Manifestations of fulminant CD8 T-cell post-transplant lymphoproliferative disorder following the administration of rituximab for lymphadenopathy with a high level of Epstein-Barr Virus (EBV) replication after allogeneic hematopoietic stem cell transplantation.

We herein report the case of a 22-year-old woman with severe aplastic anemia who underwent allogeneic hematopoietic stem cell transplantation (HSCT). After HSCT, the Epstein-Barr virus (EBV)-DNA load in the peripheral blood gradually increased, and the patient presented with a fever and lymphadenopathy on day 56 post-HSCT. Although we administered rituximab, her clinical condition worsened. After rituximab treatment, CD8 T-cells emerged and became dominant in the peripheral blood, some of which were positive on an EBV-specific tetramer analysis. However, an open biopsy of the lymphadenopathy lesions revealed the CD8 T-cells to be infected with EBV, exhibiting proliferation with oligoclonality. The patient ultimately died of multiple organ failure on day 99 post-HSCT.

Safety and clinical outcome of erythropoiesis-stimulating agents in patients with ST-elevation myocardial infarction: a meta-analysis of individual patient data.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) have been investigated in small studies in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). Erythropoiesis-stimulating agents did not show a clear effect on left ventricular function or clinical outcome, but some studies suggested an increased risk of thromboembolic events. METHODS: A systematic literature search in MEDLINE was performed, until December 2012. We included randomized clinical trials investigating the effect of ESAs in STEMI patients undergoing primary PCI, with ≥30 days of follow-up. The primary end point was a composite of all-cause mortality, myocardial infarction, and stent thrombosis after PCI. Secondary end point was all-cause mortality. RESULTS: Individual patient data were obtained from 10 of 11 trials, including 97.3% (1,242/1,277) of all patients randomized to control (n = 600) or to ESAs (n = 642). Baseline characteristics were well balanced between the treatment allocations. Mean follow-up time was 248 (±131) days. The primary end point occurred in 3.5% (20/577) in the control group and in 2.1% (13/610) in the ESA group (hazard ratio for ESAs, 0.63; 95% CI [0.31-1.27]; P = .20). Mortality occurred in 13 (2.3%) in the control group and 5 (0.8%) in the ESA group (hazard ratio for ESAs, 0.38; 95% CI [0.13-1.06]; P = .06). CONCLUSIONS: Erythropoiesis-stimulating agent administration does not result in an increased risk of adverse cardiac events in STEMI patients undergoing primary PCI. Results of ongoing studies may provide further insight to the potential beneficial clinical effects of ESAs in STEMI patients.

Involvement of cross-linked ribosomal protein S19 oligomers and C5a receptor in definitive erythropoiesis.

We performed a series of experiments under a working hypothesis that cross-linked oligomers of ribosomal protein S19 (RP S19) play an essential role in definitive erythropoiesis as a ligand of the C5a receptor of erythroblasts and macrophages. We found molecules functionally and immunologically indistinguishable from RP S19 oligomers in the extracellular fluid of porcine and guinea pig bone marrow. When an increased hematopoietic state was induced in guinea pigs by bloodletting, the bone marrow RP S19 oligomer concentration was concomitantly increased. However, when the RP S19 oligomers were immunologically neutralized or the C5a receptor was pharmacologically antagonized, hyper-erythropoiesis induced by bloodletting was prevented and the anemic state was retarded in guinea pigs. When the RP S19 oligomers were neutralized in mice after bloodletting, the reactive hyper proliferation of erythroblasts in the spleen was prevented. Proerythroblasts and erythroblasts prepared by bone marrow aspiration from healthy individuals were found to express significant levels of the C5a receptor and type 2 transglutaminase genes. Majority of erythroblasts in cord blood of healthy newborns bore the C5a receptor. Taken together, these results support our hypothesis.

Successful treatment of severe newly diagnosed immune thrombocytopenia involving an alveolar hemorrhage with combination therapy consisting of romiplostim, rituximab and vincristine.

A 51-year-old man was admitted due to a severe bleeding tendency. After he was diagnosed with immune thrombocytopenia (ITP), several therapies, including steroids, steroid pulse, vincristine and rituximab, were administered; however, the patient's bleeding symptoms were not sufficiently controllable with these treatments. Subsequently, a diffuse alveolar hemorrhage was observed. Treatment with a thrombopoietin receptor agonist, romiplostim, was initiated to prevent lethal hemorrhaging, although the efficacy of thrombopoietic receptor agonists in such emergency situations has not been elucidated. The initiation of romiplostim achieved prompt remission in platelets. This case suggests that combination therapy with romiplostim, rituximab and vincristine is effective in cases of newly diagnosed severe therapy-resistant ITP.

