Differentially Expressed Inflammatory Cell Death-Related Genes and the Serum Levels of IL-6 are Determinants for Severity of Coronaviruses Diseases-2019 (COVID-19).

Background: Inflammatory cell death, PANoptosis, has been suggested to orchestrate the lymphocyte decrement among coronavirus disease-2019 (COVID-19) patients. The main aim of this study was to examine the differences in the expression of key genes related to inflammatory cell death and their correlation with lymphopenia in the mild and severe types of COVID-19 patients. Materials and Methods: Eighty-eight patients (36 to 60 years old) with mild (n = 44) and severe (n = 44) types of COVID-19 were enrolled. The expression of key genes related to apoptosis (FAS-associated death domain protein, FADD), pyroptosis (ASC (apoptosis-associated speck-like protein containing caspase activation and recruitment domains (CARD)), the adapter protein ASC binds directly to caspase-1 and is critical for caspase-1 activation in response to a broad range of stimuli), and necroptosis (mixed lineage kinase domain-like, MLKL) genes were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay, and compared between the groups. The serum levels of interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay (ELISA) assay. Results: A major increase in the expression of FADD, ASC, and MLKL-related genes in the severe type of patients was compared to the mild type of patients. The serum levels of IL-6 similarly indicated a significant increase in the severe type of the patients. A significant negative correlation was detected between the three genes' expression and the levels of IL-6 with the lymphocyte counts in both types of COVID-19 patients. Conclusion: Overall, the main regulated cell-death pathways are likely to be involved in lymphopenia in COVID-19 patients, and the expression levels of these genes could potentially predict the patients' outcome.

Human recombinant soluble PD1 can interference in T cells and Treg cells function in response to MDA-MB-231 cancer cell line.

OBJECTIVES: PD1/PDL1 pathway targeting using antibodies shows immune related adverse events in patients with tumors. The masking of PD1 ligand by soluble human PD-1 (shPD-1) probably inhibits the PD1/PDL1 interaction between T cells and tumor cells. Accordingly, the goal of this study was to produce human recombinant PD-1-secreting cells and find out how soluble human PD-1 affects T lymphocyte function. METHODS: An inducible construct of the human PD-1 secreting gene under hypoxia condition was synthesized. The construct was transfected into the MDA-MB-231 cell line. In six groups exhausted T lymphocytes were co-cultured with transfected or non-transfected MDA-MB-231 cell lines. The effect of shPD-1 on IFNγ production, Treg cell's function, CD107a expression, apoptosis, and proliferation was assessed by ELISA and flow cytometry, respectively. RESULTS: The results of this study showed that shPD-1 inhibits PD-1/PD-L1 interaction and enhances T lymphocyte responses through a significant increase in IFNγ production and CD107a expression. In addition, in the presence of shPD-1, the percentage of Treg cells decreased, while MDA-MB-231 cell apoptosis increased. CONCLUSIONS: We concluded that the human PD-1 secreting construct induced under hypoxia condition inhibits the interaction of PD-1/PD-L1 and enhances T lymphocyte responses in tumor environments and chronic infections.

Inhibition of mouse colon cancer growth following immunotherapy with a fraction of hydatid cyst fluid.

BACKGROUND: Hydatid cyst is the larval stage of the tape worm Echinococcus granulosus which is located in human and livestock viscera. There are some scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of a fraction of hydatid cyst fluid on colon cancer tumor in BALB/c mice were investigated. MATERIALS AND METHODS: In this experimental work six groups of mice were challenged with mouse colon cancer cells. 5 days later when the sign of tumor growth in mice was seen, group 1-4 were injected with hydatid cyst fluid, the 78 kDa fraction, live protoscolices and BCG respectively. Group five was injected with alum alone and the sixth group left intact without any injection. The size of the tumor was measured and compared in all groups. Then blood samples of mice were evaluated for serum cytokine levels. RESULT: In mice injected with hydatid cyst antigens especially a fraction of hydatid cyst fluid, tumor size was smaller than the that of control groups and the difference of tumor size in cases and control groups was statistically significant. CONCLUSION: The results of this study showed that injection of mice with a fraction of hydatid cyst fluid significantly inhibits the growth of mouse colon cancer and this inhibition may be related to effect of immune response to these antigens.

