The in vitro activity of Midecamycin against various Vibrio species.

The susceptibility of various Vibrio species to Midecamycin was studied by the tube dilution method. A MIC of 100 mug per ml of Midecamycin in BHI broth was taken as an indicator of bacterial resistance. All Vibrio cholerae classic biotype strains tested, were sensitive to Midecamycin. In respectively, 87.4%, 60.7% and 89.5% of Vibrio cholerae El Tor biotype, Vibrio parahaemolyticus and Vibrio NAG strains tested, were sensitive to Midecamycin.     

Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3

BACKGROUND: Alloantibodies against human neutrophil antigen‐3 (HNA‐3) are responsible for the fatalities reported in transfusion‐related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA‐3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA‐3a or HNA‐3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA‐3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA‐3a sera reacted specifically with HNA‐3aa cells. One serum sample showed positive reaction with HNA‐3bb cells. All HNA‐3b sera recognized HNA‐3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA‐3a sera (12/12) showed positive reactivity with HNA‐3aa cells with no cross‐reactivity with HNA‐3bb cells. Again, all HNA‐3b sera reacted with HNA‐3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA‐3 allele caused by a nucleotide substitution C>T at Position 457 leading to L₁₅₃F mutation in choline transporter‐like protein‐2. This mutation impairs polymerase chain reaction with sequence‐specific primers based HNA‐3a typing. However, analysis with cells expressing F₁₅₃ isoform showed that this mutation did not alter the binding of HNA‐3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA‐3 are suitable for the detection of HNA‐3 alloantibodies allowing reliable screening of blood products.     

Bioequivalence study of two clopidogrel film-coated tablet formulations in healthy volunteers

The present study was performed to compare the bioavailability of two clopidogrel 75 mg film-coated tablet formulations (test formulation and reference formulation). This study was a randomized, single-blind, two-period, two-sequence cross-over study which included 24 healthy adult male and female subjects under fasting condition. The pharmacokinetic parameters were assessed based on the concentrations of clopidogrel (CAS 120202-66-6) parent compound. In each of the two study periods (separated by a washout of one week) a single dose of test or reference drug was administered. Plasma concentrations of the drug were determined by LC-MS/MS method. The pharmacokinetic parameters assessed in this study were area under the plasma concentration-time curve from time zero to 24 h (AUCt), area under the plasma concentration-time curve from time zero to infinity (AUCinf), the peak plasma concentration of the drug (Cmax), time needed to achieve the peak plasma concentration (t(max)), and the elimination half life (t1/2). The geometric mean ratios (90% CI) of the test drug/reference drug for clopidogrel parent compound were 95.19% (81.63-110.90%) for AUCt, 95.55% (80.50-113.42%) for AUCinf, and 100.18% (80.87-124.09%) for Cmax. The 90% confidence intervals calculated for AUCt and Cmax of clopidogrel parent compound were within the standard bioequivalence range (80-125% for AUC and Cmax). It was concluded that the two clopidogrel film-coated tablets (test and reference drug) were bioequivalent in terms of the rate and extent of absorption.     

Report on the 15th International Society of Blood Transfusion platelet immunology workshop

Background and Objectives The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet‐reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti‐HPA‐1a and serologically difficult‐to‐assess antibodies against HPA‐3, to identify whether anti‐HPA‐1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. Materials and Methods Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. Results Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA‐1a, in contrast, gave largely congruent results. Serologically difficult‐to‐assess antibodies recognizing HPA‐3a and HPA‐3b were not detected by many laboratories. For genotyping, good agreement was achieved. Conclusions Detection of HPA‐1a antibodies, titration of anti‐HPA‐1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA‐3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable.     

Low-avidity anti-HPA-1a alloantibodies are capable of antigen-positive platelet destruction in the NOD/SCID mouse model of alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen 1a (HPA‐1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti‐HPA‐1a could be overlooked by standard methods due to low avidity. Low‐avidity HPA‐1a antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low‐avidity anti‐HPA‐1a. STUDY DESIGN AND METHODS: A retrospective cohort consisting of 82 HPA‐1bb mothers of HPA‐1ab newborns with thrombocytopenia was analyzed using standard serologic methods. Maternal immunoglobulin (Ig)G fractions were investigated for low‐avidity antibodies in SPR using purified GPIIb/IIIa (HPA‐1a or ‐1b). The capability of HPA‐1a antibodies to clear platelets (PLTs) in vivo was analyzed using the NOD/SCID mouse model of alloimmune thrombocytopenia. RESULTS: HPA antibodies were detectable in sera from 68 of 82 (83%) mothers using standard serologic methods and undetectable in 14 of 82 sera. In SPR, IgG fractions of sera reacting positive in monoclonal antibody immobilization of PLT antigen (MAIPA) assay showed specific binding to an HPA‐1a flow cell (mean, 87 ± 21 resonance units [RU]). When MAIPA‐negative sera were tested in SPR, binding with low avidity was observed in 7 of 14 to HPA‐1a (mean, 31 ± 5 RU), but not to HPA‐1b flow cell (mean, 5 ± 2 RU). In vivo, low‐avidity antibodies were capable of clearing HPA‐1ab PLTs but not HPA‐1bb PLTs in a NOD/SCID mouse model. Elimination kinetics were slower than observed with MAIPA‐positive antibodies. CONCLUSIONS: Low‐avidity HPA‐1a antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbor the capability of PLT destruction in vivo.     

Single amino acid substitution in human platelet glycoprotein Ibbeta is responsible for the formation of the platelet-specific alloantigen Iy(a)

We recently described a new low-frequency platelet alloantigen on the human platelet glycoprotein (GP) Ib-IX complex, termed Iy(a), which was implicated in a severe case of neonatal alloimmune thrombocytopenia. Immunoprecipitation studies with trypsin-treated platelets indicated that the Iy(a) alloantigenic determinants are formed by the membrane-associated remnant moiety of GP Ibalpha (GP Ibalpha(r)) together with GP Ibbeta and GP IX. To elucidate the molecular basis underlying the Iy(a) alloantigen, we amplified GPIbalpha(r), GPIbbeta, and GPIX genes by polymerase chain reaction (PCR). Nucleotide-sequence analysis of these 3 genes showed a G to A transition at position 141 on GPIbbeta gene in a subject positive for Iy(a). This transition resulted in a Gly(15)Glu dimorphism on the N-terminal domain of GPIbbeta. This finding was confirmed by genotyping analysis of 6 Iy(a)-positive subjects by restriction fragment length polymorphism (RFLP) studies using NarI endonuclease. In 300 randomly selected healthy blood donors, one Iy(a)-positive individual was found. Phenotypes determined by monoclonal antibody-specific immobilization of platelet antigens assay and genotypes determined by RFLP were identical in this population. Analysis of Iy(a)-positive platelets showed that the point mutation affected neither the degree of surface expression nor the function of the GP Ibalpha-GP Ibbeta-IX complex on the platelet surface. Transient expression of the GP Ib-IX complex in CHO cells using wild-type GP Ibbeta (Gly(15)) or mutant GP Ibbeta (Glu(15)) allowed us to demonstrate that this single amino acid substitution is sufficient to induce Iy(a) epitope(s). (Blood. 2000;95:1849-1855)