Ken-ichi Sawada

Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells

OBJECTIVEnIn an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells.nnnMATERIALS AND METHODSnCord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC).nnnRESULTSnIn the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells.nnnCONCLUSIONnThis ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.

A possible role for lamivudine as prophylaxis against hepatitis B reactivation in carriers of hepatitis B who undergo chemotherapy and autologous peripheral blood stem cell transplantation for non-Hodgkin's lymphoma

Hepatitis B virus (HBV) reactivation, a well-known complication in immunosuppressed patients, can give rise to acute hepatitis and even fatal fulminant hepatitis. Three Japanese males with non-Hodgkins lymphoma (NHL) who were carriers of HBV received high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (PBSCT). To prevent HBV reactivation, all received oral lamivudine (150 mg/day), a nucleoside analogue, at the start of chemotherapy. All were treated at full-dose intensity, including corticosteroids, without modification of treatment regimens. All three patients completed the total course of chemotherapy and PBSCT, with no signs of HBV reactivation. Peripheral blood stem cell (PBSC) harvests and hematological recoveries after transplantation were not affected by lamivudine, which was continued for at least 16 weeks after transplantation. HBV-DNA and DNA polymerase levels remained negative/normal after discontinuation of lamivudine. Lamivudine effectively inhibits HBV replication and has few serious adverse effects, particularly those related to hematopoiesis. Thus, prophylactic use of lamivudine from initiation of chemotherapy deserves consideration in the treatment of HBV carriers who require immunosuppressive chemotherapy, and may prevent HBV reactivation. Bone Marrow Transplantation (2001) 27, 433–436.

HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype

Primary effusion lymphoma (PEL) has been recognized as a body‐cavity‐based lymphoma that was originally reported to be associated with human herpes virus 8 (HHV8) infection, and was frequently found in human immunodeficiency virus‐positive (HIV) patients. Here we describe an autopsy case of PEL of the peritoneal cavity in an immunocompetent patient. Cytological analysis of tumor cells within ascites revealed immunocytochemical features of keratin positivity and CD45 negativity. At autopsy, the presence of a massive volume of ascites as well as diffuse tumor cell infiltrates within the serosa of the intestine and mesenterium were observed. Tumor cells were morphologically similar to anaplastic large‐cell lymphoma, but were immunohistochemically positive for keratin and epithelial membrane antigen (EMA). They also showed no reactivity to representative lymphocyte surface markers including CD45, in addition to being negative for CD30 and p80NPM/ALK. Molecular analysis of the tumor cells revealed monoclonality of the immunoglobulin heavy‐chain gene rearrangement which demonstrated a lymphoma of the B‐cell lineage. Furthermore, HHV8 was not detected by immunohistochemical analysis, PCR or nested PCR technique. Based on these results, we consider the present case to be an HHV8‐negative PEL with keratin and EMA positivity.

Stem cell factor prevents Fas-mediated apoptosis of human erythroid precursor cells with Src-family kinase dependency

The Fas ligand (Fas-L) expressed on mature erythroblasts may induce apoptosis of more immature erythroid cells that express Fas, whereas stem cell factor (SCF) may prevent Fas-mediated cell death in hematopoietic progenitor cells. The manner in which SCF prevents Fas-mediated cell death still is unclear. Given the essential role of SCF and the potentially important involvement of the Fas/Fas-L system in the development of erythrocytes, we studied mechanisms related to SCF prevention of Fas-mediated apoptosis. We used primary cultured human erythroid colony-forming cells (ECFC) derived from CD34+ cells and enriched glycophorin A positive (GPA+) c-kit+ cells in ECFC. Apoptosis of ECFC was induced by an Fas-L mimetic monoclonal antibody CH11. DNA fragmentation and the activation of caspase-3 and caspase-8 were measured using commercially available kits. Characterization of expanded cells was performed using multiparameter flow cytometry. Lyn kinase activity was measured by enolase kinase assays. SCF inhibited the CH11-induced DNA fragmentation of ECFC as well as enriched GPA+ c-kit+ cells in ECFC, but not those of GPA+ c-kit- cells. SCF also inhibited the activation of caspase-3 and caspase-8, without downregulation of the surface expression of Fas, suggesting that SCF prevents apoptosis through uncoupling of Fas ligation from subsequent caspase activation. PP2, a specific inhibitor of Src-family kinases, antagonized the effects of SCF in preventing Fas-mediated apoptosis. We propose that SCF prevents Fas-mediated apoptosis of erythroid progenitor cells in a manner dependent on the activity of Src-family tyrosine kinases. We also identified active Lyn in erythroid cells. These data suggest the presence of a novel Src-family-dependent function of SCF in the development of erythrocytes.

