Fumihiko Ishimaru

Monitoring of human herpesviruses after allogeneic peripheral blood stem cell transplantation and bone marrow transplantation

Herpesviruses frequently cause serious complications after allogeneic bone marrow transplantation (allo‐BMT). Recent studies have shown more rapid immune reconstitution after allogeneic peripheral blood stem cell transplantation (allo‐PBSCT) compared with allo‐BMT. However, it has not been clarified whether the improved immune reconstitution after allo‐PBSCT is associated with a lower incidence of herpesvirus infections. We monitored the emergence of Epstein‐Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV‐6) and HHV‐7 DNA by a nested‐double polymerase chain reaction in peripheral blood leucocytes from 22 allo‐BMT and 16 allo‐PBSCT patients. Each virus had an unique temporal profile of detection. HHV‐6 DNA was detected most frequently at 3 weeks after transplantation, whereas CMV and EBV DNA were detected later (2–3 months). Detection rates of HHV‐6 DNA at 3 and 4 weeks after allo‐BMT were significantly higher than those after allo‐PBSCT (9/16 v 2/13 at 3 weeks, P < 0.01; 10/21 v 1/15 at 4 weeks, P < 0.01). Detection rates of the other three herpesviruses after the two types of allogeneic transplantation were not significantly different throughout observation period. Furthermore, detection of HHV‐6 DNA within the first 4 weeks was associated with delayed platelet engraftment after both allo‐BMT and allo‐PBSCT (P < 0.01). These results suggest an advantage for allo‐PBSCT over allo‐BMT in terms of suppression of HHV‐6 reactivation and prevention of subsequent complications.

Elevation of serum hepatocyte growth factor during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization.

We examined serum levels of the angiogenic factors, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), in normal donors for allogeneic peripheral blood stem cell (PBSC) transplantation. Granulocyte colony‐stimulating factor (G‐CSF) (filgrastim 400 μg/m2/d) was administered to 23 donors for 5 d and aphereses were performed on days 4 and 5. Although bFGF remained at similar levels after G‐CSF treatment, serum VEGF and HGF levels increased 1·5‐fold (n = 13; P = 0·02) and 6·8‐fold (n = 23; P < 0·0001) respectively. The serum HGF level before G‐CSF administration on day 1 correlated inversely with mobilized CD34+ cell numbers. Time course kinetics of HGF showed that on the day after G‐CSF administration (day 2), serum HGF levels increased to 3678 pg/ml. For auto PBSC mobilization with chemotherapy and G‐CSF 200 μg/m2/d (n = 8), we observed similar HGF elevation, which appeared to be dose‐dependent on the G‐CSF administered.

G-CSF reduces IFN-γ and IL-4 production by T cells after allogeneic stimulation by indirectly modulating monocyte function

Despite a 10-fold increase of T cell dose, the incidence and severity of acute GVHD following allogeneic transplantation of G-CSF-mobilized PBSC is not increased compared to BMT. Experimental murine studies demonstrate that G-CSF polarizes donor T cells toward a type 2 cytokine response. To determine whether G-CSF alters T cell cytokine responses, we investigated the effects of G-CSF administration on T cell proliferative and cytokine responses to alloantigen and Con A in nonadherent PBMC (NAC) and CD3+ T cells obtained from normal individuals before and after G-CSF administration (10 μg/kg × 4 days). Although T cell proliferative and cytokine (IFN-γ and IL-4) responses to alloantigen stimulation and Con A were significantly reduced in post-G-CSF NAC, they were restored by the removal of non-T cells from post-G-CSF NAC. Furthermore, there was less T cell alloreactivity in MLR in the presence of autologous post-G-CSF monocytes than in the presence of pre-G-CSF monocytes. This alteration was not replicated in vitro by culturing PBMC with G-CSF. These results suggest that G-CSF administration suppresses T cell proliferative and cytokine (IFN-γ and IL-4) responses to allogeneic stimulation by indirectly modulating monocyte function. Bone Marrow Transplantation (2000) 25, 1035–1040.

Hepatic graft-versus-host disease presenting as an acute hepatitis after allogeneic peripheral blood stem cell transplantation

Hepatic graft-versus-host disease (GVHD) generally presents as cholestatic jaundice, and increased serum alkaline phosphatase (ALP) is followed by hyperbilirubinemia and clinical jaundice. Currently accepted standards for evaluating the clinical severity of GVHD are based not on serum aminotransferase levels but on the serum bilirubin level. We describe a 17-year-old Japanese female who had increased aminotransferases without cholestasis on day 23 after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Liver biopsy revealed lymphocytic infiltration of the portal tracts and pericentral necrosis of the lobuli. The limiting plates were not clearly defined due to cellular infiltrates. There was periductal lymphocytic infiltration and vacuolization of the biliary epithelial cells with exocytosis, compatible with GVHD of cholangiohepatitic type. These findings indicate that acute hepatic GVHD may present as acute hepatitis and this should be included in the differential diagnosis for patients with increased aminotransferases after allogeneic stem cell transplantation. Bone Marrow Transplantation (2001) 27, 1007–1010.

