Decreased frequency of Th22 cells and IL-22 cytokine in kidney transplant patients with active cytomegalovirus infection.

BACKGROUND: The immunity of CD4+ T cell subsets against human cytomegalovirus (HCMV) is considerable due to their essential role in controlling the infection in transplant individuals. Previously explained CD4+ subsets such as T helper (Th) 1 have been proven to have a protective role against HCMV infection, while the role of the recently identified Th22 subset has not been described yet. Here, the frequency changes of Th22 cells and the IL-22 cytokine production were investigated in kidney transplant recipients with and without HCMV infection. METHODS: Twenty kidney transplant patients and ten healthy controls were enrolled in this study. Patients were categorized into HCMV + and HCMV- groups based on the HCMV DNA real-time PCR results. After isolating CD4+ T cells from PBMCs, the phenotype (CCR6+CCR4+CCR10+) and cytokine profile (IFN-γ-IL-17-IL-22+) of Th22 cells were analyzed by flow cytometry. The gene expression of Aryl Hydrocarbon Receptor (AHR) transcription factor was analyzed by real-time PCR. RESULTS: The phenotype frequency of these cells was lower in recipients with infection than in those without infection and healthy controls (1.88 ± 0.51 vs. 4.31 ± 1.05; P = 0.03 and 4.22 ± 0.72; P = 0.01, respectively). A lower Th22 cytokine profile was observed in patients with infection than in the two other groups (0.18 ± 0.03 vs. 0.20 ± 0.03; P = 0.96 and 0.33 ± 0.05; P = 0.04, respectively). AHR expression was also lower in patients with active infection. CONCLUSIONS: Overall, this study for the first time suggests that the reduced levels of Th22 subset and IL-22 cytokine in patients with active HCMV infection might indicate the protective role of these cells against HCMV.

Investigating the role of Bacillus subtilis type II toxin-antitoxin system in drought stress survival.

Toxin-antitoxin (TA) systems, present in plasmids and bacterial chromosomes, are widespread in bacteria such as Bacillus subtilis and are known to be involved in growth regulation, bacterial tolerance to environmental stress conditions as well as biofilm formation. The aim of the current study was to investigate the role of TA systems in drought condition stress in B. subtilis isolates. The presence of TA systems including mazF/mazE and yobQ/yobR in B. subtilis (strain 168) was investigated using the polymerase chain reaction (PCR) method. TA system expression at 438 and 548 g/L of ethylene glycol concentrations was evaluated using real-time PCR method and sigB gene was used as internal control. The expression rate (fold change) of mazF toxin gene treated with 438 and 548 g/L of ethylene glycol was 6 and 8.4, respectively. This indicates an increase in the expression of this toxin in drought stress condition. Also, the fold change of mazE antitoxin in the treatment with 438 and 548 g/L of ethylene glycol was 8.6 and 5, respectively. While yobQ/yobR showed a decrease in expression in 438 and 548 g/L of ethylene glycol concentrations. So that the highest expression reduction (8.3) was observed for yobQ gene at the concentration of 548 g/L of ethylene glycol. Results of this study revealed the significant role of B. subtilis TA systems in drought stress which can be considered as the resistance mechanism of this bacterium under stress conditions.

Evaluation the reactivity of a peptide-based monoclonal antibody derived from OmpA with drug resistant pulsotypes of Acinetobacter baumannii as a potential therapeutic approach.

BACKGROUND: Acinetobacter baumannii is an opportunistic and antibiotic-resistant pathogen that predominantly causes nosocomial infections. There is urgent need for development nonantibiotic-based treatment strategies. We developed a novel monoclonal antibody (mAb) against a peptide of conserved outer membrane protein A (OmpA) and evaluated its reactivity with different pulsotypes of A. baumannii. METHODS: Peptide derived from A. baumannii OmpA was conjugated to keyhole limpet hemocyanin and injected into BALB/c mice. Splenocytes of immunized mice were fused with SP2/0 myeloma cells followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, one monoclone was selected as 3F10-C9 and the antibody was tested for reaction with five different Acinetobacter pulsotypes that were resistant to carbapenem antibiotics. The affinity constant was measured by ELISA. The ELISA, western blotting, indirect immunofluorescence (IFA), and in vitro opsonophagocytosis assays were used to evaluate the reactivity of generated mAb. RESULTS: The anti-OmpA antibody reacted with the immunizing peptide and had a high affinity (1.94 × 10-9 M) for its antigen in the ELISA. Specific binding of mAb to OmpA was confirmed in Western blot. IFA assays revealed that mAb recognized specific OmpA on the pulsotypes. Opsonophagocytosis assays showed that the mAb increased the bactericidal activity of macrophage cells. The antibody function was higher in the presence of serum complement. CONCLUSIONS: The peptide-based mAb demonstrated optimal performance in laboratory experiments which may be appropriate in investigation on OmpA in Acinetobacter pathogenesis and development of passive immunization as a novel therapeutic approach.

