Abstract: The diagnostic potential of recombinant Lepp12 (rLepp12) antigen cloned from Leishmania infantum was assessed in L. donovani infections by Western blotting. Ninety-two serum samples, including 30 patients with active kala-azar (KA), 17 post-treated KA patients (KA-PT), 20 post-kala-azar dermal leishmaniasis (PKDL) patients and 25 controls, were analysed for rLepp12, rK39 and DAT positivity. All KA samples taken at pre-treatment stage were positive for Lepp12 antibodies. Seventeen of these were evaluated post treatment (KA-PT), 10 of which were found to be negative. Nine of these 10 negative cases corresponded to clinically cured patients with regressed spleen. Seven post-treatment cases were rLepp12-positive; all of them corresponded to patients who were considered clinically cured but continued to have an enlarged spleen (> or =5 cm). The majority of PKDL patients (18/20) were found to be seronegative by immunoblot test using rLepp12 antigen. The rLepp12-based Western blot diagnosed 100% of patients with visceral disease, whilst none of the control cases were found to be reactive to rLepp12. rLepp12 protein provides a useful reagent for highly sensitive and specific diagnosis of KA. Additionally, rLepp12 appears to have potential as a prognostic marker for the infection.
Abstract: The parasite Leishmania is a major cause of disease worldwide. In the past 15 years, many groups have analysed DNA-derived proteins from Leishmania. Large amounts of data obtained by these groups can be collated to direct future research into Leishmania and to find novel immunological mechanisms and information about its metabolism. International coordination will increase both the basic knowledge about Leishmania and the capacity to apply this knowledge to combat leishmaniases.
Abstract: Leishmaniases, caused by parasites belonging to Leishmania spp, constitute a vast variety of diseases, from cutaneous lesions (CL) to visceral leishmaniasis (VL). If untreated, leishmaniases can be fatal, and affect 12 million people in nearly 90 countries, presenting a worldwide public-health problem. Most diagnostic tools are not suitable for use in field conditions. There is no satisfactory chemotherapy for CL; chemotherapy for VL is efficient in most immunocompetent people, but not in immunocompromised individuals, and is toxic and costly; and chemotherapy-resistant leishmania strains have also been reported. At present, there is no vaccine against leishmaniases: vaccine development for parasitic diseases is more difficult than for most bacteria and viruses due to the complexity of the pathogen and its intricate interactions with the vertebrate host. We review the recombinant DNA-derived leishmania proteins of potential use in diagnostics, therapy, and development of vaccines, and address the question of how these proteins can aid in the fight against leishmaniases.
Abstract: BACKGROUND: We report cloning and characterization of a novel Leishmania infantum protein which we termed Lepp12, and we examine its possible implication in the interference with intramacrophage signaling pathways. RESULTS: The protein Lepp12 contains 87 amino acid sequence and exhibits 5 potential phosphorylation sites by protein kinase C (PKC). Recombinant GST-Lepp12 is phosphorylated in vitro by exogenous PKC and by PKC-like activities present in promastigote and in the myelomonocytic THP-1 cell line, indicating that at least one phosphorylation site is functional on the recombinant Lepp12. The natural Lepp12 protein is present in L. infantum promastigotes, as evidenced using specific anti-Lepp12 antibodies produced by immunopurification from acute phase VL patient sera. Interestingly, human patient sera are strongly reactive with GST-Lepp12, demonstrating immunogenic properties of Lepp12 in man, but no immune response to Lepp12 is detectable in experimentally infected animals. When isolated from promastigotes, Lepp12 migrates as two species of apparent MW of 18.3 kDa (major) and 14 kDa (minor), localizes in the nuclear fraction and appears constitutively phosphorylated. Natural Lepp12 is phosphorylable in vitro by both exogenous PKC and PKC-like activity present in THP-1 extracts. The intracellular Lepp12 transfected into THP-1 cells activates these cells to produce IL-1beta and induces an enhancing effect on PMA stimulated IL-1beta synthesis, as demonstrated using GST-Lepp12 transfectants. CONCLUSIONS: Together these results indicate that Lepp12 represents a substrate for PKC or other PKC-like activities present in the promastigote form and the host cell and therefore may interfere with signal transduction pathways involving PKC.
