Non-D Rh antibodies appearing after apheresis platelet transfusion: stimulation by red cells or microparticles?

BACKGROUND: Apheresis platelets (APs) have gained favour over whole blood-derived platelets on the presumption that they are less likely to provoke alloimmunization to red-blood-cell antigens. CASE REPORTS: Non-D Rh antibodies appeared in three patients after apheresis platelet transfusion. Anti-C and anti-E arose in two female patients with previous antigen exposure. Both anti-c and anti-E arose in a male recipient with no prior transfusion history. MATERIALS AND METHODS: Fifty APs were analysed for residual RBCs and RBC-derived microparticles, using samples obtained from a local blood centre. Cells and microparticles were quantified with a flow cytometry gating scheme, using PE-labelled anti-CD235a (glycophorin A) and FITC-labelled anti-CD41a (platelet gp IIb/IIIa) to distinguish lineage. RESULTS: Apheresis platelets were found to contain a mean of 7·5×10(6) (95% C.I. [6·3-8·5×10(6) ]) RBCs on one manufacturer's device and 5·2×10(6) (95% C.I. [4·0-6·3×10(6) ]) RBCs on another's. RBC-derived microparticles averaged 210·7×10(6) (95% C.I. [166·2-254·2×10(6) ]) on one manufacturer's device and 232·3×10(6) (95% C.I. [194·3-272·9×10(6) ]) on another's. These counts all correspond to volumes of <1 µl. CONCLUSION: Despite RBC contamination of APs below commonly accepted thresholds for Rh immunogenicity, AP transfusion can provoke non-D Rh antibody formation. RBC-derived microparticles, smaller but more numerous than RBCs, are volumetrically comparable and may be a hitherto underappreciated antibody stimulus. Further microparticle research will guide considerations of extended phenotypic matching of platelet components.

Contribution of Va24Vb11 natural killer T cells in Wilsonian hepatitis.

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patient's immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Valpha24+Vbeta11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs-negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Valpha24+Vbeta11+ NKT cells are almost equal to CD1d-restricted NKT cells. Therefore we investigated CD1d-restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highalphabetaTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highalphabetaTCRbright NKT cells expressed an invariant rat Valpha14-Jalpha281 chain, which is CD1d-restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highalphabetaTCRbright NKT cells had significant and CD1d-specific cytotoxic activity.

Fatal Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with clonal karyotype abnormality.

We report a case of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) with clonal karyotype abnormality. A 5-year-old boy was admitted to our hospital with persistent high-grade fever, hepatomegaly, and pancytopenia. Laboratory data disclosed a coagulation abnormality and severe liver damage. Clonal proliferation of EBV-infected cells was detected in the bone marrow by Southern hybridization, and bone marrow cells exhibited clonal chromosomal abnormality. Although the patient was treated with immunochemotherapy according to the HLH94 protocol, the disease recurred during the induction therapy, and the patient died of disseminated intravascular coagulopathy. Considering this aggressive and fatal clinical course, it is important to take intensive therapeutic measures if karyotype abnormality is noted in the treatment of EBV-HLH patients.

Expression of transcription factors during megakaryocytic differentiation of CD34+ cells from human cord blood induced by thrombopoietin.

Although normal megakaryocytic development has been shown to require the presence of functional GATA-1 and NF-E2 transcription factors in vivo, the roles of other members of the GATA binding factors and NF-E2 family during megakaryocytic differentiation are unclear. the present study, the expression of GATA family members, GATA-1 and GATA-2, a GATA-binding factor, EVI-1, the large subunit of NF-E2 factor, p45 and the related factors, Nrf1, Nrf2, Nrf3, BACH1, BACH2, and the small subunit of NF-E2, MAFK and MAFG has been examined in human megakaryocytic and erythroid cells by reverse transcriptase-polymerase chain reaction. CD34+ cells isolated from human cord blood were induced to unilineage megakaryocytic or erythroid differentiation in liquid suspension culture in the presense of thrombopoietin or erythropoietin, respectively. Each lineage was identified by monoclonal antibody against GPIIb/IIIa or glycophorin A. In megakaryocytic culture, p45, Nrf1, Nrf2, BACH1, MAFK and MAFG mRNAs were induced similarly to erythroid culture. Nrf3 mRNA was barely detected in both cultures. BACH2 was induced only in megakaryocytic culture, although the level of expression was low. Furthermore, the profiles of transcription factors involved in hematopoiesis, EVI-1 and Ets-1 mRNAs were induced only in megakaryocytic culture. Megakaryocytic and erythroid differentiation pathways are closely related to each other, and these two lineage cells share a number of lineage-specific transcription factors. However, the results showed that the profile of the expression of these transcription factors in megakaryocytic cells is distinct from that of erythroid lineage. The dynamic changes in the levels of different transcription factors that occur during primary megakaryocytic differentiation suggest that the levels of these factors may influence the progression to specific hematopoietic pathways.