STEM CELL FACTOR PROTECTS C-KIT+ HUMAN PRIMARY ERYTHROID CELLS FROM APOPTOSIS

OBJECTIVEnIt has been reported that stem cell factor (SCF) promotes cell survival in primary cultured human erythroid colony-forming cells (ECFC). Given the heterogeneous nature of ECFC, which may affect interpretation of the data, we purified c-kit+ ECFC and investigated the specificity and mechanisms of the anti-apoptotic effects of SCF on these cells.nnnMATERIALS AND METHODSnGlycophorin A+ (GPA+) c-kit+ cells were purified from primary cultured ECFC derived from purified human CD34+ cells. The GPA+c-kit- and nonerythroid cells were generated from the same CD34+ cells. Apoptosis of ECFC was investigated in the absence or presence of SCF and erythropoietin (EPO) in serum-free medium. DNA fragmentation was measured with enzyme linked immunosorbent assay for oligonucleosome-sized DNA, gel electrophoresis, and annexin V labeling. Characterization of expanded cells and enriched cells was performed using multiparameter flow cytometry. For Akt assay, cells were lysed and the cleared lysates subjected to SDS-PAGE followed by Western blotting.nnnRESULTSnIn GPA+c-kit+ cells, deprivation of cytokine caused rapid DNA fragmentation within 4 hours that reached a maximum at 6 hours. This was partially but clearly prevented by SCF or EPO. In contrast, no significant DNA fragmentation was seen in GPA+c-kit- and nonerythroid cells within 24 hours. PP2, a specific Src family kinase inhibitor, but not its inactive analogue PP3, reversed the anti-apoptotic effects of SCF. PP2 also inhibited SCF-induced phosphorylation of Akt.nnnCONCLUSIONnThese data indicate that SCF protects purified human GPA+c-kit+ cells from apoptosis and suggest that kit-mediated Src kinase activation is involved in Akt activation and cell survival.

Reversal of HIV-associated motor neuron syndrome after highly active antiretroviral therapy