Abstract: We tested in outbred golden hamsters the protective potential of highly immunogenic Leishmania infantum protein papLe22 which we recently identified. Immunisation was performed using papLe22 cDNA, administered as a single intramuscular injection. The level of antibodies directed against total leishmanial antigens was significantly decreased in the vaccinated hamsters as compared with the controls, indicating that the administration of papLe22 cDNA downregulated the Th2 type response and suggesting that the immune response was reoriented toward the cell-mediated type. The presence of the parasite kDNA in the peripheral blood was systematically detected as early as 3 weeks post infection in all mock-vaccinated hamsters. By contrast, in the vaccinated animals the occurrence of the episodes of Leishmania circulation was reduced by 50%. The immunisation presenting efficacy in this highly susceptible species which develop VL similar in gravity to human and canine disease should prove also efficient in naturally infected hosts. The marked decrease of the frequency of parasite circulation induced by papLe22 cDNA immunisation appears therefore important and potentially able to reduce transmission and thus to control the spread of the disease.
Abstract: In this report, a sandwhich ELISA was developed to quantify spleen and liver burdens from L. infantum-infected BALB/c mice. Amastigote antigens obtained following Nonidet P40 extraction of parasite-harbouring tissues were captured by anti-L. infantum human IgG insolubilized onto microtiter plate and subsequently revealed with anti-L. infantum F(ab)' fragments labelled with peroxidase. The method was easy to perform, precise and capable to specifically and accurately detect 5 x 10(4) amastigotes/100 mg tissue. Parasite burdens from infected BALB/c mice, in various conditions, were measured by ELISA and Giemsa-stained touch imprint reference methods, and compared. Both techniques agreed well with close values for liver burdens, but the spleen loads measured by the ELISA were, on average, 10.7 times higher than those calculated from imprints. This difference was attributed partly to the underestimation brought by Stauber's formula. However, it did not preclude the usefulness of this newly developed test, since results obtained in kinetics studies and evaluation of the efficiency of leishmanicidal drugs allowed us to draw identical conclusions.
Abstract: BACKGROUND: The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum. RESULTS: Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden. CONCLUSIONS: Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs.
Abstract: We analyzed differential responses of spleen and liver, major organ targets for viscerotropic Leishmania species, to experimental infection and examined if resistance to challenge was organ-specific. In liver, parasites were spontaneously cleared and iNOS trancripts expression paralleled that of amastigote load. In the spleen, amastigote multiplication was only partly controlled, and iNOS transcripts expression was transient. Total numbers of spleen cells, B cells, and T cells were decreased, while F4/80(+) and Mac1(+) cells were conserved. Expression of splenic MCP-1 transcripts remained constant, indicating its possible contribution to immigration of Leishmania host cells and to sustained parasite load. Spleen cells produced both, Th1- and Th2-type cytokines and Th2-type response was dominant, compatible with the sustained MCP-1 expression. Challenge experiments showed that in contrast to the liver, where initial infection conferred a progressively established immunity, in the spleen there was no induced protection against reinfection. Organ-specific resistance against challenge could be important for designing antileishmanial vaccines.
