The phospholipase A(2) inhibitor methyl indoxam suppresses diet-induced obesity and glucose intolerance in mice.

BACKGROUND AND PURPOSE: Previous results have shown that mice lacking in the group 1B phospholipase A(2) (Pla2g1b) are resistant to obesity and diabetes induced by feeding a diabetogenic high-fat/high-carbohydrate diet. This study examined the potential of using the Pla2g1b inhibitor methyl indoxam as therapy to suppress diet-induced obesity and diabetes. EXPERIMENTAL APPROACH: Male C57BL/6 mice were fed the diabetogenic diet with or without methyl indoxam supplementation. Body weight gain, fasting plasma glucose levels, glucose tolerance and postprandial lysophospholipid absorption were compared. KEY RESULTS: Wild-type C57BL/6 mice fed the diabetogenic diet without Pla2g1b inhibitor showed 31 and 69% body weight gain after 4 and 10 weeks respectively. These animals also showed elevated plasma glucose levels and were glucose intolerant. In contrast, C57BL/6 mice fed the diabetogenic diet with 90 mg.kg(-1) of methyl indoxam gained only 5% body weight after 10 weeks. These animals were also euglycaemic and displayed normal glucose excursion rates in glucose tolerance test. Methyl indoxam suppression of diet-induced body weight gain and glucose intolerance was correlated with the inhibition of Pla2g1b-mediated postprandial lysophospholipid absorption. CONCLUSIONS AND IMPLICATIONS: These results show that oral supplementation of a diabetogenic diet with the Pla2g1b inhibitor methyl indoxam effectively suppresses diet-induced obesity and diabetes in mice. This suggests that Pla2g1b inhibition may be a potentially effective oral therapeutic option for treatment of obesity and diabetes.

Homeotic control in Drosophila; the scabrous gene is an in vivo target of Ultrabithorax proteins.

The regulatory functions of transcription factors encoded by the Ultrabithorax (Ubx) gene initiate genetic programmes essential for segmental identity and morphogenesis in Drosophila. Based on the formation of DNA-protein adducts in intact nuclei and immunoselection procedure, we cloned genomic targets for Ubx proteins. One clone was studied in detail. It encompasses parts of the last intron and exon of the scabrous (sca) gene, which encodes a secreted protein involved in cellular communication during neurogenesis. Five motifs, presenting the ATTA core, which is shared by most homeodomain binding sites, were found in the nucleotide sequence of this clone. We detail here the dynamic pattern of sca transcript accumulation during embryogenesis and show that mutation of Ubx results in the ectopic transcription of sca in the first abdominal segment. We propose that a direct interaction of Ubx with cis-acting elements in sca negatively regulates the gene. Transcript localization in several combinations of deficiencies in the Bithorax complex (BX-C) indicates that sca is downregulated by abdominal A (abdA) and Abdominal B (AbdB), and suggests that it is a common target of the three genes of BX-C.

Generation of CD8 cytolytic T cells early after autologous or allogeneic bone marrow transplantation.

Longitudinal in vitro assays related to cell-mediated immunity were performed in patients following allogeneic (32) or autologous (15) bone marrow transplantation (BMT). In both groups of reconstituted patients, low CD4+/CD8+ T cell ratio and weak allogeneic mixed lymphocyte reactions were found in the first 6 months after BMT, progressively reaching values similar to controls (bone marrow donors or unrelated individuals). In contrast, a strong generation of allogeneic cytotoxic cells, assessed by the number of lytic units per 10(6) cells, was frequently found (18/38 patients tested in both groups) in the first 4 months, despite the quantitative deficit of the CD4+ subset. This in vitro differentiation was found to be independent of in vivo acute graft-versus-host disease (GVHD) and chronic GVHD in allo-transplanted patients. As also documented in autologous recipients, this observation suggests that this phenomenon could be, at least partially, related to the transplantation per se. Preliminary characterization of the effector cells indicates that they belong to the CD8+ subset and that their differentiation is interleukin-2-dependent. Experimental depletion of the CD4+ subset in normal subjects did not increase the number of lytic units in allogeneic cultures. This implies qualitative differences between BMT recipients and normal subjects, namely in CD8+ subset: i.e. that following BMT early CD8+ T cells appear to produce their own growth factor (IL-2), while in normal adult individuals, such autocrine CD8+ T cells, if present, are very rare.

