The antibody response to methyl isocyanate: experimental and clinical findings.

As a result of the industrial accident in Bhopal, India (December 1984) in which thousands of people were exposed to methyl isocyanate (MIC), concern was raised for possible long-term health effects. The well-recognized immunologic consequences of exposure to other industrial isocyanates prompted investigation of an antibody response to MIC. Using procedures which had been developed in this laboratory to evaluate isocyanate immunotoxicity, animal studies were undertaken to develop and test reagents which could be used to detect antibodies to MIC in the exposed population. Guinea pigs were injected with MIC in its reactive isocyanate form. Three weeks later, blood was drawn and serum evaluated using ELISA. To detect antibodies, an antigen was prepared by reaction of MIC with guinea pig serum albumin. Antibodies were detected in each of the four animals injected with MIC. Titers achieved were 1:5120 to 1:10,240. Inhibition assays revealed antibody specificity directed toward the MIC hapten. Analogous antigens prepared by reaction of MIC with human serum albumin were used to evaluate sera from individuals exposed in Bhopal to MIC. Antibodies were detected in 12 of 144 exposed persons. Antibodies were specific for MIC, as evidenced by inhibition assays, and belonged to the IgG, IgM and IgE classes. However, titers were generally low and transient and were found in persons having had the highest MIC exposures. Total IgE values of sera were not significantly different from those of control sera obtained from Bombay residents. The results indicate that exposure to methyl isocyanate resulted in production of specific antibodies. However, the low titers observed and the transient nature of the response suggest little health consequence should result form the antibody response.

Circulating immune complexes in chronic myeloid leukemia patients at various stages of the disease.

Circulating immune complexes (CICs) in sera from patients suffering from chronic myeloid leukemia (CML) at initial diagnosis, in 'remission', at relapse and in blastic crisis have been quantitated using fluid [125I]Clq binding assay in terms of per cent binding activity and microgram/ml aggregated human globulin (AHG) equivalents. The Clq binding activity (Clq-BA) has been compared within the groups of CML patients in different phases of the disease as well as with sera obtained from normal healthy donors. The results showed that the mean Clq-BA was significantly increased in CML patients at initial diagnosis (25.74 +/- 3.48, p less than 0.001), in relapse (53.36 +/- 6.9, p less than 0.001) and in blastic crisis (60.5 +/- 8.7, p less than 0.001) when compared to control sera. Sera of 'remission' patients showed significant decrease in Clq-BA when compared to sera collected in active phases of the disease, however, the values were still significantly higher (12.87 +/- 1.58, p less than 0.02) than those of normal healthy donors. When the levels of CICs as assessed by Clq-BA were compared with the WBC/blast counts of CML patients in chronic as well as blastic phase, it was noted that the variations in numbers of circulating leukemic cells do not correlate with the CIC levels. The significance of assessment of CIC levels in monitoring the disease in CML patients is discussed.

Natural and antibody-dependent cellular cytotoxicity in chronic myeloid leukemia patients in remission.

Non-adherent peripheral blood mononuclear cells (NA-PBMNC) from 67 chronic myeloid leukemia (CML) patients in the first and two subsequent remissions, and 23 normal healthy donors were tested for NK and ADCC activities in short term chromium release assays using K562 and antibody-coated chicken RBCs as respective targets. CML patients in remission exhibited significantly reduced NK cytotoxicity (16. 1-19.7%) compared to normal healthy donors (47.4%). Of the patients tested, 55% exhibited NK levels below the mean percent cytotoxicity--2SD (12.5%) of normal donors (low responders), while 45% exhibited NK cytotoxicity above the 12.5% level (normal responders). On the other hand, CML patients in remission showed ADCC activity comparable to that of normal healthy donors (53.3%) irrespective of whether they belonged to normal NK responder group (55.5-65.0% ADCC) or low NK responder group (39.4-48.3% ADCC). The low or normal NK responder status of CML patients was not found to be related to either progression on the disease, or the type of drug used to bring about remission, or to the period in remission at the time of testing. In-vitro treatment of effector lymphocytes with recombinant human IFN alpha resulted in augmented of NK activity in both low and normal NK responder patients. The IFN-augmented NK activity in low responder patients however remained below the normal levels.

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.

Establishment and characterization of four new squamous cell carcinoma cell lines derived from oral tumors.