Erythropoietin, but not asialoerythropoietin or carbamyl-erythropoietin, attenuates monocrotaline-induced pulmonary hypertension in rats.

Erythropoietin (EPO) has long been utilized for the treatment of renal anemia. The erythropoietin receptor (EPOR) is also expressed in the cardiovascular and central nervous systems in addition to an erythroid lineage, to provide an organoprotective role against several types of cellular stress. Pulmonary hypertension (PH) is a poor prognostic disease caused by primary and secondary pulmonary vascular injury. We observed the effects of EPO derivatives on monocrotaline-induced PH in rats on the supposition that EPO may protect small arteries from injury. Asialoerythropoietin (AEPO) lacks sialic acids in the termini of carbohydrate chains that results in rapid clearance from blood. Carbamyl-erythropoietin (CEPO) interacts with EPOR/ßc heterodimers, but not with EPOR homodimers expressed in erythroid cells. Monocrotaline-injected rats were treated with continuous intravenous injection of 2500 ng/kg/day of EPO, AEPO, or CEPO for 21 days, and lung histology, cardiac function, and mRNA expression in the lungs were examined. Wall thickening of small arteries in the lungs and PH were improved by administration of EPO, but not by its non-hematopoietic derivatives, AEPO, or CEPO. Erythropoietin administration increased mRNA expression of the anti-apoptotic molecule, Bcl-xL, and maintained expression of the CD31 antigen. We conclude that lungs may express EPOR homoreceptors, but not heteroreceptors. Adequate serum erythropoietin levels may be essential for pulmonary protective effects.

A novel synthetic derivative of human erythropoietin designed to bind to glycosaminoglycans.

To synthesize long-acting and antiangiogenic erythropoietin to be clinically applied for treatment of patients with solid tumors, we synthesized a hybrid molecule of human erythropoietin added onto the C-terminus with a heparin-binding motif of human PLGF-2 to develop a novel derivative of long-acting and antiangiogenic erythropoietin: heparin-binding erythropoietin (HEPO), and studied the characteristics of this novel erythropoietin derivative. HEPO cDNA was synthesized, expressed in insect cells, and the protein was purified using a heparin-sepharose affinity column. The erythropoietic and angiogenic effects of the partially purified protein were analyzed in vitro and in vivo. The erythropoietic activity of the protein was equivalent to natural EPO in vitro. In vivo administration of the protein to mice revealed its long-acting erythropoietic activity as expected. Administration of the protein inhibited angiogenesis in a mouse limb ischemia model. In conclusion, the heparin-binding motif of PLGF-2 may act as, so to speak, a superendostatin. This novel long-acting erythropoietin derivative may have an advantage to inhibit tumor growth while preserving hematopoietic and tissue-protective effects.

Features of Japanese patients with myelodysplastic syndrome in an aging population of Sado Island.

Myelodysplastic syndrome (MDS) is relatively common in the elderly, and aging of populations is progressing in developed nations, notably so in Japan. The major age group in Japan and Sado Island are distributed between 30 and 60 and between 50 and 80, respectively. The aim of this study was to analyze the features of MDS in the population of Sado Island to anticipate the characteristics of the disease in the near future. One-hundred and fifty-three patients (71 male, 82 female, 19-94 years old, median 73 years old) with de novo MDS between 1985 and 2005 were retrospectively evaluated. All patients were reclassified according to WHO-2001 criteria. The predictive power of the international prognostic scoring system and the WHO classification-based prognostic scoring system were evaluated. The major causes of death were leukemic transformation (38%) in refractory anemia with an excess of blasts and infection (48%) for total MDS. Age was another independent prognostic factor. Elderly patients exhibited a significantly poorer prognosis mainly due to infections such as pneumonia. Although novel remedies for MDS and hyperferremia have recently been developed, prevention of infection remains important in MDS, particularly for older patients.

Japanese case of Budd-Chiari syndrome due to hepatic vein thrombosis successfully treated with liver transplantation.

