Kohji Yoshimoto

Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial

BACKGROUND Preclinical studies have established that implantation of bone marrow-mononuclear cells, including endothelial progenitor cells, into ischaemic limbs increases collateral vessel formation. We investigated efficacy and safety of autologous implantation of bone marrow-mononuclear cells in patients with ischaemic limbs because of peripheral arterial disease. METHODS We first did a pilot study, in which 25 patients (group A) with unilateral ischaemia of the leg were injected with bone marrow-mononuclear cells into the gastrocnemius of the ischaemic limb and with saline into the less ischaemic limb. We then recruited 22 patients (group B) with bilateral leg ischaemia, who were randomly injected with bone marrow-mononuclear cells in one leg and peripheral blood-mononuclear cells in the other as a control. Primary outcomes were safety and feasibility of treatment, based on ankle-brachial index (ABI) and rest pain, and analysis was per protocol. FINDINGS Two patients were excluded from group B after randomisation. At 4 weeks in group B patients, ABI was significantly improved in legs injected with bone marrow-mononuclear cells compared with those injected with peripheral blood-mononuclear cells (difference 0.09 [95% CI 0.06-0.11]; p<0.0001). Similar improvements were seen for transcutaneous oxygen pressure (13 [9-17]; p<0.0001), rest pain (-0.85 [-1.6 to -0.12]; p=0.025), and pain-free walking time (1.2 [0.7-1.7]; p=0.0001). These improvements were sustained at 24 weeks. Similar improvements were seen in group A patients. Two patients in group A died after myocardial infarction unrelated to treatment. INTERPRETATION Autologous implantation of bone marrow-mononuclear cells could be safe and effective for achievement of therapeutic angiogenesis, because of the natural ability of marrow cells to supply endothelial progenitor cells and to secrete various angiogenic factors or cytokines.

Periostin and bone marrow fibrosis

Periostin is a secreted protein that shares structural homology with the insect axon guidance protein fasciclin 1. Periostin is expressed predominantly in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses. We have shown previously that periostin is a novel component of subepithelial fibrosis in bronchial asthma. Here, we investigated the relationship between periostin and bone marrow (BM) fibrosis. Periostin was expressed in the stroma and stromal cells of BM fibrosis specimens and to a great extent its expression levels correlated closely to the grade of fibrosis, as estimated by silver staining. However, in the present study, we found no relationship between plasma periostin levels and the extent of BM fibrosis. We also demonstrated that periostin is secreted by human BM hTERT stromal cells and that its secretion is enhanced by TGF-β, a cytokine produced by clonal proliferation of megakaryocytes and/or monocytes. These results indicate that periostin is a component of BM fibrosis and that it may play a role in the disease progression.

Recent progress in the diagnosis and therapy for veno-occlusive disease of the liver.

Veno-occlusive disease (VOD) is one of the severe complications of the liver, which may occur after hematopoietic stem cell transplantation (HSCT). Although an early diagnosis is important to initiate antithrombotic therapy before serious organ failure, the widely used clinical criteria only become clinically fulfilled at an advanced stage of disease. Liver biopsy provides useful findings for the diagnosis of VOD, however, in the later or less severe stages of VOD liver biopsy may provide false-negative sampling error because the biopsy sample may be too small to evaluate the whole liver. In addition it may be difficult to follow the clinical course with repeat biopsy in individual cases. Imaging diagnosis of VOD including gray-scale US, Doppler US, and MRI have been reported as convenient and useful. Color-Doppler US is superior because of its specificity and sensitivity. Blood sampling tests including factor VII, protein C, N-terminal propeptide for type III procollagen (P-III-P) and hyarulonic acid have predictive value, and their measurement may simply be another way to evaluate early hepatic impairment. Since no optimal treatment for VOD has been established as yet, the prophylaxis of VOD or early initiation of treatment is important. These new diagnostic approaches for VOD may provide a direction to resolve the clinical problems of VOD such as the time of initiation of therapy, the therapeutic regimen of choice, and the cessation of therapy.

