Hypoxia regulates the differentiation and anti-tumor effector functions of γδT cells in oral cancer.

Hypoxia within the tumor microenvironment (TME) is a key factor contributing to immunosuppression in tumors, co-relating with poor treatment outcome and decreased overall survival in advanced oral cancer (OC) patients. Vδ2 is a dominant subset of gamma delta T cells (γδT cells) present in the peripheral blood which exhibits potent anti-tumor cytotoxicity and is evolving as a key player of anti-cancer cellular therapy. However, the fate of γδT cells in hypoxic oral tumors remains elusive. In the present study, we compared the effect of hypoxia (1% O2 ) and normoxia (21% O2 ) on the expansion, proliferation, activation status, cytokine secretion and cytotoxicity of γδT cells isolated from OC patients and healthy individuals. Hypoxia-exposed γδT cells exhibited reduced cytotoxicity against oral tumor cells. Our data demonstrated that hypoxia reduces the calcium efflux and the expression of degranulation marker CD107a in γδT cells, which explains the decreased anti-tumor cytotoxicity of γδT cells observed under hypoxia. Hypoxia-exposed γδT cells differentiated to γδT17 [γδ T cells that produce interleukin (IL)-17] cells, which corroborated our observations of increased γδT17 cells observed in the oral tumors. Co-culture of γδT cells with CD8 T cells in the presence of hypoxia showed that programmed cell death ligand 1 (PD-L1)high γδT cells brought about apoptosis of programmed cell death 1 (PD-1)high CD8 T cells which could be significantly reversed upon blocking PD-1. Thus, future immunotherapeutic treatment modality for oral cancer may use a combined approach of blocking the PD-1/PD-L1 signaling and targeting hypoxia-inducible factor 1α, which may help in reversing hypoxia-induced immunosuppression.

Molecular variants of HPV-16 associated with cervical cancer in Indian population.

Human papilloma virus is a causative factor in the etiology of cervical cancer with HPV16 being the most prevalent genotype associated with it. Intratype variations in oncogenic E6/E7 and capsid L1 proteins of HPV 16 besides being of phylogenetic importance, are associated with risk of viral persistence and progression. The objective of this multicentric study was to identify HPV-16 E6, E7 and L1 variants prevalent in India and their possible biological effects. Squamous cell cervical cancer biopsies were collected from 6 centres in India and examined for the presence of HPV 16. Variants of HPV-16 were characterized by full length sequence analysis of L1, E6 and E7 genes in 412 samples. Similar distribution of the variants was seen from the different centres/regions, with the European variant E350G being the most prevalent (58%), followed by American Asian variant (11.4%). Fifty six changes were seen in E6 region, 31 being nonsynonymous. The most frequent being L83V (72.3%), Q14H (13.1%) and H78Y (12.1%). Twenty-nine alterations were seen in E7 region, with 12 being nonsynonymous. The most frequent being F57V (9%). L1 region showed 204 changes, of which 67 were nonsynonymous. The most frequent being 448insS (100%), and 465delD (100%), H228D (94%), T292A (85%). The identified variants some new and some already reported can disrupt pentamer formation, transcriptional regulation of the virus, L1 protein interface interaction, B and T cell epitopes, p53 degradation, and thus their distribution is important for development of HPV diagnostics, vaccine, and for therapeutic purpose.

Evaluation of immunomodulatory activity of methanolic extract of Piper betel.

Many of the disorders today are based on the imbalances of immunological processes. This necessitates the search for newer and safer immunomodulators. Thus, the objective of the present study was to explore the immunomodulatory activity of the methanolic extract of Piper betel L. (MPb) (Family: Piperaceae). The MPb consists of mixture of phenols, flavonoids, tannins and polysaccharides. Both in vitro as well as in vivo evaluation was carried out. The effects of MPb on lymphocyte proliferation, interferon-gamma receptors and the production of nitric oxide were measured in vitro. Further, the extract at different dose levels was studied in vivo for the humoral and cellular immune responses on mice immunized with sheep red blood cells. P. betel significantly suppressed phytohaemagglutinin stimulated peripheral blood lymphocyte proliferation in a dose-dependent manner. The decrease in antibody titre and increased suppression of inflammation suggests possible immunosuppressive effect of extract on cellular and humoral response in mice. Thus, the MPb could be explored extensively as a therapeutic agent to treat various immune disorders including autoimmune disorders.

