolled a cohort of 101 low-income adult participants over three years. Participants self-identified as food insecure and had previously been diagnosed with a diet-related disease. Each received a “prescription” to purchase fruits and vegetables from the local farmers market. Participants attended monthly clinic visits and attended plant-based cooking classes. Demographic, baseline and post-program outcome data (SF-36; Medical Outcomes Survey Short Form) were collected.

Results: All SF-36 subscales and composite scales showed statistical significance indicating subjectively improved quality of life/health among program graduates. SF-36 Role-Physical, Bodily Pain, Energy-Vitality and Role-Emotional subscales also demonstrated Reliable Changes (RCIs) indicating clinically significant outcomes across these domains of function. Individually, 32% and 27% of graduates exceeded the RCI for the Physical Composite Scale and Mental Composite Scales, respectfully.

Conclusion: This is the first study to apply the SF-36 RCI measure to assess generic quality of life outcomes of a fruit and vegetable prescription program based at a farmers market. It uniquely adds to the growing evidence supporting the benefits of improved access to healthy food through collaborative community interventions. Study participants reported both statistically significant and clinically meaningful improvements in functional health and QoL.

Keywords

Vegetable prescription, Clinical significance, Quality of life, Dietary behavior, Farmers market, Reliable change index

Introduction

Fruit and vegetable prescription programs such as the FVRx® have emerged as health interventions at farmers markets throughout the United States [1]. Such programs are premised on research showing a favorable link between increased fruit and vegetable consumption and markers of health [2-4]. Fruit and vegetable prescription programs also rely on emerging community food system studies examining public health collaborations designed to improve fresh produce accessibility and consumption [5,6].

The produce prescription model has been studied as a medical intervention for diet-related chronic diseases [7], as a measure of the psychosocial benefits of farmers markets [8,9], a catalyst for nutrition education [10], a vehicle for healthy food access in low-income/low-access neighborhoods [11,12] and a learning milieu for healthcare professionals [13]. Researchers have collected quantitative biological data as well as qualitative self-reported data [14,15]. Studies have also reported outcomes of improved health perception among participants [16].

Previously reported data from year-end program evaluations of the five FVRx programs in Georgia (of which our cohort was a part) conducted by Wholesome Wave Georgia showed both socioeconomic and health benefits. In 2017, this statewide data showed that, from baseline to the end of the six-month program, the percentage of program participants reporting that “food often didn’t last” and there “wasn’t money to buy more food” decreased by 79% (22% to 4%) across all sites. Further, the overall percentage of program participants reporting that they had often gone hungry “due to lack of money for food” decreased by 89% (44% to 11%) over the course of the program [17]. The 2018 program evaluation of this statewide data found that over the relatively short six-month program timeframe, Body Mass Index decreased 1.4% and waist circumference decreased 3.3% [18].

As fruit and vegetable prescription programs grow in number and design, studies must also seek to discover the clinical significance [19] resulting from community food-healthcare partnerships. In other words, do participants self-report evidence of improved quality of life upon completing these community-based programs?

This three-year study examined whether low-income/low- access adult graduates of an FVRx program experienced favorable improvement in their subjective Quality Of Life (QoL) and function. To our knowledge, this is the first study to assess subjective change in QoL and function in fruit and vegetable prescription programs based at a farmers market using the Medical Outcomes Survey – Short Form 36 (SF-36), a standard in generic quality of life outcome metrics.

Methods & Materials

Participants

On average, 34 adults enrolled in the program annually, for three years in sum total, at no personal cost. The majority of participants came from areas with low access to healthy food options, self- identified as food-insecure and had been previously diagnosed with a diet-related disease such as obesity, hypertension, diabetes, and/or heart disease.

FVRx Program Description

The program targeted change in dietary behavior among participants and included three required components: (a) monthly clinic visits, (b) weekly farmers market attendance, (c) and plant-based cooking classes. The program was a six-month per year intervention conducted over three years (2017-2019).

Outcome Measurement

We selected the SF-36 as the outcome measure. The SF-36 is widely considered a gold standard in generic quality of life metrics, quantifying disease burden, and measuring patients self-report of functional health. It has been utilized in 4000+ peer reviewed scientific publications – of which more than 400 were randomized controlled clinical trials – and judged to be a useful tool in evaluating benefits of treatment interventions [20]. It is suitable for age ≥ 14 and requires 5-10 minutes to complete. The data is summarized in two higher order factors, termed Physical Health and Mental Health Composites which are equated to patients’ attitudes regarding overall physical and mental health status. Eight subscales are generated, reflecting physical functioning, role-physical, bodily pain, general health, vitality, social functioning, role-emotional, and mental health. Each subscale attempts to quantify a patient’s attitude in specific physical or health domains. For example; the Role-physical scale measures how much a patient believes that their daily activity is limited by physical dysfunction whereas the energy-vitality scale ranges from feeling “tired and worn out” to “full of energy and pep”.

