Aberrant cytokine and VCAM-1 expression in patients with viral and non-viral related liver cirrhosis.

The study aim was to compare the alterations in the expression levels of proinflammatory and chemotactic cytokines as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-17A and IL-8, the down regulatory cytokine IL-10, in addition to the vascular cell adhesion molecule-1 (VCAM-1) gene in different groups of patients with cirrhosis due to various etiologies. This case-control study included 84 patients suffering from cirrhosis of viral and non-viral etiologies and 20 sex and age-matched healthy controls. All patients were subjected to detailed history taking, clinical examination, and liver function assessment. The expression levels of TNF-α, IL-17A, IL-8, IL-10, and VCAM-1 were assessed in peripheral blood mononuclear cells by real-time PCR. Patients with cirrhosis showed marked changes in the tested gene expression levels relative to the control group. Higher expression levels of all genes except IL-10 were seen in patients of the viral than in the non-viral groups. Most of the significant correlations of liver function parameters were observed with TNF-α in both the viral and non-viral groups, followed by IL-17A. Increased TNF-α and IL-17A presented potential risk factors for disease progression to cirrhosis of Child class C.

Regulatory T Cells and IL35 in Chronic Hepatitis C Related Cirrhosis and Hepatocellular Carcinoma.

Regulatory T cells (Tregs) play a fundamental role in maintaining immune homeostasis to balance between the tissue-damaging and protective effects of the immune response. There are strong evidences that Treg cells and their cytokines may play an important role in the induction of tolerance in the liver and progression of HCV infection. Herein, we investigated the frequency of Treg cells and interleukin 35 (IL-35) level in blood and their potential relationship to the various chronic hepatitis C (CHC) complications and outcomes. A total of 36 HCV infected patients subdivided into, CHC complicated with cirrhosis (HCV LC; n = 18), CHC complicated with hepatocellular carcinoma (HCV- HCC; n=18) and apparently healthy control group (n=18) were enrolled in this study. Treg cells percentages were determined by flow cytometric analysis and ELISA was used to measure IL35 serum levels. A significant increase in the frequency of peripheral Tregs and serum IL35 level was found in HCV HCC, and HCV LC groups compared with the control group. The frequency of peripheral Tregs and plasma (IL-35) levels were significantly positively correlated with viral load along with disease progression. We conclude that the higher percentage of Tregs and IL35 level in peripheral blood of HCV HCC and HCV LC groups compared to the control group may suggest their contribution to viral persistence and progression of HCV infection.

Concordance between peripheral and decidual NK cell subsets and killer immunoglobulin-like receptors in women with recurrent spontaneous miscarriages.

BACKGROUND: The role of decidual natural killer (dNK) cells in normal and complicated pregnancy and their relation with peripheral NK (pNK) cells remains unclear. The study aim was phenotypic analysis of pNK and dNK cells at time of miscarriage in recurrent spontaneous miscarriage (RSM) patients to assess whether measuring levels of pNK cell populations can reflect changes in dNK cells or not. METHODS: This study included 40 middle aged pregnant women in the 1st trimester subjected to evacuation because of a current miscarriage. They had a history of previous ≥ two unexplained miscarriages. Frequencies of pNK and dNK cells, based on the expression of CD56, CD16, inhibitory (CD158b) and activating (CD161) Killer immunoglobulin-like receptors (KIRs), were detected by flow cytometry. RESULTS: Percentages of CD56+ NK cells in peripheral blood and decidua were 17.5 % and 17.3 %, respectively. In both blood and decidua, CD56dim NK cells were exceeding CD56bright NK cells. The CD56dim CD16- NK cells were the predominating subset of NK cells, followed by CD56dim CD16dim. No substantial differences were detected in the levels of KIRs expression by the different NK subsets between blood and decidua. Abnormal up-regulation of both CD161 and CD158b on NK cells was observed in blood and decidua. CONCLUSION: At the time of miscarriage, patients with RSM have an extremely active immune system and an increased number of toxic NK cells both in blood and decidua. The pNK cells reflect dNK cell changes during miscarriage and may be a useful non-invasive predicting tool in reproductive failure setting.

Genetic Polymorphism and Serum Levels of RORc2 in Rheumatoid Arthritis.

