Distribution of beta-thalassemia mutations in the Indian population referred to a diagnostic center.

Hemoglobinopathies are the most commonly inherited genetic disorders in India. Population screening has identified certain communities in India with high risk of beta-thalassemia, the prevalence of carrier status in some being as high as 17%. Over a period of 6 years we have conducted DNA analysis on 1,233 carriers of 23 beta-thalassemia mutations and Hb E, using the amplification refractory mutation system. The studies included analyses of five common mutations for Asian Indians, namely IVS-I-5 (G-->C), 619 bp deletion, IVS-I-1 (G-->T), and the frameshifts at codons 8/9 (+G) and 41/42 (-TTCT). The occurrence of these was seen in 1,066 (86.45%) of the carriers referred to us, the percentage of mutations varying between 5.03-42.58%. We found codon 15 (TGG-->TAG) in 47 (3.81%) samples which was also considered a common mutation in the Indian population. Rare beta-thalassemia mutations were found in 87 (7.06%) individuals. We have designed five new primers and modified four primers for nine rare mutations. These were seen in nine (0.73%) samples. The beta-thalassemia anomaly in 33 (2.68%) carriers remained uncharacterized. State-wide and community-wide distribution patterns of mutations indicated that IVS-I-5 (G-->C) is the most common beta-thalassemia allele in the Indian population. Sindhis settled in Gujrat, and Maharashtra and Lohanas from Gujrat showed a prevalence of the 619 bp deletion mutation in 49.2 and 45.5% carriers, respectively. High frequency of the IVS-I-1 (G-->T) mutation was also found in Sindhis (25.5%), Punjabi Hindus (34.7%), and Lohanas (31.2%). These studies of mutation patterns in different communities have helped us in the quick identification of beta-thalassemia mutations for prenatal diagnosis.

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.

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.

Umbilical cord blood as a source of CD34 positive haematopoietic stem cells.

In this paper we have used a monoclonal antibody to CD34 an antigen expressed solely on stem cells, and stem cell colony assays to show that umbilical cord blood has nearly the same number of functional stem cells as compared to normal bone-marrow. The number of CD34+ve cells in cord blood being 2 to 2.7 per cent, whereas bone-marrow had 3 to 3.5 per cent. The multi-potent colony forming cells (CFU-GEMM) were 60 +/- 18 in cord blood per 2 x 10(5) mononuclear cells (MNCs), whereas normal bone-marrow had 70 +/- 10 per 2 x 10(5) MNCs. Enrichment of these stem cells on Percoll gradients was successful for normal bone-marrow but not for cord blood.

T cell responses in Hodgkin's disease: frequency distribution of IL-2 producing cells and quantitation of IL-2 produced per cell.

T cell dysfunction in Hodgkin's disease (HD) is well documented. Since interleukin-2 (IL-2) plays a pivotal role in T cell proliferation, we have investigated frequency distribution of IL-2 producing phytohaemagglutinin (PHA)-stimulated lymphocytes from HD patients compared to that of healthy donors using two limiting dilution (LD) culture systems in which autologous peripheral blood lymphocytes (PBL) and Epstein Barr Virus transformed allogeneic B lymphoblastoid cell lines (EBV-LCL) have been used as feeders. The latter provided better conditions for IL-2 production by single cells, as evident from the enhanced frequencies obtained (For healthy donors: 1/67 +/- 1545.5 using EBV-LCL and 1/1123 +/- 1.7438 using autologous PBL as feeders). The data showed significantly reduced frequency of IL-2 producing cells as well as reduced quantity of IL-2 produced per cell in HD even after using/EBV-LCL as feeders, the amount of IL-2 produced per activated responder cell in HD patients being 0.825-1.3 pg/well (p < 0.001) as compared to 1.48-2.43 pg/well in healthy donors. Thus, the EBV-LCL feeders did provide better culture conditions for estimating frequencies of functional T cells. However these cell lines were unable to restore in vitro the abnormalities in functional properties of T cells in HD.

