Na+/H+ exchange in glial cells of Necturus optic nerve

Single and double-barreled pH-sensitive electrodes were used to study intracellular pH (pHi) regulation in glial cells of Necturus optic nerve in the nominal absence of HCO3-/CO2. After the cells were acidified by the addition and withdrawal of NH4+, the pHi recovered toward the original steady-state pHi. The recovery from acidification was Na+-dependent and inhibited by 1 mM amiloride. These results suggest the existence in intact vertebrate glial cells of a Na+/H+ exchanger which functions in acid extrusion.     

Neuropeptide Y in the female reproductive tract of the rat. Distribution of nerve fibres and motor effects

The spontaneous firing of single neurones in the region of the lateral reticular nucleus was the subject of a pharmacological study employing microiontophoretic and systemic application of adrenoceptor agonists and antagonists. Both iontophoretic noradrenaline and systemic clonidine depressed neuronal firing. The depressions were consistently reversed by the alpha-2 antagonist RX781094. Other adrenergic antagonists, prazosin and sotalol, were ineffective. The results suggest the existence of alpha-2 receptors in this region of the brain.     

Occurrence of hlyA and sheA genes in extraintestinal Escherichia coli strains

The association of a hemolytic phenotype with the carriage of the alpha-hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+ E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+ sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.     

Transmission of HIV-1 infection between trophoblast placental cells and T-cells take place via an LFA-1-mediated cell to cell contact

HIV-1 vertical transmission is thought to mainly take place by virus crossing the placental barrier. However, the mechanism by which HIV-1-infects placental cells remains to be elucidated. We have found that purified cytotrophoblasts as well as trophoblastic cell lines are susceptible to infection by different HIV-1 isolates as detected by DNA-PCR and release of infectious virus, although with very low efficiency. Purified trophoblast or trophoblastic cell lines express low levels of chemokine receptors CCR-5 and CXCR-4 but not CD4 on the cell surface. To test if those molecules were used as receptors for HIV-1 infection, placental cells were pretreated with antibodies to CD4, CC-chemokines, C-X-C chemokines. None of those treatments inhibited HIV-1 infection. In contrast, we have found that HIV-1 infection of placental cells was increased in cocultures of infected T-cell blasts and placental cells. More interestingly, antibodies to beta(2) integrins and to LFA-1 were able to significantly block infection of placental cells. Cell surface expression of ICAM-1, an adhesion molecule involved in attachment of leukocytes to placenta, was upregulated in HIV-1-infected placental cells. Placental cells were able to transfer HIV-1 infection to T-cell blasts. This transmission required cell to cell contact and was also inhibited by anti-LFA-1 antibodies. In summary our results suggest that placental trophoblast could be infected by HIV-1 by a mechanism involving T cell to placental contact. Moreover, placental infection enhanced ICAM-1 expression and leukocyte adherence, an event which was required to transfer HIV-1 infection to T cells. This provides an explanation of the virus passing through the placental barrier during in utero HIV-1 vertical transmission.     

Development of DNA vaccines against hemolytic-uremic syndrome in a murine model

Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2 Delta A). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.     

Immunoglobulin M (IgM)-glycoinositolphospholipid enzyme-linked immunosorbent assay: an immunoenzymatic assay for discrimination between patients with acute toxoplasmosis and those with persistent parasite-specific IgM antibodies

In the present study we developed an enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin M (IgM) specific for glycoinositolphospholipids (GIPL) derived from tachyzoite membrane (IgM-GIPL ELISA). The sensitivity and specificity of the assay were compared with those of commercially available Toxoplasma-specific IgM serological tests, namely, immunofluorescence assay (IFA) with fixed tachyzoites and capture ELISA employing tachyzoite extracts. Our results show that all patients with acute toxoplasmosis, as determined by clinical data and conventional serological tests, were also positive by the IgM-GIPL ELISA. Interestingly, many patients that were classified as indeterminate, who had IgG with high avidity but positive results in the IgM-specific IFA and capture ELISA, were negative by the IgM-GIPL ELISA. Finally, we tested the sera from patients with rheumatoid arthritis and various parasitic infections and found no evidence of false positives in the IgM-GIPL ELISA.     

Cloning,isolation, and IgE-binding properties of Helix aspersa (brown garden snail) tropomyosin

BACKGROUND: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail. This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates. METHODS: A tropomyosin-specific cDNA encoding H. aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein. IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients. RESULTS: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84-69% identity) as well as with those from arthropods (65-62%), and less homology with vertebrate ones (56% identity). Tropomyosin reacted with 18% of the sera from patients with snail allergy. Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins. Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts. CONCLUSIONS: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity.     

Ontogeny, distribution and function of CD38-expressing B lymphocytes in mice

Analysis of expression of CD38, CD45R (B220), IgM and IgD on splenic B lymphocytes from mice of different ages demonstrated CD38 on both immature (B220(+), BCR(-)) and mature (B220(+), BCR(+)) B lymphocytes. Similarly, CD38 is expressed as early as B220 on the surface of progenitor B cells in the bone marrow. In spite of expressing of CD38 and IgM, neonatal B cells, in contrast to the adult, failed to proliferate to either anti-CD38 or anti-IgM cross-linking when IL-4 was present. They did, however, respond to LPS and anti-CD40, and by 2 weeks of age they began to respond to anti-CD38 and anti-IgM, reaching adult B cell levels by 4 weeks. Although the distribution of CD38 on adult B cells from most different lymphoid compartments was broadly similar, significantly higher levels of CD38 were expressed on peritoneal B lymphocytes. A detailed analysis, using IgM / IgD ratio and staining with anti-CD5 confirmed that B1 lymphocytes were expressing a high level of CD38. Interestingly, both immature B cells and peritoneal B1 lymphocytes were unresponsive to anti-CD38. However, they were activated by LPS or anti-CD40.