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.

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.

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.

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.

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.

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.

Increased spontaneous, tumor necrosis factor receptor- and CD95 (Fas)-mediated apoptosis in cord blood T-cell subsets from Turner's syndrome.

Increased spontaneous as well as TNF-alpha-induced and CD95-mediated apoptosis were observed in CD4+ and CD8+ T cells from the cord blood of a patient with Turner's syndrome as compared to normal cord blood. Increased apoptosis was associated with an increased expression of TNFR-1, TNFR-2, and CD95L and decreased expression of cIAP1 and FLIP(L). No significant difference was observed in the expression of Bcl-2 family members (Bcl-2, Bax) between Turner's syndrome cord blood and normal cord blood lymphocytes. This study demonstrates that increased apoptosis of T-cell subsets in Turner's syndrome occurs via the death receptor pathway and may play a role in the pathogenesis of immunological defects associated with Turner's syndrome.

Biochemical and molecular basis of thimerosal-induced apoptosis in T cells: a major role of mitochondrial pathway.

The major source of thimerosal (ethyl mercury thiosalicylate) exposure is childhood vaccines. It is believed that the children are exposed to significant accumulative dosage of thimerosal during the first 2 years of life via immunization. Because of health-related concerns for exposure to mercury, we examined the effects of thimerosal on the biochemical and molecular steps of mitochondrial pathway of apoptosis in Jurkat T cells. Thimerosal and not thiosalcylic acid (non-mercury component of thimerosal), in a concentration-dependent manner, induced apoptosis in T cells as determined by TUNEL and propidium iodide assays, suggesting a role of mercury in T cell apoptosis. Apoptosis was associated with depolarization of mitochondrial membrane, release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria, and activation of caspase-9 and caspase-3, but not of caspase-8. In addition, thimerosal in a concentration-dependent manner inhibited the expression of XIAP, cIAP-1 but did not influence cIAP-2 expression. Furthermore, thimerosal enhanced intracellular reactive oxygen species and reduced intracellular glutathione (GSH). Finally, exogenous glutathione protected T cells from thimerosal-induced apoptosis by upregulation of XIAP and cIAP1 and by inhibiting activation of both caspase-9 and caspase-3. These data suggest that thimerosal induces apoptosis in T cells via mitochondrial pathway by inducing oxidative stress and depletion of GSH.

Role of adhesion molecules in recruitment of Vdelta1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients.

The mechanism responsible for tissue specific localization of gammadelta T cell subsets is not well understood. In order to explain the sequestration of specific gammadelta T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer, we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules was observed. In vitro activated gammadelta T cells showed dominant expression of LFA-1 (CD11a), VLA-alpha4 (CD49d), intermediate expression of VLA-alpha5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and alphaEbeta7 (CD103). It was observed that the gammadelta T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells, whereas they adhere to fibroblast cells using LFA-1, VLA-alpha4 and VLA-alpha5. Vdelta1 T cell subsets from the peripheral blood gammadelta T cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-alpha4, VLA-alpha5, L-selectin and alphaEbeta7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vdelta2 T cells. Flow cytometric analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vdelta1+ gammadelta T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important role in the recruitment and retention of Vdelta1 T cells in the tumor milieu.

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.

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.

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.

gammadelta T cells lyse autologous and allogenic oesophageal tumours: involvement of heat-shock proteins in the tumour cell lysis.

T cells expressing gammadelta receptors were isolated from the peripheral blood of oesophageal cancer patients and analysed for their potential to lyse tumour targets. Immunophenotyping by flow cytometry showed that the dominant population of gammadelta T cells expressed the Vgamma9 and the Vdelta2 T cell receptor, and a minor population expressed the Vdelta1 receptor. Cytotoxicity assays revealed that activated gammadelta T cells lysed Daudi Burkitt's lymphoma and K562 cells. Lysis of autologous oesophageal tumours was higher than of allogenic tumours. Anti-hsp60 and anti-hsp70 mAb significantly inhibited the cytotoxicity of gammadelta T cells to both autologous and allogenic oesophageal tumours. Surface expression of hsp60 and hsp70 on oesophageal tumours and Daudi cells was demonstrated by flow cytometry. In conclusion, gammadelta T cells isolated from the peripheral blood of oesophageal cancer patients have the ability of kill oesophageal tumour cells. The lysis of tumour targets by the gammadelta T cells is brought about via recognition of heat-shock proteins expressed on the surface of tumour cells. gammadelta T cells isolated from the peripheral blood may have applications in adoptive immunotherapy of oesophageal cancer.

Enhanced expression of a transmembrane phosphotyrosine phosphatase (LAR) in keratoconus cultures and corneas.