DICER-AS1 lncRNA: A putative culprit in the pathogenesis of gastric cancer.

The nuclear factor-κB (NF-κB) has a pivotal role in the pathogenesis of several cancers including gastric cancer. We have recently reported dysregulation of a number of NF-κB-associated lncRNAs in a variety of human disorders including breast cancer and coronary artery disease. In the current study, we evaluated expression of five NF-κB-associated lncRNAs (CHAST, ADINR, DICER1-AS1, HNF1A-AS1 and NKILA) and two NF-κB-associated-mRNA coding genes (CEBPA and ATG5) in gastric cancer tissues and their paired non-cancerous tissues using real time PCR method. Expression of DICER-AS1 was significantly down-regulated in gastric cancer tissues compared with the corresponding non-cancerous tissues (Expression ratio = 0.23, P value = .01). Expressions of other genes were not significantly different between these two sets of samples. Relative expression of DICER1-AS1 in cancer tissues versus non-cancerous tissues tended to associated with histological grade (P = .05). Tumoral expression levels of NKILA, ADINR, CEBPA and HNF1A-AS1 were significantly higher in patients with positive family history of cancer compared with those without such history (P values = .03, 0.02, 0.02 1nd 0.03, respectively). Besides, expression levels of NKILA, ADINR, DICER1-AS1, CEBPA, CHAST, HNF1A-AS1 and ATG5 were lower in H. pylori-infected tissues (P values = .01, 0.02, 0.03, 0.01, 0.004, 0.004 and 0.04, respectively). The lowest tumoral expression of DICER1-AS1 was detected in stage II cancers, while the highest expression of this lncRNA was reported in a single stage I tumor tissue. Similar pattern of expression was detected for ATG5. Significant pairwise correlations were demonstrated between expression levels of NF-ƙB-associated genes in both gastric cancer tissues and non-cancerous tissues. Expression levels of DICER1-AS1 had sensitivity and specificity values of 63.3% and 63.3% in differentiating between tumoral and non-tumoral tissues (Estimate criterion>6.96, J = 0.27, P value = .01, AUC = 0.67). Although previous studies have reported involvement of NF-κB pathway in the pathogenesis of gastric cancer, among the reported lncRNAs associated with this pathway, we could only detect differential expression of DICER1-AS1 between tumoral and non-tumoral tissues. Thus, the mechanism underlying dysregulation of this pathway might be different among various cancers.

Delamanid and bedaquiline resistance patterns in Mycobacterium tuberculosis in Iran: A cross-sectional analysis.

Introduction: The surge of multidrug-resistant TB (MDR-TB) in Iran poses a significant challenge to global healthcare. The introduction of delamanid (DLM) and bedaquiline (BDQ), two potent antimycobacterial drugs, marks a crucial advance. Nevertheless, as resistance in Mycobacterium tuberculosis is on the rise in Iran and resistance to these newer medications is emerging, investigations in this field are of utmost importance. Methods: In this cross-sectional study, 38 MDR-TB strains were collected from five distinct regional TB laboratories in Iran. The clinical isolates were confirmed as M. tuberculosis using the phenotypic tests and IS6110-based PCR assay. Drug susceptibility testing (DST) for isoniazid, rifampicin, ethambutol, DLM, and BDQ was performed using WHO-approved methods. Sequencing was used to investigate genetic mutations in DLM (ddn, fgd1) and BDQ (Rv0678, atpE, pepQ) genes associated with resistance. Results: Among the 38 collected MDR-TB isolates, 7 (18.5 %) exhibited resistance to DLM, while all remained susceptible to BDQ. Analysis of the sequencing data revealed that the ddn gene exhibited the highest number of mutations in DLM-resistant isolates, including 18 nonsynonymous mutations and 1 indel leading to frameshift mutations. A common mutation, Gly81Ser, was present in 4 of the DLM-resistant isolates (4/7; 57.1 %). A synonymous mutation, T960C, in the fgd1 gene was uniformly found in DLM-resistant samples. Notably, no significant mutations were observed in the atpE, Rv0678, or pepQ genes in any of the BDQ-susceptible isolates. Conclusions: Our study underscores the emergence of DLM resistance in a subset of MDR-TB isolates in Iran, primarily associated with mutations in the ddn gene. This emphasizes the ongoing necessity for TB drug resistance surveillance and research. While BDQ remains efficacious, the emergence of DLM resistance is a concerning development, warranting further exploration into resistance mechanisms and the formulation of effective TB control strategies.

