Immune privilege in the anterior chamber of the eye: alloantigens and tumour-specific antigens presented into the anterior chamber simultaneously induce suppression and activation of delayed hypersensitivity to the respective antigens.

Allografts placed into the anterior chamber of the eye enjoy prolonged and sometimes permanent survival while similar grafts are promptly rejected if transplanted to non-privileged sites. The immunological privilege of the anterior chamber is due, in large part, to the systemic suppression of delayed-type hypersensitivity (DTH) that is induced by anterior chamber presentation of alloantigens and is termed anterior chamber-associated immune deviation (ACAID). Although many categories of antigens elicit ACAID, strong tumour-specific transplantation antigens (TSTA) do not induce ACAID and instead, provoke potent DTH responses following anterior chamber presentation. The present study sought to determine if the anterior chamber were simultaneously confronted with these two categories of antigens, which systemic immune response would prevail: DTH or suppression of DTH? The results show that inoculation of minor histocompatibility alloantigens and TSTA into the anterior chamber induced both afferent and efferent suppression of specific anti-alloantigen DTH responses, yet simultaneously induced normal anti-TSTA DTH responses. Both responses (i.e. DTH and suppression) were transferable with lymphoid cells.


Phenotypic and ultrastructural characterization of an epithelial cell line established from rat thymic cultures.

An epithelial cell line (TE-R 2.5) was established from a long-term culture of rat thymic epithelium. Its epithelial nature was confirmed using anti-cytokeratin (CK) monoclonal antibodies (mAb) and electron-microscopy. TE-R 2.5 cells were reactive with K 8.13, K 8.12, CK 8, R-MC 18, R-MC 19 and Mar 3 mAb and bind Ulex europaeus agglutinin I. Based on the results of this study it was concluded that they possess the phenotype of subcapsular/perivascular or medullary epithelium. This was in accordance with Western blot analysis of water-insoluble cell extracts showing the presence of 56,000, 52,000, 50,000 and 48,000 MW CK polypeptides. In addition, TE-R 2.5 cell line coexpressed CK and vimentin (a 57,000 MW polypeptide) which was demonstrated using dual immunohistochemistry and Western blot analysis. Electron microscopy demonstrated that TE-R 2.5 cells have all the characteristics of hypertrophic thymic epithelial cells (TEC) localized in situ exclusively in the medulla and thus further characterized this line as a type of medullary TEC. Finally, TE-4F10 mAb raised against an antigen of TE-R 2.5 cells selectively stained a subset of medullary TEC in situ including Hassall's corpuscles indicating again the medullary origin of this TEC line.


Monoclonal antibodies to the murine VLA-6 alpha-chain trigger homotypic lymphocyte aggregation.

Very Late Antigen-6 (VLA-6) is a laminin receptor found on T cells, macrophages and platelets and may function as an activation antigen. Here we describe that two monoclonal antibodies (mAb) targeting the VLA-6 alpha-chain are capable of inducing homotypic aggregation of murine T-cell lines (3A9 and C10 cells). 3A9 and C10 cells are of the memory phenotype (CD4+, CD8-, CD44+, CD45+) and express VLA-6 abundantly. The VLA-6-induced aggregation is temperature dependent, energy requiring, and involves the cytoskeleton. In addition, divalent cations (Ca2+ and Mg2+) are also necessary for aggregation. The VLA-6-induced aggregation is not inhibited with mAb targeting the LFA-1/ICAM-1, VLA-4/VCAM-1 and CD2/LFA-3 adhesion pairs. We conclude that VLA-6 has a broader function and can serve as an activation molecule, triggering homotypic aggregation of T-cell subsets.


Interleukin-1 receptor antagonist in inflammatory exudate cells of rabbits. Production, purification and determination of primary structure.

A rabbit interleukin-1 (IL-1) inhibitor in inflammatory peritoneal exudate cells was purified to apparent homogeneity. This inhibitor was extracted from exudate cells of the 24-hr stage of casein-induced peritoneal inflammation and purified using isoelectrofocusing (IEF), gel filtration, followed in this order by high-performance liquid chromatography (HPLC) steps with hydroxylapatite and anionic ion exchanger. The purified factor showed a single band on silver-stained SDS-PAGE. This molecule of MW 19,000 and pI 5.5 inhibited the binding of both IL-1 alpha and beta to receptors on a thymoma cell line, EL-4 and a B-cell line, 70Z/3. We determined its primary structure by a combination of peptide chemistry and molecular cloning. The inhibitor was synthesized as a precursor composed of 177 amino acids and was processed to a mature molecule of 143 amino acids. The N-terminal amino acid of the mature inhibitor was N-acetyl-methionine residue. The deduced amino acid sequence of the inhibitor showed a 77% homology to the human IL-1 receptor antagonist (IL-1Ra) and essentially the same mode of action as seen with human IL-1Ra. We consider that this inhibitor is a rabbit counterpart of human IL-1Ra, although there are differences with respect to the molecular structure; the N-terminus of the mature rabbit IL-1Ra at a position of nine amino acids downstream from that of human IL-1Ra.


Inhibition of interleukin-1 beta mRNA expression and interleukin-1 alpha and beta secretion by a specific human recombinant interleukin-1 receptor antagonist in human peripheral blood mononuclear cells.

The transcription and translation of interleukin-1 (IL-1) may have a pleiotropic effect on the immune system and inflammatory diseases. Recently it has been reported that human monocytes not only produce IL-1 but also induce, with adherent IgG, the secretion of an IL-1 receptor antagonist (IL-1Ra), which can play an essential in vivo and in vitro role in the regulation of IL-1 activity. Recombinant human (rh) IL-1Ra is structurally similar to IL-1 beta but with no IL-1-like activity, and specifically binds to the IL-1 receptor. To more fully evaluate and clarify the inhibitory effect of rhIL-1 receptor antagonist on IL-1 we have studied the influence of rhIL-1Ra on IL-1 transcription and translation. In this report we show that IL-1 beta mRNA from peripheral blood mononuclear cells (PBMC) is strongly inhibited (66%) when rhIL-1Ra (250 ng/ml) was added to cultured cells activated with lipopolysaccharide (LPS) (100 ng/ml) for 4 hr, determined with the slot blot analysis. The addition of exogenous rhIL-1 beta to the cell culture diminished the inhibitory effect (44%). Moreover, we report that the block of IL-1 mRNA transcription consequently leads to the inhibition of IL-1 alpha and IL-1 beta secretion in human PBMC, as measured by ELISA method. In fact, herein we show that LPS activates human PBMC to secrete IL-1 beta and IL-1 alpha, an effect inhibited, in a dose-dependent fashion by rhIL-1Ra (0.025-250 ng/ml) in an overnight incubation. Since IL-1 is a strong inducer of IL-1 synthesis in vivo and in vitro, in our study we used rh IL-1 alpha to stimulate the secretion of IL-1 beta in human PBMC. This activation, carried out overnight, also provoked the release of IL-1 beta in a dose-dependent manner, which was strongly inhibited by rhIL-1Ra used at different concentrations (0.025-250 ng/ml). The inhibitory effect exerted by IL-1Ra on human PBMC IL-1 mRNA transcription and the down-regulation of secretion of IL-1 beta stimulated by IL-1 alpha, may contribute to therapeutic effects in inflammatory diseases such as rheumatoid arthritis and other autoimmune diseases.(ABSTRACT TRUNCATED AT 400 WORDS)


Suppression by Trypanosoma cruzi of T-cell receptor expression by activated human lymphocytes.

The immunosuppression that develops during Chagas' disease and African sleeping sickness is thought to facilitate survival of the causative agents in their mammalian hosts. Whereas a number of manifestations of immunosuppression manifested during the course of these diseases has been reported in patients and animals, the mechanisms by which they are induced remain obscure. An in vitro system in which phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBMC) were co-cultured with purified Trypanosoma cruzi or T. brucei rhodesiense was used in the present work to establish whether these organisms were able to alter the capacity of activated helper/inducer (CD4+) or cytotoxic/suppressor (CD8+) cells to express T-cell receptor (TcR). Suppressed interleukin-2 receptor (IL-2R), known to be caused by both the trypanosomes and supernatants containing their secretion products, was the independent parameter used to demonstrate the occurrence of immunosuppression in all experiments. We found marked reductions in the percentage of TcR+ cells in T. cruzi-containing cultures as early as 18 hr after PHA stimulation. This alteration was still readily demonstrable after 72 hr of culture, i.e. when last tested for. Suppressed TcR expression occurred concomitantly with reduced levels of CD4 or CD8 molecules on the surface of helper/inducer and cytotoxic/suppressor T lymphocytes, respectively, indicating that the parasite had induced more than one alteration in the same cells. These effects were reproduced when the trypanosomes were separated from the PBMC by a 0.45 micron pore size filter or when filtrates from T. cruzi suspensions substituted for the parasite in the cultures, indicating that TcR suppression was mediated by a parasite secretion product(s). Interestingly, neither T. b. rhodesiense nor filtrates of suspensions of this organism altered significantly the level of TcR expression in cultures in which suppressed IL-2R expression by activated human T cells took place. Thus despite sharing the ability to impair IL-2R expression, T. cruzi and T.b. rhodesiense appear to differ in other mechanisms by which they affect human T-cell function. If occurring in infected hosts, the alterations that T. cruzi causes in the expression of TcR, CD4, CD8 and IL-2R--all molecules playing important roles in lymphocyte activation--could contribute to the development of the immunosuppression observed during the acute phase of Chagas' disease.


Protection against Chlamydia psittaci in mice conferred by Lyt-2+ T cells.

A murine model was used to study the respective roles of L3T4+ and Lyt-2+ T cells in protection against Chlamydia psittaci. Donor mice were intravenously (i.v.) infected with 1 x 10(5) plaque-forming units (PFU) per mice of live C. psittaci. One month after inoculation, splenic cells from donors were transferred into syngenic recipients (5 x 10(7) cells/mouse). As measured by splenic colonization on Day 6 after i.v. challenge (1 x 10(5) PFU/mouse), transfer with primed (untreated) cells conferred a 3 log protection in this model. In vitro treatment, before transfer, of splenic cells with anti-Lyt-2 monoclonal antibody (mAb) and complement, markedly impaired the protection in comparison with control mice transferred with primed untreated cells, whereas treatment with anti-L3T4 mAb did not reduce the transferred protection. Resistance to a reinfection with C. psittaci was also studied after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. One month after primary infection, mice were treated with anti-L3T4 or anti-Lyt-2 mAb and challenged thereafter (i.v., 1 x 10(5) PFU). The splenic colonization on Day 6 after challenge demonstrated that treatment with anti-Lyt-2 mAb impaired resistance against a subsequent infection with C. psittaci. Treatment with anti-L3T4 mAb in vivo had no effect on protection, as previously described in vitro. The mechanisms by which Lyt-2+ T cells could participate in the elimination of bacteria were discussed.


