Evaluation of Myeloid-derived Suppressor Cells in the Blood of Iranian COVID-19 Patients.

The cytokine storm and lymphopenia are reported in coronavirus disease 2019 (COVID-19). Myeloid-derived suppressive cells (MDSCs) exist in two different forms, granulocyte (G-MDSCs) and monocytic (M-MDSCs), that both suppress T-cell function. In COVID-19, the role of chemokines such as interleukin (IL)-8 in recruiting MDSCs is unclear. A recent report has correlated IL-8 and MDSCs with poor clinical outcomes in melanoma patients. In the current study, we evaluated the frequency of MDSCs and their correlation with serum IL-8 levels in severe COVID-19 patients from Iran. Thirty-seven severe patients (8 on ventilation, 29 without ventilation), thirteen moderate COVID-19 patients, and eight healthy subjects participated in this study between 10th April 2020 and 9th March 2021. Clinical and biochemical features, serum, and whole blood were obtained. CD14, CD15, CD11b, and HLA-DR expression on MDSCs was measured by flow cytometry. COVID-19 patients compared to healthy subjects had a greater frequency of M-MDSCs (12.7±13.3% vs 0.19±0.20%,), G-MDSCs (15.8±12.6% vs 0.35±0.40%,) and total-MDSCs (27.5±17.3% vs 0.55±0.41%,). M-MDSC (16.8±15.8% vs 5.4±4.8%,) and total-MDSC (33.3±18.5% vs 17.3±13.3%) frequency was higher in non- ventilated compared to moderate COVID-19 subjects. Serum IL-8 levels were higher in patients with COVID-19 than in normal healthy subjects (6.4±7.8 vs. 0.10±00 pg/mL). Ventilated patients (15.7±6.7 pg/mL), non-ventilated patients (5.7±2.7 pg/mL) and moderate patients (2.8±3.0 pg/mL) had significantly different levels of IL-8.  A negative correlation was found between the frequency of G-MDSCs and the international normalized ratio (INR) test (r=-0.39), and between the frequency of total-MDSCs and oxygen saturation (%) (r=-0.39). COVID-19 patients with severe non-ventilated disease had the highest levels of M-MDSCs. In addition to systemic MDSCs, lung, serum IL-8, and other inflammatory biomarkers should be measured.

The miR-196a SNP Rs11614913 but not the miR-499 rs37464444 SNP is a Risk Factor for Non-small Cell Lung Cancer in an Iranian Population.

Background: Globally, lung cancer represents a major cause of cancer-related deaths. The regulation of gene expression is modulated by small noncoding RNAs called miRNAs that can act as both tumor suppressors and oncogenes. The maturation, expression and binding to target mRNAs is affected by single nucleotide polymorphisms (SNPs) in miRNA genomic regions thereby contributing to cancer susceptibility. SNPs Rs11614913 in miR196a and Rs3746444 in miR-499 are implicated in the development of cancers such as non-small cell lung cancer (NSCLC) in non-Arabic subjects. Materials and Methods: A small cohort of 204 participants including 104 lung cancer patients and 100 non-cancer controls subjects were enrolled into the study. The allele frequencies were determined by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) and their correlation with lung cancer risk was determined. Results: The miR-196a rs11614913 polymorphism increased the risk of NSCLC (CC vs. TT+TC: OR= 2.26, 95%CI= 1.28 - 3.98, P= 0.0046) in a dominant genetic model. No statistically significant association was found between the miR-499 rs37464444 polymorphism and NSCLC. Conclusion: The rs11614913 polymorphism in miR-196a, but not the miR-499 rs37464444 polymorphism, increased the risk of NSCLC. Further studies with larger sample sizes in correlation with functional outcomes at the cellular level should be undertaken.