Low serum lipids suggest severe bone marrow failure in children with aplastic anemia.

BACKGROUND: Significantly low serum lipid levels are occasionally seen at the time of diagnosis in children with aplastic anemia (AA). The aim of the present study was to clarify the pathologic and clinical significance of pretreatment serum lipid levels in AA. METHODS: A questionnaire seeking precise data about AA in pediatric patients, including the initial laboratory data at the time of onset of AA and the clinical course of these patients, was sent to 18 institutes in Japan; 13 institutes responded to the questionnaire. In this retrospective study, data concerning hematologic examination and serum lipids were available for analysis in 127 children with AA. Serum lipoprotein patterns were analyzed using conventional agarose electrophoresis in eight patients. In order to elucidate the cause of hypolipidemia in AA, we assayed serum macrophage colony stimulating factor (M-CSF), which is well known to have apparent cholesterol-lowering activity, by using an enzyme-linked immunosorbent assay in seven patients with hypocholesterolemia and compared the results with those obtained in patients with iron-deficiency anemia (IDA). RESULTS: We found that pretreatment total cholesterol (TC) and triglyceride levels in the serum correlated well with counts of both nucleated cells and hemopoietic cells in the bone marrow (BM) and were inversely correlated with the lymphocyte ratio in both the BM and peripheral blood. Patients with serum TC lower than 150 mg/dL showed a poor response to any form of therapy except BM transplantation. There was no difference in the serum lipoprotein patterns between the controls and patients examined. The serum M-CSF level was significantly higher in patients with TC levels lower than 150 mg/dL compared with controls. CONCLUSIONS: These results indicate that the pretreatment serum lipid level may reflect hematopoietic activity within the BM and can help to predict the therapeutic response of each case of AA to treatment with immunosuppressive drugs, corticosteroids and anabolic steroids. These results also indicate that M-CSF may be one of the contributing causes of the hypocholesterolemia that occurs in both AA and IDA.

[Clinical studies of tazobactam/piperacillin (TAZ/PIPC) in pediatric patients].

Tazobactam/Piperacillin (TAZ/PIPC) is a newly developed intravenous antibiotics, in which TAZ, a new potent inhibitor of beta-lactamases, is combined with PIPC, a well-established beta-lactam antibiotics, at the ratio of 1:4. In this study, we clinically evaluated efficacy of the drug in 14 pediatric patients with various infections, and pharmacokinetic study was applied to 3 patients. Range of age was from 1-month to 15 1/4-year. Patients consisted of 9 cases of pneumonia, 3 urinary tract infection, 1 acute otitis media, and 1 left sacroiliitis with sepsis. Standard dose of TAZ/PIPC was 50 mg/kg/dose and administered 2-4 times per day with intravenous injection or drip infusion. Two cases of pneumonia were excluded because of non-bacterial infection. Nine causative pathogens including 3 Gram-positive cocci and 6 Gram-negative bacilli were detected in 7 patients, of which 5 Gram-negative strains produced bete-lactamase. All of cases showed 100% of efficacy rate and bacteriological eradication rate. It was noted that beta-lactamase-producing E. coli and B. catarrhalis were eradicated efficiently by TAZ/PIPC, which should be resistant to PIPC alone according to MIC data. Non-serious diarrhea and discomfort of back with nausea were observed in one each patients as side effects. Both of side effects were transient, and improved with anti-diarrheic agent or cessation of the drug, respectively. As abnormal laboratory test results, moderate increases of the eosinophils and platelets counts as well as moderate elevation of the transaminases were observed in 2 separate patients. Pharmacokinetics study showed that Cmax, T1/2, and AUC were similar to the data reported in adult patients. Urinary recovery rate in the first 6 hours also resemble the data from adult patients. Based on above results, TAZ/PIPC is a useful agents pediatric infections by beta-lactamase producing strains also.

