Association between Toll-Like Receptor 3 (TLR3) rs3775290, TLR7 rs179008, TLR9 rs352140 and Chronic HCV.

BACKGROUND: Inconsistent results were reported on the association of TLRs polymorphisms with the risk of HCV infection and HCV-related diseases. OBJECTIVE: to assess the relation between TLR3 rs3775290, TLR7 rs17900 and TLR9 rs352140 SNPs and chronic HCV in the Egyptian cohort and to study their relation to interferon response. METHODS: TLR3 rs3775290, TLR7 rs179008 and TLR9 rs352140 gene polymorphisms were typed by RFLP for 100 patients with chronic HCV and 25 with HCC in addition to 100 healthy controls. RESULTS: A significant higher frequency has been found for the CT genotype of TLR3 rs3775290 in chronic HCV infection (p < 0.001) and CC genotype and the combined genotype CC-AT-GA ♀ in controls (p < 0.001). Non-significant associations have been found for studied SNPs and HCC and response to interferon and also the viral load or the degree of fibrosis, however, the higher HCV viral load and the higher grade of fibrosis were associated with treatment failure (p < 0.001). CONCLUSION: The heterozygous CT genotype of TLR3 rs3775290 may be a susceptibility risk factor for chronic HCV infection and the homozygous CC and the combined CC-AT-GA ♀ genotypes may be protective. The HCV viral load and the grades of liver fibrosis could be considered a risk factor for interferon treatment failure. It seems that the studied SNPs have no role in HCC development or failure of treatment. However, the small sample size is a limiting factor of the present study when interpreting the negative associations and that the current used cohort does not permit such conclusion. ABBREVIATIONS: cHCV=chronic Hepatitis C virus, HCC=hepatocellular carcinoma, TLR=Toll like Receptor, RFLP=Restriction Fragment Length Polymorphism, SNP=Single Nucleotide Polymorphism, IFN-α= interferon alpha.

HLA-class II alleles in Egyptian patients with hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) is linked to environmental, dietary, and life style factors. Its incidence and distribution vary widely among ethnic groups, sex, and geographic regions. HBV and HCV Infection, liver cirrhosis, male gender, and old age are important risk factors of HCC. Variability in outcome following exposure, and the clustering of HCC within families raise the possibility that genetic factors are also involved in susceptibility to HCC. The Major Histocompatibility Complex (MHC) plays a key role in anti-virus and tumor defense. HLA polymorphism is implicated in conferring genetic susceptibility to a large number of immune-mediated diseases, including some cancers. The association between HLA class II antigen and HCC in different ethnic populations that has been reported is controversial. Therefore, the aim of this work was to study the association between HLA class II-DRB1 and DQB1 polymorphism and HCC in Egyptian patients and to investigate their role as risk factors for the development of HCC. METHODS: HLA-class II (DRB1 and DQB1) typing was done by SSP for 100 subjects; 50 patients suffering from HCC (45 males and 5 females) with age range 40-64 years (51.16 years (y) +/- 6.16); and 50 normal healthy control subjects. RESULTS: 1. A significantly increased frequency of DRB1*04, and DQB1 *02 in HCC patients versus control group (p = 0.016, and 0.032, respectively) was found; 2. A significantly decreased frequency of DQB1*06 (p = 0.032) was found; 3. A significantly increased frequency of DRB1*07 (odds ratio (OR) = 4.929) was found; and 4. A significantly decreased frequency of DRB1*15 (OR = 0.316) was seen. In conclusion, while some alleles are significantly associated with HCC (DRB1*04, DQB1*02) and others are not associated (DQB1*06); therefore, it can be concluded that the DRB1*04 and DQB1*02 alleles might be risk factors for the occurrence of HCC (OR = 4.373 and 3.807, respectively), and DQB1*06 may be a protective allele (OR = 0.259).

IRF5, PTPN22, CD28, IL2RA, KIF5A, BLK and TNFAIP3 genes polymorphisms and lupus susceptibility in a cohort from the Egypt Delta; relation to other ethnic groups.