Decreased serum levels of angiotensin converting enzyme (ACE)2 and enhanced cytokine levels with severity of COVID-19: normalisation upon disease recovery.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Circulating soluble angiotensin-converting enzyme (sACE2)2, the main receptor for SARS-CoV-2, together with components of the renin-angiotensin system promote infection and disease severity. OBJECTIVE: This pilot study followed the time-course of sACE2 levels in relation to systemic cytokines in severe and moderate COVID-19 patients treated with remdesivir/dexamethasone in combination. METHODS: Peripheral blood was obtained upon admission from 30 patients (12 with moderate disease and 18 with severe disease) and 14 patients with PCR-confirmed mild COVID-19. Severe and moderate patients were treated with remdesivir (200mg/first day and 100mg/day for the remaining days) and dexamethasone (100mg/day). 6 healthy control subjects (HC) were also enrolled. Serum interleukin (IL)-6 and IL-8 and sACE2 levels were measured by ELISA at baseline and during treatment in severe and moderate patients and at baseline in mild and HCs. RESULTS: Baseline sACE2 levels were lower in severe (p = 0.0005) and moderate (p = 0.0022) patients than in patients with mild COVID-19 and in HC (p = 0.0023 and p = 0.0012 respectively). Treatment significantly increased sACE2 levels in patients with moderate disease (p = 0.0156) but only 50% of patients with severe disease showed enhanced levels compared to baseline. Systemic IL-6 and IL-8 levels were higher in all patient groups compared with HC and were not significantly affected over time or by remdesivir/dexamethasone treatment for 5 days. CONCLUSION: Serum sACE2 levels increase in severe COVID-19 patients as they recover over time whilst circulating cytokines are unaffected. Future studies should link these results to clinical outcomes.

Evaluation of Lymphocyte Subtypes in COVID-19 Patients.

Background: Although many aspects of the COVID-19 disease have not yet been clarified, dysregulation of the immune system may play a crucial role in the progression of the disease. In this study, the lymphocyte subsets were evaluated in patients with different severities of COVID-19. Materials and Methods: In this prospective study, the frequencies of peripheral lymphocyte subsets (CD3+, CD4+, and CD8+ T cells; CD19+ and CD20+ B cells; CD16+/CD56+ NK cells, and CD4+/CD25+/FOXP3+ regulatory T cells) were evaluated in 67 patients with confirmed COVID-19 on the first day of their admission. Results: The mean age of patients was 51.3 ± 14.8 years. Thirty-two patients (47.8%) were classified as severe cases, and 11 (16.4%) were categorized as critical. The frequencies of blood lymphocytes, CD3+ cells, CD25+FOXP3+ T cells, and absolute count of CD3+ T cells, CD25+FOXP3+ T cells, CD4+ T cells, CD8+ T cells, and CD16+56+ lymphocytes were lower in more severe cases compared to the milder patients. The percentages of lymphocytes, T cells, and NK cells were significantly lower in the deceased patients. (p= 0.002 and p= 0.042, p=0.006, respectively). Conclusion: Findings of this cohort study demonstrated that the frequencies of CD4+, CD8+, CD25+FOXP3+ T cells, and NK cells differed in the severe cases of COVID-19. Moreover, lower frequency of T cells and NK cells could be predictors of mortality in these patients.

Effectiveness of Borage plus syrup on COVID-19 patients in intensive care units.

Introduction: COVID-19 (coronavirus disease-2019) still causes a high rate of death globally with no definite curative treatment described. The traditional plant Borage (Borago officinalis L.) is a good source of gamma-linolenic (GLA). We hypothesized that Borage plus syrup (BPS) would be beneficial in severe COVID-19 patients within an intensive care unit (ICU) setting. Materials and methods: A pilot single center, randomized trial with no placebo was undertaken. A total of 60 PCR-positive severe COVID-19 participants admitted to ICU from June 2020-December 2020 at Masih Daneshvari Hospital Tehran-Iran gave informed consent. The participants were randomly assigned to either Borage Plus Syrup (BPS, 5 ml for 5 days) (n = 30) or standard care (IFN-ß and favipiravir) as a control group (n = 30). Pao2/Fio2, serum ferritin, CRP, bilirubin, IL-6, TNF-α, ALT, AST, PCT and serum IL-8 was measured upon admission and on release. Results: All the measured parameters decreased significantly with BPS treatment. In the control group, most parameters significantly improved apart from AST and PCT. In addition, the suppression of serum TNF levels in the BPS group was greater than that seen in the control group (P ≤ 0.05). Moreover, the length of ICU stay was significantly lower in the BPS group compared with the control group (P ≤ 0.05). Conclusion: Our study shows that addition of BPS to the standard treatment regime of COVID-19 patients in ICU improved outcomes and reduced the length of ICU treatment. Natural products could be considered as new approaches for reducting the harmful consequences of COVID-19.

Potential diagnostic value of pleural fluid cytokines levels for tuberculous pleural effusion.

