Sayed F. Abdelwahab

Calcium Signaling through Phospholipase C Activates Dendritic Cells To Mature and Is Necessary for the Activation and Maturation of Dendritic Cells Induced by Diverse Agonists

ABSTRACT Calcium is an important second messenger in the phospholipase C (PLC) signal transduction pathway. Calcium signaling is involved in many biological processes, including muscle contraction, cellular activation, and cellular proliferation. Dendritic cell (DC) maturation is induced by many different stimuli, including bacterial lipopolysaccharide (LPS), bacterial toxins, inflammatory cytokines, prostaglandins, as well as calcium mobilization. In the present study, we determined the role of the PLC signal transduction pathway in the activation and maturation of human monocyte-derived DCs (MDDCs) induced by diverse agonists. We found that signaling through PLC activates MDDCs to mature and is necessary for LPS, cholera toxin, dibutyryl-cyclic AMP, prostaglandin E2, and the calcium ionophore A23187 to induce MDDC maturation. The results of the present study along with the results of other studies indicate that multiple signaling pathways are involved in the activation of DCs and that inhibition of any of these pathways inhibits the maturation of DCs.

Risk factors for hepatitis C virus infection among Egyptian healthcare workers in a national liver diseases referral centre.

Little is known about the prevalence of hepatitis C virus (HCV) among healthcare workers (HCW) in Egypt, where the highest worldwide prevalence of HCV exists. The prevalence of HCV, hepatitis B virus and Schistosoma mansoni antibodies was examined in 842 HCWs at the National Liver Institute in the Nile Delta, where >85% of patients are HCV antibody-positive. The mean age of HCWs was 31.5 years and they reported an average of 0.6±1.2 needlesticks/HCW/year. The prevalence of anti-HCV, hepatitis B surface antigen (HBsAg) and co-infection was 16.6%, 1.5% and 0.2%, respectively. HCV-RNA was present in 72.1% of anti-HCV-positive HCWs, and all but one subject were infected with HCV genotype 4. Schistosoma mansoni antibodies were present in 35.1%. The anti-HCV rate increased sharply with age and employment duration, but not among those with needlestick history. After adjusting for other risk factors, the anti-HCV rate was higher among older HCWs [P<0.001; risk ratio (RR) = 1.086, 95% CI 1.063-1.11], males (P=0.002; RR=1.911, 95% CI 1.266-2.885) and those with rural residence (P<0.001; RR=2.876, 95% CI 1.830-4.52). Occupation (P=0.133), duration of employment (P=0.272) or schistosomal antibody positivity (P=0.152) were not significant risk factors for anti-HCV positivity. In conclusion, although one in six HCWs had been infected with HCV, the infections were more likely to be community-acquired and not occupationally related.

Pasteurella multocida Toxin Activates Human Monocyte-Derived and Murine Bone Marrow-Derived Dendritic Cells In Vitro but Suppresses Antibody Production In Vivo

ABSTRACT Pasteurella multocida toxin (PMT) is a potent mitogen for fibroblasts and osteoblastic cells. PMT activates phospholipase C-β through Gqα, and the activation of this pathway is responsible for its mitogenic activity. Here, we investigated the effects of PMT on human monocyte-derived dendritic cells (MDDC) in vitro and show a novel activity for PMT. In this regard, PMT activates MDDC to mature in a dose-dependent manner through the activation of phospholipase C and subsequent mobilization of calcium. This activation was accompanied by enhanced stimulation of naïve alloreactive T cells and dominant inhibition of interleukin-12 production in the presence of saturating concentrations of lipopolysaccharide. Surprisingly, although PMT mimics the activating effects of cholera toxin on human MDDC and mouse bone marrow-derived dendritic cells, we found that PMT is not a mucosal adjuvant and that it suppresses the adjuvant effects of cholera toxin in mice. Together, these results indicate discordant effects for PMT in vitro compared to those in vivo.

Strong Hepatitis C Virus (HCV)–specific Cell-mediated Immune Responses in the Absence of Viremia or Antibodies Among Uninfected Siblings of HCV Chronically Infected Children

BACKGROUND Cell-mediated immune (CMI) responses to hepatitis C virus (HCV) antigens in adults without seroconversion or viremia are biomarkers for prior transient infection. We investigated HCV-specific CMI responses in seronegative children living with HCV-infected siblings. METHODS Children 3-18 years of age living with HCV-infected siblings were screened for HCV antibodies and HCV RNA. Peripheral blood mononuclear cells (PBMCs) were evaluated for HCV-specific CMI responses by interferon γ (IFN-γ) enzyme-linked immunospot assay using 3 recombinant HCV protein antigens. Flow cytometry phenotypically characterized IFN-γ-secreting cells. RESULTS Forty-five seronegative children and 5 seropositive viremic siblings had functionally viable PBMCs. Among the 45 seronegative siblings, 15 (33.3%) had positive HCV-specific IFN-γ responses, and subsequent RNA testing revealed that 3 were viremic. Compared with the 5 seropositive viremic children, the median number of HCV-specific spot-forming units was significantly higher in the 12 seronegative aviremic children (P = .002) and in the 3 seronegative viremic children (P = .025). Flow cytometric analysis revealed that IFN-γ was synthesized mainly by CD4(+) T cells. CONCLUSION Strong HCV-specific CD4(+) T cell responses were detectable in higher frequency among seronegative, aviremic children compared with persistently infected siblings. Further studies are needed to determine whether these immune responses are protective against HCV infection.