Expression of minor histocompatibility antigen, HA-1, in solid tumor cells.

Background. Minor histocompatibility antigen (mHag) induces and mounts graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Among several mHags, HA-1 is one that acts alone and is the most studied. It is suggested that HA-1 may be one of the immunodominant antigens inducing not only graft-versus-host disease but also graft-versus-malignancy effects. There are some reports that mHag HA-1–specific cytotoxic T lymphocytes generated from an HA-1–negative donor can lyse HA-1–positive leukemic cells. However, the tissue distribution of HA-1 has been described as restricted to the cells of the hematopoietic lineage. Methods. We examined the HA-1 expression in peripheral blood mononuclear cells (PBMNC), leukemia/lymphoma cell lines, solid tumor cell lines, and paired samples of tumor and normal tissues from individual cancer patients by quantitative reverse-transcription polymerase chain reaction. Results. We found that mRNA of HA-1 is expressed in all leukemia/lymphoma cell lines and PBMNC. Most of the leukemia/lymphoma cell lines have the same levels of HA-1 expression as a leukemia/lymphoma cell line, Raji. The expression levels of human PBMNC were 14- to 19-fold higher than those of Raji. Among 32 solid tumor cell lines, 7 showed >50% expression levels compared with Raji. Conclusions. HA-1 expression in the mRNA level is higher in cells of hematopoietic origin, but this tissue distribution is not strictly restricted. Some solid tumor cells and tissues express HA-1 gene equal to hematopoietic cells.

Activated human umbilical cord blood dendritic cells kill tumor cells without damaging normal hematological progenitor cells.

Apart from their role as antigen presenting cells, human peripheral blood monocyte and CD34+ cell‐derived dendritic cells (DC), have been demonstrated to exert cytotoxicity against some tumor cells, and their tumoricidal activity can be enhanced by some stimili. However, there have been no reports concerning the tumoricidal activity of human cord blood dendritic cells (CBDC). In this article, we report that human cord blood monocyte‐derived DC acquire the ability to kill hematological tumor cells, after activation with lipopolysaccharide (LPS) or γ‐interferon (IFN‐γ), associated with the enhanced TNF‐α‐related apoptosis‐inducing ligand (TRAIL) expression in CBDC cytoplasm. The CD14‐positive cells collected from cord blood were induced to CBDC in vitro. After activation with IFN‐γ for 12 h, CBDC exhibited cytotoxicity against HL60 and Jurkat cells, while activation with LPS induced cytotoxicity against Daudi and Jurkat cells. However, both LPS‐ and IFN‐γ‐stimulated CBDC showed no cytotoxic activity against normal CD14‐negative cord blood mononuclear cells. The formation of umbilical cord hematopoietic progenitor colonies, identified as burst‐forming unit‐erythroid and colony‐forming unit granulocyte‐macrophage, was not inhibited by stimulated or unstimulated CBDC. IFN‐γ or LPS stimulation enhanced intracellular but not cellular surface TRAIL, and neither intracellular nor cellular surface tumor necrosing factor‐α and Fas Ligand as analyzed by flow cytometry. Our results show that activated CBDC can serve as cytotoxic cells against hematological tumor cells without damaging the normal hematopoietic progenitor cells. (Cancer Sci 2005; 96: 127–133)

High-dose Chemotherapy with Hematopoietic Stem Cell Transplantation is Effective for Nasal and Nasal-type CD56+ Natural Killer Cell Lymphomas

CD56+ natural killer (NK) cell lymphomas occur frequently in the nasal and nasopharyngeal regions and carry a poor prognosis. We have studied seven cases with NK-cell lymphomas. These lymphomas showed the following immunophenotype: CD56+, CD2+, sCD3 and Epstein-Barr virus-encoded small RNAs (EBERs)+. Six patients had localized (stage I or II) disease involving the nasopharyngeal region, while one had stage III disease. One patient with stage I disease achieved a complete remission (CR) after treatment with involved-field irradiation, but subsequently relapsed and died. The remaining six patients received combination chemotherapy as primary treatment: five patients with localized stage I or II disease and one patient with advanced stage III disease. Responses to initial chemotherapy were generally poor. These six patients received a variety of salvage chemotherapy regimens, but never achieved a CR. Subsequently, four of six patients showed a highly aggressive clinical course and died of disseminated disease within 1 year from the diagnosis. Three of six patients received high-dose chemotherapy supported by syngeneic, autologous or allogeneic peripheral blood stem cell transplantation. Two of the three transplant patients achieved a CR and are now surviving in continuous CR. Our clinical experience suggests that myeloablative high-dose chemotherapy and bone marrow rescue by hematopoietic stem cell transplantation may be an effective salvage treatment modality for refractory NK-cell lymphomas and could be considered as a part of the initial therapy for these patients.