Abstract: We report here the characterization of a novel Leishmania infantum protein termed papLe22 (22-kDa potentially aggravating protein of Leishmania). A positive clone from a cDNA library was identified by serum of a visceral leishmaniasis (VL) patient. Full-length cDNA obtained using rapid amplification of cDNA ends-PCR codes for a 22-kDa protein. In L. infantum promastigotes an endogenous nuclear protein of 14-kDa electrophoretic mobility was found by using an antiserum prepared against the fusion protein glutathione S-transferase-papLe22. Its expression was also shown in L. infantum amastigotes and in Leishmania major and Leishmania guyanensis promastigotes. VL patients' sera showed anti-papLe22 immunoglobulin M (IgM) and IgG reactivities, indicating that a primary response against the leishmanial protein papLe22 accompanied acute VL manifestations. Specific IgG levels were correlated with patients' clinical status. The presence of IgG1, IgG2, and IgG3 subclasses suggested a mixed Th1- and Th2-type response; there was no correlation between subclass reactivity and the disease course. The recombinant papLe22 specifically activated interleukin-10 production by VL patients' peripheral blood mononuclear cells collected at diagnosis and after treatment-induced cure, indicating its contribution to VL pathogenesis and concomitant immunosuppression and its potential role in the reactivation of latent parasites. As a dominant immunogen, papLe22 might be used as a vaccine component, provided that the vaccination protocol directs the response toward the Th1 pattern.
Abstract: Visceral leishmaniosis (VL) due to Leishmania infantum (L. chagasi) is a lethal disease if untreated, but asymptomatic L. infantum infections have been reported previously. A better understanding of parasite transmission, dissemination, and survival in the human host is needed. The purpose of this study was to assess whether L. infantum circulated in peripheral blood of subjects with no history of VL. Sera from 565 blood donors were screened by Western blotting to detect Leishmania-specific antibodies and identify individuals with probable past exposure to Leishmania. Seropositivity was found in 76 donors whose buffy coats were examined by PCR and direct culture. The parasite minicircle kinetoplast DNA was amplified from blood samples of nine donors. Promastigotes were detected by culture in blood samples from nine donors. Only two donors were PCR and culture positive. These results indicate that L. infantum circulates intermittently and at low density in the blood of healthy seropositive individuals, who thus appear to be asymptomatic carriers. Implications for the safety of blood transfusion are discussed.
Abstract: In the immunocompetent host, visceral leishmaniasis (VL) is a fatal disease if untreated. In immunosuppressed patients, VL is an opportunistic infection for which there is no effective treatment for relapses. Here we report on the long-term activity of orally administered hexadecylphosphocholine (HDPC) against established Leishmania infantum infection in BALB/c mice. HDPC is a synthetic phospholipid with antiproliferative properties that has been extensively studied for its cancerostatic activity. Its short-term leishmanicidal effects in mice recently infected with viscerotropic Leishmania species have been previously reported. First, we show that 5 days of oral therapy with HDPC (20 mg/kg of body weight/day) led to amastigote suppression in the liver and the spleen of 94 and 78%, respectively (versus 85 and 55% suppression by meglumine antimonate in the liver and spleen, respectively), in mice infected 6 weeks before treatment and examined 3 days after the end of treatment. These results demonstrate the short-term efficacy of HDPC against an established Leishmania infection. Next, the long-term efficacy of HDPC was examined. In HDPC-treated mice both the hepatic and splenic amastigote loads were significantly reduced (at least 89%) 10, 31, and 52 days after the end of the treatment. In the treated mice, the increase of the splenic load was significantly slower than that in the untreated mice, demonstrating that the HDPC-exerted inhibition of Leishmania growth persisted for at least 7 to 8 weeks. Orally administered HDPC--the safe doses and side effects of which are at least partially known--appears to be a promising candidate for the treatment of VL.
Abstract: Leishmania parasites persist in their vertebrate host after the treatment-induced clinical cure and in the asymptomatic infection. They confer resistance to reinfection but represent a risk of occurrence of acute leishmaniosis in immunosuppressed conditions. We examined the effects of prolonged dexamethasone administration on a chronic Leishmania infantum infection. Splenic T cell populations from the long-term-infected BALB/c mice were reduced by 55%, whereas those from uninfected controls were depleted by 85%. The ability of the remaining spleen cells to produce IL-2, IFN-gamma, IL-4 and TNF-alpha after in vitro specific stimulation decreased twofold, and the specific anti-leishmanial antibodies declined 3- to 5-fold. Liver, spleen and bone marrow are the main L. infantum targets in natural and experimental infections. Three-fold increase of amastigote burden was evidenced in the spleen, after dexamethasone administration was prolonged for over 2 months. No reactivation of Leishmania proliferation was disclosed in the liver and bone marrow. These results show a decreased sensitivity of splenic T cells to dexamethasone in a chronic Leishmania infection and a distinct response of the Leishmania-infected target organs to the dexamethasone-induced immunosuppression.