A 22-year-old Japanese woman was found to have severe esophageal varices and then suffered from hepatic encephalopathy. She was diagnosed with Budd-Chiari syndrome (BCS) due to hepatic vein (HV) thrombosis accompanied by portal vein thrombosis without inferior vena cava (IVC) obstruction. Latent myeloproliferative neoplasm (MPN) lacking the JAK2-V617F mutation was considered to be the underlying disease. Liver transplantation was strikingly effective for treating the clinical symptoms attributable to portal hypertension. Although thrombosis of the internal jugular vein occurred due to thrombocythemia, which manifested after transplantation despite anticoagulation therapy with warfarin, the thrombus immediately disappeared with the addition of aspirin. Neither thrombosis nor BCS has recurred in more than 4 years since the amelioration of the last thrombotic event, and post-transplant immunosuppression with tacrolimus has not accelerated the progression of MPN. In Japan, IVC obstruction, which was a predominant type of BCS, is suggested to have decreased in incidence with recent improvements in hygiene. The precise diagnosis of BCS and causative underlying diseases should be made with attention to the current trend of the disease spectrum, which fluctuates with environmental sanitation levels. Because the stepwise strategy, including liver transplantation, has been proven effective for patients with pure HV obstruction in Western countries, this strategy should also be validated for utilization in Japan and in developing countries where HV obstruction potentially predominates.

Hypocellularity and insufficient expression of angiogenic factors in implanted autologous bone marrow in patients with chronic critical limb ischemia.

The aim of this study was to identify the clinical parameters of absolutely poor-prognosis patients with chronic critical limb ischemia (AP-CLI). Sixteen no-option CLI patients with arteriosclerosis obliterans: ASO (nine) and non-ASO patients (seven) treated with bone marrow-mononuclear cell implantation (BMI) were analyzed. There were three AP-CLI patients (all ASO). The mRNA expression of several angiogenic factors in the implanted cells was analyzed in comparison with normal donor bone marrow. To observe the response of bone marrow components to hypoxia, normal bone marrow cells were cultured for 24 h in 2.5% O(2), and mRNA expression of angiogenic factors were measured. AP-CLI patients exhibited extraordinary low bone marrow cellularity as well as the percentage of CD34-positive cells. Among angiogenic factors, only VEGF expression was maintained in response to HIF-1, while other factors such as HGF, Ang-1, PLGF, and SDF-1 decreased in the implanted bone marrow cells of the patients with CLI compared to normal bone marrow cells. HIF-1 and all of the five angiogenic factors increased in vitro in response to hypoxia. Thus it is highly likely that angiogenic factors except VEGF do not respond to chronic ischemia in bone marrow in vivo. An organ-protection system against tissue ischemia may be applied for acute hypoxia, but it may be insufficient for chronic ischemia.

Free heme is a danger signal inducing expression of proinflammatory proteins in cultured cells derived from normal rat hearts.

Endogenous molecules from damaged tissue act as danger signals to trigger or amplify the immune/inflammatory response. In this study, we examined whether free heme induced pro-inflammatory proteins in cultured cells derived from normal hearts and investigated the cells targeted by heme, together with its mechanism of action in these cells. We cultured collagenase-isolated heart-derived cells from normal rats and examined whether free heme induced pro-inflammatory proteins, reactive oxygen species (ROS) production and NF-κB activation, by quantitative RT-PCR, ELISA and flow cytometry. Free heme increased mRNA of various pro-inflammatory proteins in cultured cardiac resident cells (CCRC) (at least 100-fold) and induced intracellular ROS formation. Approximately 85-90% of CCRC are fibroblast/smooth muscle cells and 10-15% are CD11bc-positive macrophages; therefore to examine individual target cells, macrophage-deleted (CD11bc-negative) CCRC, primary cultured cells (cardiac fibroblasts, arterial smooth muscle cells and cardiac microvascular endothelial cells) and macrophage cells lines (NR8383) were similarly treated. Free heme activated NF-κB and induced expression of some pro-inflammatory proteins, including IL-1 and TNF-α in NR8383. On the other hand, macrophage-deleted CCRC strongly increased expression of these proteins on treatment with IL-1 or TNF-α, but not free heme. Induction of expression of pro-inflammatory proteins by free heme was not inhibited by intracellular ROS reduction, but by protease and proteasome inhibitors capable of regulating NF-κB. These data suggest that free heme strongly induces various pro-inflammatory proteins in injured hearts through NF-κB activation in cardiac resident macrophages and through cross-talk between macrophages and fibroblast/smooth muscle cells mediated inter alia by IL-1, TNF-α.

Myelodysplastic syndrome with Ph negative monosomy 7 chromosome following transient bone marrow dysplasia during imatinib treatment for chronic myeloid leukemia.

