Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) +49A>G (rs231775) gene polymorphism is not associated with COVID-19 severity and mortality in an Iranian population.

Introduction: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates T cell immune responses as an immune activation inhibitor. Literature reviews suggest that COVID-19 is associated with dysregulation of the inflammatory immune response. The purpose of the present hospital-based case-control study was to evaluate the genetic association of the CTLA4 +49A > G (rs231775) Single Nucleotide Polymorphism (SNP) with COVID-19 severity and mortality among the Iranian people. Method: Genomic DNA of peripheral blood nuclear cells was extracted from the 794 COVID-19 patients and 167 control individuals. The polymorphic site of rs231775 was genotyped using the PCR-RFLP technique. Also, to identify whether this genetic variation was related to CTLA-4 mRNA expression, total RNA was extracted from 178 COVID-19 patients and 70 controls. The mRNA levels of CTLA-4 were determined using real-time PCR. Result: There were no statistically significant differences found in the genotype and allele frequencies among the different genetic models with regards to the severity and mortality of COVID-19. Furthermore, there was no significant association between rs231775 genotypes and CTLA-4 mRNA expression in patients. Conclusion: Our findings demonstrated that SARS-CoV-2 infection is not associated with rs231775 in the Iranian people. More investigations are crucial to show how this genetic variation affects other ethnic groups. Given the importance of CTLA-4 in regulating immune responses, further studies are recommended to examine other CTLA-4 SNPs and the function of this gene in COVID-19 patients.

Catalase-gold nanoaggregates manipulate the tumor microenvironment and enhance the effect of low-dose radiation therapy by reducing hypoxia.

Radiotherapy as a standard method for cancer treatment faces tumor recurrence and antitumoral unresponsiveness. Suppressive tumor microenvironment (TME) and hypoxia are significant challenges affecting efficacy of radiotherapy. Herein, a versatile method is introduced for the preparation of pH-sensitive catalase-gold cross-linked nanoaggregate (Au@CAT) having acceptable stability and selective activity in tumor microenvironment. Combining Au@CAT with low-dose radiotherapy enhanced radiotherapy effects via polarizing protumoral immune cells to the antitumoral landscape. This therapeutic approach also attenuated hypoxia, confirmed by downregulating hypoxia hallmarks, such as hypoxia-inducible factor α-subunits (HIF-α), vascular endothelial growth factor (VEGF), and EGF. Catalase stability against protease digestion was improved significantly in Au@CAT compared to the free catalase. Moreover, minimal toxicity of Au@CAT on normal cells and increased reactive oxygen species (ROS) were confirmed in vitro compared with radiotherapy. Using the nanoaggregates combined with radiotherapy led to a significant reduction of immunosuppressive infiltrating cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T-regs) compared to the other groups. While, this combined therapy could significantly increase the frequency of CD8+ cells as well as M1 to M2 macrophages (MQs) ratio. The combination therapy also reduced the tumor size and increased survival rate in mice models of colorectal cancer (CRC). Our results indicate that this innovative nanocomposite could be an excellent system for catalase delivery, manipulating the TME and providing a potential therapeutic strategy for treating CRC.

Association of programmed cell death 1 (PD-1) gene polymorphism (rs10204525) with COVID-19 severity and mortality: A case-control study in the Iranian population.

