Rubén López-Revilla

Intragastric and intraperitoneal administration of Cry1Ac protoxin from Bacillus thuringiensis induces systemic and mucosal antibody responses in mice

The spore-forming soil bacterium Bacillus thuringiensis produces parasporal inclusion bodies composed by delta-endotoxins also known as Cry proteins, whose resistance to proteolysis, stability in highly alkaline pH and innocuity to vertebrates make them an interesting candidate to carrier of relevant epitopes in vaccines. The purpose of this study was to determine the mucosal and systemic immunogenicity in mice of Cry1Ac protoxin from B. thuringiensis HD73. Crystalline and soluble forms of the protoxin were administered by intraperitoneal or intragastric route and anti-Cry1Ac antibodies of the major isotypes were determined in serum and intestinal fluids. The two forms of Cry1Ac protoxin administered by intraperitoneal route induced a high systemic antibody response, however, only soluble Cry1Ac induced a mucosal response via intragastric. Serum antibody levels were higher than those induced by cholera toxin. Systemic immune responses were attained with doses of soluble Cry1Ac ranging from 0.1 to 100 microg by both routes, and the maximal effect was obtained with the highest doses. High anti-Cry1Ac IgG antibody levels were detected in the large and small intestine fluids from mice receiving the antigen via i.p. These data indicate that Cry1Ac is a potent systemic and mucosal immunogen.

Intranasal, rectal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis induces compartmentalized serum, intestinal, vaginal and pulmonary immune responses in Balb/c mice

Recently we discovered that the Cry1Ac protoxin of Bacillus thuringiensis administered to Balb/c mice intraperitoneally (i.p.) or intragastrically is a systemic and intestinal immunogen as potent as cholera toxin. To further characterize the mucosal immunogenicity of Cry1Ac we additionally tried the intranasal (i.n.) and rectal routes and used enzyme-linked immunoassays to determine anti-Cry1Ac antibody responses in the serum as well as in vaginal and tracheobronchial washes and in the fluids of the large and the small intestine. Immunization by the i.p., i.n. and rectal routes induced IgM, IgG and IgA antibodies in all the mucosal surfaces analyzed, but the magnitude and predominant isotype of each response depended on the route used and the mucosal site analyzed. These data extend our findings on the striking mucosal immunogencity of Cry1Ac and provide additional evidence on the compartmentalization of the mucosal immune system.

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyers patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.

The UGA3-GLT1 intergenic region constitutes a promoter whose bidirectional nature is determined by chromatin organization in Saccharomyces cerevisiae

Transcription of an important number of divergent genes of Saccharomyces cerevisiae is controlled by intergenic regions, which constitute factual bidirectional promoters. However, few of such promoters have been characterized in detail. The analysis of the UGA3‐GLT1 intergenic region has provided an interesting model to study the joint action of two global transcriptional activators that had been considered to act independently. Our results show that Gln3p and Gcn4p exert their effect upon cis‐acting elements, which are shared in a bidirectional promoter. Accordingly, when yeast is grown on a low‐quality nitrogen source, or under amino acid deprivation, the expression of both UGA3 and GLT1 is induced through the action of both these global transcriptional modulators that bind to a region of the bidirectional promoter. In addition, we demonstrate that chromatin organization plays a major role in the bidirectional properties of the UGA3‐GLT1 promoter, through the action of an upstream Abf1p‐binding consensus sequence and a polydAdTtract. Mutations in these cis‐elements differentially affect transcription of UGA3 and GLT1, and thus alter the overall relative expression. This is the first example of an intergenic region constituting a promoter whose bidirectional character is determined by chromatin organization.