Progression from myelodysplastic syndrome with monosomy 7 to acute monoblastic leukemia with MLL gene rearrangement.

We report a case of myelodysplastic syndrome (MDS) with the 11q23 translocation at its leukemic transformation. Southern blot analysis demonstrated that the MLL gene on chromosome 11 was rearranged during the progression from MDS to acute leukemia. The clinical observation in this case supports the notion that leukemic transformation involves multiple cytogenetic evolutionary progresses, and that MLL gene rearrangement corresponds to the final step of leukemogenesis.

Human small Maf proteins form heterodimers with CNC family transcription factors and recognize the NF-E2 motif.

The transcription factor NF-E2, a heterodimeric protein complex composed of p45 and small Maf family proteins, is considered crucial for the regulation of erythroid gene expression and platelet formation. To facilitate the characterization of NF-E2 functions in human cells, we isolated cDNAs encoding two members of the small Maf family, MafK and MafG. The human mafK and mafG genes encode proteins of 156 and 162 amino acid residues, respectively, whose deduced amino acid sequences show approximately 95% identity to their respective chicken counterparts. Expression of mafK mRNA is high in heart, skeletal muscle and placenta, whereas mafG mRNA is abundant in skeletal muscle and is moderately expressed in heart and brain. Both are expressed in all hematopoietic cell lines, including those of erythroid and megakaryocytic lineages. In electrophoretic gel mobility shift assays binding to NF-E2 sites was found to depend on formation of homodimers or heterodimers with p45 and p45-related CNC family proteins. The results suggest that the small Maf family proteins function in human cells through interaction with various basic-leucine zipper-type transcription factors.

Naturally occurring dominant negative variants of Stat5.

Stat5 was initially identified as a prolactin-induced member of the signal transducer and activator of transcription (Stat) family in sheep. However, Stat5 is also activated in the response to a variety of cytokines. In mice, and possibly in other species, there exist two Stat5 genes (Stat5a and Stat5b) that encode proteins of 92 and 94 kDa that are 95% identical. In the studies described here, we demonstrate that naturally occurring carboxyl-truncated, variant Stat5 proteins of 77 and 80 kDa exist and that these proteins are inducibly tyrosine phosphorylated in the response to several cytokines and form heterodimers with the full-length, wild-type proteins. Using expression constructs encoding truncated forms, we demonstrate that the truncated forms can be tyrosine phosphorylated and bind DNA. Surprisingly, the tyrosine phosphorylation of the carboxyl-truncated forms is considerably more stable than that of the wild-type proteins. Overexpression of a carboxyl-truncated Stat5a in cells resulted in the specific inhibition of the transcriptional activation by interleukin-3 of the genes for oncostatin M (Osm) and the cytokine-inducible, SH2 domain-containing gene (Cis), both of which have been shown to be normally regulated by Stat5. Although Stat5 dominantly suppressed the induction of these genes, no effects on cell proliferation were observed. Together, the results demonstrate the natural existence of potentially dominantly suppressive variants of Stat5 and implicate the carboxyl domain of Stats in transcriptional regulation and functions related to dephosphorylation.

Abundant expression of erythroid transcription factor P45 NF-E2 mRNA in human peripheral granurocytes.

Transcription factor NF-E2 is crucial for regulation of erythroid-specific gene expression. p45 subunit of NF-E2 contains a basic-leucine zipper domain and dimerizes with the small Maf family protein to form functional NF-E2 complex. While p45 expression was shown to be restricted to erythroid cells, megakaryocytes and mast cells in hematopoietic lineage, we found in this study that p45 mRNA is abundantly transcribed in the granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in granulocyte fraction of human peripheral blood cells. As neutrophils occupy approximately 92% of the cells in this fraction, the cells expressing p45 is most likely to be neutrophils. p45 mRNA is also expressed in HL-60 promyelocytes, albeit the expression level is much lower than that of the granulocyte fraction. HL-60 cells were found to express mafK mRNA, indicating the presence of genuine NF-E2 complex in the cells. Although p45 mRNA is transcribed from two different promoters, aNF-E2 promoter and fNF-E2 promoter, in erythroid and megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 megakaryocytic lineage cells, p45 mRNA is transcribed only from aNF-E2 promoter. The expression of p45 mRNA in the neutrophils declined rapidly after transfer of the cells to in vitro culture and G-CSF could not sustain the expression from the down-regulation, suggesting the E2 may also participate in the regulation of neutrophil-specific gene expression.