Masahiro Masuya

Diagnostic Value of PCR Analysis of Bacteria and Fungi from Blood in Empiric-Therapy-Resistant Febrile Neutropenia

ABSTRACT This study aimed to assess the clinical utility of PCR for the analysis of bacteria and fungi from blood for the management of febrile neutropenic patients with hematologic malignancies. Using a PCR system able to detect a broad range of bacteria and fungi, we conducted a prospective pilot study of periodic analyses of blood from patients following intensive chemotherapy. When fever occurred, it was treated with empirical antibiotic therapy, basically without knowledge of the PCR results. In 23 febrile episodes during the neutropenic period, bacteria were detected by PCR in 11 cases, while the same species were identified by blood culture in 3 cases. In 10 out of 11 PCR-positive cases, fever could be managed by empirical therapy. In the empirical-therapy-resistant case, the identification of Stenotrophomonas maltophilia by PCR led to improvement of fever. No fungi were detected by PCR in febrile cases, while Aspergillus fumigatus was detected in one afebrile patient, several days before a clinical diagnosis was made. In subsequent sporadic PCR analyses in 15 cases of febrile neutropenia, bacteria were detected by both PCR and blood culture in 7 cases and by PCR alone in 6. Fungi were not detected. While fever was improved by empirical therapy in 12 out of the 13 PCR-positive cases, the identification of Pseudomonas aeruginosa by PCR in one therapy-resistant case contributed to the successful treatment of persistent fever. Our results indicate that PCR analysis of bacteria from blood provides essential information for managing empirical-therapy-resistant febrile neutropenia.

A potential activity of valproic acid in the stimulation of interleukin-3 mediated megakaryopoiesis and erythropoiesis

OBJECTIVE Although the anticancer activities of histone deacetylase (HDAC) inhibitors have been studied, a role for HDAC in normal hematopoiesis has not been clearly defined. Previous studies have shown that the potent HDAC inhibitor FK228 stimulates interleukin (IL)-3-mediated erythropoiesis. Here, we examined whether the widely used valproic acid (VPA) affects megakaryopoiesis as well as erythropoiesis. MATERIALS AND METHODS CD34(+) cells were incubated in serum-free or serum-containing cultures with cytokines, with or without VPA. RESULTS In the serum-free cultures containing IL-3+stem cell factor (SCF), VPA significantly increased generation of CD61(+)GPA(-) megakaryocytic and a CD61(+)GPA(+) mixture of megakaryocytic and erythroid precursors from CD34(+) hematopoietic precursors at a pharmacological concentration (100 microg/mL). The increase in generation of megakaryocytic and erythroid precursors by VPA was confirmed by replating cultured cells with thrombopoietin+SCF and erythropoietin+SCF, respectively. VPA was as potent as FK228. In cultures with granulocyte-macrophage colony-stimulating factor+SCF, where CD61(-)GPA(+) erythroid precursors were mostly developed, VPA mainly enhanced the generation of CD61(-)GPA(+) erythroid precursors. In serum-containing cultures, only low numbers of CD61(+) or GPA(+) cells were developed with IL-3+SCF. Nevertheless, a substantial number of these cells were generated with VPA. Furthermore, these stimulating effects of VPA were observed by incubating CD34(+) cells from patients with myelodysplastic syndrome. Quantitative reverse transcription polymerase chain reaction showed that VPA enhanced GATA-2, but not GATA-1, messenger RNA expression with IL-3+SCF. CONCLUSIONS These results indicate a novel role for VPA in enhancing the potential of IL-3 to stimulate megakaryopoiesis as well as erythropoiesis and suggest a new therapeutic approach of epigenetic therapy for hematological disease.

Human bone marrow stromal cells simultaneously support B and T/NK lineage development from human haematopoietic progenitors: a principal role for flt3 ligand in lymphopoiesis

The regulation of human early lymphopoiesis remains unclear. B‐ and T‐lineage cells cannot develop simultaneously with conventional stromal cultures. Here we show that telomerized human bone marrow stromal cells supported simultaneous generation of CD19+CD34lo/−CD10+cyCD79a+CD20+/−VpreB− pro‐B cells and CD7+CD34+ CD45RA+CD56−cyCD3− early T/Natural Killer (NK) cell precursors from human haematopoietic progenitors, and the generation of both lymphoid precursors was promoted by flt3 ligand (flt3L). On the other hand, stem cell factor or thrombopoietin had little or no effect when used alone. However, both acted synergistically with flt3L to augment the generation of both lymphoid precursors. Characteristics of these lymphoid precursors were evaluated by gene expression profiles, rearrangements of IgH genes, or replating assays. Similar findings were observed with primary human bone marrow stromal cells. Notably, these two lymphoid‐lineage precursors were generated without direct contact with stromal cells, indicating that early B and T/NK development can occur, at least in part, by stromal cell‐derived humoral factors. In serum‐free cultures, flt3L elicited similar effects and appeared particularly important for B cell development. The findings of this study identified the potential of human bone marrow stromal cells to support human early B and T lymphopoiesis and a principal role for flt3L during early lymphopoiesis.