Sirs: Neurological disease occurs frequently in patients infected with the human immunodeficiency virus (HIV) [7]. Although less common than encephalopathy, a variety of neuromuscular disorders associated with HIV infection have been reported [1]. To our knowledge, there are few documented cases of motor neuron syndrome (MNS) complicating HIV infection [2–4, 10, 11]. On the other hand, the antireteroviral combination regimens that contain protease inhibitors have had a great impact on the natural history of HIV infection. There are reports of complete or partial resolutions of active opportunistic disease and HIV associated dementia after the initiation of highly active antiretroviral therapy (HAART) [6, 9]. The clinical efficacy of protease-inhibitor based treatment for patients affected by HIV-associated MNS remained to be elucidated. We treated a patient with HIV-associated MNS whose regression of neurological abnormalities following the initiation of HAART was obvious. In July 1999 a 42-year-old heterosexual HIV-infected Japanese woman was admitted to the Hospital of Hokkaido University School of Medicine for a 1 month history of dysphagia, dysarthria and dysphonia. She had been diagnosed as being HIV positive in 1990 and retrovir, didanosine and lamivudine were prescribed when she was seen at another hospital. She had refused treatment since 1997. On admission she was afebrile, alert and was cooperative during the usual general examinations. Neurologically there were moderate dysarthria and dysphonia, dysphagia, decreased masseter strength, decreased gag reflex, proximal limb symmetric weakness, and muscular atrophy and weakness of both sternocleid muscles. Her tongue was atrophied, with obvious fasciculation. Eye muscles were normal and there was no evidence of a retinopathy. Tendon jerks could be elicited both in the upper and lower extremities. Babinski responses were not elicited. Coordination and all sensory modalities were preserved. She denied sphincter problems, her gait was normal, and vital capacity on admission was 2.8 l/min with a negative inspiratory force of –50 cmH2O. Chest radiography, all laboratory data on serum, including human T lymphocytotropic virus I, creatine kinase, thyroid function test, vitamin B12 and folate level, bacterial, mycobacterial, viral (including detection of pp65 cytomegalovirus, CMV, antigen) and fungus tests yielded negative results. The CD4+ T-cell count was 107x106/l with a CD4-CD8 ratio of 0.15. The plasma HIV–1 RNA (Amplicor HIV–1 Monitor assay, Roche Molecular Systems, Branchburg, N. J., USA) was 44,510 copies/ml. Enhanced magnetic resonance imaging of the brain revealed no abnomalities. CSF obtained by lumbar puncture contained 29 lymphocytes/mm3 without atypia, a slightly raised protein concentration (126 mg/dl, normal below 45 mg/dl) and a glucose concentration of 46 mg/dl (the corresponding serum glucose concentration of 98 mg/dl). CSF cultures for bacteria, mycobacteria, and fungi were negative. Specific polymerase chain reaction in the CSF, including HIV, JC virus, CMV, and varicella zoster virus were not carried out. Nerve conduction studies showed normal motor ulnar, peroneal, tibial motor velocity. Distal motor latencies and F wave responses were not delayed. Needle electromyography was not performed. The diagnosis of HIVassociated MNS was made. Stavudine (60 mg daily), lamivudine (300 mg daily), and nelfinavir (2500 mg daily) were prescribed. After 4 weeks of anti-HIV therapy the HIV load was undetectable in the plasma, and CD4+ cell counts increased to 165x106/l. She showed substantial improvement in motor function within 8 weeks of beginning the drug regimen. In particular there was a significant improvement in dysphagia and masseter strength (Fig. 1). After 7 months of HAART she remains in good clinical condition with minimal neurological deficits including equivocal dysarthria, tongue atrophy and fasciculation. She is able to perform her daily living activities without effort. Retroviral infections may cause motor neuron pathology in both laboratory animals and humans as a result of various mechanisms [5]. However, optimal treatment for humans with HIV-related MNS has not been well-defined. The woman whom we report appears to be the first patient reported of effective HAART with HIV-associated MNS. Four of five HIV-associated MNS patients in the literature died, and the fifth progressed to a complete quadriplegia without HAART [2–4, 10, 11]. Autopsy was carried out three patients, one of whom showed evidence of myeloradiculopathy [11], one AIDS encephalopathy, vacuolar myelopathy LETTER TO THE EDITORS

Large scale purification of human blood CD34+ cells from cryopreserved peripheral blood stem cells, using a nylon-fiber syringe system and immunomagnetic microspheres

Isolation of large numbers of human peripheral blood CD34+ cells could lead to therapeutic applications, including purging of malignant cells from blood cell transplantations, purging of T cells from allogeneic bone marrow, and even blood cell transplantation. This procedure has limitations if there are not sufficient numbers of progenitor cells in the leukapheresis concentrates available for selection after detection of tumor cells in apheresis products. Use of frozen/thawed peripheral blood mononuclear cell (PBMC) samples would make feasible pooling of two or even more stem cell harvests collected at different time points and the total number of CD34+ progenitor cells available would increase. We established an efficient method for purification of CD34+ cells from cryopreserved apheresis products, using a nylon-fiber syringe system and immunomagnetic microspheres. We compared purity, recovery rate and clonogenicity of CD34+ cells purified from fresh (nu2009=u200922) and cryopreserved apheresis products (nu2009=u200914), using a nylon-fiber syringe system and immunomagnetic microspheres. The purity of CD34+ cells from cryopreserved products was less than that from fresh products (85.9u2009±u200914.4% vs 94.6u2009±u200910.0%), but the recovery rate of CD34+ cells and colony-forming cells was comparable between fresh and cryopreserved products. One patient underwent grafting with peripheral blood CD34+ cells selected after freezing, with good success. Therefore, these cells are capable of rapidly reconstituting hematopoiesis after high-dose chemotherapy. Bone Marrow Transplantation (2000) 26, 787–793.

