Association between IL28B rs12979860 single nucleotide polymorphism and the frequency of colonic Treg in chronically HCV-infected patients.

The IL28B gene is associated with spontaneous or treatment-induced HCV viral clearance. However, the mechanism by which the IL28B single nucleotide polymorphism (SNP) affects the extra-hepatic HCV immune responses and its relationship to HCV pathogenesis have not been thoroughly investigated. To examine the mechanism by which IL28B affects HCV clearance. Forty Egyptian patients with chronic HCV infection receiving an Interferon/ribavirin treatment regimen were enrolled into this study. There were two groups: non-responders (NR; n = 20) and sustained virologic responders (SVR; n = 20). The initial plasma HCV viral loads prior to treatment and IL28B genotypes were determined by quantitative RT-PCR and sequencing, respectively. Liver biopsies were examined to determine the inflammatory score and the stage of fibrosis. Colonic regulatory T cell (Treg) frequency was estimated by immunohistochemistry. No significant association between IL28B genotypes and response to therapy was identified, despite an odds ratio of 3.4 to have the TT genotype in NR compared to SVR (95 % confidence interval 0.3-35.3, p = 0.3). Patients with the TT-IL28Brs12979860 genotype (unfavorable genotype) have significantly higher frequencies of colonic Treg compared to the CT (p = 0.04) and CC (p = 0.03) genotypes. The frequency of colonic Treg cells in HCV-infected patients had a strong association with the IL-28B genotype and may have a significant impact on HCV clearance.

Association of colonic regulatory T cells with hepatitis C virus pathogenesis and liver pathology.

BACKGROUND AND AIM: Forkhead box protein P3 (FoxP3)(+) regulatory T (Treg ) cells play a fundamental role in maintaining the balance between the tissue-damaging and protective immune response to chronic hepatitis C (CHC) infection. Herein, we investigated the frequency of Treg cells in the colon and their potential relationship to the various CHC outcomes and hepatic histopathology. METHODS: Colonic biopsies were collected from three groups with CHC: treatment naïve (TN; n = 20), non-responders (NR; n = 20), sustained virologic response (SVR; n = 20), and a fourth healthy control group (n = 10). The plasma viral loads and cytokines levels were determined by quantitative real-time polymerase chain reaction, and ELISA, respectively. Liver biopsies were examined to assess inflammatory score and fibrosis stage. Colonic Treg frequency was estimated by immunohistochemistry using confocal microscopy. RESULTS: A significant increase in the frequency of colonic Treg was found in TN, and NR groups compared with the control and SVR group. The frequency of colonic Treg , plasma interleukin (IL)-10 and IL-4 levels were significantly positively correlated with viral load and negatively correlated with METAVIR inflammatory score, and fibrosis stages. CONCLUSION: Colonic Treg cells are negatively correlated with liver inflammation and hepatitis C virus (HCV) viral load, which suggests a strong linkage between gut-derived Treg cell populations and HCV infection.

Frequency of regulatory B cells phenotypes in breast cancer patients in Egypt.

Accumulating evidence has indicated that immune regulatory cells are involved in the establishment of the anti-tumor activity, however; the role of regulatory B cells (B-regs) in breast cancer (BC) remains unclear. This study intended to assess the frequency of peripheral B-regs phenotypes in patients with BC, and to determine the relation between these phenotypes and the patient's clinicopathological characters. The expressions of the immune cell populations were analyzed by four-color flow cytometry in 40 naïve BC patients and 10 age-matched apparently healthy individuals as controls attending the department of Clinical Oncology and Nuclear Medicine at Assiut University Hospitals. The percentages of B-regs phenotypes CD19+IL10+ and CD19+CD24hiCD27+IL10+ were higher in BC patients than in the controls. The percentage of CD19+IL10+ B cells phenotype was significantly associated with the HER-2 expression levels, T, and N stages of BC. In conclusion, high percentage of B-regs phenotypes CD19+IL10+ and CD19+CD24hiCD27+IL10+ in BC patients indicates a possible role in immune suppression during the development of BC.