RORc2 is the master transcription factor of T helper 17 cells. We aimed to evaluate whether RORc2 genetic polymorphism and serum levels have association with the risk and activity of rheumatoid arthritis (RA). RORC genetic polymorphisms were investigated by real time PCR. Serum RORc2 protein levels were determined by enzyme-linked immunosorbent assay. Protective effects of rs370515 CT, rs370515 CT + TT, rs3828057 CT, rs3828057 CT+TT and rs9826 GG genotypes were detected. The genotype-phenotype analysis showed no significant differences in the disease activity score 28 (DAS 28) under the recessive versus dominant genotypes. RORc2 protein serum levels were significantly higher in RA patients than controls (P= 0.001) and had a positive correlation with DAS-28. In conclusions, RORC genetic polymorphisms correlate with the risk but not activity of RA, whereas RORc2 serum levels have a positive correlation with both risk and activity of RA.

Occult Hepatitis B Infection Among Blood Donors in Al Azhar University Hospital, Upper Egypt: The Current Status After 25 years of Vaccine Introduction.

Occult hepatitis B virus infection (OBI) is a challenging topic with much debate regarding its clinical and virological relevance. The reliance on anti HBc seropositivity for diagnosis of OBI is still controversial. We aimed to determine the frequency of OBI among Egyptian blood donors, the role of anti HBc and HBe Ag as predictors of OBI and the possible risk factors. A total of 300 randomly selected hepatitis B surface antigen (HBsAg) negative blood donors at the Blood Bank of Al Azhar University Hospital in Assiut were included. Measurement of liver enzymes and screening for HBV core antibodies (anti HBc) and Hepatitis e antigen (HbeAg) were done. Quantitative HBV DNA measurement was achieved by real time polymerase chain reaction with a detection limit of 20 IU/mL after DNA extraction from the peripheral blood mononuclear cells (PBMC). The prevalence of OBI was 3.7% (11/300) among the donors and the majority had low level viremia (63.6%). Anti HBc was detected in 20.7% (62/300) (group I). OBI was detected in 14.5% (9/62) in group I compared to 0.84% (2/238) in the anti-HBc Ab negative donors (group II). Anti HBc had a sensitivity and specificity of nearly 82% for detection of OBI with a high negative predictive value (99.16%). HBe Ag was detected in only 1.6% (1/62) of group I. There were no statistical significant differences regarding the liver enzymes, demographic data or risk factors between group I and II and even between cases of OBI and those without. We conclude that OBI exists in an alarming percentage among Egyptian blood donors. Anti HBc should be introduced in the routine blood screening. Negative anti HBc results ensures safe blood, while positive results need nucleic acid confirmation especially if given to high risk recipients. More in-depth evaluation of the immunization program is needed.

Role of T-Helper 9 Cells in Chronic Hepatitis C-Infected Patients.

Hepatitis C virus is a hepatotropic virus that is transmitted parenterally. Viral infections are usually associated with modulations of the immune cells, leading to enhanced viral survival and spreading, and accordingly, life-threatening complications. Recently, it has been proposed that a new subset of T-helper, named T-helper 9, is involved in the pathogenesis of different immunopathological conditions, such as allergies, tumors, and viral infections. Some studies reported a protective role, and others described a pathogenic potential for the T-helper 9 cells. Here, we present evidence that T-helper 9 cells are dynamically increased with increasing the pathogenic strategy for hepatitis C virus (HCV). Furthermore, viral clearance is associated with a decrease in T-helper 9. The increase in T-helper 9 was paralleled with an increase in its receptor expression. Taken together, our data suggest that T-helper 9 cells play an important role in the pathogenesis of HCV, and is directly associated with HCV-related complications.

The efficacy of hepatitis B vaccination program in upper Egypt: Flow cytometry and the evaluation of long term immunogenicity.

Anti-HBs levels wanes with time. Many studies discussed the B cell response to HBV vaccine. However, the data about memory T cell response are limited. To evaluate the efficacy of hepatitis B vaccine via evaluating anti-HBs levels and HBsAg specific memory T-lymphocytes through descriptive study. The study was conducted in a tertiary care setting. This study included 440 vaccinated persons during infancy. Group I: 6 to less than 10 years old; Group II: 10 to less than 14 years old; Group III: 14 to less than 17 years old; Group IV: 17 years old. The serum samples were screened for HBV markers. Cytokines secretion by HBsAg-specific memory CD45RO(+) CD4(+) T cells was measured after in vitro culture using flow cytometry. The mean titer of anti-HBs was higher in group I in comparison to others (P-value = 0.000 for each). IFN-γ and IL-4 secreted by memory CD4(+) T cells were positive in all with anti-HBs >100 mIU/ml, while positive in 87% and 75% of participants with anti-HBs <10 mIU/ml and positive in 73% and 32% of participants with absent anti-HBs. The percentage of cells secreting IFN-γ and those secreting IL-4 were higher among participants with serum anti-HBs >100 mIU/ml than those having <10 mIU/ml or absent (P < 0.001 for each). Anti-HBs positivity decreased with time since childhood vaccination. Breakthrough infections are rare in vaccinated persons. Hepatitis-B vaccine is efficient in controlling HBV infection. Flow cytometry is a useful tool to assess the long term persistence of T cell memory after childhood vaccination. J. Med. Virol. 88:1567-1575, 2016. © 2016 Wiley Periodicals, Inc.