Four cell lines were established from squamous cell carcinomas (SCC) of the oral cavity. Cell lines AW 13516 and AW 8507 were derived from poorly differentiated SCC and epidermoid carcinoma of the tongue respectively. Cell line AW 10498 was derived from moderately differentiated SCC of the lower alveolus, and AW 9803 grew from a well-differentiated SCC of a retromolar trigone. The cultures showed typical epithelial cell morphology, numerous mitotic figures, occasional multinucleated giant cells, individual cell diskeratosis and nuclear and nucleolar abnormalities. The cell lines AW 13516 and AW 8507 were fast growers with a doubling time of 35.5 h and 31.9 h, respectively, which was independent of the initial seeding density. Cell lines AW 10498 (doubling time 52.2 h) and AW 9803 (doubling time 66 h) showed slower growth and had shorter doubling times at higher seeding densities. The presence of cytokeratins was detected in all the four cell lines by using polyclonal antikeratin antisera in indirect immunofluorescence and in Western blotting. None of the cell lines expressed major histocompatibility complex (MHC) class II antigens. MHC class I antigens were expressed by three cell lines but not by AW 9803. Flow cytometric analysis of DNA content and chromosomal studies suggested the presence of polyploidy and aneuploidy in all the four cell lines. Ultrastructural studies revealed typical epithelial cell features, such as the presence of desmosomes, tonofilaments and keratin bundles.

Effects on muscle of a toxin from Indian cobra (Naja naja naja) venom.

The mode of action of a purified toxin from Naja naja naja (Indian cobra) venom was investigated in frog rectus abdominis muscle, chick biventer cervicis muscle, cat tibialis anterior muscle (close-arterial) and in both innervated and denervated rat diaphragm muscle preparations. The toxin inhibited the acetylcholine responses of rectus abdominis muscle. The inhibition was antagonized by neostigmine and increasing concentrations of acetylcholine, suggesting a competitive binding of the toxin to cholinergic receptors. The toxin, even at high doses, did not produce depolarizing contractures in chronically denervated diaphragm, biventer cervicis muscle and rectus muscle preparations. In both cat tibialis anterior and denervated diaphragm muscles, the toxin abolished the acetylcholine sensitivity of the muscles at a faster rate than its effects on muscle contraction, suggesting a preferred action on the motor end-plate. A well-maintained tetanic contraction and very poor post-tetanic potentiation was observed in all preparations treated with toxin, indicating an atypical Wedensky inhibition. Anti-curare agents, such as K+ and Ca2+, were ineffective in antagonizing the curare-like neuromuscular block in phrenic nerve-diaphragm preparations. A frequency-independent neuromuscular block observed in these nerve-muscle preparations was suggestive of the absence of a possible presynaptic effect. These results demonstrate that although the neurotoxin in some cases can imitate d-tubocurarine, its neuromuscular blocking activity is different from that of curare in many respects.

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.

Antisphingolipid antibodies in the sera of leprosy patients.

Earlier we reported the presence of significant levels of antigalactocerebroside (GalC) antibodies in the sera of leprosy patients. This study corroborates the above result and also gives evidence for the presence of antibodies to the nonpolar ceramide (Cer) moiety of GalC. AntiCer antibody titres were higher as compared to antiGalC antibodies in all categories of leprosy. The specificity of antibodies directed to the Cer moiety was confirmed using Lactosyl-BSA and neutralization assays. Statistically significant and positive correlations were observed between antiGalC and antiCer antibodies. Responsiveness factors were computed using natural logarithmic transformation of the variables.

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.

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.

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.

Determination of type specific antigenic reactivities and quantitation of murine mammary tumor virus by ELISA.

Type specific differences associated with the antigenic determinants of exogenous and endogenous murine mammary tumor viruses (MuMTVs) isolated from strain C3H(Jax) and ICRC were demonstrated by competition enzyme linked immunosorbent assay (ELISA). In this assay, highly purified intact MuMTV from the milk of strain C3H(Jax) was conjugated to the enzyme penicillinase using glutaraldehyde. IgG fraction of antiserum prepared against disrupted C3H (Jax) MuMTV was used in the competition ELISA. The assay was highly reproducible, specific to MuMTV antigens and detected 6-9 ng of the viral proteins. For competition, MuMTV preparations from corresponding endogenous MuMTV carrying subline C3H(Mect) and ICRCf were also used. The assay demonstrated the type specific antigenic reactivities inherent in the MuMTVs isolated from C3H(Jax) and ICRC strains of mice. The competition ELISA was also used to quantitate MuMTV specific proteins in tissue extracts and milk plasma.