Procedures

Prior to enrollment, we outlined the program’s attendance expectations and incentive component before participants signed informed consent forms. Volunteer healthcare providers (i.e., physicians, nurses, registered dietitians), accompanied by first-year medical students, collected baseline program-related information to include demographics, SF-36 surveys, and other data not reported herein during the initial visit. Self-reporting surveys were issued with literacy support when needed. Clinicians provided healthy eating and lifestyle education and then wrote a “prescription” for each participant to redeem for only fruits and vegetables at the farmers market. The prescription had a no-cash value of $7/person/week for four weeks each month, based on family size. For example, an adult participant with a family of four received redeemable market tokens equivalent to
$28/week ($112/month).

Participants returned to the clinic monthly where volunteer clinicians offered encouragement, provided general health and nutrition education, and renewed prescriptions. Participants attended a weekly farmers market to redeem their prescriptions for fruits and vegetables, sold by local farmers, and attended a minimum of four required plant-based cooking classes at no cost. Health educators at the farmers market provided nutrition information and cooking demos for the general public, of which participants were a part. Farmers also provided recipes and preparation tips to help demystify lesser-known seasonal fruits and vegetables.

When absent from the program, participants reported schedule conflicts, illness and lack of transportation as the most common reasons for not attending. If absent from the farmers market three weeks in a row, and/or if non-compliant with clinic or cooking class attendance, participants were listed as non-completers and unenrolled. An average 26% of participants were recorded as non-completers. Participants who completed the above-mentioned requirements and the post-SF-36 on (or within one month of) the sixth clinic visit were considered program “graduates”/completers.

Statistical Analysis

Descriptive statistics of demographic and SF-36 outcome data at baseline and at six-month follow-up were calculated. Per Protocol (PP) and Intent to Treat (ITT) analyses using SPSS 26.0 statistical package were employed to investigate change in SF-36 scores occurred after participation in the FVRx six-month program. Within subject paired sample t-tests were used to examine if statistical differences existed in each of the eight subscales or either of the two composite scales on the SF-36 after six months of FVRx program participation relative to baseline. Given the exploratory nature of this study, a conventional p-value of ≤0.05 was selected to denote statistical significance. Adjunctively, RCI also were calculated for each participant’s SF-36 subscales and composite summary scales to appreciate where clinically significant change may have also occurred. RCI calculations have been validated using normative data for general populations and several disease states [21].

Results

Of the 101 participants enrolled, 75 completed the program and had a full data set. There were no significant differences between completers and non-completers in any measured demographic, health, or socioeconomic variable. Our average participant was a female (82%) African American (76%) who earned less than $2000/month (74%) and lived in a low access area. A majority were uninsured or covered by a federal insurance program (68%) and received supplemental benefits (64%) (Table 1).

Table 1: Demographics.

Demographics

% of Sample

Age 18-29

7.90%

30-39

11.9%

40-49

19.8%

50-59

34.70%

60+

25.7%

Sex Male Female

17.80%

82.2%

Race Hispanic,

6.9%

Asian, Asian American

1%

American Indian, Alaskan Native

3%

Black, AA, or Carib. American

76.20%

Hawaiian, Pacific Islander

13.90%

Other

5.90%

Educational background

10.90%

< High school diploma

19.80%

High school or GED certificate

36.60%

Some college or tech school

15.80%

2 year college or tech degree

12.90%

4 year college or tech degree

4%

>4 college degree

Employment status

Student

2%

Working part-time

10.90%

Working full-time

25.70%

Not employed, or homemaker

15.80%

On disability

27.70%

Retired

12.90%

“Other”

5%

Health Insurance

Uninsured

23.80%

Medicated or Medicare

44.60%

Insured through employer

21.80%

Insured, private insurance

4%

Other

5%

Income level

<$1000/month

38.60%

$1001 – $1300/month

19.80%

$1301 – $1700/month

4%

$1701 – $2000/month

11.90%

$2001 – $2400/month

6.90%

$2401 – $2700/month

5.90%

$2701 – $3000/month

5%

$3001 – $3400/month

5%

> $3401

3%

Supplemental benefits

No

35.60%

Received

64.40%

Average (SD) # of people aged 0-17 Living in home: 1.5 (1.6), ranging from 0 to 9.

Note 1: Final sample size was N = 101, reflecting 35 participants from year 2017, 39 from year 2018, and 27 from year 2019.

All eight of the SF36 individual subscales and both of the composite scales showed statistical significance in a favorable direction implicating improved self-reported quality of life/health direction after program participation. This was true in both the PP (all P-values ≤0.002) and ITT (all p-values ≤0.028) analyses (Table 2 and Figure 1).

Table 2: Descriptive Statistics and Over-Time Comparisons for SF-36.