Antigen-presenting characteristics of peritoneal cells of Salmonella enteritidis 11RX-infected mice.

Investigation of the possibility that infection with intracellular bacterial parasites such as Salmonellae may modulate the function of antigen-presenting cells (APC) revealed no major change in APC function of peritoneal cells (PC) harvested from the peritoneal cavity of mice 1-3 days after intraperitoneal immunization with S. enteritidis 11RX. Analysis of the phenotype of the Salmonella-primed T cells which responded when cultured with PC from either normal or infected mice and Salmonella-antigen showed that only L3T4+ T cells proliferated. This was also true when PC from normal and infected mice were compared for their ability to induce allogeneic responses; both L3T4+ and Lyt-2.2+ T cells were induced to proliferate, with the majority belonging to the class I restricted, Lyt-2.2+ phenotype. Significant levels of alloantigen-specific Lyt-2.2+ cytotoxic T-cell activity were also induced in both types of cultures. However, a minor population of adherent cells which inhibited Salmonella antigen-specific T-cell proliferation in vitro was detected in peritoneal cell suspensions harvested 3 days after intraperitoneal immunization with S. enteritidis 11RX. Further characterization of these peritoneal cells revealed that they also inhibited the induction of in vitro T-cell responses to alloantigens. It is likely that the cells mediating these inhibitory effects belonged to a macrophage-like subset.


Heterogeneity of human blood monocyte: two subpopulations with different sizes, phenotypes and functions.

Human blood mononuclear cells from normal adults were collected after density-cut centrifugation and monocytes were then isolated by removal of lymphocytes using the techniques of E-rosetting and cell adhesion. The purified monocytes were further analysed by velocity sedimentation, and two distinct subpopulations with different cell sizes were obtained. The larger monocytes were 17.0 +/- 1.8 microns in diameter with a mean sedimentation rate (SR) of 7.0 +/- 0.6 mm/hr, while the smaller monocytes were 9.5 +/- 0.8 microns in size and 4.1 +/- 0.2 mm/hr in SR. The population ratio of larger:smaller cells was approximately 2:1 (66 +/- 2.8%:34 +/- 1.6%). Both cell populations exhibited a high positive rate (> 98%) in both the non-specific esterase and the peroxidase stain. However, the larger cells had much higher phagocytic activity than the smaller ones. Furthermore, the expression of monocyte-associated antigens was also different between these two subpopulations. Thus, while most of the larger monocytes (98%) could be recognized by monoclonal antibodies MY7 and OKM1, only some (35 and 61%, respectively) of the smaller monocytes could react with those antibodies. In addition, the larger monocytes secreted a significant amount of monokines including interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2) and their production increased in proportion to the level of stimulation by bacterial lipopolysaccharide (LPS), whereas the production of monokines by the smaller monocytes remained at low levels and did not respond to LPS stimulation. These results reveal the existence of phenotypic and functional heterogeneity in human blood monocytes.


Retention of tissue-specific phenotype in a panel of colon carcinoma cell lines: relationship to clinical correlates.

A panel of eight cell lines has been derived from colon carcinomas. These cell lines have both been characterized according to standard criteria of growth rate, response to mitogens (epidermal growth factor and basic fibroblast growth factor), xenograft growth and growth in soft agar, and according to the ability of the cells to express epitopes known to be expressed by cells in the normal intestinal mucosa. The expression of epitopes present in columnar (absorptive) cells has been assessed using a panel of monoclonal antibodies to brush border peptidases and disaccharidases, villin and brush border-specific peptides. Goblet cell epitopes have been determined by monoclonal antibodies to mucin and carcinoembryonic antigen. An antibody to chromogranin was used to identify endocrine cells. Using these antibodies we found that all the cell lines reacted with at least one of the antibodies to columnar cells. Similarly, varying proportions of cells in six of the eight cell lines stained with antibodies to mucin. None of the cells expressed chromogranin. Expression of a differentiated colonic phenotype, as measured from antibody staining, did not correlate with measurements of malignancy, such as the ability of the cells to grow in soft agar or as xenografts. Similarly, there was no correlation between retention of a colonic phenotype and the initial pathological stage of the tumour from which the cell lines were derived.


Differential eicosanoid synthesis by murine fetal thymic non-lymphoid cells.

The temporal patterns of synthesis of prostaglandin (PG)E2 and PGI2 by organ-cultured fetal thymic lobes and the cell population(s) responsible for synthesis of such products within the murine fetal thymus have been investigated. Embryonic day 14 thymic lobes were organ-cultured in defined media for 14 days and the media were collected every 24 h and replaced with fresh media. Collected media were processed for quantitation of either PGE2 or PGI2. Lobes were also cultured in 2'-deoxyguanosine (1.35 mmol/L) to produce an enriched non-lymphoid population. The per cent cyclooxygenase-positive cells within non-lymphoid cell-enriched lobes as well as the capacity of such lobes to synthesize either PGE2 or PGI2 were determined and compared with that of intact thymic lobes. Results demonstrate that fetal thymic lobes, in vitro, differentially synthesize PGI2 and synthesize PGE2 at a constant rate. Moreover, lobes enriched for non-lymphoid cells contain a greater percentage of cyclooxygenase-positive cells and synthesize increased amounts of eicosanoids per 10(4) cells compared with controls.


Parasite-induced changes to localized erythrocyte membrane deformability in Plasmodium falciparum cultures.

The effect of intra-erythrocyte development of the Plasmodium falciparum parasite on local deformability of human erythrocyte membranes was studied by aspiration of cells into 0.56 micron diameter pores in polycarbonate filters and examination, after fixing, with a scanning electron microscope. As the aspiration pressure increased, the erythrocyte membrane was extruded into the filter pores. The pressure dependence of the protrusion length and the minimum pressure required to produce any deformation provided measures of the membrane shear and the bending moduli, respectively. At the trophozoite and, to a greater extent, schizont stage of development, host cell membrane deformability was significantly decreased. There was no appreciable difference between uninfected and ring-infected erythrocytes.


Limiting dilution analysis in leprosy.

Peripheral blood mononuclear cells (PBM) obtained from leprosy patients and healthy controls were cultured with Mycobacterium leprae and the control antigens, BCG and SKSD. Parallel cultures were supplemented with additional interleukin-2 (IL-2). On the basis of the level of response to M. leprae, leprosy patients could be divided into low, intermediate and high responders. The addition of IL-2 resulted in enhanced proliferation to antigen only by cells from intermediate responders. This effect was neither antigen specific nor was it confined to cells from leprosy patients. When limiting dilution analyses were performed on cells from 26 patients across the leprosy spectrum, no M. leprae-reactive lymphocytes were detected in cells from subjects with lepromatous disease. The precursor frequency for cultures containing M. leprae plus IL-2 was no greater than that of cultures containing IL-2 alone, thereby excluding the possibility of clonal anergy reversible with IL-2. This was observed in both untreated patients and those on long-term treatment, which made sequestration of antigen-reactive cells within leprosy lesions an unlikely explanation. On the other hand, M. leprae-reactive lymphocytes were detected in patients with tuberculoid and borderline tuberculoid disease and in two subjects with borderline lepromatous leprosy in type I reversal reaction. IL-2 reactive cells were detected in all patients regardless of clinical classification. Three 'suppressor' curves were obtained but were not confined to cells from lepromatous patients. Taken together, these findings suggest that the non-responsiveness to M. leprae characteristic of the great majority of multibacillary patients is due to an absence of antigen-sensitive T cells.


D11, a novel monoclonal antibody specific for human mature macrophages and peripheral blood monocytes.

A new monoclonal antibody designated Mab D11 is described, which shows a restricted reactivity to cells of the monocyte/macrophage system. When tested by light and electron microscopic immunoperoxidase methods, Mab D11 specifically reacts with blood monocytes and stains resident macrophages in a wide variety of human tissues; it does not mark the macrophages of other species, i.e., rat, swine and mouse. Antigen-presenting cells, e.g., Langerhans cells, are Mab D11 negative. Mab D11 reveals the antigen on cryostat and paraffin tissue sections. Ultrastructurally the antigen recognized by Mab D11 in all macrophage types studied is located on the plasma membrane and within cytoplasmic structures including lysosomes. On immunoblotting, Mab D11 detects the 125-kDa antigen in human liver and the 135-kDa protein in tumours of histiocytic origin. The similarity of Mab D11 to known "pan-macrophage" monoclonal antibodies is discussed.


Interferon-gamma is unable to increase monocyte and neutrophil-mediated nonspecific cytotoxicity induced by immune complexes.

We have previously demonstrated that normal human neutrophils and monocytes triggered by immune complexes (IC) are able to destroy non-sensitized target cells through the activation of a nonspecific cytotoxic mechanism (NSC), that is dependent on the generation of reactive oxygen intermediates (IRO). In the present study, we analyze the ability of interferon-gamma (IFN-gamma) to modulate NSC. Our results indicate that, despite the ability of IFN-gamma to increase both the generation of superoxide anion and hydrogen peroxide by phagocytic cells and the expression of the high-affinity 72-kDa Fc gamma RI, it is completely unable to increase NSC mediated either by neutrophils or monocytes. These data suggest that there is no correlation between cytotoxicity and the ability of phagocytic cells to release superoxide anion and/or hydrogen peroxide. They also indicate that Fc gamma RI is not involved in the induction of NSC. To further analyze this point, we studied the ability of two monoclonal antibodies (mAb), specific for different epitopes of Fc gamma RI, to inhibit NSC. These mAb strongly inhibited ADCC mediated by untreated monocytes or IFN-gamma treated monocytes and neutrophils. On the other hand, they were completely unable to inhibit NSC mediated by untreated or IFN-gamma treated cells.


Identification of C-terminal peptides of bovine beta-casein that enhance proliferation of rat lymphocytes.

A casein polypeptidic fraction, obtained from a pepsin-chymosin digestion of caseins, showed a mitogenic effect on primed lymph node (LN) cells and unprimed spleen cells of rats. A biologically active C-terminal sequence of bovine beta-casein (residues 192-209) was characterized. The corresponding synthetic peptide had a stimulatory effect on primed LN cells but failed to enhance proliferation of spleen cells. We prepared two chymosin digests (PA and PB) of bovine beta-casein which contained, respectively, 80% and 95% of the sequence including residues 193-209. They induced a significant proliferative response in both LN and spleen cells. It is therefore possible that other active peptides in the PA preparation may be involved in mitogenic activity.