Distribution of subpopulations of T lymphocytes in Hodgkin's disease.

In this paper, two aspects of T cell subsets are investigated. Splenic mononuclear cells (MNC) of untreated Hodgkin's disease (HD) patients have been investigated for proportion of immunoregulatory T gamma and T mu cells from 8 uninvolved spleens and 13 involved splenic tissues from HD patients with splenic involvement. As controls, two normal spleens from accident cases were used. It was found that irrespective of the involvement of splenic tissue in the disease process, the HD spleens showed lower percentage of T gamma cells (23.77 +/- 1.03) and higher sequestration of T mu cells (31.6 +/- 2.13) compared to normal spleens (42.5% and 13% resp.). However, there was no significant difference in the total T cell percentages of HD and normal spleens (49.1 +/- 1.13% and 52% resp.). The results therefore indicated the possibility of abnormal sequestration and traffic of T cell subsets in HD. We have also reported here a comparison between T cell subsets from the PBMNC of treated and untreated HD patients and normal healthy donors as assessed by the FcR markers and monoclonal antibodies of Leu series. It was found that the abnormality in T cell subsets could be demonstrated by FcR markers, while Leu 2a and Leu 3a reactivities did not differ in HD and normal PBMNC. The subset proportions identified by two tests did not tally with each other.

Studies on natural killer cells in chronic myeloid leukemia patients in remission.

In our earlier studies, the natural killer (NK) cell mediated cytotoxicity was found to be impaired in 55% of chronic myeloid leukemia (CML) patients in remission (low NK responders), while the rest exhibited normal range of cytotoxicity (normal NK responders). In the present investigations probable factors contributing to the impaired NK activity of low NK responder CML patients have been analyzed. Peripheral blood lymphocytes (PBL) from both low and normal NK responder CML patients possessed normal percentages of HNK-1+, CD3+ and CD8+ cells. The proportion of HNK-1+ cells and CD8+ cells in low density fractions (LDF) and high density fractions (HDF) respectively of Percoll separated PBL from low and normal NK responder CML patients and healthy donors was comparable. However, the NK activity of LDF cells of low NK responder CML patients was much lower. Also, HDF cells from low NK responder CML patients exhibited a regulatory effect on NK cytotoxicity of PBL from healthy donors. Therefore, it is likely that the presence of suppressor cells and perhaps an intrinsic inability of HNK-1+ cells may together contribute to the impaired NK cytotoxicity of low NK responder CML patients.

In vitro induction of chronic myeloid leukemia associated immune reactivity in normal human lymphocytes by xenogeneic immune RNA.

Peripheral blood lymphocytes from normal human donors were treated with immune RNA (IRNA) prepared from lymphoid tissues of guinea pigs immunized with chronic myeloid leukemia (CML) cells, normal leukocytes (WBCs) and normal bone marrow (BM) cells, in order to study whether IRNA can confer specific immunoreactivity on normal lymphocytes. The IRNA treated lymphocytes were further exposed to solubilized membrane antigens from CML cells, normal WBCs and BM cells in a criss-cross fashion. The migration inhibition factor produced by these lymphocytes was tested in an in vitro leukocyte migration inhibition assay using normal leukocytes. The results indicated that IRNA prepared from guinea pigs immunized with all the three cell types could transfer the immune reactivity to normal cells in response to the respective antigens. The WBC IRNA incubated lymphocytes did not react with BM cell and CML cell antigens. A potent transfer of specific reactivity was shown by CML IRNA. IRNA produced against BM cells and CML cells demonstrated considerable cross reactivity perhaps due to shared immature cell antigens.

Subpopulations of human T lymphocytes in patients with Hodgkin's disease before and after treatment.

Circulating T gamma and T mu cells enumerated in the peripheral blood of 51 untreated Hodgkin's disease (HD) patients and 66 treated HD patients tested after few months to more than 3 years of remission, brought about by radiation and/or chemotherapy. 53 age matched normal healthy individuals were studied as controls. Untreated HD patients showed significant increase in T gamma cells (p less than 0.001) and decrease in T mu cells (p less than 0.001) when compared to normal donors. The abnormal percentages of T cell subsets did not correlate with the severity of the disease. A progressive partial restoration in the proportion of these two subsets of T lymphocytes was seen in the disease free condition. However, even after 3 years of remission the recovery was not equivalent to controls. There was no correlation between the recovery of T gamma and T mu cells with the modality of treatment.