Drug Interaction Between Itraconazole and Bortezomib: Exacerbation of Peripheral Neuropathy and Thrombocytopenia Induced by Bortezomib

Study Objective. To investigate whether a drug interaction exists between bortezomib and the cytochrome P450 (CYP) 3A4 inhibitor itraconazole and/or the CYP2C19 inhibitor lansoprazole that results in increased severity of bortezomib‐induced peripheral neuropathy and thrombocytopenia.

Effect of washing solution on platelet counts following transfusion with twice‐washed platelets: a single‐patient experience

Dear Sir, For patients with plasma component deficiencies immunized to the deficient plasma component, transfusion after multiple washings of the components to reduce plasma concentration is required to prevent transfusion reactions. We report the case of an 8-year-old boy undergoing chemotherapy for acute lymphoblastic leukaemia, who developed a severe transfusion reaction even after red cell concentrates (RCCs) had been washed once. Serum examination revealed haptoglobin deficiency and haptoglobin antibodies. Further transfusion reactions were prevented by washing the platelet concentrates (PCs) twice before transfusion, but the clinical outcomes differed depending on the washing solution used. Transfusion of PCs washed twice with a novel additive solution (M-SOL), or washed once with anticoagulant citrate dextrose-buffered saline (A-SOL) followed by M-SOL, resulted in adequate platelet count increments. However, transfusion of PCs washed twice with A-SOL failed to increase the platelet count, despite prompt transfusion. This case suggests that the choice of washing solution may be important when multiple cycles of washing are required before transfusion. Patients occasionally develop severe transfusion reactions to plasma-containing PCs, but such adverse reactions can usually be prevented by reducing the plasma concentration of the PCs (Heddle et al., 1999; Azuma et al., 2009). Saline with anticoagulant citrate dextrose solution (ACD-A) has been used to wash PCs in Japan, but is not a good preservative at low plasma concentrations (Heddle et al., 1994; Vo et al., 2001). A range of factors such as pH have been found to affect significantly the function and viability of PCs, and various additive solutions have, therefore, been developed (Gulliksson, 2003, 2007). M-SOL is a newly developed additive solution

The effectiveness of measuring for fragmented red cells using an automated hematology analyzer in patients with thrombotic microangiopathy.

Thrombotic microangiopathy (TMA) or thrombotic thrombocytopenic purpura (TTP) is a life-threatening syndrome characterized by increased number of fragmented red cells (FRCs) and thrombocytopenia. FRCs can be measured using the recently developed automated hematology analyzer XE-2100. The normal range for FRCs is 0% to 0.205%, as determined by the automated hematology analyzer XE-2100. The FRC count is significantly elevated in patients with TMA associated with liver transplantation, bone marrow transplantation, or TTP. In patients with TMA after liver transplantation, the FRC count is significantly higher than in those without TMA. In receiver operating characteristic analysis for the diagnosis of TMA, the area under the curve is 0.986, suggesting that FRC is a useful marker for the diagnosis of TMA. When the cutoff value of FRC for TMA is 1.2%, the sensitivity is 90% and the specificity is 96%, indicating that FRC is the most useful screening test for the diagnosis of TMA.

An efficient method for single hematopoietic stem cell engraftment in mice based on cell-cycle dormancy of hematopoietic stem cells

OBJECTIVE To develop an efficient method for single hematopoietic stem cell (HSC) transplantation for high-level hematopoietic engraftment. MATERIALS AND METHODS We combined single-cell sorting with short-term culture of putative HSCs. Mouse bone marrow cells that had been highly enriched for HSCs were individually deposited into a 96-well culture plate and incubated in the presence of mouse c-kit ligand and either mouse interleukin-11 or human recombinant granulocyte colony-stimulating factor. One week later, the resulting clones of cells were individually transplanted into lethally irradiated recipients. We also carried out time-course analysis of proliferation of the individual clones. Finally, we used micromanipulation of the paired progenies of the single cells and studied self-renewal and differentiation potentials of HSCs again in combination with transplantation. RESULTS There was a correlation between clone size at day 7 of culture and engraftment at 2 months post-transplantation. Small clones, such as those consisting of <15 cells, often showed high-level multilineage engraftment, while clones consisting of > or =40 cells showed very low levels of engraftment. Daily observation of cell divisions of individual clones revealed that some HSCs are in the G(0) state for as long as 1 week, despite the presence of permissive cytokines. Studies using micromanipulation of paired progenies documented the ability of an HSC to generate two HSCs, as well as asymmetric cell divisions. CONCLUSIONS Single-cell sorting combined with short-term culture of individual putative HSCs provides an efficient method for single HSC transplantation. Analyses of the kinetics of individual HSCs provided direct evidence for HSC cell-cycle dormancy, self-renewal, and expansion.

Deletion of chromosome arm 15q in a case of minimally differentiated hypoplastic AML-M0.

Deletion of the long arm of chromosome 15 is known as a rare but recurrent chromosomal abnormality in myeloid malignancies. We report a novel case of minimally differentiated hypoplastic acute myeloid leukemia (AML M0) in a patient who initially had a normal karyotype, but clonal interstitial deletion of chromosome 15, del(15)(q11.2q22), coincided with increment of leukemic cells a year later. We also summarize 18 published cases with myeloid malignancies and this chromosomal abnormality.