Role of ATG16LI (rs2241880) and Interleukin 10 (rs1800872) Polymorphisms in Breast Cancer Among Egyptian Patients.

This study was performed to determine the role of autophagy-related 16-like 1 (ATG16L1, rs2241880) and IL10 (rs1800872) polymorphisms in the susceptibility to and early prediction of breast cancer in Egyptians. The study included 50 breast cancer patients and 50 apparently healthy controls. The PCR-RFLP technique was used to detect ATG16L1 (rs2241880) and IL10 (rs1800872) genotypes. IL10 level was determined in serum by ELISA. The mean age of the patients was 54.2 years. Among the patients, 80% had no family history for breast cancer, 70% were postmenopausal, and 72% exhibited grade II tumors. Metastasis was detected in 18% of the patients, and 6% of the cases exhibited triple-negative receptor (TNR) status. In the ATG16LI (rs2241880) gene, the GG genotype frequency was significantly higher in patients than in controls (14% in patients versus 2% in controls, P =0.02), and no metastasis was observed in patients with the AA genotype (P=0.03). In the IL10 (rs1800872) gene, the A allele was observed in 30% of patients and 23% of controls, but the difference was insignificant (P=0.26). Also, the prevalence of the AA genotype was 8% in patients and 4% in controls (P=0.54). Serum IL10 levels were higher in patients than in controls (P < 0.001). Within the patient group, individuals with the IL10 (rs1800872) AA genotype showed significantly higher serum IL10 levels than those with the CC and CA+CC genotypes (P =0.03 and 0.04, respectively). In conclusion, in Egyptian breast cancer patients, the GG genotype of ATG16LI (rs2241880) may be associated with increased disease risk, and the AA genotype could be protective against metastasis.

Evaluation of autophagy-related genes in Egyptian systemic lupus erythematosus patients.

Disturbances in autophagy are known to be implicated in autoimmune disorders. Many studies have connected polymorphisms in autophagy-related gene 5 (ATG-5) to systemic lupus erythematosus (SLE). Our aim was the determination of the expression level of ATG-5, Beclin-1 and microtubule-associated protein-light chain 3 (LC-3) in Egyptian SLE patients to investigate the impact of disturbances in autophagy genes on the incidence and progression of the disease. Also, we investigated the incidence of single nucleotide polymorphism (SNP) rs573775 in ATG-5 gene among Egyptian SLE patients. Our results showed that the mean levels of Beclin-1, LC-3 and interleukin (IL)-10 transcripts were significantly higher in SLE patients compared to healthy controls. The previous transcripts were positively correlated with SLE Disease Activity Index (SLEDAI). Beclin-1 and LC-3 transcripts were negatively correlated to complement component 3 (C3) levels. Only LC-3 transcripts were negatively correlated to complement component 4 (C4). The rs573775 SNP of ATG-5 with the variant allele was significantly associated with disease susceptibility, conferring a higher risk of SLE development. This variant allele was more prevalent in patients below 30 years, patients with anemia and in patients with anti-double-stranded DNA (dsDNA), confirming the essential role of ATG-5 polymorphism in the susceptibility of Egyptian patients to SLE.

Effect of ciprofloxacin and N-acetylcysteine on bacterial adherence and biofilm formation on ureteral stent surfaces.

