Circulating fibrocytes correlate with the asthma control test score.

BACKGROUND: Bronchial asthma is characterised by airway inflammation and remodelling with a decline of lung function. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that play important roles in the pathogenesis of airway remodelling. Several clinical parameters are currently being used in routine clinical practice to assess outcome of therapy in asthma including frequency of rescue with short-acting ß2-agonist and the asthma control test. In this study, we hypothesised that asthma control test is associated with circulating levels of fibrocytes in bronchial asthma. METHODS: There were 20 patients with asthma and seven healthy controls. The number of CD45(+)Collagen I(+) circulating fibrocytes was assessed in the peripheral blood by flow cytometry. RESULTS: The number of circulating fibrocytes was significantly increased in asthma patients with moderate and severe disease compared to controls, and it was inversely correlated with % forced expiratory volume in one second and % forced vital capacity (%FVC). The frequency of inhalation of short-acting ß2 agonist and the asthma control test score was significantly and inversely correlated with the number of circulating fibrocytes. CONCLUSION: The results of this study showed that the number of circulating fibrocytes is inversely correlated with clinical asthma control parameters, further supporting the relevance of measuring circulating fibrocytes as a marker of clinical control in bronchial asthma.

Efficiency of diversion of the first aliquot of blood and prestorage leukoreduction for preventing bacterial contamination in red blood cell concentrates assessed using a rapid polymerase chain reaction-based bacterial detection system.

OBJECTIVES: Sepsis caused by the bacterial contamination of blood products is a major infection risk associated with blood transfusion. Diversion of the initial 25 mL of blood and prestorage leukoreduction were implemented in Japan in 2007 for all donated blood products. We assessed the efficacy of these new collection procedures in preventing bacterial contamination of red blood cell (RBC) concentrates. METHODS: Broad-range 16S ribosomal RNA polymerase chain reaction was used to determine bacterial contamination in segment samples of RBCs before and after implementation of the new collection procedures. To evaluate whether these new procedures reduced bacterial contamination, we compared bacterial contamination rates of blood samples from diversion pouches with those of segment samples from the same donor's RBCs. RESULTS: The rate of bacterial contamination of RBCs before implementation of the new collection procedures was 1.27%. Most of the isolated bacteria were Staphylococcus epidermidis or Propionibacterium acnes. After implementation, this rate was significantly reduced to 0.10%. Of the 233 whole blood samples obtained from the Mie Red Cross Blood Center, 1.72% of blood samples from diversion pouches were contaminated, but no bacterial contamination was detected in segment samples from the same donor's RBCs after prestorage leukoreduction. CONCLUSIONS: The new collection procedure significantly reduced bacterial contamination of RBC concentrates.

Non-DNA-binding Ikaros isoform gene expressed in adult B-precursor acute lymphoblastic leukemia.

Ikaros, a zinc finger transcription factor, is essential for lymphoid development. Mutant mice expressing dominant-negative Ikaros gene (Ikaros) isoforms develop an aggressive form of lymphoid malignancies. We examined the expression of Ikaros isoforms in 11 leukemic cell lines and adult acute lymphoblastic leukemia cells from 36 patients with B-precursor acute lymphoblastic leukemia (pre-B ALL) and nine with T-precursor acute lymphoblastic leukemia (pre-T ALL), using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In one pre-B ALL cell line, INC cells, and primary leukemic cells from 16 patients with pre-B ALL, we found the predominant expression of a non-DNA-binding Ikaros isoform, Ik-6. However, Ik-6 was not detected in pre-T ALL cells. All of pre-B ALL cells expressing Ik-6 were CD10(+), whereas CD10(-) pre-B ALL cells did not express Ik-6. The expression of Ik-6 was not related to karyotype abnormalities such as t(9;22) and t(4;11). Proteins from the cells that expressed Ik-6 alone failed to bind to the Ikaros protein-specific binding sequence in DNA. Ikaros proteins lacking the DNA binding sequences were detected in the cytoplasm but not in the nucleus of the cells. When INC and primary pre-B ALL cells that express Ik-6 alone were irradiated and cultured in the absence of serum, these cells produced functional Ikaros isoforms, Ik-1 and Ik-2. Purified CD19(+) CD10(-) and CD19(+) CD10(+) cells from normal human bone marrow did not express Ik-6. The predominant expression of Ik-6, which is the result of post-transcription dysregulation, is characteristic of adult pre-B ALL, especially CD10(+) pre-B ALL.

c-kit gene expression in CD7-positive acute lymphoblastic leukemia: close correlation with expression of myeloid-associated antigen CD13.

