Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

Activation of human platelets by C5a-stimulated neutrophils: a role for cathepsin G.

Human platelets can be stimulated by recombinant human fifth component of complement (rhC5a) in the presence of human neutrophils. After challenge with N-formyl-Met-Leu-Phe or rhC5a, concentrated neutrophils release cathepsin G into the supernatant. The concentrations of cathepsin G recovered by titration of the enzymatic activity correlate with the capability of these supernatants to induce platelet stimulation as measured by serotonin release. Cathepsin G purified from neutrophil granules triggered platelet aggregation and serotonin release independent of arachidonic acid metabolites and platelet-activating factor formation. A concentration of 100 nM of cathepsin G, which was reached in the surrounding space of activated neutrophils, induced a 50% platelet stimulation. Three distinct antiproteinases were tested against cathepsin G-induced platelet activation. Z-Gly-Leu-Phe-CH2Cl, a specific inhibitor of cathepsin G enzymatic activity, proved to be nonspecific in our biological system. By contrast, alpha 1-antichymotrypsin and alpha 1-antitrypsin displayed specific activities. The physiological specific inhibitor of cathepsin G, alpha 1-antichymotrypsin, was the most potent and was used in the rhC5a-induced neutrophils-mediated platelet activation. A complete inhibition was achieved, showing that release of cathepsin G from neutrophils accounts for platelet activation. Such a chain of events involving C5a, neutrophils, cathepsin G, and platelets may be of relevance in certain inflammatory states, particularly the adult respiratory distress syndrome.

In vivo effects of ovalbumin-conjugated muramyl peptides on the anti-ovalbumin IgE and IgG responses in mice.

The effects of pretreatments of BALB/c mice with several conjugates of MDP and MDP-Lys to ovalbumin before immunization with ovalbumin (OA) were tested on the anti-OA IgE responses. Pretreatment with MDP-Lys-OA, but not with MDP-OA, induced an inhibition of the anti-OA primary and secondary responses, as measured by passive cutaneous anaphylaxis (PCA) and also by mast cell degranulation. The inhibition by pretreatment with MDP-Lys-OA was obtained whether it was administered in Freund's incomplete adjuvant (FIA) or in saline. This IgE suppression was accompanied by an enhancement of IgG2a and IgG2b anti-OA antibodies, with no change in the specific IgG1 levels. Loss of antigenicity of OA, detected by the lack of degranulation of peritoneal mast cells sensitized by IgE anti-OA, was observed in the MDP-Lys-OA but not in the MDP-OA conjugates. This loss of antigenicity appears to correlate with the ability of the conjugate to induce suppression of the specific IgE response.