Baseline

Follow-up

Paired Sample

Paired Sample

RCI

t-test

t-test

N = 101

N = 75

with case-wise

with missing

SF-36 Variable

AVG (SD)/Min-Max

AVG (SD)/Min-Max

deletiona

values replacedb

+

Physical Functioning

60.8 (28.5)/0-100

66.1 (26.1)/10-100

-3.16 (p = .002)

-2.23 (p = .028)

Role-Physical

47.5 (43.1)/0-100

66.7 (39.1)/0-100

-3.99 (p < .001)

-4.96 (p < .001)

Yes

Bodily Pain

47.6 (25.8)/0-100

57.6 (25.0)/0-100

-3.68 (p < .001)

-4.44 (p < .001)

Yes

General Health

51.8 (20.1)/5-100

61.5 (19.0)/20-97

-5.36 (p < .001)

-6.00 (p < .001)

Energy-Vitality

45.6 (20.6)/0-90

57.1 (19.7)/10-100

-5.29 (p < .001)

-6.01 (p < .001)

Yes

Social Functioning

64.5 (25.2)/0-100

77.2 (22.3)/13-100

-5.10 (p < .001)

-5.28 (p < .001)

Role-Emotional

58.8 (42.2)/0-100

80.4 (36.0)/0-100

-4.10 (p < .001)

-5.40 (p < .001)

Yes

Mental Health

71.6 (18.3)/28-100

77.1 (14.7)/40-100

-3.41 (p = .001)

-3.53 (p = .001)

PCS Composite

37.4 (11.7)/13-65

41.1 (10.8)/12-66

-4.78 (p < .001)

-4.28 (p < .001)

MCS Composite

47.3 (10.5)/28-68

52.5 (9.4)/25-68

-4.98 (p < .001)

-5.58 (p < .001)

aPer protocol analysis, in that IF data was missing at time 2, THEN entire case was deleted. Total of 26 cases had missing data at time 2 resulting in a final sample of N = 75.
bIntention to treat analysis, in that all 101 participants data was included with missing data at time 2 imputed by using the average of the group. N = 101.
Note 2: N = total sample size. AVG = computed average. SD = Standard Deviation. Min = Minimum Value. Max = Maximum Value. RCI + = Reliable Change Index (Jacobson &Truax, 1991) was exceeded, indicating that as a group a reliable change in overall obtained scores at follow-up relative to baseline had occurred.

PEP-1-2-107-g001

Figure 1. SF-36 Overtime Comparison.

As a cohort, the Role-Physical, Bodily Pain, Energy-Vitality and Role-Emotional scales all exceeded established SF-36 RCI for clinically meaningful change. Individually, 32% and 27% of graduates were judged to have demonstrated reliable change on the SF-36 Physical Composite Scale (PCS) and Mental Composite Scale (MCS), respectively (Figure 2).

PEP-1-2-107-g002

Figure 2. SF-36 Reliable Change.

Discussion

Fruit and vegetable prescription programs rely on emerging studies of community food systems. Farmers markets may provide uniquely collaborative spaces in which community health improvements can occur. Fruit and vegetable prescription programs at farmers markets are also premised on research showing a favorable link between increased fresh produce consumption and markers of health. However, little research has been published to date that has investigated the impact of participation in such programs on self- reported quality of life and function.

The present study highlights several important findings. First, the majority (75%) of participants were able to complete a six-month FVRx program requiring monthly clinic in-person check-ins and attendance in several cooking classes. The relatively low attrition rate supports the contention that this kind of programming is both viable and well regarded by study participants who have low access to healthy food options, are food insecure, and are challenged by medical morbidities including obesity, hypertension, diabetes, and/or heart disease. Second, these results highlight that after completion of the program, study participants self-reported experiencing improved physical functioning, bodily pain, general health, level of energy/pep, social functioning, and mental health while also experiencing less impact on their ability to perform expected roles because of emotions or physical challenges (all results were statistically significant). Third, over and above the aforementioned statistically significant differences, appreciation of RCIs also would highlight that clinically meaningful change in a favorable direction occurred in the areas of bodily pain experienced, level of energy/pep, and ability to perform expected roles.

Several study limitations are evident. First, as a cohort study, this endeavor was limited by lack of a control or comparison group as well as some variations in the year-to-year program requirements and implementation. We also recognize that while the socioeconomic mix of program cohorts, educators and farmers is not specifically addressed in this study, it likely represents a significant influence on its outcomes and would be a valuable area for future study. Future studies should also include a control or comparison group and be powered/ designed to estimate quality adjusted life years and cost effectiveness.

In summation, our study adds to the growing body of literature supporting fruit and vegetable prescription programs and expands knowledge of the benefits of increasing access to healthy food and wellness education within the milieu of farmers markets. The convergence of partners involved in providing these community- based collaborative health programs represents a behavioral health treatment approach with a wide variety of players and motivations. We believe increasing access to these types of programs should be a national priority.

Acknowledgements

Dozens of community members gave voluntary time as program participants; we appreciate their commitments. We also thank Augusta Locally Grown for program coordination; Wholesome Wave Georgia for program funding & design; Augusta District Dietetic Association for volunteer clinicians; Medical College of Georgia  & Augusta University for volunteer students; Harrisburg Family Healthcare for hosting; St Luke United Methodist for transportation; EAT Local CSRA & Icebox Ministries for cooking education; Women in Philanthropy, Community Foundation of the CSRA, Good Neighbor Ministries, Women’s Health of Augusta and Reid Memorial Presbyterian for education program funding. Finally, we thank all the farmers of the Veggie Park Farmers Market.