Safety and bioactivity studies of Jasad Bhasma and its in-process intermediate in Swiss mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Bhasma, Ayurvedic medicinal preparations, are prepared using herbs and minerals on following long iterative procedures. However, industrially mercury and sulphur are more commonly used to prepare bhasma from its raw material. The end point of this iterative procedure is mainly judged by the traditional tests specifying physical appearance of the powders. They fail to give better idea about chemical nature of the material. Moreover, the differences in biological activity of final product verses intermediate are not addressed. AIM OF THE STUDY: To compare the physicochemical as well as biological properties of the Jasad bhasma and its in-process intermediate using modern science methods. MATERIALS AND METHODS: The Jasad bhasma and its in-process intermediate are characterized for their physicochemical properties using electron microscopy, x-ray diffraction and CHNS(O) analysis. The biological effects of both the preparations are then studied. The bioaccumulation of zinc, effect on liver antioxidant status, liver and kidney function (by conventional tests as well as SPECT: Single Photon Emission Computed Tomography), effect on blood cells and effect on immune system are studied in mice model, Swiss albino. Since bhasma is given with an accompaniment (anupan), all the bioactivity studies were carried out by administering the preparation with and without Amala powder (Phyllanthus emblica L., fruit, dry powder) as anupan. RESULTS: The XRD results accompanied with Rietveld analysis indicate that the final bhasma is mainly oxide of zinc, whereas the intermediate is mainly sulphide of zinc. The animal studies show that the bhasma as well as its intermediate do not lead to any bioaccumulation of zinc in major organs, when administered with and without anupan. Both, bhasma and intermediate do not cause any deleterious effects on kidney and liver as indicated by blood biochemistry and SPECT studies. However, the intermediate perturbs antioxidant status more and affects the platelet turnover, in comparison with bhasma. On 28day treatment, the bhasma treated animals show prominence of TH1 mediated immune response whereas, intermediate treated animals show prominence of TH2 mediated immune response. CONCLUSION: A set of simple modern microscopy and diffraction techniques can affirmatively identify in-process intermediate from the final preparation. These can be used to decide the end point of long and iterative preparation methods in accordance with modern science practices. The differences in physicochemical properties of particles from the two preparations reflect in their different biological effects. Moreover, the bhasma affects several components of biological systems which again in-turn interact with each other, which emphasizes the need of multifaceted studies in this field.

Role of NF-kappaB signaling pathway in increased tumor necrosis factor-alpha-induced apoptosis of lymphocytes in aged humans.

In human aging, lymphocytes display increased sensitivity to tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. TNF-alpha induces both survival and apoptotic signals. The survival signal is mediated by the activation of NF-kappaB. Although a role of certain proapoptotic molecules in aging has been reported, a role of altered NF-kappaB signaling pathway has not been explored in detail. In this study, we have compared TNF-alpha-induced activation of NF-kappaB, phosphorylation of IkappaBalpha, and the expression of IKKbeta between lymphocytes from young and aged humans. Furthermore, we have explored a role of IKKbeta in increased susceptibility of lymphocytes from aged humans to TNF-alpha-induced apoptosis. Lymphocytes from aged humans displayed decreased activation of NF-kappaB, reduced phosphorylation of IkappaBalpha, and decreased expression of IKKbeta. In addition, overexpression of IKKbeta in lymphocytes from aged humans normalized TNF-alpha-induced apoptosis to the level of young subjects. These data suggest a deficiency of NF-kappaB signaling pathway and a role of IKKbeta, at least in part, for increased sensitivity of lymphocytes from aged humans to TNF-alpha-induced apoptosis.

Place of birth and risk of gallbladder cancer in India.

