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Summary Background Fungal infections are a major complication of neutropaenia following chemotherapy. Their early diagnosis is difficult, and empirical antifungal treatment is widely used, and uses of less toxic drugs that reduce breakthrough infection are required. Objective We conducted a multicentre, open‐label, randomised, non‐inferiority trial to compare the safety and efficacy of intravenous itraconazole (ivITCZ) and liposomal amphotericin B (LAmB) as empirical antifungal therapy in patients with haematological malignancies with neutropaenia and persistent fever. Methods Patients with haematological malignancies who developed fever refractory to broad‐spectrum antibacterial agents under neutropaenia conditions were enrolled. Patients were randomised for treatment with LAmB (3.0 mg/kg/d) or ivITCZ (induction: 400 mg/d, maintenance: 200 mg/d). Results Observed overall favourable response rates of 17/52 (32.7%) and 18/50 (36.0%) in the LAmB and ivITCZ groups, with a model‐based estimate of a 4% difference (90% CI, −12% to 20%), did not fulfil the statistical non‐inferiority criterion. In the LAmB group, there were two cases of breakthrough infection and five cases of probable invasive fungal disease, whereas in the itraconazole group, neither breakthrough infection nor probable invasive fungal disease occurred. Patients in the ivITCZ group had significantly fewer grade 3‐4 hypokalaemia‐related events than LAmB group patients (P

Background: Regulatory T cells (Treg) play important roles in onset of graft-versus-host disease (GVHD) and chronic GVHD (cGVHD) in elderly people is more frequent than in younger people after allogeneic hematopoietic stem cell transplantation (HCT). However, little is known about the differences in the number and function of Treg and natural killer (NK) cells in elderly and young patients. In this study, we examined the cause of the difference in incidences of cGVHD in these patient populations by measuring Treg and NK cells over time after HCT. Patients and Methods: The subjects were 65 consecutive patients aged >18 years who underwent HCT for different hematologic diseases at our hospital between December 2008 and December 2018 and survived for >100 days after HCT. The median age was 57 years (range: 19-73 years), and those aged ≥58 (n=33) and ≤57 (n=32) were classified as elderly and younger patients, respectively. Evaluated variables included recipient and donor characteristics, and transplant-related factors, including conditioning regimen, donor type, degree of match, and GVHD prophylaxis. After obtaining informed consent, blood was drawn from all patients on day 100 and at 12-month intervals thereafter. Analysis of CD4+CD25+Foxp3+ Treg cells, CD3+CD4+ T cells, CD3+CD8+ T cells, CD19+ B cells, and CD3-CD56+ NK cells was performed using flow cytometry, and the absolute numbers of these cells were calculated. NK activity was measured by the standard 51Cr release assay at an effector:target (E:T) ratio of 20:1. Results: The incidence of grades II-IV acute GVHD was 29.2% at 100 days. There was no difference in the incidence of acute GVHD between elderly and younger patients [HR=0.85, 95% CI 0.34-2.09, p=0.72]. However, the overall incidence of cGVHD was 37.1%. In univariate analysis, the incidence of cGVHD in elderly patients was significantly higher than that in younger patients [HR=3.61, 95% CI 1.13-11.59, p=0.031]. Elderly patients had a significantly lower percentage of B cells than younger patients at 24 months (13.4±9.6% vs. 27.0±15.6%, p=0.024) and 36 months (16.4±10.3% vs. 31.1±11.2%, p Conclusion: The percentage and absolute number of Treg cells in elderly patients were significantly lower than those in younger patients, whereas CD8+ T cells were significantly higher in elderly patients after HCT. The absolute number of Treg cells tended to reconstitute one year after HCT in younger patients, but tended to decrease gradually in elderly patients, with no recovery even three years after HCT. In contrast, there was no change in NK cells. Similar results were obtained in an analysis excluding patients who developed cGVHD. These results suggest that a difference in immunological reconstitution between elderly and younger patients may explain the difference in the incidence of cGVHD between these patients. This may be useful in determining the choice of therapy with consideration of the immunologic mechanism. Disclosures Suzumiya: Celgene, Kyowa Kirin, Chugai-Roche, Eisai, Takeda, Celltrion, SymBio, Astellas, Ono, AstraZeneca, Ootsuka, Taiho, Mundi, Dainihon-Sumitomo: Research Funding.

Background: The immunological graft-versus-leukemia effects associated with chronic myeloid leukemia (CML) are well-established. Both regulatory T cells (Treg) and natural killer (NK) cells play a role in the maintenance of CML. In this study, we examined changes in the number of circulating Treg and NK cells and the NK activity in CML patients before and after treatment with a tyrosine kinase inhibitor (TKI). Our goal was to evaluate the clinical significance of this treatment on these two different cell types. Moreover, the levels of these cells suggest that there is a need to make the TKI more effective as an immunotherapy. Patients and Methods: Treg and NK cells were characterized and quantified by flow cytometry in 27 newly diagnosed CML patients, 19 newly diagnosed patients with other myeloproliferative neoplasms (MPN), and 15 healthy controls (HC-group). CML patients, who were diagnosed as chronic phase and initially treated with a TKI between September 2007 and July 2017 and were followed up for more than 12 months were enrolled in the study. The molecular response was analyzed by quantitative PCR of the BCR-ABL fusion genes at approximately 6-month intervals after the initiation of treatment. After obtaining informed consent, peripheral blood (PB) was drawn from all patients at their initial diagnosis and at 6-month intervals thereafter. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard 51Cr release assay at an effector:target (E:T) ratio of 20:1. Results: The median age for CML patients was 65 years (range: 44-85), and 17 out of 27 of the patients were male. The median period of observation was 656 days. After 12 months, 12 CML patients (MMR-group) had a major molecular response (MMR), and 15 patients (NR-group) did not have a MMR. Treg% values in the PB before treatment in the CML-group (0.39±0.30%) were significantly lower than that in the HC-group (0.70±0.29%, p Conclusions: The absolute number of Treg cells in the CML patients decreased remarkably after treatment. The absolute number and the percentage of Treg cells decreased remarkably after treatment especially among the CML patients in the MMR-group. Although the number of NK cells did not change in the MMR-group compared to that in the HC-group, the NK activity was significantly lower and tended to approach the value of the normal control group after treatment. These data suggest that patients with CML have a more profound remission by treatment with a TKI resulting in decreased levels of Treg cells and increased NK activity. Immunological control via T cells and NK cells is very important in the treatment of CML. Disclosures Suzumiya: Chugai-Roche: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Pfizer: Research Funding; SymBio: Research Funding; Ono: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Shire Japan: Speakers Bureau; Taiho: Research Funding, Speakers Bureau; Toyama Chemical: Research Funding; Celltrion: Research Funding; Takeda: Research Funding, Speakers Bureau; Zenyaku Kogyo: Consultancy; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sumitomo Dainioppon: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Ohtsuka: Speakers Bureau.