Immunoprecipitation of mycobacterial antigens with sera from patients with leprosy.

Pooled sera from leprosy patients across the clinical spectrum, tuberculosis patients and healthy individuals were tested for their reactivity with antigens of Mycobacterium leprae and a panel of cultivable mycobacteria by immunoprecipitation technique. Sera from lepromatous leprosy patients demonstrated exclusive reactivity with the 26-kDa protein of M. tuberculosis H37Ra, 28-kDa protein of M. kansasii, 45-kDa protein of M. smegmatis, and 158, 40 and 14 kDa proteins of M. phlei. Sera from patients with borderline tuberculoid leprosy, tuberculoid leprosy, tuberculosis and health individuals failed to identify these antigens. Our studies indicate that analysis and characterization of immunodominant antigenic epitopes present on proteins of cultivable mycobacteria, sharing cross-reactive epitopes with M. leprae may prove to be important in the serodiagnosis of multibacillary leprosy as well as for developing vaccines for immunotherapy of leprosy.

Immunoreactivity of mycobacterial strain ICRC and Mycobacterium leprae antigens with polyclonal and monoclonal antibodies.

ICRC, a cultivable mycobacterium, is undergoing clinical trials as an antileprosy vaccine in India. In the present study, we have investigated the crossreactivity between antigens of the mycobacterial strains of ICRC and Mycobacterium leprae using polyclonal and monoclonal antibodies in a radioimmunoprecipitation assay. It was observed that polyclonal anti-ICRC and anti-M. leprae antibodies showed predominant reactivity to a 21-kDa protein of the mycobacterial strain ICRC and the 21- and 14-kDa proteins of M. leprae. Crossreactivity between the antigens of the mycobacterial strains ICRC and M. leprae was established further by using M. leprae-specific monoclonal antibody WML06 (reacting with the 14-kDa protein of M. leprae), which identified the 21- and 14-kDa proteins of the mycobacterial strain ICRC. Thus, our studies demonstrate that the 14-kDa protein of M. leprae, which is known to harbor T- and B-cell epitopes, shares crossreactive antigenic determinants with the 21- and 14-kDa proteins of the mycobacterial strain ICRC. We believe that such proteins may provide important reagents for designing subunit vaccines and for determining skin-test reagents.

Lymphocyte proliferation, IFN-gamma production and limiting dilution analysis of T-cell responses to ICRC and Mycobacterium leprae antigens in leprosy patients.

Lymphocyte proliferative responses and interferon-gamma (IFN-gamma) production after stimulation with antigens of ICRC, Mycobacterium leprae, and purified protein derivative (PPD) were assessed in leprosy patients and healthy donors. The patients studied were newly diagnosed as having lepromatous leprosy (LL), multidrug therapy (MDT) responders (MDT-R LL), MDT nonresponders (MDT-NR LL), borderline lepromatous (BL), and borderline tuberculoid/tuberculoid (BT/TT) leprosy. The tuberculoid leprosy patients showed increased lymphocyte proliferation and IFN-gamma production in response to stimulation with ICRC, M. leprae, and PPD antigens compared to other groups of LL patients and healthy donors. Although lymphocytes from LL patients showed low responses to ICRC and M. leprae antigens, their responses to PPD were not grossly affected. MDT-R LL patients showed higher lymphocyte proliferative responses and IFN-gamma production after stimulation with ICRC and PPD but not with M. leprae antigens. Tuberculoid leprosy patients showed higher T-cell frequencies to ICRC and M. leprae antigens compared to MDT-R LL and MDT-NR LL patients. The increased lymphocyte proliferative responses to ICRC observed in the MDT-R LL patients was reflected in the increased T-cell frequency to ICRC compared to M. leprae.

Modulation of tumour associated antigen expressed on human squamous cell carcinoma cell lines by recombinant interferon-alpha.