Deficient transformation of murine trisomy 16 fetal liver cells by the Abelson and J2 viruses.

Mouse trisomy 16 (Ts16), an animal model for human Down syndrome (trisomy 21), exhibits severe abnormalities in the development of lymphoid and myeloid cells. Whereas fetal liver cells from diploid mice can be easily immortalized by retroviral transformation with Ab1-MuLV or J2 virus, fetal livers from Ts16 mice contain significantly fewer transformable cells. Infection of Ts16 fetal liver cells by Ab1-MuLV results in a 52- and 12-fold reduction in the frequency of transformation at days 17 and 18 of gestation, respectively. By contrast, the efficiency of transformation with J2 virus, another retrovirus known to transform fetal liver cells, is only mildly (factor 2-3) affected. The Ig gene rearrangements of Ts16 and diploid retrovirally transformed fetal liver cell lines do not differ from one another. This suggests that there is a deficiency in the early stem cell compartment, rather than in the development of pre-B cells.


Rabbit single domain antibodies specific to protein C expressed in prokaryotes.

VDJ genes were amplified by the polymerase chain reaction from mRNA isolated from peripheral blood B cells of rabbits immunized with protein C. The amplified genes were cloned into a lambda phage expression vector and packaged. A library of 6 x 10(5) recombinant phages was screened with labelled protein C and 30 positive clones were found. Three of them were plaque purified and the affinity of the single domain antibodies to the antigen determined to be 10(6)-10(7) l M-1. The data indicate the feasibility of generating single domain antibody, specific to protein antigen, from rabbit.


Immunosuppression induced by hepatic portal venous immunization spares reactivity in IL-4 producing T lymphocytes.

Immunization of naive or specifically primed C3H/HEJ with irradiated B10.BR spleen cells via the hepatic portal vein leads to an antigen specific decrease in the proliferative and cytotoxic response to B10.BR antigen assayed in vitro (and to increased graft survival of B10.BR grafts in vivo). This effect seems to be mediated in the main by a decrease in IL-2 production from CD4+ T lymphocytes of mice given antigen by the portal route, which is in turn caused by a decreased precursor frequency of IL-2-producing cells. No clear decrease in IL-4 production was seen. Hepatic APC isolated from mice receiving antigen via the portal vein were unable to induce IL-2 production from a C3H/HEJ anti-B10.BR cell line in vitro, in contrast to splenic APC derived from the same mice. Even when antigen was given by conventional (systemic) intravenous routes (in this case via the lateral tail vein) hepatic APC isolated from those mice were unable to stimulate IL-2 production from this cell line. Furthermore, 24 h exposure of a cell line to antigen pulsed hepatic APC left those cells refractory to a subsequent restimulation with antigen presented by splenic APC. Spleen lymphoid cells from primed mice challenged in vivo with B10.BR liver cells (i.v.) were similarly unable to produce IL-2 on rechallenge in vitro with irradiated B10.BR spleen cells, though no defect was seen if in vivo challenge was with B10.BR spleen cells. These data imply that presentation of multiple minor cell surface antigens by hepatic APC leads to specific anergization of IL-2 producing T cells, in a fashion which seems to be distinct from that previously reported as due to 'veto-like' activity.


Perforin gene expression in stimulated human peripheral blood T cells studied by in situ hybridization and northern blotting analysis.

In situ hybridization was used here to monitor the mRNA level of the pore-forming protein perforin in mitogen-stimulated primary peripheral blood human T cells. In situ hybridization was performed using sense and antisense ribonucleotide probes specific for this granule mediator. After IL-2 treatment, an increase in perforin mRNA could be detected by 4 h; they peaked at 12 h, and decreased after 24 h. The perforin mRNA was also induced in T cells treated with a combination of phorbol ester PMA plus lectin or OKT3 mAb. This latter induction followed slower kinetics, peaking at 48 h. For all three mitogens used, even at peak induction times less than 10% of T cells were labeled with perforin probe. Similar patterns of mRNA expression were observed for both unprimed T cells and lectin-primed T blasts. The induction response of mRNA due to IL-2 stimulation is probably mediated by the IL-2 receptor p75 chain since its mRNA was upregulated by IL-2 with a kinetics comparable to that associated with an increase of perforin mRNA. The p55 IL-2 receptor chain increased much more slowly than p75.


Effect of beta 2-microglobulin on immunoglobulin production.

Chronic renal failure patients display high serum levels of beta 2-microglobulin (beta 2-M) and a pronounced defect in immunoglobulin production. In this light, the present study was conducted to investigate whether beta 2-M might influence the rate of antibody synthesis. Peripheral blood mononuclear cells (PBMC) from healthy subjects were cultured in the presence of beta 2-M (80 mg/l) for 7 days. Immunoglobulin concentrations were measured by ELISA-techniques in culture supernatants. Basal IgM levels were 390 +/- 10 ng/ml and increased to 940 +/- 216 ng/ml in the presence of beta 2-M. After PWM stimulation, IgM concentrations were 2654 +/- 614 ng/ml without and 3750 +/- 914 ng/ml with beta 2-M. IgG and IgA production by PBMCs was not influenced by beta 2-M in the culture medium. Likewise, the generation of beta 2-M by PBMCs was unchanged in the presence of exogenous beta 2-M in the medium. In terms of clinical relevance, serum beta 2-M levels of 28 uremic patients were correlated with capacity of their PBMCs for immunoglobulin production in vitro. However, there was no apparent correlation between beta 2-M serum levels and immunoglobulin synthesis in vitro. Taken together, beta 2-M seems to have a stimulatory effect on IgM synthesis in vitro. Whether this has some clinical meaning in patients with chronic renal failure remains to be investigated.


Anti-leukocyte antibodies as a consequence of HIV infection in HIV+ individuals.

Antibodies (Ab) that induce antibody-dependent cell-mediated cytotoxicity (ADCC) against non-T lymphocytes, anti-HLA class II specific Ab and anti-PMN were tested in hemophilic (He) patients who were alloimmunized because they had received replacement treatment with blood derivates and become infected with HIV, as well as in those who remained seronegative. In addition, the serum reactivity of spouses of HIV+ individuals and their children was studied to determine the effect of HIV infection in the absence of concomitant alloimmunization. The results of this study indicate that ADCC Ab were already present in HIV- He, suggesting the influence of alloimmunization. Their titer increased after appearance of HIV disease. While low reactivity against class II antigens was observed in HIV- He, activity augmented sharply after HIV infection and increased further with disease progression. Anti-PMN reactivity followed a similar pattern. Anti-class II, ADCC Ab and anti-PMN were also detected in the asymptomatic HIV+ spouses of HIV+ patients in titers that were similar to those of asymptomatic HIV+ He. In children born to HIV+ mothers in whom HIV infection was confirmed, anti-class II, ADCC Ab and anti-PMN reactivity were also observed, and activity increased after the onset of disease. These results suggest that induction of anti-leukocyte Ab occurs in the absence of massive allostimulation after HIV infection. HIV infection may enhance preexisting class II and anti-leukocyte response in allostimulated individuals.


Extracellular diaphorase-like activity as a marker of cytolytic damage to cells.

A new variant of the enzyme release colorimetric method for NK/LAK cytotoxicity is described. Kinetic determination of diaphorase released from damaged target cells (TC) provided an exact measurement of the lytic activity of effector cells (EC). A polyenzyme testing system permitted the use of a wide spectrum of TC. The percentage cytotoxicity and the kinetics of the process coincide, within experimental error, with data from staining with Trypan blue. The method is simple and convenient and can be applied to study cell-mediated cytotoxicity (CMC).


H2 genotype and IgE immune response to Fel dI, the cat major allergen, in mice.

Mice of 6 strains were immunized with a highly purified Fel dI allergen adsorbed to alum. Their ability to display a significant IgE response was detected via passive cutaneous anaphylaxis (PCA) tests, performed in rats. Linkage of the responsiveness to the H2 genotype is not totally obvious. IgE response can be delayed, particularly in the SJL strain (H2-s histocompatibility allele), and lacking in the C57 B1/6 strain (H2-b). Mice with H2-k, H2-d alleles and the B6D2F1 hybrid H2-b/d are good responders, leading to the hypothesis that the IgE response to Fel dI may be related to the H2-d allele. For all good responders, individual variations are rather important. All our observations show that mice perfectly mimic human hypersensitization to the cat major allergen Fel dI, at least where the IgE response is concerned.


In vitro bioassay for type E prostaglandins based on their NK immunodepressing activity.

A biological assay for PGE in commercial preparations, or secreted by tumor cells in culture fluid was developed on the basis of the immunosuppressive effect of PGE on the cytotoxic activity of NK cells. The assay is simple, rapid and convenient for detecting PGE cell secretion, either spontaneous or induced by various signals. The sensitivity of the bioassay is limited by the sensitivity of NK cells to the immunosuppressive activity of PGE (i.e., about 10(-8) M).


Radioresistant cells of thymus--producers and targets of thymocyte growth factor and their possible role in postradiation restoration of thymus.

In the present study the association between the damaging and the stimulating action of radiation on the cells of thymus (in particular intrathymic T-lymphocyte precursors, TLP) is analyzed. The radioresistance and possibility to secrete humoral products, in particular, the thymocytes growth factor, THGF, is assessed. The possible role of THGF in the postradiation restoration of thymus is also discussed.


Down-regulation of murine B lymphocyte growth: arrest of B cells in G1 underlies immunosuppression induced by an IgM antibody.

Previous studies have demonstrated that culture supernatants of LPS-stimulated murine splenic B lymphocytes (BLPSSN) are able to inhibit the growth of freshly isolated B cells via an IgM antibody. In this work we investigated the progress of LPS-activated B lymphocytes through the cell cycle in the presence of this antibody. We found that the regulatory IgM did not affect the entry of LPS-stimulated B lymphocytes into G0*, as assessed by the increased expression of I-A antigens. Events that characterize the early G1 phase (G1A), such as cell enlargement and increased RNA synthesis, also occurred in the presence of the antibody. In contrast, events which mark the G1B phase, such as further cell enlargement and late RNA synthesis were inhibited. Moreover, a significant portion of the cells failed to incorporate [3H]thymidine and did not progress through S, G2, or M, as revealed by their DNA content. Therefore, our work points toward a well-defined stage of the early G1 phase at which the antibody inhibits the progression of B lymphocyte activation. This result shows a new insight into the mechanism of antibody-mediated down-regulation of polyclonal B cell responses.