Background: It is important to determine the changes in both donor T cells and B cells on immune reconstitution following allogeneic hematopoietic stem cell transplantation (HCT). It is well-known that Treg cells play important roles in the prevention of T-cell-mediated graft-versus-host disease (GVHD) and in promoting tumor escape from T-cell-dependent immunosurveillance. However, very little is known about the number and function of both CD4+CD25+Foxp3+ regulatory T (Treg) cells and CD3-CD56+ natural killer (NK) cells and about NK activity during immune reconstitution. In this study, we examined changes in the number of circulating Treg and NK cells in the peripheral blood (PB) and the NK activity of HCT patients at various times after HCT, and we evaluated the clinical significance of these findings relative to patient survival. Patients and Methods: We evaluated 29 consecutive patients (ages >18) without GVHD who underwent HCT for different hematologic diseases between December 2008 and April 2017. Treg and NK cells were characterized and quantified by flow cytometry from these 29 patients and 15 healthy controls. Evaluated variables included recipient and donor characteristics, transplant-related factors, including conditioning regimen (myeloablative conditioning or reduced-intensity conditioning), donor type (matched sibling donor, unrelated donor, other relative (haploidentical), or cord blood transplant), degree of match (8/8, 7/8, 6/8, or haploidentical), and GVHD prophylaxis. Patients who were followed up for more than 12 months were enrolled in the study. After obtaining informed consent, blood was drawn from all patients on the day of engraftment and at day 100 and at 12-month intervals thereafter. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard 51Cr release assay at an effector: target (E: T) ratio of 20:1. Results: The median patient age was 58 years (range: 19-71 years), and 21 out of 29 of the patients were male. At 100 days, the percentage of B cells (2.5±6.0%) and absolute numbers of CD8+ T cells (269.7±284.8/μL), CD4+T (22.83±292.4/μL) cells and NK cells (248.3±229.1/μL) were significantly lower than those at 2 years (20.9±11.6%, 747.2±648.4/μL, 588.0±607.3/μL, 558.1±336.2/μL, p Conclusion: The percentage and the absolute number of Treg cells in HCT patients were significantly lower than those in the normal control group, whereas CD8+ T cells was significantly higher in the patients within 2 years after HCT than that in the normal control group. The % Treg cells did not recover even at 3 years after HCT, rather it tended to be somewhat lower. In contrast, there was no change in the NK cells. These results suggest that the immunological reconstitution has not been achieved even at 3 years after transplantation and that the immunological consequences of GVHD are maintained. The antitumor effect is also maintained. On the other hand, NK cells recover at an early stage. The roles of these different immune cells after HCT require further investigation. Disclosures Suzumiya: Eisai: Research Funding, Speakers Bureau; Pfizer: Research Funding; Celltrion: Research Funding; Taiho: Research Funding, Speakers Bureau; Sumitomo Dainioppon: Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; SymBio: Research Funding; Bristol Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Chugai-Roche: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Zenyaku Kogyo: Consultancy; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Ono: Speakers Bureau; Ohtsuka: Speakers Bureau; Shire Japan: Speakers Bureau.

Aims: Although bone marrow cells are reported to migrate to the liver under circumstances of severe liver injury, the bone marrow cell type and the mechanisms in this process, remain to be clarified. We examined the involvement of hepatocyte growth factor (HGF) in this process and the cell type of migrated hematopoietic cells by HGF. Methods: The CD34+ cells and colony forming cells in the peripheral blood were examined in HGF transgenic, recombinant HGF-administered, and HGF-expressing adenovirus-administered mice. The cell type mobilized by HGF was examined by the percentages of donor cells in the peripheral blood of the recipient mice transplanted with Lin-c-kit+Sca-1+CD34+ cells and those with Lin-c-kit+Sca-1+CD34- cells. Expression of stem cell factor (SCF) was examined after the addition of HGF in MS-5 stromal cells. The numbers of the cells which were mobilized from bone marrow and recruited into liver by HGF were assessed using green fluorescence fluorescent (GFP)-chimera mice. Results: Mobilized CD34+ cells and colony forming cells in the peripheral blood were increased by HGF treatment. The cells mobilized by HGF were mostly Lin-c-kit+Sca-1+CD34+ cells. Recruitment of bone marrow cells into liver was not suppressed in MMP-9-/- mice. Expression of SCF was induced by HGF in MS-5 stromal cells. However, expression of CXCR4, SDF-1, MMP-9 or VCAM-1 was not changed. The numbers of GFP-positive cells in liver 1 month after treatment by HGF was greater than that by G-CSF. Conclusion: The results of the present study suggest that HGF mobilizes and recruits hematopoietic progenitor cells from bone marrow into the liver through SCF-mediated mechanism.

The efficacy and safety of high-dose chemotherapy with tandem autologous peripheral blood stem cell transplantation (auto-PBSCT) were evaluated in a multicenter clinical study of patients with advanced multiple myeloma. Eligible patients (n = 40) were consecutively enrolled in the phase I/II study and received 2-4 cycles of vincristine-adriamycin-dexamethasone regimen. The responding patients underwent PBSC harvesting following high-dose cyclophosphamide and filgrastim administration. The first auto-PBSCT (n = 32) following high-dose melphalan (200 mg/m(2)) was performed within 2 months of PBSC harvesting; the second auto-PBSCT (n = 28) was scheduled 3-6 months later. Treatment-related mortality was 2.5% (n = 1) throughout the protocol. Grade 4 nonhematologic toxicity occurred in 12.5 and 14.3% of the first and second auto-PBSCT patients, respectively. All but one patient (who died) achieved hematopoietic recovery. For the 28 patients completing the second auto-PBSCT, the results were favorable with a response rate of 65% (complete response rate = 27.5%, n = 11); the five-year progression-free survival and overall survival were 20.3 and 66.5%, respectively. In conclusion, high-dose chemotherapy with tandem auto-PBSCT is feasible and safe with a favorable response rate in treating advanced multiple myeloma in Japan.