Expression of human c-kit proto-oncogene and interleukin-7 receptor (IL-7R) in acute lymphoblastic leukemia (ALL) cells expressing CD7 was examined by Northern-blot analysis and reversed transcription polymerase chain reaction (RT-PCR) assay in relation to the phenotypes. Leukemic cells from four out of 12 CD7+ ALL patients, all of which fulfilled the criteria of ALL in the FAB classification, expressed c-kit genes. Surface CD3 (sCD3) was absent in all of these cases, while cytoplasmic CD3 (cCD3) was found in the two sCD3- cases. CD3 epsilon transcripts were detected in one of the sCD3- cCD3- cases. IL-7R genes were transcribed in the three cases with c-kit gene expression. In addition, there was a good correlation between c-kit gene expression and myeloid associated antigen CD13 positivity of the leukemic cells. None of the patients with c-kit gene expression had mediastinal tumor. Our results show that leukemic cells in a proportion of CD7+ ALL express receptors for cytokines that are secreted by bone marrow stromal cells. Ligands for c-kit genes and IL-7 could play an important role for the regulation of proliferation and differentiation of T-cell progenitors in bone marrow.

Cellular characteristics of chronic myelocytic leukemia basophilic crisis cells: phenotype, responsiveness to and receptor gene expression for various kinds of growth factors and cytokines.

Leukemic cells from a patient with chronic myelocytic leukemia (CML) basophilic crisis were examined in an in vitro clonogenic assay using recombinant human hematopoietic growth factors to elucidate the proliferative and differentiative behaviors. More than 90% of the leukemic cells showed the morphologic characteristics of basophils and were positive for CD11b and CD13. The phenotype of the leukemic cells was different from that of mast cells. In the clonogenic assay using various recombinant growth factors, the leukemic cells were responsive to interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), but not to granulocyte-CSF (G-CSF), erythropoietin (Epo), or IL-4. IL-5 showed synergistic effects on colony formations induced by both IL-3 and GM-CSF. Transcripts of the GM-CSF receptor alpha chain gene were detected in the leukemic cells, but transcripts of the IL-4 receptor gene were not. Furthermore, c-kit and IL-7 receptor genes were expressed in the leukemic cells. Our results suggest that the differentiation pathway of basophils is different from that of mast cells, even though the receptor gene for stem cell factor (c-kit) was expressed on the basophilic leukemic cells, as it was on mast cells.

Structural alterations of the p53 gene in human leukemias.

We have investigated alterations of the p53 gene in human leukemias by polymerase chain reaction-mediated restriction fragment length polymorphism analysis. This method detects the codon 72 polymorphism of the p53 gene, allowing identification of loss of heterozygosity (LOH) of the p53 gene. In this study, at least two specimens were obtained from each patient to compare the allele status at different points of clinical course. Of 21 cases examined 7 were heterozygous at this polymorphic site and were evaluable for LOH study. Only one patient of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) lost the heterozygosity in the specimen of her last relapse. Leukemic cells from her repeated relapses including the last one revealed to have the clonally rearranged immunoglobulin H chain gene of the same size, indicating that alteration of the p53 gene in this patient might account for the lethal relapse as a clonal evolution event. Northern-blot analysis of the p53 gene showed that one case of CD7(+) ALL had altered p53 mRNA. Overall, it is demonstrated that alterations of the p53 gene might be involved in the pathogenesis or progression of at least some human leukemias, although the alterations in leukemias seemed to be not as frequent as in solid tumors.

G-CSF accelerates the cell-cycling of hematopoietic progenitor cells and abrogates the deceleration by TNF alpha.