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Observational Study of the Effect of the Hydroxychloroquine/Azithromycin Combination in Patients Hospitalized for a Severe Form of COVID-19

DOI: 10.31038/JPPR.2020322

 

What is Already Known about This Subject?

The discussion on the efficiency of the hydroxychloroquine/azithromycin combination remains open. To date, no randomized trial has demonstrated the efficiency of this combination in patients with a severe form of COVID-19.

What This Study Adds?

We report the results of a non-randomized observational study compared hydroxychloroquine/azithromycin combination to any antiviral treatment for patients with a severe form of COVID-19. We warn of the difficulty of interpreting these results in the absence of randomization.

Abstract

Following the demonstration in vitro of the efficiency of a synergistic effect of hydroxychloroquine (HCQ) associated with azithromycin (AZI) against the SARS-CoV-2, some studies have aimed to evaluate its efficiency in a clinical setting. We present the results of a non-randomized observational study of patients admitted for a severe form of COVID-19 disease who have been treated by HCQ/AZI after collegial physicians’ decision. Of the 306 patients included (average age: 72.8 years), 53 received the HCQ/AZI association. Univariate analysis shows in non-survivors a higher average age, more severe clinical signs on admission (lung invasion rate> 50%, Dyspnea and creatinine>133µmol/L) and more comorbidities (cerebrovascular accident, chronic kidney disease, immunodeficiency).We evaluated the efficiency of the HCQ/AZI treatment on a population (n=96) with comparable characteristics (age, risk factor, gravity…). If mortality of the patients treated with HCQ/AZI seems different in this sub-study population (HCQ/AZI: 0% vs. Other: 8%), the methods of the study and its size do not allow the identification of a statistically significant difference (p=0.122).

At this time of the epidemic, the HCQ/AZI must be evaluated in a randomized trial at the right and safe dosage.

Introduction

At the end of 2019, a new coronavirus, designated SARS-CoV-2, was causing an epidemic of respiratory diseases in China. The city of Wuhan (China) went into full confinement on January 23, 20201. On March 12, 2020, the World Health Organization (WHO) declared the COVID-19 pandemic. The importance of the number of symptomatic patients and the particular gravity of some of them lead the Hospitals to reorganize in emergencies on an almost daily basis both at the structural, organizational and medical level. Among the antiviral treatments likely to be effective on the symptoms of SARS-CoV-2, three molecules or associations stood out: remdesivir, the fixed combination ritonavir/lopinavir and (hydroxy)chloroquine associated or not with azithromycin [1].

In mid-March, remdesivir and the ritonavir/lopinavir combination were announced as a complete disruption by the laboratories producing them. The French Ministry of Health then supervised the off-label use of hydroxychloroquine, conditioning it to severity criteria and subject to a collegial decision by the medical profession. We present the results of a monocentric, observational, retrospective study aimed at evaluating the efficiency of the hydroxychloroquine/azithromycin combination on the COVID-19 disease.

Methods

All patients hospitalized at our center for a severe form of COVID-19 disease were included in the study.The diagnosis of COVID-19 disease combined the recording of symptoms (fever, caugh, fatigue, myalgia, headache, dyspnea, diarrhea, nausea, vomiting, anorexia, anosmia, aguesia, dizziness, fall, hypoxemia) with a SARS-CoV-2 reverse-transcriptase–polymerase-chain-reaction (RT-PCR) and/or standard low dose CT imaging.The following characteristics were sought in the study population: age, body mass index (BMI), active or history of smoking, chronic kidney/heart/respiratory disease, hypertension, diabetes mellitus, dyslipidemia, cerebrovascular accident and immunodeficiency (including cancer under treatment). The home drugs treatment of interest were sought: angiotensin-converting-enzyme (ACE) inhibitors and angiotensin-receptor blockers (ARBs) calcium-chanel inhibitor, diuretics, betablockers, corticosteroids and aspirin. We have listed antivirals under evaluation:remdesivir, hydroxychloroquine (HCQ), azithromycin (AZI), ritonavir/lopinavir.The severity of the COVID-19 stage of the patients included was assessed on the basis of at least one of the following criteria: dyspnea, O2 saturation ≤93%, radiological pulmonary infiltrates >50% at 24-48 hours of admission [2].

HCQ/AZI

The dosage of HCQ/AZI was: HCQ (D1-D10; 600mg per day in 3 doses) combined with azithromycin (D1: 500mg in 1 dose; D2-D5: 250mg per day). Only patients not on dialysis, with no contraindication to HCQ/AZI treatment (e.g. severe kidney failure, widening of the QTc wave) received treatment with HCQ/AZI after collegial physician decision. All patients on HCQ/AZI underwent an electrocardiogram before initiating treatment. Once treatment was started, an electrocardiogram was performed daily during hospitalization, as well as a determination of serum potassium, magnesemia and blood sugar. In order to reduce the biases linked to the care of patients, to the comorbidities, to the typology of the medical service (e.g. palliative care, geriatry) and to the contraindication of HCQ/AZI treatment (dialysis), we have make a sub-study. In this sub-study only patients treated in internal medicine were included and we have excluding dialysis patients.

