Telomerase Expression Related with Poor Immune Response to HCV Core Antigen in Egyptian HCV Patients' PBMCs.

Background: Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. Aim: This study aimed to assess serum human telomerase enzyme (hTERT) levels and their relation to the progression of liver disease. Also, it aimed to assess the effect of hepatitis C virus (HCV) core protein on memory T-cells in HCV patients with or without HCC and the correlation between memory cell phenotype and the progression of the disease in the same patients. Methods: HTERT level in serum was assessed through relative quantitative RT-PCR. Flow cytometric analysis was used to assess T-cell responsiveness (as IFN- γ secretion) before and after stimulation with HCV core protein and the memory CD8+ cell phenotype using several differentiation markers. Results: HTERT was found to be increased in a stepwise manner upon comparing its level in controls, chronic hepatitis patients, cirrhotic patients, and HCC patients. T-cells showed a similar manner of stepwise decrease in response (decreased IFN- γ secretion) in HCC patients compared to HCV patients without HCC and controls. Also, late differentiated memory cells (CD8+, CD27-, CD28-, CD45RA+, and CCR7-) were depleted in HCC patients compared to HCV patients without HCC. Conclusion: These results suggest a negative correlation between hTERT and IFN- γ secretion by T-cells in HCV patients and that this relationship, along with the depletion of late differentiated memory cells, could help the progression of liver disease to HCC.

Assessment of Interferon Gamma-Induced Protein 10 mRNA Release Assay for Detection of Latent Tuberculosis Infection in Egyptian Pediatric Household Contacts.

OBJECTIVES: Current diagnostic tests for tuberculosis (TB) in children living in low-endemic countries are limited by low specificity and the inability of the current tests to differentiate between active TB and latent TB infection (LTBI). This study aimed to evaluate the blood IP-10 mRNA expression level to detect LTBI in Egyptian pediatric household contacts (PHC). METHODS: TB-specific IP-10 and IFN-γ mRNA levels were assessed by real-time quantitative PCR (RT-qPCR) in 72 Egyptian PHC of active pulmonary TB cases. All study participants were also assessed by Tuberculin Skin Test (TST) and Quantiferon gold in tube (QFN-GIT) assay. RESULTS: IP-10 and IFN-γ mRNA expression levels were significantly higher in PHC with active TB or LTBI than TB negative (p < 0.0001). The level of IP-10 mRNA expression was significantly higher in PHC with active TB than LTBI (p = 0.0008). In contrast, there was no significant differences in the IFN-γ mRNA expression between PHC with active TB compared to LTBI (p = 0.49). The sensitivity and specificity of the IP-10 RT-qPCR were 94.2% and 95.2%, respectively, in PHC with active TB compared to 85.7% and 81.8% in PHC with LTBI. The negative and positive predictive values and accuracy of IP-10 RT-qPCR for distinguishing active TB from LTBI were 85.2%, 58.3%, and 72.6% respectively. CONCLUSION: Blood IP-10 mRNA expression level may be a potential diagnostic marker to help distinguish active TB from LTBI in PHC.

Assessment of Human Papillomavirus Infection and Risk Factors in Egyptian Women With Breast Cancer.

Numerous risk factors for breast cancer (BC) have been identified. High-risk human papilloma virus (HR-HPV) is the etiological agent of cervical cancer and in some cases of head and neck cancer, specifically oropharyngeal cancer, but the role of HR-HPV in evoking neoplasia in BC is still unclear. In this study, all women above the age of 18 visiting the oncology clinic at Al-Azhar university hospital and Ain Shams specialized hospital between the period of February 2017 and March 2018 were invited to participate. We determined the prevalence of HR-HPV genotypes 16, 18, and 31 in breast tissue samples from 72 women with treatment-naïve BC and 15 women with benign breast lesions (BBL) by quantitative real-time PCR (qRT-PCR) and primer sets targeting the E6 and E7 regions. High-risk human papilloma virus DNA was detected in 16 of 72 (22.2%) BC cases (viral load range = 0.3-237.8 copies/uL) and 0 of 15 women with BBL. High-risk human papilloma virus was detected in 14 of 16 (87.5%), 2 of 16 (12.5%), and 0 of 16 (0%) for genotypes 16, 18, and 31, respectively. Forty-three age-matched healthy Egyptian women were enrolled as controls for assessment of local risk factors that can be used to initiate a strategy of BC prevention in Egypt. Assessment of the risk factors demonstrated that low education level, passive smoking, lack of physical activity, family history of cancer, and use of oral contraception were significant risk factors for BC. In conclusion, our results lead us to postulate that HR-HPV infection may be implicated in the development of some types of BC in Egyptian women. In addition, identification of local risk factors can support practical prevention strategies for BC in Egypt.