We examined the effects of granulocyte colony-stimulating factor (G-CSF) on cell-cycling of hematopoietic progenitors in serum-free methylcellulose clonal cultures. Serial observations of the cultures showed hastening of growth of colonies by G-CSF, as determined by evaluating the time for individual colonies of 20 cells to reach 40 cells. G-CSF did not affect the incidence of proliferating cells in each developing colony. Cell-cycle analysis revealed that addition of G-CSF to cultures led to a decrease in the percentage of cells in the G1 phase of the cell-cycle, thereby indicating that G-CSF can modulate the cell-cycle of hematopoietic progenitors mainly by shortening the period of the G1 phase. Tumor necrosis factor alpha (TNF alpha) exerted opposite effects on cell-cycling of hematopoietic progenitors to those seen with G-CSF. G-CSF abolished the inhibitory effects of TNF alpha on the cell-cycling of hematopoietic progenitors. These observations indicate positive and negative regulatory roles of C-CSF and TNF alpha, respectively, and their interactions in the regulation of cell-cycling of hematopoietic progenitors.

Distinctive actions of interleukin-13 and interleukin-4 on growth of hematopoietic progenitors.

We examined the effects of interleukin (IL)-13 and IL-4 on growth of hematopoietic progenitors from 5-fluorouracil (5-FU)-treated mice, using an in vitro culture system. IL-13 or IL-4 alone failed to support colony formation by enriched marrow cells. IL-4 but not IL-13 in combination with IL-11 yielded a significant number of colonies. Neither IL-4 nor IL-13 affected colony formation supported by IL-3. When tested with two-factor combinations, IL-4 but not IL-13 suppressed the formation of colonies including multilineage colonies in the absence of IL-11. The inhibitory effects of IL-4 were not lineage-specific. Delayed addition experiments demonstrated that IL-4 is inhibitory in the early stage of colony formation. Effects of IL-4 on colony formation by pooled blast cells derived from 5-day culture of post-5-FU marrow cells was not evident. These findings indicate that IL-4 but not IL-13 is a negative regulator of hematopoietic progenitors, suggesting distinctive roles for IL-4 and IL-13 in early hematopoiesis.

Successful autologous peripheral blood stem cell transplantation for relapsed intravascular lymphomatosis.

We describe a case of relapsed intravascular lymphomatosis (IVL) successfully treated with autologous PBSCT. A 59-year-old Japanese female patient with IVL who had achieved CR after six courses of biweekly CHOP therapy developed lymphoma. She achieved a second CR after six courses of modified biweekly CHOP therapy, followed by autologous PBSCH and high-dose chemotherapy (CBDCA, VP-16, MCNU, CY) with PBSCT. There has not been any evidence of recurrence 48 months after PBSCT. Our case suggests that PBSC is acceptable as a source for stem cell rescue in IVL.

Aggressive neoplastic plasma cell growth with MLL gene rearrangement after high-dose therapy with autologous stem cell support for multiple myeloma.

We report a case of a patient with IgA kappa multiple myeloma (MM) mobilized with etoposide and subsequently receiving high-dose melphalan (HDM) with stem cell support. She relapsed rapidly post transplantation. Southern blot and fluorescent in situ hybridization analysis showed MLL gene rearrangement in the myeloma cells, which was not detected in the sample at diagnosis or in the PBSC harvested with etoposide plus G-CSF. These observations suggest that clonal rearrangement of the MLL gene is caused by etoposide. Patients with MM undergoing HDM with stem cell rescue may be at an increased risk of not only secondary leukemia, but also secondary genetic abnormalities in myeloma cells, especially those receiving priming with etoposide for peripheral blood stem cell collection.

Analysis of clonal relationship using single-cell polymerase chain reaction in a patient with concomitant mantle cell lymphoma and multiple myeloma.

We report a case of concomitant mantle cell lymphoma (MCL) and multiple myeloma (MM) in which we investigated the possibility of a clonal relationship. A 76-year-old man was diagnosed with MCL [immunoglobulin (Ig)M,D-kappa; stage IVB] and MM (IgG-kappa; stage I). Ig heavy chain (IgH) gene complementarity-determining region 3 in DNA from both the MCL tumor and from single MM cells from bone marrow smears was amplified to investigate whether there was a clonal relationship between MCL and MM. Sequence analysis revealed no clonal relationship between MCL and MM in our patient.

Rearrangement patterns of immunoglobulin heavy chain (IgH) and light chain genes in acute lymphoblastic leukemia and chronic myelocytic leukemia lymphoid crisis cells showing oligoclonal IgH gene rearrangements.