Clinical Outcomes

Critical disease defined by death or transfer to intensive care unit (ICU) and death alone were the two main outcomes.

Statistical Analysis

Continuous data are summarized as mean (standard deviation) and categorical data as frequency (percentage). For univariate comparisons T-test and Chi-2 tests were used as appropriate to compare differences between non-critical and critical disease or survivor and non-survivor. The multivariate analysis used logistic regression ajusted on age, sex, body mass index (BMI), chronic heart disease, hypertension, diabetes mellitus, chronic kidney disease, hemodialysis, chronic lung disease (asthma or chronic obstructive lung disease), personal history of stroke, current or former tobacco use, treatment with immunosuppressive drugs (including anticancer drugs), creatine and C-reactive protein (CRP) levels, percentage of lung affected on CT-scan, low oxygen saturation, dyspnea, oxygen flow. A two-sided p value < 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 26.0 package. Neither patients nor the public were involved in the conception or conduct of the study.

Results

Between March 15 and April 15, 2020, 314 patients were admitted into the different wards. Eight patients were transferred to another hospital and were considered loss of follow-up: 6 were transferred to another region to vacant intensive care beds and 2 were taken care of for extracorporeal membrane oxygenation. The median age was 75.6 ± 14.5 years. The number of days between the onset of symptoms and hospitalization was 6.6±7.8 days. Men were a little more represented (54%).

Comorbidities

Table 1 shows major comorbidities recorded on admission. The most common were chronic heart disease (71%), hypertension (58%), dyslipidemia (36%), chronic kidney disease (27%), diabetes mellitus (26%), immunodeficiency (21%) and chronic respiratory disease (19%). Eighty nine percent of the patients have comorbidity with an average number of comorbidities of 4.1±2.3. Three per cent (8/306) of patients were current smokers and 10% were former smokers.It can be noted that 26% of patients have a BMI> 30kg/m2.

Table 1: Demographic findings of patients on admission.

Demographic data

Total
(N=306)

Survivor
(N=231)

Non-survivor
(N=75)

p-value *

Age – yr

75.6 ± 14.5

72.8 ± 14.7

84.0 ± 9.9

<0.0001

Sex (male sex)

54%

55%

49%

0.394

Weight (kg)

74.8 ± 15.8

76.3 ± 15.7

70.2 ± 15.4

0.007

Body mass index (kg/m2)

26.8 ± 5.0

27.2 ± 5.0

25.6 ± 4.9

0.03

BMI<25 kg/m2 (%)

59%

61%

50%

0.11

BMI 25–29.9 kg/m2 (%)

32%

34%

28%

0.396

BMI≥30 kg/m2 (%)

26%

27%

22%

0.376

Nursing home resident (%)

13%

10%

23%

0.003

Severity and mortality

Death (%)

25%

0%

100%

ICU transfer (%)

3%

3%

3%

0.871

Critical cases (Death or transfer to ICU) (%)

27%

3%

100%

<0.0001

Time from symptom onset to admission (days)

6.6 ± 7.8

7.2 ± 7.5

4.5 ± 8.5

0.013

Length of stay in hospital (days)

11.3 ± 10.0

12.2 ± 9.4

8.5 ± 11.3

0.005

Time between admission and ICU transfer (days)

1.1 ± 1.7

1.2 ± 1.7

1.0 ± 1.9

0.802

Comorbidities (%)

Hypertension

58%

55%

67%

0.065

Diabetes mellitus

26%

27%

25%

0.797

Chronic heart disease

71%

67%

84%

0.005

Dyslipemia

36%

33%

44%

0.094

Chronic respiratory disease

19%

20%

15%

0.276

Chronic kidney disease

27%

23%

43%

0.001

Dialysis

4%

3%

7%

0.232

Immunodeficiency

21%

16%

36%

<0.0001

Cerebrovascular accident

20%

16%

32%

0.002

At least one comorbidity

89%

87%

97%

0.009

Smoking status (%)

Current smoker

3%

3%

1%

0.424

Former smoker

10%

9%

15%

0.134

Home drug treatments (%)

ACE inhibitors

20%

19%

24%

0.31

ARBs

22%

22%

21%

0.955

Calcium-chanel inhibitor

23%

22%

24%

0.729

Diuretics

27%

22%

44%

<0.0001

Betablockers

31%

27%

44%

0.007

Corticosteroids

5%

3%

11%

0.008

Aspirin

30%

25%

45%

0.001

*p-value univariate statistical test between Survivor VS Non-Survivor. – : statistical test non applicable.