We describe a 60-year-old Japanese patient with chronic myeloid leukemia (CML) who developed myelodysplastic syndrome (MDS) with Ph negative monosomy 7 chromosome following transient bone marrow dysplasia during imatinib treatment. Most cases that developed chromosomal abnormality in Ph negative cells during imatinib therapy were reported to have less clinical implications, while rare cases developed MDS/AML. The present case suggested that metaphase karyotype analysis and bone marrow examination should be performed for the long term follow-up under imatinib treatment in cases showing cytopenia. The results also suggested that monosomy 7 in Ph negative cells may be an indicator of a poor prognosis.

Clinical value of assessing the response to imatinib monitored by interphase FISH and RQ-PCR for BCR-ABL in peripheral blood for long-term survival of chronic phase CML patients: results of the Niigata CML-multi-institutional co-operative clinical study.

This retrospective analysis investigated the prognostic value of monitoring the response to imatinib using peripheral blood (PB) samples and the impact of the response on outcome in 133 patients with chronic myeloid leukemia (CML). We divided the response into 3 categories according to the results of neutrophil (N)-FISH and BCR-ABL transcript levels in PB; more than a 3-log reduction [major molecular response (MMR)], between a 2-log and 3-log reduction or negative with N-FISH [complete cytogenetic response equivalent (CCyRe)], N-FISH positive or less than a 2-log reduction (non-CCyRe). The median follow-up was 5.46 years. At 5 years, the overall survival (OS) rate and progression-free survival (PFS) rate were 94.4 and 92.0%, respectively. The estimated rate of the CCyRe and MMR were 81.7 and 67.1%, respectively. 106 patients achieving the CCyRe had significantly better OS and PFS than 27 patients without achieving the CCyRe. Patients with MMR had significantly better survival free from death, progression, imatinib withdrawal and a loss of the CCyRe, than patients whose response level remained in the CCyRe without achieving MMR until 18 months. Our observation suggests that the response level of the CCyRe on PB serve as a prognostic indicator, and achieving MMR provides stable long-term survival.

Generation of antigen-specific cytotoxic T lymphocytes using a leukemic plasmacytoid dendritic cell line as antigen presenting cells.

Establishment of a leukemia plasmacytoid dendritic cell line (PMDC05) and intra-lineage transformation from pDCs to mDCs in PMDC05 has been reported. In this paper, we show the applicability of PMDC05 for cellular immunotherapy. By stimulation with LPS, PMDC05 showed enhancement in expression of antigen presentation-associated surface molecules and production of cytokines (IL-12p70 and TNF-α). The antigen presenting ability was markedly increased in PMDC05 stimulated with LPS. By co-culturing of CD8(+) T cells with LPS-stimulated and WT1/CMVpp65 peptide-pulsed PMDC05, WT1/CMVpp65 tetramer(+) cytotoxic T lymphocytes were efficiently generated. These findings reveal the applicability of PMDC05 in cellular immunotherapy for tumor and severe viral infections.

WT1 peptide vaccination in a CML patient: induction of effective cytotoxic T lymphocytes and significance of peptide administration interval.

Although antigen-specific immune responses including cytotoxic T cells (CTLs) against antigen peptide could be enhanced after tumor antigen peptide vaccinations, the immune responses do not necessarily result in a decrease or eradication of tumor cells in the vaccination trials. We focused on whether antigen-specific CTLs could be damaged by the repeated stimulation of antigenic peptide and whether regulatory T (Treg) cells would be increased by the administration of WT1 peptide. We administered WT1 peptide 22 times over 18 months in a CML patient who was being treated with imatinib. Although WT1 peptide administration every 2 weeks did not show any beneficial effects on the minimal residual disease (copies of bcr-abl transcripts), the transcripts remarkably decreased to the level of major molecular response after changing the administration interval of WT1 peptide from 2 to 4 weeks. An ex vivo study demonstrated that re-stimulation with WT1 peptide made WT1-specific T cells less reactive to WT1 tetramers and the impaired reactivity of CTLs lasted at least for 1 week. In addition, the cytotoxicity of the T cells was hampered by re-stimulation. Treg cells increased up to more than fivefold at the end of the WT1 administration period. The present findings suggested that the administration of the peptide every 4 weeks is superior to every 2 weeks. In addition, the findings that Treg cells increased gradually in accordance with the duration of WT1 peptide administration revealed the significance of manipulating Treg cells for establishing an efficient tumor antigen peptide vaccination.