Successful non-myeloablative stem cell transplantation for a heavily transfused woman with severe aplastic anemia complicated by heart failure

A 30-year-old Japanese woman weighing 35 kg with severe hemochromatosis due to multiple transfusions was referred to our clinic for treatment of severe aplastic anemia (SAA). The patient had heart failure with an ejection fraction of 36% requiring diuretics and a severe liver dysfunction with an indocyanine green clearance rate of 18%, as well as other transfusion-related complications such as chronic hepatitis due to hepatitis C virus and diabetes mellitus. She was treated with a non-myeloablative preparative regimen that included fludarabine monophosphate (Flu, 120 mg/m2), cyclophosphamide (CY, 1200 mg/m2) and antithymocyte globulin (ATG, 15 mg/kg) followed by allogeneic peripheral blood stem cell transplantation (PBSCT) from her HLA-matched sister. The regimen was well tolerated, and engraftment rapidly occurred without any therapy-related complications. Chimerism analysis on day 14 after transplant showed reconstitution with 100% donor cells. She no longer needed transfusion after day 23 and has been well in 90% Karnofsky status at 4 months post transplant. The clinical course of this patient indicates that this preparative regimen enables SAA patients with severe organ failure to safely undergo allogeneic stem cell transplantation. Bone Marrow Transplantation (2001) 28, 783–785.

Stem Cell Factor Regulation of Fas-Mediated Apoptosis of Human Erythroid Precursor Cells

Multiple cytokines regulate the development of erythrocytes. Increasing attention has been directed to the possible role of Fas and its cognate ligand (Fas-L), a subject of wide interest. Documentation of in vitro data supports the role of Fas and Fas-L in erythropoiesis. Several laboratories, including ours, investigated the opposing actions of erythropoietin (EPO) and stem cell factor (SCF) on Fas-mediated cell death of the erythroid cells. Only circumstantial in vivo evidence has accumulated concerning the issue. There are several reports suggesting that Fas-mediated cell death may have a role in some pathological conditions. Results of the accumulating findings and possible implications in clinical hematology are summarized in this review.

Effective high-dose chemotherapy combined with CD34 + -selected autologous peripheral blood stem cell transplantation in a patient with cutaneous CD30-negative large T cell lymphoma

Generalized multiple cutaneous tumors developed in a 60-year-old Japanese man. Skin biopsy revealed atypical large T lymphocytes infiltrating the dermis. CD30 staining was negative in the tumor cells. The diagnosis of CD30-negative cutaneous large T cell lymphoma was made. Axial and inguinal lymphadenopathy was present, but there was no evidence of bone marrow involvement. Seven cycles of chemotherapy and local electron beam irradiation were administered and complete remission (CR) was attained. As CD30-negative cutaneous large T cell lymphoma has a poor prognosis despite intensive chemotherapy, high-dose chemotherapy followed by CD34+-selected autologous peripheral blood stem cell transplantation (CD34+-APBSCT) was prescribed. The clinical course after CD34+-selected APBSCT was complicated with CMV infection occurring twice but administration of ganciclovir resolved the symptoms. He has remained in CR for 16 months after CD34+-APBSCT. This appears to be the first case report of CD34+-APBSCT in a patient with CD30-negative cutaneous large T cell lymphoma. Bone Marrow Transplantation (2000) 25, 1315–1317.