The aim of this study was to evaluate the effect of ciprofloxacin (CIP), N-acetylcysteine (NAC) alone and in combination on biofilm production and pre-formed mature biofilms on ureteral stent surfaces. Two strains each of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebseilla pneumoniae, Pseudomonas aeruginosa and Proteus vulgaris, recently isolated from patients undergoing ureteral stent removal and shown to be capable of biofilm production, were used in this study. The inhibitory effects of ciprofloxacin, N-acetylcysteine and ciprofloxacin/N-acetylcysteine combination were determined by static adherence assay. Ciprofloxacin (MIC and 2 MIC) and N-acetylcysteine (2 and 4 mg/ml) inhibited biofilm production by > or = 60% in all tested microorganisms. Disruption of pre-formed biofilms of all tested microorganisms was found to be > or = 78% in the presence of ciprofloxacin (MIC and 2 MIC) and > or = 62% in the presence of N-acetylcysteine (2 and 4 mg/ml), compared to controls. Ciprofloxacin/N-acetylcysteine showed the highest inhibitory effect on biofilm production (94-100%) and the highest disruptive effect on the pre-formed biofilms (86-100%) in comparison to controls. N-acetylcysteine was found to increase the therapeutic efficacy of ciprofloxacin by degrading the extracellular polysaccharide matrix of biofilms. These data are statistically significant. The inhibitory effects of ciprofloxacin and N-acetylcysteine on biofilm production were also verified by scanning electron microscope (SEM). In conclusion, Ciprofloxacin/N-acetylcysteine combinations have the highest inhibitory effect on biofilm production and the highest ability to eradicate pre-formed mature biofilms.

HLA-B*08 Carry a Risk for Type 1 Diabetes among Cow's Milk Exposed Egyptian Infants and Unmarked Linkage Disequilibrium with DR3-DQA1*05-DQB1*02 Haplotype.

Type 1 diabetes (T1D) is an autoimmune disease associated with multiple genetics and environmental factors. The aim of the study is to determine the frequency of HLA-B*08 and HLA-B*39 and its linkage disequilibrium with common risk haplotypes DR3-DQA1*05-DQB1*02, and DR4-DQA1-03-DQB1*0302 among T1D Egyptian infants. And assess different environmental factors as early exposure to cow's milk, exclusive breast feeding, mode of delivery and low birth weight. Sixty eight diabetic infants and 120 healthy controls were studied. HLA-DQB1, and DQA1 alleles were identified using homogeneous PCR and oligonucleotide hybridization assays. HLA-B*08 and HLA-B*39 genes were identified using multiplex PCR. The results showed that early exposure to cow's milk before 6 months carry a significant risk for T1D (16% in patients versus 6.6%in control group, P value=0.03). HLA-B*08 frequency was significantly higher among T1D infants than in control group (14.5% in diabetic infants versus 5%in control group, P value=0.024). DR3-DQA1*05-DQB1*02, and DR4-DQA1-03-DQB1*0302 were significantly higher in diabetic infants than controls (P value < 0.001 and 0.004 respectively). HLA-B*08 gene was found in (15.5%) of DR3-DQA1*05-DQB1*02 positive cases while in control group it was found in (13.5%) (P value=0.8). In conclusion, HLA-B*08 gene carry a risk for T1D in Egyptian infants, while DR3-DQA1*05-DQB1*02 haplotype lacks linkage disequilibrium with HLA-B*08 among T1D infants. Further studies are needed to determine which HLA-B gene is strongly linked to DR3-DQA1*05-DQB1*02 haplotype in T1D infants other than HLA-B*08 and HLA-B*18.

Plasma Levels of Interleukin-35 and its Association with Clinical Features of Breast Cancer Patients at Assiut University Hospitals.

Interleukin-35 (IL-35), is a recently identified cytokine that belongs to the IL-12 family, it is a potent anti-inflammatory and immunosuppressive cytokine which was first recognized to be produced by regulatory T cells (Tregs) cells, and recently was found to be produced by regulatory B cells (Bregs). The study aimed at determining whether plasma levels of IL-35 are associated with clinical characteristics of breast cancer (BC) patients. The study included 40 patients with breast cancer (BC), and 10 matched controls. The IL-35 cytokine was measured in plasma using ELISA. Results showed that plasma IL-35levels were significantly higher in BC than healthy controls (P˂ 0.05), and were significantly associated with BC grade 2 and HER-2 over expression level "3+", suggesting that plasma IL-35 levels may be associated with the development and progression of BC.

Divergent susceptibilities to AAV-SaCas9-gRNA vector-mediated genome-editing in a single-cell-derived cell population.