Our earlier studies have shown that monoclonal antibody (Mab) 3F8E3 generated against a head and neck cancer cell line LICR-LON-HN2 showed reactivity with squamous cell carcinoma (SCC) irrespective of the tissue of origin and identified antigens on SCC cell lines AW 13516 and AW 8507. The affinity constants (Ka) for binding of Mab 3F8E3 to AW 13516 and AW 8507 cell lines were 6.2 x 10(8) and 4.3 x 10(8) mol/l and it identified 6.8 x 10(4) and 3.77 x 10(4) sites/cell, respectively, as determined by Scatchard analysis. Treatment of both the cell lines with recombinant human interferon-alpha (rHu-IFN alpha) increased the binding affinity of the Mab but did not increase the number of binding sites on the SCC cell lines. Shedding of antigen recognised by the Mab in the culture supernatant of the cell lines was reduced after rHu-IFN alpha treatment. The results suggest that rHu-IFN alpha may bring about a firm anchorage of the tumour associated antigen on the SCC cells. Cells modulated with rHu-IFN alpha may serve as better targets for assessing cell mediated as well as Mab mediated cytotoxicity in oral cancer patients.

Modulatory effect of cytokines on natural killer and antibody dependent cellular cytotoxicity directed to squamous cell carcinoma targets by lymphocytes from oral cancer patients.

Cells from solid tumours are generally poor targets for natural killer (NK) cytotoxicity and antibody dependent cellular cytotoxicity (ADCC). In this paper, we have analysed NK cytotoxicity and ADCC mediated by peripheral blood mononuclear cells from healthy individuals and oral cancer patients before and after modulation with recombinant interleukin-2 (rIL-2), on target cells derived from two squamous cell carcinoma (SCC) cell lines prior to and after treatment with recombinant interferon-alpha (rIFN alpha). Target SCC cell directed monoclonal antibody 3F8E3 was used in ADCC. The results showed that the unmodulated SCC cells were poor targets for NK and ADCC compared to standard targets (K562 cells and chicken red blood cells, respectively). Modulation of targets alone with rIFN alpha showed moderate increase in their susceptibility while rIL-2 treated effectors could significantly lyse even unmodulated targets. Combined treatment of targets with rIFN alpha and effectors with rIL-2 showed additive enhancement in NK and ADCC activity against SCC cells. Lymphocytes from treated patients with recurrent disease could not efficiently lyse SCC targets even after combined modulation.

Impairment in cellular protein phosphorylation by mitogen activated lymphocytes from patients with Hodgkin's disease.

T cell activation process in patients of Hodgkin's disease was studied in terms of cellular protein phosphorylation following interaction of T lymphocytes with mitogen PHA. Peripheral blood lymphocytes from Hodgkin's disease patients and healthy donors, labelled with [32P] were activated with PHA. The cell lysates were subjected to SDS-PAGE, 2-dimensional gel analysis and were autoradiographed. It was observed that lymphocytes from both Hodgkin's disease patients and healthy donors followed similar time kinetics of phosphorylation. Nine of the eleven major protein bands, resolved on SDS-PAGE in the molecular weight range of 15.7-98 kD showed reduced phosphorylation (ratios of densitometric readings taken after and before stimulation) compared to that of healthy donors. Isoelectric focusing of these major protein bands in 2-dimensional gels further resolved them into about 27 proteins. Most of these showed increased phosphorylation in lysates of activated lymphocytes from healthy donors compared to that of Hodgkin's disease patients. The results showed a defect even at an early stage in terms of inadequate cellular protein phosphorylation.

Major proteins of mycobacterial strain ICRC and Mycobacterium leprae, identified by antibodies in sera from leprosy patients and their contacts.

