Antiplatelet activity of dipyridamole in non anticoagulated whole blood

Dipyridamole has been reported to inhibit platelet aggregation in citrate anticoagulated whole blood (WB). However, citrate may alter the response of platelets and/or the effect of antiplatelet drugs. The present study evaluates the "ex vivo" effect of dipyridamole, two hours after a single dose (3 mg/Kg) in 25 normal subjects in non-anticoagulated (native) WB and in WB anticoagulated with citrate or hirudin. We have used the BASIC anticoagulated with citrate or hirudin. We have used the BASIC wave as analytical method, which can evaluate the early steps of platelet activation with collagen in less than 1 min after venoclysis, thus allowing the study in native WB. The results show that dipyridamole significantly inhibits (p less than 0.001) platelet activation to collagen in citrated WB (66%) while the drug's effect is much lower (21%) and non-significant if evaluated in native or hirudine anticoagulated WB. These results suggest that citrate or low calcium concentration amplify the drug's platelet inhibitory action in WB and, therefore, the laboratory results may overestimate the drug's effect "in vivo".     

HLA-B44 subtyping in a Spanish population: further evidence of Caucasian population diversity

HLA-B44 is the most frequent HLA-B allele in Caucasian populations. Several B44 subtypes, B*4402-B*4406, have been identified in individuals with this ethnic origin. Mismatches among B44 subtypes have been described as major targets for allogeneic responses in bone marrow transplantation. We have developed a PCR-SSO method, based on a B12- specific DNA amplification of exon 2 through exon 3 and subsequent non radioactive hybridization with eight probes, which allow us to discriminate all B12 homozygous combinations. We applied this method to determine the frequency of B44 subtypes in a Spanish population, as well as their HLA-A.-C.-DRB1,-DRB3/DRB4/DRB5.-DQA1 and -DQB1 associated haplotypes. A total of 141 healthy unrelated Spanish individuals and 31 B44-bearing haplotypes were investigated. Four B44 alleles were identified, B*4402 (33%), B*4403 (66%), B*4404 (0.7%), and B*4405 (0.7%). Haplotype analysis showed a clear differentiated distribution pattern for the two major B44 subtypes. B*4402 is associated with Cw5 (11/13) and A2 antigens (10/13). In contrast, B*4403 is mainly found together with DRB1*0701 (14/16). An inverted B*4402/B*4403 frequency in comparison with other European and North American Caucasian populations, revealed the existence of an extended haplotype diversity between populations of the same ethnic origin. Apart from anthropological studies, high resolution typing for HLA class I antigens presenting molecular polymorphism will be of great relevance in unrelated bone marrow transplantation.     

Survey of rotavirus G and P types associated with human gastroenteritis in São Paulo, Brazil, from 1986 to 1992.

Rotavirus strains causing gastroenteritis in Brazilian children were characterized by PCR-based typing assays. In addition to strains bearing the major human G and P types, large numbers of strains bearing P3 (M37-like), P6 (HCR3-like), untypeable P and G types, and complex mixtures of P and G types not previously recognized were present in the community.     

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector

Summary. We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4µg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.Authors contributed equally to the results presented in this report.     

Effect of cyclosporine on the response of normal human lymphocytes to cytomegalovirus in vitro.

Lymphocytes from healthy volunteers seropositive for cytomegalovirus (CMV) demonstrated strong lymphoproliferative responses to CMV-infected and glutaraldehyde-fixed foreskin fibroblasts (CMVFFx) when cocultured for 6 days. Addition of the immunosuppressive drug cyclosporine (CsA) to the cultures resulted in a 10-fold reduction (P less than 0.001) in counts per minute of [3H]thymidine uptake. The proliferative response to noninfected fixed fibroblasts was also reduced 10-fold. Kinetic studies showed an inhibition of the lymphoproliferative response and not an alteration in the time course kinetics in CsA-treated cultures. Cytotoxicity to CMV-infected and unfixed fibroblasts by lymphocytes primed by CMVFFx in the presence of 0.5 micrograms of CsA per ml was significantly reduced (P less than 0.01) as compared with untreated cultures but remained significantly above background level (P less than 0.01). The cytotoxic response was still present but reduced at concentrations of greater than or equal to 1.0 micrograms/ml. Cytotoxicity to noninfected fibroblasts by CMVFFx-primed lymphocytes could be eliminated by as little as 0.5 micrograms of CsA per ml. Stimulation of lymphocytes by CMVFFx but not by noninfected fixed fibroblasts resulted in the in vitro generation of suppressor cells. CsA at a final concentration of 1.0 or 2.5 micrograms/ml did not significantly impair the induction of cells capable of suppressing the lymphoproliferative response of fresh autologous mononuclear cells to CMVFFx. The findings described above may have important clinical implications in that a degree of protective immunity to CMV-infected cells is maintained even in the presence of CsA.     

Accuracy of a commercial enzyme-linked immunosorbent assay for CagA in patients from Brazil with and without gastric carcinoma

We validated a commercial enzyme-linked immunosorbent assay for the detection of anti-CagA antibodies in Brazilian patients with Helicobacter pylori infection. The test presented high sensitivity (97.4%) and specificity (88.9%) when employed in patients without gastric carcinoma. However, in gastric carcinoma patients, the test was neither sensitive nor specific enough to detect cagA-positive H. pylori infection.     

An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish

A hitherto unrecognised lethal toxin from the extracellular products (ECP) of Aeromonas hydrophila is described. The pure toxin was 300 times more toxic than the crude ECP and is the most toxic substance so far described from this bacterium, with a minimum lethal dose of 0.05 micrograms g-1 fish. The toxin had high acetylcholinesterase activity and occurred in native ECP as a monomeric 15.5 kDa polypeptide. The purified toxin had five isoelectric focusing forms ranging from pl 4.45 to 4.70. The ECP of each of six strains of A. hydrophila isolated from fish possessed acetylcholinesterase activity suggesting that the toxin is common in this species. The toxin was not a cytolysin and produced no gross pathology in injected fish. Its enzymic nature, low lethal dose, lack of tissue pathology and its apparent narcotic effect suggest that this toxin may act upon the central nervous system of the fish.     

Group‐specific component (GC) in curiaú and pacoval,two african‐derived brazilian populations

The group‐specific component (GC) system is of interest in anthropological genetic studies because the distribution of its subtypes distinguishes among major ethnic groups. The GC system was analyzed in Curiaú and Pacoval, two remnant Quilombo populations (African‐derived populations) from the Brazilian Amazon. There was no significant statistical difference in allelic frequencies between the two populations or between them and three other African‐derived Brazilian populations (Mimbó, Sítio Velho, and Gaucinha in Northeastern Brazil). These populations share similarities among themselves and with African populations (high frequencies of GC*1F and lower frequencies of GC*1S), which may reflect the influence of a high level of African contribution to their formation, but there is a clear difference between them and Europeans and South American Indians. It is suggested that the GC system is a useful marker for studying relationships between single populations and major ethnic groups, but does not discriminate between populations which share the same parental stock. Am. J. Hum. Biol. 13:718–720, 2001. © 2001 Wiley‐Liss, Inc.