Impact of common functional polymorphisms in renin angiotensin system genes on the risk of renal parenchymal scarring following childhood urinary tract infection.

BACKGROUND: Pathogenesis of renal parenchymal scarring (RPS) after acute pyelonephritis (APN) is unclear. The risk of RPS varies markedly among individuals, suggesting a genetic role. OBJECTIVE: To investigate a possible role of common polymorphisms in renin angiotensin system genes in APN-associated RPS in children. PATIENTS AND METHODS: This study included 104 APN children and 300 controls. APN was diagnosed by urine culture and typical findings on 99Tc-DMSA scans. Voiding cystourethrogram tested the presence of vesicoureteral reflux (VUR). Follow-up DMSA scans were performed 4-6 months later to identify new RPS. Angiotensin converting enzyme gene I/D, angiotensin II receptor type-1 A1166C and angiotensinogen M235T polymorphisms were genotyped. RESULTS: New RPS developed in 44.2% (46/104) of children with APN. VUR was diagnosed in 35.6% (37/104) of APN cases. RPS developed in 73% of cases of VUR. The D allele of ACE gene I/D polymorphism was significantly more common in APN cases with RPS (73.91%) than non-RPS (58.6%) and controls (54.5%) (p = 0.021, p = 0.002, respectively). The AGTR-1 A1166C A allele was significantly more common in VUR than the non-reflux children (91.9% versus 76.1%; p = 0.005). VUR, in contrast to the D allele (OR 6.1, 95% CI 0.878-19.7; p = 0.05), was an independent risk factor for RPS. DISCUSSION: ACE gene D allele is associated with a twofold increase in RPS risk, which could be a result of a functional effect to increase tissue levels and activity of ACE during APN. However, D allele failed to qualify as an independent risk and its RPS association could be dependent on other co-factors, such as TGFß1 activation, or the D-allele might link with recently discovered functional polymorphisms at the 5' end of the ACE gene. Although VUR is an independent risk for RPS, it is not clear whether this is due to exposure of the kidneys to infected urine, or VUR-associated dysplasia. In contrast with published literature, we noted higher rates of RPS and high-grade VUR, suggesting a more aggressive VUR course or local unawareness of APN. Our study has its limitations; the small number of VUR children, and the clinical and ethical difficulties of testing VCUG and DMSA in controls. CONCLUSIONS: ACE gene D allele is associated with, but cannot independently predict, RPS in children. VUR is an independent risk for post-pyelonephritic scarring. AGTR-1 1166A/C polymorphism is associated with occurrence, but not progression, of VUR.

Regulatory T cell subsets in children with systemic lupus erythematosus.

The aim of this work was to quantify CD4(+)CD25(+)Foxp3(+) T cells (Tregs) in Egyptian children with SLE and to correlate these findings with their disease activity scores and drug therapy. We enrolled 37 Egyptian children with active SLE. Disease activity was assessed by measuring serum levels of anti-dsDNA antibody and by the SLEDAI scores. Twenty healthy children were also enrolled as normal controls. The CD4+CD25+, CD4+CD25(bright), and CD4+CD25(dim) cells in patients were significantly increased in comparison to controls. There was no significant difference in the Foxp3 gated on CD4+CD25(bright) and CD4+CD25(dim), but there was a significant increase when gated on CD4+CD25- and whole CD4+ cells in patients than controls. There was no significant difference among patients with different degrees of activity on different lines of treatments and their outcomes as regards all studied values. There was no significant correlation between SLEDAI score and any of the studied parameters except for a significant negative correlation with gated lymphocytes. There is increased expression of Foxp3 in CD4+ T cells mostly CD25- in Egyptian children with active SLE under corticosteroid treatment regardless of disease activity.

Histological study on effect of mesenchymal stem cell therapy on experimental renal injury induced by ischemia/reperfusion in male albino rat.