BACKGROUND: Programmed cell death 1 (PD-1), as a negative immune regulator, regulates the activation of T cells and maintains the immune system's homeostasis. Previous studies suggest that the effective immune response against COVID-19 contributes to the outcome of the disease. The present study aims to evaluate whether the PD-1 rs10204525 polymorphism is associated with PDCD-1 expression and COVID-19 severity and mortality in the Iranian population. METHODS: The PD-1 rs10204525 was genotyped in 810 COVID-19 patients and 164 healthy individuals as a control group using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Moreover, we assessed the expression of PDCD-1 in peripheral blood nuclear cells by real-time PCR. RESULTS: Regarding disease severity and mortality, no significant differences were detected between study groups in alleles and genotypes frequency distribution under different inheritance models. We found that the expression of PDCD-1 was significantly lower in COVID-19 patients with AG and GG genotypes than in the control group. Regarding disease severity, mRNA levels of PDCD-1 were significantly lower in moderate and critical patients carrying AG genotype than in control (P = 0.005 and P = 0.002, respectively) and mild (P = 0.014 and P = 0.005, respectively) individuals. Additionally, the severe and critical patients with GG genotype displayed a significantly lower level of PDCD-1 compared with the control (P = 0.002 and P < 0.001, respectively), mild (P = 0.004 and P < 0.001, respectively), and moderate (P = 0.014 and P < 0.001, respectively) ones. Regarding disease mortality, the expression of PDCD-1 was significantly lower in non-survivor COVID-19 patients with GG genotype than in survivors. CONCLUSION: Considering the lack of significant differences in PDCD-1 expression in different genotypes in the control group, lower expression of PDCD-1 in COVID-19 patients carrying the G allele suggests the impact of this single-nucleotide polymorphism on the transcriptional activity of PD-1.

Is COVID-19 severity unrelated to antinuclear antibodies?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces an overreaction of the immune system, resulting in the production of auto-antibodies. Several studies have reported that autoantibodies are prevalent in COVID-19 patients. In our study, antinuclear antibodies were evaluated in patients with COVID-19. We examined 131 sera from patients (>17-year-old) with confirmed COVID-19. Samples were collected prior to receiving any medication and antinuclear antibodies (ANA) levels were measured by the indirect immunofluorescence (IIF) method. Furthermore, the immunoblotting test was used to determine the presence of anti-nuclear antigen antibodies. The IIF-ANA test was positive in 36.4% (48/131) of patients. Overall, non-ICU patients had higher IIF-ANA titers than ICU patients. In particular, ICU patients had fewer nuclear, cytoplasmic, and mitotic IIF-ANA antibodies than non-ICU patients. In conclusion, COVID-19 patients frequently have ANA possibly reflecting the immune dysregulation due to several damaged cells by SARS-CoV-2 virus.

Immunophenotyping characteristics of COVID-19 patients: Peripheral blood CD8+ HLA-DR+ T cells as a biomarker for mortality outcome.

INTRODUCTION: The goal of this study was to identify biomarker(s) to assign risk of mortality in COVID-19 patients to improve intensive care unit (ICU) and coronary care unit  management. A total of 100 confirmed COVID-19 patients admitted at Imam Khomeini Hospital in Tehran, were compared to 70 control subjects. Peripheral blood leukocyte was studied using staining reagents included CD3, CD4, CD8, HLA-DR, CD19, CD16, and CD56. The immunophenotyping analysis was evaluated using the FACSCalibur instrument. To investigate the cell density of lung infiltrating T cells, postmortem slides of needle necropsies taken from the lung tissue of 3 critical patients were evaluated by immunohistochemistry staining. The number of lymphocyte subpopulations was significantly lower in COVID-19 patients than in the control group. Regarding the disease severity, the absolute count of T, NK, and HLA-DR+ T cells were significantly reduced in severe patients compared to the moderate ones. The critical patients had a significantly lower count of CD8-HLA-DR+ T cells than the moderate cases. Regarding the disease mortality, based on univariate analysis, the count of HLA-DR+ T, CD8- HLA-DR+ T, and CD8+ HLA-DR+ T cells was associated with mortality in COVID-19 patients. Receiver operating characteristic curve analysis showed the count of CD8+ HLA-DR+ T cells is the best candidate as a biomarker for mortality outcome. Furthermore, pulmonary infiltration of T cells in the lung tissue showed only slight infiltrations of CD3+ T cells, with an equal percentage of CD4+ and CD8+ T cell subpopulation in the lung tissue. These findings suggest that close monitoring of the value of CD8+ HLA-DR+ T cells in COVID-19 patients may be helpful to identify high-risk patients. However, further studies with larger sample size are needed.

Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines.