The role of unfolded protein response-associated miRNAs in immunogenic cell death amplification: A literature review and bioinformatics analysis.

Immunogenic cell death (ICD) is a type of cellular death that is elicited in response to the specific types of anti-cancer therapies and enhances the anti-tumor immune responses by the combination of antigenicity and adjuvanticity of dying tumor cells. There is a well-established interlink between endoplasmic reticulum stress (ERS) and ICD elicited by anti-cancer therapies. Most recent evidences support that unfolded protein response (UPR)-associated miRNAs can be key players in the ERS-induced ICD. Hence, in the present study, we conducted a literature review on the role of these miRNAs and associated molecular pathways that may regulate ICD. We first collected UPR-associated miRNAs that promote ERS-induced apoptosis and then focused on microRNAs (miRNAs) that promote ERS-induced apoptosis via PERK/eIF2α/ATF4/CHOP pathway activation, as the main core for ICD and release of damage-associated molecular patterns. To better identify PERK/eIF2α/ATF4/CHOP pathway-inducing miRNAs that can be used as potential therapeutic targets for improving ICD in cancer treatment, we did a comprehensive bioinformatics analysis and network construction. Our results showed that "pathways in cancer", "MAPK signaling pathway", "PI3K-Akt signaling pathway", and "Cellular senescence", which correlate with UPR components and ERS induction, were among the significant signaling pathways related to the target genes of these miRNAs. Furthermore, a protein-protein interaction (PPI) network was constructed, which revealed the involvement of the PPI-extracted hub genes in the regulation of proliferation and apoptosis. In conclusion, we propose that these types of miRNAs can be considered as the potential cancer therapy options for better induction of ICD in combination with other ICD inducers.

Diagnostic Value of Natural Killer Cells, CD56+ CD16+ Natural Killer Cells, NLRP3, and Lactate Dehydrogenase in Severe/Critical COVID-19: A Prospective Longitudinal Study According to the Severe/Critical COVID-19 Definitions.

Innate immunity, as the first line of defense of our immune system, plays a crucial role in defending against SARS-CoV-2 infection and also its immunopathogenesis. We aim to investigate the immune status of natural killer (NK) cells, natural killer T (NKT) cells, and NLRP3 gene expression in COVID-19 patient blood samples. The immunophenotype of NK cell subsets and NKT cells was detected by flow cytometry and the expression of NLRP3 gene assessed by reverse transcriptase real-time polymerase chain reaction in 44 COVID-19 patients and 20 healthy individuals. The percentage of most of NK cell subpopulation and NKT cells was significantly decreased in COVID-19 patients. The percentage of CD56dim CD16- NK cell subsets, and NLRP3 gene expression increased. The percentage of total NK cells, CD56+ CD16+ NK cells, and NLRP3 gene expression had acceptable sensitivity and specificity for assisting diagnosis of severe/critical COVID-19. O2 saturation% and lactate dehydrogenase levels showed valuable diagnostic value to identify critical cases. The declined NK and NKT cells in COVID-19 patients and enhanced NLRP3 gene expression were associated with disease severity. Total NK cells, CD56+ CD16+ NK cells, and NLRP3 gene expression might be used as meaningful indicators for assisting diagnosis of severe/critical COVID-19.

The Role of Cobalamin on Interleukin 10, Osteopontin, and Related MicroRNAs in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Considering how vitamin B12 or cobalamin affects the immune system, especially inflammation and the formation of the myelin sheath, it appears as a complementary therapy for MS by affecting some signaling pathways. Recently diagnosed MS patients were divided into two groups (n=30). One group received interferon-beta (IFN-ß or Avonex), and another received IFN-ß+B12 for six months. Blood samples were taken before and after treatments.  Interleukin (IL)-10 and osteopontin (OPN) levels in the plasma were determined by the enzyme-linked immunosorbent assay (ELISA) method, and the expression of microRNA (miR)-106a, miR-299a, and miR-146a by real-time PCR. IFN-ß neither changed the IL-10 plasma levels nor miR106a and miR-299a expression, but it led to a remarkable decrease in OPN concentration and enhancement in let-7c and miR-146a expression. There was a significant decrease in IL-10, OPN plasma levels, miR-106a expression, and a substantial increase in let-7c and  miR-146a expression in IFN-ß+B12, treated group. There was no correlation between IL-10 and OPN with related miRNAs in the two treatment groups. Our study indicated that B12 could be a complementary treatment in MS that may influence the disease improvement.