Life-threatening bleeding and acquired factor V deficiency associated with primary systemic amyloidosis

Acquired factor X deficiency has been described in patients with amyloidosis but acquired factor V deficiency is quite rare. We report here a case of life-threatening bleeding and acquired factor V deficiency associated with primary amyloidosis. A 50-year-old man who had no previous hemorrhagic diathesis was referred to our hospital because of recurrent epistaxis, gingival bleeding and hemospermia. The laboratory examination revealed that both the prothrombin time (PT) and the activated partial thromboplastin time (aPTT) were significantly prolonged, and factor V activities were markedly decreased to 14-39% of the normal value. Other coagulation factors such as fibrinogen, prothrombin, factor VII, factor VIII, factor IX and factor X were subnormal and normal. Transaminases were slightly elevated but serological tests of hepatitis B and hepatitis C were negative. Mild hepatosplenomegaly was noted without sign of liver cirrhosis. The PT and aPTT obtained 8 years ago when he received a cholecystectomy due to cholecystitis were both normal. Specific assays for the detection of factor V inhibitor were repeatedly performed but no factor V inhibitor was found. Furthermore, a significant recovery of the infused factor V was noted shortly after an intravenous administration of 5-10 U fresh frozen plasma, but it did not last more than 6 h. Melena, bleedings into the left shoulder and buttock, and finally mortal retroperitoneal hemorrhage developed despite repeated infusions of large amounts of fresh frozen plasma. Acquired factor V deficiency associated with primary amyloidosis was suspected but histological diagnosis was not obtained because of the severe bleeding tendency. Autopsy revealed hepatosplenomegaly and massive deposits of AL amyloid in the liver, spleen, heart and other parenchymal organs. Perivascular amyloid deposition and factor V deficiency are both thought to be the cause of the severe hemorrhagic tendency seen in this patient.

Overexpression of novel short isoforms of Helios in a patient with T-cell acute lymphoblastic leukemia.

OBJECTIVE In previous studies, we demonstrated overexpression of the dominant-negative isoform of the transcription factor Ikaros, Ik-6, in patients with blast crisis of chronic myelogenous leukemia and B-cell acute lymphoblastic leukemia. In the present study, we analyzed expression of the Ikaros family genes Ikaros, Aiolos, and Helios in a panel of human T-cell leukemia/lymphoma cell lines and bone marrow samples of patients with T-cell acute lymphoblastic leukemia. MATERIALS AND METHODS We performed reverse transcriptase polymerase chain reaction, sequencing analysis, immunoblotting, and Southern blotting. RESULTS We found overexpression of novel short isoforms of Helios (Hel-5 and Hel-6) in the HD-Mar cell line. Southern blot analysis suggested that there might be a small deletion in the Helios locus of HD-Mar. In addition, we observed decreased expression of more than one Ikaros family gene in 3 of 9 patients with T-cell acute lymphoblastic leukemia. Moreover, one of the patients overexpressed novel short isoforms of Helios (Hel-7 and Hel-8). CONCLUSION This study provides the first evidence of an Ikaros family member (other than Ikaros) of which novel short isoforms become overexpressed in human leukemia.

Allogeneic peripheral blood stem cell transplantation for the treatment of chronic active Epstein–Barr virus infection

The prognosis of chronic active Epstein–Barr virus infection (CAEBV) is very poor. We describe a 24-year-old male with severe CAEBV who was treated with allogeneic peripheral blood stem cell transplantation (allo-PBSCT). On admission, EBER-1 in lymphocytes infiltrating the liver, EBV-DNA in peripheral blood mononuclear cells (PBMC) and monoclonal NK cell proliferation were confirmed. After unsuccessful chemotherapy, he received an allo-PBSCT from his HLA-identical sister. Although he died of pulmonary hemorrhage on day +19, EBV-DNA was undetectable by PCR in PBMC, and the post-mortem liver showed no EBER-1-positive lymphocytes. This experience suggests that EBV-positive lymphocytes in CAEBV may be eradicated by allo-PBSCT, thereby raising the possibility of a new treatment modality. Bone Marrow Transplantation (2000) 26, 805–808.