BACKGROUND: Relapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics. METHODS: CRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated. RESULTS: Although with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed. CONCLUSION: Enrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.

Interpretation of Hematological, Biochemical, and Immunological Findings of COVID-19 Disease: Biomarkers Associated with Severity and Mortality.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spread rapidly all over the world in late 2019 and caused critical illness and death in some infected patients. This study aimed at examining several laboratory factors, especially inflammatory and immunological mediators, to identify severity and mortality associated biomarkers. Ninety-three hospitalized patients with confirmed coronavirus disease 2019 (COVID-19) were classified based on disease severity. The levels of biochemical, hematological, immunological, and inflammatory mediators were assessed, and their association with severity and mortality were evaluated. Hospitalized patients were mostly men (77.4%) with an average (standard deviation) age of 59.14 (14.81) years. The mortality rate was significantly higher in critical patients (85.7%). Increased serum levels of blood sugar, urea, creatinine, uric acid, phosphorus, total bilirubin, serum glutamic-oxaloacetic transaminase, serum glutamic-oxaloacetic transaminase, lactic dehydrogenase, C-reactive protein, ferritin, and procalcitonin were significantly prevalent (p=0.002, p<0.001, p<0.001, p=0.014, p=0.047, p=0.003, p<0.001, p<0.001, p<0.001, p<0.001, P<0.001, and p<0.001, respectively) in COVID-19 patients. Decreased red blood cell, hemoglobin, and hematocrit were significantly prevalent among COVID-19 patients than healthy control subjects (p<0.001 for all). Troponin-I, interleukin-6, neutrophil/lymphocyte ratio (NLR), procalcitonin, and D-dimer showed a significant association with the mortality of patients with specificity and sensitivity more than 60%. Age, sex, underlying diseases, blood oxygen pressure, complete blood count along with C-reactive protein, lactic dehydrogenase, procalcitonin, D-dimer, and interleukin-6 evaluation help to predict the severity and required management for COVID-19 patients. Further investigations are highly recommended in a larger cohort study for validation of the present findings.

Alteration in serum levels of immunoglobulins in seriously eye-injured long-term following sulfur-mustard exposure.

INTRODUCTION: Sulfur mustard (SM) is a potent toxic agent that cause local and systemic changes in the human body such as dysregulation of the immunological system. This gas affects different organs such as lungs, skin, eyes and the gastrointestinal tract. METHODS: 128 veterans with SM-induced eye injuries were examined and compared to 31 gender- and age-matched healthy controls. Serum levels of IgM, IgE, IgA, IgG, and IgG subclasses were measured using ELISA method. RESULTS: There was no significant difference in IgM level between two groups with abnormal and normal ocular conditions except for those having bulbar conjunctiva-limbal ischemia and bulbar conjunctiva-hyperemia abnormalities. There were not significant difference in IgA, IgE, and IgG levels between two groups with and without ocular problem also between study groups. IgG1 level in some ocular abnormalities were significantly lower than the healthy control groups. IgG2 level in SM-exposed participants with stromal abnormality was higher in the SM-exposed groups without this problem. IgG2 levels in the exposed group with some ocular problems were significantly increased compared with control. IgG3 level in all patients did not reveal any significant changes compared with the controls except the fundus abnormality. IgG4 level was not significantly different between two groups with normal and abnormal ocular conditions. Nonetheless, IgG4 level in the exposed participants with some ocular abnormalities significantly increased compared with the controls. CONCLUSION: The results showed SM exposure could alter immunoglobulins level compared with healthy controls and the changes of IgG2 and IgG1 levels were associated with some ocular problems.

Alteration in inflammatory mediators in seriously eye-injured war veterans, long-term after sulfur mustard exposure.