To investigate the clinical role of the soluble form of c-kit receptor (s-kit) in patients with acute myeloid leukemia (AML), we determined the levels of serum s-kit and expression of c-kit antigens and mRNA in leukemic cells. The serum s-kit level was measured using ELISA assay in 30 AML patients and 20 normal controls. C-kit antigens of leukemic blasts were stained immunohistologically, and c-kit mRNA was detected by RT-PCR. The serum s-kit level in M1 and M2 were significantly increased (p

Human bone marrow–derived mesenchymal stem cells (BM-MSCs) are expected to be a potential source of cells for transplantation. Although recent reports have shown that isolated MSCs can differentiate into hepatocytes, the efficiency of differentiation is insufficient for therapeutic application. To circumvent this problem, it is necessary to understand the mechanisms of hepatic differentiation of human BM-MSCs. Hepatocyte nuclear factor 3β (HNF3β), a forkhead/winged helix transcription factor, is essential for liver development. In the present study, we established a tetracycline (Tet)-regulated expression system for HNF3β in UE7T-13 BM-MSCs. HNF3β expression significantly enhanced expression of albumin, α-fetoprotein (AFP), tyrosine amino transferase (TAT) and epithelial cell adhesion molecule (EpCAM) genes. The differentiated cells showed hepatocyte-specific functions including glycogen production and urea secretion. During treatment with the Tet-on system for 8 days, over 80% of UE7T-13 cells turned out to express albumin. Furthermore, the combination of Tet with basic fibroblast growth factor (bFGF) efficiently induced the genes such as albumin and TAT, which are associated with maturity of hepatocytes; however, it suppressed genes such as AFP and EpCAM, which are associated with immaturity of hepatocytes, suggesting that Tet-induced HNF3β expression sensitizes BM-MSCs to bFGF signals. Finally, the results of the present study suggest that down-regulation of Wnt/β-catenin signals caused by translocation of β-catenin to cytoplasmic membrane is associated with hepatic differentiation of human BM-MSCs. Conclusion: HNF3β expression induced efficient differentiation of UE7T-13 human BM-MSCs. (HEPATOLOGY 2008;48:597–606.)

We report the case of a 74-year-old man with Fontaine stage IV chronic arteriosclerosis obliterans who had been suffering from inveterate giant skin ulcers on the dorsum and heel of the right foot. As conventional medical treatments had not improved these ulcers and surgical treatment was considered unfeasible, amputation of the right lower limb below the knee appeared to represent the only option. The patient was admitted to Tottori University Hospital to attempt a new angiogenic therapy using auto-mononuclear cell transplantation to avoid amputation. On admission, neither right ankle blood pressure nor transcutaneous partial pressure of oxygen at the right toe were detectable. The patient had a history of multiple cerebral infarctions, and collection of mononuclear cells from bone marrow was considered too difficult, so collection of peripheral blood mononuclear cells was selected. Transcutaneous partial pressure of oxygen and skin temperature in the treated limb started to improve from 2 weeks after implantation. Ulcer size was recognizably reduced by 1 month after treatment. Partial auto-skin implantation on the right heel was performed 2 months after treatment, and the giant skin ulcer was finally completely covered. No adverse effects were noted during follow-up lasting 1 year. These results suggest that peripheral blood mononuclear cell implantation may offer a suitable alternative rescue therapy for patients with critical limb ischemia whose general condition is not good.

For more than a decade it was believed that hematopoietic stem cells express CD34. However, this dogma was recently challenged by the observation that stem cells of normal adult mice are CD34-. In order to clarify the controversy, we carried out systematic examination of stem cells by using C57BL/6 mice that are congenic for Ly-5. As reported previously, stem cells in the normal adult mice were CD34-. However, stem cells stimulated in vivo by 5-fluorouracil injection or in vitro by a combination of interleukin-11 and steel factor were CD34+. The activated CD34+ stem cells reverted to CD34- when the recipients' marrow achieved steady state. The majority of G-CSF-mobilized stem cells also were CD34+ and reverted to CD34- under steady-state conditions. Most recently, we examined the developmental changes of stem cell CD34 expression. In order to gain information on the total population of stem cells we prepared CD34+ and CD34- populations of mononuclear cells without prior enrichment and studied their engrafting potentials. All stem cells from perinatal to 5-week-old mice were CD34+. In 7-week-old mice CD34- stem cells began to emerge, and the majority of the stem cells were CD34- in the 10- and 20-week-old mice. An estimated 20% of the adult stem cells expressed CD34. These observations provide insight into the current controversy regarding CD34 expression by adult hematopoietic stem cells.

Thrombopoietin (TPO) is the growth factor for megakaryocytes and platelets, however, it also acts as a potent regulator of stem cell proliferation. To examine the significance of TPO expression in proliferation of hepatic oval cells, the effect of adenovirus-mediated TPO gene transfer into livers of the Solt-Farber model, which mimics the condition where liver regeneration is impaired, was examined. Hepatic TPO mRNA peaked its expression at 2 days after gene transduction and then gradually decreased. The peripheral platelet number began to increase at 4 days (P0.05) and reached its plateau at 9 days (P0.01). Oval cells expressed c-Mpl, a receptor for TPO as well as immature hematopoietic and hepatocytic surface markers such as CD34 and AFP. The proliferating cell nuclear antigen-positive oval cells in rats into which adenovirus-TPO gene was transferred at 7 and 9 days were significantly greater than those in adenovirus-LacZ gene transferred (P0.05, each), and the total numbers of oval cells in the adenovirus-TPO gene transferred at 9 and 13 days were also significantly greater than those in adenovirus-LacZ gene transferred (P0.05, each). Expression of SCF protein was increased at 4, 7, and 9 days by TPO gene administration and that of c-Kit was increased at 4 and 7 days. These data suggest that adenovirus-mediated TPO gene transfer stimulated oval cell proliferation in liver as well as increasing peripheral platelet counts, emphasizing the significance of the TPO/c-Mpl system in proliferation of hepatic oval cells.

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.

Serum soluble interleukin-6 receptors (sIL-6R) have been demonstrated to play an important role in hematopoiesis. We report here that serum sIL-6R levels reflect proliferative kinetics of the progenitors after stimulation by chemotherapy plus granulocyte colony-stimulating factor. Serum sIL-6R were serially evaluated in 26 courses of peripheral blood (PB) stem cell collections in 16 patients using enzyme-linked immunosorbent assay. Expressions of IL-6R and CD34 on PB mononuclear cells were examined by flow cytometric analysis and expressions of IL-6R mRNA were examined by reverse transcriptase polymerase chain reaction. There were no significant differences between the serum sIL-6R levels on day 0 in patients (27.8+/-2.1 ng/ml, mean +/- SEM) and those in controls (27.5+/-1.5 ng/ml). Following chemotherapy the serum sIL-6R levels were significantly decreased, reaching a minimal level on day 14 (22.3+/-1.2 ng/ml, p0.01) and then significantly increased to above the baseline levels on day 21 (32.0+/-2.1 ng/ml, p0.01). Similar oscillations in the number of white blood cells, IL6R+ cells, CD34+ cells and colony-forming unit-granulocyte/macrophage (CFU-GM) in PB could be observed and the peak expression of mRNA was compatible with the expression of antigen. Serum sIL-6R levels on day 17 and 19 were positively correlated with the number of CD34+ cells, IL-6R+ cells, CFU-GM in PB and the number of collected CD34+ cells in leukapheresis products. In addition, when comparing the 2 groups divided by the number of prior chemotherapies, the status of disease or dose of the mobilizing regimen, the serum sIL-6R levels were significantly increased after day 17 in the group that received fewer courses of prior chemotherapy, the group in complete remission and the group of high-dose chemotherapy. These findings indicated that sIL-6R levels do not reflect the hematopoietic ability in the steady state, or the capability of the hematopoiesis after stimulation. Thus, sIL-6R levels may be a marker for the timing of PBSC collection or the prediction of the number of collected CD34+ cells.