Hepatitis C Virus-Specific Cell-Mediated Immune Responses in Children Born to Mothers Infected with Hepatitis C Virus

Objective To investigate the association between hepatitis C virus (HCV)-specific cell-mediated immunity (CMI) responses and viral clearance in children born to mothers infected with HCV. Study design A cross-sectional study of children from a mother-infant cohort in Egypt were enrolled to detect CMI responses to recombinant core and nonstructural HCV antigens (nonstructural segments NS3, NS4a/b, and NS5 of the HCV genome) using an interferon-gamma enzyme-linked immunospot assay. Children born to mothers with chronic HCV were enrolled into 3 groups: transiently viremic (n = 5), aviremic (n = 36), and positive control (n = 6), which consisted of 1 child with chronic HCV from this cohort and another 5 children with chronic HCV from a companion study. Children without HCV born to mothers without HCV (n = 27) served as a negative control group. Wilcoxon rank sum test was used to compare the magnitude of CMI responses between groups. Results None of the 6 control children who were positive for HCV responded to any HCV antigen, and 4 (80%) of 5 children with transient viremia responded to at least one HCV antigen, compared with 5 (14%) of 36 and 3 (11%) of 27 children in the aviremic and negative control groups, respectively. Children with transient viremia elicited stronger responses than did negative controls (P = .005), positive controls (P = .011), or children without HCV viremia (P = .012), particularly to nonstructural antigens. Conclusions HCV-specific CMI responses were significantly higher in magnitude and frequency among transiently infected children compared with those persistently infected. This suggests CMI responses may be associated with past viral clearance and can identify children at high risk of infection, who can be targeted for health education, screening, and follow-up.

Incidence of hepatitis C virus infection among Egyptian healthcare workers at high risk of infection.

BACKGROUND Hepatitis C virus (HCV) is a global health threat with Egypt having the highest worldwide prevalence. Evaluation of the efficacy of a preventive HCV vaccine, such as those currently in Phase I/II trials, requires a cohort with a high-risk exposure to HCV. OBJECTIVE To identify a reliable cohort for evaluating preventive HCV vaccines, we studied HCV incidence among HCW in a hospital where almost 85% of patients are HCV-infected. STUDY DESIGN Of 717 HCW negative for HCV-antibodies (anti-HCV) at baseline, 651 were followed up and tested for seroconversion twice annually for an average of 504 ± 154 days. Those reporting a needle-stick injury were additionally tested for both HCV antibodies and RNA monthly for a total of four months. RESULTS Two subjects (0.31%) had anti-HCV and HCV-RNA seroconversion with an overall incidence of 2.04/1000 person-years and a 4.8% incidence among the 21 subjects who reported a needle-stick injury. Two additional subjects had viremia without detectable anti-HCV. Two of the four subjects were among 21 with reported needle-stick injuries (9.5%) and another had surgery. All four were nurses providing direct patient care. CONCLUSIONS Our results show that both transient and persistent viremia were detectable in this high-risk cohort of HCW and suggest that absence of anti-HCV in two of the subjects may be due to low-dose viral exposures. These data indicate that HCV infections acquired from documented injuries during direct patient care are frequent in Egypt and can guide selection of eligible HCW suitable for preventive HCV vaccine trials.

Hepatitis C Virus-Multispecific T-Cell Responses without Viremia or Seroconversion among Egyptian Health Care Workers at High Risk of Infection