Analysis of clonality of tumour infiltrating lymphocytes in breast cancer and uveal melanoma.

Fresh tumour infiltrating lymphocytes (TILs) from 6 uveal melanomas and 4 breast cancers were analysed by flow cytometry with a panel of 6 monoclonal antibodies to V beta regions of the T cell receptor (V betas 5a, 5b, 5c, 6, 8a and 12a). With a single exception where one TIL sample lacked V beta 12a, lymphocytes from both tumour and blood contained cells reactive with all 6 probes, and no probe was highly dominant or missing. The proportions of reactive cells differed between tumour and blood within each patient. The data indicate that while tumour infiltrating lymphocytes have a capacity to locate selectively within the tumour they nonetheless comprise a population expressing a diversity of TCR V beta genes.


Skin response to tuberculin in pig foetuses and germ-free piglets.

Cellular response to intradermally administered PPD (2 TU) was demonstrated in pig foetuses of various ages and in germ-free piglets. CD2+, CD4+, CD8-, 86D-, SLA-D- T lymphocytes were the predominant cells in the skin tuberculin reaction in both foetuses and germ-free animals. Reactive T cells were observed as early as in mid-gestation, whereas SLA-D+ (porcine MHC class II) cells appeared only in older foetuses. Polymorphonuclear leukocytes were never observed in the PPD reaction.


Sequence and chromosome assignment to 11p13-p12 of human RAG genes.

The recombination-activating genes RAG-1 and RAG-2 are required for V(D)J DNA rearrangements at loci for immunoglobulin and T cell receptor genes. We isolated the human RAG-2 gene and determined its nucleotide sequence. Mapping analysis of RAG-1 and RAG-2 genes on human chromosomes by fluorescence in situ hybridization indicated that the genes are located on chromosome 11p13-p12. RAG-1 and RAG-2 do not seem to be linked to any of the primary immunodeficiencies for which defective genes have already been mapped.


A fluorescent molecular rotor probes the kinetic process of degranulation of mast cells.

A confocal fluorescence microscope was used to study the exocytotic secretory processes of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its quantum yield with decreasing intramolecular rotation. Here, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramatically in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D (10 microM). However, fluo-3 fluorescence intensities still increased after addition of compound 48/80.


A monoclonal antibody directed against the sporozoite stage of Plasmodium vivax binds to liver parenchymal cells.

The circumsporozoite (CS) protein of malaria parasites is a major surface protein of the sporozoite stage. In the process of investigating the immunogenicity of this protein in the Plasmodium vivax complex, we found that a monoclonal antibody (mAb) directed against the CS protein of isolates of P. vivax recognizes New World monkey hepatocytes and human hepatoma cells HepG2A16 in Western blot and by immunoelectron microscopy. The mAb NVS3 binds to the amino acid sequence AGDR, which is also shared with the alpha 3 domain of the human and primate major histocompatibility complex class I. In addition, in vitro experiments suggest that the binding of the mAb NVS3 to hepatocytes from Saimiri monkey enhances the invasion or development of malaria sporozoites. These results form the basis for investigating the relationships between parasite surface proteins and host-cell receptors.


Anti-TCR-V beta 6 breaks tolerance in female AKR, Mlsa mice inducing foetal lethality.

Immunisation with anti-TCR-V beta 6 of female AKR mice prior to and during syngeneic pregnancy resulted in neonatal lethality and eventually in abortion or foetal resorption. The sera of the hyperimmunised mothers were shown to have anti-H-2k and anti-Mlsa autoantibodies and were cytotoxic to H-2k targets in vitro and also blocked Mlsa-induced mixed lymphocyte reactions. These observations are discussed herein.


Mhc-DRB diversity of the chimpanzee (Pan troglodytes).

Fifty-four chimpanzee Patr-DRB and five human HLA-DRB second exons were cloned and sequenced from thirty-five chimpanzees and four human B-cell lines and compared with known Mhc-DRB sequences of these two species. Equivalents of the HLA-DRB1*02, -DRB1*03, -DRB1*07 allelic lineages and the HLA-DRB3, -DRB4, -DRB5, -DRB6, and -DRB7 loci were all found in the chimpanzee. In addition, two chimpanzee Patr-DRB lineages (Patr-DRBX and -DRBY) were found for which no human counterparts have been described. None of the Patr-DRB sequences is identical to known HLA-DRB sequences. The Patr-DRB1*0702 and HLA-DRB1*0701 alleles are the most similar sequences in a comparison between the two species and differ by only two nucleotides out of 246 sequenced. Equivalents of the HLA-DRB1*01, -DRB1*04, and -DRB1*09 alleles were not found in our sample of chimpanzees. A per locus comparison of the number of Patr-DRB alleles with the HLA-DRB alleles shows that the Patr-DRB3, -DRB4, -DRB5, and -DRB6 locus are, thus far, more polymorphic than their human homologs. The polymorphism of the Patr-DRB1 locus seems to be less extensive than that reported for the HLA-DRB1 locus. Nevertheless, the Patr-DRB1 locus seems to be the most polymorphic of the Patr-DRB loci. Phylogenetic analyses indicate that the HLA-DRB1*09 allele may have originated from a recombination between a Mhc-DRB5 allele and the DRB1 allele of a Mhc-DR7 haplotype. Although recombination seems to increase the diversity of the Patr-DRB alleles, its contribution to the generation of Patr-DRB variation is probably low. Hence, most Patr-DRB diversity presumably accumulated via recurrent point mutations. Finally, two distinct Patr-DRB haplotypes are deduced, one of which (the chimpanzee equivalent of the HLA-DR7 haplotype) is probably older than 6-8 million years.


Trans-species origin of Mhc-DRB polymorphism in the chimpanzee.

Trans-specific evolution of allelic polymorphism at the major histocompatibility complex loci has been demonstrated in a number of species. Estimating the substitution rates and the age of trans-specifically evolving alleles requires detailed information about the alleles in related species. We provide such information for the chimpanzee DRB genes. DNA fragments encompassing exon 2 were amplified in vitro from genomic DNA of ten chimpanzees. The nucleotide sequences were determined and their relationship to the human DRB alleles was evaluated. The alleles were classified according to their position in dendrograms and the presence of lineage-specific motifs. Twenty alleles were found at the expressed loci Patr-DRB1, -DRB3, -DRB4, -DRB5, and at the pseudogenes Patr-DRB6, -DRB7; of these, 13 are new alleles. Two other chimpanzee sequences were classified as members of a new lineage tentatively designated DRBX. Chimpanzee counterparts of HLA-DRB1*01 and *04 were not detected. The number of alleles found at individual loci indicates asymmetrical distribution of polymorphism between humans and chimpanzees. Estimations of intra-lineage divergence times suggest that the lineages are more than 30 million years old. Predictions of major chimpanzee DRB haplotypes are made.


Evolution of the major histocompatibility complex: a hundred-fold amplification of MHC class I genes in the African pigmy mouse Nannomys setulosus.

The genome of the African murine rodent Nannomys setulosus was found to harbor several thousand major histocompatibility complex (MHC) class I genes instead of the 30-40 genes found in conventional laboratory mice, which are mostly of Mus musculus domesticus origin. Other genes of N. setulosus, either functionally or physically linked to class I genes, are not amplified. Amplified genes derive from as few as three ancestors and amplification has likely occurred after the divergence of the two Nannomys species, N. setulosus and N. minutoides, which took place about three million years ago. Amplified genes are mostly pseudogenes. Statistical analysis of dinucleotide frequencies leads us to propose that inactivation of the genes has occurred through the repeat induced mutation process, a possible "newcomer" in the evolution of the MHC.


Transcription and diversity of immunoglobulin lambda chain variable genes in the rat.

In order to determine the extent of the repertoire of the immunoglobulin light chain V-region locus (Igl-V) in the laboratory rat (Rattus norvegicus), we constructed a specifically primed cDNA library from lipopolysaccharide-stimulated DA strain rat spleens. The library was screened with a rat Igl-C2-specific probe, and 33 clones containing identifiable V regions were sequenced, of which 19 sequences are presented here. In addition to one sequence (Igl-V1) which was already known, and is closely related to the two known mouse V lambda gene segments, clones encoding representatives of three new, distantly related, rat Igl-V subfamilies were found, namely Igl-V2, Igl-V3, and Igl-V4. At least two of these sub-families, Igl-V2 and Igl-V3, contain multiple members as well as restriction fragment length polymorphism variants, indicating the presence of at least 10-15 Igl-V gene segments (including some pseudogenes) in the rat genome. An additional ten clones contained no rearranged V region, although they showed a correct J-C splice, suggesting the presence of cryptic transcriptional promoters between J lambda and the 3'-most Igl-V gene segment. Phylogenetic tree reconstruction based on amino acid sequence alignments showed at least three of the four rat Igl-V sequences clustering with distinct human Igl-V genes. Thus, although rats express lambda-bearing Ig at levels no higher than mice, the rat Igl-V locus is considerably more complex than that of laboratory mice, and its diversity reflects the products of gene duplications which predate the time of primate/rodent divergence.


Transmembrane and cytoplasmic domain sequences demonstrate at least two expressed bovine MHC class I loci.

We have used the polymerase chain reaction to amplify cDNA from expressed bovine major histocompatibility complex class I genes. Sequences obtained from transmembrane and cytoplasmic domains were used to identify the number of expressed alleles. Data from three animals suggest that there are four major expressed alleles, representing the products of two (or more) loci. We have also demonstrated the presence of an alternatively spliced mRNA, which has been observed in five animals. The alternative splicing removes exon 7 (the major site of class I phosphorylation), which predicts a truncated molecule with a cytoplasmic portion 16 amino acids shorter than usual. This phenomenon was detected for only a single class I allele within each individual.


Two subtypes of HLA-B51 differing by substitution at position 171 of the alpha 2 helix.

Newly defined antigens of the B5, B35 cross-reacting group have been found in Japanese and North American Indians. Nucleotide sequencing of the alleles encoding the Japanese B5.35 antigen and the variant B5 antigen from the Piman Indians show them to be identical. This new allele, B*5102, differs from B*5101 by a single nucleotide substitution that changes residue 171 from histidine to tyrosine. Residue 171, which is part of the alpha 2 helix, is believed to contribute directly to peptide interaction in the A pocket of the binding groove and is either histidine or tyrosine in all HLA-A, B, C heavy chains. Tyrosine 171 is shared by B*5102, B*3501, B*3502, and B*5301 and must be responsible for the serological cross-reactivities of these molecules not shared with B*5101. Stimulation of lymphocytes from a B*5101 positive donor with B*5102 positive cells failed to generate cytotoxic T cells with specificity for the difference between these molecules. However, one out of five clones of cytotoxic T cells raised against B*5101 failed to lyse targets expressing B*5102. Substitution of histidine for tyrosine at residue 171 affected recognition of HLA-B35-restricted human minor histocompatibility antigen-specific T cell clones.