Hepatitis virus infection through virus reactivation has a high risk of mortality in patients with hematological malignancies receiving chemotherapy. We examined the incidence of both hepatitis B virus (HBV) and hepatitis C virus (HCV) infection and severe liver dysfunction (alanine aminotransferase >ten times the normal upper limit and total bilirubin >5 mg/dl) during chemotherapy in 268 patients with hematological malignancies. Eight patients (3.0%) were infected with HBV and 22 patients (8.2%) were infected with HCV. One patient (0.4%) was infected with both HBV and HCV. HBV- or HCV-infected patients showed severe liver dysfunction at a significantly higher incidence than non-infected patients (11/31 (35.5%) vs. 0/237 (0%), p

The effects of activation of adult murine stem cells on their expression of CD38 were studied using a murine transplantation model. First, the published finding that the majority of long-term engrafting cells from normal adult steady-state marrow are CD38+ was confirmed. Next, it was determined that the majority of stem cells activated in vivo by injection of 5-fluorouracil (5-FU) or mobilized by granulocyte colony-stimulating factor are CD38−. Stem cells that were activated in culture with interleukin-11 and steel factor were also CD38−. Previous studies have shown that expression of CD34 by adult stem cells is also modulated by in vivo or in vitro activation. To determine whether there is reciprocal expression of CD38 and CD34, 4 populations of post–5-FU marrow cells were analyzed. The majority of the stem cells were in the CD38−CD34+ fraction. However, secondary transplantation experiments indicated that when the bone marrow reaches steady state, the majority of the stem cells become CD38+CD34−. In addition, the minority populations of CD34+ stem cells that occur in steady-state bone marrow are CD38−. This reversible and reciprocal expression of CD38 and CD34 by murine stem cells may have implications for the phenotypes of human stem cells.

Background: Regulatory T cells (Treg) play an important role in immune surveillance of malignancies. Treg expansion occurs frequently in high-risk myelodysplastic syndrome (MDS) and in progression of this disease. Azacitidine (AZA) is increasingly used for treatment of MDS. In this study, we examined changes in the number of circulating Treg in peripheral blood of patients with MDS treated by AZA and we evaluated the clinical significance of these changes. Patients and Methods: Patients with MDS and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) according to the WHO 2008 classification who were initially treated between January 2015 and December 2015 and followed up for more than six months were enrolled in the study. AZA was given intravenously at a dose of 75 mg/m2 daily for 5 or 7 consecutive days every 4 weeks. The response was assessed using the IWG response criteria for MDS. A total of 29 patients with AML-MRC (n=5), refractory anemia with excess blasts (RAEB) (n=7), refractory cytopenia with multilineage dysplasia (RCMD) (n=16), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (n=1) were included in the study. IPSS was intermediate-1 (n=15), intermediate-2 (n=6), and high (n=8). Eighteen patients did not need treatment, including transfusion (untreated group). Eleven patients were treated with AZA (AZA group). After obtaining informed consent, blood was drawn from all patients at initial diagnosis. In the 11 patients treated with AZA, blood was also drawn after 6 completed cycles. CD4+CD25+Foxp3+ Treg cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry and the absolute numbers of these cells were calculated. Results: The median age was 74 years (range: 44-88) and 22 of the patients were male. The median period of observation was 283 days. In the AZA group, 3 patients died and one had recurrence (AZA-ineffective group), and 7 reached CR or PR (AZA-effective group). The absolute number of Treg was higher in AML/MRC + MDS/MPN than in RCMD (32.5 ± 19.6 vs. 16.4 ± 11.9 /μL, p=0.0234). Treg% values in peripheral blood before treatment in the AZA (1.17±0.80%), AZA-effective (1.15±0.54%), and AZA-ineffective (1.21±1.24%) groups were all significantly higher than that in the untreated group (0.49±0.26%, p=0.0022, p=0.0004, p=0.0241, respectively). Similarly, the absolute numbers of Treg were significantly higher in the AZA (31.9±17.0 /μL), AZA-effective (24.3±11.5 /μL), and AZA-ineffective (45.1±18.2 /μL) groups than in the untreated group (14.9±8.1 /μL, p=0.0011, p=0.0296, p=0.00003, respectively), and significantly higher in the AZA-ineffective group than in the AZA-effective group (p=0.0434). After 6 months of treatment in the AZA group, there were significant decreases in %Treg (1.17±0.80% to 0.65±0.48%, p=0.0373) and absolute number of Treg (31.9 ± 17.0 to 19.7 ± 13.6 /μL, p=0.0258). These changes were not significant in the AZA-effective group, but the absolute number of Treg significantly decreased in the AZA-ineffective group (45.1 ± 18.2 to 16.23 ± 9.54 /μL, p=0.0194). No Treg values after treatment differed significantly from those in the untreated group. The count of CD8+ T cells was unchanged by AZA treatment, but was moderately higher in the AZA group compared to the untreated group (378.6 ± 504.6 vs. 205.4 ± 140.2 /μL, p=0.0455). The counts of CD3-CD56+ NK cells did not differ significantly between the untreated group and the AZA group before treatment. However, after AZA treatment, this count became significantly higher than that in the untreated group (311.0 ± 263.5 vs. 159.2 ± 105.5 /μL, p = 0.0371). The count of NK cells did not reach a high level in the AZA-ineffective group, but was significantly higher (300.9 ± 183.5 /μL, p=0.0030) in the AZA-effective group. Conclusion: The number of Treg in MDS cases requiring treatment was higher than that in MDS cases for which treatment was unnecessary. In particular, Treg was high in MDS with a poor prognosis. Treg was decreased by treatment with AZA and became equivalent to that in the untreated group. In contrast, there was increase in the number of NK cells after AZA treatment, which was found to be particularly high in cases with a good prognosis. These results suggest that an immunological mechanism associated with the prognosis of MDS may be improved by treatment with AZA. Further examination of this mechanism and the influence of AZA on the immunological effect is required. Disclosures No relevant conflicts of interest to declare.

