Shobha Sehgal

A genetic analysis of HIV-1 from Punjab, India reveals the presence of multiple variants.

ObjectiveTo determine the extent of HIV-1 genetic variation in Indian patients. Design: To avoid any bias in selecting viral variants, HIV-1 DNA was amplified directly from the peripheral blood mononuclear cells of patients and sequenced. Genetic similarity between Indian sequences and other geographic isolates was analysed by phylogenetic analysis algorithms. MethodsA fragment encompassing the C2/V3-V5 regions of HIV-1 gp120 was amplified from the lymphocyte DNA of 12 Indian patients. Multiple clones from each patient were sequenced. Nucleotide sequences encompassing about 650 base pairs were aligned for the Indian and other geographically distinct isolates. Inter-isolate relationships were analysed by means of distance, parsimony and neighbour-joining algorithms. ResultsNucleotide sequence comparisons showed low interpatient variation. Amino-acid comparisons revealed a high degree of homology between Indian sequences in this study and those studied earlier. On distance and parsimony trees, most of the Indian sequences clustered together as subtype C. However, sequences from three patients also showed significant homologies and phylogenetic clustering outside of subtype C. ConclusionsThe predominant strain of HIV-1 in India belongs to subtype C and little interpatient nucleotide sequence divergence in the majority of cases suggests recent spread of HIV-1 in this region. This study also presents the first evidence for non-C subtypes in the Indian population with two epidemiologically linked samples remaining unclassified for any existing env subtype. The presence of variant subtypes in Indian patients sheds light on the transmission routes of HIV-1 to India and emphasizes the need to include these sequences in vaccine development strategies.

Immune responses in patients with HIV infection after vaccination with recombinant Hepatitis B virus vaccine

BackgroundPatients with HIV infection are at risk of co-infection with HBV, as the routes of transmission are shared and thus immunization with HBV vaccine could be protective in them. The aim of the present study was to assess the efficacy of recombinant vaccine in treatment-naive HIV positive patients and healthy controls, and to dissect out differences if any, in different limbs of immune response.MethodsForty HIV positive patients and 20 HIV negative controls, negative for HBsAg, HBsAbs and HBcAbs were vaccinated with three doses of 40μg and 20μg of vaccine respectively. Patients were divided into high CD4 and low CD4 group based on CD4+ lymphocytes of 200 and < 200/mm3 respectively. Group II consisted of healthy controls. Detection of phenotypic markers was done by flowcytometry. Cytokine estimation was done by sandwich ELISA. HBsAbs were estimated in serum by ELISA.ResultsAfter vaccination, CD4+, CD8+ and CD3+ cells increased significantly in all the groups. There was no increase in NK cell activity in patients with high CD4+ lymphocytes and only a marginal increase in patients with low CD4+ lymphocytes (170 to 293/mm3) whereas a marked increase was observed in controls (252 to 490/mm3). After vaccination, although an increase in memory cells was observed in HIV positive patients, yet HBsAb levels were significantly lower than controls (P < 0.05) indicating a functional defect of memory cells in HIV/AIDS patients. Basal IFN-γ levels were also significantly lower in HIV/AIDS patients (P < 0.01). Although the levels increased after vaccination, the peak level remained lower than in controls. HBsAb titers were much lower in HIV positive patients compared to controls. (High CD4+ group: 8834 mIU/ml, low CD4+ group: 462 mIU/ml Vs. Controls: 16,906 mIU/ml). IL-4 and IL-10 were low in patients.ConclusionDespite a double dose in patients, IL-4 and IL-10, which regulate antibody response, were also lower in patients, and this together with low CD4+ counts and lack of T help, accounted for low HBsAb levels. Vaccination in patients with CD4+ lymphocytes < 50/mm3 was ineffective. Thus early immunization is advocated in all HIV positive patients at a stage when they are still capable of mounting an adequate immune response

Primary immunodeficiencies in India: a perspective

Although primary immunodeficiency diseases (PIDs) were first reported in India in the 1970s, those diagnoses were based predominantly on clinical presentations—very limited immunological analyses were performed. Therefore, the validity of many early reports of PIDs may be questionable. However, in the last 10–15 years, diagnoses of PIDs have been based on flow cytometric analysis and, in a few cases, by mutational analysis. In India, PIDs in adults are markedly underreported. We present data from two major centers where diagnosis of PID has been focused primarily in children. We highlight some of the limitations and challenges in the diagnosis and therapy of PID, and more recent efforts to establish PID Centers of Excellence and a national PID registry in India.

Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and monocyte-derived macrophages

BackgroundSeveral subtypes of HIV-1 circulate in infected people worldwide, including subtype B in the United States and subtype C in Africa and India. To understand the biological properties of HIV-1 subtype C, including cellular tropism, virus entry, replication efficiency and cytopathic effects, we reciprocally inserted our previously characterized envelope V3–V5 regions derived from 9 subtype C infected patients from India into a subtype B molecular clone, pNL4-3. Equal amounts of the chimeric viruses were used to infect T-lymphocyte cell lines (A3.01 and MT-2), coreceptor cell lines (U373-MAGI-CCR5/CXCR4), primary blood T-lymphocytes (PBL) and monocyte-derived macrophages (MDM).ResultsWe found that subtype C envelope V3–V5 region chimeras failed to replicate in T-lymphocyte cell lines but replicated in PBL and MDM. In addition, these chimeras were able to infect U373MAGI-CD4+-CCR5+ but not U373MAGI-CD4+-CXCR4+ cell line, suggesting CCR5 coreceptor utilization and R5 phenotypes. These subtype C chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium inducing (NSI) phenotypes. More importantly, the subtype C envelope chimeras replicated at higher levels in PBL and MDM compared with subtype B chimeras and isolates. Furthermore, the higher levels subtype C chimeras replication in PBL and MDM correlated with increased virus entry in U373MAGI-CD4+-CCR5+.ConclusionTaken together, these results suggest that the envelope V3 to V5 regions of subtype C contributed to higher levels of HIV-1 replication compared with subtype B chimeras, which may contribute to higher viral loads and faster disease progression in subtype C infected individuals than other subtypes as well as rapid HIV-1 subtype C spread in India.

Lack of serological specificity of recombinant heat shock protein of Leishmania donovani.

In order to identify a specific recombinant antigen of Leishmania donovani with potential use for diagnosis, a cDNA library was constructed in lambda ZAP II expression vector. On screening the cDNA library using pooled sera from Indian patients with kala azar, 20 antibody reactive clones were identified. These were subcloned into pBluescript phagemid by an in vivo excision procedure. The molecular weights of the expressed recombinant proteins varied from 15 to 70 kDa and the cDNA insert sizes varied from 0.5 kb to the largest size of approximately 2.0 kb which was designated as the E2b clone. The nucleotide sequencing revealed that 50% of the clones had sequence homology to the heat shock protein gene of L. donovani. The serological studies conducted with the kala azar positive sera and sera from healthy laboratory workers using the recombinant protein from the E2b clone and having sequence homology to Ldhsp 70, indicated that although all the kala azar sera were positive, 12 of 20 healthy individuals also showed antibodies against the recombinant hsp70, indicating that this antigen is not suitable for serological diagnosis of kala azar.

High incidence of Epstein Barr virus infection in childhood acute lymphocytic lukemia: a preliminary study.