OBJECTIVE: Recombinant adeno-associated virus (AAV)-based vectors are characterized by their robust and safe transgene delivery. The CRISPR/Cas9 and guide RNA (gRNA) system present a promising genome-editing platform, and a recent development of a shorter Cas9 enzyme from Staphylococcus aureus (SaCas9) allows generation of high titer single AAV vectors which carry both saCas9- and gRNA-expression cassettes. Here, we used two AAV-SaCas9 vectors with distinct GFP-targeted gRNA sequences and determined the impact of AAV-SaCas9-gRNA vector treatment in a single cell clone carrying a GFP-expression cassette. RESULTS: Our results showed comparable GFP knockout efficiencies (40-50%) upon a single low-dose infection. Three consecutive transductions of 25-fold higher doses of vectors showed 80% GFP knockout efficiency. To analyze the "AAV-SaCas9-resistant cell population", we sorted the residual GFP-positive cells and assessed their permissiveness to super-infection with two AAV-Cas9-GFP vectors. We found the sorted cells were significantly more resistant to the GFP knockout mediated by the same AAV vector, but not by the other GFP-targeted AAV vector. Our data therefore demonstrate highly efficient genome-editing by the AAV-SaCas9-gRNA vector system. Differential susceptibilities of single cell-derived cells to the AAV-SaCas9-gRNA-mediated genome editing may represent a formidable barrier to achieve 100% genome editing efficiency by this vector system.

Extra-hepatic infection of hepatitis C virus in the colon tissue and its relationship with hepatitis C virus pathogenesis.

Extra-hepatic compartments might contribute to hepatitis C virus (HCV) persistence and extra-hepatic manifestations. Therefore, we investigated HCV infection in colonic tissue in patients with chronic hepatitis C (CHC) and its relationship with HCV pathogenesis. Colonic biopsies were collected from three groups with CHC infection: treatment naïve (TN; n=12), non-responders (NR; n=10) to anti-HCV therapy (pegylated interferon-α and ribavirin) and sustained virologic response (SVR; n=10) and from a fourth healthy control group (n=10). Liver biopsies were examined to assess inflammation and fibrosis. HCV infection and colonic T regulatory (Treg) frequency were detected by immunohistochemistry. HCV core and NS3 proteins were detected in B cells and macrophage/monocytes of 42 % and 25 % of TN and 50 % and 30 % of NR, respectively, but not in SVR or control group. The numbers of cells expressing HCV proteins were positively correlated with both HCV viral load and colonic Treg frequency. A significant negative correlation between HCV-expressing cells with both liver inflammation and fibrosis was identified. Our study provides evidence that HCV can infect B cells and macrophages of the colon. The correlations between HCV infection in colonic tissue and HCV viral load and liver pathology underline the significance of this extra-hepatic infection in HCV pathogenesis and response to therapy.

Is Foxp3 a good marker for regulatory T cells?

To track the changes in the tested Treg markers especially Foxp3 following activation to determine whether data of human studies using Foxp3 in evaluation of Tregs are reliable or not. Four-colour flow cytometry analysis was carried out to calculate the percentages of Tregs before and after lymphocyte activation. Foxp3 expression by CD4(+)CD25(+)* and CD4(+)CD25(high) T cells increased after T cell activation. A moderate negative correlation was observed between the percentage of each of CD4(+)CD25(+)Foxp3(+)IL10(+) or CD4(+)CD25(high) Foxp3(+)IL10(+) T cells and the percentage of CD4(+)CD25(+) T cells "after activation" and a weak negative correlation was similarly observed between the percentage of CD4(+)CD25(-)Foxp3(+)IL10(+) T cells and the percentage of CD4(+)CD25(+) T cells "after activation". A moderate negative correlation was observed between the percentage of each of CD4(+)CD25(+)Foxp3(+)IL10(+), CD4(+)CD25(high)Foxp3(+)IL10(+) or CD4(+)CD25(-) Foxp3(+)lL10(+) T cells and the percentage of CD4(+)CD25(high) T cells "after activation". CD4(+)CD25(high) T cell subpopulation expressed a significantly higher level of intracellular Foxp3 compared with CD4(+)CD25(low) and CD4(+)CD25(-) T cells subpopulations. In conclusions, Foxp3 is a good marker of Tregs especially if panels of markers were used for their identification. CD4(+)CD25(high) Foxp3(+) T cell subpopulation mostly represents Tregs and thus should be the one targeted in Treg studies.