OBJECTIVE: To replicate a single nucleotide polymorphism (SNP) of known genes for lupus (IRF5 rs10488631, PTPN22 rs2476601, BLK rs2736340 and TNFAIP3 rs5029939) and other autoimmune diseases (CD28 rs1980422, IL2RA rs2104286 and KIF5A rs1678542) on a newly studied Egyptian cohort to investigate the genetic disparity with different studied ethnic groups in relation to lupus susceptibility. METHODS: 170 Egyptian patients from Egypt Delta with SLE and 241 matched healthy controls were genotyped by Taqman real time PCR for the selected SNPs. RESULTS: The results revealed significant association with IRF5 (p<0.0001) and PTPN22 (p=0.008) and insignificant association with KIF5A, CD28, IL2RA, BLK and TNFAIP3 genes. CONCLUSIONS: This study may provide an additional evidence for the association between IRF5 and PTPN22 and lupus susceptibility and may exclude it for CD28, IL2RA, and KIF5A.

HLA-DQB1* alleles and genetic susceptibility to type 1 diabetes mellitus.

AIM: To determine human leukocyte antigen (HLA)-DQB1 allele association with susceptibility to type 1 diabetes (T1D) and to clinical and laboratory findings. METHODS: This study was conducted on 85 unrelated Egyptian children with T1D recruited consecutively from the Pediatric Diabetes Endocrinology outpatients Clinic; Mansoura University Children's Hospital, Egypt. Patient mean follow up period was 2.5 years. Patients were subdivided according to level of HbA1c (optimal/suboptimal control < 8.5% and poor control ≥ 8.5%). The control group consisted of 113 unrelated age- and sex-matched healthy subjects without T1D or other autoimmune diseases. Genomic DNA extraction was done for all subjects using a DNA isolation kit. HLA-Class II-DQB1 allele typing was carried out with a polymerase chain reaction-sequence-specific oligonucleotide probe using a INNO-LiPA HLA-DQB1 update kit. RESULTS: Significant differences were detected between Egyptian patients with T1D and control groups in the frequencies of DQB1*02 [44.4% vs 18.6%, corrected P value (Pc) < 0.001] and DQB1*03 (41.2% vs 24.4%, Pc < 0.001). Significant differences were also observed between control groups and T1D patients in the frequencies of DQB1*05 (14.6% vs 7.2%, P = 0.029) and DQB1*06 (34.1% vs 7.2%, P < 0.001). However, after correction for multiple comparisons, the significance was retained for HLA-DQB1*06 (Pc < 0.001) but lost for HLA-DQB1*05. HLA-DQB1*0201, *0202, *030201 were positively associated with T1D (Pc = 0.014, Pc < 0.001, and Pc < 0.001 respectively), while HLA-DQB1*060101 was negatively associated (Pc < 0.001) with the condition. Although the HLA-DQB1 alleles 030101 and 050101 were significantly higher in controls (P = 0.016, P = 0.025 respectively), both of them lost statistical significance after correction of P value. The frequency of the HLA-DQB1 genotypes 02/02, 02/03, and 03/03 was higher in T1D patients, and the frequency of the genotypes 03/06, 05/06, and 06/06 was higher in controls, these differences being statistically significant before correction. After correction, the genotypes 02/02, 02/03 in T1D, and the genotypes 03/06, 06/06 in controls were still significant (Pc = 0.01, Pc < 0.001, Pc < 0.001, and Pc = 0.04, respectively). Non-significant associations were found between the frequency HLA-DQB1 alleles and genotypes in T1D in relation to the grade of diabetic control, Microalbuminuria, age, gender, age of presentation, weight, height, frequency of diabetic ketoacidosis (P = 0.42), serum cholesterol, and fasting and post-prandial level of C-peptide (P = 0.83, P = 0.9, respectively). CONCLUSION: The Current work suggests that HLA-DQB1 alleles *030201, *0202, *0201, and genotypes 02/03, 02/02 may be susceptibility risk factors for development of T1D in Egyptian children, while the HLA-DQB1*060101 allele, and 03/06, 06/06 genotypes may be protective factors. HLA-DQB1 alleles and genotypes do not contribute to microalbuminuria or grade of diabetic control.

Effect of genetic polymorphisms on the development of secondary failure to sulfonylurea in egyptian patients with type 2 diabetes.

