Differences in the Expression Levels of SARS-CoV-2 Spike Protein in Cells Treated with mRNA-Based COVID-19 Vaccines: A Study on Vaccines from the Real World.

Comirnaty (BNT162b2) and Spikevax (mRNA-1273) COVID-19 vaccines encode a full-length SARS-CoV-2 Spike (S) protein. To evaluate whether the S-protein expressed following treatment with the two vaccines differs in the real-world context, two cell lines were treated for 24 h with two concentrations of each vaccine, and the expression of the S-protein was evaluated using flow cytometry and ELISA. Vaccines were obtained from three vaccination centers in Perugia (Italy) that provided us with residual vaccines present in vials after administration. Interestingly, the S-protein was detected not only on the cell membrane but also in the supernatant. The expression was dose-dependent only in Spikevax-treated cells. Furthermore, the S-protein expression levels in both cells and supernatant were much higher in Spikewax-than in Comirnaty-treated cells. Differences in S-protein expression levels following vaccine treatment may be attributed to variations in the efficacy of lipid nanoparticles, differences in mRNA translation rates and/or loss of some lipid nanoparticles' properties and mRNA integrity during transport, storage, or dilution, and may contribute to explaining the slight differences in the efficacy and safety observed between the Comirnaty and Spikevax vaccines.

Overexpression of Potential Markers of Regulatory and Exhausted CD8+ T Cells in the Peripheral Blood Mononuclear Cells of Patients with B-Acute Lymphoblastic Leukemia.

B-acute lymphoblastic leukemia (B-ALL) is one of the most common pediatric cancers, wherein regulatory T cells (Treg) and exhausted CD8+ T cells may be important in its development and maintenance. In this bioinformatics study, we evaluated the expression of 20 Treg/CD8 exhaustion markers and their possible roles in patients with B-ALL. The mRNA expression values of peripheral blood mononuclear cell samples from 25 patients with B-ALL and 93 healthy subjects (HSs) were downloaded from publicly available datasets. Treg/CD8 exhaustion marker expression was normalized with that of the T cell signature and correlated with the expression of Ki-67, regulatory transcription factors (FoxP3, Helios), cytokines (IL-10, TGF-ß), CD8+ markers (CD8α chain, CD8ß chain), and CD8+ activation markers (Granzyme B, Granulysin). The mean expression level of 19 Treg/CD8 exhaustion markers was higher in the patients than in the HSs. In patients, the expression of five markers (CD39, CTLA-4, TNFR2, TIGIT, and TIM-3) correlated positively with Ki-67, FoxP3, and IL-10 expression. Moreover, the expression of some of them correlated positively with Helios or TGF-ß. Our results suggested that Treg/CD8+ T cells expressing CD39, CTLA-4, TNFR2, TIGIT, and TIM-3 favor B-ALL progression, and targeted immunotherapy against these markers could be a promising approach for treating B-ALL.

Thrombotic events with or without thrombocytopenia in recipients of adenovirus-based COVID-19 vaccines.

COVID-19, the severe acute respiratory syndrome, is one of the major emergencies that have affected health care systems. Drugs and oxygen are only partially effective in saving lives in patients with severe COVID-19, and the most important protection from death is vaccination. The widespread use of COVID-19 adenovirus-based vaccines has provided evidence for the occurrence of rare venous thrombotic events including cerebral venous thrombosis and splanchnic venous thrombosis in recipients of Vaxzevria and Jcovden vaccines and the review focus on them. One year ago, thromboses in Vaxzevria recipients have been associated with thrombocytopenia in the presence of antibodies to platelet factor 4 and have been called vaccine-induced immune thrombotic thrombocytopenia (VITT). The incidence of VITT is equal to 9-31 events per one million doses of vaccines as evaluated by health agencies worldwide and is higher in female and young vaccine recipients. More recently, by using the European EudraVigilance database, it has been demonstrated that the incidence of thrombosis in recipients of adenovirus-based vaccines is 5-10 fold higher than that of VITT and 7-12 fold higher than observed in the recipients of Comirnaty, an mRNA-based vaccine, suggesting that adenovirus-based vaccines cause not only VITT but also thrombosis without thrombocytopenia (non-VITT thrombosis). The incidence of the vaccine-dependent non-VITT thrombosis is different in the adenovirus-based vaccines and the VITT/non-VITT incidence ratio depends on the severity of thrombosis and is inversely related to the age of the recipients. The possible causes and clinical implications of non-VITT thrombosis in vaccine recipients are discussed.

