The immunoregulatory axis (programmed death-1/programmed death ligand-1) on CD4+ T cells in lupus nephritis: association with vitamin D and chemokine C-X-C motif ligand 12.

Lupus nephritis (LN) is one of the most serious complications of systemic lupus erythematosus (SLE). Multiple immunomodulatory mechanisms contribute to the pathogenesis of LN. A deep understanding of the immunopathogenesis of LN is essential to identify optimal molecular targets, as most immunotherapeutic algorithms are still based on unselective drugs. The study aimed to elucidate the possible association of vitamin D deficiency with the programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) axis and inflammatory response in patients with LN, as well as the relationship between the PD-1/PD-L1 axis and chemokine C-X-C motif ligand 12 (CXCL12). Flow cytometry was used to determine the frequencies of CD279 (PD-1) and CD274 (PD-L1) in the peripheral CD3+CD4+ cell population of persons with LN. Furthermore, ELISA was used to detect serum CXCL12 and vitamin D concentrations. A distinct decrease of PD-1 and a significant increase of PD-L1 was demonstrated in patients with LN compared with either SLE patients with no LN or healthy controls. The PD-1/PD-L1 axis was negatively correlated with different disease parameters. Vitamin D deficiency and insufficiency were more prevalent in patients with LN than in controls, being significantly associated with disease activity and inversely associated with the PD-1/PD-L1 expression. Moreover, CXCL12 was negatively correlated with the PD-1/PD-L1 axis and vitamin D concentration. The findings suggest an involvement of the PD-1/PD-L1 axis in lupus nephritis, which might serve as a potential highly selective therapeutic target that is more effective but less toxic. In addition, restoring adequate vitamin D levels in lupus nephritis could be a possible simple measure to control inflammatory immune responses.

Low-dose interleukin-2-loaded nanoparticle effect on NK and T-reg cell expression in experimentally induced type 1 diabetes mellitus.

INTRODUCTION: Type 1 diabetes mellitus is an autoimmune disorder characterized by inflammatory damage to pancreatic ß cells resulting in loss of insulin secretion. In autoimmune type 1 diabetes mellitus (T1D) natural killer cells (NK) initiate pancreatic islets cell lyses in autoimmune T1D. Loss of T regulatory cells (Treg) at disease onset facilitates the activation and accumulation of NKs in the pancreatic microenvironment. A proper low-dose interleukin 2 (IL-2) could enhance Tregs and enforce control and regulation of pro-inflammatory NKs. AIM: This relation needs to be studied to improve therapeutic strategies aimed at resetting the balance between Tregs and proinflammatory cells. MATERIAL AND METHODS: We used novel formulations of low-dose IL-2 loaded on chitosan nanoparticles. The study included 116 T1D BALB/c mice experimentally induced by streptozotocin, divided into groups. Their splenocytes were maintained in a short-term culture for assessment of expression of CD4+FOXP3+ Treg and NKp46+NK by both flow cytometry and enzyme-linked immunoassay (ELISA). Morphological, immunohistochemical, and morphometrical analyses were done. In vitro suppressor assay was used to assess the suppressor effect of Treg cells after exogenous IL-2 treatment. RESULTS: NK cell expression, NKp46 level, and NK cell functions were modulated more in mice injected with IL-2-loaded chitosan nanoparticles than in other groups. A statistical inverse correlation was found between Treg and NK cell expression in IL-2-loaded chitosan with 0.3 µIU (p = 0.047), and this correlation was related to FOXP3 expression on Treg cells. The modified expression of NK and NKp46 was noticed in mice injected with 0.3 µIU for longer duration (3 weeks) (p < 0.001), but the NK functions did not show any significant changes with prolonged treatment. CONCLUSIONS: Prolonged administration of low-dose IL-2 results in the vigorous expression of NKp46, indicating a significant role of Tregs in NK stimulation and motivation. Low-dose IL-2 selectively modulates NKp46 NK and FOXP3+ Tregs and increases their expression.

The interplay of interleukin-17A and breast cancer tumor microenvironment as a novel immunotherapeutic approach to increase tumor immunogenicity.