We investigated leukemic cells with multiple immunoglobulin heavy chain (IgH) gene rearrangements from nine B-precursor cell acute lymphoblastic leukemia (ALL) patients and three chronic myelocytic leukemia lymphoid crisis (CML.Ly-BC) patients in order to determine detailed recombination patterns of the variable (V), diversity (D), and joining (J) region genes. Southern blot study, using DNA probes for DQ52 and 5'D region genes, was useful to distinguish VDJ recombination from DJ recombination at the level of each allele. Leukemic cells from seven out of eight CD10-positive ALL patients showed biallelic VDJ recombinations. Rearrangements of Ig kappa genes were found in only one case. Leukemic cells from all of the CML.Ly-BC patients had a DJ/(V)DJ IgH genotype. These findings suggest that the multiple IgH gene rearrangements in B-precursor cell ALL occurred as a consequence of continuing V-(V)DJ rearrangements after neoplastic transformation, and were closely related to the stage of bone marrow B-precursor cell differentiation. Multiple IgH gene rearrangements in CML.Ly-BC might take place earlier in the process of IgH gene rearrangements than is the case in B-precursor cell ALL. In this sense, the genotypic oligoclonality observed in ALL and CML.Ly-BC should be regarded not as 'true', but as 'pseudo' oligoclonal leukemia.

Efficient ex vivo generation of human dendritic cells from mobilized CD34+ peripheral blood progenitors.

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.

Non-Hodgkin's lymphomas with chromosomal translocations involving 3q27 band and immunoglobulin gene loci: report of two cases.

Two cases of non-Hodgkin's lymphoma (NHL) of B-cell origin with reciprocal chromosome translocations involving the 3q27 band and loci for the immunoglobulin genes are described. In case 1, [t(3;14)(q27;q32)], the 14q32 band for the immunoglobulin heavy chain gene was involved, and in case 2, [t(3;22)(q27;q11)], the reciprocal breakpoint was the 22q11 band where the immunoglobulin lambda light chain gene is located. Although the two patients shared some clinical features, the lymphomas varied in histologic phenotypes; in case 1, the lymphoma being a diffuse large cell subtype, whereas in case 2, the lymphoma being the high grade small noncleaved cell lymphoma. Thus, the consistent association of the 3q27 translocation with a specific subtype of NHL remains to be determined. Southern blot analysis of the lymphoma cells from the two patients showed rearrangements and deletions in immunoglobulin genes with features which cannot be explained by normal immunoglobulin-generating mechanisms and which suggest that the translocations involved these loci at the molecular level. Cloning of the breakpoint using DNA probes from the immunoglobulin genes would prove a novel oncogene at the 3q27 band.

Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia.

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9;22)(q34;q11) that was detected in all metaphases, a t(11;17)(q23;q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bcr and abl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13, CD19, CD7, and CD41. Immunogenotypically, some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia.

Bcl-2 in cell-cycle regulation of hematopoietic cells by transforming growth factor-beta1.

We reported that several growth factors regulate the doubling time of hematopoietic progenitor cells by modulating the time required to pass through the G1 phase. As recent studies revealed the link between cell death and cell-cycle progression, we asked if cell death regulators such as Bcl-2 play a role in regulating the cell-cycle of hematopoietic cells by growth factors. Among growth factors, transforming growth factor-beta1 (TGF-beta1), a negative regulator of hematopoiesis, was chosen. When a large number of cells was required for analysis, we used IL-3-dependent Ba/F3 cells instead of primary hematopoietic progenitor cells because the response of Ba/F3 cells to TGF-beta1 was similar to that of primary hematopoietic progenitor cells. TGF-beta1 decelerated the cell-cycling of hematopoietic cells by inducing a delay in G1 to S phase transition, an event associated with increase in the level of Bcl-2 as well as p27, a cyclin/cyclin-dependent kinase inhibitor. In experiments using Ba/F3 cells with the potential to produce Bcl-2 in an inducible manner, Bcl-2 apparently functions upstream of p27. The effects of TGF-beta1 on Bcl-2 and p27 expression as well as cell growth were abrogated by c-kit ligand. These findings suggest that Bcl-2 plays a crucial role in regulating the cell-cycle of hematopoietic progenitor cells.

Role for C-MPL and its ligand thrombopoietin in early hematopoiesis.