OBJECTIVE: This study investigated the possibility that genetic factors, such as polymorphism of K inward rectifier subunit (Kir6.2), E23K, and Arg(972) polymorphism of insulin receptor sub-strate-1 (IRS-1), may predispose patients to sulfonylurea failure. METHODS: A total of 100 unrelated Egyptian patients with type 2 diabetes were recruited. They were divided into two equal groups: group I consisted of patients with secondary failure to sulfonylurea (hemoglobin A(1c) ≥ 8% despite sulfonylurea therapy) while group II consisted of patients whose condition was controlled with oral therapy. RESULTS: Of all the patients, 45% and 14% were carriers of the K allele and Arg(972) variants respectively. The frequency of the K allele was 34% among patients with diabetes that was controlled with oral therapy and 56% among patients with secondary failure to sulfonylurea. The frequency of the Arg(972) IRS-1 variant was 6% among patients with diabetes controlled with oral therapy and 22% among patients with secondary failure. CONCLUSION: The E23K variant of the Kir6.2 gene and Arg(972) IRS-1 variants are associated with increased risk for secondary failure to sulfonylurea.

Relation of Cag-A-positive Helicobacter pylori strain and some inflammatory markers in patients with ischemic heart diseases.

Recently, a potential link between infectious agents and athero-sclerosis has been suggested. H. pylori strains bearing the cytotoxin associated gene A (Cag-A) provoked a heightened inflammatory response in vivo and showed stronger relation to gastric complication of this infection. The association between Cag-A positive strain and vascular diseases producing conflicting results. So, the present study aimed to estimate the seroprevalence of H. pylori Cag-A positive strains as a risk factor among different groups of ischemic heart disease and to study its interaction with high sensitivity CRP (hs-CRP) and IL6 as inflammatory host responses. The present study was conducted on anti H. pylori IgG positive 60 ischemic heart disease (IHD) patients and 20 apparently healthy individuals as a control group. IHD patients were classified into 3 groups: (group I) with acute myocardial infarction, unstable angina pectoris patients (group II), chronic stable angina pectoris patients (group III). For all patients and control groups serum anti Cag-A IgG, IL6, hs-CRP, CK, CKMB, LDH, AST and Lipid profile were estimated. IL6 and hs-CRP levels were increased in groups I, II and III as compared with group IV (P < 0.001) with positive correlation between IL6 and hs-CRP in groups I, II and III (P < 0.05). The percentage of anti Cag-A positive cases was similar among the patient groups, but significantly higher than in the control group. Thus, infection with Cag-A positive H. pylori strain may play a role as a risk factor in development of ischemic heart diseases through provocation of high inflammatory response or through other mechanism. Therefore eradication of this infection is important as it is much less expensive than long term treatment of the other risk factors.

Vascular endothelial growth factor, p53, and the H-ras oncogene in Egyptian patients with bladder cancer.

AIM: To evaluate the relationship between vascular endothelial growth factor (VEGF), p53, and the H-ras oncogene and different clinicopathological parameters in Egyptian patients with Schistosoma-associated transitional cell carcinoma of the bladder. METHODS: The study included 50 patients with transitional cell carcinoma for whom radical cystectomy and urinary diversions were carried out. VEGF and p53 protein expressions were evaluated with an immunohistochemical staining method, and H-ras oncogene mutations were analyzed with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. RESULTS: High grade tumors revealed higher p53 immunostaining than low grade tumors (P = 0.016). p53 and VEGF protein expressions, as well as H-ras oncogene mutations, had an insignificant impact on patient outcomes (P = 0.962, P = 0.791, and P = 967, respectively). Cancer extension to regional lymph nodes was associated with poor outcomes (P = 0.008). CONCLUSION: VEGF, p53 and the H-ras oncogene have no relation to patient survival and outcome in Schistosoma-associated transitional cell carcinoma.

Transforming growth factor-beta 1 in diabetic nephropathy.