Based on its known role in mediating tumor progression and the correlation with poor response to chemotherapy, we hypothesized that blocking interleukin-17A (IL-17A) by anti-IL-17 monoclonal antibodies might have the ability to suppress programmed death-ligand-1 (PD-L1) and to modulate the expression and function of myeloid-derived suppressor cells (MDSCs) in BC microenvironment. We also compared the apoptotic activity of anti-IL-17 with those acquired from our previous work on monoclonal antibodies against IL-6. The influence of anti-IL-17 was investigated in two doses on localized freshly resected tissues from 50 patients with BC. Results revealed increased IL-17A in BC tumor tissues versus surrounding tissues. Additionally, PD-L1 expression was inhibited in cultures treated with both doses of anti-IL-17. Frequencies of MDSCs were reduced in those cultures with anti-IL-17 with reduced suppressive activity. The induced apoptosis in the tumor cells was significantly increased. Anti-IL-17 antibodies effect was related to late stages, vascular metastasis, and hormonal status. Results of the current work suggest a promising role for anti-IL-17 monoclonal antibodies in enhancement of anti-tumor immunological activity in BC, potentially involving suppression of immune checkpoint PD-L1 and MDSCs inhibition with a marked response in aggressive disease.

Modulation of Memory B Cell Phenotypes and Toll-Like Receptor-7 in Chronic Hepatitis C Virus Infection During Direct-Acting Antiviral Interferon-Free Therapy: Correlation with Interleukin-7.

Hepatitis C virus (HCV) infection is a major worldwide problem with the highest incidence rates in Egypt. It affects B cells that serve as reservoirs for persistent HCV, resulting in phenotypic B cell alterations. Interleukin-7 (IL-7) is a cytokine with antiviral activity, important for B cell physiology. In addition, B cell-intrinsic toll-like receptor-7 (TLR7) signaling is required for optimal B cell responses during chronic viral infection, and the deficiency of TLR7 in B cells is sufficient to significantly impact antibody responses. Based on their known immunomodulatory effects, we hypothesized that direct-acting antiviral interferon-free therapy may affect TLR7 expression and the exhausted peripheral B cell compartment with the possibility of their restoration in patients who achieved a sustained virological response and their correlation to IL-7 level. This prospective study was accomplished on 80 Egyptian HCV patients and 75 controls. Frequencies of peripheral B cell subsets, TLR7 gene expression, TLR7 protein, and serum IL-7 levels were investigated by flow cytometry, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. B cell subpopulations were exhausted and partially restored among HCV patients after receiving treatment, but not recovered with regard to activated mature or resting memory B cells. Almost all responders to direct antiviral drugs showed upregulation of TLR7 gene expression and correlated with the frequency of memory B cell, but not with IL-7. Moreover, IL-7 was not significantly different between groups although correlated with immature transitional B cells. Results may indicate the interplay between TLR7 and B cells during remission or progression of HCV. Thus, TLR7 could be used as a promising biomarker for assessment of antiviral treatment efficacy among chronically infected HCV patients, and that targeting TLR7 may be used as a potential prophylactic and/or therapeutic agent during chronic HCV as well as immune-potentiation of memory B cells.

TGF-ß Enhances the Anti-inflammatory Effect of Tumor- Infiltrating CD33+11b+HLA-DR Myeloid-Derived Suppressor Cells in Gastric Cancer: A Possible Relation to MicroRNA-494.