Relations of regulatory T cells with hepatitis markers in chronic hepatitis B virus infection.

To assess regulatory T cells (Treg) in chronic hepatitis B (CHB) infected patients and to evaluate the presence of a possible relation between them and hepatitis B markers, flow cytometry analysis was carried out to calculate the percentages of Tregs, Tregs secreting IL-10 and CD4(+) T cells secreting interferon-γ (IFN-γ) and enzyme-linked immunosorbent assay was used to detect hepatitis B virus (HBV) markers in 59 patients and 32 healthy controls. CD4(+)CD25(+), CD4(+)CD25(+)Foxp3(+), CD4(+)D25(high), CD4(+)CD25(high)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) T cells and Treg cells secreting IL-10 were higher in CHB patients than in healthy controls. CD4(+)CD25(+), CD4(+)CD25(-), and total CD4(+)T cells secreting IFN-γ were generally lower in CHB patients than in healthy controls. Fair correlations were observed between CD4(+)CD25(+)Foxp3(+) T cells and alanine aminotransferase (ALT) levels and between HBsAb and both CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(high)Foxp3(+) T cells. CD4(+)CD25(+) T cells were significantly higher in CHB virus infected patients positive for HBeAg than in those negative for HBeAg and a good correlation was observed between CD4(+)CD25(+) T cells and HBeAg. Fair negative correlations were observed between CD4(+)CD25(high) T cells and both HBeAb and HBcAb. These data suggest that Tregs contribute to viral persistence. It was not possible to say that Tregs were the cause of immune suppression in this group of patients.

Chlamydia trachomatis: methods of identification and impact on semen quality.

Chlamydia trachomatis infection is considered to be one of the most common sexually transmitted diseases. It is currently unclear whether chlamydial infection causes pathological conditions of the male accessory glands has consequences for male infertility. To determine the frequency of C. trachomatis Infection among infertile men with leukocytospermia using different diagnostic techniques such as the detection of secretory IgA antibodies (Abs) in seminal plasma by enzyme-linked immunosorbent assay (ELISA), plasmid DNA by polymerase chain reaction (PCR) and direct detection of elementary body by flowcytometric analysis in seminal fluid. To assess the relationship between C. trachomatis infection and semen quality hence male infertility. Seventy five infertile male patients with leukocytospermia and 25 apparently healthy age matched fertile men were included as controls. Routine semen analysis and LeucoScreen test were done for each patient and control. Detection of C. trachomatis secretory IgA in seminal plasma by ELISA and detection of plasmid DNA by PCR and elementary body by flowcytometric analysis in semen samples were performed. Primary and secondary infertility were detected in 55 (73.3%) and 20 (26.7%) of patients, respectively. Sperm concentration and sperm motility (A+B) were statistically significant lower in patients with leucocytospermia than control group (P < 0.0001). Sperm concentration in patients with pus cells more than 3 x 10(6)/ml was statistically significant lower than those with pus cells less than 2 x 10(6) /ml. ELISA-detected IgA Abs against C. trachomatis in patients seminal plasma were positive in 20 (26.7%) and equivocal in 5 (6.6%) patients. Flowcytometric analysis of semen sample for C. trachomatis was positive in 35 (46.6%) patients and C. trachomatis plasmid DNA detection by PCR was positive in 23 (30.7%) patients. In conclusions, Detection of C. trachomatis antibodies of IgA type by ELISA in seminal plasma appears to be as specific as PCR in diagnosis of C. trachomatis in seminal fluid. High detection rate of C. trachomatis by flowcytometry was observed. Concerning the effect of C. trachomatis on routine semen characteristics, no significant obvious changes could be detected. Further studies for the assessment of sperm viability and DNA integrity are recommended.