BACKGROUND AND OBJECTIVES: Acute kidney injury (AKI) represents a major clinical problem with high mortality and limited treatment protocols. This study was planned to evaluate the therapeutic effectiveness of bone marrow - derived mesenchymal stem cells (BM-MSCs) in a rat model of ischemia/reperfusion (I/R) AKI. METHODS AND RESULTS: This study was carried out on thirty adult male albino rats. Animals were divided equally into three groups. Group I (control sham-operated group) (n=10), were subdivided equally into two subgroups; Ia and Ib. The experimental group (n=20) were all subjected to I/R injury by clamping both renal pedicles for 40 minutes. Half of the I/R animals did not receive MSC therapy (group II) [non-MSC treated group]. The other half of the I/R animals received single intravenous injection of PKH26 labelled BM-MSCs immediately after removal of the clamps and visual confirmation of reflow (group III) [MSC treated group]. Animals were sacrificed 24 hrs (subgroups IIa & IIIa) and 72 hrs (subgroups IIb & IIIb) after intervention. Serological measurements included serum urea and creatinine. Kidney specimens were processed for H&E, PAS and PCNA. Mean % of renal corpuscles with affected glomeruli, mean % of affected tubules, mean area % of PAS-positive reaction and mean area % of PCNA immunoreactivity were measured by histomorphometric studies and statistically compared. MSCs-treated group exhibited protection against renal injury serologically and histologically. CONCLUSIONS: Results of the present study suggest a potential reno-protective capacity of MSCs which could be of considerable therapeutic promise for cell-based management of clinical AKI.

Influence of dipyridamole and its combination with NO donor or NO synthase inhibitor on adjuvant arthritis.

The anti-arthritic and anti-inflammatory effects of dipyridamole and the possible involvement of NO in the dipyridamole action are not yet clear. The aim of this study was to evaluate the effects of dipyridamole alone and in combination with either the nitric oxide donor, sodium nitroprusside (SNP) or the non-selective nitric oxide synthase inhibitor, L-NG- monomethyl arginine (L-NMMA), on pathogenesis of adjuvant-induced arthritis model in rats. The results of the present work showed that prophylactic administration of dipyridamole alone and dipyridamole administration in combination with either low dose of SNP or L-NMMA significantly ameliorated pathogenesis of adjuvant arthritis in rats as evidenced by significant decrease in arthritis index, hind paws volume, loss of body weight, hyperalgesia compared with control vehicle (1% DMSO) treated adjuvant arthritic rats. Inflammatory cellular infiltrate in synovium of ankle joint and pannus formation were also markedly inhibited. Interleukin-10(IL-10) levels were significantly increased in these groups of animals. In contrast, a high dose of SNP counteracted the anti-inflammatory and anti-arthritic effects of dipyridamole. The inhibitory effect of therapeutic administration of dipyridamole alone on adjuvant arthritis syndrome was not significantly different from that of vehicle administration. In conclusion, dipyridamole has prophylactic but not therapeutic anti-arthritic and anti-inflammatory effects that appear to be dependent on inhibition of NO synthase. A synergistic combination between dipyridamole and NO synthase inhibitor or low dose of NO donor may have prophylactic and therapeutic values in autoimmune diseases like RA.

Comparison of different phenotypic and molecular methods for detection of methicillin-resistant Staphylococcus aureus in intensive care patients.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) has been problematic ever since its discovery. This work was performed to evaluate the effectiveness of different phenotypic methods for MRSA detection in intensive care patients. Three hundred and eighty-nine specimens from 100 patients were inoculated onto mannitol salt agar. All S. aureus isolates were examined by four selective culture media [ORSAB, MSO, MSA-Cefox, OAS], two disc diffusion methods [CDD and ODD], and MicroScan panel for MRSA. Polymerase chain reaction (PCR) for mecA gene was performed as the gold standard. S. aureus isolates were revealed from 56 patients, 41 of them were found to be MRSA by PCR. CDD yielded the best sensitivity (97.6%), followed by ODD and MSA-Cefox (92.7%). CDD, MSA-Cefox and OAS showed the best specificity (100%). In conclusion, MSA-Cefox and CDD showed improved sensitivity and excellent specificity compared to other methods. It is advisable to use the two methods for MRSA detection if PCR is not available.

Enhancement of the anti-inflammatory and anti-arthritic effects of theophylline by a low dose of a nitric oxide donor or non-specific nitric oxide synthase inhibitor.