BACKGROUND: Accumulation of myeloid-derived suppressor cells (MDSCs) constitutes a key mechanism of tumor immune evasion in gastric cancer (GC). Therefore, searching for more accurate prognostic factors affecting their immunosuppressive role has become a growing interest in cancer immunotherapy research. Increased expression of microRNA-494 was noticed in MDSCs from tumor-bearing mice, suggesting another new therapeutic objective for cancer treatment. It was also discovered that tumor-derived transforming growth factor beta (TGF-ß) is responsible for the up-regulation of microRNA-494 in MDSCs. The purpose of this study was to address the effect of recombinant (rTGF-ß) on the anti-inflammatory activity of MDSCs in GC and its possible association with micro-RNA-494 expression in tumor tissue. METHODS: Freshly obtained GC tumor tissue samples and peripheral blood were used for isolation of CD33+11b+HLADR- MDSCs cells from 40 GC patients and 31 corresponding controls using flow cytometry. MDSCs were co-cultured with isolated autologous T cells to assess proliferation and cytokine production in the presence and absence of rTGF-ß. Real-time PCR and Enzyme linked immunosorbent assay were used to evaluate tumor expression of miRNA-494 and TGF-ß respectively. RESULTS: Results showed that rTGF-ß markedly increased the suppressive ability of tumor MDSCs on proliferation of autologous T cells and interferon gamma production. However, no inhibitory effect was observed for MDSCs from circulation. In addition, infiltration of MDSCs in tumors is associated with the prognosis of GC. MiRNA-494 was also extensively expressed in tumor samples with a significant correlation to MDSCs. CONCLUSION: These results indicate that tumor-derived MDSCs but not circulatory MDSCs have an immunosuppressive effect on T cells, potentially involving TGF-ß mediated stimulation.  Results also suggest a role for miRNA-494 in GC progression. Therefore, control of TGF-ß and miRNA-494 may be used as a treatment strategy to downregulate the immunosuppressive effect of MDSCs.
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The novel role of IL-37 to enhance the anti-inflammatory response of regulatory T cells in patients with peripheral atherosclerosis.

OBJECTIVES: Regulatory T cells (Tregs) mediate immunomodulation and protect against atherosclerosis. It is considered that reducing the amount of pro-inflammatory mediators could be achieved by enhancing the anti-inflammatory response, and this may be considered one of the main targets for therapy development. The inhibitory cytokines secreted by Tregs mainly include interleukin-10 (IL-10) and transforming growth factor-beta (TGF-ß). Based on its known immunosuppressive involvement with other inflammatory disorders, we hypothesized that the newly characterized cytokine interleukin-37 (IL-37) might be associated with the inhibitory functions of Treg in atherosclerosis. Immune regulatory functions of IL-37 have not been completely clarified. Accordingly, we speculated that IL-37 might play a regulatory role in the immunosuppression of Tregs in atherosclerotic disease. METHODS: Real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to test gene expression and protein levels of IL-37 in peripheral blood and localized freshly resected arterial tissues from 84 patients with peripheral arterial occlusive disease and 50 non-atherosclerotic subjects. Results were correlated to disease hallmarks. We also evaluated the ability of recombinant IL-37 to modulate Treg cytokine secretion and T cell inhibition in relation to atherosclerotic disorder in vitro.Results: Our results revealed that IL-37 was increased in patients with chronic lower limb atherosclerotic ischemia, compared to non-atherosclerotic controls. In addition, the expression levels of circulating IL-37 correlated with disease severity of chronic lower limb ischemia. Supplementation with rIL-37 augmented levels of released IL-10 and TGF-ß in supernatants of T cells co-cultured with Tregs in the enrolled patients.Conclusions: Results suggest a role for IL-37 in mediating anti-inflammatory functions in the atherosclerotic process, potentially involving enhancement of Treg inhibitory function and anti-inflammatory cytokine secretion with a particularly marked direct response in severe disease.

Changes and significance of T helper-9 cells and interleukin-9 in patients with atherosclerotic chronic lower limb ischemia: Effect on IL-17 release.