Human immunity to Schistosoma mansoni: observations on mechanisms, and implications for control.

This review summarizes the personal experiences of the authors and their colleagues during ten years of field and laboratory studies on human immunity to Schistosoma mansoni infections. There is evidence for the very slow development with age of an acquired resistance to reinfection (demonstrable after chemotherapy of the primary infection) distinguishable from a lack of reinfection due to reduced exposure. The implications of this immunity for the design of chemotherapy programs targeted at infected schoolchildren are discussed. Observational studies on the mechanisms of immunity have demonstrated a marked helminthocidal capacity of eosinophils. Subsequent correlative studies have indicated a role of IgM and IgG2 "blocking" antibodies in maintaining the continued susceptibility of young children, and of IgE antibodies in mediating protection in older individuals. Some problems in studying human immunity, and the implications for vaccine development, are also discussed.


Environmental, genetic and immunological factors in human resistance to Schistosoma mansoni.

The design of programs for the control of endemies requires the knowledge of the principal factors that determine parasite transmission and infection levels in exposed populations. In the studies summarized in this article, the role of environmental and host specific factors in the infection by S. mansoni have been evaluated. It is shown that a limited number of factors actually influences infection intensity: water contacts, age, and sex were shown to account for 20 to 25% of infection variance, while 35 to 40% of it was accounted for by the effect of a major codominant gene. A remarkable fact is the important weighting (around 55% of the variance) of factors (the major gene and age) that influence human capacities of resistance. This observation strongly supports control measures aimed at increasing human resistance, such as vaccination. The effect of age on the development of resistance has now been observed in several studies on S. mansoni or S. haematobium. It is, therefore, a constant finding in schistosomiasis infections that resistance develops extremely slowly requiring a long period of exposure to the parasite and repeated infections. These studies provide strong incentives to increase efforts in the evaluation of the immune response of subjects living in endemic areas. Such evaluations are necessary to define vaccine and vaccination programs, and they are also urgently needed to evaluate the effects of chemotherapy on the development of immunity in children and adolescents, as well as on the persistence of protective immunity in adults. Immunological studies begin to provide a clearer picture of the role of acquired immunity in human protection against S. mansoni. It is increasingly clear that the slow development of resistance in children, as well as its alteration in certain age groups, are related to the maturation of parasite specific immunity and its alteration by specific immune factors. Thus, the development of resistance is associated with the maturation of IgE-dependent immunity, whereas blocking Ab may interfere in children and adolescents with the expression of full resistance. This finding raises the question as to whether a vaccine could include major allergens without triggering the well-known deleterious side effects associated with hypersensitivity reactions. The absence of such reactions in subjects with high parasite-specific IgE levels who are exposed to daily infections suggests that this may be feasible.


Experimental models of immunization against schistosomes: lessons for vaccine development.

Schistosomiasis is a debilitating, and sometimes deadly, parasitic infection that afflicts hundreds of millions of people living in developing countries. One of the best hopes for control of this disease is vaccine development. Studies on experimental models of attenuated vaccines have proven that high levels of protective immunity can be achieved. In these systems, resistance has been shown to be directed against the migrating larval stages of the parasite and to have both cellular and humoral components. Several candidate vaccine immunogens have been identified on the basis of antibody reactivity. However, the level of protection induced by immunization with nonliving vaccines has at yet not approached the level observed with attenuated infection. Current challenges to vaccine development include identification of protective T cell immunogens, determination of ways to strengthen the immunogenicity of isolated parasite antigens, and development of methodologies to selectively stimulate protective, as opposed to ineffective or even detrimental, immune responses.


Linkage between monokine production and regulation of the negative surface charge density of human monocytes.

The regulation of the negative surface charge density of human monocytes was investigated with the help of the synthetic glycolipid analogue BAY R 1005. This compound is incorporated into the outer membrane of isolated monocytes during 24 hours of incubation. After this time the electrophoretic mobility (EM) of monocytes is unchanged at 0.95 x 10(-4) (cm2 V-1 s-1) and remains unchanged even under conditions where non-treated monocytes increase their EM up to 1.1 x 10(-4) (cm2 V-1 s-1). In addition BAY R 1005 stops differentiation of monocytes to macrophages, it triggers monokine production and abolishes monocyte suppressor activity and spreading capability. The results show that BAY R 1005 affects intracellular features. In connection with earlier investigations of the regulation of the negative surface charge density of human monocytes (1,2) the study suggests that monokine production and maintenance of the EM of monocytes are linked.


Surface matrix binding alters murine peritoneal mononuclear phagocyte TNF-alpha and IL-6 induction.

Interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS) are important promotors of mononuclear phagocyte (MO) activation. Signals derived from binding to a surface matrix also participate in promoting the activation process of MO. In this study, we examined the relative contribution of adherence in augmenting murine MO activation for cytokine production. Kinetic studies compared the production and secretion of tumor necrosis factor-alpha (TNF) and interleukin-6 (IL-6) by MO cultured as adherent monolayers to those of MO cultured as suspended cells in teflon vessels. All cells were maximally stimulated in vitro with IFN-gamma and LPS prior to analysis. Immunoprecipitation analysis of protein and RNA slot blots showed that both secreted protein and mRNA representing TNF and IL-6 are delayed two to six hours in nonadherent MO cultures compared to adherent MO cultures. Moreover, data from bioassays confirmed that these cytokines were completely functional in both systems examined. Although IFN-gamma/LPS were able to stimulate production and secretion of TNF and IL-6 in the nonadherent cells, without cell-matrix interaction, the process was significantly delayed. These data support the hypothesis that the physical event of adherence significantly facilitates the production of specific cytokines by activated MO.


A flow microsphere inhibition immunoassay (FMII) for detecting paratope binding anti-idiotypic antibodies.

A simple, rapid and reproducible flow microsphere inhibition immunoassay (FMII) has been developed to detect the ability of paratope specific anti-idiotypic antibody (anti-Id or AB2) to inhibit antigen binding to the corresponding paratope of the Id (AB1). To evaluate the FMII as a measurement of paratope binding anti-Id, an avian model was used to produce Id and anti-Id antibodies for the study. Both antibody to bovine serum albumin (BSA Id) and anti-BSA Id were produced in white leghorn chickens and affinity isolated from egg yolks. The anti-BSA Id samples were incubated with BSA Id coated microspheres, then without rinsing, fluoresceinated BSA (BSA-FITC) was added for a short incubation period and the resulting decrease in fluorescent intensity was used to calculate the extent of inhibition. For validation, statistical comparisons of the line equations generated by BSA dilution curves and anti-BSA Id dilution curves were performed. Replications within each ligand were not significantly different which indicated the assay was reproducible for determining the presence of paratope reactive anti-BSA Id used in this model.


A simple and inexpensive glucose oxidase substrate system for enzyme immunoassay.

A simple and inexpensive glucose oxidase substrate system was developed for enzyme immunoassay. This system is based on the formation of a coloured complex between polyvinyl alcohol or starch and iodine produced from iodide in the presence of hydrogen peroxide generated by the glucose oxidase reaction. The rate of iodine production was enhanced by the supplementation of molybdate. In an enzyme immunoassay for anti-Salmonella antibodies using glucose oxidase as the indicator enzyme, the molybdate-enhanced polyvinyl alcohol- and starch-glucose-iodide substrate systems were as sensitive as a conventional glucose oxidase assay system employing horseradish peroxidase as a secondary enzyme and a suitable hydrogen-donating chromogen.


The detection of a novel neutrophil-activating peptide (ENA-78) using a sensitive ELISA.

Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides (less than 10 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C) and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophil-activating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GRO alpha; 52%), and IL-8 (22%). In addition, ENA-78 appears to activate neutrophils through the IL-8 receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.


Tense ascites redefined: renal consequences of tense ascites in decompensated cirrhotics.

Cirrhotics with tense ascites fail to achieve increased diuresis in the supine position. To assess the role of inferior vena cava compression in this phenomenon, we studied cirrhotics with mild to moderate (n = 11) and tense (n = 2) ascites, and patients with membranous inferior vena cava obstruction (n = 2) before and after balloon dilatation, in the sitting, supine and 10 degrees head down tilted positions for 2 hours each. Urinary output (p < 0.005), creatinine clearance (p < 0.025) and sodium excretion (p < 0.025) increased in cirrhotics with mild to moderate ascites in the supine position, and further in the head down position. Similar changes occurred in patients with inferior vena cava membrane. In cirrhotics with tense ascites, these parameters did not change significantly in the supine position, but increased in the head down position. We conclude that failure to augment diuresis in the supine position in cirrhotics with tense ascites is not due to inferior vena cava obstruction alone but is probably also due to compression of the collateral vessels. This clinical observation may serve as a criterion for diagnosing tense ascites.


Magnetic resonance imaging in pancreatic diseases: comparison with other imaging techniques.

Magnetic resonance imaging (MR) was performed in 14 healthy subjects and 16 patients with pancreatic disease. All the 16 patients were subjected to ultrasonography (USG), computed tomography (CT) and MR while endoscopic retrograde cholangiopancreatography (ERCP) was performed in 10 cases. In one patients with adenocarcinoma and two with gastrinoma, MR demonstrated abnormalities while USG and CT were normal. MR was, however, inferior to ERCP in demonstrating ductal abnormalities in chronic pancreatitis. Our initial experience suggests that MR is superior to other imaging modalities in the diagnosis and staging of pancreatic tumors; however, it is inferior to ERCP in the diagnosis of pancreatitis.


Gastrointestinal endoscope disinfection practices in India: results of a national survey.

A questionnaire was sent to 435 endoscopy centers in the country to obtain information regarding current endoscopic disinfection practices. Of these, 133 (30.6%) centers responded. Adequate disinfection (a minimum exposure to 1% glutaraldehyde for 2 minutes) before the start of endoscopy sessions and between procedures was practised in 61 (46%) and 45 (34%) centers respectively. The proportion of centers practising adequate disinfection was similar among those performing < 1500 and > 1500 endoscopies/year (50% vs 36%; p = ns). Twenty two (17%) centers used some additional precautions in patients with hepatitis B virus infection or immunocompromised states. Two (1.5%) of the 133 centers performing upper GI endoscopies and eight (18%) of the 44 centers performing ERCP examinations reported occurrence of infections following these procedures. Allergic reactions to disinfectants were reported by five (4%) centers. We conclude that only about a third of the gastroenterology centers in the country are practising adequate endoscope disinfection routinely.