BACKGROUND AND PURPOSE: Although there are many new specific phosphodiesterase inhibitors with anti-inflammatory activity, none have yet reached the market because of their low therapeutic efficacy. Our study was aimed to evaluate the anti-inflammatory and anti-arthritic effect of an established phosphodiesterase inhibitor, theophylline, and to investigate the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP) or NO synthase inhibitor, L-N(G)-monomethyl arginine (L-NMMA) on its actions. EXPERIMENTAL APPROACH: The effects of theophylline alone and combined with SNP or L-NMMA on the pathogenesis of adjuvant-induced arthritis in rats were evaluated. KEY RESULTS: Prophylactic or therapeutic doses of theophylline significantly ameliorated the pathogenesis of adjuvant arthritis in rats as evidenced by a significant decrease in the arthritis index, hind paws volume, ankle joint diameter, fever, body weight loss and hyperalgesia in a dose-dependent manner. Inflammatory cellular infiltrate in synovium of ankle joint and pannus formation were also markedly inhibited. Interleukin-10 (IL-10) levels were significantly increased in arthritic rats given theophylline alone or in combination with either SNP or L-NMMA. Co-administration of a low dose of SNP or L-NMMA enhanced significantly the anti-inflammatory and anti-arthritic effect of theophylline. In contrast, a high dose of SNP counteracted the anti-inflammatory and anti-arthritic effects of theophylline. CONCLUSIONS AND IMPLICATION: These findings confirm the anti-inflammatory and anti-arthritic activities of theophylline and suggest a new approach to enhance the anti-inflammatory and anti-arthritic effects of theophylline would be to administer it in combination with a low dose of a NO donor or a non-specific NO synthase inhibitor.

Dual effect of nitric oxide donor on adjuvant arthritis.

The effect of medical use of NO donors on the pathogenesis of arthritis is still yet unclear. We investigated the effects of the NO donor, sodium nitroprusside (SNP), on the pathogenesis of adjuvant-induced arthritis in rats. Rats were given SNP intraperitoneally either from day 5 to day 14 (as a prophylactic protocol) or from day 16 to day 25 (as a therapeutic protocol) after inoculation of adjuvant. SNP administration, whether prophylactic or therapeutic, in doses of 0.1 and 1 mg/kg/d significantly aggravated pathogenesis of adjuvant arthritis in rats. SNP-treated rats showed significant (P<0.05) increase in arthritis index, hind paw volume, ankle joint diameter and hyperalgesia compared with control adjuvant arthritic rats. However, in adjuvant rats given the smallest dose of SNP (0.01 mg/kg/d), arthritis index, volume of hind paws, ankle joint diameter, body weight loss, and hyperalgesia were significantly lower than that of control adjuvant rats. After 30 d of the induction of adjuvant arthritis, TNF alpha levels exhibited insignificant changes either in control adjuvant rats or in rats given SNP compared with control non adjuvant rats. IL-10 levels in adjuvant control rats and adjuvant rats given 1 mg or 0.1 mg/kg/d from day 15 to day 25 were significantly lower than that of control non adjuvant rats. Histopathology examination of ankle joint showed that large doses of SNP (1 mg or 0.1 mg/kg/d) increased the mononuclear cells infiltration and erosion of cartilage induced by adjuvant while the infiltration of the inflammatory cells in the synovium of adjuvant rats treated with 0.01 mg/kg/d was minimal and the pannus was inhibited with alleviation of erosion of articular cartilage. Prophylactic small dose of SNP improved the histological status more than the therapeutic small dose. The present work reveals that SNP administration, either prophylactic or therapeutic, was deleterious in higher doses. However, the smallest dose used 0.01 mg/kg/d attenuates joint inflammation, hyperalgesia and body weight loss in adjuvant arthritic rats. These results suggest that small dose of NO donor may exert partial protective effects while the safety of the clinical use of NO donors, in higher doses, in patients with rheumatoid arthritis is questioned.

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.

Oxidative stress as a common mediator for apoptosis induced-cardiac damage in diabetic rats.

AIM: To investigate the possible role of oxidative stress as a common mediator of apoptosis and cardiac damage in diabetes. MATERIALS AND METHODS: This experimental work was conducted on 5 groups of Wistar rats. Group I was the control group. Diabetes type 1 was induced in other groups (by streptozotocin) and animals received insulin or vitamin E (300 mg /kg body weight), both insulin and vitamin E, or no treatment for 4 weeks according to their group. At the end of the study, serum and cardiac tissues were examined for biochemical parameters of cardiac function, oxidative stress and apoptosis. Electron microscopy pictures of cardiac tissue were also evaluated for signs of cardiac damage RESULTS: Markers of oxidative stress, apoptosis, inflammation as well as manifestations of cardiac damage as assessed by electron microscopy were significantly decreased in rats treated with both insulin and vitamin E when compared with untreated diabetic rats or rats treated with either insulin or vitamin E alone CONCLUSION: Administration of both vitamin E and insulin was effective in reducing markers of oxidative stress and apoptosis and improving parameters of cardiac function in experiments animals. Antioxidants might prove beneficial as an adjuvant treatment in addition to insulin in type 1 diabetes associated with manifestations of cardiac complications.