Indirect ELISA using recombinant nonstructural protein 3D to detect foot and mouth disease virus infection associated antibodies.

Foot-and-mouth disease (FMD) is a highly infectious disease of transboundary importance. Routine biannual vaccination along with surveillance activities is seen as the principal to control FMD in India. Non-structural protein (NSP) based immunoassays are the test of choice for the differentiation between infected and vaccinated population. In this study, 3D protein of FMD virus was expressed in Escherichia coli and an indirect ELISA (I-ELISA) was developed to detect 3D-antibodies in the infected bovines. 3D I-ELISA demonstrated comparable diagnostic sensitivity (97.6%) but lower specificity (80.8%) as compared to the in-house r3AB3 I-ELISA. However, the specificity values varied significantly for naïve and vaccinated samples and were observed to be 98.42% and 76.93%, respectively. A moderate degree of concordance (88.5%) was observed between the overall results of two ELISAs. 3D I-ELISA displayed a considerably lower specificity in uninfected vaccinated samples, thereby suggesting against its application for serosurveillance in intensively vaccinated population. However by virtue of its high diagnostic sensitivity and longer duration of persistence of 3D-antibody post-infection, 3D I-ELISA could be adopted for seroepidemiological investigations in regions not practicing vaccination and could be extended to susceptible species which are generally not covered by vaccination.

Diagnostic potential of recombinant nonstructural protein 3B to detect antibodies induced by foot-and-mouth disease virus infection in bovines.

Detection of antibodies to nonstructural proteins (NSP) of foot-and-mouth disease virus is the preferred diagnostic method to differentiate infected from vaccinated animals. In India, an endemic region practising preventive biannual vaccination, 3AB3 indirect ELISA (r3AB3 I-ELISA) has been employed as the primary screening test for serosurveillance. However, because of the variability observed in the immune response to the NSPs, the likelihood of detecting or confirming an infected animal is increased if an antibody profile against multiple NSPs is considered for diagnosis. In this study, all three copies of NSP 3B were expressed in a prokaryotic system to develop an indirect ELISA (r3B I-ELISA). At the decided cutoff of 40 percent positivity, the diagnostic sensitivity and specificity of the r3B I-ELISA were estimated to be 92.1% (95% CI: 89.0-94.5) and 98.1% (95% CI: 96.9-98.8), respectively, as compared to 97.04% and 95.04% for r3AB3 I-ELISA. Although r3B I-ELISA displayed lower sensitivity compared to the screening assay, which could possibly be attributed to additional relevant B-cell epitopes in the carboxy-terminal half of the 3A protein, the former achieved considerably higher specificity on repeatedly vaccinated animals. NSP antibodies could be detected from 10 to as late as 998 days postinfection in experimental calves. Substantial agreement in the test results (90.6%) was found between the two ELISAs. The r3B I-ELISA, when used in conjunction with the r3AB3 I-ELISA as an integrated system, can potentially augment the efficiency and confidence of detection of infected herds against the backdrop of intensive vaccination.

VEGFa/VEGFR2 autocrine and paracrine signaling promotes cervical carcinogenesis via ß-catenin and snail.

VEGF secretion into the tumor microenvironment by cancer cells regulates several oncogenic signaling pathways and cancer-regulated angiogenesis. VEGFR receptors are exclusively present on endothelial cells to maintain their biological homeostasis. The acquisition of unique VEGFR2 receptor and VEGFa in cervical cancer (CC) cells reflects VEGFa/VEGFR2 autocrine machinery. Given the critical role of VEGFR2 in endothelial cell proliferation, migration, and angiogenesis, we explored its function in CC epithelial-mesenchymal transition (EMT) and stemness. Here we report that VEGFR2 regulates cancer-induced angiogenesis and EMT-linked stemness in CC cells via AKT/GSK3ß/ß-catenin and Snail pathway. Receptor tyrosine kinase inhibitor (RTKi) of VEGFR, Pazopanib (PAZ), shows potential anti-VEGFR2 activity and inhibits VEGFa induced metastatic events such as migration, invasion, and anoikis resistance in CC cells. Similarly, PAZ also attenuates cancer-regulated angiogenesis by inhibiting VE-cadherin internalization in endothelial cells followed by inhibition of endothelial cell migration. Selective depletion of VEGFR2 ligand VEGFa in CC cells also attenuates EMT, metastatic events, and inhibition of cancer-induced angiogenesis. In addition, blocking of VEGFR2 signaling in CC cells via PAZ or shRNA alters the formation of cervical tumorspheres (TS) and their successive generation. Collectively, inhibition of functional VEGFa/VEGFR2 autocrine and paracrine axis ceases the cancer-promoting events in cervical cancer cells. Based on the finding in this study, this oncogenic pathways could be used as a potential therapeutic target in a clinical setting with conventional radio-chemotherapy to achieve synergistic killing of CC cells.

Acute kidney injury in late pregnancy in developing countries.