In order to confirm the effect of interferon-α (IFN-α) in inducing a prolonged duration of the chronic phase (CP) on patients with chronic myelogenous leukemia (CML), we retrospectively compared the duration of CP between patients who continued on IFN-α and the patients in whom IFN-α was discontinued before the blast phase. Of the 32 patients not pretreated for CML in the early CP who received IFN-α therapy, 25 continued on IFN-α while seven discontinued the therapy (side effects, 5; resistance, 1; patient's refusal, 1). Only four of the 25 patients in whom IFN-α was continued (16.0%) progressed to the blast phase or accelerated phase, but six of the seven patients who discontinued IFN-α (85.7%) progressed to the blast phase or accelerated phase. Fourteen of the 25 patients who continued on IFN-α therapy showed cytogenetic response (complete cytogenetic response, 3; minimal cytogenetic response, 11) whereas 11 patients showed no cytogenetic response. However, non-responders showed a longer duration of CP than the patients whom IFN-α was discontinued. Although elderly patients showed a high incidence of side effects, and some patients progressed early after the beginning of IFN-α therapy, our data clearly demonstrated that in accordance with previous large multi-centric randomized studies the continuation of IFN-α, even in low doses, prevents disease progression.

Introduction: As the population continues to advance in age, it is necessary to examine aggressive treatment for elderly patients with malignant lymphoma. However, chemotherapy can also reduce the health-related quality of life (QOL) in such patients. Therefore, we examined the impact of a change of treatment on elderly patients 80 years or older and their QOL. Methods: We enrolled 39 elderly malignant lymphoma patients, aged 80 years or older (median age, 85 years), who were treated in our department between September 2007 and September 2014. We administered a questionnaire survey to assess the QOL in 19 cases using SF36. We re-administered the survey to about 11 cases one year after the initial examination. As controls, we selected 78 patients with malignant lymphoma who were younger than 80 years. Results: The histological diagnosis was determined by lymph node biopsy in 34 cases, but in 5 cases, the diagnosis remained unknown because of the lack of superficial lymphadenopathy; in such cases the diagnosis was made using bone marrow, pleural effusion or ascitic fluid. Chemotherapy was administered to 34 patients. Eleven of 34 patients died; in ten patients the cause of death was recurrence or refractoriness to chemotherapy, and treatment-related death only occurred in one case. Eight patients exhibited partial response or greater after chemotherapy, but were lost to follow-up as they moved to a local care facility. With respect to the histological classification, there were 19 cases of diffuse large B-cell lymphoma (DLBCL), six cases of follicular, small lymphocytic, or mantle cell lymphoma, two cases of peripheral T-cell lymphoma, four cases of the high-grade group and three cases of Hodgkin's disease. We examined 18 cases of newly diagnosed DLBCL. Eleven cases were treated with dose-modified R-CHOP therapy, and five cases with R-miniCHOP therapy from 2012; two cases underwent palliative care. The International prognostic index (IPI) was High for nine cases, High-intermediate for four cases, Low-intermediate for four cases and Low for one case. The median duration of observation was 487 days, and the remission rate was 87%. The 2-year survival rate was 73%. Eight patients exhibited disease-free survival throughout the follow-up period, and 5 patients ultimately died. One of the fatalities was due to an infection as a consequence of small intestinal perforation after chemotherapy. After the chemotherapy was considered efficacious, six cases changed hospital to a local nursing facility, and were lost to follow-up. With respect to the QOL, while there was no apparent decrease in the social function compared with the patients who were younger than 80 years, physical function (PF) was impaired due to neuropathy (p=0.0006). Furthermore, after the chemotherapy, the mental component summary showed an upward trend for recovery of vitality and mental health (p=0.04), but the physical and role-social components remained impaired and were still below the normal-based scoring. Discussion: Thirty-four of 39 elderly patients aged 80 years or older received chemotherapy, and only one case experienced a treatment-related death. However, after chemotherapy, several cases intended to change the hospital to local nursing facilities, and it was not possible to investigate the prognosis, including recurrence. We also reported cases for which agreement was not obtained with respect to invasive biopsies, i.e., an abdominal operation. While the patients were stable psychologically, there was a severe decrease of physical function with respect to the QOL. Chemotherapy-induced neuropathy is included among the causative processes, and we should weigh the choice of chemotherapeutic agents that do not contribute to severe neuropathy. With sufficient supportive care, the chemotherapy could be administered safely and would be anticipated to be efficacious. However, invasive biopsy indicated an impossible case, and decrease of physical function, due to side effects, lowered the QOL. It is anticipated that future clinical studies will consider these factors. Disclosures No relevant conflicts of interest to declare.

Although bone marrow cells are reported to migrate to the liver under circumstances of severe liver injury, the bone marrow cell type and the mechanisms in this process, remain to be clarified. We examined the involvement of hepatocyte growth factor (HGF) in this process and the cell type of migrated hematopoietic cells by HGF.The CD34(+) cells and colony forming cells in the peripheral blood were examined in HGF transgenic, recombinant HGF-administered, and HGF-expressing adenovirus-administered mice. The cell type mobilized by HGF was examined by the percentages of donor cells in the peripheral blood of the recipient mice transplanted with Lin(-)c-kit(+)Sca-1(+)CD34(+) cells and those with Lin(-)c-kit(+)Sca-1(+)CD34(-) cells. Expression of stem cell factor (SCF) was examined after the addition of HGF in MS-5 stromal cells. The numbers of the cells which were mobilized from bone marrow and recruited into liver by HGF were assessed using green fluorescence fluorescent (GFP)-chimera mice.Mobilized CD34+ cells and colony forming cells in the peripheral blood were increased by HGF treatment. The cells mobilized by HGF were mostly Lin(-)c-kit(+)Sca-1(+)CD34(+) cells. Recruitment of bone marrow cells into liver was not suppressed in MMP-9-/- mice. Expression of SCF was induced by HGF in MS-5 stromal cells. However, expression of CXCR4, SDF-1, MMP-9 or VCAM-1 was not changed. The numbers of GFP-positive cells in liver 1 month after treatment by HGF was greater than that by G-CSF.The results of the present study suggest that HGF mobilizes and recruits hematopoietic progenitor cells from bone marrow into the liver through SCF-mediated mechanism.