Spermatozoa Induce Maternal Mononuclear Cells for Production of Antibody with Cytotoxic Activity on Paternal Blood Mononuclear Cells.

OBJECTIVE: The maternal immune response to paternal antigens is induced at insemination. We believe that pregnancy protective alloantibodies, such as anti-paternal cytotoxic antibody (APCA), may be produced against the paternal antigens in the context of stimulated immunity at insemination and that they increase during pregnancy. APCA is necessary for pregnancy. It is directed towards paternal human leucocyte antigens (HLAs) and has cytotoxic activity against paternal leucocytes. The present study aims to determine whether APCA is produced by the maternal peripheral blood mononuclear cells (PBMCs) in contact with the husband's spermatozoa and to evaluate the relation of APCA production with HLA class I and II expressions by spermatozoa in fertile couples. MATERIALS AND METHODS: This cross-sectional study included 30 fertile couples with at least one child. The maternal PBMCs were co-cultured with the husband's spermatozoa and the supernatant was assessed for the presence of IgG by ELISA. Cytotoxic activity of the supernatant on the husband's PBMCs was assessed by the complement-dependent cytotoxicity (CDC) assay. RESULTS: IgG was produced in all co-cultures, and the mean level of supernatant IgG was 669 ng/ml. The cytotoxic activity of the supernatant was observed in all the supernatant obtained from the co-cultures. The mean percentage of APCA in supernatant was 73.93%. CONCLUSION: Based on the results of this study it can be concluded that APCA may be a natural anti-sperm antibody (ASA), which can be produced during exposure to spermatozoa and may have some influence before pregnancy. Further research is required to determine the role of APCA before pregnancy.

The impact of neutrophil extracellular trap from patients with systemic lupus erythematosus on the viability, CD11b expression and oxidative burst of healthy neutrophils.

BACKGROUND: NET (neutrophil extracellular trap) has been shown to directly influence inflammation; in SLE (systemic lupus erythematosus), it is reportedly a plausible cause for the broken self-tolerance that contributes to this pathology. Meanwhile, the role of NET is not easily explicable, and there is a serious discrepancy in the role of NET in SLE pathology and generally inflammation; in particular, the interactions of neutrophils with NET have been rarely inspected. This study evaluates the effect of NET on neutrophils in the context of SLE. The neutrophils were incubated by the collected NET (from SLE patients and healthy controls) and their expression of an activation marker, viability and oxidative burst ability were measured. RESULTS: The level of cell mortality, CD11b expression and the oxidative burst capacity were elevated in NET-treated neutrophils. Also, the elevation caused by the SLE NET was higher than that produced by the healthy NET. CONCLUSION: The decreased neutrophil viability was not due to the increase in apoptosis; rather, it was because of the augmentation of other inflammatory cell-death modes. The upregulation of CD11b implies that NET causes neutrophils to more actively contribute to inflammation. The increased oxidative burst capacity of neutrophils can play a double role in inflammation. Overall, the effects induced by NET on neutrophils help prolong inflammation; accordingly, the NET collected from SLE patients is stronger than the NET from healthy individuals.

The Upregulation of hsa-mir-181b-1 and Downregulation of Its Target CYLD in the Late-Stage of Tumor Progression of Breast Cancer.

Some microRNAs are usually dysregulated in the cancers and influencing tumor behavior and progression. Hsa-miR-181b-1 and its target CYLD are involved in regulating the inflammatory pathways. This study aimed to investigate the expression levels of hsa-mir-181b-1 and CYLD in a cohort of breast tumor tissues and normal adjacent tissues to assess their association with breast cancer stages. A total number of 60 breast samples including cancerous and normal adjacent tissue specimens were collected. After pathological study, the expression of hsa-mir-181b-1 and CYLD were measured by qRT-PCR method. The hsa-mir-181b-1 expression level was significantly increased in breast tumor tissues compared to the controls. This increase was associated with the disease progression. Conversely, CYLD expression level was decreased in tumor samples compared to normal samples, significantly. ROC curve data added other prestigious information of hsa-mir-181b-1 and CYLD by defining cancer and healthy tissues with high specificity and sensitivity at a proposed cutoff point. Also, bioinformatic enrichment for the possible targets of mature sequence of "hsa-mir-181b-5p" was performed. Computational analysis showed the five most significant pathways including metabolic, cancer, calcium signaling, PI3K-Akt signaling and focal adhesion pathways which may be influenced by hsa-mir-181b-1. Thus, we suggested hsa-mir-181b-1 and CYLD might be involved in the pathogenesis of breast cancer and could be considered as two biomarkers for prediction, prognosis and diagnosis of the stages of the breast cancer.

