Vaccinia virus expressing ICP47 : a novel platform for cancer vaccines highlighting tumor epitopes and hiding viral antigens

Background Recombinant poxviruses expressing tumor associated antigens (TAAs) are evaluated since 20 years as immunogenic vaccine vector in clinical trials. Some caveats for using recombinant viral vector are due to either prior systemic immunity to poxviruses or immunodominance of viral antigens which may reduce the induction of immune response against weaker tumor antigens. To address this issue, we developed a recombinant vaccinia virus (VV) expressing herpes simplex virus (HSV) type I protein ICP47. This protein down-regulates MHC class-I antigen presentation by blocking the transporter associated with antigen processing (TAP), which translocates peptides, generated by proteasomal protein degradation, into the endoplasmic reticulum for loading onto MHC class I molecule. Methods HSV-US12 gene, coding for ICP47 was introduced into wild type vaccinia virus (VV) and into r.VV expressing MART-1/Melan-A27-35 HLA-A201 ER-targeted epitope. Following infection with non-replicating recombinant virus, effect of ICP47 expression on cell surface MHC-class-I, MHC class-II and co-stimulatory molecules was characterized by antibody staining and FACS analysis. Human T-lymphocytes were stimulated in vitro with autologous CD14+ cells infected with r.VV-US12, r.VV-Mart-US12 or control virus. Responsiveness of specific CD8+ and CD4+ T cells to viral proteins and to recombinant epitopes was monitored by MHC-multimer staining and cytokines (IFNγ & IL-2) expression analysis. In order to highlight in vitro the immunogenic advantage provided by ICP47 blockade of viral epitopes, T cells were pre-sensitized with WT-VV infected APC and then used as responders to r.VV-Mart-US12 or r.VV-Mart infected APC. Results Cells infected with HSV-US12-r.VV, demonstrated a decreased ability of presenting MHC class-I antigens to CD8+ T cells whereas MHC-class-II restricted presentation to CD4+ T cells remained unaffected. Cell surface expression of CD80 as a costimulatory molecule and CD44 as cell adhesion molecule was not affected in cells infecied with VV expressing ICP47. Co-expression of ER-Melan-A/Mart-127-35 appeared to partially compensate for the ICP47 related surface MHC class-I down-regulation and preserve a strong capacity to induce CTL response against the TAA derived peptide. The differential prime-stimulation for Mart epitope, in conditions where clearance of infected APC by VV specific CTL could become a limiting factor, showed significantly enhanced CTL responses to Mart stimulation as characterized by IFN-γ and IL-2 gene expression in cultures primed with r.VV-Mart-US12 as compared to cultures primed with r.VV-Mart. Conclusion Thus, viral vectors expressing ICP47 confirmed a diminished TAP-dependant processing of endogenous class-I restricted epitopes while the immunogenic potential of recombinant epitopes directly targeted to the ER was enhanced. Such reagents could become of high relevance especially in multiple-boost vaccine protocol required in cancer immunotherapy

Correlation between Gene Polymorphisms of Prostaglandin D Receptor and Severity of Asthma in Children

Background: Patients, their families and the community suffer a heavy burden when it comes to asthma because it is both common and possibly life-threatening. It has been postulated that arachidonic acid's most prevalent cyclooxygenase metabolite, prostaglandin D2 (PGD2), is a mast cell activation marker in asthma. Objective: To evaluate association between PGDR-441 polymorphism with risk factors, laboratory characteristics, and severity of asthma in children. Methods: In this cross-sectional study, Forty Egyptian children were genotyped using allele specific polymerase chain reaction (AS-PCR) to assess single nucleotide polymorphism of PTGDR2 receptors. Selected cases were classified according to GINA guidelines and spirometrically assessed to evaluate pulmonary functions. Results: There is significant difference between mild, moderate and severe asthma regarding total IgE level(P1<0.001). 68.0% of the studied patients had Homogenous PGDR2(TT) and 32% had Heterozygous PGDR2(TC). There were no statistically significant associations between PGDR2 Polymorphisms and both of asthma risk factors and laboratory characteristics. There was a statistically significant difference between PGDR2 Polymorphisms and bronchial asthma severity of the studied patients. Heterozygous PGDR2 was associated with more severe bronchial asthma. Conclusion: Our study showed a strong relationship between polymorphism of PTGDR2 receptor and severity of bronchial asthma

Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool

BACKGROUND: Monitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals. METHODS: WB (300 microl) was incubated at 37 degrees C with specific antigens, in the form of peptides or commercial vaccines for 5-16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4 degrees C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes. RESULTS: Spiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 microl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1beta (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-gamma gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination. CONCLUSION: This method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in "field" studies with difficult access to laboratory facilities

Role of Circ-ITCH Gene Polymorphisms and Its Expression in Breast Cancer Susceptibility and Prognosis

Introduction/Objective: Breast cancer (BC) is the first leading cause of cancer-related mortality in females worldwide. We have investigated the correlation between circ-ITCH gene polymorphisms, circ-ITCH expression, and their effect on β-catenin levels and BC development. Methods: Participants included 62 BC and 62 controls matched in terms of age. The circ-ITCH polymorphisms rs10485505 and rs4911154 were genotyped using whole blood samples. In addition, mRNA expression analysis of circ-ITCH was performed on BC tissues. The β-catenin levels in serum samples were measured using ELISA. Results: The qRT-PCR results demonstrated that circ-ITCH was significantly downregulated in BC compared to normal healthy tissues. The genotype distribution of rs10485505 and rs4911154 were significantly associated with BC risk. For rs10485505, genotype CT and TT were significantly associated with an increased BC risk. In contrast, there was a significant association between rs4911154, genotypes GA and AA, and an increased BC risk. Regarding the rs10485505 genotype, carriers of the T allele frequently have a poor prognosis compared to carriers of the CC genotype. Serum β-catenin in the BC patients’ group was significantly higher than in the control group. The relative expression levels of circ-ITCH were remarkably decreased in the BC samples of patients carrying the A allele at rs4911154 compared to patients with a GG genotype. Conversely, β-catenin protein levels were increased in patients carrying the A allele, while rs10485505 genotype carriers of the CT and TT genotypes showed downregulation of circ-ITCH expression fold compared to the CC genotype. Contrarily, β-catenin levels markedly increased in TT and CT genotypes compared with the CC genotype. Conclusions: Our research showed that the rs10485505 polymorphism (T allele) and the rs4911154 polymorphism (A allele) are associated with the risk and prognosis of BC. This finding may be due to the effect on the level of circ-ITCH mRNA expression in BC tissues as well as the level of β-catenin in BC patients

Assessment of blood glucose variability by continuous monitoring as a therapy guide for patients with diabetic nephropathy on hemodialysis

Background Plasma glucose variability may confer a risk for development of chronic diabetic complications. Glycosylated hemoglobin (HbA1c) reflects average glucose level but not glucose variability, which is measured by mean amplitude of glycemic excursions (MAGEs) and continuous glucose monitoring (CGM). Aim To study glucose variability using CGM/MAGE compared with sugar profiles and to assess their value as a therapy guide in patients with diabetic nephropathy on hemodialysis. Patients and methods Group 1 included 50 patients with type 2 diabetes mellitus (T2DM) without diabetic nephropathy. Group 2 included 50 patients with T2DM with diabetic nephropathy. Group 3 included 50 patients with T2DM with diabetic nephropathy on hemodialysis. Measurements of fasting blood glucose, postprandial blood glucose, HbA1c, and glucose variability by MAGE and CGM were done. Results CGM showed significant blood glucose variability (amplitude>130 mg/dl in 40 patients=80% using CGM and in 45 patients=90% using MAGE) in dialysis group (group 3) in comparison with glucose variability in nondialysis groups (group 1+group 2) (amplitude>130 mg/dl in 20 patients=20% using either CGM or MAGE). Group 1 showed significant correlations between either CGM or MAGE and all sugar profiles. Group 2 showed significant correlations between CGM and MAGE with either fasting or postprandial blood glucose but not with HbA1c, whereas group 3 showed nonsignificant correlations between either CGM or MAGE and any of sugar profiles. Conclusion CGM/MAGE have high specificity and sensitivity to measure variability of sugar levels, especially in patients with diabetic nephropathy on hemodialysis or not, in which HbA1c may not be a reliable tool

Gene polymorphisms of Patatin-like phospholipase domain containing 3 (PNPLA3), adiponectin, leptin in diabetic obese patients.