Out-patient esophageal dilation: an experience in 130 patients using Savary-Gilliard dilators.

Experience of outpatient esophageal dilation using Savary-Gilliard dilators in 130 patients during a 17 month period is reported. The strictures were radiation induced (48), tumoral stenoses (40), anastomotic (16) and due to other causes (26). Both fluoroscopy and endoscopy were used in 58%, endoscopy alone in 23% and fluoroscopy alone in 19% of patients for placement of the guide wire. One hundred and twenty six stricture dilations (97%) were technically successful. Eighty one (62%) patients could be dilated to 14 mm or more. One hundred and nineteen (94%) patients were dilated in one or two sessions. Among the 109 patients who followed up, dilation was successful in providing adequate dysphagia relief in 97 patients and facilitated the performance of other therapies in 9 patients. Major complications included severe continuous chest pain (1 case), hematemesis (2), fever (4) and transient stridor (2). The patient with persistent pain and 9 patients undergoing other therapies required hospitalization. There were no perforations or death. We conclude that esophageal dilation with Savary-Gilliard dilators is safe and effective even in tumors and post-radiation strictures. It can be performed on an outpatient basis in a majority of patients.


Endoscopic sclerotherapy versus propranolol in prevention of recurrent variceal bleeding in patients with child's B and C cirrhosis: a preliminary report.

Thirty two patients with cirrhosis of the liver of Child's B and C class and an episode of endoscopically proven variceal bleed were randomly assigned to receive endoscopic sclerotherapy (EST) or oral propranolol for the prevention of recurrent upper gastrointestinal bleeding. EST was performed at 3 week intervals using 1% polidocanol intravariceally, till eradication of varices. Propranolol dose was adjusted to reduce the resting heart rate by 25% of the basal value (mean +/- SD, 194.3 +/- 63.9 mg/day) Two patients in the propranolol group were excluded within 48 hours due to side effects of the drug. Thirty patients (EST-16, propranolol-14) completed the trial. Patients were followed up for a maximum of 480 days. Mean follow-up in the EST and propranolol groups was 217 and 243 days respectively. The median bleeding free intervals were 480 and 194 days and number of rebleeding episodes was eight and 16 respectively in the EST and propranolol groups (both p = ns). Our study suggests a trend in favor of EST in preventing variceal rebleeding in patients with hepatic cirrhosis who belong to Child's B and C classes.


Comparative study of omeprazole and famotidine in the treatment of duodenal ulcer.

In a double blind, multicenter, parallel group clinical trial in patients with symptomatic duodenal ulcers, 129 patients were randomized to receive either omeprazole 20 mg once daily (n = 65) or famotidine 40 mg once daily (n = 64) for 2 weeks, and if the ulcers were not healed, for a total of 4 weeks. Seventy four percent of these receiving omeprazole had healed ulcers after 2 weeks compared with 34.3% of those receiving famotidine (p < 0.001). At 4 weeks, the respective figures were 97.3% and 77.6% (p < 0.001). After 2 weeks of treatment, only 11.1% and 29.8% of omeprazole and famotidine treated patients respectively had day time pain (p < 0.02). Diary cards (successfully completed by 2/3rd of patients) showed that omeprazole treated patients required smaller amounts of antacids (p = ns). Over the first two weeks, ulcer healing rate was similar in smokers and non- smokers. No significant side effects were reported in either group. Omeprazole 20 mg/day provides more rapid relief of symptoms and heals a greater proportion of duodenal ulcers at 2 and 4 weeks than famotidine 40 mg/day.


A study of Helicobacter pylori in north Indian subjects with non-ulcer dyspepsia.

A prospective case control study was conducted in 50 patients with non-ulcer dyspepsia (NUD) and 10 age and sex matched controls to determine the prevalence of Helicobacter pylori in patients with NUD and to correlate symptoms, histology and presence of H pylori in gastric biopsies. Endoscopic biopsies from antrum and fundus were subjected to urease test and histological examination. On histology, H pylori was identified in 27 (54%) NUD patients and in one of 10 controls. Urease test was positive in 31 (62%) antral and 21 (42%) fundal biopsies in patients with NUD and in only one (10%) of 10 antral biopsies in the control group. On histology, gastritis was present in the antrum in 46 (92%) NUD patients and in the fundus in 40 (80%) cases; of these, 27 (54%) and 15 (30%) had H pylori in antral and fundal biopsies respectively. The severity of antral gastritis correlated with the density of H pylori (p < 0.05).


Frequency of hepatitis B, C and D and human immunodeficiency virus infections in multi-transfused thalassemics.

Of forty multi-transfused thalassemia patients (26 males, 14 females; mean age 8.1 +/- 5.3 years, range 1-35) with no clinical or biochemical evidence of liver disease, HBsAg, anti-hepatitis C virus and anti-human immunodeficiency virus antibodies were present in 18 (45%), 7 (17.5%) and 1 (2.5%) cases respectively. Three of the 18 (16.7%) HBsAg positive patients were anti-delta antibody positive. Our results indicate that more than 50% of multi-transfused thalassemia patients show serological evidence of one or more of hepatitis B, C and D and human immunodeficiency virus infection.


Coarctation of inferior vena cava in situs inversus totalis.

A woman aged 28 years with situs inversus totalis presented with chronic Budd-Chiari syndrome with symptoms of 3 years' duration. The left sided inferior vena cava was occluded below the diaphragm above a patent morphologic right hepatic vein. A dorsal cavoatrial bypass was attempted but abandoned due to bleeding from extensive collaterals. The occurrence of coarctation of the inferior vena cava in situs inversus totalis lends support to the view that it is a congenital condition and occurs due to disturbance in fusion of the hepatic segment of the inferior vena cava and the hepatocardiac channel.


Periampullary carcinoma in a young female with situs inversus.

Periampullary carcinoma in a young female with situs inversus viscerum is reported. Endoscopy was difficult because of the altered anatomy.


Intestinal endometriosis.

Two cases with intestinal endometriosis, one with a right iliac fossa mass and the other with subacute intestinal obstruction, are presented. In both the cases, the diagnosis was not suspected clinically and was reached only on histological examination of the resected specimens.


Intussuscepting ileal hemangioma with perforation.

A rare case of solitary intussuscepting capillary hemangioma in the distal ileum with perforation and peritonitis is presented. The diagnosis was made only at surgery for presumed acute intestinal obstruction.


Intermittent intravenous pulse cyclophosphamide treatment in systemic lupus erythematosus.

To determine the efficacy and safety of intermittent intravenous pulse cyclophosphamide in patients of severe systemic lupus erythematosus (SLE), 50 patients having severe/refractory lupus nephritis, vasculitis or neuropsychiatric manifestations were treated with 3 weekly pulses of cyclophosphamide for 6 such pulses. This treatment was found to be associated with significant and sustained improvement during a 2 yr follow up with respect to the mean renal activity score, individual renal parameters (proteinuria, erythrocyturia, and serum creatinine levels), focal neurological manifestations, vasculitic lesions, antinuclear antibody titers, complement component C3, anti-dsDNA antibodies levels and ESR. There was a sustained decrease in the overall mean disease activity score, and the mean daily dose of prednisolone (pretreatment 32.62 mg daily to 3.75 mg daily after 24 months). There was a significant decline in the percentage and absolute B cell count after 7, 14 and 21 days of this treatment. Effect on other lymphocyte subsets (CD3+, CD4+ and CD8+) was not marked. Pulse cyclophosphamide could therefore be an effective and less toxic form of treatment in patients with SLE having severe lupus nephritis, focal neurological lesions or vasculitis.


In vitro effect of intravenous immunoglobulin supplementation on serum opsonic activity in tracheostomised patients.

An in vitro study of the effect of immunoglobulins (mainly IgG) on opsonophagocytic activity of polymorphs was carried out in 17 tracheostomised patients admitted in medical intensive care unit of our hospital. The opsonic and phagocytic activities were tested against Staphylococcus aureus by modified polymorphonuclear leucocyte overlay method; and serum IgG and serum IgM levels were estimated by single radial immunodiffusion technique. As compared to healthy volunteers, opsonophagocytic activity was significantly lower in tracheostomised patients. However, this activity improved markedly after immunoglobulin supplementation (P less than 0.01). The same degree of enhancement was also observed in normal controls.


Alloimmunisation to platelet transfusions in the Indian patients.

Twenty nine patients of acute and chronic leukemia undergoing chemotherapy and receiving blood transfusions (BT) and platelet transfusions (PT) from random donors (3-20 PT from 4-56 donors) were followed up for alloimmunisation using the platelet immunofluorescence test. Two women patients aged 65 and 28 yr reacted positive. Both patients had received multiple BT but no PT at the time of testing. Both were parous women. Our results point to the need to test for alloimmunisation prior to starting PT in parous women who have received multiple BT, although a study on larger number of patients is necessary for confirmation.


Epidemiological study of coronary heart disease in Gujaratis in Delhi (India).

A community based survey of coronary heart disease (CHD) was carried out in Gujarati families settled in Delhi. The number of adults surveyed in the age group 25-64 yr was 1317. CHD was diagnosed either on the basis of clinical history supported by documentary evidence of treatment in the hospital or at home or on ECG evidence in accordance with the Minnesota Code. The prevalence rate of CHD on clinical history was 25.1 (28.2 in males and 22.4 in females) per 1000 adults (25-64 yr). The prevalence rates were slightly lower in Gujaratis than the general Delhi urban population. The prevalence rate based on both clinical history and ECG criteria was estimated at 66.8 as compared to 96.8/1000 in general urban Delhi population. The risk factors for CHD such as socio-economic status, family history, obesity, smoking, physical activity and hypertension were studied. The mean and 5th, 50th and 95th percentile values of blood lipids were also estimated in CHD patients and compared with the control group. Hypertension ranked the leading risk factor. Prevalence rate of CHD was higher in the upper socioeconomic group. The positive correlation of higher levels of serum lipids e.g., total cholesterol, low density lipoprotein cholesterol (LDL-C) and triglyceride with CHD was confirmed.


A rapid survey on substance abuse disorders in the urban slums of New Delhi.