Induction of humoral immune responses and inhibition of metastasis in mice by a VEGF peptide-based vaccine.

OBJECTIVES: Blocking of vascular endothelial growth factor (VEGF) plays a pivotal role in inhibition of metastasis and is a target for development of anti-angiogenic agents. In this study, a peptide-based vaccine was designed and its potential for induction of humoral immune responses, generation of neutralizing antibodies, inhibition of tumor growth and metastasis was determined. MATERIALS AND METHODS: With online bioinformatics tools, a fragment of the VEGF-A was selected for a peptide-based vaccine. To enhance its antigenicity, the peptide was conjugated with Keyhole limpet hemocyanin and used to immunize mice. Then, the polyclonal anti-VEGF antibody titer was measured and its effect on proliferation of HUVEC cell line was investigated by MTT assay. Finally, we checked the effect of the peptide on tumor growth, metastasis, and survival rates in a mouse model of cancer. RESULTS: The bioinformatics analysis of the selected region confirmed dis-similarity of the peptide with any other human protein and its acceptable antigenicity to stimulate a tumor-specific humoral response. Anti-VEGF antibody titers were significantly greater in vaccinated mice than in controls. IgG antibody from mice immunized with recombinant VEGF-A inhibited HUVEC proliferation (P<0.0001). Tumors in vaccinated mice were significantly smaller than those in controls. Moreover, metastasis was reduced and survival rates increased in the vaccinated group. CONCLUSION: Production of high-titer antibody against the peptide vaccine indicated that the peptide has the potency to be used as a peptide-based vaccine for humoral inhibition of tumor growth and metastasis. The efficacy of the peptide should be further tested in primate models.

NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency.

BACKGROUND: Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1. METHODS: Functional characterization of the NOX1 variant included ROS generation, wound healing, 2-dimensional collective chemotactic migration, single-cell planktonic migration in heterologous cell lines, and RNA scope and immunohistochemistry of paraffin-embedded patient tissue samples. RESULTS: Using exome sequencing, we identified a stop-gain mutation in NOX1 (c.160C>T, p.54R>*) in patients with pediatric-onset IBD. Our studies confirmed that loss-of-function of NOX1 causes abrogated ROS activity, but they also provided novel mechanistic insights into human NOX1 deficiency. Cells that were NOX1-mutant showed impaired wound healing and attenuated 2-dimensional collective chemotactic migration. High-resolution microscopy of the migrating cell edge revealed a reduced density of filopodial protrusions with altered focal adhesions in NOX1-deficient cells, accompanied by reduced phosphorylation of p190A. Assessment of single-cell planktonic migration toward an epidermal growth factor gradient showed that NOX1 deficiency is associated with altered migration dynamics with loss of directionality and altered cell-cell interactions. CONCLUSIONS: Our studies on pediatric-onset IBD patients with a rare sequence variant in NOX1 highlight that human NOX1 is involved in regulating wound healing by altering epithelial cytoskeletal dynamics at the leading edge and directing cell migration.

Can the Decreased Expression of Human Leukocyte Antigen Class â and â ¡ by Spermatozoa Lead to Recurrent Spontaneous Abortion?