CONTEXT: Within India, the incidence of gallbladder cancer (GBC) is characterized by marked geographical variation; however, the reasons for these differences are unclear. AIMS: To evaluate the role of place of birth, length of residence, and effect of migration from high- to low-risk region on GBC development. SETTINGS AND DESIGN: Population-based cancer registries (PBCRs); case-control study. SUBJECTS AND METHODS: Data of PBCRs were used to demonstrate geographical variation in GBC incidence rates. A case-control study data examined the role of birth place, residence length, and effect of migration in etiology of GBC. STATISTICAL ANALYSIS: Rate ratios for different PBCRs were estimated using Chennai Cancer Registry as the reference population. Odds ratios (ORs) for developing GBC in a high-risk region compared to a low-risk region and associated 95% confidence interval (CI) were estimated through unconditional logistic regression models using case-control study. RESULTS: GBC shows marked variation in incidence with risk highest in Northeast regions and lowest in South India. OR of 4.82 (95% CI: 3.87-5.99) was observed for developing GBC for individuals born in a high-risk region compared to those born in a low-risk region after adjusting for confounders. A dose-response relationship with increased risk with increased length of residence in a high-risk region was observed (OR lifetime 5.58 [95% CI: 4.42-7.05]; Ptrend ≤ 0.001). The risk persisted even if study participant migrated from high- to low-risk region (OR = 1.36; 95% CI: 1.02-1.82). CONCLUSIONS: The present study signifies the importance of place of birth, length of stay, and effect of migration from high- to low-risk region in the development of GBC. The data indicate role of environmental and genetic factors in etiology of disease.

Characterization of domains of the phosphoriboprotein P0 of Plasmodium falciparum.

Antibodies against the amino-terminal domain of the Plasmodium falciparum P0 phosphoriboprotein were detected extensively in immune people living in malaria endemic areas of India. It has been shown earlier that specific antibodies raised against the PfP0N domain (17-61 amino acid) of the PfP0 protein inhibit P. falciparum growth in vitro. To study the properties of the rest of the protein, the remaining 61-316 amino acids on the carboxy-side of the PfP0 protein were expressed as a glutathione-S-transferase fusion protein (PfP0C). Antibodies raised against PfP0C identified the 38 kDa P0 protein on a parasite Western blot analysis. An ELISA assay using both the PfP0N and PfP0C fusion proteins showed no reactivity with malaria patient sera samples, but showed extensive reactions with the immune sera. Antibodies against both the PfP0C and PfP0N domains were raised in rabbits and different inbred strains of mice. T-cells from immunized mice showed lymphoproliferation when presented with PfP0 protein domains. IgG from both anti-PfP0N and anti-PfP0C sera inhibited the growth of P. falciparum in vitro in a concentration dependent manner. The IgG did not show any significant effect on the growth of intraerythrocytic stages, but specifically inhibited re-invasion of red cells. Merozoites and sexual stages showed surface reactivity to both anti-PfP0N and anti-PfP0C antibodies in immunofluorescence assays. These properties strongly indicate PfP0 as a possible target for invasion-blocking antibodies.

Immunoreactivity of anti K562 monoclonal antibodies with leukemic cells of myeloid lineage.

Three monoclonal antibodies (MAb) raised against K562 cells (erythroleukemic cell line) reacting specifically with myeloid maturation related antigens, were characterized. MAb 4.3 (IgG2bk) reacted with promyelocytes, metamyelocytes and myelocytes, MAb 4.6 (IgG3k) also identified the myeloid blasts, while MAb 4.8 (IgG1k) reacted with metamyelocytes. The affinity constant of the MAbs ranged from 0.8 X 10(7) -3.9 X 10(8) M-1 and the binding sites on K562 cells varied from 8 X 10(4)-4 X 10(5). In competition RIA MAbs 4.6 and 4.8 competed with each other for binding sites on K562 cells. In Western blot analysis the MAbs reacted with 66,000 mol.wt and 84,000 mol.wt peptides on K562 cells and 97,000 mol.wt peptide on chronic myeloid leukemia (CML) cells. These MAbs may help in immunophenotyping of myeloid leukemias.

Frequency analysis of proliferating and cytotoxic T cells in livers and peripheral blood of patients with chronic hepatitis B.

Frequencies of proliferating and cytotoxic lymphocytes from liver biopsy samples and peripheral blood of chronic hepatitis B (CHB) patients and control subjects were monitored by limiting dilution analysis. Precursor frequencies of proliferating T lymphocytes were not significantly different in the liver and peripheral blood compartments of patients and controls. Moreover, similar frequencies of natural killer cells and cytotoxic T lymphocytes were observed in the peripheral blood of patients and controls. A higher frequency of cytotoxic T cells (1 of 22) compared to NK cells (1 of 306) was observed in liver tissues of CHB patients. Dual colour flow cytometric analysis revealed the presence of both CD4+ HLA-DR+ and CD8+ HLA-DR+ T cells in the liver tissues. These results suggest that in livers of CHB patients not only activated CD8+ T cells but also activated CD4+ T cells may play a significant role in the pathogenesis of chronic hepatitis B.