Anticancer Activity of Ethnopharmacological Plants of Golestan Province/Iran against AGS, HT-29 and KYSE-30 Cell Lines through Promoting the Apoptosis and Immunomodulatory Effects.

The anticancer and immunomodulatory effects of medicinal plants from Golestan province, as a promising source of cancer therapy against gastrointestinal cancer cell lines, were investigated in this study. The ethanolic root/aerial part extracts of 9 medicinal plants were screened for their cytotoxicity against normal mouse fibroblast cells (L-929) and three human cancer cell lines including gastric adenocarcinoma (AGS), colorectal adenocarcinoma (HT-29), and esophagus adenocarcinoma (KYSE-30) by performing MTT assay to determine the IC50 of the extracts. The in-vitro antioxidant activity, total phenolic (TPC), and total flavonoid content (TFC) of extracts was evaluated. Flow cytometry and Real-Time PCR were used for apoptosis assay and evaluation of expression of some genes involved in cell signaling; TLR-4, AKT, ERK1/2, and NFκB. Out of the 9 plant extracts screened, Arctiumlappa root (ALR), showed the most potent cytotoxicity against AGS, KYSE-30, and HT-29 cells with IC50 values of 10, 200, and 2030 µg/mL, respectively. In addition, ALR exerts high TPC (215.8 ± 0.3 mg GAE/g), TFC (69.03 ± 0.7 mg QUE/g) and high radical scavenging activity with IC50 (1250 ± 0.1 µg/mL) in DPPH method. Also, ALR stimulates TLR-4 signaling, increased apoptosis, and decreased cancer cell attachment to the surface compared to the untreated cells. This plant, with a strong cytotoxic effect on cancer cells as well as increased apoptosis and its effect on molecules involved in TLR4 signaling as the immunomodulatory effect can be a suitable candidate for in-vivo studies in the future for cancer therapy.

Blood clots and bleeding events following BNT162b2 and ChAdOx1 nCoV-19 vaccine: An analysis of European data.

The involvement of viruses and SARS-CoV-2 in autoimmune diseases is well known. The recent demonstration that ChAdOx1 nCoV-19 Covid-19 (AstraZeneca) vaccine (ChA) favors the production of anti-platelet factor 4 (anti-PF4) antibodies, blood clots, and thrombocytopenia raises the question of whether other anti-CoViD-19 vaccines favor the same patterns of events. We assessed the frequency of severe adverse events (SAEs) documented in the EudraVigilance European database up to April 16, 2021 related to thrombocytopenia, bleeding, and blood clots in recipients of ChA compared to that of recipients of the BNT162b2 Covid-19 (Pfizer/BioNTech) vaccine (BNT). ChA administration was associated with a much higher frequency of SAEs in each AE Reaction Group as compared with that elicited by BNT. When considering AEs caused by thrombocytopenia, bleeding and blood clots, we observed 33 and 151 SAEs/1 million doses in BNT and ChA recipients, respectively. When considering patients with AEs related to cerebral/splanchnic venous thrombosis, and/or thrombocytopenia, we documented 4 and 30 SAEs and 0.4 and 4.8 deaths/1 million doses for BNT and ChA recipients, respectively. The highest risk following ChA vaccination is in young people and, likely, women of reproductive age, as suggested by hypothesized scenarios. In conclusion, the immune reaction promoted by ChA vaccine may lead to not only thrombocytopenia and cerebral/splanchnic venous thrombosis but also other thrombotic and thromboembolic SAEs. These events are not favored by BNT vaccine. Our study may help in the evaluation of the benefit/risk profile of the ChA vaccine considering the epidemic curve present in a country.

Combination of 5-fluorouracil and Lipopolysaccharide Synergistically Induces Cytotoxicity and Apoptosis in MCF-7 Human Breast Cancer Cells.

