Spatio-temporal differences in presentation of CD8 T cell epitopes during HBV infection

Distinct populations of hepatocytes infected with HBV or only harboring HBV-DNA integrations coexist within an HBV chronically infected liver. These hepatocytes express HBV antigens at different levels and with different intracellular localizations but it is not known whether this heterogeneity of viral antigen expression could result in an uneven hepatic presentation of distinct HBV epitopes/HLA class-I complexes triggering different level of activation of HBV-specific CD8+ T cells.Using antibodies specific to two distinct HLA-A*02:01/HBV epitope complexes of HBV nucleocapsid and envelope proteins, we mapped their topological distribution in liver biopsies of two anti-HBe+ chronic HBV (CHB) patients. We demonstrated that the core and envelope CD8+T cell epitopes were not uniformly distributed in the liver parenchyma but preferentially located in distinct and sometimes mutually exclusive hepatic zones. The efficiency of HBV epitope presentation was then tested in vitro utilizing HLA-A*02:01/HBV epitope-specific antibodies and the corresponding CD8+ T cells, in primary human hepatocyte and hepatoma cell lines either infected with HBV or harboring HBV-DNA integration. We confirmed the existence of a marked variability in the efficiency of HLA-class I/HBV epitope presentation among the different targets that was influenced by presence of IFN-γ and availability of newly-translated viral antigens. In conclusion, HBV antigen presentation can be heterogeneous within an HBV-infected liver. As a consequence, CD8+ T cells of different HBV specificities might have different antiviral efficacy.Importance The inability of patients with chronic HBV infection to clear HBV is associated with defective HBV-specific CD8+ T cells. Hence, the majority of immunotherapy developments focus on HBV specific T cell function restoration. However, knowledge of whether distinct HBV-specific T cells can equally target all the HBV-infected hepatocytes of a chronically infected liver are lacking. In this work, analysis of CHB patient liver parenchyma and in vitro HBV infection models shows a non-uniform distribution of HBV CD8+ T cells epitopes that is influenced by presence of IFN-γ and availability of newly-translated viral antigens. These results suggest that CD8+ T cells recognizing different HBV epitopes can be necessary for efficient immune therapeutic control of chronic HBV infection

A comparative study of bacterial agents in eggs, with or without eggshell's contamination that produced in Tabriz.

Zoonotic bacterial diseases are considered as the most important human infectious diseases. In this category, a disease that transfer and infect human through food has an special role. Among foods with animal origin, eggs due to their application as food ingredient in food products, like sauces, spices, ice creams and other food products that may use in half cooked or crude forms, always has the potential to transfer microbial pathogens to human. This study is about to prove that presence of remainder feces on eggshells leads the penetration of bacterial agents into egg yolk. A total of 120 daily egg samples (with and without fecal contamination) were collected from henneries around Tabriz and transferred immediately to microbiology laboratory. After performing microbial examinations (using BHI Broth, BHI Agar, Blood Agar, Selenite F, Tetrationate, XLD,SS Agar),Gram Staining, Oxidase and Catalase tests, bacteria of eggshells and egg yolks has been identified. Out of 120 eggs, 15.83% bacterial contaminants were found in egg yolks samples. Among them, 73.68%  were gram negative and 26.31% were gram positive. Among gram negatives, Pseudomonas with occurrence of 8.3% and in gram positives group, Bacillus with abundance of  4.16% were the most frequent bacterias. Also in samples collected from eggshells, 99.16% of the eggs demonstrated bacterial contamination which 23.55% of positive samples were among gram negative group and 76.44% were gram positive. The most contribution of eggshell gram negative bacterias were relevant to Pseudomonas, E.coli, Proteus and Citrobacter with respectively 18.33%,10.83%,5.83% and 4.16% rations. Among gram positive bacteria that isolates form eggshell samples for the most frequent bacterias, were Bacillus, Staphylococccus, Streptococcus, Rhodococcus and Micrococcus with respectively 80.00%, 34.16%, 15%, 9.16% and 7.5% rations. Use and consumption of eggs with shells contaminated with faces in food products is a serious risk factor for human health and can lead to zoonotic bacterial diseases by transferring bacterial pathogens