Sera from leprosy patients across the clinical spectrum, healthy contacts, tuberculosis patients, and healthy donors were tested for their reactivity with antigens of mycobacterial strain ICRC (a cultivable mycobacterium) and Mycobacterium leprae by immunoprecipitation technique. Using M. leprae antigens, it was not possible to distinguish between reactivities of sera from lepromatous, borderline lepromatous, borderline tuberculoid, and tuberculoid leprosy patients. All these sera identified M. antigens with molecular masses of 47, 36, 21, and 14 kDa. When the same sera were tested for their reactivities with antigens of mycobacterial strain ICRC, several differences were observed. The 21-kDa antigen of mycobacterial strain ICRC was exclusively precipitated by sera from all lepromatous leprosy patients and from those undergoing erythema nodosum leprosum reaction. Sera from all the other donors tested failed to identify the 21-kDa antigen of mycobacterial strain ICRC. The 14-kDa protein of mycobacterial strain ICRC was identified by sera from a few lepromatous leprosy patients (5 of 26) and all their contacts. Our studies indicate that antigens present on cultivable mycobacteria rather than species-specific antigens may prove to be useful in the serodiagnosis of leprosy.

Analysis of regulation of T-cell responses by soluble inhibitory factors from the sera of patients with Hodgkin's disease.

Soluble inhibitory factors (SIF) have been demonstrated in the sera of cancer patients, which interfere with the T-cell activation process. We have shown that the major contributory factor to the inhibitory effect of sera from patients with Hodgkin's disease (HD) could be the soluble form of Interleukin-2 receptors (sIL-2R). The parameters studied to show the presence of SIF include (i) inhibiton of mitogen-induced proliferation; (ii) status of high- and low-affinity IL-2R; and (iii) internalization of IL-2-IL-2R complex, by lymphocytes from healthy donors activated with mitogen in presence of HD sera. Parameters studied to show the inhibitory role of sIL2R include (i) quantitation of sIL-2R in HD sera; (ii) effect of high-sIL-2R-containing sera on mitogen-induced proliferation and detection of IL-2 in activated lymphocyte culture supernatants; (iii) effect of exogenous IL-2 supplementation; and (iv) abrogation of inhibitory activity of sIL-2R-containing sera after passing them through IL-2 affinity columns. Our results show that 6/23 HD sera tested had high inhibitory activity (greater than 50% inhibition of mitogen-induced proliferation). The SIF did not affect expression of high- and low-affinity IL-2 receptors, or internalization of the complex by activated lymphocytes. Ten of the 15 sera tested showed significantly high levels of sIL-2R. Pooled sera with high sIL-2R content inhibited mitogen-induced proliferation of normal lymphocytes with a concomitant reduction in IL-2 activity in the lymphocyte culture supernatants. When supplemented with exogenous IL-2, there was a partial recovery of the inhibitory effect. When sIL-2R containing serum pool was passed on IL-2 affinity columns, the inhibitory effect was reduced. The eluted "sIL-2R" adsorbed on the IL-2 column showed anti-proliferative effect.

Mitogen induced proliferation and cytokine production by lymphocytes from leprosy patients.

In this paper we have assessed the mitogen responses of leprosy patients and healthy donors in terms of proliferation and cytokine production (Interleukin 2 and Interferon gamma). The patients investigated included untreated and multidrug therapy non-responsive LL patients and MDT responsive LL and TT patients. The mitogen responses of untreated and multidrug therapy non responsive LL patients were not significantly different from those of the healthy donors. It was interesting to note that TT and multidrug therapy responsive LL patients showed higher responses to mitogens than healthy donors as assessed by all three parameters. The results suggest a correlation of increased mitogenic responses with improvement in clinical status in leprosy.

Impairment in proliferation, lymphokine production and frequency distribution of mitogen-responsive and interleukin-2-producing cells in Hodgkin's disease.