Establishment and characterization of immortal hepatocytes derived from various transgenic mouse lines

The potential of three genetic changes introduced into mice by the transgenic or knockout technology aimed at immortalizing hepatocytes in vitro and concomitantly preserving their differentiated hepatic functions was analyzed. Six hepatocyte lines were isolated from neonatal and adult transgenic mice expressing either IgEGF (a secretable variant of hEGF) or SV40 T antigen in the liver and from neonatal and adult p53 knockout (KO) mice and have been subcultured >150 times in serum-free, arginine-deficient medium. Only in SV40 T antigen transgenic lines profiles of mRNAs encoding serum proteins, transcription factors, and liver-specific enzymes were similar to those found in livers and primary hepatocytes. Accordingly, these cells displayed basal and inducible expression of CYP proteins as well as testosterone metabolizing activities. Thus, either knockout of the p53 gene or expression of SV40 T antigen or of IgEGF imparts immortality to hepatocytes in vitro, but only SV40 T antigen expression is compatible with the concomitant long-term preservation of differentiated liver functions.

Progressive paralysis associated with diffuse astrocyte anaplasia in Δ202 mice homozygous for a transgene encoding the SV40 T antigen

A convenient transgenic astrocytoma model in Δ202 mice, homozygous for a construct encoding the early region of the SV40 virus genome, is described. In the offspring of crosses between Δ202 mice heterozygous for the transgene nearly 60% were transgenic; one third of these developed progressive paralysis starting in the hindlimbs at approximately 35 days of age and died at 90 ± 30 days of age. In affected mice proliferating‐non‐neuronal cells immunostained with antibodies to the GFAP, an astrocyte marker, whose number increased with age were found in the white matter of the brain, cerebellum and spinal cord, and progressive degeneration and necrosis of spinal motoneurons was observed that may explain the paralysis. The early onset and reproducible time course of the neurological disease suggest that homozygous Δ202 mice, whose proliferating astrocytes appear to damage spinal motoneurons, are a useful model to study astrocyte differentiation, function and tumorigenesis.

Metronidazole and Mebendazole Pretreatments Suppress the Antiamebic Recognition of Lamina propia Lymphocyte Supernatants from the Small and Large Intestine in Intraperitoneally Immunized Balb/c Mice

We have previously demonstrated that intraperitoneal (i.p.) immunization of mice with glutaraldehyde-fixed trophozoites (GFT) of Entamoeba histolytica elicites strong local and systemic immune responses (1). One of the most common drugs used against a wide variety of anaerobic protozoan parasites and anaerobic bacteria is metronidazole, whose toxicity has been reviewed. The main side effects of metronidazole are headache, nausea, dry mouth, and metallic taste. Other more severe symptoms are experienced only occasionally (2). Although related chemicals have caused blood dyscrasias, only temporary neutropenia, reversible after discontinuation of therapy, occurs with metronidazole (3). Mebendazole is a versatile antihelminthic agent. Benzimidazoles produce many biochemical changes in susceptible nematodes; there is strong evidence, however, that the primary action of these drugs is to inhibit microtubule polymerization by binding to b -tubulin. Mebendazole does not cause significant systemic toxicity in routine clinical use, this probably resulting from its low systemic bioavailability. Rare side effects in patients treated with high doses of mebendazole include allergic reactions, alopecia, reversible neutropenia, agranulocytosis, and hypospermia (4). In a previous work (5), we analyzed by ELISA the antiamebic antibody response in the sera and intestinal samples of metronidazole–mebendazole pretreated, and nontreated GFT-immunized mice. We found that the GFT-immunized mice pretreated with metronidazole and mebendazole showed a significantly lower IgM and IgG serum response and a significant IgM and IgA response in intestinal fluids and supernatants of lamina propia lymphocytes from the large intestine than nontreated GFT i.p.-immunized mice. Moreover, we found that sera of metronidazole–mebendazole pretreated mice showed very low recognition of amebic proteins by Western blot. These data suggest that metronidazole and mebendazole treatment suppresses both the mucosal and systemic humoral immune response. The purpose of this work was to determine the effect of a 7-day 2.5 mg/per dose metronidazole and mebendazole (1:1) pretreatment in Balb/c mice immmunized i.p. with GFT by analyzing lamina propia lymphocyte supernatants from the small and large intestine and the systemic antiamebic immune response.