Abstract: OBJECTIVE: To discriminate cases of visceral leishmaniosis (VL) following a primary infection from cases originating in a reactivation of a latent Leishmania infection and to assess the impact of CD4+ T-cell counts on the occurrence of VL in patients with HIV disease. METHODS: We searched by Western blotting for the presence of Leishmania infantum-specific antibodies in the sera of 236 HIV-positive patients. We performed a follow-up of antileishmanial serology and analysed the evolution of the CD4+ T-cell counts for 14 HIV-positive VL patients and for 18 HIV-positive Leishmania-seropositive patients without VL. RESULTS: This study (1) showed that the VL disease/Leishmania infection ratio in HIV-positive individuals is high (1 : 10); (2) discriminated between a primary Leishmania infection (five patients who converted from Leishmania-seronegative to Leishmania-seropositive) and a reactivation of a latent infection (seven patients); (3) showed that HIV-positive individuals with dramatically low CD4+ T-cell counts maintained or generated a specific antileishmanial antibody production; (4) demonstrated that the primary-VL appeared at significantly higher (P = 0.028) CD4+ T-cell levels than the reactivation-VL; (5) documented the existence of HIV-positive Leishmania-seropositive individuals who despite a severe and prolonged immunosuppression did not develop VL (eight of 18). CONCLUSION: Our data stress the utility of the follow-up by Western blotting for an early diagnosis of VL, and therefore an early treatment, for HIV-positive patients living in endemic areas. They suggest that in a latent Leishmania infection supplementary control mechanism(s) might operate in addition to the T-cell-mediated response, and provide a further example of non-appearance of an opportunistic infection despite a severe reduction in CD4+ T cells.
Abstract: The survival of Leishmania, which encounter drastic changes of environment during their life-cycle, requires regulation and control of ionic concentrations within the cell. We analysed the influence of growth stage, ionic composition of the medium, heat and acidic stress on 86Rb+ influx in L. infantum promastigotes. Proliferating promastigotes exhibited faster and higher 86Rb+ uptake than stationary cells. Cl- anion did not have any effect, but in the presence of physiological concentration of HCO3-, 86Rb+ uptake was significantly increased. This enhancing effect was only partially inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), a blocker of ion-translocating ATPases. 86Rb+ influx was abolished by N-ethylmaleimide (NEM), indicating a major contribution of plasma membrane transporters. Heat shock and acidic shock notably decreased 86Rb+ influx. Our data provide indirect evidence that an energy-dependent system which brings K+ in, such as K+/H(+)-ATPase evidenced by Jiang et al. (1994), is active in Leishmania in different environments. Mechanism(s) other than ion-translocating ATPases occur, at least in the presence of HCO3-, and their contribution to K+ pathways varies in different environmental conditions.
Abstract: We investigated in BALB/c mice the influence of the immunological environment created by the nematode Trichinella spiralis on the course of visceral leishmaniasis due to Leishmania infantum. On the day of Leishmania inoculation (day 0), mice, T. spiralis infected 7 days earlier, presented increased gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-5 mRNA levels locally and systemically and increased the potential of spleen cells to synthesize IFN-gamma and IL-4 after activation in vitro. Eighteen days after Leishmania inoculation (day 18), corresponding to the acute phase of leishmaniasis, the hepatic amastigote burden in mice coinfected with L. infantum and T. spiralis (LT mice) was significantly lower (P < 0.001) than that in mice infected with L. infantum only (L mice). IFN-gamma and IL-4 mRNAs were overexpressed in livers of LT and L mice. On day 70, corresponding to the chronic phase, the splenic amastigote load was significantly lower (P = 0.004) in LT mice than it was in L mice. Splenic IFN-gamma transcripts were overexpressed in both L and LT mice. After Leishmania-specific in vitro stimulation, cytokine production was enhanced in both groups, but spleen cells from L mice produced significantly more IFN-gamma than did spleen cells from LT mice. Our data (i) generalize previous results indicating the lack of a clear-cut correlation between the outcome of murine visceral leishmaniasis and the type of cytokine pattern and (ii) demonstrate that in LT mice, leishmaniasis takes a markedly milder course than it does in L mice, providing information on the potential consequences of coinfection in a mammalian host.
