Glycogen synthase kinase-3 (GSK-3) inhibition induces apoptosis in leukemic cells through mitochondria-dependent pathway.

The roles of glycogen synthase kinase-3 (GSK-3) in cell survival and apoptosis are controversial. We examined the effect of a specific GSK-3 inhibitor (SB-415286) on the regulation of leukemic cells proliferation and apoptosis. SB-415286 (40 µM) induced cell growth inhibition, ß-catenin stabilization, cell cycle arrest in G(2)/M phase, cyclin B1 downregulation, and apoptosis in leukemic cell lines KG1a, K562, and CMK. Blocking the death receptor pathway by using a specific inhibitor of caspase-8, did not inhibit SB-415286-induced apoptosis. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Furthermore, we found that depolarization of mitochondria membrane caused by GSK-3 inhibition is regulated by dephosphorylation of anti-apoptotic protein Bcl-2 and downregulation of Bcl-xL. Thus, inhibition of GSK-3-induced apoptosis of leukemic cells could be an attractive target for treatment of leukemia.

Accurate Rh phenotype determination by reticulocyte mRNA typing shortly after multiple transfusions.

Alloimmunization is a common phenomenon after transfusion, with an estimated incidence of 0.5% increasing to 20-60% in chronically transfused patients. In recently transfused patients, serological typing can be hampered by mixed field agglutination. We established RT-PCR methods for RHD, RHC/c and RHE/e typing using mRNA from reticulocytes. Molecular typing was performed soon after 51 separate mismatched transfusion events involving 30 patients. Accurate identification of the transfused patients' phenotype was confirmed in all cases. Reticulocyte maturation studies revealed that temperature is a crucial parameter for transition into mature red blood cells.

A screening and intervention program aimed to reduce mortality and serious morbidity associated with severe neonatal alloimmune thrombocytopenia.

The study's objective was to identify HPA 1a-negative women and to offer them an intervention program aimed to reduce morbidity and mortality of neonatal alloimmune thrombocytopenia (NAIT). HPA 1 typing was performed in 100 448 pregnant women. The HPA 1a-negative women were screened for anti-HPA 1a. In immunized women, delivery was performed by Cesarean section 2 to 4 weeks prior to term, with platelets from HPA 1a-negative donors reserved for immediate transfusion if petechiae were present and/or if platelet count was less than 35 x 10(9)/L. Of the women screened, 2.1% were HPA 1a negative, and anti-HPA 1a was detected in 10.6% of these. One hundred seventy pregnancies were managed according to the intervention program, resulting in 161 HPA 1a-positive children. Of these, 55 had severe thrombocytopenia (< 50 x 10(9)/L), including 2 with intracranial hemorrhage (ICH). One woman with a twin pregnancy missed the follow-up and had one stillborn and one severely thrombocytopenic live child. In 15 previous prospective studies (136 814 women) there were 51 cases of severe NAIT (3 intrauterine deaths and 7 with ICH). Acknowledging the limitation of comparing with historic controls, implementation of our screening and intervention program seemed to reduce the number of cases of severe NAIT-related complications from 10 of 51 to 3 of 57.

Evaluation of a new flow cytometric HPA 1a screening method. A rapid and reliable tool for HPA 1a screening of blood donors and pregnant women.

We have evaluated a flow cytometric screening method for identification of HPA la negative individuals, using a commercially available monoclonal anti-CD61 antibody specific for the HPA 1a allotype, and compared the method with an ELISA based method for HPA la phenotyping and two methods for PCR genotyping. HPA 1a phenotyping by fluorochrome conjugated monoclonal anti-HPA la and analysis by flow cytometry is a rapid, reliable and inexpensive technique, suitable for screening purposes.

Rapid and reliable genotyping of human platelet antigen (HPA)-1, -2, -3, -4, and -5 a/b and Gov a/b by melting curve analysis.

BACKGROUND: The probability for occurrence of neonatal alloimmune thrombocytopenic purpura (NAITP) depends largely on the frequency of each individual phenotype in various populations. In caucasians, antibodies to human platelet antigen (HPA)-1a are the major cause of neonatal alloimmune thrombocytopenic purpura, whereas in the Japanese population, antibodies to HPA-4b is most frequently involved in NAITP. Conventional PCR techniques for platelet antigen genotyping rely on sequence-specific primers (SSPs) and detection by gel electrophoresis, a method which is laborious and time consuming. New PCR technology, measuring the match of a hybridization probe with its target and thereby allowing simultaneous detection of both alleles, provides an efficient tool for genotyping of the HPA systems. STUDY DESIGN AND METHODS: A total of 105 healthy blood donors were genotyped for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b with new primers and probes designed for mutation detection by melting curve analysis (using LightCycler technology). Donor DNA was independently genotyped by an allele-specific assay, using SSPs, in a reference laboratory. RESULTS: There was full concordance between the two genotyping methods, and genotype frequencies were comparable with previous studies in caucasians. CONCLUSION: We present rapid and reliable detection systems for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b based on mutation detection of both alleles simultaneously by melting curve analysis. As the Gov system has been reported to have similar frequency of involvement in alloimmune thrombocytopenia as HPA-5, the opportunity for genotyping should aid the diagnosis of such patients.