Patients with tuberculous pleural effusion (TPE) or malignant pleural effusions (MPE) frequently have similar pleural fluid profiles. New biomarkers for the differential diagnosis of TPE are required. We determined whether cytokine profiles in the PE of patients could aid the differential diagnosis of TPE. 30 patients with TPE, 30 patients with MPE, 14 patients with empyema (EMP) and 14 patients with parapneumonic effusion (PPE) were enrolled between Dec 2018 and 2019. The levels of interleukin (IL)-6, IL-18, IL-27, CXCL8, CCL-1 and IP-10 were determined in PE by ELISA along with measurements of adenosine deaminase (ADA). The best predictors of TPE were combined ADA.IL-27 [optimal cut-off value = 42.68 (103 U ng/l2), sensitivity 100%, specificity 98.28%], ADA [cut off value 27.5 (IU/l), sensitivity 90%, specificity 96.5%] and IL-27 [cut-off value = 2363 (pg/ml), sensitivity 96.7%, specificity 98.3%, p ≤ 0.0001]. A high level of IL-6 [cut-off value = 3260 (pg/ml), sensitivity 100%, specificity 67.2%], CXCL8 [cut-off value = 144.5 (pg/ml), sensitivity 93.3%, specificity 58.6%], CCL1 [cut-off value = 54 (pg/ml), sensitivity 100%, specificity 70.7%] and IP-10 [cut-off value = 891.9 (pg/ml), sensitivity 83.3%, specificity 48.3%] were also predictive of TPE. High ADA.IL-27, ADA and IL-27 levels differentiate between TPE and non-TPE with improved specificity and diagnostic accuracy and may be useful clinically.

Evaluation Expression of miR-146a and miR-155 in Non-Small-Cell Lung Cancer Patients.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is the major type of lung cancer. MicroRNAs (miRNAs) are novel markers and targets in cancer therapy and can act as both tumor suppressors and oncogenes and affect immune function. The aim of this study was to investigate the expression of miR146a and miR155 in linked to blood immune cell phenotypes and serum cytokines in NSCLC patients. METHODS: Thirty-three NSCLC patients and 30 healthy subjects were enrolled in this study. The allele frequencies of potential DNA polymorphisms were studied using polymerase chain reaction (PCR)-restriction fragment length polymorphism (PCR-RFLP) analysis in peripheral blood samples. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of miR-146a and miR-155 in peripheral blood mononuclear cells (PBMCs). Serum cytokine (IL-1ß, IL-6, TNF-α, TGF-ß, IL-4, IFN-γ) levels were determined by ELISA. The frequency of circulating CD3+CTLA-4+ and CD4+CD25+FOXP3+ (T regulatory cells/Treg) expression was measured by flow cytometry. RESULTS: miR-146a was significantly downregulated in PBMC of NSCLC patients (P ≤ 0.001). Moreover, IL-6 and TGF-ß levels were elevated in NSCLC patients (P ≤ 0.001, P ≤ 0.018, respectively). CD3+ CTLA-4+ and Treg cells frequencies were higher in patients than in control subjects (P ≤ 0.0001, P ≤ 0.0001, respectively). There was a positive correlation between miR-155 and IL-1ß levels (r=0.567, p ≤ 0.001) and a negative correlation between miR-146a and TGF-ß levels (r=-0.376, P ≤ 0.031) in NSCLC patients. No significant differences were found in the relative expression of miR-146a and miR-155, cytokine levels or immune cell numbers according to miR-146a and miR-155 (GG/GC/CC, TT/AT/AA) genotypes. However, there was a positive correlation between miR-146a and IL-1ß levels (r=0.74, P ≤ 0.009) in GG subjects and a positive correlation between miR-146a expression and CD3+CTLA4+ cell frequency (r=0.79, P ≤ 0.01) in CC genotyped subjects. Conversely, a negative correlation between miR-146a expression and Treg cell frequency (r=-0.87, P ≤ 0.05) was observed with the GG genotype. A positive correlation between miR-155 and IL-1ß expression (r=0.58, p ≤ 0.009) in the TT genotype and between miR-155 expression and CD3+CTLA-4 cell frequency (r=0.75, P ≤ 0.01) was observed in the AT genotype. CONCLUSIONS: The current data suggest that the miR-146a expression in PBMC and serum TGF-ß and IL-1ß levels may act as blood markers in NSCLC patients. Further study is needed to elucidate the link between immune cells and serum miR146 at early disease stages.

T Helper Cell Subsets in the Pleural Fluid of Tuberculous Patients Differentiate Patients With Non-Tuberculous Pleural Effusions.

