Intestinal bicarbonate secretion in cystic fibrosis mice.

Gene-targeted disruption of the cystic fibrosis transmembrane conductance regulator (CFTR) in mice results in an intestinal disease phenotype that is remarkably similar to bowel disease in cystic fibrosis patients. In the intestinal segment downstream from the stomach (i.e., the duodenum), CFTR plays an important role in bicarbonate secretion that protects the epithelium from acidic gastric effluent. In this report, we examine the role of CFTR in cAMP-stimulated bicarbonate secretion in the murine duodenum and the mechanisms of acid-base transport that are revealed in CFTR knockout (CF) mice. Ion substitution, channel blocker and pH stat studies comparing duodena from wild-type and CF mice indicate that CFTR mediates a HCO(3)(-) conductance across the apical membrane of the epithelium. In the presence of a favorable cell-to-lumen HCO(3)(-) gradient, the CFTR-mediated HCO(3)(-) current accounts for about 80% of stimulated HCO(3)(-) secretion. Exposure of the duodenal mucosa to acidic pH reveals another role of CFTR in facilitating HCO(3)(-) secretion via an electroneutral, 4,4'-diisothiocyanato-stilbene-2,2' disulfonic acid (DIDS) sensitive Cl(-)/HCO(3)(-) exchange process. In CF duodenum, other apical membrane acid-base transporters retain function, thereby affording limited control of transepithelial pH. Activity of a Cl(-)-dependent anion exchanger provides near-constant HCO(3)(-) secretion in CF intestine, but under basal conditions the magnitude of secretion is lessened by simultaneous activity of a Na(+)/H(+) exchanger (NHE). During cAMP stimulation of CF duodenum, a small increase in net base secretion is measured but the change results from cAMP inhibition of NHE activity rather than increased HCO(3)(-) secretion. Interestingly, a small inward current that is sensitive to the anion channel blocker, 5-nitro-2(3-phenylpropyl amino)-benzoate (NPPB), is also activated during cAMP stimulation of the CFTR-null intestine but the identity of the current is yet to be resolved. Studies to identify the proteins involved in non-CFTR mediated HCO(3)(-) secretion are on-going and potentially will provide targets to correct deficient HCO(3)(-) secretion in the CF intestine.

A novel Leishmania infantum recombinant antigen which elicits interleukin 10 production by peripheral blood mononuclear cells of patients with visceral leishmaniasis.

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.

Molecular cloning of a CYP1A cDNA from the teleost fish Dicentrarchus labrax.

A cDNA encoding for cytochrome P450 1A has been cloned in the marine teleost fish Dicentrarchus labrax. This fish, common in the Mediterranean, was chosen since it is considered a good sentinel species. Moreover, biomarkers of exposure to organic contaminants (such as EROD) are often measured in this species and make it possible to evaluate the quality of waters. For cloning purposes, RNAs were extracted from the liver of benzo[a]pyrene (BaP)-treated animals and used as template in degenerate RT-PCR. The cDNA product was cloned and used for the design of highly stringent primers that were utilized in Rapid Amplification of cDNA Ends (RACE) PCR. The cloned cDNA hybridizes with a 2.7 kb mRNA which is induced by treatment of the fish with BaP, a classical CYP1A inducer. The closest sequences found in data banks belong to fish CYP1A.

Evaluation of biomarkers in caged fishes and mussels to assess the quality of waters in a bay of the NW Mediterranean Sea.

Specimens of sea bass (Dicentrarchus labrax) were placed in cages for 1 month in spring and autumn at different locations in the Bay of Cannes (NW Mediterranean). Biochemical markers evaluated were: ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities in fish livers and acetylcholinesterase (AChE) activity in fish muscle. EROD and GST activities were higher in front of the outlet for the wastewater plant of Cannes and in the harbour than outside the marina. High EROD and GST activities may be induced by petrol hydrocarbons and/or polychlorinated biphenyls (PCBs). AChE was low in the muscles of the fish caged in the harbour compared with samples from the other cages. Low AChE activity could suggest the presence of organophosphorus and carbamate compounds in seawater from the harbour. Mussels (Mytilus galloprovincialis) were caged off Cannes for the same periods as the fishes. Heavy metal, metallothionein (MT) concentrations and lysosomal membrane stability were evaluated in the digestive gland of the mussels. Results show low heavy metal and MT concentrations, implying low metal concentrations in the surrounding waters. High lysosomal membrane stability revealed a good physiological status of these animals after caging. The whole set of data indicates that seawater in the Bay of Cannes appeared to be unpolluted as regards pollutants which may induce the measured biomarkers, except in restricted areas.

Progression of visceral leishmaniasis due to Leishmania infantum in BALB/c mice is markedly slowed by prior infection with Trichinella spiralis.

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.