Ascorbic Acid and α-Tocopherol in the Inactivated SARS-CoV-2 Vaccine Formulation: Induction of the Th1 Pattern in Aged Mice.

Aging is physiologically associated with a decline in the function of the immune system and subsequent susceptibility to infections. Interferon-gamma (IFN-γ), a key element in the activation of cellular immunity, plays an important role in defense against virus infections. Decreased levels of IFN-γ in the elderly may explain their increased risk for viral infectious diseases such as COVID-19. There is accumulating evidence that ascorbic acid (vitamin C [VitC]) and α-tocopherol together help improve the function of the immune system in the elderly, control infections, and decrease the treatment duration. A SARS-CoV-2 strain was isolated from a patient and then cultured in the Vero cell line. The isolated and propagated virus was then inactivated using formalin and purified by the column chromatography. The inactivated SARS-CoV-2 was formulated in the Alum adjuvant combined with VitC or α-tocopherol and/or both of them. The vaccines were injected twice to young and aged C57BL/6 mice. Two weeks later, IFN-γ, IL-4, and IL-2 cytokines were assessed using ELISA Kits. Specific IgG and IgG1/IgG2a were assessed by an in-house ELISA. In addition, the expression of PD1 and TERT genes in the spleen tissue of the mice was measured using real-time PCR. IL-4 and IFN-γ cytokines showed a significant increase in both aged and young mice compared with the Alum-based vaccine. In addition, our results exhibited a significant decrease and increase in specific total IgG and the IgG2a/IgG1 ratio, respectively. Furthermore, the vaccine formulated in α-tocopherol + VitC led to decreased PD1 and increased TERT gene expression levels. In conclusion, our results demonstrated that α-tocopherol + VitC formulated in the inactivated SARS-CoV-2 vaccine led to a shift toward Th1, which may be due to their effect on the physiology of cells, especially aged ones and changing their phenotype toward young cells.

Gene expression analysis of SOCS, STAT and PIAS genes in lung cancer patients.

One of the most devastating diseases with the highest prevalence and mortality rate worldwide is lung cancer. Non-small cell lung cancer (NSCLC) is the subtype of lung cancer in 85% of cases. In this work, the expression levels of the STAT, SOCS and PIAS family genes involved in angiogenesis, proliferation and differentiation were examined. Using QRT-PCR technique, the expression level of STAT3 gene was assessed and tumor tissue samples had higher expression than normal tissue. In addition, the histological grade of adenocarcinoma was associated with the increase in STAT3 gene expression. The expression of the SOCS1 and SOCS2 genes in tumors was measured to be 0.58-fold and 0.36-fold lower than in healthy samples adjacent to the tumor, but this reduction in expression was not significant. In addition, when examining the relationship between the expression of SOCS1 and 2 and the clinical features of tumor samples, there was a significant decrease in the expression of the SOCS1 and 2 genes in the adenocarcinoma subtype. Compared to neighboring tumor samples, the expression of PIAS1 in the tumors was not different with controls. Our research revealed that tissue samples from adenocarcinoma had higher levels of STAT3 expression. Taken together, the mentioned genes can be suggested as possible targets for further studies in NSCLC.

Larvicidal Effects of Metabolites Extracted from Nocardia and Streptomyces Species against the Forth Larval Stage of Anopheles stephensi (Diptera: Culicidae).