Background The developmental changes of Ni2+-sensitivity to automaticity of Nkx2.5-positive cells derived from mouse embryonic stem cell have been identified, suggesting developmental regulation of expressing Ni2+-sensitive T-type Ca2+ channel, although the mechanism of the change has not been fully studied. Methods and Results Transcripts of Cav3.2, Cav3.1 and Cav1.2 genes of beating Nkx2.5-positive cells, which encode the Ni2+-sensitive T-type Ca2+ channel, Ni2+-insensitive T-type Ca2+ channel, and L-type Ca2+ channel, respectively, were investigated by real-time reverse-transcriptase-polymerase chain reaction, and the current density of each channel was measured by patch-clamp techniques at the early and late stages of differentiation. The expression of the Cav3.2 transcript predominated in the early stage whereas those of Cav3.1 and Cav1.2 transcripts were upregulated in the late stage, which was consistent with the change in each current density, suggesting the expression of channel proteins is largely determined at the transcriptional level. Conclusion The results indicate that the mechanism of change of Ni2+-sensitivity is partly, if not completely, the subtype switch of T-type Ca2+ channel from Cav3.2 to Cav3.1 at the transcriptional level, and that the expression of the L-type Ca2+ channel might have an attenuating effect on Ni2+-sensitivity to automaticity in the late stage of differentiation. (Circ J 2005; 69: 1284 - 1289)

A 65-year-old woman with essential thrombocythemia (ET) was admitted to hospital where she was diagnosed as acute myocardial infarction (AMI). Because of abundant thrombus of right coronary arteries, percutaneous transluminal coronary recanalization by administration of urokinase was selected as the reperfusion therapy, resulting in successful revascularization with Thrombolysis in Myocardial Infarction grade III coronary flow. The maximum creatine kinase reached 507 IU/L, and left ventriculography performed at 1 month after initiation of both anticoagulant and antiplatelet therapies revealed reduced motion in the inferior wall with an ejection fraction of 57%. Despite good recovery of left ventricular function, bleeding complications, such as epistaxis or ecchymoma, which did not require blood transfusion, occurred during the clinical course. Because ET causes not only thrombus formation but also bleeding tendency, it is very important to carefully follow-up any clotting abnormality in AMI patients with ET.

We examined the incidence of hepatitis C virus (HCV) infection in 42 patients with acute leukemia before and after blood donor screening for the HCV Ab using the first-generation assay and the second-generation assay. Although 14 (93%) of the 15 patients before screening and 6 (50%) of the 12 patients after screening by the first-generation assay became seropositive for HCV RNA, none of the 15 patients became seropositive after screening by the second-generation assay. These findings indicate that the second-generation assay is effective in preventing HCV infection in multitransfused patients with acute leukemia.

Objective The mechanisms of mobilization of hematopoietic stem cells (HSC) from bone marrow to peripheral blood (PB) by cytokines are poorly understood. One hypothesis is that cytokines disrupt cytoadhesive interactions of stem cells with bone marrow stroma. The soluble portion of c-kit (s-kit) binds stem cell factor (SCF) and can specifically block the ability of SCF to bind HSC. Materials and methods To examine stem cell mobilization by s-kit, we prepared PB mononuclear cells from s-kit– or granulocyte colony-stimulating factor (G-CSF)–treated mice and assayed their colony-forming abilities and their long-term reconstituting abilities by transplantation into lethally irradiated Ly-5.2 congenic mice. Results We confirmed the published findings that human recombinant s-kit can block SCF-stimulated hematopoietic colony growing. We then found that s-kit could mobilize colony-forming cells from bone marrow to PB, and we found long-term reconstitution cells in the PB from s-kit–treated mice. The majority of s-kit–mobilized stem cells were in the CD34 + cell population. We also tested the additive effect between G-CSF and s-kit. The mean percentages of donor cells in the mice transplanted with Lin − cells from the G-CSF–treated mice and the G-CSF/s-kit–treated mice were 44.6% and 64.8%, respectively ( p =0.028). Conclusions These findings demonstrate that stem cells with long-term engraftment capabilities can be mobilized by s-kit, and that s-kit combined with G-CSF treatment leads to significant enhancement of engraftment efficiency, suggesting mobilization via disruption between c-kit and SCF as the mechanism.

We report a case of a 41-year-old man with acute myelogenous leukemia who developed fulminant hepatitis from reactivation of trace hepatitis B virus (HBV) 2 months after complete remission. Although he became positive for HB surface antigen at the onset of fulminant hepatitis, he had been negative for HBV serum markers, and only HBV DNA was detected by polymerase chain reaction (PCR) amplification on admission. The original stocks of serum samples from all blood donors were tested again for HBV DNA by PCR, and all samples were negative. This case demonstrates that testing for HBV DNA by PCR is necessary before chemotherapy, because silent HBV carriers are rare and fulminant hepatitis may be induced by chemotherapy in patients with hematologic malignancies.

Objective It has been reported that fetal murine hematopoietic stem cells are CD34 + , whereas adult stem cells are CD34 − . We sought to delineate the developmental changes of CD34 expression by hematopoietic stem cells and carried out systematic analysis of long-term engrafting cells in the bone marrow and/or blood of perinatal, juvenile, and adult mice. Materials and Methods To obtain information on the total population of stem cells, we prepared CD34 + and CD34 − populations of mononuclear cells without prior enrichment and assayed their long-term reconstituting abilities by transplantation into lethally irradiated Ly-5 congenic mice. Results All stem cells from perinatal to 5-week-old mice were CD34 + . In 7-week-old mice, CD34 − stem cells began to emerge, and the majority of the stem cells were CD34 − in the 10- and 20-week-old mice. Approximately 20% of adult stem cells expressed CD34. Conclusions Developmental changes of CD34 expression from the positive to the negative state takes place between 7 and 10 weeks of age for the majority of murine stem cells. Approximately 20% of adult stem cells remain CD34 + . These observations provide insight into the current controversy regarding CD34 expression by adult hematopoietic stem cells and suggest that the majority of stem cells in human umbilical cord blood and bone marrow of young children are CD34 + .

Controversy has existed about CD34 expression by hematopoietic stem cells. We recently reported that CD34 expression reflects the activation state of stem cells by using a murine transplantation model. It has been generally held that mobilized blood stem cells express CD34.However, it has also been reported that mobilized stem cells and progenitors are in G0/G1 phases of the cell cycle. To address the state of CD34 expression by the mobilized stem cells, we again used the mouse transplantation model. We prepared CD34− and CD34+ populations of nucleated blood cells from granulocyte colony-stimulating factor–treated Ly-5.1 mice and assayed each population for long-term engrafting cells in lethally irradiated Ly-5.2 mice. The majority of the stem cells were in the CD34+population. The CD34 expression by mobilized stem cells was reversible because re-transplantation of Ly-5.1 CD34− marrow cells harvested from the Ly-5.2 recipients of CD34+-mobilized stem cells 8 months posttransplantation revealed long-term engraftment. These results may support the use of total CD34+ cells in mobilized blood as a predictor for engraftment and CD34 selection for enrichment of human stem cells.