In diabetic nephropathy the extent of matrix accumulation in both glomeruli and the interstitium correlates strongly with the degree of renal insufficiency and proteinuria. Factors responsible for the deposition and accumulation of extra cellular matrix material within the kidney are therefore of considerable interest. Such factors include the potent fibrotic cytokine TGF-beta. We measured serum TGF-beta1 in patients with various stages of diabetic nephropathy, and correlated its level with different biochemical parameters. The study was conducted on: Group I: 30 patients with diabetic nephropathy (Subgroup IA: 20 patients with microalbuminuria; Subgroup IB: 10 patients with overt nephropathy), Group II: 19 diabetic patients without nephropathy (positive control), Group III: 20 healthy volunteers (negative control). Serum creatinine, Fasting and postprandial blood glucose, Fasting serum cholesterol, Glycated haemoglobin (HbA1c), Microalbumin estimation in urine, Serum TGF-beta1 estimation were done for all the studied groups. Our results showed a statistically significantly higher serum TGF-beta1 level in patients with diabetic nephropathy versus diabetic patients without nephropathy (mean +/- SD, 47.66 +/- 21.92 and 27.07 +/- 15.46 respectively) (P<0.001). Also in patients with diabetic nephropathy versus healthy controls (mean +/- SD, 47.66 +/- 21.29 and 27.05 +/- 8.95 respectively) (P<0.001). While serum TGF-beta1 concentrations were almost similar in diabetic patients without nephropathy and in healthy controls. Serum TGF-beta1 was statistically significantly higher in patients with overt nephropathy versus patients with microalbuminuria (mean+/-SD, 73.5 +/- 2.41 and 34.9 +/- 12.41) (P<0.001). Serum TGF-beta1 was significantly positively correlated with albumin excretion rate, fasting and postprandial blood glucose levels, serum cholesterol and HbA1c, these correlations were only found in diabetic patients with nephropathy but not in those without nephropathy or the control group. (r=0.86, P<0.001, r=0.444, P<0.05, r=0.375, P<0.05, r=0.532, P <0.01, r=0.696, P<0.001 respectively. HbA1c was found to be predictor of 68% of changes of serum TGF-beta1 (P<0.001) and serum cholesterol was predictor of 73% of changes of serum TGF-beta1 concentration (P<0.01). In conclusion, our results suggest that TGF-beta1 may play a key role in the development and progression of diabetic nephropathy. Accordingly, it may be also directly implicated in the functional deterioration of the kidney functions seen in patients with diabetic nephropathy, therefore beside proper glycemic control, strategies aiming at antagonizing TGF- beta1 for example by the use of specific antibodies or a specific inhibitor of TGF-beta1 may help to prevent the development or attenuate the progression of nephropathy in diabetic patients.

Proinflammatory cytokines (IL-12 and IL-18) in immune rheumatic diseases: relation with disease activity and autoantibodies production.

Interleukin-18 (IL-18) and its inducer IL-12 have multiple biological activities that are important in generating Th1 responses and inflammatory tissue damage. We investigated serum concentration of the novel proinflammatory Th1 cytokine; IL-18, and its inducer IL-12 in patients with immune rheumatic diseases. Group I comprised32 patients of systemic lupus erythmatosus (SLE), Group II comprised 36 patients of rheumatoid arthritis (RA). Group III comprised 9 patients (2 patients of Behcet, 2 patients of Dermatomyositis, 2 patients of Sicca syndrome, one patient of Scleroderma, and 2 patients of Mixed connective tissue disease). Group IV is a control group consists of 21 sex and age matched healthy subjects and correlated their levels with autoantibody concentration (ANA and ds-DNA), clinical grades and SLE disease activity index (SLEDAI). Serum IL-18, IL-12, ANA and ds-DNA were measured by enzyme immuno sorbent assay. IL-18, IL-12 and ANA were significantly higher in the three studied groups than in the control group (IL-18; P < 0.001 in the three groups, IL-12; P = 0.019, P = 0.002, and P = 0.006, and ANA; P < 0.001, P = 0.002,and P = 0.006, respectively).ds-DNA was significantly higher in SLE patients than in control group (P < 0.001). There were significant positive correlations between; A) levels of IL-18,and both ANA and ds-DNA in SLE patient (r = 0.41,P = 0.001, r = 0.58 and P=0.001 respectively); and B) IL-18 and ANA in both RA and group III patients (r = 0.32, P = 0.005, r = 0.61and P = 0.022 respectively). Also, there were significant positive correlation between the levels of IL-18 and clinical grades of the three groups (r = 0.60,P = 0.001, r = 0.79,P = 0.001, r = 0.78 and P= 0.001 respectively). In SLE patients , IL-18 concentration shows significant positive correlation with SLEDAI score (r = 0.76, P = 0.001). In conclusion, the elevation of proinflammatory cytokines (IL-18 and IL-12 ) may trigger the inflammatory process in immune rheumatic diseases and IL-18 is correlated with disease activity

IL-13 gene expression in patients with atopic dermatitis: relation to IgE level and to disease severity.