T-cell receptor gamma and delta gene rearrangements in T-cell acute lymphoblastic leukemia in Indian patients.

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal lymphoid malignancy and junctional sequences of rearranged T-cell receptor (TCR) represent the best suitable marker to study clonality in these patients. A sensitive, non-radioactive, and rapid approach of PCR coupled with heteroduplex analysis was used to analyse clonality of TCR gamma and delta gene rearrangements in 26 Indian T-ALL patients. Amongst TCR gamma gene family, VgammaI-Jgamma1.3/2.3 sequences were most utilized (53.9%) while from TCRdelta repertoire Vdelta1-Jdelta1 sequences were preferentially rearranged (23.1%) in these patients. 19.2% of Indian T-ALL patients demonstrated both clonal TCR gamma and delta gene rearrangements along with surface expression of TCRgammadelta. Although the majority of T-ALL patients showed surface expression of TCRalphabeta, the small fraction (19.2%) of TCRgammadelta+ T-ALL represent a distinct subgroup which needs further evaluation.

Analysis of DNA ploidy and expression of tumour-associated antigens on human oral carcinomas xenografted in nude mice.

Human squamous cell carcinomas (SCC) of the oral cavity were successfully established as xenografts in nude mice. Tumours with higher malignancy scores and involvement of lymph nodes in patients were more readily accepted as xenografts in nude mice. The xenografted tumours were characterised with respect to morphology, histology, DNA index and expression of tumour-associated antigens (TAA). Flow cytometric analysis of cellular DNA content revealed that many of the xenografts retained the parent tumour DNA pattern while some of the xenografts showed progression to aneuploidy. All the xenografted tumours expressed TAA recognised by monoclonal antibody (MAb) 3F8E3. On Western blotting, MAb 3F8E3 recognised proteins of molecular weight 62-64 kDa on parent and xenografted tumours. In general, the xenografts reflect many of the characteristics of the tumours from which they were derived and may provide a useful model for investigating newer approaches of treatment and diagnosis.

Genetic haplotyping of ataxia-telangiectasia families localizes the major gene to an approximately 850 kb region on chromosome 11q23.1.

The genotyping data given localize the major A-T gene to an approximately 850 kb region. They also localize the group A A-T gene (ATA) to a region that contains the approximately 850 kb region. They are compatible with linking A-TFresno to 11q22-23. NBS-V2 does not link to this region. Four non-linking families contain only single affecteds, suggesting that these may be spontaneous mutations rather than evidence for an A-T gene outside the 11q22-23 region. Finally, two other non-linking families contain recombinant haplotypes that are compatible with a second A-T gene at 11q22-23, slightly distal to the approximately 850 kb region. However, convincing evidence for a second gene is still lacking.

Development, characterization & potential clinical use of monoclonal antibodies against human alphafoetoprotein.

Four anti-alphafoetoprotein (AFP) monoclonal antibodies (MAb) were raised in the laboratory and characterized using ELISA and immunodot assays. The affinity constants of the MAbs, analysed by scatchard plots, ranged from 3.1 X 10(8) to 2.15 X 10(9) M/l. Epitope analysis using competition RIA indicated that MAb 5E2D7 and 5E2E3 recognize different epitopes on AFP. This combination was used to set up a two site sandwich ELISA with HRPO conjugated 5E2D7. AFP values in sera of hepatocellular carcinoma patients and pregnant women were quantitated using sandwich ELISA. The anti-AFP MAbs showed strong reactivity when tested on hepatoma tissue sections using immunoperoxidase technique.

Murine mammary tumor virus expression during mammary tumorigenesis in ICRC mice.

The expression of murine mammary tumor virus (MuMTV) antigens was examined in the mammary epithelium of strains ICRC and C3H/Jax mice during sequential events leading to mammary tumorigenesis, employing immunoperoxidase technique and competition radioimmunoassays (RIA). The MuMTV antigens could be localized in the lobule alveolar structures of normal, lactating, preneoplastic and neoplastic mammary epithelium as well as in metastatic nodules of mammary tumors of ICRC force breeders. A graded increase in staining reaction was evident with increase in cellularity of the mammary gland due to age and parity. These observations compared well with the quantitation of MuMTV antigens by RIA. The levels of MuMTV antigens in the mammary glands of ICRC mice of different experimental groups were consistently lower than those in C3H/Jax mice. The differential response of the mammary glands to ovariectomy observed in these two strains was reflected in the MuMTV expression.

