Highlights Patients with COVID-19 show a prominent Th1 and Th17 cytokine profile. COVID-19 induces the expression of TGF-β. TGF-β can be used as a predictive factor of disease severity in patients with COVID-19.

Background: Cell-mediated immunity including T-cells (T helper and cytotoxic) plays an essential role in efficient antiviral responses against coronavirus disease-2019 (COVID-19). Therefore, in this study, we evaluated the ratio and expression of CD4 and CD8 markers in COVID-19 patients to clarify the immune characterizations of CD4 and CD8 T-cells in COVID-19 patients. Methods: Peripheral blood samples of 25 COVID-19 patients and 25 normal individuals with similar age and sex as the control group were collected. White blood cells, platelets, and lymphocytes were counted and CD4 and CD8 T lymphocytes were evaluated by flow cytometry. Results: The number of white blood cells, lymphocytes, and platelets were reduced significantly in COVID-19 patients (P < 0.05). The difference in CD4:CD8 ratio, CD4 T-cell frequency, CD8 T-cell frequency, and CD4 mean fluorescence intensity (MFI) was not significant between COVID-19 patients and healthy individuals (P > 0.05); however, the CD8 MFI increased significantly in COVID-19 infected patients (P < 0.05). Conclusion: Although, there is no significant difference in the ratio of CD4 to CD8 between two groups, the expression level of CD8 in COVID-19 patients was significantly higher than the normal individuals. This result suggested that the cellular immune responses triggered by COVID-19 infection were developed through overexpression of CD8 and hyperactivation of cytotoxic T lymphocytes.

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The human tumor necrosis factor α (hTNF‐α) is an important pro‐inflammatory cytokine which plays critical roles in inflammatory diseases such as rheumatoid arthritis (RA). The anti‐TNF‐α proteins can reduce symptoms of RA. Due to limitations of protein‐based therapies, it is necessary to find new anti‐TNF‐α agents instead of common anti‐TNF‐α proteins. Therefore, the aim of the current study was to identify a new DNA aptamer with anti‐hTNF‐α activity. The protein systematic evolution of ligands by exponential enrichment (SELEX) process was used for identifying DNA aptamers. Anti‐hTNF‐α aptamers were selected using dot blot, real‐time PCR, and in vitro inhibitory assay. The selected aptamers were truncated in two steps, and finally, a dimer aptamer was constructed from different selected truncates to improve their inhibitory effect. Also, Etanercept was used as a positive control to inhibit TNF‐α, in comparison to the designed aptamers. After 11 rounds, four aptamers with anti‐hTNF‐α inhibitory effect were identified. The truncation and dimerization strategy revealed a new dimer aptamer with 67 nM Kd, which has 40% inhibitory effect compared with Etanercept (60%). Overall, the dimerization and truncation aptamers could improve its activity. With regard to the several limitations of anti‐TNF‐α proteins therapies including immunogenicity, side effects, and cost‐intensive, a new designed anti‐hTNF‐α dimer aptamer could be considered as a potential therapeutic and/or diagnostic agent for hTNF‐α‐related disorders.

Introduction: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a safe, convenient, and low-cost technique with high applications for the measurement of cell proliferation rate in researches and clinical laboratories. Our aim was to evaluate the proliferation rate of human peripheral blood mononuclear cells (PBMCs) and production rate of Tumor necrosis factor alpha (TNF-α) by these cells after various mitogens stimulation in different situations. Methods: The MTT test was performed with various concentrations of mitogens including concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA) on the PBMCs. The cells were incubated for 24, 48, 72, and 96 hours in the culture medium and TNF-α cytokine assay was performed on the supernatant of the cultured splenocytes using the enzyme-linked immunosorbent assay (ELISA) method. Results: The optimal time and incubation of the PBMCs with the mixture of PHA-ConA were 5 μg/mL and 72 hours, respectively. The TNF-α level increased significantly after PHA-ConA and PHA stimulation. Conclusion:The results showed that the mixture of PHA-ConA (at the concentration of 5 μg/ mL) can give rise to the optimal results on stimulation of the PBMcs using the MTT assay after 72 hours incubation.