The effect of vitamin E- selenium injection on serumic levels of Albumin, Protein, Calcium, Phosphorous, Alkaline phosphates, Alaninamonitransferas and Aspartataminotransferas in the Arabian horse

This study was performed to evaluate the effect of vitamin E and Selenium injection on serumic levels of albumin, protein, calcium, phosphorous, alkaline phosphates, alaninamonitransferas and aspartataminotransferas in the Arabian stallions. In this study 12 Arabian horses were selected in 2 groups of 4 and 5 years old. In each group, horses were allocated to two sub groups of controls and treatment, each of them containing three animals. In the treatment group, 1cc/30 kg vitamin E and selenium was injected intramuscularly in 2 day intervals for 6 days, while in the control group the same amounts of normal saline (0.9%) was injected. Blood samples of all animals were collected from the jugular vein at days 2, 4 and 6 and their sera were harvested by centrifuging and the levels of the parameters were measured using commercial kits and the spectrophotometric method. The results of this study indicated that there was no significant difference on mean serumic albumin, phosphorous, alkaline phosphates, alaninamonitransferas, and spartataminotransferas in the studied animals while mean serumic protein values of 4 year old horses indicated a significant difference following the third vit E – Selenium injection (

The infection status of Mycobacterium avium paratuberculosis in traditional dairy cattle farms in Moghan region

Mycobacterium avium paratuberculosis is the causative agent of John's disease which is a remedial chronic disease in all ruminants and is important from economical viewpoint. In this study, a total of 86 fecal samples from suspected dairy cattle were obtained from 11 traditional dairy farms in Moghan region. All samples were evaluated by direct microscopic examination. Subsequently, milk sample of the related cattle were tested by PCR technique. Twenty samples from positive and 10 specimens of negative samples in direct microscopic assay were selected randomly for PCR examination. Among the 86 samples, 51 (59%) samples were positive, while, 35 (41%) samples were found as negative by microscopic assay. From 20 positive samples, 19 (95%) samples showed positive result by PCR, however, among negative samples, 3 (30%) samples were positive in PCR assay. Results revealed that there is a direct relation between contamination of fecal and milk samples. Moreover, due to the correlation between the results of microscopic examination of fecal samples and PCR assay of milk specimens, direct microscopic evaluation of feces could be performed prior to PCR-based detection of Mycobacterium avium paratuberculosis in milk samples. According to the results, high contamination rate of Mycobacterium avium paratuberculosis was found in milk samples. On the other hand, duo to possible etiological role of this bacterium in the development of Crohn’s diseases in human, it should be considered as a serious concern indeed

Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions

Background Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. Result We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. Conclusion Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells

Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells

T-cell immunoglobulin mucin 3 (Tim3) is an immune checkpoint receptor that plays a central role in chimeric antigen receptor (CAR) T cell exhaustion within the tumor microenvironment. This study was aimed to evaluate the effects of targeted-knockdown of Tim3 on the antitumor function of anti-mesothelin (MSLN)-CAR T cells. To knockdown Tim3 expression, three different shRNA sequences specific to different segments of the human Tim3 gene were designed and co-inserted with an anti-MSLN-CAR transgene into lentiviral vectors. To investigate the efficacy of Tim3 targeting in T cells, expression of Tim3 was assessed before and after antigen stimulation. Afterwards, cytotoxic effects, proliferative response and cytokine production of MSLN-CAR T cells and Tim3-targeted MSLN-CAR T cells were analyzed. Our results showed that activation of T cells and MSLN-CAR T cells led to up-regulation of Tim3. Tim3 knockdown significantly decreased its expression in different groups of MSLN-CAR T cells. Tim3 knockdown significantly improved cytotoxic function, cytokine production and proliferation capacity of MSLN-CAR T cells. Our findings indicate that targeted knockdown of Tim3 allows tumor-infiltrating CAR T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby altering the tumor microenvironment from immunosuppressive to immunosupportive via mitigated Tim3 signaling

MicroRNA-124 Enhances T Cells Functions by Manipulating the Lactic Acid Metabolism of Tumor Cells