BACKGROUND: Sulfur mustard (SM) exposure produces extensive systemic and ocular adverse effects on the victims. One of the most important effects is immunological insults that can lead to other organ damages, including the eyes. METHODS: In this descriptive study, 128 SM-exposed veterans with severe eye injury were compared with 31 healthy controls. Tear levels of tumor necrosis factor (TNF)-α and serum concentrations of interleukin (IL)-1α, IL-1ß, IL1Ra, IL-6, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Fas Ligand (FasL) were compared between the two groups. RESULTS: Meibomian gland dysfunction (MGD); tear breakup time (TBUT < 10″); and conjunctival, limbal, and corneal abnormalities were more frequent among the cases (MS-exposed veterans) than the controls. Ocular involvement was mild in 14.8%, moderate in 24.2%, and severe in 60.9% of the cases. Serum levels of IL-1α and FasL were significantly higher among the cases than among the controls (P < 0.001 and P = 0.037, respectively). Also, a significant decrease was observed in serum and tear levels of TNF-α in the cases as compared with controls (P < 0.001, P < 0.001, respectively). Serum levels of FasL were significantly higher in cases with severe ocular involvement than in the controls (P = 0.03). Nonetheless, serum levels of IL-1ß, IL-1Ra, IL-1α/IL-1Ra, and IL-6 were not significantly different between the two groups. CONCLUSION: Serum levels of IL-1α and FasL may cause different ocular surface abnormalities in SM-exposed patients. Lower tear TNF-α concentration may be due to lower serum levels of this cytokine in these patients.

Tear and serum MMP-9 and serum TIMPs levels in the severe sulfur mustard eye injured exposed patients.

INTRODUCTION: Sulfur mustard (SM) intoxication produces local and systemic changes in the human body. In this study, the relationship between tear and serum matrix metalloproteinase (MMP)-9 and serum tissue inhibitors of metalloproteinases (TIMPs) are assessed in serious eye-injured SM-exposed casualties. METHODS: A group of 128 SM-exposed patients with serious ocular injuries in three subgroups (19 mild, 31 moderate, and 78 severe cases) is compared with 31 healthy controls. Tear and ocular status and serum MMPs and MMP-9/TIMPs complex levels were evaluated using enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum level of MMP-9 was significantly higher in the SM-exposed group compared to the control group (P = 0.009). Mean serum MMP-9 level in the SM-exposed group with ocular abnormalities was significantly higher than that in the SM-exposed group without ocular abnormalities. SM-exposed people with corneal calcification had significantly higher serum MMP-9/TIMP-1 level compared to the SM-exposed ones without this problem (P = 0.045). The SM-exposed group with severe ocular injuries had significantly higher MMP-9/TIMP-1 than the controls (P = 0.046). The SM-exposed group had significantly lower levels of MMP-9/TIMP-4 complex than the controls (P < 0.001). The SM-exposed group with tear meniscus and fundus abnormality had significantly higher MMP-9/TIMP-4 levels than the SM-exposed group without these problems (P = 0.009 and P = 0.020). CONCLUSION: Serum MMP-9 level had increased in SM-exposed groups with ocular problems, while TIMP-1 and TIMP-2 levels had remained unchanged. Serum TIMP-4 drastically decreased in SM-exposed group, which clearly explains the severity of the systemic and ocular damages.

Serum and sputum levels of IL-17, IL-21, TNFα and mRNA expression of IL-17 in sulfur mustard lung tissue with long term pulmonary complications (28 years after sulfur mustard exposure).

BACKGROUND: Iranian veterans who had exposed to Sulfur Mustard (SM) suffer from long term complications such as Chronic Obstructive Pulmonary Disease (COPD) and bronchiolitis obliterate (BO). Th17 cells product IL-17A, IL-17F, IL-21, and IL-22. They have important roles in chronic inflammatory diseases. Also, TNFα has a major part in pathobiological processes of COPD. In this study, we evaluate the serum and sputum levels of IL-17, IL-21, TNF-α, and mRNA expression of IL-17 in the lung tissue of the patients 28 years after SM exposure. MATERIAL AND METHOD: The cytokine levels of IL-17, IL-21 and TNFα were measured by ELISA method in serum and sputum of 455 SM-exposed and 123 unexposed people participated in Sardasht-Iran Cohort Study (SICS) of chemical victims. The mRNA expression of IL-17 was evaluated with qRT-PCR in lung biopsies (SM-exposed =52, control =33). Analyses of all data were accomplished with the SPSS software with P value ≤05. RESULT: The results show the sputum level of IL-17 in the exposed group decreased significantly compared to control group (P = 0.007) and Veterans and Martyrs Affair Foundation (VMAF) assessment was significantly lower in abnormal/exposed than normal/exposed group (P = 0.042). There were no significant differences between control and exposed groups in serum level of IL-17; also serum and sputum levels of IL-21, TNF-α, and IL-17 mRNA expression. CONCLUSION: Conclusively, The IL-17 level decreased in the exposed group. This decline could cause by mutation on transcription factors like Signal transducer and activator of transcription 3 gene (STAT3) or CCL20 as a chemokine.