The purpose of the study was to identify genes that are differentially expressed in normal versus keratoconus corneas. Total RNA isolated from corneal stromal cell cultures was reverse-transcribed and then amplified by the polymerase chain reaction (PCR) using defined, arbitrary primers. The products were displayed on polyacrylamide gels and bands that were differentially expressed were excised, re-amplified and subcloned. The resulting clones were sequenced and utilized as probes for Northern blots with cultured cell RNA or Southern blots of corneal cDNA. One of the products that appeared to be more highly expressed in keratoconus cultures and corneas displayed 100% homology with leukocyte common antigen related protein (LAR), a transmembrane phosphotyrosine phosphatase. Western analyses and immunohistochemistry with monoclonal and/or polyclonal antibodies to LAR were used to examine keratocyte cultures and fresh frozen normal, keratoconus and pseudophakic bullous corneas. We identified a gene product with 100% homology to LAR that is expressed at the RNA level in keratoconus corneas and cell cultures but is found only at low or undetectable levels in normal cultures and normal and pseudophakic bullous keratopathy (PBK) corneas. By Western blotting and immunofluorescence with specific LAR antibodies, the protein was identified in keratoconus stromal cell cultures but not in normal cultures. When fresh frozen tissue was examined, LAR protein was localized to numerous stromal cells throughout central keratoconus corneas, while no central staining was seen in normal or bullous keratopathy corneas. LAR, a transmembrane phosphotyrosine phosphatase, is more highly expressed in keratoconus corneas and stromal cell cultures as demonstrated by differential display, Northern analyses, immunohistochemistry and Western blotting.

Human gamma delta T cells recognize heat shock protein-60 on oral tumor cells.

In the present investigations, gammadelta T cells were isolated from the peripheral blood of oral cancer patients and analyzed for their immunophenotype and cytotoxic potential. Flow-cytometric analysis revealed a dominant population expressing Vgamma9 and Vdelta2 T-cell receptors. In a 4-hr 51Cr-release assay, activated gammadelta T cells showed specific cytotoxicity against Daudi Burkitt's lymphoma cells and fresh oral tumor cells. Cold target competition assays demonstrated that gammadelta T cells recognize a common ligand on Daudi and oral tumor cells. Expression of heat shock protein 60 (hsp60) molecules was detected on the surface of Daudi as well as oral tumor cells by flow cytometry and immunoprecipitation of surface biotinylated cells by anti-hsp60 monoclonal antibody (MAb). Such MAbs brought about a significant inhibition of cytotoxicity of gammadelta T cells against Daudi and oral tumor cells. The results suggest that gammadelta T cells isolated from the peripheral blood of oral cancer patients have the ability to lyse oral tumor cells. The lysis of oral tumor cells occurs via recognition of hsp60 on the surface of oral tumor cells.

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.

CAND3: a ubiquitously expressed gene immediately adjacent and in opposite transcriptional orientation to the ATM gene at 11q23.1.

Using a magnetic beads-mediated cDNA selection procedure and a fetal brain expression library, we identified a transcriptional unit within a cosmid positive for the marker D11S384. Pursuit of its full-length cDNA led to the cloning of the third candidate gene (CAND3) we studied in our quest for the ataxia-telangiectasia (A-T) gene, ATM. CAND3 spans approximately 140 kb of genomic DNA and is located immediately centrimeric to ATM, with 544 bp of DNA separating the two genes. CAND3 encodes two ubiquitously expressed transcripts of approximately 5.8 kb and approximately 4.6 kb that are divergently transcribed from a promoter region common to ATM. Nucleotide sequence was determined for one of its alternately spliced transcripts. The predicted protein has 1175 amino acids and is novel in sequence, with only weak homologies to transcriptional factors, nucleoporin protein, and protein kinases, including members of the phosphatidylinositol 3-kinase (PI-3 kinase) family. Although neither homology to ATM nor any mutation of CAND3 in A-T patients has been found, the head-to-head arrangement of CAND3 and ATM, with expression of both housekeeping genes from a common stretch of 544 bp intergenic DNA, suggests a bi-directional promoter possibly for co-regulation of biologically related functions. YACs, BACs, cosmids, and STSs are defined to aid in further study of this gene.

Ability of lymphokine-activated killer cells to lyse mycobacteria-infected cells.

Lymphokine-activated killer (LAK) cells were generated by interleukin-2 activation of peripheral blood lymphocytes obtained from lepromatous leprosy (LL) patients and healthy individuals. The ability of LAK cells to lyse targets (macrophages and T-24, a bladder carcinoma cell line) infected with mycobacteria (Mycobacterium leprae and mycobacterial strain ICRC) was assessed in a 51 chromium-release assay. It was observed that LAK cells generated from LL patients and healthy individuals could preferentially lyse M. leprae or ICRC-pulsed macrophages and T-24 cells, compared to non-pulsed targets. The ability of LAK cells to kill intracellular mycobacteria was demonstrated in colony forming assays. These results indicate a promising role for LAK cells in immunotherapy of leprosy.