Peritoneal macrophages from C57BL/6 mice orally immunized with Toxoplasma gondii antigens in association with cholera toxin possess an enhanced ability to inhibit parasite multiplication

International audienc

A constitutive nitric oxide synthase modulates insulin secretion in the INS-1 cell line

We provide immunocytochemical evidence that the neuronal isoform of constitutive NO synthase (cNOS) is expressed in the rat insulinoma cell line INS-1. Furthermore, using N omega -nitro-L-arginine methyl ester (L-NAME), a pharmacological inhibitor of cNOS activity, we show that this enzyme is implicated in the modulation of insulin secretion in INS-1 cells. Indeed, in the presence of 2.8 MM glucose, L-NAME induced a specific and dose-dependent increase in insulin release, suggesting that cNOS exerts an inhibitory tone on basal insulin secretion. Moreover, L-arginine, the physiological substrate of cNOS, significantly reduced the marked enhancing effect Of L-NAME on insulin release and to a lesser extent, at low concentrations, that of 10 mM KC1. L-NAME also potentiated the insulin secretion stimulated by 5.5 and 8.3 mM glucose, but in this case, its effect was not reduced by L-arginine. In conclusion, our data show that the neuronal isoform of cNOS exerts a negative modulation on insulin secretion in INS-1 cells, confirming the previous results obtained in the isolated perfused rat pancreas or pancreatic islets. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved

Monoclonal antibody 667 recognizes the variable region A motif of the ecotropic retrovirus CasBrE envelope glycoprotein and inhibits Env binding to the viral receptor

Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D(57), is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes

Counter-Protective Role for Interleukin-5 during Acute Toxoplasma gondii Infection

The role of interleukin-5 (IL-5) during Toxoplasma gondii infection was investigated by comparing disease progression in IL-5 gene deficient (IL-5−/−) mice and their wild-type (WT) counterparts on a C57BL/6 background. IL-5−/− mice infected orally with T. gondii were less susceptible to infection than WT mice as demonstrated by reduced mortality rates. Consistent with this data, orally infected IL-5−/− mice had less severe pathological changes in their small intestines than WT mice at 8 days postinfection. At this time, splenocytes and mesenteric lymph node cells derived from IL-5−/− mice produced levels of IL-12, interferon-γ (IFN-γ), IL-4, IL-10, and nitric oxide (measured as nitrite) similar to those derived from WT mice when stimulated with Toxoplasma lysate antigen. However, peak serum IL-12 and IFN-γ levels (at days 6 and 8, respectively) were significantly higher in IL-5−/− mice than in WT mice. In addition, WT mice but not IL-5−/− mice had raised levels of eosinophils in their peripheral blood between days 5 and 8 following infection. Oral administration of Nω-nitro-l-arginine methyl (from day 4 postinfection) increased mortality rates in both IL-5−/− and WT mice, indicating a protective role for nitric oxide during the early stages of oral T. gondii infection. In comparison with oral infection, no difference in mortality was observed between IL-5−/− and WT mice following intraperitoneal infection with T. gondii, with all mice surviving until 35 days postinfection. Similarly, no significant differences were observed in the severity of the meningitis, perivascular cuffing, or number of microglial nodules or parasites in the brains of intraperitoneally infected mice. Together, these results demonstrate a detrimental role for IL-5 during the early stage of oral infection with T. gondii which is associated with increased small-intestine pathology, eosinophilia, and reduced plasma IL-12 and IFN-γ levels