Immunophenotypes and cytotoxic functions of lymphocytes in patients with hepatocellular carcinoma.

AIMS AND BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia. Immunological mechanisms are thought to play an important role in the control of tumor progression. The immune responses in HCC patients are poorly understood. In the present study, the proliferation and cytotoxic functions of lymphocytes from tumor tissues and peripheral blood of HCC patients were analysed. Simultaneously, the microcultures were phenotyped in order to determine the involvement of different lymphocyte subsets in mediating the cytotoxic function. METHODS: The frequencies of proliferating and cytotoxic lymphocytes from three tumor tissues and peripheral blood from ten HCC patients and nine healthy individuals were assessed by limiting dilution microculture analysis. These microcultures were phenotyped by single and dual color flow cytometry using monoclonal antibodies specific for CD4, CD8, CD56 and HLA-DR markers. RESULTS: The precursor frequencies of both proliferating and cytotoxic lymphocytes were found to be comparable in the peripheral blood of HCC patients and healthy individuals. Compared to peripheral blood, a marked reduction in the precursor frequencies of proliferating and cytotoxic lymphocytes was observed in the tumor tissues of HCC patients. In the tumor tissues, a significantly higher frequency of cytotoxic T cells compared to natural killer cells was observed. Dual color flow cytometric analysis revealed increased percentages of CD8+ HLA-DR+ lymphocytes compared to CD4+ HLA-DR+ cells in the tumor tissues. CONCLUSIONS: Our results suggest that depressed immune responses at the tumor site might be responsible for the escape of tumor cells from the immune surveillance of the host.

Characterization of tumor-associated antigens on human oral squamous cell carcinomas using monoclonal antibody 3F8E3.

Tumor associated antigen (TAA) on oral squamous cell carcinoma (SCC) was characterized using the monoclonal antibody (MAb) 3F8E3. Flow cytometric analysis revealed a varying degree of reactivity of MAb 3F8E3 to TAA on oral tumor cells. Pretreatment of SCC cells with pronase and trypsin annulled the reactivity of MAb 3F8E3. Sodium metaperiodate (NaIO4) and neuraminidase marginally enhanced the binding of 3F8E3 on oral SCC cells. The studies indicate that the TAA recognized by MAb 3F8E3 on oral tumors is a protein moiety. On Western blotting MAb 3F8E3 showed reactivity to proteins with a molecular weight of 60-66 kDa on oral tumor lysates. MAb 3F8E3 reacted strongly to recombinant human hsp60 and 70 in ELISA. The results suggest that MAb 3F8E3 may react to an epitope expressed on a family of heat shock proteins.

Decreased functional response to Toll like receptor ligands in patients with oral cancer.

Patients with oral cancer (OC) show dysregulation of variety of anti tumor immune responses. To assess the role of Toll like receptor (TLR) signaling in peripheral blood lymphocytes (PBL) from OC patients, we analyzed the expression of TLR2, TLR3, TLR4 and TLR9 on various lymphocyte subsets. Results revealed an increased expression of TLRs on unconventional T cells (like γδ T cells, NKT cells and CD4(+)CD8(+) T cells) as compared to conventional αß T cells. Functional studies using TLR ligands (CpG, Poly I:C, LPS and Pam3CSK4) showed defects in the TLR mediated signaling in PBLs of OC patients. Proliferation of OC PBLs in response to stimulation with TLR ligands was significantly decreased. TLR ligand induced IFN-γ production by PBLs from OC patients were low as compared to HI. Stimulation with TLR ligands upregulated the levels of activation markers (CD25 and CD69) on PBLs from HI but not from OC patients. TLR ligands CpG, Poly I:C, LPS and Pam3CSK4 significantly augmented the tumor directed cytotoxic response of PBLs from HI but not from OC patients. Our data suggests that impairment of TLR function on PBLs may be another strategy adopted by tumor cells to dampen tumor directed immune responses.

Type II phosphatidylinositol 4-kinase ß is an integral signaling component of early T cell activation mechanisms.

The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase ß in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase ß with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase ß in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase ß with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase ß is a key component in early T cell activation signaling cascades.