Home Drug Treatments

Home drug treatments included beta-blockers (31%), aspirin (30%), diuretics (27%), calcium channel blockers (23%), ARBs (22%), ACE inhibitors (20%) and corticosteroids (5%). No patient was treated by non-steroidal anti-inflammatory drugs (Table 1).

Severity of the Clinical Case

At the admission, 43% of the patients presented a C-reactive protein >100mg/L, 43% an oxygen saturation £93%, 21% a creatinine>133µmol/L and 7% of the patient presented a proportion of lung invasion rate>50% (average percentage of invasion: 29%±18) (Table 2). Among the 306 patients eligible for analysis, 250 had a lung CT scan and 213 a RT-PCR.

Symptoms

The most common symptoms were dyspnea (67%), fever (60%), fatigue (59%), cough (51%), diarrhea (30%), myalgia (18%) and dizziness/fall (17%). Only 6% of patients reported ageusia/anosmia on admission (Table 2).

Table 2: Clinical, laboratory and radiographic findings of patients on admission.

Symptoms at onset of illness (%)

Total
(N=306)

Survivor
(N=231)

Non-survivor
(N=75)

p-value*

Fever

60%

62%

55%

0.266

Cough

51%

52%

51%

0.898

Fatigue

59%

58%

60%

0.812

Myalgia

18%

20%

11%

0.058

Headache

8%

9%

4%)

0.184

Dyspnea

67%

64%

77%

0.028

Diarrhea

30%

33%

21%

0.058

Nausea or vomiting

9%

11%

1%

0.008

Anorexia

11%

12%

11%

0.809

Anosmia or ageusia

6%

7%

1%

0.054

Dizziness. fall

17%

16%

19%

0.657

Hypoxemia (oxygen saturation < 93%)

43%

40%

55%

0.024

Inpatient drug treatments (%)

Hydroxychloroquine with azithromycin

17%

22%

4%

<0.0001

Hydroxychloroquine alone

2%

1%

3%

0.417

Azithromycin alone

4%

5%

1%

0.184

Lopinavir with Ritonavir

4%

4%

5%

0.593

Oxygen therapy

87%

86%

91%

0.269

Invasiveventilation

9%

9%

9%

0.858

Laboratory findings

PCR confirmed diagnosis (%) n=213

79%

75%

90%

0.023

C-reactive protein (mg/L)

101.2 ± 83.6

95.9 ± 83.5

118.0 ± 82.3

0.05

CRP<5 mg/L (%)

5%

6%

1%

0.108

CRP<100 mg/L (%)

57%

60%

47%

0.051

CRP 100–199 mg/L (%)

28%

25%

36%

0.075

CRP≥200 mg/L (%)

15%

14%

17%

0.64

Creatinine (µmol/L)

115.7 ± 110.4

103.6 ± 93.2

154.6 ± 147.6

0.001

Creatinine ≥ 133µmol/L (%)

21%

17%

33%

0.003

Radiologic findings (n=250)

Percentage of lung affected on the CT scan (%)

29 ± 18

28 ± 17

31 ± 22

0.253

Proportion of lung affected on the CT scan > 50%(%)

7%

5%

14%

0.028

Typical CT-scan (%)

14%

15%

7%

0.125

Bilateral pulmonary infiltration (%)

46%

48%

36%

0.120

Ground-glass opacities (%)

29%

33%

14%

0.008

Analysis of Non-survivors as Compared with Survivors

Tables 1 and 2 show the distribution of demographic characteristics and coexisting conditions among survivors and non-survivors. Non-survivors were older (p<0.0001) and had a greater prevalence of chronic heart disease (p=0.005), cerebrovascular accident (p=0.002), chronic kidney disease (p=0.001), and immunodeficiency (p<0.0001). Among medication, aspirin (p=0.001), corticosteroids (p=0.008), betablockers (p=0.007) and diuretics (p<0.0001) were more commonly used by non-survivor patients. The non-survivors were faster hospitalized after the first symptoms (4.5±8.5 days vs. 7.2±7.5 days). Among non-survivors mortality rate was of 25% and transfer to ICU was of 3%. Laboratory and radiologic findings for non-survivors reveal fewer signs at admission of renal dysfunction (creatinine≥ 133µmol/L; p<0.003) and lung invasion rate (>50%; p<0.028). The multivariate analysis of factors associated with non-survival reveals that increasing age was a strong predictor of in hospital mortality after adjusting for major comorbidity (per additional year, OR 1.083; p=0.003) (Figure 1). Stroke history (OR:2.983; p=0.027), immunodeficiency (OR:4.665; p=0.002) and percentage of the lung affected on the CT scan were also statistically associated with increased hospital mortality (OR:1.032 per additional percent of lung affected; p=0.009). The other factors don’t show a significant implication on the patient outcomes.

JPPR-3-2-315-g001

Figure 1. Mortality by age.