Several studies have been conducted to find suitable combinations of drugs to increase the efficacy of chemotherapy and reduce the resistance of tumor cells to treatment. Lipopolysaccharide (LPS), as a ligand for Toll-like receptor 4 (TLR-4), can modify immune responses in different cancers. Although multiple studies have been performed in this area, the effect of LPS on tumor cells remains controversial. In the present study, the cytotoxic effects of 5-fluorouracil (5-FU), with or without LPS, were evaluated in human breast cancer cell line (MCF-7) on apoptosis and gene expression in downstream signaling pathways. MCF-7 was obtained from the Pasteur Institute of Iran. The effects of LPS and 5-FU on cytotoxicity, apoptosis, and gene expression in NF-κB, ERK, and AKT signaling pathways were evaluated by MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and qRT-PCR, respectively. Our findings showed that LPS alone did not significantly affect cytotoxicity or apoptosis, compared to the control cells (untreated cells), while combined with 5-FU, it caused a significant increase in the apoptosis of cancer cells and decreased cell viability. It was also concluded that LPS in combination with 5-FU increased TLR-4 expression and down-regulated gene expression in NF-κB, ERK, and AKT pathways (p=0.001). Although the role of LPS in tumor inhibition or progression remains controversial, our findings suggest that LPS can be considered a novel complementary approach intranslational oncology research of breast cancer therapy.

Correlation Between HMGB1 and TLR4 Expression in Sinonasal Mucosa in Patients With Chronic Rhinosinusitis.

OBJECTIVES: Chronic rhinosinusitis (CRS) is one of the most common inflammations in the upper airway. Despite the wide prevalence of CRS, the pathogenesis of this disease is poorly understood. Several components of the innate immune system may play a significant role in CRS, including Toll-like receptor 4 (TLR4), TLR9, and high-mobility group box 1 protein (HMGB1). This study was conducted to determine the expression of TLR4, TLR9, HMGB1, and pNFκ-B p65 in paraffin-embedded blocks of patients with CRS with nasal polyps compared with those of the control group. METHODS: Twenty-six formalin-fixed, paraffin-embedded samples from patients with confirmed CRS and 26 patients undergoing septoplasty due to anatomic variations and no other inflammatory nasal diseases as the control group were assessed. Expression patterns of HMGB1, TLR9, TLR4, and pNFκ-B p65 genes were examined using real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). Statistical analyses were performed with SPSS and analyzed using unpaired 2-tailed t tests or 1-way analysis of variance. RESULTS: Real-time PCR showed that the expression level of HMGB1 messenger RNA was significantly increased in the tissues of patients with CRS compared with controls (P < .05). The other 3 genes were also upregulated in the patients, but were not significant compared with control. Analysis of the Pearson correlation coefficient (r) revealed a significant positive correlation between HMGB1 and TLR4 (r = 0.79, P < .05) in patients and negative correlation between TLR4 and NfκB in the control group (r = 0.94; P < .05). CONCLUSIONS: Both HMGB1 and TLR4 are increased in the paranasal sinus mucosa of patients with CRS. These results suggest a possible contribution of HMGB1 and its internal receptor (TLR4) in the pathophysiology of CRS.

Tim-3 Up-regulation in Patients with Gastric Cancer and Peptic Ulcer Disease

Background: T-cell immunoglobulin and mucin domain protein-3 (Tim-3), an inhibitory immunoregulatory receptor, has been recently implicated in tumor biology and tumor-associated immune suppression. In the present study, expression of Tim-3 was evaluated in gastric cancer (GC) and peptic ulcer disease (PUD) at both mRNA and protein levels. Methods: A total of 133 gastric tissue biopsies, comprising 43 from GC cases, 48 from PUD and 42 from non-ulcer dyspepsia (NUD) serving as controls were collected. Additionally, non-neoplastic adjacent tissue biopsies were also obtained from 6 patients with GC. Infection with Helicobacter pylori was determined by the rapid urease test for all participants and H&E staining was conducted for GC and PUD patients. Tim-3 relative mRNA expression was determined by SYBR Green based Real-Time PCR using ß-actin as a reference gene. Tim-3 protein expression was also studied by immunohistochemistry in 7 GC, 7 PUD and 10 NUD tissue samples. Results: Tim-3 was expressed at higher levels in GC (p=0.030) and PUD (p=0.022) cases compared to he NUD group. Among paired samples obtained from gastric cancer patients, tumor tissues showed elevated Tim-3 expression (p=0.019) in comparison with adjacent non-neoplastic biopsies. Tim-3 mRNA findings were supported by detection of more Tim-3 protein in cancerous (p=0.002) and ulcerative (p=0.01) tissues than in controls. Tim-3 was similarly expressed in H. pylori positive and negative cases.Conclusion: Higher Tim-3 expression in patients with gastric cancer and peptic ulcer implies that it might be involved in immune regulation and establishment of these gastrointestinal diseases. Targeted immunotherapy by blocking of inhibitory receptors like Tim-3 could be a promising approach for gastric cancer treatment.