OBJECTIVES: Atherosclerosis is considered as a chronic inflammatory disorder where the central role of T cells in its pathogenesis is well known. T helper-9 cells have a distinctive effect upon the inflammatory processes. They stimulate macrophages via secretion of their cytokine interleukin-9. Based on its known involvement with other inflammatory disorders, we hypothesized that interleukin-9 might be associated with the inflammatory limb of peripheral atherosclerotic disease. METHODS: We tested this hypothesis on peripheral blood mononuclear cells (PBMCs) and freshly resected arterial tissues from 84 patients with peripheral arterial occlusive disease (PAOD) and 50 non-atherosclerotic subjects. A number of experimental methods were used including flow cytometry analysis of T helper-9 cells using anti-CD3, anti-CD4, and anti-interleukin-9monoclonal antibodies as well as real-time polymerase chain reaction for the assessment of gene expression of interleukin-9. In addition, circulating serum levels of interleukin-9 were measured using enzyme linked immunosorbent assay. We also evaluated the ability of recombinant interleukin-9 to modulate IL-17 release in cultured isolated CD3+ T cells with relation to atherosclerotic disorder in vitro. RESULTS AND CONCLUSIONS: Here we report increased percentages of T helper-9 cells and interleukin-9 levels in patients with chronic lower limb atherosclerotic ischemia, compared to healthy controls. Through investigation of different atherosclerotic patient populations with different disease stages, we found elevated interleukin-9 level both systemically and within the lesion and increased expression of cells in severe disease stages. The current study also revealed enhanced expression of mRNA levels of interleukin-9 within the atherosclerotic lesion when compared with non-atherosclerotic vessels. Levels of released IL-17 in CD3+ T cell culture supernatants supplemented with interleukin-9 were significantly positively correlated in the enrolled patients. The results suggest a role for T helper-9 cells and IL-9 in atherosclerotic process, potentially involving IL-17-mediated mechanisms. Indeed, we found that interleukin-9 promoted IL-17 release in PBMCs, with a particularly marked response in severe disease.

Study of Toll­Like Receptor 4 Gene Polymorphisms in Colorectal Cancer: Correlation with Clinicopathological Features.

Polymorphisms of Toll-like receptor 4 (TLR4) as a key player in cell proliferation, apoptosis, and angiogenesis have been linked to colorectal cancer (CRC) in different populations. We aimed in this study to determine genetic associations of TLR4 variants with CRC progression in Egyptian patients. Genotype and allelic frequencies of Asp299Gly (rs4986790) and Thr399Ile (rs4986791) were compared between 127 CRC patients and 141 healthy Egyptians using restriction fragment length polymorphism, and were correlated to clinicopathological findings. Results revealed that the variant alleles (G of Asp299Gly) and (T of Thr399Ile) were significantly associated with CRC among Egyptians. Confirmed by haplotype analysis, AT and GT haplotypes were more frequent in CRC patients than controls with increased CRC odds (OR = 3.54 and 3.45, 95% CI = 1.48-8.48 and 1.09-10.83, respectively). In addition, the G allele of Asp299Gly SNP was observed to be significantly associated with progressive CRC, including stage IV (P = .001), grade III (P = .025), N2 lymph nodes (P = .020), and metastasis (P = .001). On the other hand, Thr399Ile variant did not show any association with tumor behavior. Taken together, we conclude a significant association of Asp299Gly and Thr399Ile variants with the risk of development of CRC in Egypt. Asp299Gly variant, but not the Thr399Ile variant, may serve as a biomarker of this disease progression in Egyptian population.

Effect of low dose IL-2 loaded chitosan nanoparticles on natural killer and regulatory T cell expression in experimentally induced autoimmune type 1 diabetes mellitus.

INTRODUCTION: Natural killer cells (NK) initiate pancreatic islets cell lyses in autoimmune type 1 diabetes mellitus (T1D). Loss of T regulatory cells (Treg) at disease onset facilitates activation and accumulation of NKs in the pancreatic microenvironment. A proper low dose interleukin 2 (IL-2) could enhance Tregs and enforce control and regulation of pro-inflammatory NKs. This relation needs to be studied to improve therapeutic strategies aimed at resetting the balance between Tregs and proinflammatory cells. MATERIAL AND METHODS: We used novel formulations of low dose IL-2 loaded on chitosan nanoparticles. The study included 116 T1D BALB/c mice experimentally induced by streptozotocin, divided into groups. Their splenocytes were maintained in a short-term culture for assessment of expression of CD4+Foxp3+ Treg and NKp46+NK by both flow cytometry and enzyme linked immunoassay (ELISA). In vitro suppressor-assay was used in order to assess the suppressor effect of Treg cells after exogenous IL-2 treatment. RESULTS: NK cell expression, NKp46 level and NK cell functions were modulated in mice injected with IL-2 loaded chitosan nanoparticles than other groups. A statistical inverse correlation was found between Treg and NK cell expression in IL-2 loaded chitosan with (0.3 µIU) (p = 0.047) and this correlation was related to Foxp3 expression on Treg cells. The modified expression of NK and NKp46 was noticed in mice injected with (0.3 µIU) for longer duration (three weeks) (p < 0.001) but the NK functions did not show any significant changes with prolonged treatment. CONCLUSIONS: Low dose (0.3) µIU IL-2 nanoparticles effectively modulated NK and NKp46 expression. It selectively modulates the suppressive activity of Tregs indicating a significant role of Tregs in NK activation and function by controlling the availability of IL-2 in the microenvironment.