Proto-oncogene c-mpl is structurally homologous with the hematopoietic growth factor receptor superfamily. The ligand for c-mpl was purified and its gene cloned. Extensive functional studies revealed that the ligand for c-mpl plays a crucial role in megakaryocytopoiesis and platelet production, hence, this ligand proved to be the long-sought hematopoietin, thrombopoietin (TPO). We briefly review here the role for TPO in early hematopoiesis, based on our in vitro data obtained using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) but not TPO alone supported the growth of murine primitive hematopoietic progenitors. Studies on lineage expression indicated that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of primitive hematopoietic progenitors. TPO also hastened IL-3-dependent growth of progenitors by shortening the time required for cell-cycling. While size of the colonies did not differ between colonies supported by IL-3 alone and those supported by IL-3 plus TPO, the incidence of megakaryocyte-containing colonies in cultures supported by IL-3 plus TPO was higher than that in cultures supported by IL-3 alone. Taken together, TPO as a single factor can support the survival of hematopoietic progenitors and TPO synergizes with SF or IL-3 to be active on early multipotential hematopoietic progenitors. These findings suggest that the function of TPO initially thought to be restricted to the megakaryocytic lineage proved to be greater in hematopoiesis. Reports from other laboratories regarding the involvement of TPO in early hematopoiesis are also discussed.

New modified activated partial thromboplastin time and prothrombin time methods using a synthetic chromogenic substrate in combination with diazotization.

A chromogenic substrate, H-D-Phe-Pip-Arg-pNA (S-2238) is a highly specific substrate to thrombin and releases p-nitroaniline (pNA) by the action of thrombin. We describe new modified APTT and PT methods using S-2238 in combination with the diazotization of pNA. In the modified APTT method, 100 microliter citrated plasma (diluted to 10-fold), 90 microliter 1 mM S-2238, 100 microliter 20 mM CaCl2 and 100 microliter Actin were mixed in an ice-bath, then incubated for 8 min at 37 degrees C. The reaction was stopped, and the generated pNA was diazotized by adding the following solutions sequentially: 975 microliter 0.04% sodium nitrite, 975 microliter 0.3% ammonium sulfamate and 975 microliter 0.07% N-(l-naphthyl)-ethylenediamine dihydrochloride. Diazotization changed pNA from yellow to pink. Then, absorbance at 545 nm was read, and values were expressed as thrombin units/ml plasma. In the modified PT method, 100 microliter citrated plasma (diluted to 20-fold), 90 microliter 1 mM S-2238 and 200 microliter tissue thromboplastin-C solution were mixed and processed as above. Correlations of the present modified APTT and APTT methods, and of modified PT and PT methods were significant (r = 0.426, p less than 0.01 and r = 0.561, p less than 0.01, respectively).

Plasma concentration of itraconazole and its antifungal prophylactic efficacy in patients with neutropenia after chemotherapy for acute leukemia.

Patients with acute leukemia are at high risk of fungal infection, suggesting that a preventive strategy is required. Fourteen patients receiving intensive chemotherapy for acute leukemia were studied to evaluate antifungal prophylaxis using itraconazole, and the plasma concentration of the drug was measured to determine its relationship to clinical efficacy. The median age of the patients was 50 years (range, 25 to 79 years), and all patients had neutropenia (less than 500 neutrophils/&mgr;l) which had lasted a median of 16 days (range, 4 to 30 days). Itraconazole was given orally at a dose of 200 mg (four capsules of 50 mg) once daily for at least 14 days. An H2-receptor antagonist was also given to prevent chemotherapy-induced gastrointestinal toxicity. Trough plasma concentrations of itraconazole and its metabolite, hydroxyitraconazole, were determined by reverse-phase high-performance liquid chromatography. The mean concentrations of itraconazole and hydroxyitraconazole on day 10 were 300 +/- 96 ng/ml (range, 131-428 ng/ml) and 776 +/- 369 ng/ml (range, 320-1582 ng/ml), respectively. There were marked inter-patient variations in both concentrations. No side effects were observed in the patients, and there was no definite fungal infection episode during this study. Daily oral administration of 200 mg of itraconazole appears to be effective as prophylaxis against fungal infection in neutropenic patients with acute leukemia. However, there were marked individual variations in the itraconazole plasma concentrations, which suggests that the plasma concentration should be monitored in patients with a risk of low absorption of this drug to adjust the dose given to an adequate level.