Characterization of the light chain-restricted clonal B cells in peripheral blood of HCV-positive patients.

To investigate the association between hepatitis C virus (HCV) and B cell proliferation, we searched for the clonal B cells by flow cytometric analysis of the surface immunoglobulin kappa (κ):lambda (λ) light chain ratios of the circulating B (CD19+) cells in 240 HCV-positive patients and 150 negative controls with liver diseases. Clonal B cells with light chain restriction (κ:λ ratio >3:1 or <1:2) were analyzed for CD5 expression and the presence of monoclonal immunoglobulin heavy-chain (IGH) gene rearrangements and the t(14;18) chromosomal translocation. Clonal B cells were detected in 7 cases with HCV (2.9%), but was never detected in the controls (p < 0.05). Of the 7 cases, all had monoclonal IGH gene rearrangements and one had the t(14;18) chromosomal translocation. These HCV-related clonal B cells are not uniform in the intensity of CD5 expression and showed no increase in the frequencies of CD5+ population compared with non-clonal B cells. No “chronic lymphocytic leukemia-phenotype” cells were found. The loss of clonality was observed in 2 cases treated with interferon and in one case treated with splenectomy. The longitudinal study is required to determine whether these circulating clonal B cells progress to lymphoproliferative disorders in future or not.

Demonstration of reversed flow in segmental branches of the portal vein with hand-held color Doppler ultrasonography after hematopoietic stem cell transplantation

Summary:Hepatic veno-occlusive disease (VOD) is a severe complication of hematopoietic stem cell transplantation (SCT). When monitored with hand-held color Doppler ultrasonography during day −7 to +35 around SCT, reversed blood flow in the segmental branches of the portal vein was detected in nine of 56 patients who had undergone SCT. Three of nine patients had clinical evidence of VOD, but six patients did not fulfill the criteria for diagnosis of VOD initially. Two patients progressed to clinical VOD at a later date and the reversed portal flow disappeared with or without treatment for VOD in the other four patients. Monitoring for reversed portal flow with color Doppler ultrasonography may be a useful tool for the early diagnosis of VOD, and may improve prognosis by allowing early initiation of treatment.

Colour Doppler ultrasonography of a segmental branch of the portal vein is useful for early diagnosis and monitoring of the therapeutic course of veno‐occlusive disease after allogenic haematopoietic stem cell transplantation

We report two cases in which visualization of the segmental branch of the hepatic portal vein with the colour Doppler ultrasonography (US) technique was useful for the early diagnosis of veno‐occlusive disease. The change in blood flow in the segmental branch of the portal vein occurred 5 and 6 d before the clinical criteria were fulfilled in the two cases. Reverse flow in the segmental branch began partially in the liver at first, and then spread to the whole liver several days later. All the US findings in both cases disappeared after thrombolytic therapy.

Simultaneous Hepatic Relapse of Non-Hodgkin’s Lymphoma and Hepatocellular Carcinoma in a Patient with Hepatitis C Virus-Related Cirrhosis

We report a 66-year-old man with hepatitis C virus (HCV)-related cirrhosis and simultaneous hepatic relapse of non-Hodgkin’s lymphoma (NHL) and of hepatocellular carcinoma (HCC). Although the liver is frequently involved by NHL, hepatic colocalization of NHL and HCC is rarely detected by imaging techniques. HCV has been suggested to be lymphotrophic as well as hepatotrophic, and therefore has attracted speculation about a causative role in some cases of lymphoma. The patient had a past history of cutaneous diffuse large B cell lymphoma (DLBCL) in concurrence with HCC 32 months previously. Complete remission (CR) had been maintained for both diseases until February 2004, when ultrasonography and computed tomography (CT) showed multiple liver tumors. Two of these, appearing hyperattenuating in the arterial phase of contrast-enhanced CT, were diagnosed histopathologically as HCC, and treated with radiofrequency ablation. The other tumors, hypoattenuating in the portal phase CT, were diagnosed histopathologically as DLBCL, and treated with cyclophosphamide, tetrahydropyranyl-Adriamycin, vincristine and prednisolone (THP-COP) in combination with rituximab. CR was achieved for both DLBCL and HCC. Given the previously demonstrated immune system tropism and perturbation by HCV, the virus might have contributed to the occurrence of the NHL as well as the HCC.