The results are presented of a rapid survey screening method of general population for identifying dependent and non dependent drug abusers, especially in vulnerable groups such as slum dwellers. The results showed that with the help of instrument with modified DSM III and interviewing only the heads of the households it was possible to get reliable estimates of dependence disorder in the community. This method can be of assistance to health planners for a quick assessment of the magnitude of the problem leading to better allocation of funds and developing services for the affected population. The method is an improvement on key informant technique and a full fledged survey.


Comparative influence of vanillin & capsaicin on liver & blood lipids in the rat.

The influence of vanillin (0.15 mg %) and capsaicin (0.3 mg %) on liver and blood lipids in growing female Wistar rats was determined, when fed along with a normal diet. Vanillin, like capsaicin, decreased the serum triglycerides and the triglycerides associated with lipoproteins viz., VLDL+LDL and HDL with a tendency to lower liver triglycerides as well. Cholesterol and phospholipids in liver and serum were unaffected. The accumulation of triglycerides in serum after Triton WR-1339 blocking was higher in animals fed a vanillin supplemented normal or hypertriglyceridemic diet than in the corresponding controls. The accumulation of triglycerides in serum with capsaicin supplementation with either diet was much higher than with vanillin supplementation.


Comparative study on determination of alpha-hydroxybutyrate dehydrogenase, lactate dehydrogenase isoenzyme LDH-1 by adsorption on DEAE Sephadex A-50 & electrophoresis.

Serum alpha-hydroxybutyrate dehydrogenase (HBDH) and lactate dehydrogenase isoenzyme (LDH-1) levels by two techniques viz., adsorption on DEAE Sephadex A-50 and by electrophoresis were estimated in healthy subjects and patients with testicular tumours. The correlation coefficient between HBDH and LDH-1 by adsorption technique was found to be r = 0.8. HBDH was found to have a value almost twice that of LDH-1 by adsorption and electrophoretic technique and the activity of LDH-1 obtained by two methods and of HBDH in normals and patients with testicular tumours could be converted using a factor of 0.557. The quantitative method used for determination of HBDH is reliable, accurate, simple and rapid and therefore has better value in a clinical setting than electrophoresis and adsorption techniques which are laborious and time consuming.


Computed tomographic evaluation of pineal calcification.

A prospective study to ascertain the incidence of normally calcified pineal gland, was carried out in 1000 consecutive patients from different parts of Uttar Pradesh (India), undergoing cranial computed tomography for reasons other than a pineal or parapineal pathology. A total of 167 (16.70%) patients were found to have calcified pineals. Of these 128 were males and 39 females. The incidence rose from 1.16 per cent in the first decade to 31.88 per cent above the age of 50 yr. The percentage incidence of normal pineal calcification was lower than that seen in the Western population. No significant difference was found between men and women in any age group. Although calcification appeared as early as the first decade, this percentage was significantly lower than in the higher age groups. Significantly higher incidence rates were seen in the second decade, third decade and sixth decade onwards.


Antimutagenic activity of plumbagin in Ames Salmonella typhimurium test.

This paper reports the antimutagenic activity of plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) against certain known chemical mutagens in a standard mutagenicity test system of Ames using S. typhimurium strains. Plumbagin by itself did not show any mutagenic effect, whereas it reduced significantly the mutagenic effect of 4-nitrophenylene diammine, phenyl hydrazine and sodium azide in test strains of S. typhimurium, suggesting that plumbagin possessed antimutagenic activity.


Ultrastructural study of mixed growth hormone & prolactin secreting pituitary adenomas.

An ultrastructural study was done on 15 mixed growth hormone (GH) and prolactin (PRL)-secreting pituitary adenomas surgically removed from acromegalic patients with hyper-prolactinaemia, in order to see whether the 2 hormones were present in the same cell or in different cells. Double labelling immunogold technique was used for simultaneous ultrastructural localization of GH and PRL. It was found that each neoplastic cell in these 15 tumours (30 to 50 cells were studied in each case) contained 4 populations of granules viz., (i) granules positive for only GH; (ii) granules positive for only PRL; (iii) granules positive for both GH and PRL; and (iv) granules negative for both GH and PRL (unlabelled). Though the relative percentage of these 4 types of granules varied from cell to cell even within the same tumour, the major population (49.9 to 96%) was constituted by the mixed granules showing labelling for both GH and PRL. Almost all the cells examined from each tumour appeared to be mammosomatotrophs. Thus, the study indicated that mammosomatotroph adenomas are perhaps more common among mixed GH and PRL--secreting pituitary adenomas than previously believed. It could be important to recognize these tumours from the therapeutic point of view.


Incidence of abnormalities in sperm morphology as an indicator of its fertilizing potential.

A retrospective analysis was carried out to assess the correlation of sperm morphology with the occurrence of fertilization in vitro of human oocytes. Seventy women who were subjected to in vitro fertilization procedure were taken up for this study. The cytological smears of semen were evaluated for the morphological characteristics of spermatozoa. Instances where in vitro fertilization did not occur (group II) had significantly (P less than 0.001) higher morphological abnormalities (head defects) when compared with those where fertilization occurred in vitro (group I). The results indicate that evaluation of sperm morphology with special reference to head abnormalities has diagnostic potential in human in vitro fertilization.


Neutralization potency of Russell's viper venom toxoid antivenom, as compared with standard antivenom.

Rabbits were immunized with gamma (gamma) irradiated Russell's viper venom toxoid, adsorbed to aluminium phosphate adjuvant. Antibody (raised against toxoid inoculation) titer was compared to a commercial antivenom on the basis of its ability to neutralize hemorrhagic, necrotic and lethal effects of viper venom. Toxoid immunization (on day 0, 15 and 30) produced antivenom which showed approximately one-third antilethal, half antihemorrhagic and antinecrotic titers as compared to those of the commercial hyperimmunized, concentrated horse antivenom.


DNA analysis of breast cancer by flow cytometry & correlation with other prognostic parameters.

Flow cytometric estimation of DNA content (ploidy and S-phase fraction--SpF) was done on breast cancer tissues from 171 patients. Twenty eight per cent of the tumours were diploid and 72 per cent were aneuploid. SpF was measurable in 82 DNA histograms; of these 22.4 per cent had SpF less than 10 per cent, 34.1 per cent had SpF between 10-20 and 43.5 per cent had SpF greater than 20 per cent. The mean SpF of the measurable histograms was 19.01 per cent with a range 1.78 to 45.19 per cent. A significant correlation between DNA ploidy and SpF was observed (P less than 0.01). Eighty nine per cent of diploid tumours had SpF less than 10 per cent and 73 per cent of aneuploid tumours had SpF greater than 20 per cent. A significant correlation was also found between ploidy and SpF and oestrogen receptor (ER) status of the tumours (P less than 0.05) and between SpF and progesterone receptor (PgR) status of the tumours (P less than 0.05), but not between ploidy and PgR status of the tumours. A significant direct correlation was observed between SpF and tumour grade (P less than 0.05), but not between ploidy and tumour grade. No correlation was observed between DNA ploidy and SpF and tumour type, tumour size, axillary lymph node involvement, age and menopausal status of the patients. Although the incidence of breast cancer is one-third of that reported in the Western countries, there is apparently no biological difference between the various parameters studied.


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.


Isolation & characterization of angiogenic factors from cultured cells & conditioned media.

Angiogenic factors were isolated by ion exchange chromatography from three established cell lines viz., HEp 2, HeLa and CHO, a primary culture of mouse mammary adenocarcinoma and from conditioned media of HEp2 and cultured mammary adenocarcinoma cells. The angiogenic activity was assayed by chicken chorioallantoic membrane assay. The angiogenic factor eluted at 0.5 M, exhibited lambda max at 258 +/- 1 nm, contained protein and nucleic acid. The cellular angiogenic factor showed a ratio of 1:1 for protein and nucleic acid whereas the secreted angiogenic factor had 3-5 parts of protein to 1 part of nucleic acid. The angiogenic factor from HEp2 and CHO cells did not bind to heparin-agarose. Microheterogeneity of the angiogenic factors was established by SDS-PAGE. Antiserum raised against the cellular angiogenic factor from HEp2 cells, showed a titre of 1:1600 by ELISA. The angiogenic factor was directly localized on whole cells by ELISA. Cellular as well as secreted angiogenic factors crossreacted with the antibody. Neutralizing effect of the antiserum on induction of angiogenesis was detected on chicken chorioallantoic membrane.


Immunohistochemical, ultrastructural & immunoelectron microscopic study of glial fibrillary acidic protein in corpora amylacea.

Brain tissues from 10 patients (of non-neurological disease) were studied for the presence of corpora amylacea (CA) using light microscopy (LM), immunohistochemistry (IH) for localisation of glial fibrillary acidic protein (GFAP) and transmission electron microscopy (TEM). Immunoelectron microscopy (IEM) by post-embedding technique using colloidal gold was also performed in two of these patients for more precise localisation of GFAP. Three types of immunoreactivity were noted by IH under LM; some CA were completely negative for GFAP (type III), while others showed positivity, which was either diffuse (type I) or confined to the periphery (rim positivity-type II). TEM showed variable sizes in electron dense material in the centre associated with different amounts of glial filaments (GFs) at the periphery. Thus the different types of IH staining appeared to corroborate with the presence and amount of GFs in CA. The sensitive technique of IEM confirmed the presence of GFAP in all CA irrespective of their IH typing at LM. It is suggested that CA formation in astrocytes is associated with progressive fragmentation and disintegration of GFs with resulting increase in the accumulation of electron dense GFAP-negative material. As more and more of GFs get incorporated and disintegrated, it results in increase in the size of the CA. Thus, the present study clearly demonstrates that GFAP in the GFs contributes to the composition of CA.


Energy intake & resting metabolic rate of young Indian men & women.

Energy intake, body weight and resting metabolic rate (RMR) were measured on seven days consecutively, in 20 men and 14 women on ad libitum food intake under free-living conditions. These measurements were repeated in 8 men and 9 women twice with an interval of two months. Mean energy intake (EI) as measured by 'Weightment' method, was 2820 +/- 577 kcal/d in men and 1611 +/- 147 kcal/d in women. EI showed significant differences between weeks (P less than 0.01, CV = 13.1% in men and 9.5% in women) and between subjects (P less than 0.05, CV = 13.7% in men and 9.8% in women). Intra individual variance contributed up to 60 to 70 per cent of the total variance in EI in both the groups. RMR measured by using Oxylog, varied between the three periods (CV = 6% in men and 5.9% in women) and between subjects (CV = 5.7% in men and 12.3% in women). Mean RMR of both men and women was lower than the predicted value by FAO/WHO/UNU equation. Estimate of mean 24 h energy expenditure, using activity diary and factorial method, in the first round was 2458 +/- 144 kcal in men and 1655 +/- 172 in women, resulting in an energy balance of 308 kcal in men and -44 kcal/d in women. The pattern of activity remained the same in the three rounds for both groups. There were no significant changes in weight (CV-1.6%) or per cent body fat during the study period (P greater than 0.1).