Anti-interleukin 2 receptor monoclonal antibodies. Respective role of epitope mapping and monoclonal antibody-receptor interactions in their antagonist effects on interleukin 2-dependent T cell growth.

Functional studies, using mainly interleukin 2 (IL2)-dependent growth of human T cell lines or clones but also mixed lymphocyte cultures and mitogen T cell activation, allowed a collection of locally produced anti-IL2 receptor monoclonal antibodies (mAb) to be classified. They fell into two groups: one with strong to moderate inhibition of IL2, the other without any detectable functional activity in in vitro assays. Direct and sequential immunoprecipitation as well as peptide mapping confirm that all the mAb recognize the same surface molecule. The parameters responsible for such functional dichotomy were characterized: the main parameter was found to be linked to the epitopic cluster recognized on the molecule by the mAb. All functional mAb pertained to a given epitopic cluster and all the nonfunctional ones to an alternative cluster. Studies on mAb receptor and IL2 receptor interactions confirmed these findings and strongly suggest that functional mAb interact with a region on the IL2 receptor identical or very close to the site of ligand-receptor interaction. These data could facilitate the choice of mAb to be used in therapeutical approaches in vivo when ethical objections could be overcome by appropriate committees.

HLA-SB in the south of France. Correlation between locally derived and reference typing reagents.

The HLA-D region of the Major Histocompatibility Complex has been subdivided since 1978 (Mawas et al. 1978) into two subregions separable by recombination: a telomeric subregion (closer to HLA-B), coding for the classical HLA-DR or Dw specificities (Mawas et al. 1980) as well as for the more recent MT series (Park et al. 1980); and a centromeric subregion (closer to GLO), coding for a new series of alleles provisionally named SB (for secondary B cell antigens) (Shaw et al. 1980, 1981a). Reagents allowing the identification of six independent alleles have been characterized in two laboratories (Charmot et al. 1980 and Shaw et al. 1980, 1981b) using the technology of primed lymphocytes typing (Sheehy et al. 1975; Mawas et al. 1975). The existence of this new locus is supported by the following arguments: population studies by Shaw demonstrating five traits distinct from DR behaving as alleles (Shaw et al. 1981b), analysis of two informative SB/DR recombinant families (Mawas et al. 1978; Mawas et al. 1980; Shaw et al. 1981a), and, finally, studies of mutants showing independent loss of DR expression without loss of SB expression (Kavathas et al. 1981). The present report summarizes the HLA-SB typing of 109 unrelated individuals from the South of France and segregation studies in 14 unrelated families; a first attempt to correlate local "SB" reagents with the NIH reference standards is presented.

Expansion of human lymphocyte populations expressing specific immune reactivities. III. Specific colonies, either cytotoxic or proliferative, obtained from a population of responder cells primed in vitro. Preliminary immunogenetic analysis.

Human alloreactive cell lines were maintained in culture over prolonged periods of time using conditioned medium. Primed lymphocyte typing reactivity was observed in these T cell lines for only 1 mo, but these T cell lines have remained for more than 7 mo highly and specifically cytotoxic. Using as growth promoter an irradiated autologous feeder consisting of irradiated peripheral blood lymphocytes and the lectin leucoagglutinin, we have derived by limiting dilution cloning of in vitro primed allogeneic combinations, primary colonies (or primary clones) with monofunctional immune reactivities: either cytotoxic (the rarest observed) or PLT reactive (the majority of the colonies). Furthermore, each monofunctional primary colony when tested for PLT or CML reactivity on a panel of unrelated PBL, always showed a restricted specificity when compared to the original primed population. The PLT reactivity of each of the primary clones was short lasting in contrast to their growth potential. The CML reactivity of the primary clones, as for the T cell lines, was long lasting as was their growth potential.