Abstract: Leishmanial antigens which stimulate T lymphocytes from primed individuals may be candidates for a vaccine. We recently found a significant concordance between the humoral response specific for two proteins from Leishmania infantum promastigotes, p14 and p18, and a positive leishmanin delayed-type hypersensitivity reaction, testifying to the occurrence of cell-mediated immunity. In this communication, we describe a partial characterization of these antigens and an in vitro analysis of their capacity to activate primed human T cells. We showed, by immunofluorescent staining and through analysis of subcellular fractions by Western immunoblotting, that in stationary-phase promastigotes, p14 and p18 were located only in the parasite nuclei; in the middle of the log phase, a transitory and only weak expression outside the nucleus was detected. We then showed that p14 and p18 antigens shared a common epitope(s). Finally, we analyzed the in vitro proliferation and interleukin-2 production induced by leishmanial proteins in human peripheral blood mononuclear cells from sensitized subjects. We showed that in some individuals who have been exposed to L. infantum the specific response to the whole lysate was mostly due to the nuclear antigens. We demonstrated directly the capacity of nitrocellulose-bound p14 and p18 to activate in vitro all of the tested primed peripheral blood mononuclear cells, which contrasted with a lack of stimulatory activity of other membrane-bound leishmanial proteins. Taken together, our results suggest that an antigenic determinant(s) dominant for some individuals might exist on both antigens.
Abstract: Western blot analysis of sera from 32 patients with acute clinical leishmaniasis due to Leishmania infantum showed the simultaneous presence of antibodies against 4 antigens with molecular masses of 18, 21, 23, 31 kDa. The simultaneous presence of these 4 antigens was specific to the clinical disease and it was not detected in 47 sera from asymptomatic individuals living in the leishmaniasis endemic area of Alpes-Maritimes (southern France) or in 37 sera from patients with other protozoan infections.
Abstract: 2',3'-didehydro-2',3'-dideoxythymidine (D4T) is a thymidine analogue with potent anti-HIV activity in vitro and is currently being investigated therapeutically in patients with advanced HIV infection. We describe a first one-step competitive ELISA method developed for D4T measurement. Anti-D4T rabbit antibodies were raised against a D4T hemisuccinate-bovine serum albumin immunogen. A D4T-hemisuccinate-horseradish peroxidase conjugate and a monoclonal anti-rabbit IgG antibody insolubilized onto a microtiter plate were used as a tracer and capture system, respectively. The method was capable of detecting 2 ng/ml of D4T in cell cultures and 20 ng/ml of D4T in plasma samples previously separated in microconcentrator devices. Cross-reactivity analysis showed that thymidine, D4T monophosphate, or azidothymidine, were weakly recognized by the ELISA and that thymine or other nucleosides were unreactive. The test was successfully used for the quantification of D4T in cell extracts from CEM or Molt 4 cell lines cultured with D4T and in the plasma of patients with advanced HIV infection, receiving D4T therapy. Moreover this ELISA could be used for the indirect quantification of D4T phosphorylated intracellular metabolites previously separated by reverse phase HPLC and hydrolyzed with alkaline phosphatase.
Abstract: In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.