Obesity leads a crucial importance in metabolic disorders, as well as type 2 diabetes mellitus. Our present study was designed to assess the potential role of irisin, adiponectin, leptin and gene polymorphism of PNPLA3, leptin and adiponectin as predictive markers of diabetes associated with obesity. One hundred eighty subjects were distributed to three groups including; healthy non-diabetic non obese volunteers as a control group, diabetic non obese group, and diabetic obese group (n = 60 for each group). Fasting blood samples of all groups were collected to determine fasting blood glucose, insulin levels, insulin resistance, total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triacylglycerol, irisin, adiponectin, leptin; as well as, polymorphism of PNPLA3, adiponectin and leptin. The results showed that glucose, insulin resistance, total cholesterol, irisin, leptin, LDL-C, triacylglycerol concentrations were significantly increased, however, insulin, HDL-C, adiponectin were significantly decreased in diabetic obese patients in relation to diabetic non-obese patients as well as in healthy volunteers. The polymorphism of PNPLA3 rs738409 was linearly related to irisin and leptin but was not related with circulating concentrations of adiponectin. We concluded that increased irisin and leptin levels can predict the insulin resistance in obese patients. Moreover, patients who have mutant genotype of PNPLA3 I148 gene (rs738409) C>G, ADIPOQ gene (rs266729) G>C and LEP gene (rs2167270) G>A showed a significant higher susceptibility rate for DM in obese people than those with wild type. This could be considered as an adjustable retort to counter the impact of obesity on glucose homeostasis

Study of non-organ-specific antibodies in children with Genotype 4 chronic Hepatitis C

Background/Aim: Adult studies established a relationship between hepatitis C virus (HCV) infection and the presence of non-organ-specific antibodies (NOSAs). Most studies were carried out on genotypes 1 and 2. Only a few studies addressed that issue in pediatrics. No studies have been carried out on autoimmunity and genotype 4 in children. We aim to investigate NOSAs in 80 Egyptian children with chronic HCV infection along with studying the underlying genotype of HCV, and correlating autoimmunity with the epidemiological, clinical, biochemical, and virological features. Materials and Methods: HCV-RNA was assayed by the polymerase chain reaction and viral genotypes were determined. NOSAs were measured and liver biopsies were taken for histopathological examination. Results:Genotype 4 was the only detected genotype in the included 80 patients. Anti-smooth muscle antibodies (ASMA) were the only detected antibodies in 32 (40%) patients, always with V specificity (vessels only) at titers ranging from 1:20 and 1:160. Anti-nuclear antibodies (ANA) and liver-kidney microsomal antibodies-1 (LKMA-1) were not detected in any of our patients. Epidemiologic and clinical features did not significantly differ between autoantibody-positive and -negative patients. Among biochemical features, significantly high levels of total bilirubin, albumin, immunoglobulins, alkaline phosphatase, and gamma-glutamyl transpeptidase were found in the antibody-positive group. Conclusion: Genotype 4 HCV is the prevailing genotype in Egyptian children with chronic HCV infection. A consistent proportion of these children with chronic HCV infection circulate non-organ-specific autoantibodies. The prevalence of ASMA and the absence of ANA and LKMA-1 might be related to the unique situation in Egypt with unique prevalence of genotype 4. More studies are warranted on larger pediatric population to validate these findings

Assessment of Glutathione Peroxidase-1 (GPX1) Gene Expression as a Specific Diagnostic and Prognostic Biomarker in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a malignant tumor of the mesothelial lining of the thorax. It has been related to frequent exposure to asbestos. Diagnosis of malignant pleural mesothelioma is considered a criticizing problem for clinicians. Early diagnosis and sufficient surgical excision of MPM are considered the cornerstone success factors for the management of early MPM. Glutathione peroxidase-1 (GPX1) is an intracellular protein found to be extensively distributed in all cells, and it belongs to the GPX group. In the current study, we included ninety-eight patients with MPM that underwent surgery at the Zagazig University Hospital in Egypt. We assessed GPX1 gene expression level as it was thought to be related to pathogenicity of cancer in a variety of malignant tumors. We observed a significant elevation in GPX1-mRNA levels in MPM relative to the nearby normal pleural tissues. It was found to be of important diagnostic specificity in the differentiation of MPM from normal tissues. Moreover, we studied the survival of patients in correlation to the GPX1 expression levels and we reported that median overall survival was about 16 months in patients with high GPX1 expression levels, while it was found to be about 40 months in low GPX1 levels