High production of lactic acid is a common feature of various tumors. Lactic acid is an immunosuppressive molecule with crucial roles in tumor cells' immune escape, which could largely be attributed to its negative effects on the T cells present in the tumor microenvironment (TME). Strategies that decrease the glycolysis rate of tumor cells could enhance immunosurveillance and limit tumor growth. Pyruvate kinase M2 (PKM2) is a key enzyme in the glycolysis pathway, and it plays a vital role in lactic acid buildup in the TME. MicroRNA (miR)-124 has been shown to be able to decrease tumor cell lactic acid synthesis indirectly by reducing PKM2 levels.In this study, we first overexpressed miR-124 in the tumor cells and evaluated its effects on the PKM2 expression and lactic acid production of the tumor cells using quantitative real-time polymerase chain reaction (qRT-PCR) and spectrophotometry, respectively. Then, we cocultured miR-124-treated tumor cells with T cells to investigate the effects of miR-124 overexpression on T cell proliferation, cytokine production, and apoptosis.Our results demonstrated that miR-124 overexpression could significantly reduce the amount of lactic acid produced by tumor cells by manipulating their glucose metabolism, which led to the augmented proliferation and IFN-gamma production of T cells. Moreover, it rescued T cells from lactic acid-induced apoptosis.Our data suggest that lactic acid is a hindering factor for T-cell-based immunotherapies; however, manipulating tumor cells' metabolism via miR-124 could be a promising way to improve antitumor responses of T cells

Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR) Expressing T Cell

Chimeric antigen receptor (CAR) T cell therapy is considered as an encouraging approach for the treatment of hematological malignancies. However, its efficacy in solid tumors has not been satisfying, mainly in the immunosuppressive network of the tumor microenvironment and paucity of appropriate target antigens. Mesothelin (MSLN) is a tumor-associated antigen (TAA) expressed in numerous types of solid tumors such as gastrointestinal, ovarian, and pancreatic tumors. Owing to high expression in tumor cells and low expression in normal tissues, MSLN-targeted therapies like monoclonal antibodies have been previously developed. In the present study, a CAR T cell harboring the second-generation of a fully human anti-MSLN-CAR construct containing CD3 zeta and 4-1BB signaling domains was produced and it was functionally evaluated against an MSLN-expressing cell line. The findings showed potent, specific proliferation, cytotoxic activity, and interleukin (IL)-2, Tumor necrosis factor-(TNF) alpha, and Interferon-(IFN) gamma production in an antigen-dependent manner. Cytotoxic activity was shown in effector-to-target ratio from 1:1 to 20:1, but the most adequate efficacy was observed in the ratio of 10:1. Non-specific activity against MSLN negative cell line was not observed. Our data demonstrated that primary human T cells expressing fully human MSLN-CAR construct are effective against MSLN-expressing cell lines in vitro, suggesting this MSLN-CAR construct as a potential therapeutic tool in a clinical setting

Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR) Expressing T Cell

Chimeric antigen receptor (CAR) T cell therapy is considered as an encouraging approach for the treatment of hematological malignancies. However, its efficacy in solid tumors has not been satisfying, mainly in the immunosuppressive network of the tumor microenvironment and paucity of appropriate target antigens. Mesothelin (MSLN) is a tumor-associated antigen (TAA) expressed in numerous types of solid tumors such as gastrointestinal, ovarian, and pancreatic tumors. Owing to high expression in tumor cells and low expression in normal tissues, MSLN-targeted therapies like monoclonal antibodies have been previously developed. In the present study, a CAR T cell harboring the second-generation of a fully human anti-MSLN-CAR construct containing CD3ζ and 4-1BB signaling domains was produced and it was functionally evaluated against an MSLN-expressing cell line. The findings showed potent, specific proliferation, cytotoxic activity, and interleukin (IL)-2, Tumor necrosis factor-(TNF) α, and Interferon-(IFN) γ production in an antigen-dependent manner. Cytotoxic activity was shown in effector-to-target ratio from 1:1 to 20:1, but the most adequate efficacy was observed in the ratio of 10:1. Non-specific activity against MSLN negative cell line was not observed. Our data demonstrated that primary human T cells expressing fully human MSLN-CAR construct are effective against MSLN-expressing cell lines in vitro, suggesting this MSLN-CAR construct as a potential therapeutic tool in a clinical setting