Introduction In decades past, patients with chemotherapy were treated with severed sterile or low-bacteria diets, and after chemotherapy, diet control was sometimes continued for some months. Sterile diets do not have a place in diet management today because of improved antimicrobial therapies, lack of evidence to demonstrate efficacy. Nonetheless, some form of diet restriction to minimize acquisition of organisms from food sources and food handlers is common. We investigated the influence of teaching for diet restrictions on quality of life (QOL) during and after chemotherapy in patients with hematological malignancies. Methods From September 2007 to May 2013 at Yonago Medical Center, all patients with chemotherapy received the education about dietary restriction using the diet and food safety guideline for the hematopoietic stem cell transplantation (HSCT) patients of The Japan Society for Hematopoietic Cell Transplantation. And those patients are advised to follow diet precautions for 6 months after chemotherapy and allogeneic graft patients with HSCT for 6 months after termination of immunosuppressive drugs. Patients and caregivers require education on food safety practices before chemotherapy were started. 95 patients were investigated in this study. We performed the questionnaire to the patients twice (initial and follow-up surveys) at intervals of one year in May, 2013 from April, 2012. 21 patients were in the period of dietary restriction (initial), and one year afterward (follow-up) (phase 1), and 74 patients were in the time of dietary restriction being canceled (initial), and one year afterward (follow-up) (phase 2). We divide the dietary feeling over dietary restriction into observance, pain, illusion, and uneasiness and evaluated four scores of each. To assess QOL, the Medical Outcomes Study Form-36 was used. We analyzed changes in dietary feeling scores, QOL scores, and body mass index (BMI) between initial and follow-up, and between phase 1 and phase 2. We confirmed that scores of the dietary feeling showed significantly correlation to QOL scores, carrying out the questionnaire to 90 outpatients with hematological disorders who did not received chemotherapy. Results The patients included 10 patients with acute myeloid leukemia, 7 with myelodysplastic syndrome, 3 with acute lymphoblastic leukemia, 3 with chronic lymphoid leukemia, 10 with multiple myeloma, 60 with malignant lymphoma, and 2 with others. 29 patients underwent HSCT. In phase 1, the Role-Physical (RP), Bodily Pain (BP), and Social Functioning (SF) scores increased significantly (all, P Conclusion From the period of dietary restriction to one year afterward of dietary restriction being canceled, QOL scores and the pain by dietary restriction has improved. After that time, in one year observation, an improvement of those scores was not found. The correlation between the changes of QOL and those of dietary feeling was not found except only BP. On the other hand, scores of the dietary feeling showed significantly correlation to QOL scores of the patients without chemotherapy. By diet and food safety education by nurses, the patients who receives chemotherapy understood the necessity for diet restriction. Our results suggest that dietary restriction has not directly influenced lowering QOL of patients with chemotherapy. If diet and food safety education is required for a chemotherapy, we may perform dietary restriction positively, although, it cannot predict from this study what restriction can prevent infection. In the future, the dietary restriction should make it clear at what kind of it is required. Disclosures: No relevant conflicts of interest to declare.

Cell plasticity of bone marrow stem cells to hepatocytes is known, however, the details are still unclear. hepatocyte growth factor (HGF) promotes an increase in liver stem cells in severely injured liver, but intervention to bone marrow stem cell is unclear. We examined a role of HGF in bone marrow stem cell-mediated liver regeneration in order to obtain effective liver regeneration. First, we found that the phenotype of stem cells, which can differentiate into hepatocytes, is Lin−c-kit+Sca-1+CD34− in bone marrow or Lin−c-kit+Sca−1+CD34+ in peripheral blood mobilized by G-CSF. We transplanted single Lin−c-kit+Sca-1+CD34− bone marrow cell harvested from male EGFP mouse to female wild type mouse, and then, using this GFP-chimera-mouse, we found that bone marrow origin GFP+ and Y chromosome+ hepatic cells were present in liver after acute liver damage. Next, single Lin−c-kit+Sca-1+CD34+ peripheral blood cell, which was mobilized in peripheral blood by administration of G-CSF, was transplanted into the portal vein of the wild type mouse which was given hepatic damage. We found that the GFP-positive cells also expressed albumin. Second, we investigated whether the HGF can mobilize stem cells from bone marrow to peripheral blood. In peripheral blood of HGF transgenic mice, 1.1% developed CD34+ cells and 20±3 colony forming cells of 1X106 peripheral blood mononuclear cells were shown. Colony forming cells were found in the mouse into which an HGF-expressing adenovirus was administered. After injection of rHGF to mice, a significant time-dependent increase of percentage of CD34+ cells in the PB was noted at the first 3 hours and CD34+ cells were increased in dose-dependent manner of rHGF and reached plateau level at 100 m/kg. The mice having transplantation with PB cells from 100 mg HGF-treated mice for 4days showed engraftment 2 months after transplantation. Upon activation of rHGF in mouse MS-5 stromal cells, phosphorylation c-Met and SCF were up-regulated, while VCAM -1, MMP-9, SDF -1 or CXCR4 were not changed. SCF level in conditioned media was also increased after the HGF stimulation. Finally, we examined whether the bone marrow stem cells in PB mobilized by HGF transdifferentiate into hepatocytes. Using GFP-chimera-mice given acute liver injury after administration of retorolusine and CCl4, the levels of GFP+ cells in liver of GFP-chimera-mice 2 months after treatment by PBS and HGF were 2.2±1.4% and 12.7±3.6%, respectively (p