Frequency of CD4+ regulatory T cells, CD8+ T cells, and human papilloma virus infection in Egyptian Women with breast cancer.

Several subsets of regulatory CD4+ T cells (CD4+ Tregs) have been described in peripheral blood and tumor microenvironment of breast cancer (BC) patients and may play a role in the progression of BC. High-risk human papilloma virus (HR-HPV) has a causal role in cervical, head, and neck tumors but the role of HR-HPV in evoking neoplasia in BC is still unclear. In this study we assessed the prevalence of CD4+CD25+ FOXP3+ regulatory T cells (CD4+Tregs) and CD3+ CD8+ T cells by flow cytometry in peripheral blood from a total of 55 Egyptian women, including 20 treatment-naïve BC, 15 with breast benign lesions (BBL), and 20 healthy volunteers (HV). HR-HPV genotypes type 16, 18, and 31 were investigated in breast tissue from all BC and BBL patients using Real-Time PCR. HR-HPV was detected in 4/20 (20%) and 0/15 (0%) BC and BBL patients respectively. The frequency of CD4+ Tregs was significantly higher in BC compared to BBL and HV, (P < 0.001). In addition, we observed a significantly higher frequency of CD3+ CD8+ T cells in peripheral blood of patients with late stage III BC compared to early stage I and II BC (P = 0.011). However, there was no significant association between the ratio of CD8+ T cell to CD4+ Tregs frequencies and the expression of Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER2). These results lead us to postulate that the association between the frequency of CD4+ Tregs and CD8+ T cells in the peripheral blood may be a prognostic or predictive parameter in Egyptian women with BC. In addition, HR-HPV infection may be implicated in the development of some types of BC in Egyptian women.

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection.

Molecular diagnosis of Entamoeba spp. versus microscopy in the Great Cairo.

Amoebiasis is a human disease produced by Entamoeba histolytica which causes widespread mortality and morbidity worldwide through diarrheal disease and abscess establishment in parenchymal tissues such as liver, lung, and brain. The true prevalence of infection is unknown for most areas of the world due to the difficulty to characterise Entamoeba histolytica versus other non-pathogenic amoebas with identical morphology, as Entamoeba dispar, and Entamoeba moshkovskii. To overcome microscopy misidentification issues, we tested a nested multiplex polymerase chain reaction (PCR) and a real-time PCR on 194 stool samples collected from incoming dysentery patients in Cairo hospitals diagnosed with E. histolytica by microscopy. Nested PCR showed only 20 (10.3%) samples positive to E. histolytica and 17 (8.7%) to E. dispar. The real-time PCR detected only 19 and 11 samples positive to E. histolytica and E. dispar respectively, showing less sensitivity than the nested PCR. The data show that prevalence of E. histolytica in Cairo is lower when specific diagnosis methods are used instead of traditional microscopy, allowing to differentiate between morphologically identical human amoebas species.

Detection of Cryptosporidium parvum and Cryptosporidium hominis in human patients in Cairo, Egypt.