Relationship between intracellular calcium and airway reactivity in guinea pigs.

The present study was carried out to examine the relationship between intracellular free calcium ion concentrations and its regulatory enzymes, sodium potassium adenosine triphosphatase (Na(+),K(+)-ATPase) and calcium adenosine triphosphatase (Ca(2+)-ATPase), with airway reactivity to inhaled histamine in guinea pigs. Forty-nine guinea pigs were included in this study. Of these, 34 animals responded to histamine bronchoprovocation challenge in vivo with a greater than 35% fall in specific airways conductance and were labeled as "reactive," and the remaining 15 were "nonreactive." The dose of histamine producing a 35% fall in specific airways conductance was labeled as ED(35) SGaw. The animals were then sacrificed, and the following biochemical measurements were carried out: intracellular free calcium ion concentrations [Ca(2+)](i) in leukocytes and isolated tracheal smooth muscle cells, activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase in tracheal homogenate, and plasma levels of lysophosphatidylcholine (LPC). Reactive guinea pigs showed significantly higher [Ca(2+)](i) and Na(+),K(+)-ATPase and Ca(2+)-ATPase activities. Airway reactivity (ED(35) SGaw) had significant negative correlation with [Ca(2+)](i), with activities of each of the ATPases and with plasma lysophosphatidylcholine. It is concluded that the level of [Ca(2+)](i) is an important determinant of airway reactivity. Intracellular calcium levels modulate airway response to histamine with higher levels being associated with greater reactivity.

Liposome entrapped allergen reduces plasma histamine in sensitized mice.

Immunotherapy of allergic diseases is associated with problems of adverse systemic reactions. We have shown earlier that liposome entrapped allergen (LEA) is effective in inducing IgG response and restricting IgE response in immunized mice. This mode of treatment may be more effective and safer if it can prevent anaphylaxis. To determine this feature, mice were administered allergen preparations repeatedly and later challenged with the same allergen. Mice given liposomal preparation showed lower specific IgE response as compared to the mice given free allergen or alum adsorbed allergen of Artemisia scoparia. Specific IgG response was higher in mice immunized with LEA. The mice immunized with liposomal preparation survived whereas others injected with free allergen or alum adsorbed allergen died probably due to anaphylaxis. High levels of histamine were observed in mice injected with free allergen as compared to the mice injected LEA. The increase in plasma histamine level may be the cause of anaphylaxis during allergen challenge. In conclusion, LEA could be used as a safe and effective mode of immunotherapy for allergy diseases, since it reduces plasma histamine levels considerably thereby reducing the chances of anaphylaxis.

A study on antigenic and allergenic changes during storage in three different biological extracts.

The stability of three allergens common in tropical countries was evaluated under different storage conditions. Prosopis juliflora (PJ), Rhizopus nigricans (RN), and wheat dust (WD), were taken as representatives of various groups of allergens viz, pollen, fungi and dust. The extracts were stored in buffer containing phenol (0.4%) or glycerol (50%) at temperatures ranging from 4-55 degrees C for 15 to 60 days. Protein content of PJ extract was reduced remarkably when it was stored at 40 degrees C for 45 days. Thin layer isoelectric focusing and rocket immunoelectrophoresis of PJ showed that certain antigenic proteins degrade rapidly even at 25 degrees C as early as day 15. However, two to three proteins of PJ remain stable at a higher temperature (40 degrees C) for two months. Relative radioallergosorbent test (RAST) inhibition showed substantial loss of allergenic activity in all the three extracts, when stored at higher temperatures (25-55 degrees C) even for short durations, i.e., 15 days. Extracts (PJ and RN) containing 50% glycerol were found to be stable, retaining more than 50% activity, even when stored at 55 degrees C for 40 days, while extracts without glycerol lost more than 75% of their allergenic activity. However, addition of glycerol did not change the stability of wheat dust allergenic extract. The present findings indicate that allergenic extracts behave differently when stored. Hence, the stability of each extract should be determined individually.

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.