The HLA-D system: at least two loci and four distinct phenotypic traits per haplotype. Introduction to component typing in families and population by primed lymphocyte typing.

Using a number of intrafamilial PLTs raised against identical HLA haplotypes it has been possible to construct a model in an informative family defining the HLA-D region as a genetic system. This system consists of at least two regions separated by a recombination between HLA-D and GLO. In relation to the site of recombination, a minimum of one centromeric and three telomeric components can be identified per haplotype. - Fourteen PLTs raised and defined within the family were subsequently tested in a Caucasian population (n = 84) and in 13 unrelated, complete families. - It is concluded that the hypothetical model proposed for the HLA-D regions as a genetic system of linked loci, coding at the cell surface for associated but distinct components (at least four per haplotype), allows for typing of the components of the HLA-D system of any given haplotype. Serological typing of HLA-D components should, in the near future, provide a more convenient way of establishing component phenotypes than the present use of primed lymphocyte typing reagents. Among the components isolated, some have a high association with the classic alleles defined either by homozygous typing cells or DR serology. Others form the basis of cross-reactivity but their presence does not interfere with standard typing. Others, however, seem by their mere presence to be responsible for false assignments. - The concept of HLA-D as a genetic system clarifies many of the inconsistencies observed with a one-locus system.

Production, expansion, and clonal analysis of T cells with specific HLA-restricted male lysis.

A cytotoxic T cell (CT) lines grown as a population (CT line) was initiated from the peripheral blood lympocytes (PBL) of a female aplastic anemia patient who was known to express CT that were able to lyse HLA-A2-positive male cells. The anti-H-Y HLA-A2-restricted cytotoxic activity could be maintained over prolonged periods of time. The CT lines could be expanded and maintained in culture for >65 d by the use of mitogens and irradiated feeder cells. Out of 68 cultures obtained after cloning of the CT lines, 43 showed varying, but always specific, anti-H-Y HLA-A2-restricted lytic capacity on a per-cell basis. We could show that the cloned cultures were composed of >80% T cells that carry the HLA-A, -B, -C, and also the HLA-DR antigens identical to the original PBL.

Split of HLA-D into two regions alpha and beta by a recombination between HLA-D and GLO. I. Study in a family and primed lymphocyte typing for determinants coded by the beta region.

In a family with a paternal recombinant child between HLA-D/Dr and GLO, PLT have been raised between HLA-A-B-C-D-DR and Bf identical sibs. Since these sibs differ only within a region (beta) between HLA-D/DR and GLO and the centromere, these PLTs allow the typing of two new determinants differing from HLA-D/DR, possibly alleles at a new locus mapping in a region outside HLA-D/DR. A difference limited to this beta region can induce a weak primary and a strong secondary MLR.

Expansion of human lymphocyte populations expressing specific immune reactivities. II. A comparison of immune reactivities in human T lymphocyte lines derived from allogeneically primed cultures and maintained with lectins or conditioned medium.

In a previous paper, lectins were shown to allow a strong expansion of in vitro primed cells; among the lectins tested, PKW but not PHA (nor Con A) was found to reactivate CTLs. We then asked two further questions: Could one maintain a long term expansion of human T cells using iterative lectin stimulation? And, if so, could the immune reactivities, once expressed in the original primed cells, also be maintained? We report here that lectins remain potent mitogens for primed cells, and allow good expansion over many cycles, provided fresh irradiated cells (autologous or not to the responder) are added to the cultures. When PKW is used as the iterative mitogen, both the specific proliferation (PLT) and the specific cytotoxicity are maintained after each cycle. When PHA is the iterative stimulant, only the specific proliferation (PLT) is maintained, while no measurable cytolysis is present. However, if iteratively PHA stimulated primed cells are now cultured in the absence of PHA, even without additional specific stimulation, cytolytic activity is recovered from the cultures. In parallel, primed cells iteratively expanded using conditioned medium appear to retain both their specific lytic function and their specific proliferative function. Preliminary data suggest that the cloning efficiency when using lectins is higher than when using conditioned medium.