Expression of epithelial cancer-related antigens in hematologic malignancies applicable for peptide-based immunotherapy.

Recent advances in tumor immunology have resulted in identification of many epithelial cancer-related antigens and peptides applicable to specific immunotherapy. The authors investigated whether these peptides, which are being studied clinically, could be appropriate target molecules for treatment of patients with hematologic malignancies. The majority of hematologic malignant cells studied expressed five different epithelial cancer-related antigens. Cytotoxic T lymphocyte (CTL) precursors reactive to these antigen-derived peptides were detected in peripheral blood mononuclear cells (PBMCs) of the majority of HLA-A24+ patients, and the mean number of peptides recognized by CTL precursors was 2.4 per patient, ranging from 0 to 8 among the 10 peptides tested. These peptide-stimulated PBMCs exhibited HLA-A24-restricted cytotoxic activity against hematologic malignant cells but not against blastoid T cells. More importantly, these peptide-stimulated PBMCs exhibited cytotoxicity against freshly prepared autologous malignant cells in an HLA-A24-restricted manner. These results may provide a scientific basis for the use of these peptides from epithelial cancer-related antigens in specific immunotherapy for patients with hematologic malignancies.

Promyelocytic crisis of chronic myelogenous leukaemia during imatinib mesylate treatment.

An untreated 66-year-old woman with chronic myelogenous leukaemia (CML) in the chronic phase was initially given imatinib mesylate, rapidly achieving a good cytogenetic response with treatment. However, acute promyelocytic leukaemia complicated by a disseminated intravascular coagulation occurred 9 months after beginning imatinib treatment. Promyelocytic crisis of CML was diagnosed by demonstration of both BCR/ABL and PML/RARα chimeric genes in leukaemic cells by karyotypic and fluorescence in situ hybridization analysis. Clonal evolution with addition of the PML/RARα translocation may have arisen in the early chronic phase of CML, with expansion of this clone during imatinib treatment. Promyelocytic crisis of CML is rare; furthermore, we know of no previous report of promyelocytic crisis occurring during treatment with imatinib.

Determination of Thrombopoietin‐Derived Peptides Recognized by Both Cellular and Humoral Immunities in Healthy Donors and Patients with Thrombocytopenia

Thrombopoietin (TPO) is a cytokine that promotes megakaryocytopoiesis and thrombopoiesis and is considered a drug suitable for patients with thrombocytopenia. However, unexpected severe thrombocytopenia has developed in some healthy individuals participating in phase I clinical trials with a pegylated recombinant human megakaryocyte growth factor (PEG‐rHuMGDF) that contained the first 163 amino acids of endogenous TPO, which resulted in hampering the further development of clinical trials. Autoimmune responses to PEG‐rHuMGDF, which cross‐reacted with endogenous TPO, were suggested to be involved in this rare but severe adverse event, although the immunogenic epitopes have not yet been determined. To better understand the molecular basis of such autoimmune reactions, we investigated the reactivity of 18 TPO‐derived peptides with HLA‐A2–binding motifs to plasma and T cells, both from patients with thrombocytopenia (n = 24) and from healthy donors (HDs) (n = 24). Four peptides, including those possessing amino acids in receptor‐binding sites, were preferentially reactive to plasma from at least 20% of the patients, whereas one peptide at position 101–109 was equally reactive to those of the patients and the HDs. Each of the five peptides had the ability to induce peptide‐specific cytotoxic T lymphocytes (CTLs) in both groups, albeit with less frequency among the patients. More important, each of these five peptides had the ability to induce HLA‐A2–restricted and peptide‐specific CTL activity reactive to cells that produce TPO. These results may provide new insights to gain a better understanding of autoimmune reactions to TPO.