Abstract: We have reported recently that colchicine and other microtubule-disrupting agents stimulated interleukin-1 (IL-1) alpha and beta synthesis in human monocytes. In this study we found that unexpectedly colchicine failed to stimulate the expression of two other potent immune mediators, namely tumor necrosis factor-alpha and interleukin-6. Remarkably, taxol which induces stable microtubule bundles, antagonized the colchicine but not the LPS-induced IL-1 synthesis. These results suggested that the colchicine-mediated IL-1 induction was generated by microtubule disassembly. We next demonstrated that microtubule disruption triggered an elevation of intracellular levels of cAMP and a subsequent stimulation of protein kinase A. The use of different protein kinase inhibitors supported a role of the PKA, but not the PKC, in the colchicine-induced IL-1 production. Furthermore, elevation of intracellular cAMP levels by 8-bromo-cAMP, forskolin, or cholera toxin potentiated the effect of suboptimal concentration of colchicine on IL-1 synthesis. However, these agents alone were unable to induce IL-1 synthesis. Therefore, our data indicate that the cAMP/protein kinase A-signaling pathway is necessary but not sufficient to generate IL-1 synthesis by microtubule disruption. Thus, microtubule-disrupting drugs appear as useful tools to further characterize the molecular events which regulate IL-1 production in human monocytes.
Abstract: The T cell surface molecule CD2 forms, with its counter-receptor CD58 (LFA-3), a powerful adhesion/activation pathway. There is some evidence that CD2 might bind more than a single ligand. Chinese hamster ovary cells (CHO) expressing human CD59 after cDNA transfection (CD59+CHO) form rosettes with human T cells; these rosettes are inhibited in a dose-dependent fashion by the CD59 monoclonal antibody (mAb) H19 and by the CD2 mAb O275 known to block natural rosettes, but not by the CD2R mAb D66, a poor rosette blocker. CD2+CHO transfectants form rosettes with human erythrocytes; these rosettes are inhibited by the CD59 mAb H19 in addition to CD2 mAb O275 and CD58 mAb; murine thymoma cells expressing human CD2 form rosettes with CD59+CHO cells that again are blocked by CD59 H19 and by CD2 O275 mAb. In a marked contrast with H19, two others CD59 mAb, YTH 53.1 and MEM 43, which react with a different epitope on CD59, led to a 50%-70% increase of the number of cells forming rosettes. In addition to rosette experiments, the binding of 125I-labeled CD59 molecules to CD2+CHO cells was specifically inhibited by unlabeled CD59 molecule and CD2 mAb. Furthermore, the binding of CD59 molecules to resting T cells induced expression of CD2R epitopes. These results directly show that CD2 binds CD59 and that subtle molecular changes occur upon binding.
Abstract: We have generated stable Chinese hamster ovary (CHO) cell transfectants expressing either CD58 or CD59 or both molecules to compare their respective parts played in T cell adhesion and activation. Using a rosetting assay, we have shown the following: 1) The CD59 molecule was directly responsible for adhesive interaction between human T cells and CD59+ CHO transfectants. CD59-mediated adhesion induced 12 +/- 2% (mean +/- SEM, n = 25) of rosettes. 2) The CD58 molecule expressed on CD58+ CHO transfectants induced 29 +/- 6% (mean +/- SEM, n = 8) of rosettes. 3) Double transfected CD58+CD59+ CHO cells formed up to 80% of rosettes, largely exceeding the sum of rosettes formed by single transfectants, thus disclosing at least an additive and possibly a synergic action of both molecules in mediating adhesion to T cells. Culturing purified human T cells in the presence of fixed CHO transfectants and submitogenic doses of PHA + rIL-1 alpha showed that: 1) CD59+ CHO transfectants induced sevenfold T cell proliferation enhancement, demonstrating the direct involvement of the CD59 molecule in T cell activation; 2) CD58+ CHO transfectants induced 20-fold T cell proliferation increase; and 3) the enhancement induced by CD58+CD59+ CHO cells was more than 40-fold. These results suggest that CD58 and CD59 molecules present on the surface of accessory cells might exert synergic function in T cell adhesive interactions and in the stimulation of T cell activation.