Background: Tuberculous pleural effusion (TPE) is one of the most common forms of extrapulmonary tuberculosis (Tb). Patients with TPE or malignant pleural effusions (MPE) frequently have a similar lymphocytic pleural fluid profile. Since the etiology of PE in various diseases is different, identifying the cellular components may provide diagnostic clues for understanding the pathogenesis. Objective: We determined the frequency of T helper (Th) subtypes in the PEs for differentiation of Tb and non-Tb patients. Methods: Thirty patients with TPE, 30 patients with MPE, 14 patients with empyema (EMP), and 14 patients with parapneumonic effusion (PPE) were enrolled between December 2018 and December 2019. Five-milliliter fresh PE in tubes containing heparin as an anticoagulant was obtained from patients. The frequencies of CD4+IL-9+, CD4+IL-22+, CD+IL-17+, and regulatory T-cells CD4+CD25+ FOXP3+ (Treg) were determined by flow cytometry. Results: Treg cells have a lower frequency in TPE patients [4.2 (0.362-17.24)] compared with non-TPE patients [26.3 (3.349-76.93, p < 0.0001)]. The frequency of CD4+IL-9+ cells was significantly lower in TPE patients [3.67 (0.87-47.83)] compared with non-TPE groups [13.05 (1.67-61.45), p < 0.0001]. On the contrary, there was no significant difference in the frequency of CD4+IL-17+ and CD4+IL-22+ cells between TPE and non-TPE patients (p = 0.906 and p = 0.2188). Receiver-operator curve (ROC) analysis demonstrated that CD4+CD25+FOXP3+ T cells [optimal cutoff value = 13.6 (%), sensitivity 90%, specificity 75.86%] could be considered as predictor for TPE. However, adenosine deaminase [cutoff value 27.5 (IU/l), sensitivity 90%, specificity 96.5%] levels had an even greater predictive capacity. Conclusion: ADA, Treg cells, and CD4+IL-9+ cells may differentiate TPE from non-TPE patients. However, these results need validation in an independent large cohort.

Increased Serum Levels of Soluble TNF-α Receptor Is Associated With ICU Mortality in COVID-19 Patients.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) has infected over 112M patients and resulted in almost 2.5M deaths worldwide. The major clinical feature of severe COVID-19 patients requiring ventilation is acute respiratory distress syndrome (ARDS) possibly associated with a cytokine storm. Objectives: To elucidate serum levels of TNF-α and soluble TNF-Receptor 1 (sTNFR1) in patients with severe and mild COVID-19 disease as determinants of disease severity. Methods: We determined serum TNF-α and sTNFR1 concentrations in 46 patients with laboratory-confirmed COVID-19 (17 patients with severe disease within the intensive care unit [ICU] and 29 non-severe, non-ICU patients) and 15 healthy controls upon admission using ELISA. Subjects were recruited between March-May 2020 at the Masih Daneshvari Hospital Tehran, Iran. Results: Serum levels of sTNFRI were significantly higher in ICU patients (P<0.0001) and non-ICU patients (P=0.0342) compared with healthy subjects. Serum sTNFR1 were significantly higher in ICU patients than in non-ICU patients (P<0.0001). Serum TNF-α levels were greater in ICU and non-ICU patients than in the healthy subjects group (p<0.0001). The sTNFRI concentration in ICU (r=0.79, p=0.0002) and non-ICU (r=0.42, p=0.02) patients positively correlated with age although serum sTNFRI levels in ICU patients were significantly higher than in older healthy subjects. The sTNFRI concentration in ICU patients negatively correlated with ESR. Conclusions: The study demonstrates higher sTNFRI in ICU patients with severe COVID-19 disease and this be a biomarker of disease severity and mortality. Future studies should examine whether lower levels of systemic sTNFR1 at admission may indicate a better disease outcome.