Reliability of skinfold calipers as a tool for measuring body fat in human beings.

Triceps skinfold thickness measured in ten adults aged 19 to 50 yr by a Holtain skinfold caliper, was compared with subcutaneous fat thickness obtained on cross-sections of the left upper arm by computerized axial tomography (CAT). The thickness of subcutaneous fat was measured on CAT scan film directly with the inbuilt computer graphic technique. The measurements taken on CAT scan film were found to be significantly higher (by 21-45%; P less than 0.001) than those by the skinfold calipers. Our findings suggest that skinfold calipers may not be very accurate tools for the assessment of body fat in human beings. Therefore, its use in the determination of body composition, for which great precision is required calls for re-thinking.


Use of rat jejunum for assay of acetylcholine.

Experiments were carried out to determine the advantage in using isolated rat jejunum in the assay of acetylcholine (ACh) in preference to other tissues. Rat jejunum was found to be sensitive to ACh 0.01 micrograms, 5-hydroxytryptamine (5HT) 0.2 to 0.5 microgram and least sensitive to histamine. (Ach greater than 5HT greater than histamine).


Analysis of the major surfactant-associated proteins in human lung.

The protein component of human pulmonary surfactant was analysed. A purified surfactant preparation, after delipidation, was subjected to gel filtration on Sephadex G-200. The proteins present in the surfactant were categorised by means of SDS-polyacrylamide gel electrophoresis into serum and non-serum components. Molecular masses determination showed the presence of three sub-groups with molecular masses of 60-68 kDa, 28-36 kDa and 10-18 kDa, respectively. Antiserum generated against 28-36 kDa protein strongly reacted with the purified surfactant and amniotic fluid, while it did not show any cross reactivity with other groups of proteins and serum in a double diffusion immunoprecipitation assay. We propose that this protein is the major non-serum surfactant-associated protein present in human lung surfactant.


Ascorbic acid status in uremics.

Plasma levels of ascorbic acid (AA) and dehydroascorbic acid (DHA) were estimated in 27 patients of end stage renal failure (ESRF) on standard conservative therapy (group A) and 9 patients of ESRF on maintenance haemodialysis (MHD; group B). Fourteen healthy subjects matched for age and sex served as control (group C). The dietary intake of vitamin C was significantly decreased in group A than in group B compared to control. Similarly, plasma AA was significantly lowered to 0.801 +/- 0.283 mg per cent in group A compared to 1.421 +/- 0.47 mg per cent in control. While it was just lowered to 1.058 +/- 0.272 mg per cent in group B. Although plasma level of DHA was raised to 0.243 +/- 0.486 mg per cent and 0.166 +/- 0.54 mg per cent in groups A and B respectively, the increase was not statistically significant. In our present study, the DHA/AA ratio was found to be inversely proportional to the plasma AA. Further, this ratio has been claimed to be a better indicator of overall reducing atmosphere (i.e., profile of vitamin C) of the body.


Auditory evoked response in patients of diabetes mellitus.

Brainstem auditory evoked response (BAER) and the mid latency response (MLR) were recorded in 25 diabetic men and compared with a group of 25 control subjects. The threshold of hearing was higher (P less than 0.001) in the patients. The BAER peak latencies were also significantly prolonged in both the ears; however, the degree of prolongation was more prominent in the right ear. The interpeak latencies of waves I-III were also prolonged (P less than 0.01) in the left ear, with non significant increase noticed in the right ear also. The wave V amplitude was lower (P less than 0.05) in diabetics. The MLR, on the other hand, was not affected in patients. These findings suggest functional impairment in the auditory pathway in patients of diabetes mellitus.


Prevalence & pathogenesis of hypophosphatemia in ventilated patients.

Hypophosphatemia was found in 12 (43%) patients within 24-36 h of mechanical ventilation in a group of 28 patients in an intensive care unit. The hypophosphatemic patients had a significant lowering of renal phosphorus threshold (P = 0.005) and inappropriate phosphaturia. These abnormalities resolved once ventilation was discontinued. Serum parathyroid hormone (PTH) levels were unaltered in this group of patients suggesting that some factor other than PTH was responsible for hypophosphatemia. This study has demonstrated that abnormalities of renal phosphate handling can cause hypophosphatemia in ventilated patients.


Secondary immune defects in non-Hodgkin's lymphoma.

Changes in the humoral, cellular and phagocytic components of the immune system were investigated in 44 biopsy proven, untreated patients of non-Hodgkin's lymphoma (NHL). There was significant decrease in the total lymphocyte number, their subsets (T and B) and the T helper (T mu) population. Impaired leukocyte migration inhibition response and DNA synthesis following stimulation by phytohaemagglutinin were noted. Significant reduction of serum IgA was found in patients of both diffuse and nodular NHL. Phagocytic activity and nitroblue tetrazolium (NBT) reduction were unaltered, but chemotaxis was significantly reduced and bactericidal activity showed variable results.


Characterization of G6PD Rohini--a new class III Indian variant.

A new Indian G6PD variant was detected in a 15 yr old Maratha male during a population screening programme in high school children in Bombay (India). The propositus and two family members having the same variant were apparently healthy. This enzyme variant has a mild erythrocyte G6PD deficiency and a slow electrophoretic mobility. It is characterized by a high Michaelis-Menton constant for G6P, a bimodal curve for pH optima and a slight decrease in the thermostability. The rate of utilization of substrate analogue is similar to that of normal. These observations suggest identification of a new class III variant, designated as G6PD Rohini.


Treatment of intractable spasticity in spinal cord injured patients.

Spasticity and flexor spasms can be most incapacitating in SCI victims. Muscle relaxants, physiotherapy and elimination of triggering factors must be tried before opting for peripheral surgery or alcohol block. The choice of alcohol block or peripheral surgery depends in whether damage to the spinal cord is complete or incomplete. Results of both the procedures are satisfactory in rightly chosen patients. Alcohol block is a simple, safe and effective method of treating spasticity in the patients of complete paraplegia. The effect is immediate and almost permanent. However, alcohol block is contra-indicated in the patients of incomplete paraplegia where peripheral surgery is a better choice.


Diabetes mellitus in Libya: a retrospective study.

The records of the Diabetes Hospital, Tripoli, Libya for the years 1961-1983 were examined and the available particulars relating to 24,962 diabetic patients (12,486 males + 12,476 females) registered during this period were obtained. The highest number of male and female diabetic patients were in the age group 40-50 years and the highest number of obese diabetic patients also belong to this age group. There was a gradual increase in the number of diabetic patients visiting the Diabetes Hospital with the highest number in 1983. The increase has been both in the type I and type II patients. In 1983, 15% of the diabetic patients belong to type I diabetes. Family history of diabetes appears to be an important factor in the onset and manifestation of type II diabetes. Patients with diabetes in the family have a lower mean body mass index and fasting blood glucose level as compared with those of patients without known case of diabetes in the family. This may be due to greater alertness, early diagnosis of the disease and steps taken to control blood glucose levels and body weight in the case of patients with family history of diabetes.


Unusual manifestations of myxoedema.

The lesser known manifestations of myxoedema encompass a wide spectrum of neurologic, psychiatric, endocrinologic and autoimmune pathology that mask obvious clinical diagnosis. Interesting head and neck presentations are discussed. The relationships with the Pendred and Down syndromes raise hereditary and genetic considerations.


Mechanisms of action of known human carcinogens.

Mutational mechanisms can be proposed for most, if not all, known human carcinogens. Many of these are electrophilic or metabolically activated to reactive molecules which can alter DNA, causing genetic damage and different types of mutations. Even some human carcinogens previously proposed to be nongenotoxic (e.g., hormones and asbestos) exhibit mutational activity in assays for chromosomal mutations. Since such chemicals are usually inactive in the Salmonella assay and other assays for gene mutation, more emphasis has been placed on their nonmutational mechanisms. Clear evidence exists that these carcinogens can alter gene expression and stimulate cell proliferation by epigenetic mechanisms. Such properties are undoubtedly important in their carcinogenic activity. Although they are less well studied, DNA reactive, genotoxic carcinogens also alter gene expression and increase cell turnover by epigenetic mechanisms. These findings are consistent with the current understanding of the molecular basis of multistep carcinogenesis. Most common human cancers evolve as the result of multiple mutational events. The molecular basis of these mutations is varied, and they include point mutations, deletion mutations, chromosomal rearrangements, gene amplification and chromosomal losses and gains. Therefore, different mutational activities of carcinogens can influence the carcinogenic process at different steps. Influences on gene expression and cell proliferation are also important in allowing clonal expansion of preneoplastic cells and in disrupting the suppressive effects of surrounding normal cells on preneoplastic cells (Dotto et al., 1988). The mechanisms of action of human carcinogens, and very probably many rodent carcinogens, include both genetic and epigenetic processes. Carcinogenesis is a multistep, multigenic, multicausal process (Barrett, 1987b), so both epigenetic and genetic factors are probably important.(ABSTRACT TRUNCATED AT 250 WORDS)


Use of short-term tests in determining the genotoxicity or nongenotoxicity of chemicals.

This article brings together the several current approaches to the use of short-term tests for identifying carcinogens and the several meanings of the term 'genotoxic'. Examples of genotoxic and nongenotoxic carcinogens and of genotoxic and nongenotoxic mechanisms of carcinogenesis are discussed. Emphasis is placed on the determination of genotoxicity as it affects evaluation of the mechanism of action of a new rodent carcinogen.


Sex hormones and cancer.

There are few instances in which a clear effect of hormones on cancer risk is known and in which the effects of those hormones on the cells concerned are also known. The best examples are the relationships between sex hormones and cancer in women. The effects of sex hormones both on the risk for endometrial cancer and on the cells of the endometrium are well understood, and the evidence strongly suggests that hormones act by altering the rate of cell division. The same mechanism may explain the relationships between sex hormones and the risk for breast cancer, but our understanding of cancers at this site is incomplete. Less still is known about the mechanisms of the effects of sex hormones on other hormone-related cancers, such as those of the ovary and cervix. Most sex hormones are not genotoxic.