Antiviral Treatment

Fifty-three patients received the HCQ/AZI association, 13 the lopinavir/ritonavir association, 12 azithromycin alone and 5 HCQ alone.The HCQ/AZI have been administered an average of 7.9±3.1 days of treatment. Among 16 patients who did not receive the 10 days of treatment, the causes were: transfer to ICU (N=6), widening of the QTc wave (N=6; occurring between 1 and 8 days after initiation of treatment), renal failure (N=1), death (N=2; after 1 and 3 days of treatment), pseudo-cerebellar syndrome attributed to viral damage combined with HCQ (N=1). Thepatients treated by HCQ/AZIhad severe form of COVID-19 (9% of lung affected on CT-scan), were younger patient (13 years) and tend to have more comorbidities except diabetes and immunodeficiency.Percentage of lung affected on CT-scan was the only variable statistically associated with death or ICU transfer (OR:1.033 [1.002-1.064], p=0.039) among patients admitted into the two internal medicine ward where HCQ-AZI were used. The sub-study (N=96) which evaluate the efficacy of treatment with HCQ/AZI for the patients admitted into the two internal medicine units included 96 patients. There was no significant difference in demographic, comorbidities, smoking status, home drug treatment and laboratory/radiologic findings between the treated and untreated population. If the mortality of patients treated with HCQ/AZI seems different, this result is not statistically significant (0% vs. 8%; p=0.122).

Discussion

The COVID-19 situation worldwide, as of 1 July 2020 based on the data transmitted by the different countries is 10 446 353 cases of COVID-19 including 511 037 deaths [1]. If the number of new cases seems to decrease in Europe, the world wide situation continues to worsen. The average incubation period for COVID-19 is 5 days [3]. The interval from symptom onset the hospital admission is 6, days in our study. The average age of patients in our study (75.6 years) is higher than the studies currently published (47-73 years) with a male preponderance3. The clinical manifestation of COVID-19 is various. We found standard symptoms in hospitalized patients included: dyspnea, fever, asthenia, cough, diarrhea, muscle ach and dizziness/fall. Anosmia ageusia may be identified in only 6% of patients. If comorbidities presented by 89% of our patients are among the most common (hypertension, diabetes mellitus), they are present in a remarkably high rate of our patients (chronic heart disease>70%, chronic kidney disease > 25%, immunodeficiency>20%) [4,5]. Obesity seems to play a special role affecting more than 25% of our patients. Obesity or excess ectopic fat deposition are suspected of unifying risk factor for severe COVID-19 infection, reducing both protective cardiorespiratory reserve as well as potentiating the immune dysregulation that appears, at least in part, to mediate the progression to critical illness and organ failure in a proportion of COVID-19 patients [6]. However, it should be noted that it does not appear as a factor of death. Concerning the long-term treatments taken by the patient, the patients treated with aspirin, diuretics, betablockers or corticoids are significantly higher in the non-survivor group. But, it is difficult to attribute excess mortality to these treatments, since they are generally used to manage the comorbidity previously mentioned. The search for a predictive sign of worsening of a patient’s condition at admission is a major issue in medical management. There is an excess mortality in patients more older (Figure 1), patients who on admission have dyspnea, oxygen saturation £ 93%, a lung invasion rate> 50% or a creatinine> 133 µmol/L. These elements are warning signs for the physician supported by other publications on the subject [7]. On the other hand, on the advanced side on antiviral treatment, there has been no notable progress to date. Among the treatments discussed, remdesivir, the ritonavir/lopinavir and HCQ/AZI combination were the first treatments offered in France. For reasons essentially of availability, hydroxychloroquine associated or not with azithromycin has become one of the only therapeutic options in the absence of any evidence of effectiveness. Gautretet al. published the first result of the HCQ/AZI efficiency in COVID-19 patients [8]. They demonstrated on a series of 80 patients an undetectability of viral load at the nasopharyngeal level in 83% of patients on day 7, 93% on day 8 [9]. Respiratory samples are negative in 97.5% of patients on day 5. However, patients in this study had a low rate of comorbidity for an average age of 52.5 years. They do not appear to be the most at risk of complications given the current state of knowledge. Thus, only 53.8% (43/80) had a CT-scanner lung disease and 15% (12/80) had oxygen need.This population appears to have a relatively mild impairment compared to our study population. In agreement with the relative viral RNA load reduction, in vitro tests show a cytopathic effect of SARS-CoV2 could be observed in only 16% (5/31) wells at 60 h post infection after HCQ/AZI exposition as compared to 100% (13/13) in positive controls [9]. Rosenberg et al. publish an observational study to report adverse events and mortality of HCQ and/or azithromycin among patients with COVID-19 (Number of patient: 735 with HCQ/AZI, 271 with HCQ, 211 with azithromycin, 221 any treatment – similar age:61.4 to 65.5 average) [10]. Cardiac arrest was more frequent in patients who received HCQ with azithromycin, compared with patients who received neither drug, even after adjustment. Moreover, treatment with HCQ, azithromycin, or both, compared with neither treatment, was not significantly associated with differences in in-hospital mortality. Mahevas et al report a study carried out on 181 patients requiring oxygen therapy excluding patients transferred to ICU [11]. In their study, 15 patients were treated with the HCQ/AZI combination, none were transferred to ICU and none died.