Atopic dermatitis (AD) is a chronic inflammatory skin disease frequently associated with an increased serum IgE level. T helper cells are thought to play an important role in the pathogenesis of AD. It is commonly believed that allergens activate Th2 cells, and it is likely that the cytokines produced by Th2 cells are crucial factors in the induction and maintenance of the disease. IL-13 is one of the cytokines that are produced by Th2 lymphocytes and, like IL-4, it can induce the production of IgE. In order to evaluate its role in the pathogenesis of AD, IL-13 mRNA expression was studied in peripheral blood of patients with different degrees of AD and compared with healthy subjects. Also, we correlated its level of expression with the level of serum IgE and with the severity of the disease. EDTA blood was obtained from 25 patients (divided into three groups ranged from mild to severe AD) and 12 normal subjects as a control group. We examined the blood sample for IL-13 mRNA expression using RNA extraction technique, RT-PCR, PCR amplification using primers specific for IL-13 and beta- actin (as internal control) this is followed by visualization of the expressed bands using gel electrophoresis and DNA marker. Serum IgE level was detected using an ELISA kit. Our results revealed that, IL-13 mRNA is significantly expressed in patients with AD as compared to normal control (P<0.001). IL-13 mRNA shows higher level of expression in severe AD group in comparison with both moderate and mild groups (P = 0.05). Serum levels of IgE showed highly significant increase in patients with AD as compared with the control group (p=0.019), its level is significantly higher in severe AD group versus moderate and mild AD groups (P=0.009 and 0.022, respectively). There is a highly significant positive correlation between serum levels of IgE and the levels of IL-13 mRNA expression in all AD groups (P=0.001). In conclusion, the high level of IL-18 mRNA expression in AD, and its correlation with serum level of IgE and with severity of disease indicates that IL-13 is involved in the pathogenesis of the disease and is an important in vivo IgE inducer.

Evaluation of natural killer cells as diagnostic markers of early onset neonatal sepsis: comparison with C-reactive protein and interleukin-8.

This study was conducted on thirty-seven neonates and healthy neonates (sixteen full term and fourteen preterm). The study aimed at revealing the role played by the NK cells in neonatal sepsis and evaluating the sensitivity of NK cell number and cytotoxicity as diagnostic markers in infants with suspected early neonatal sepsis compared with the circulating cytokine IL-8 and CRP levels. All samples of peripheral blood lymphocytes were subjected to determination of CD16 and CD56 positive cells using flow cytometry and NK cytotoxicity using the standard 4h 51Cr release assay. Sera were separated to measure IL-8 using ELISA. Determination of CRP, using turbdimetric assay, as well as blood cultures were done for patient's group only. Out of the 37 cases of suspected early neonatal sepsis, 16 were given final diagnosis of sepsis. Seven infants (43.8%) in the sepsis group had culture-proven diagnosis, one of which had meningitis. The median CRP value was significantly higher in sepsis group (88 mg/L; range: 17-159 mg/L) compared with that in non-septic group (15.4 mg/L; range: 7.6-23.2 mg/L, p < 0.001) only 12-60 h after admission. On the other hand, newborns in the sepsis group had significantly higher serum levels of IL-8 (median 310 pg/mL; range: 37-583 pg/ml) at study entry than that in the non septic group (median 63 pg/mL; range: 32-94 pg/ml, P < 0.001). On admission, the NK activity, rather than the number of CD16 and CD56 positive cells was much affected where NK cytotoxicity was significantly lower in sepsis group (3.4 +/- 2.1%, range 0.9-7%) than that of the nonseptic group (18.3 +/- 6.7%: range 10.7- 25.3%, p < 0.01) and healthy neonates (23.8 +/- 4.7%: range 12.2-32.3%, p < 0.001). We may conclude that defective NK cell activity rather than NK cell number plays an important role in susceptibility to early onset neonatal sepsis. Evaluation of NK cytotoxicity as a marker in early diagnosis of neonatal sepsis reveals that the sensitivity, specificity and predictive values of reduced NK cytotoxicity (10% killing) was higher than both of CRP and IL-8, either individually or in combination. Additionally, reduced NK cytotoxicity showed high correlation with the severity and outcome of neonatal sepsis. Our data raise the possibility that the addition of NK cell activity to the standard work-up of critically ill patients with suspected sepsis could increase diagnostic certainty and generate an improved patient management.