Pharmacokinetic and pharmacodynamic analysis of cyclosporine A (CsA) to find the best single time point for the monitoring and adjusting of CsA dose using twice-daily 3-h intravenous infusions in allogeneic hematopoietic stem cell transplantation.

Pharmacological study is predictably effective in establishing an optimal monitoring strategy for the usage of cyclosporine A (CsA) to prevent graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation recipients. Pharmacokinetic profiling of 33 recipients administered CsA twice daily by 3-h intravenous infusion revealed that levels peaked 2-3 h after the start of infusion, and an exponential decline of CsA concentrations after the termination of infusion was observed. The correlation between the area under the curve (AUC(0-12)) and CsA concentration at various time points after infusion revealed that C (2) and C (3) correlated best with AUC(0-12) (r (2) = 0.725), while the trough concentration correlated poorly. Ex vivo T cell stimulation followed by intracellular cytokine detection with flow cytometry revealed that the capacity of T cells to produce cytokines upon stimulation was inversely proportional to the CsA concentration, and reached a minimum at about 700 ng/mL with a marginal decrease above this concentration. Extrapolation using the regression equations of this study and the data from our retrospective study leads to the assumption that the dose adjustment of CsA based on maintaining the C (3) concentration above 800 ng/mL may effectively prevent acute GVHD. To confirm this assumption, a prospective clinical study is required.

Establishment of culturing system for ex-vivo expansion of angiogenic immature erythroid cells, and its application for treatment of patients with chronic severe lower limb ischemia.

Angiogenesis therapy by bone marrow-mononuclear cell implantation (BMI) has been utilized. We found that erythroid cells played an essential role in angiogenesis by BMI. We then tried to establish a novel cell therapy by implantation of ex vivo expanded immature erythroblasts cultured from hematopoietic stem/precursor cells. Immature to mature erythroblasts were purified from human bone marrow, and mRNA expression were analyzed. Strongly expressed VEGF and PLGF in immature erythroid cells decreased according to erythroid maturation. To expand very immature erythroid cells, we established a two-step culturing system, i.e., bone marrow cells were cultured in the presence of Flt-3L, SCF and TPO for 7 days, and the cells were further cultured in the presence of SCF, IGF-I and EPO for an additional 7 days. The in vivo angiogenic effects of implantation of the ex vivo expanded cells were stronger than that of BMI in mouse limb ischemia model. Three patients with severe chronic lower limb ischemia accompanied by Burger's disease or collagen arteritis were enrolled in a pilot clinical trial of the novel cell therapy by transplantation of ex-vivo expanded immature erythroid cells. In the clinical trial, most clinical symptoms such as rest pain and skin ulcers improved in 4 weeks, and did not recur in the one-year follow-up. No adverse events were observed in any of the patients. Moreover this novel cell therapy required only a small amount of bone marrow collection. Further enrollment of patients with chronic severe lower limb ischemia is necessary to confirm the efficacy and safety of this novel cell therapy, and to estimate the necessary amount of bone marrow aspirate.

WT1 peptide vaccination in combination with imatinib therapy for a patient with CML in the chronic phase.

Although tyrosine kinase inhibitors is effective for dramatically reducing CML cells, it might be difficult to eradicate completely the CML stem cells. We aimed to clarify the safety and effects of WT1 peptide vaccination in combination with imatinib therapy for a CML patient. A 51 year-old male with CML in CP, who showed a resistance against imatinib therapy for 2.5 years, began to be treated with 9 mer modified-type WT1 peptides in combination with standard dose of imatinib. Although every 2-week-administration of WT1 peptides for 22 weeks did not show definite effects on the quantification of bcr-abl transcripts, by changing the administration from every 2 weeks to 4 weeks bcr-abl transcripts decreased remarkably. After 11 months of every 4-week-administration of the peptides and 12 months post cessation of the peptides bcr-abl transcripts achieved to the level below detection by RQ/RT-PCR (complete molecular response). WT1/MHC tetramer(+)CD8(+) CTLs, which appeared after the second administration of WT1 peptides and remained more than 15 in number among 10(6) CD8(+) T cells throughout the administration of WT1 peptides, are still present in the blood on 14th month post cessation of the peptides. An in vitro study as to the cytotoxicity of lymphocytes induced by mixed lymphocyte peptide culture demonstrated that cultured lymphocytes possessed cytotoxicity against WT1 expressing leukemia cells and the cytotoxicity was WT1-specific and MHC class I restricted. The present study showed that WT1 peptide vaccination in combination with TKI is feasible and effective in the therapy for imatinib-resistant CML.