Background: Larvicidal agents can be produced using microbial resources, which are environmentally friendly, biodegradable, and economical. The study's goal was to evaluate the larvicidal activity of metabolites isolated from Nocardia (N. fluminea, N. soli and N. pseudobrasiliensis) and Streptomyces (S. alboflavus) bacterial species against Anopheles stephensi. Methods: Four metabolites isolated from Nocardia and Streptomyces strains were exanimated for larvicidal activity. The experiments were performed for 24, 48, and 72 hours. 300, 350, 400, 450, 500, 550, and 600 µl of Actinobacteria metabolites were added to 100 cc of dechlorinated water. Fourth-stage larvae were placed in dechlorinated water as a control. LC50 and LC90 were calculated using toxicity data and analyzed. Results: All metabolites had a statistically significant influence on mosquito larvae (P< 0.05). At 24, 48, and 72 hours, the LC50 for N2 (N. fluminea) was 417, 386, and 370 ppm, respectively, and the LC90 was 650, 595, and 561 ppm. Moreover, LC50 for N4 (N. soli) was 389, 376, and 347 and LC90 were 591, 565, and 533 and LC50 for N5 (N. pseudobrasiliensis) was 390, 357, and 341 ppm and LC90 were 589, 532 ppm. In addition, LC50 for S921 (S. alboflavus) was 484, 416, and 382 ppm, and LC90 was 701, 612, and 574 ppm. Conclusion: The four bacterial metabolites tested in our study were found to have a notable effect on the mortality rate of Anopheles stephensi larvae, indicating their potential as natural larvicides. This is an effective technique for controlling Anopheles stephensi that has no detrimental environmental impact.

Evaluation of PLGA nanoparticles containing outer membrane proteins of Acinetobacter baumannii bacterium in stimulating the immune system in mice.

Background and purpose: Acinetobacter baumannii (A. baumannii) is known as a pathogen with antibiotic resistance, causing respiratory infections. PLGA has been approved for use in vaccines as well as drug delivery. This study was performed to evaluate PLGA nanoparticles containing the outer membrane proteins (OMPs) of A. baumannii in stimulating the mice's immune system and improving pneumonia. Experimental approach: Double emulsion solvent evaporation technique was used. The properties of the obtained nanospheres were determined using a zetasizer, FTIR, and AFM devices. Nanoparticles were administered to mice BALB/c by applying the intramuscular route. ELISA was used to measure the amounts of immunoglobulins produced; also, an opsonophagocytic killing assay was used to measure the effectiveness of immunoglobulins. Immunized mice were then challenged with live A. baumannii through the lungs; their internal organs were also removed for bacteriological studies. Findings/Results: The prepared particles were 550 nm in diameter with a negative surface charge. The production of the OMPs specific IgG was much higher in the group receiving nanoparticles containing antigen as compared to those getting pure antigen. The immunoglobulins produced against nanoparticles were superior to those developed against pure antigens. Mice that received the new nanovaccine were more resistant to pneumonia caused by this bacterium than those that received pure antigen. Conclusion and implication: Overall, it can be said that PLGA nanoparticles could deliver their internal antigens (OMPs) well to the immune system of mice and stimulate humoral immunity in these animals, thus protecting them against pneumonia caused by A. baumannii.

Prodigiosin induced the caspase-dependent apoptosis in human chronic myelogenous leukemia K562 cell.

BACKGROUND AND PURPOSE: Chronic myeloid leukemia (CML) as a myeloproliferative disease is characterized by increased cellularity of bone marrow. Implementing the latest treatment protocols is currently accompanied by serious and life-threatening side effects. There are worldwide attempts to find new effective and potent therapeutic agents with minimal side effects on CML patients. This in vitro study was carried out to discover the potential antiproliferative and apoptotic effects of naturally produced prodigiosin (PDG) on K562 cells as an accepted model of CML. EXPERIMENTAL APPROACH: The anti-proliferative effect of PDG was measured by MTT assay. To highlight the mechanism of cytotoxicity, the apoptotic cell death pathway was investigated by morphological and biochemical assessments. The dual acridine orange/ethidium bromide staining technique and western blotting method were applied to assess the mechanism of the potential apoptotic impact of PDG on K562 cells. FINDINGS/RESULTS: PDG-induced time- and concentration-dependent anti-proliferative effects were revealed with an estimated IC50 value of 54.06 µM. The highest cell viability reduction (60%) was recorded in cells, which were exposed to 100 µM concentration. Further assays demonstrated that in the dual acridine orange/ethidium bromide staining method the cell population in the late apoptosis phase was increased in a concentration-dependent manner, which was confirmed with remarkable DNA fragmentation. CONCLUSION AND IMPLICATIONS: We found that the PDG-induced apoptosis in K562 cells is mediated through the caspase-3 activation both in mRNA and protein levels. Our results suggest that PDG could be a potent compound for further pharmacokinetic and pharmacodynamics studies in the in vivo model of CML.