BACKGROUND & OBJECTIVE: Unexplained recurrent spontaneous abortion (URSA) is defined as an unknown cause of occurrence of three or more clinically detectable pregnancy losses before 20 weeks of gestation, but it occurs presumably as a result of the immune system dysfunctions. We supposed that the disruption of semen or spermatozoa might be responsible for the dysfunction of the immune system in women with URSA. Semen and spermatozoa (as antigens) induce female reproductive tract (FRT) immunity. This stimulated immunity is necessary for pregnancy occurrence. The disruption of semen or spermatozoa can be a result of altering a variety of surface molecules on spermatozoa, especially polymorphic human leukocyte antigen (HLA) molecules or antigens. Despite the importance of HLA antigens in reproduction, to the best of our knowledge, no one has studied the relation of HLA expression between spermatozoa and URSA. Therefore, this paper aims to assess this relation. METHODS: Semen samples were collected from 15 URSA couples and 20 normal couples. After purification of normal spermatozoa, the HLA class I and II expressions were evaluated by flow cytometry methods. RESULTS: Results showed that the expression of both HLA class I and II by spermatozoa, in URSA couples, was significantly less than the control couples. CONCLUSION: The decreased expression of polymorphic HLA class Ⅰ and Ⅱ by spermatozoa can be related to URSA occurrence.

Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils.

OBJECTIVE: Currently, the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of the cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have a relatively equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 h of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry. RESULTS: Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.

Comparison of Expression Levels of miR-29b-3p and miR-326 in T Helper-1 and T Helper-17 Cells Isolated from Responsive and Non-responsive Relapsing-remitting Multiple Sclerosis Patients Treated with Interferon-beta.

T helper type 1 (Th1) and Th17 Cells with distinct cytokine profiles including interferon-gamma (IFN-γ) and interleukin 17 (IL-17) have a pivotal role in neuroinflammation and myelin destruction in the central nervous system (CNS) in MS. MicroRNA-29b (MiR-29b) and miR-326 contribute to regulating Th1 and Th17 differentiation and altered expression of the miRNAs could be associated with response to treatment in multiple sclerosis (MS). Therefore, our study aimed to evaluate the percentage of Th1 and Th17 and determining the expression levels of miR-29b-3p and miR-326 in these lymphocyte subpopulations between responsive and non-responsive to interferon beta (IFN-ß) therapy in relapsing-remitting multiple sclerosis (RRMS) patients. The present study was performed on 40 RRMS patients following treatment with IFN-ß. The percentage of Th1 cells and Th17 cells were determined by flow cytometry in responsive and non-responsive patients. The expression levels of miR-29b-3p and miR-326 were assessed in Th1 and Th17 cells by quantitative polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the plasma levels of IFN-γ and IL-17A. No significant difference was observed in the percentage of Th1 and Th17 cells as well as the expression levels of miR-29b-3p and miR-326 (in Th1 and Th17, respectively) in treated patients. Also, we did not find any significant difference in IFN-γ and IL-17A plasma concentration between responsive or non-responsive to IFN-ß therapy in patients with RRMS. IFN-ß may regulate other miRNAs in Th1 and Th17 cells than miR29b-3p and miR-326 in MS patients.

Fc Receptor-Like 1 as a Promising Target for Immunotherapeutic Interventions of B-Cell-Related Disorders.

BACKGROUND: Human B-cell responses are regulated through synergy between a collection of activation and inhibitory receptors. Fc receptor-like (FCRL) molecules have recently been identified as co-receptors that are preferentially expressed in human B-cells, which may also play an important role in the regulation of human B-cell responses. FCRL1 is a member of the FCRL family molecules with 2 immunoreceptor tyrosine-based activation motifs (ITAMs) in its cytoplasmic tail. This study aimed to investigate the regulatory roles of FCRL1 in human B-cell responses. MATERIALS AND METHODS: The regulatory potential of FCRL1 in human B-cell through knockdown of FCRL1 expression in the Ramos and Daudi Burkitt lymphoma (BL) cell lines by using the retroviral-based short hairpin ribonucleic acid (shRNA) delivery method. The functional consequences of FCRL1 knockdown were assessed by measuring the proliferation, apoptosis, and the expression levels of Bcl-2, Bid, and Bax genes as well as phosphoinositide-3 kinase/-serine-threonine kinase AKT (PI3K/p-AKT) pathway in the BL cells, using the quantitative real-time polymerase chain reaction (PCR) and flow cytometry analysis. The NF-κB activity was also measured by the enzyme-linked immunosorbent assay (ELISA). RESULTS: FCRL1 knockdown significantly decreased cell proliferation and increased apoptotic cell death in the BL cells. There was a significant reduction in the extent of the Bcl-2 gene expression in the treated BL cells compared with control cells. On the contrary, FCRL1 knockdown increased the expression levels of Bid and Bax genes in the treated BL cells when compared with control cells. In addition, the extent of the PI3K/p-AKT expression and phosphorylated-p65 NF-κB activity was significantly decreased in the treated BL cells compared with control cells. CONCLUSIONS: These results suggest that FCRL1 can play a key role in the activation of human B-cell responses and has the potential to serve as a target for immunotherapy of FCRL1 positive B-cell-related disorders.