The treatment of patients was done according to modalities (collegial doctor’s decision) and within deadlines close to ours (7 days after the onset of symptoms). The mortality reported in this study is lower than ours. This can be attributed to the exclusion of patients without ICU need and the average age of the population (60 years vs. 75 years), even if the predictors of aggravation are relatively similar (CRP, percentage of pulmonary invasion). Interestingly, the authors put into perspective the importance of treatment with an antiviral during the virus contamination phase. However, HCQ exhibits immunomodulatory properties through its action on interleukin 1,2 and 6, TNFa and the inhibition of toll-like receptors. This explains its effectiveness in certain autoimmune pathologies which in the case of the inflammatory storm identified in certain forms of COVID-19 could be of theoretical interest. Nevertheless, the latest published clinical data, consistent with our series, point out that during the inflammatory phase (usually on the second week of symptoms) this treatment should have any effectiveness. Side effects, particularly of the heart, in patients with fragility induced by viral infection remain a warning point for physician using the HCQ/AZI combination. In our cohort of treated patients 11.3% (6/53) had heart rhythm problems. As rhythm disturbances were not systematically sought in the untreated group, it is difficult to conclude on an increase in disorders due to COVID-19 disease or to antiviral treatment. A study of 201 patients demonstrated that Baseline QTc intervals did not differ between patients treated with chloroquine/HCQ (monotherapy group) vs. those treated with combination group (chloroquine/HCQ and azithromycin) (440.6 ± 24.9 ms vs. 439.9 ± 24.7 ms, p = 0.834) [12]. More recently Mehraet al. presented a multinational registry analysis carried out on the largest population studied to date (96 032 patients; mean age 53.6±17.6; oxygen saturation <94%; 19.9% of non-survivors patient) [13]. This article which seemed to make everyone agree was retracted a few days after its publication by its authors. However we should note that the authors reported an excess mortality in patients treated with combination of HCQ (main dosage 587mg±128mg during 4.3±2.0 days) with a macrolide (azithromycin or clarithromycin; dosage not communicated) as well as a de-novo ventricular arrhythmia in 8.1% of patients treated (versus 0.3% of untreated patients). We find a similar percentage of ventricular rhythm disorders (11.3% vs. 8.1%) in our patients with a more advanced age (75 years vs. 54 years), a comorbidity rate, predictors of severity and duration longer treatment (7.9 vs. 4.3 days). However, we did not find an increase in the mortality of patients treated with the HCQ/AZI combination. In view of these various elements, it remains necessary to remain cautious. The need for QT interval monitoring remains necessary. We draw attention to the difficulty to interpret the results of a non-randomized observational study. Indeed, in the absence of randomization, the mortality comparison data in the group treated by HCQ/AZI and the group not treated are impacted by many biases. Thus, if mortality seems different in the total study population (HCQ/AZI: 6% vs. Other: 28%) and in the sub-study population (HCQ/AZI: 0% vs. Other 8%), it is distorted by populations that are not comparable (age, risk factor) and different treatment methods desired or not by physicians. We no longer observe any significant difference after identifying and taking into account the biases inherent in this type of study. As a conclusion, the question of the efficiency and safety of the HCQ/AZI combination in the treatment of COVID-19 disease remains open pending the results of randomized clinical studies (Figure 1 and Tables 1-3).

Table 3: Multivariate analysis of factors associated with death.

Demographic data

Adjusted OR (CI 95%)

p-value**

Age – yr

1.083(1.028-1.142)

0.003

Sex (male sex)

0.52 (0.216-1.25)

0.144

Body mass index (kg/m2)

0.971 (0.886-1.063)

0.518

Nursing home resident

1.758 (0.457-6.757)

0.411

Comorbidities

Hypertension

1.923 (0.758-4.881)

0.169

Diabetes mellitus

0.566(0.201-1.591)

0.281

Chronic heart disease

1.032 (0.34-3.129)

0.955

Chronic respiratory disease

0.759 (0.274-2.103)

0.595

Dialysis

2.045 (0.295-14.149)

0.469

Immunodeficiency

4.665 (1.73-12.58)

0.002

Cerebrovascular accident

2.983 (1.13-7.875)

0.027

Smoking status

 

Current smoker

3.564 (0.31-40.92)

0.307

Former smoker

1.448 (0.389-5.393)

0.581

Symptoms at onset of illness

Dyspnea

1.332 (0.521-3.406)

0.549

Hypoxemia (oxygen saturation < 93%)

0.936 (0.383-2.284)

0.884

Laboratory findings

Creatinine (µmol/L)

1.002 (0.999-1.006)

0.182

Radiologic findings

Percentage of lung affected on the CT scan (%)

1.032 (1.008-1.057)

0.009

Conflict of Interest

The authors declare they have no conflict of interest.

Ethics Approval

The study has been approved by the ethics committee of the GroupeHospitalier Saint Vincent (GRE 2020-01). Written informed consent has been waived in light of the urgent need to collectclinical data.

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