INTRODUCTION Epstein Barr virus (EBV) has a unique association with several human malignancies, especially lymphoproliferative disorders, mainly lymphomas in adults. There is paucity of data pertaining to EBV association with various cancers in India . OBJECTIVE The study aims to investigate the association of EBV in childhood leukemia. MATERIAL AND METHODS Patients attending pediatric oncology services of the referral center have been included in the study. Twenty-five consecutive pediatric patients with acute lymphocytic lukemia (ALL) were subjected to EBV studies employing sensitive polymerase chain reaction followed by hybridization for presence of Bam H1-W region of EBV genome and detection of anti Z EBV replication activator (ZEBRA) antibodies using Western blot. Positive control included a case of Burkitts lymphoma and infectious mononucleosis each. Raji cells were used as positive control with each test. RESULTS The PCR for EBV was positive in 8/25 patients of ALL. Western blot test using anti ZEBRA antibodies was positive in 5/25(20%) cases of ALL. Considering PCR as the gold standard, 32% of the children with ALL had evidence of active EBV replication. The positive controls were consistently positive. None of the 30 healthy laboratory controls, 22 age matched disease controls, 12 cases of AML and 15 cases of multiple myeloma were positive either by PCR or Western blots assays (P < 0. 01). There was no statistically significant correlation between duration of therapy and EBV positivity (P > 0.05). CONCLUSION These studies indicate that a significant number of patients with ALL show evidence of active EBV replication.

A monoclonal antibody cytolytic to androgen independent DU145 and PC3 human prostatic carcinoma cells

While a range of therapeutic products is available for androgen‐dependent prostatic cancer, no specific intervention modality exists for androgen‐independent prostatic cancer. The objective of this research was to explore whether epitopes exist on androgen‐independent prostatic DU145 cancer cells, which could be susceptible to cytotoxic action of specific antibodies.

Blood Transfusion Related HBV and HIV Infection in a Patient with SLE

We described the course of a young man with SLE who developed hepatitis B virus and human immunodeficiency virus infections through contaminated blood tranfusion. He presented with severe SLE, improved on treatment and then developed hepatic failure which responded to conservative treatment. He now has AIDS and the SLE and HBV infection are quiescent.

Clinical profile of 516 children affected by HIV in a tertiary care centre in northern India: 14 years of experience

Increasing numbers of children affected by HIV are being recognised in northern India. The present study aimed to evaluate the clinical profile of 516 children affected by HIV at the Pediatric Allergy and Immunology Unit, Advanced Pediatric Center, Postgraduate Institute of Medical Education and Research, Chandigarh, India, during the period January 1994 to May 2008. In total, 454 children (327 boys and 127 girls) infected by HIV were analysed. The median age at presentation was 54 months. Of these children, 401 (88.3%) acquired the infection vertically and 26 (5.7%) acquired it through transfusion of blood/blood products. Moreover, 156 children (34.4%) were asymptomatic at presentation to hospital. Common clinical features included fever (36.6%), respiratory infections (31.7%), lymphadenopathy (30.0%), hepatosplenomegaly (21.8%) and diarrhoea (18.1%); 299 children (65.9%) were malnourished. Triple drug antiretroviral therapy was initiated in 205 children. Children receiving such therapy showed significant improvement in clinical and immunological parameters. Furthermore, follow-up rates improved markedly following free supply of the drug. Therapy was very well tolerated. To conclude, physicians looking after children need to be familiar with the varying clinical presentation of HIV infection. To the best of our knowledge, this is the largest paediatric series on HIV infection from a single centre from any developing country.

Immune functions in splenectomized thalassaemic children

A prospective study to assess the immune functions in splenectomized thalassaemic children. Children were those registered in the Thalassemia major. There were 10 splenectomized children (Group 1), 10 nonsplenectomized children and 6 age-matched control (Group 3). All children were shown to be HIV seronegative. The mean concentrations of serum IgG and IgA were higher in Group 1 as compared to Groups 2 and 3 but the differences were not statistically significant. Nitroblue tetrazolium (NBT) dye reduction by stimulated polymorphonuclear leukocytes was normal in both study and control groups and the differences were not statistically significant. However, NBT reduction in the unstimulated state was much higher in Group 2 as compared to Groups 1 and 3. Phytohaemagglutinin induced mitogen proliferation was normal in all 3 groups. Children in Group 1 not only had a significantly higher absolute lymphocyte count but also had a lower CD4JCD8 ratio as compared to Groups 2 and 3. Splenectomy does appear to alter the immune status of thalassemic children but the exact mechanism by which this occurence is not clear.