The mechanisms of mobilization of hematopoietic stem cell (HSC) from bone marrow (BM) to peripheral blood (PB) by cytokines are poorly understood. The number of circulating HSC can be dramatically increased, or mobilized, by a wide variety of stimuli including hematopoietic growth factors, chemotherapy, and chemokines. Recent studies suggest that hematopoietic proteases, such as matrix metalloproteinase-9 (MMP-9), enable BM repopulating cells to translocate to circulating pool. In this study we investigated the mechanism of stem cell mobilization by the hepatocyte growth factor (HGF). First, we examined the effects of exogenous HGF on mobilizing HSC from BM to PB. To investigate engraftment of the mobilized cells from BM, 0.1mg/kg HGF was injected into Ly-5.1 mice every 24 hours for 4 days. Lin− cells in PB were collected 3 hours after the last injection of HGF and then injected into lethally-irradiated Ly-5.2 C57BL/6 mice. Two months after transplantation, the level of engraftment was assessed by analysis of donor (Ly-5.1) cells in the nucleated cells of the PB of recipient mice. The mean percentage of donor cells in mice transplanted was 1.8%, whereas that in the mice transplanted with untreated PB cells was 0%. Second, we investigated the mechanisms of HSC-mobilization by HGF from BM to PB. After determining through RT-PCR analysis that Lin− BM cells express the tyrosine kinase receptor c-met and that Lin− CD34− c-kit+ Sca-1+ BM cells does not express c-met, we speculated that the HGF/c-Met involved in HSC mobilization, focusing on the stromal cells. The murine stromal cells were cultured and stimulated with HGF under a serum-free medium condition. Phosphorylation of c-Met protein and activation of stem cell factor (SCF) in stromal cells were induced after HGF treatment. However, MMP-9 was not simultaneously activated after the HGF stimulation. The levels of SCF in supernatant began to increase 1 hour after HGF-stimulation. These in vitro data suggest that HGF can effectively promote the release of SCF from stromal cells, resulting in the mobilization effect of HGF. Third, to investigate engraftment of the mobilized cells by HGF, G-CSF, or PBS in male MMP-9 −/− mice, PB mononuclear cells were collected from HGF-, G-CSF-, or PBS-treated male MMP-9−/− mice, and then injected into lethally-irradiated female wild-type mice. Two months after transplantation, the presence of Sry gene in BM in transplanted female mice was confirmed by PCR analysis, showing the existence of the chimera. We found the Sry gene in the mice transplanted with PB cells from HGF- and G-CSF-treated mice, but not in the mice transplanted with PBS-treated PB cells. HGF and G-CSF can mobilize HSC from BM to PB in MMP-9−/− mice. These findings demonstrate that stem cells with long-term engraftment capabilities can be mobilized by HGF, and that the activation of SCF induced by HGF/c-Met from stromal cells is involved in the mobilization mechanisms of HGF but not MMP-9.

How hematopoietic stem cells (HSC) in bone marrow (BM) contribute to liver regeneration remains to be resolved. The mechanisms which mobilize HSC from BM to peripheral blood (PB) and govern their homing to the liver are unknown. Hepatocyte growth factor (HGF) is locally and generally increased following liver injury, suggesting that it promotes proliferation, adhesion, and survival of hepatocytes. PB stem cells mobilized by cytokines are widely used for clinical transplantation. However, it is not known if HGF can mobilize HSC to PB or if it has the capability to differentiate HSC into hepatocytes. In this study we examined whether HGF can mobilize HSC from BM to PB using the murine stem cell transplantation model. First, we found that HGF transgenic mice, which have high serum levels of HGF concentration, had many colony-forming cells in PB, suggesting HGF increases circulation of HSC and progenitor cells. After determining through RT-PCR analysis that lineage (Lin) − BM cells express the tyrosine kinase receptor c-MET, we speculated that the c-MET-HGF axis modulates the recruitment of HSC from BM to PB. We also examined the effects of exogenous HGF in mobilizing stem cells from BM to PB. To determine whether HGF can mobilize HSC to PB, we investigated the expression of CD34 using flow cytometric analysis. The CD34+ cells in PB mobilized by HGF increased in a dose-dependent pattern and reached a plateau at 0.1mg/kg of recombinant HGF administration. Significant increases in CD34+ cells in PB were noted at 3h after HGF infusion. The continual administration of HGF every 24h increased the CD34+ cells in PB to maximum levels at 4 days. Finally, the absolute number of CD34+ cells in PB after HGF administration was as much as the number of those cells after administration at 12-hour intervals subcutaneously with 0.125mg/kg of recombinant human granulocyte-colony stimulating factor (G-CSF) for 4 consecutive days. To investigate engraftment of the mobilized cells to BM, 0.1mg/kg HGF was injected into Ly-5.1 mice every 24h for 4 days. Lin− cells in PB were collected 3h after the last injection of HGF and then injected into lethally-irradiated Ly-5.2 C57BL/6 mice. Two months after transplantation, the level of engraftment was assessed by analysis of donor (Ly-5.1) cells in the nucleated cells of the PB of recipient mice. The mean percentage of donor cells of mice transplanted with Lin− cells from HGF-treated mice was 1.8%, whereas that of the mice transplanted with untreated PB cells was 0%. Multilineage engraftment was confirmed by the presence of the Thy-1+ cells, B220+ cells, and Mac-1/Gr-1+ cells. When we tested the CD34 expression of the stem cells transplanted, the majority of the cells expressed CD34. Then we tracked single Lin−, Sca-1+, c-kit+, CD34+ PB cells from G-CSF-treated transgenic-enhanced green fluorescent protein (EGFP) mice that were injected into spleen of the CCl4-induced liver-injured B6 mice along with 500 Lin−, Sca-1+, c-kit+, CD34+ PB cells from G-CSF-treated normal B6 mice. Two months later, donor-derived GFP+ cells were identified among recipient hepatocytes in liver-injured mice using immunohistochemistry for GFP. These findings demonstrate that stem cells with long-term engraftment capabilities can be mobilized by HGF, and that HSC in PB mobilized by HGF are capable of differentiating into hepatocytes, suggesting HGF contributes to liver regeneration partly by mobilizing HSC to PB.

症例は31歳女性. 23歳時より家族性大腸腺腫症(FAP)と診断され経過観察中に,頸部・咽頭・縦隔神経鞘腫を合併し, 31歳時に汎血球減少を認め,骨髄異形成症候群(MDS (RA))の合併と診断された. FAPの癌化とMDSの発症には遺伝子の関与が推測され,本症例は発癌構の解明に貴重な症例と考えられた.

Serum soluble interleukin-2 receptor (sIL-2R) levels were determined in patients with chronic myeloproliferative disorders (CMPD): 18 with chronic myelogenous leukemia in chronic phase (CML in CP), seven with CML in accelerated phase (AP) or blastic crisis (BC), six with polycythemia vera (PV), eight with essential thrombocythemia (ET), one with primary myelofibrosis (PMF), and 50 controls. The mean (+/-S.E.M.) levels were higher in CMPD than in controls (CML in AP or BC, 2693 +/- 694 U/ml, P < 0.0001; CML in CP, 792 +/- 63 U/ml, P < 0.0001; PV 553 +/- 89 U/ml, P < 0.05; ET, 449 +/- 56 U/ml; PMF, 628 U/ml vs. controls, 395 +/- 25 U/ml). Patients with CML in CP had significantly higher serum sIL-2R levels than patients with ET (P < 0.005), and levels were markedly elevated in AP and BC (P < 0.001). Serum sIL-2R levels were positively correlated with WBC count and lactic dehydrogenase in CMPD, and in CML in CP. Serum sIL-2R levels in CMPD were negatively correlated with RBC and platelet counts. Serum sIL-2R levels were significantly lower in patients with CML in CP who showed a cytogenetic response after interferon (IFN) therapy than in those who showed no response (P < 0.05). These findings suggest that a high serum sIL-2R level reflects the leukocyte growth in CMPD and is useful both for differentiating CML from other CMPD and for predicting the response to IFN therapy in CML.