Cryptosporidium is a significant cause of diarrheal disease in developing and industrialized nations. Cryptosporidium hominis and Cryptosporidium parvum are the main agents of cryptosporidiosis in humans. In Egypt, very little is known about genetic structure of Cryptosporidium spp. Therefore, this study was designed to examine samples from sporadic cases of cryptosporidiosis in Egyptians in order to identify the species involved in infection as well as the transmission dynamics and distribution of the parasite in the Great Cairo area. A total of 391 human faecal samples were collected, between May 2008 and March 2009, from ten public hospitals in Great Cairo. Initial screening by immunochromatographic detection kit "the Stick Crypto-Giardia; Operon" showed 23 possible positive cases. Twenty of them were confirmed by microscopic examination. PCR was performed by amplification of the oocyst wall protein (COWP) gene where 18 out of 23 samples were positive, one not detected by microscopy. Cryptosporidium genotyping was performed by RFLP analysis of PCR products of the diagnosis PCR. Only 15 samples rendered a digestion pattern. The genotyping distribution was nine cases showing C. hominis genotype, three showing C. parvum genotype and three showing mixed infection by C. hominis and C. parvum. The data showed an elevated prevalence of C. hominis (80.0%), the most anthroponotic species, suggesting a human-human transmission. Furthermore, the presence of up to 40% of samples infected with C. parvum shows that further investigations are required to determine the subgenotypes of C. parvum to clarify the mode of transmission in order to improve the control measures.

A study of the role of IL-12 in pulmonary tuberculosis using the whole blood flowcytometry technique.

Pulmonary tuberculosis remains a major health problem. It is caused by Mycobacterium tuberculosis, which elicits a T-cell dependent immune response, initiated by monocytes through a large number of cytokines of which interleukin-12 is thought to play a critical role in initiation and regulation of T-helper (Th-1) like responses. To better understand the role of IL-12 in pulmonary tuberculosis patients, intracellular IL-12 in peripheral blood-derived monocytes was examined by flowcytometery. The percentage of monocytes producing IL-12 was measured after invitro stimulation of heparinized whole blood with mycobacterial protein antigens (culture filtrate). Of the 22 active tuberculosis patients, 17 were recent cases and 5 recurrent cases. Healthy controls were 14 individuals with detectable reaction to purified protein derivative (PPD+) and 14 without detectable reaction to PPD. The role of different factors affecting disease outcome such as treatment, age, gender, smoking, severity of disease and presence of other complications on the percentage of monocytes producing IL-12 was studied. Recurrent TB patients had a higher number of monocytes producing IL-12 in unstimulated cultures compared to other groups (P < 0.001). However, after in vitro stimulation there was a significant decrease in the number of monocytes producing IL-12 in recurrent TB patients as compared to recently diagnosed TB patients and healthy PPD+ individuals (P < 0.001). Antituberculosis chemotherapy was the only factor that had significant effect on the percentage of monocytes producing IL-12 (p < 0.05) while other studied factors did not show significant effect (p > 0.05). It is concluded that IL-12 plays a prominent regulatory role in tuberculosis.

Human natural killer T cells (NKT), NK and T cells in pulmonary tuberculosis: potential indicators for disease activity and prognosis.

One third of the world's population is infected with Mycobacterium tuberculosis (MTB). However, active disease can develop only in a small percentage, when the immunity is weakened. The acquired immune response to MTB is primarily mediated by T cells. Natural killer (NK) cells play a central role in innate immunity to microbial pathogens. Human NKT cells have characteristics of both T and NK cells and also exhibit antimycobacterial activity. This work aimed to enumerate T, NK and NKT cells in active pulmonary TB compared with healthy controls and to study the correlation between these cells with different factors affecting prognosis of pulmonary TB as disease severity, complications or associated diseases, antitubrculosis chemotherapy, and age & gender. Of the 22 active tuberculosis patients examined, 17 were recent cases and 5 recurrent. Healthy controls were divided into 14 individuals with detectable reaction to purified protein derivative (PPD+) and 14 individuals without detectable reaction to PPD-. The percentages of T, NK and NKT cells in erythrocyte-lysed whole blood samples were determined using flowcytometry. The percentage of NKT cells was significantly higher among the recently diagnosed MTB cases as compared with both PPD+ (P < 0.01) and PPD- (P < 0.01) healthy controls, while no significant difference could be found in the percentages of T or NK cells among these groups. However, comparing recurrent cases with recently diagnosed cases showed a significant difference only in the percentage of T cells (P < 0.01). There was also a significant difference in the percentage of T cells according to severity of disease (P < 0.01) and in the association of diabetes mellitus (P < 0.01). Age, gender and treatment with antituberculosis chemotherapy had no effect on the percentages of T, NK or NKT cells. It is concluded that T and NKT cells play an important role in immunity against TB. In active pulmonary tuberculosis, increased T cell count points to severity of the disease, while their reduced count predicts bad prognosis. Human NKT cell count is a marker of disease activity. Enumeration of these cells in peripheral blood can be used as a non-invasive prognostic indicator for patients with active pulmonary TB.