ABSTRACT Hepatitis C virus (HCV)-specific cell-mediated immunity (CMI) has been reported among exposed individuals without viremia or seroconversion. Limited data are available regarding CMI among at-risk, seronegative, aviremic Egyptian health care workers (HCW), where HCV genotype 4 predominates. We investigated CMI responses among HCW at the National Liver Institute, where over 85% of the patients are HCV infected. We quantified HCV-specific CMI in 52 seronegative aviremic Egyptian HCW using a gamma interferon (IFN-γ) enzyme-linked immunospot assay in response to 7 HCV genotype 4a overlapping 15-mer peptide pools covering most of the viral genome. A positive HCV-specific IFN-γ response was detected in 29 of 52 HCW (55.8%), where 21 (40.4%) had a positive response for two to seven HCV pools and 8 (15.4%) responded to only one pool. The average numbers of IFN-γ total spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMC) (± standard error of the mean [SEM]) in the 29 responding and 23 nonresponding HCW were 842 ± 141 and 64 ± 15, respectively (P < 0.001). Flow cytometry indicated that both CD4+ and CD4− T cells produced IFN-γ. In summary, more than half of Egyptian HCW demonstrated strong HCV multispecific CMI without viremia or seroconversion, suggesting possible clearance of low HCV exposure(s). These data suggest that detecting anti-HCV and viremia to determine past exposure to HCV can lead to an underestimation of the true disease exposure and that CMI response may contribute to the low degree of chronic HCV infection in these HCW. These findings could have strong implications for planning vaccine studies among populations with a high HCV exposure rate. Further studies are needed to determine whether these responses are protective.

Protective role of humoral immune responses during an outbreak of hepatitis E in Egypt

Although the seroprevalence of hepatitis E virus (HEV) is approximately 80% in adult Egyptians living in rural areas, symptomatic HEV-caused acute viral hepatitis (AVH) is sporadic and relatively uncommon. To investigate the dichotomy between HEV infection and clinical AVH, HEV-specific immune responses in patients with symptomatic and asymptomatic HEV infection during a waterborne outbreak in Egypt were examined. Of 235 acute hepatitis patients in Assiut hospitals screened for HEV infection, 42 (17.9%) were acute hepatitis patients confirmed as HEV-caused AVH; 37 (88%) of the 42 patients were residents of rural areas, and 14 (33%) were from one village (Kom El-Mansoura). Another 200 contacts of AVH cases in this village were screened for HEV and 14 (7.0%), all of whom were family members of AVH cases, were asymptomatic HEV IgM-positive. HEV infections in this village peaked during the summer. Asymptomatic HEV seroconverters had significantly higher levels of epitope-specific neutralising (p=0.006) and high avidity (p=0.04) anti-HEV antibodies than the corresponding AVH cases. In conclusion, naturally acquired humoral immune responses appear to protect HEV-exposed subjects from AVH during an HEV outbreak in Egypt.

Schistosomiasis Does Not Affect the Outcome of HCV Infection in Genotype 4-Infected Patients

Although reports suggest that Schistosoma mansoni increases hepatitis C virus (HCV) morbidity and chronicity, its impact on HCV spontaneous resolution is not clear. HCV genotype, viral load, abdominal ultrasonographic findings, and HCV-specific cell-mediated immunity (CMI) were examined among 141 healthcare workers infected with HCV (68 workers with and 73 workers without S. mansoni). HCV genotype 4 was dominate, and viral loads were 2.62 ± 0.69 × 10(6) and 4.24 ± 1.4 × 10(6) IU/mL among patients with and without coinfection, respectively (P = 0.309); 23.5% with and 32.9% without coinfection had spontaneously resolved HCV infection (P = 0.297). Interferon-γ spot-forming cells/10(6) peripheral blood mononuclear cells among responding viremic patients with and without coinfection were 716 ± 194 and 587 ± 162, whereas among aviremic patients, it was 794 ± 272 and 365 ± 36 (P > 0.05), respectively. In conclusion, there was no statistical difference in HCV spontaneous resolution, viral load, liver pathology, or CMI in patients with or without S. mansoni coinfection, suggesting that it did not impact the outcome of HCV infection.

Hepatitis B virus genotype D predominates HBsAg-positive egyptian blood donors and is mainly associated with a negative HBeAg serostatus.

Objectives: Hepatitis B virus (HBV) infection is a global health burden. In this regard, Egypt has an intermediate HBV seroprevalence. HBV is classified into ten different genotypes (A-J) with different geographic distributions. Genotype D is the most prevalent in the Middle East. Limited data are available about HBV genotyping among Egyptian blood donors, particularly in Upper Egypt. We examined the seroprevalence of HBV among 12,000 blood donors attending the blood transfusion services center in Minia Governorate, Upper Egypt. Methods: HBsAg and HBeAg were examined by ELISA while HBV-DNA was examined by PCR. HBV genotyping was conducted by restriction fragment length polymorphism. Results: HBsAg was detected in 237 donors (1.98%). The HBV-DNA of 50 donors with the highest HBsAg OD was examined for the HBV genotype. All 50 DNA-positive samples were of genotype D. 82% of the DNA-positive donors were males, coinciding with their representation in the cohort. ALT levels were normal in 88% of genotyped subjects, while 84% of them were HBeAg negative. Conclusion: Taken together, these data indicate that HBV genotype D is the predominant genotype among HBsAg-positive blood donors in Upper Egypt and was in >80% of the subjects associated with a negative HBeAg serostatus.