Serum cytokine levels of COVID-19 patients after 7 days of treatment with Favipiravir or Kaletra.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) has infected 86,4 M patients and resulted in 1,86 M deaths worldwide. Severe COVID-19 patients have elevated blood levels of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor (TNF)α, IL-8 and interferon (IFN)γ. OBJECTIVE: To investigate the effect of antiviral treatment serum cytokines in severe COVID-19 patients. METHODS: Blood was obtained from 29 patients (aged 32-79 yr) with laboratory-confirmed COVID-19 upon admission and 7 days after antiviral (Favipiravir or Lopinavir/Ritonavir) treatment. Patients also received standard supportive treatment in this retrospective observational study. Chest computed tomography (CT) scans were evaluated to investigate lung manifestations of COVID-19. Serum was also obtained and cytokines levels were evaluated. 19 age- and gender-matched healthy controls were studied. RESULTS: Anti-viral therapy significantly reduced CT scan scores and the elevated serum levels of C-reactive protein (CRP) and lactate dehydrogenase (LDH). In contrast, serum levels of IL-6, IL-8 and IFNγ were elevated at baseline in COVID-19 subjects compared to healthy subjects with IL-6 (p = 0.006) and IL-8 (p = 0.011) levels being further elevated after antiviral therapy. IL-1ß (p = 0.01) and TNFα (p = 0.069) levels were also enhanced after treatment but baseline levels were similar to those of healthy controls. These changes occurred irrespective of whether patients were admitted to the intensive care unit. CONCLUSION: Antiviral treatments did not suppress the inflammatory phase of COVID-19 after 7 days treatment although CT, CRP and LDH suggest a decline in lung inflammation. There was limited evidence for a viral-mediated cytokine storm in these COVID-19 subjects.

Update on Immunology of COVID-19 Disease and Potential Strategy for Controlling.

Coronavirus disease 2019 (COVID-19) is caused by a novel form of the coronavirus that caused severe acute respiratory syndrome (SARS). SARS-CoV-2 raised in China and has broadcast to 261 countries globally. SARS-CoV-2 a member of ß-coronavirus family and has an almost matching genome sequence to a bat coronavirus, pointing to the bat as the natural host before it was transmitted to humans. SARS-CoV-2 uses the same receptor, angiotensin-converting enzyme 2 (ACE2) as that used by SARS-CoV and principally infects the respiratory tract. The clinical symptoms of COVID-19 patients include fever, cough and fatigue whilst small populations of patients have gastrointestinal symptoms. The old people and people with underlying metabolic and cardiovascular diseases are more affected to infection and have worse outcomes. These may be associated with acute respiratory distress syndrome (ARDS) and a cytokine storm. In this review, we discuss the pathogenesis and clinical characteristics of disease and the pharmacologic approaches that may control COVID-19.

The miR-146a SNP Rs2910164 and miR-155 SNP rs767649 Are Risk Factors for Non-Small Cell Lung Cancer in the Iranian Population.

Background: Lung cancer is one of the leading causes of death worldwide. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and may act as both tumor suppressors and as oncogenes. The presence of single nucleotide polymorphisms (SNPs) inside the miRNA genomic region could affect target miRNA maturation, expression, and binding to its target mRNA and contribute to cancer development. Previous studies on the SNPs Rs2910164 in miR-146a and Rs767649 in miR-155 showed association with non-small cell lung cancer (NSCLC) development. Thus, the aim of this study was to detect any correlation between those SNPs in Iranian NSCLC patients. Methods: In a small cohort study, 165 NSCLC patients and 147 noncancer controls were enrolled between Apr 2015 and Sep 2019 at the Masih Daneshvari Hospital, Tehran, Iran. Allele frequencies from the genomic DNA of blood cells were studied using PCR-RFLP and their association with the risk of lung cancer was evaluated. Results: The rs2910164C allele (OR = 1.56, 95% CI = 1.10-2.21, p = 0.012) and CC genotype (OR = 2.93, 95% CI = 1.07-7.9, p = 0.034, respectively) were associated with a significantly increased risk for lung cancer compared to that for the GG genotype. When patients were stratified according to smoking exposure, no association with rs2910164 variants was found. The AT genotype (OR = 0.57, 95% CI = 0.33-0.99, p = 0.048) and the A allele frequency (OR = 0.58, 95% CI = 0.35-0.98, p = 0.043) in rs767649 were lower in NSCLC patients in comparison with the control group. In addition, the rs767649 AT genotype frequency in smoking controls was higher than in smoking NSCLC patients (OR = 0.44, 95% CI = 0.21-0.90, p = 0.024). No association was found between rs2910164 and rs767649 variants and stage or type of NSCLC. Conclusion: Our finding suggests that miR-146a rs2910164 and miR-155 rs767649 polymorphisms may be considered as genetic risk factors for the susceptibility to NSCLC in the Iranian population. However, a larger multicenter study across Iran is needed to confirm these findings.