The expression of human leukocyte antigen by human ejaculated spermatozoa.

BACKGROUND: After coitus and insemination, an inflammatory response is evident in the female reproductive tract (FRT). Semen contains a variety of immune-activating components that have a major role in the induction of an immune response in the FRT. One of the most important is (human leukocyte antigen) HLA molecules which are present in soluble form in seminal plasma and in membrane form on the surface of cells (such as epithelial and leukocytes) existing in semen. Nevertheless, there is considerable debate over the expression of HLA antigens by human spermatozoa. Considering the critical role of HLA molecules in reproduction and the induction of an immune response, it is very important to clearly define HLA expression by spermatozoa and the role of these molecules in sperm morphology, motility, and strength to fertilize an egg. Therefore, the objective of this study was to determine HLA expression by ejaculated spermatozoa. The results of this study will facilitate the design of future studies. METHOD: Semen samples were collected from 50 healthy men with normal semen status by masturbation after 2-3 days of sexual abstinence. After purification of normal spermatozoa, HLA class I & II expression was evaluated by quantitative real-time PCR and flow cytometry methods. RESULTS: The results showed the expression of both HLA class I & class II by spermatozoa. The results also showed that the expression of HLA class Ⅱ was significantly more than HLA class Ⅰ. CONCLUSION: Spermatozoa express both HLA class I & class II molecules.

Producing Soluble Human Programmed Cell Death Protein-1: A Natural Supporter for CD4+T Cell Cytotoxicity and Tumor Cells Apoptosis.

BACKGROUND: Programmed cell death protein-1 (PD-1)/PD-L1 pathway is one of the immune checkpoint pathways involved in the regulation of the immune responses and the suppression of anti-tumor defense. PD-1/PD-L1 blocking antibodies improve immune responses such as cytotoxic activity of CD8+/CD4+T cells and increase mortality of tumor cells as well; however, their use is accompanied by adverse side effects. OBJECTIVES: We aimed to produce a native blocker of human PD-1/PD-L1, for developing T cells cytotoxicity and tumor cells apoptosis. MATERIALS AND METHODS: We designed and cloned soluble human PD-1-GFP-pcDNA3.1/hygro construct in Escherichia coli strain TOP10 cells and then transfected this construct into the HEK cells. The concentration of the secreted shPD-1 in the supernatant was measured and the supernatant was used for blocking PD-L1 on the MDA-MB-231 cells. The cytotoxicity of CD8+/CD4+T cells and the apoptosis of MDA-MB-231 cells, under the influence of shPD-1 in the co-culture of T cells with the MDA-MB-231 cells, were evaluated using flow cytometry technique. RESULTS: The GFP expression in the transfected cells illustrated the successful designing, transfection, and production of shPD-1. Soluble human PD-1 concentration in the supernatant of the transfected HEK cells was significantly higher than the untransfected cells. In addition, shPD-1 significantly blocked PD-L1 on the MDA- MB-231 cells, improved the cytotoxicity of CD4+T cells, and increased the apoptosis of MDA-MB-231 cells. CONCLUSION: Overall, increased CD4+T cell cytotoxicity and tumor cells apoptosis under the influence of shPD-1, confirmed the effectiveness of shPD-1 as a natural blocker of PD-L1and as an augmenter of the anti-tumorimmune responses.

Decreased Toll-like Receptor (TLR) 2 and 4 Expression in Spermatozoa in Couples with Unexplained Recurrent Spontaneous Abortion (URSA).