INTRODUCTION: The data directly evaluating acute renal failure (ARF) in third trimester of pregnancy from Indian subcontinent are scanty. This study analyzes the clinical spectrum of ARF with respect to total birth in third trimester of pregnancy. MATERIAL: All pregnant women after the 28th week of pregnancy or in early postpartum period (up to 7 days) admitted to our hospital between August 2006 and August 2008 were screened for clinical evidence of ARF. Pregnant women with clinical diagnosis of ARF in third trimester were included in this study. RESULTS: Of the 4758 pregnant women in third trimester, ARF developed in 85 cases (1 in 56 births). Preeclampsia, puerperal sepsis, and intrauterine death were responsible for ARF in 35.29, 24.7, and 16.67% of cases, respectively. Postpartum hemorrhage and antepartum hemorrhage were the causes of ARF in 10.59 and 8.29% of patients, respectively. Acute fatty liver of pregnancy was noted in one patient. Complicated preeclampsia (hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, eclampsia, and uterine hemorrhage) was associated and higher incidence of ARF. Live birth occurred in 61.2% of patients with vaginal delivery in 70% cases. Renal cortical necrosis was diagnosed in two cases. Overall, mortality was 20%. The puerperal sepsis contributed 41% of total death. CONCLUSION: ARF complicated 1.78% of total delivery in third trimester of pregnancy. Preeclampsia was the most common cause of ARF followed by puerperal sepsis. In contrast to the developed countries, incidence of ARF is still very high in late pregnancy in the developing countries. Overall mortality was 20% with highest (33%) mortality in puerperal sepsis group.

Olaparib modulates DNA repair efficiency, sensitizes cervical cancer cells to cisplatin and exhibits anti-metastatic property.

PARP1 trapping at DNA lesion by pharmacological inhibitors has been exploited in several cancers exhibiting defects in DNA repair mechanisms. PARP1 hyperactivation is involved in therapeutic resistance in multiple cancers. The role of PARP1 in cervical cancer (CC) resistance and implication of PARP inhibitor is yet to be elucidated. Our data demonstrates significantly higher expression of PARP1 in primary cervical tumors and CC cell lines SiHa and ME180. Upon cisplatin treatment CC cells display significant overexpression of PARP1 and its hyperactivation. PARP inhibitor olaparib shows significant anti-proliferative effect on CC cells and drive loss of clonogenic survival and enhanced cell death in combination with cisplatin. PARP inhibited cells show delay in resolution of γH2A.X foci and prolonged late S and G2-M phase arrest resulting in apoptosis. Further, PARP inhibition disrupts the localization of base excision repair (BER) effector XRCC1 and non-homologous end joining (NHEJ) proteins Ku80 and XRCC4. Due to disrupted relocation of repair factors, cisplatin induced stalled replication forks collapse and convert into double strand breaks (DSBs). Interestingly, PARP inhibition also shows anti-migratory and anti-invasive properties in CC cells, increases anchorage independent cell death and induces anoikis. Collectively, our data demonstrates therapeutic potential of PARP inhibitor in cervical cancer.

Nested Multiplex (NMPCR) Detection of Human Papillomavirus (HPV) 16 and 18 in Pre-invasive Lesions and its Implication in Screening of Carcinoma Cervix (CaCx).

INTRODUCTION: Carcinoma cervix (CaCx) is a preventable disease and is caused by certain high risk Papillomaviruses. In the present study, our aim was to investigate the utility of Nested Multiplex Polymerase Chain Reaction (NMPCR) in detecting Human Papillomavirus (HPV) 16 and 18 in cervical scrapes/biopsy samples and to correlate with cervical cytology/ histopathology findings. METHODS: A total of 119 females were subjected for Papanicolaou smear examination of cervical scrapes and/or histopathological examination of cervical tissues. These samples were also subjected to nested multiplex PCR targeting HPV 16/ 18 specific E6/7 gene sequences. RESULTS: HPV 16/18 were detected in 33.6% (40/119) cases included in the study. The overall HPV 16/ 18 positivity among cases with Negative for Intraepithelial Lesion or Malignancy, Low grade Squamous Intraepithelial Lesion, and High grade Squamous Intraepithelial Lesion was observed to be 20.8%, 44%, and 66.7% respectively. Positivity for HPV 16 in cases with Squamous Cell Carcinoma (SCC) was found to be 80%. HPV positivity among subjects reported with reactive cellular changes, a sub category of Negative for Intraepithelial Lesion or Malignancy, was observed to be 26.6%. CONCLUSION: HPV 16 and 18 positivity in cases reported with different stages of pre invasive lesions of CaCx, particularly in the subcategory reactive cellular changes of Negative for Intraepithelial Lesion or Malignancy, indicates that NMPCR detection of HPV 16/ 18 may be used as a screening tool for CaCx in conjunction with Papanicolaou smear examination.

Truncated recombinant non-structural protein 2C-based indirect ELISA for FMD sero-surveillance.

Foot-and-mouth disease (FMD) is a transboundary animal disease caused by foot-and-mouth disease virus. In India, systematic preventive vaccination using inactivated trivalent (O, A and Asia 1) vaccine is the strategy being adopted to control FMD. The use of non-structural protein (NSP)-contaminated inactivated vaccine raises concerns over differentiation of infected and vaccinated animals (DIVA) by NSP based immunoassays. However, 2C being a membrane associated protein usually remain absent in vaccine formulations and thus, anti-2C response is one of the most reliable indicator of the FMDV infection. In this study, 34 amino acids from N-terminus of 2C protein were removed to eliminate membrane-binding amphipathic helicase activity for the expression of recombinant protein in soluble form. Truncated 2C (2Ct) was utilized for development of an indirect ELISA (I-ELISA) for bovine and the developed 2Ct I-ELISA was validated using a panel constituting of serum of naïve, vaccinated and infected animals. The assay was compared with the in-house r3AB3 I-ELISA and the overall concordance was 85.31%. The diagnostic sensitivity and specificity of the 2Ct I-ELISA were 92.9% and 94.0%, respectively. The apparent prevalence of anti-2C antibodies for random bovine samples tested by the developed assay was 23.7%. The developed ELISA will help in augmenting the sensitivity of detection if used in combination with r3AB3 I-ELISA for sero-surveillance.