Analysis of T cell responses against hepatitis C virus genotype 4 in Egypt.

BACKGROUND/AIMS: Hepatitis C virus (HCV) remains the most common cause of chronic liver disease in Egypt. Despite the high prevalence of HCV and Schistosoma mansoni (S. mansoni) in Egypt, the effect of co-infection on the immune response against HCV genotype 4a has not been extensively examined. METHODS: We evaluated the HCV 4a-specific responses against the core and non-structural 5B proteins in chronic HCV with or without S. mansoni co-infection in 38 volunteers from Egypt. RESULTS: HCV 4a-specific responses were detected in 8/15 and 13/23 individuals with HCV alone or with concomitant schistosomiasis, respectively. Despite the alteration in the Th1 cytokine profile caused by schistosomiasis, the overall immune response rate against HCV was not affected (P=0.11). Seven individuals demonstrated HCV-specific responses against conserved regions of the Core that were previously identified for genotypes 1, 2 and 3 despite differences in HLA class I distribution. CONCLUSIONS: Egyptian patients infected with HCV genotype 4 can mount HCV-specific T cell responses, both CD4+ and CD8+ T cell-mediated, despite the prevalence of concomitant schistosomiasis. These findings suggest that S. mansoni co-infection may not represent a major obstacle to developing an HCV vaccine in this population.

Local expression of tumor necrosis factor-alpha and interleukin-4 mRNA in different types of warts.

The natural history of warts shows considerable variations between individuals, ranging from spontaneous regression to prolonged persistence. The level of cytokines participate in the immune response to human papilloma virus. This work investigates local expression of T-helper1 (Th1)/ T-helper2 (Th2)/ cytokines in a trial to disclose the different immunological mechanisms affecting the natural history of warts. A total of thirty patients suffering from different types (common, plane, plantar and anogenital) of viral warts were included. An excision biopsy was used to assess tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) expression. Local expression of TNF-alpha (Th1) and IL-4 (Th2) in wart tissue was determined using in-situ hybridization technique. The cytokines probes used in this assay detect messenger RNA (mRNA) in wart tissue. Local expression of TNF-alpha mRNA was higher in viral wart patients compared to healthy control (P < 0.01), while local expression of IL-4 was not significantly higher in patients compared to control (P > 0.05). Statistical analysis was done to determine whether variations in cytokine mRNA expression depend on wart location or clinical types. In conclusion, immunoreactivity to warts is likely to be associated with a predominant Th-1 or mixed Th-1/ Th-2 cytokine mRNA expression profile. However, it does not appear to be a simple causal relationship between expression of a Th-1 pattern and clearance.

HCV-specific CD27- CD28- memory T cells are depleted in hepatitis C virus and Schistosoma mansoni co-infection.

Factors that influence the generation and maintenance of memory CD8+ T cells are not fully understood. The homeostasis of memory T cells is highly dynamic and tightly regulated by various stimuli, including cytokines and antigen-major histocompatibility complex ligands. We characterized the hepatitis C virus (HCV)-specific CD8+ T-cell responses in a cohort of HCV-infected individuals with or without Schistosoma mansoni co-infection from Egypt. We observed a significantly decreased CD27- CD28- (late differentiated) memory T-cell population in the HCV co-infected individuals compared to those with HCV infection alone. In contrast, there was no significant difference in the CD27+ CD28+ (early differentiated) memory T cells between the two groups. Analysis of human cytomegalovirus-specific CD8+ T-cell responses in the same individuals failed to reveal a similar pattern of altered memory T-cell differentiation. Thus, S. mansoni co-infection targets a specific subset of memory CD8+ T cells in HCV infection.