Th17/Treg cells imbalance and their related cytokines (IL-17, IL-10 and TGF-ß) in children with autism spectrum disorder.

We aimed in this study to investigate a possible involvement of Th17/Treg cells imbalance in autism spectrum disorders (ASD). Using flowcytometry to determine circulating Th17 and Treg cells percentages, RT- PCR and ELISA for cytokine expression, we demonstrated that Th17/Treg balance in ASD children was significantly skewed toward a Th17 response compared to their control. Th17 cells and the ratio of Th17/Treg cells had a significantly positive correlation with disease severity whereas Treg cells had a negative correlation. The imbalance of Th17, Treg cells and their related cytokines may play a vital role in the progression of the disease.

Association of the polymorphisms of TRAF1 (rs10818488) and TNFAIP3 (rs2230926) with rheumatoid arthritis and systemic lupus erythematosus and their relationship to disease activity among Egyptian patients.

AIM OF THE STUDY: Recent studies demonstrated the association of tumor necrosis factor α-induced protein 3 (TNFAIP3) (rs2230926) and tumor necrosis factor receptor associated factor 1 (TRAF1) (rs10818488) with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in different populations. We aimed at determining whether they confer susceptibility to SLE and RA in Egyptian population and if there is any relation to disease activity and auto-antibodies profile. MATERIAL AND METHODS: A case-control study involving 105 individuals with RA, 90 with SLE and 75 healthy controls was performed using TaqMan genotyping assay for two SNPs that showed the best evidence of association in the previous Caucasian studies. RESULTS: We detected significant differences in G allele frequency of TNFAIP3 (rs2230926) with SLE (p = 0.017(*)) and RA (OR = 2.333; 95% CI: 1.103-4.935, p = 0.023(*)) and association with RA disease activity (< 0.001). The A allele of TRAF1 was significantly increased in RA compared to controls(p = 0.049) and with RA activity (p = 0.001), while TRAF1 polymorphism did not exhibit any significant difference in the frequencies of genotypes or alleles in SLE and control (p = 0.280). CONCLUSIONS: TNFAIP3 is a susceptibility gene to SLE and RA in the Egyptian population and is correlated to disease activity and the presence of autoantibodies while TRAF1 polymorphisms increase the risk of RA but not to SLE in Egyptian populations.

Association of DNA repair genes XRCC1 (Arg399Gln), (Arg194Trp) and XRCC3 (Thr241Met) polymorphisms with the risk of breast cancer: a case-control study in Egypt.

UNLABELLED: Various DNA damage, induced by endogenous and exogenous factors, is handled through DNA repair pathways such as X-ray repair cross-complementing protein (XRCC). Genetic variations in these pathways may have a joint or additive effect on various types of cancer, including the risk of breast cancer (BC). AIM: To evaluate the association of three single-nucleotide polymorphisms (SNPs) Arg399Gln, Arg194Trp, and Thr241Met in DNA repair genes XRCC1 and XRCC3 on the risk of BC, and to assess their interaction with risk factors and prognostic markers in a case-control study in Egypt. METHODS: We detected the studied SNPs using polymerase chain reaction-restriction enzyme polymorphism (PCR-RFLP) in peripheral blood from 100 BC patients and 75 healthy females. RESULTS: The dominant model of inheritance of Arg399Gln and Arg194Thr revealed an increase in BC risk of odds ratio (OR) of 3.56, 95% confidence interval (CI)=1.22-10.39, p=0.017 and OR: 4.45, 95% CI=2.35-8.45, p<0.001 respectively. However, there was no clear interaction between the studied SNPs and the known risk factors, or tumor criteria. No association between the Thr241Met genotype and BC risk was observed. CONCLUSION: XRCC1 Arg399Gln and Arg164Trp variant genotypes are associated with an increased risk of BC in Egyptian females.