In this paper, we have correlated the ability of peripheral blood lymphocytes (PBL) from Hodgkin's Disease patients to proliferate in response to a mitogen, phytohaemagglutinin (PHA), with production of lymphokines interleukin-2 (IL-2) and interferon gamma (IFN gamma), accumulating in the activated lymphocyte culture supernatants. We have also studied the frequency distribution of PHA-responsive and IL-2-producing T cells from PBL using limiting-dilution analysis. We observed that the levels of IL-2 and IFN gamma in the supernatants of activated lymphocytes from patients with Hodgkin's disease were significantly reduced compared to those of healthy donors. Substage-B patients showed marked reduction in the ability to produce IFN gamma. Levels of IL-2 and IFN gamma in the culture supernatants of PBL from Hodgkin's disease patients correlated positively with proliferative responses, when analysed by linear regression (r = 0.79 and r = 0.60 respectively). However, production of the two lymphokines by activated lymphocytes from the same patients did not correlate (r = +0.04). Further, the frequencies of PHA-responsive cells and IL-2-producing cells in the PBL of patients with Hodgkin's disease (ranges 1/111-1/554 and 1/3009-1/6709 respectively) were also less than those of the healthy donors (ranges 1/80-1/181 and 1/761-1/1828 respectively). Proliferation, IL-2 production in bulk cultures and frequencies of PHA-responsive and IL-2-producing cells correlated well in individual healthy donors. Whereas, one patient (BC 11,214) with a frequency of PHA-responsive cells within normal limits had a very low frequency of IL-2-producing cells. Taken together, the results indicate abnormalities in cytokine production and frequency distribution of cells required for amplification of immune response in patients with Hodgkin's disease.

Natural killer and lymphokine activated killer cell functions in Hodgkin's disease.

We report the natural killer (NK) and lymphokine activated killer (LAK) cell activities in peripheral blood lymphocytes (PBL) from untreated patients with Hodgkin's disease (HD) and from healthy donors. The frequency of LAK cell precursors was also studied using limiting dilution analysis (LDA). About 75% of the HD patients had normal NK activity. There was a higher percentage of low NK responders (mean percent NK activity of healthy donors--2 SD) in patients with lymphocyte depletion histologic grade of the disease and those who were in clinical stage IV, suggesting a correlation of decrease in NK activity with poor prognosis. We found efficient LAK activity against the NK-sensitive K562 cells and NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumour targets in both low and normal NK responder HD patients, irrespective of the histopathological grade and clinical stage of the disease. In concordance with their good LAK cell activity, HD patients showed a frequency distribution of LAK cell progenitors in the PBL comparable to that of healthy donors.

Natural killer-cell-mediated and antibody-dependent cellular cytotoxicity in leprosy.

We have assessed the natural killer (NK) cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) in the peripheral blood lymphocytes (PBL) from untreated lepromatous leprosy (LL) patients, LL patients on multidrug therapy (MDT) with favorable responses (MDT-R), LL patients clinically classified as nonresponders to MDT (MDT-NR), treated tuberculoid leprosy (TT) patients, and healthy donors. NK cytotoxicity was modulated by treating the PBL with recombinant interferon-alpha (IFN-alpha) and recombinant interleukin-2 (IL-2). The mean percent NK cytotoxicity of untreated LL patients (15 +/- 3), treated MDT-R patients (20 +/- 4), and treated MDT-NR patients (12 +/- 4) was significantly lower than that of TT patients (39 +/- 6) and healthy donors (37 +/- 5). Treatment of effectors with IL-2 or IFN-alpha enhanced NK cytotoxicity in 5 of 6 untreated LL patients, 6 of 6 treated MDT-R LL patients, 4 of 5 and 3 of 5 treated MDT-NR LL patients, respectively, and 5 of 8 and 3 of 8 treated TT patients, respectively. Although PBL from TT patients showed initial NK activity comparable to that of healthy donors, fewer TT patients showed modulation of NK activity by IL-2, and IFN-alpha to a lesser extent. The ADCC activity was lower in untreated LL patients compared to treated patients, while TT patients had normal ADCC activity. The results indicate that although LL patients show lowered spontaneous cytotoxicity, it can be modulated favorably by lymphokines.