Abstract: To determine whether autoantibodies to the insulin receptor may represent markers of Type 1 (insulin-dependent) diabetes, the prevalence of such antibodies was investigated in sera of 60 newly diagnosed untreated Type 1 diabetic patients. A sensitive assay, based on enzyme linked immunosorbent assay has been set up which detects antibodies to the insulin receptor irrespective of their potentially inhibiting effect on insulin binding. Moreover, this method allows easy determination of the immunoglobulin class involved in the anti-receptor activity. Among the 60 sera examined, only one was found to contain anti-insulin receptor autoantibodies (IgG class). In view of our data, we conclude that autoantibodies to the insulin receptor are infrequent findings in Type 1 diabetes of recent onset.
Abstract: Insulin signal transmission through the plasma membrane was studied in terms of relationship between basal autophosphorylation of the beta-subunit and the ability to bind insulin by the alpha-subunit of the insulin receptor. In a cell free system, receptors phosphorylated on tyrosine residues in the absence of insulin were separated from non-phosphorylated receptors using antiphosphotyrosine antibodies. Insulin binding assays were then performed on basally autophosphorylated and on non-phosphorylated receptors. We found that the tyrosine phosphorylated receptors, which corresponded to 25% of the total number of receptors, were accountable for 60-80% of insulin binding. Scatchard representation of binding data has shown that the plot corresponding to tyrosine phosphorylated receptors was localized above, and was steeper than the plot corresponding to non-phosphorylated receptors. These data make it likely that the conformation of alpha-subunit which favours ligand binding is connected to the conformation of beta-subunit which favours phosphate reception on tyrosine residues. Reciprocally, the high-affinity conformation of insulin receptor seems to become stabilized by basal autophosphorylation.
Abstract: Properties of oligomeric states of the insulin receptor were analyzed by polyacrylamide gel electrophoresis in nondenaturing buffer conditions (ND-PAGE). Partially purified insulin receptors resolve in ND-PAGE as three distinct species: (i) the fast electrophoretic mobility, low molecular mass form manifests intense labeling by iodinated insulin and shows basal and insulin-stimulated autophosphorylation; (ii) the middle, intermediate mobility form exhibits strong labeling by iodinated ligand but does not possess the capacity to be autophosphorylated; (iii) the slow mobility, highest molecular mass form necessitates covalent binding with iodinated hormone to withstand electrophoresis and shows autophosphorylation enhanced by insulin. This receptor form is more heavily labeled by phosphorylation than the low form. At 22 degrees C, binding and autophosphorylation do not appear to be time dependent. At 37 degrees C, binding and autophosphorylation of low and high species attain a maximum after 15 min and then decrease as time of incubation with insulin is prolonged to 120 min; the middle species exhibits a much slower association rate, and its labeling by iodinated hormone becomes more intense with time. Our data show that in cell-free systems insulin receptors appear in various oligomeric states and that the highest molecular mass oligomer exhibits the most pronounced autophosphorylation. This is compatible with the concept that insulin receptor oligomerization provides a mechanism for transmembrane signaling.
Abstract: An 11 day-old neonate presented with bilateral renal venous thrombosis in the course of Salmonella Ibadan infection. 36 hours thrombolytic treatment with urokinase was associated with an immediate favorable outcome. Heparin and antibiotic treatment were pursued for 3 weeks. A 6 month follow-up and results of renal ultrasonography, urography and renal scanning allow to assess recovery, with restoration ad integrum for both kidneys.
Abstract: Co-administration of ketoprofen was found in 4 of 118 cycles of high-dose methotrexate (MTX) analysed retrospectively in thirty-six patients. All 4 cycles were characterised by severe MTX toxicity, which was fatal in three cases. Simultaneous administration of ketoprofen was associated with prolonged and striking enhancement of serum MTX levels. There were no abnormalities in MTX kinetics or evidence of MTX toxicity when ketoprofen was given at least 12 h after completion of high-dose MTX infusion. The kidney may be the site of drug interaction. This high-risk association between MTX toxicity and ketoprofen may also apply to other non-steroidal anti-inflammatory drugs.