Studies have shown that toll-like receptors (TLRs) play some important roles in reproductive processes such as ovulation, spermatogenesis, sperm capacitation, fertilization, and pregnancy to the best of our knowledge, no study has evaluated the expression and role of these molecules and their impairment in spermatozoa; accompanied by pregnancy complications such as recurrent spontaneous abortion (RSA). Therefore, this study investigates the alteration of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) expression in spermatozoa in men whose spouse have unexplained RSA. Fifteen fertile couples and fifteen couples with unexplained recurrent spontaneous abortion (URSA) were included in this study. The level of TLR2 and TLR4 expression in untreated and lipopolysaccharide (LPS) or PAM3CYS in treated spermatozoa were examined by flow cytometry. The results showed reduced expression of TLR4 in untreated spermatozoa and decreased LPS or PAM3CYS levels in treated spermatozoa in the URSA group compared to the control group. No significant differences were found in TLR2 expression of untreated spermatozoa in RSA and control groups. After the treatment of spermatozoa with LPS, the TLR2 expression was decreased in both groups. After the treatment of spermatozoa with PAM3CYS, the level of TLR2 expression was significantly increased in the URSA group; while no significant differences were shown in the control group in comparison to untreated spermatozoa. We have concluded that decreased TLR4 expression and a differently increased TLR2 expression in response to ligand treatment in spermatozoa is associated with URSA.

Effect of hydatid cyst antigens on inhibition of melanoma cancer growth in mouse model.

Cancer is the main cause of death in the developed countries. There are some scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. Hydatid cyst is the larval stage of Echinococcus granulosus, which causes hydatidosis in human and livestock. We have already shown that vaccination of mice with hydatid cyst crude antigens and subsequently challenge them with cancer cells, causes inhibition of melanoma cancer growth. In this study, therapeutic effects of hydatid cyst antigens on C57/black mice that had already been challenged with melanoma tumor were investigated. In this experimental study, 6 groups of C57 black mice were subcutaneously inoculated with melanoma cancer cells (line B16F10) in PBS inside their chest site. After 2 weeks case groups were injected with hydatid cyst fluid, a fraction of cyst fluid, live protoscolices or BCG.  control groups were injected with alum alone and other control group was left intact without any intervention. The size of each tumor was measured in all mice. Blood samples were also taken to estimate Interleukin-2 (IL-2), Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ) and Interleukin-4 (IL-4) levels. Treatment of mice bearing melanoma cancer with hydatid cyst antigens resulted in inhibition of tumor growth and the difference between mean size of tumor in case and control groups was statistically significant. Also, according to our results mean level of measured cytokines between case and control groups was statistically different. Hydatid cyst antigens have anti-melanoma activities and this effect may be related to immune response to parasite antigens.

Evaluation of the effect of TIM-3 suppression by miR-498 and its effect on apoptosis and proliferation rate of HL-60 cell line.

BACKGROUND: Acute Myeloid Leukemia (AML) is a Cancer of hematopoietic stem cells with a rapid progression. TIM-3 is expressed on leukemic stem cells (LSCs) in most types of AML and might have a positive effect on maintenance of malignant phenotype. MicroRNAs play important roles in either cancer progression or suppression. In this study were evaluated, the inhibitory effect of miR-498 on TIM-3 expression and its impact on proliferation and survival of HL-60 cell line. METHODS: Firstly, the probable inhibitory effect of miR-498 on TIM-3 expression was predicted. HL-60 cells were cultured and expression of TIM-3 was induced on the cells using phorbol miristate acetate. The cells were transfected with miR-498 and expression level of TIM-3 were measured using with q-RT-PCR and flow cytometry methods. In addition, the effect of suppression of TIM-3 expression in HL-60 cell line was analyzed with apoptosis and cell proliferation assays. RESULTS: Bioinformatics analyses predicted that miR-498 has high ability to silence TIM-3 gene expression. Our experiments confirmed that miR-498 was able to strongly silence TIM-3 expression (68% silencing) in HL-60 cell line (P < 0.002). Also, the cells with suppressed expression of TIM-3 had a lower proliferation and higher apoptosis rates. CONCLUSION: Based on our results, the miR-498 can effectively suppress TIM-3 expression in the AML cell line. TIM-3 suppression, in turn, inhibits malignant cell proliferation and induces its apoptosis. Collectively, suppression of TIM-3 by miR-498 can be considered as a potential powerful way for treatment of AML.