Serum Concentration of Thyroid Hormones Long-Term after Sulfur Mustard Exposure.

BACKGROUND: Despite several reports on the clinical manifestations of sulfur mustard (SM) intoxication, there is no study on serum concentrations of thyroid hormones long-term after SM exposure. In this study, the changes in thyroid functioning parameters 20 yr after SM exposure were evaluated. METHODS: This study is a part of a larger historical cohort study conducted in 2007 following 20 years of the exposure to SM, called Sardasht-Iran cohort study (SICS). We (SICS) comprised an SM-exposed group from Sardasht City, West Azerbaijan Province, Iran (n=169 as hospitalized group and n=203 as non-hospitalized exposed group); and control participants were selected from Rabat, a town near Sardasht (n=126). Peripheral blood samples were taken in fasting state and then the sera were separated. T4, T3, TSH, antithyroglobulin (anti-Tg), and antithyroid peroxidase (anti-TPO) concentrations in the sera were measured by the ELISA method. RESULTS: The mean of T3 concentration was significantly higher in the exposed than control group (0.88 ± 0.26 nmol/L vs 0.8 ± 0.25 nmol/L, P<0.001). The levels of TSH, T4, and T3up were not significantly different between the exposed and control groups. Thyroglobulin level was significantly higher in the exposed non-hospitalized group (56.07 ± 140.22 µg/L vs 17.66 ± 41.49 µg/L, P=0.004), but the level of anti-Tg and anti-TPO showed no significant differences between the two groups. CONCLUSION: More studies are needed on the alterations in thyroid hormones, their gene expressions, and mechanisms involved in SM exposure to clarify the causes of these alterations.

Peripheral blood mononuclear cellular viability and its correlation with long-term pulmonary complications after sulfur mustard exposure.

INTRODUCTION: Sulfur mustard (SM) as a chemical warfare agent has short- and long-term complications on its victims. Complications of exposure to SM depend on the level of contamination. Long-term pulmonary complications are the most serious problems. Recent evidence has shown that absorbed SM can be conducted to other tissues by the bloodstream. In this study, we evaluated the long-term effects of SM on the vital activity of peripheral blood mononuclear cells (PBMCs) in SM-exposed patients with long-term pulmonary complications. MATERIALS AND METHODS: Our study samples were 110 patients with long-term pulmonary complications in the SM-exposed group and 109 unexposed individuals in the control group. After clinical examination and pulmonary function tests, the severity of pulmonary complications was classified. Also, the participants' peripheral blood was taken into EDTA-treated Vacutainer tubes. Then, the complete blood count (CBC) was calculated, and PBMCs was purified from whole blood using Ficol-Paque gradient method, finally, the vital activity was assessed by MTT assay. RESULT: The vital activity of PBMCs in the SM-exposed group with the mitogen was significantly lower than that in the control group (P = 0.016). Whereas, there was no significant difference in the viability of PBMCs without the mitogen between two groups. Furthermore, hematologic findings indicated that the SM-exposed group had a significant increase in the total count of WBC, neutrophil, MCV, and HCT values but the lymphocyte count and MCHC value were significantly lower than those in the control group. CONCLUSION: Exposure to SM even after a long time, can affect hematologic parameters and vital activity of PBMCs.