The in vitro cellular response of human lymphocytes to trinitrophenylated autologous cells: HLA-D restriction of proliferation but apparent absence of HLA restriction of cytolysis.

Primary as well as secondary proliferative and cytotoxic responses to 2,4,6-trinitrophenyl (TNP)-modified autologous human cells have been studied. Proliferative responses have been obtained both by primary (peak on day 6) and secondary (peak on day 2--3) stimulation. Both responders and nonresponders were found among the panel of unrelated individuals tested. All responders in a secondary reaction also gave significant primary responses. Intrafamilial studies showed that the ability to restimulate a proliferative response followed the major histocompatibility complex haplotype of the responder; in some cases, the two haplotypes differed in their ability to restimulate. Using unrelated individuals typed for HLA-A, B and C, as well as HLA-D and DR, proliferation was shown to occur only when the unrelated stimulator shared HLA-D region products with the responder. In contrast, no HLA restriction was found in cell-mediated lympholysis (CML) (neither in primary nor in secondary responses) in most cases. The data suggest that the observed killing is independent of sensitization. Both responders and nonresponders in proliferation yielded high levels of lysis; no increase of lysis was found in kinetic studies; most allogeneic CML combinations were highly lytic for the TNP-modified responder cells at a time when the lysis of the specific allogeneic target is negligible. These preliminary data suggest that the killing observed might be different from classical T cell-mediated lympholysis.

Specific inhibition of human lymphocyte responses by primed autologous lymphocytes. I. Evaluation of MLR inhibition as a model for suppression.

Human lymphocytes from person A, primed for 10 to 14 days in MLC against lymphocytes from person B, inhibit specifically the proliferative response to B by fresh (i.e., unprimed) lymphocytes of A. Gamma-irradiated (2000 R) primed lymphocytes likewise inhibit specifically, although less strongly. Cells of A, primed with cells of B and then irradiated, usually can inhibit the response of A to cells of any individual sharing HLA-D antigens with B, and the effect tends to be independent of the number of stimulating cells. We also often see inhibition of responses to cells sharing HLA-A and -B antigens with person B, but this effect tends to be lost when the number of stimulating cells is increased. Similarly, at low doses, cells primed for HLA-D antigen a appear not to inhibit the response to an irrelevant HLA-D antigen b on the same stimulating cell. At higher doses of primed cells, even the response to the irrelevant antigen is inhibited. These data suggest to us that at least two mechnaisms may be involved: one directed at the stimulating cell (most likely cell-mediated cytolysis), and predominant at high ratios of primed cells to stimulating cells; the other directed at specific clones of responding cells, and predominant at low ratios.

A weak human MLR locus mapping at the right of a crossing-over between HLA-D, Bf and GLO.

An unexpected MLR reaction has been observed between three HLA-identical sibs; it consists of bidirectional positive MLR between identical female twins and a sister. No argument for a lymphoid mosaic could be found, although twins were frequent in the family; similarly no HLA-A/B or HLA-B/D recombinant could be demonstrated. The MLR, although weak, was highly reproducible. PLTs could be raised between the sibs, without an apparent segregation in this family nor in five other families, but such PLTs discriminated well between the positive and negative controls. In the absence of any proof that such a weak MLR locus could be on another chromosome than chromosome 6, two lines of argument are indirect evidences that such a locus could be indeed on chromosome 6: one of the sibs differs from the two others for two markers outside HLA--D--DR--Bf: glyoxalate (GLO) and red blood group P.