Atsushi Yasumoto

A Retrospective Analysis of Allogeneic Hematopoietic Stem Cell Transplantation for Adult T Cell Leukemia/Lymphoma (ATL): Clinical Impact of Graft-versus-Leukemia/Lymphoma Effect

Adult T cell leukemia/lymphoma (ATL) is a highly aggressive T cell malignancy, and has a poor prognosis. Recently, allogeneic-hematopoietic stem cell transplantation (allo-HSCT) has been suggested to improve the outcome. We retrospectively analyzed 15 patients with ATL who had received allo-HSCT in 2 institutions in Hokkaido, Japan. The median age of the patients was 57 years. The estimated 3-year overall survival (OS) and progression-free survival (PFS) rates were 73.3% and 66.7%, respectively. Calcineurin inhibitor dosage was reduced and administration was discontinued abruptly in 6 of the 15 patients for disease control; as a result, 4 (66.7%) of the 6 patients achieved complete response (CR) or partial response. Therefore, a graft-versus-leukemia/lymphoma (GVL) effect might be induced by discontinuation of immunosuppression. Thirteen of the 15 patients were followed up by monitoring HTLV-1 proviral DNA levels. In 10 of the 11 patients with positive HTLV-1 proviral DNA before allo-HSCT, HTLV-1 proviral DNA became undetectable at least once after allo-HSCT, and only 1 of the 5 patients in whom HTLV-1 proviral DNA became detectable after allo-HSCT relapsed. Compared to the results of past studies, these results show that allo-HSCT greatly improved the prognosis of ATL and suggest a contribution of the induction of a GVL effect.

Porcine Model of Hemophilia A

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.

Degradation of cross-linked fibrin by leukocyte elastase as alternative pathway for plasmin-mediated fibrinolysis in sepsis-induced disseminated intravascular coagulation

An alternative pathway for fibrinolysis that comprises leukocyte elastase and its interaction with the plasminogen activator-plasmin system has been suggested. Plasma levels of cross-linked fibrin degradation product by leukocyte elastase (e-XDP) were significantly increased in patients with sepsis induced disseminated intravascular coagulation (DIC) compared with healthy subjects (18.6±19.9 vs 0.58±0.47U/mL, p<0.001). Twenty seven unique spots were identified from e-XDP dominant patients by immune-purification and two-dimensional difference gel electrophoresis, and they contained fibrinogen Bβ-chain derived fragments Bβ Asp-164, Ser-200, Gln-301, Ala-354, Ile-484 and γ-chain derivatives γ Val-274 at their amino-termini by acquired and processed tandem mass spectrometer. The Sequential Organ Failure Assessment Scores in patients with e-XDPs levels 3-10U/mL were significantly lower than those with e-XDPs levels -3U/mL, 10-30U/mL, and 30- U/mL. The adjusted odds for 28-day mortality rate in patients with e-XDP levels less than 3U/mL (hazard ratio, 4.432; 95% CI, 1.557-12.615 [p=0.005]) were significantly higher than those in patients with e-XDP levels of 3-10U/mL. These data suggest that leukocyte elastase might contribute to the degradation of cross-linked fibrin in sepsis-induced DIC.

Intra-articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII-deficient mice

Summary.  Background:  Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice.

Increased number of CD16+CD56dim NK cells in peripheral blood mononuclear cells after allogeneic cord blood transplantation

In the present study, we investigated subpopulations of natural killer (NK) cells and the expression of stimulatory and inhibitory NK receptors after adult blood and bone marrow transplantation (BBMT) and cord blood transplantation (CBT). There were significant increases in CD16(+)CD56(dim) cell proportion and in absolute number in peripheral blood mononuclear cells (PBMC) during a period of 4-9 months after CBT compared with these in normal PBMC, cord blood (CB), and in PBMC after BBMT. Also, increased numbers of CD16(+)CD56(dim) NK cells were sustained in some patients until 4 years after CBT. This CD16(+)CD56(dim) cell subset after CBT exhibited decreased expression of NKG2A compared with that in CB and increased expression of NKG2C. Purified CD16(+)CD56(dim) cells from patients 8-9 months after CBT exhibited significantly higher levels of cytolytic activity against K562 than did purified CD16(+)CD56(bright) cells and also whole PBMC. The CD16(+)CD56(dim) cell subset with a high level of cytolytic activity significantly increased after CBT, and these cells may be responsible for NK cell-mediated immunity after CBT.

Prognosis of patients with core binding factor acute myeloid leukemia after first relapse

Core binding factor acute myeloid leukemia is known to have a favorable prognosis, however, there have been no detailed analyses on prognostic factors after first relapse. Using a nationwide database, we retrospectively analyzed core binding factor acute myeloid leukemia patients who relapsed after being treated with chemotherapy alone during their first complete remission. Of a total of 397 patients who were diagnosed with core binding factor acute myeloid leukemia, 208 experienced a first relapse, and analyses were performed in 139 patients for whom additional data were available. In the entire cohort, the overall survival rate after relapse was 48% at 3 years. By multivariate analysis, younger age at diagnosis, a longer interval before relapse, and inv(16) were shown to be independently associated with better survival after relapse. Although there was no significant difference in survival after relapse between patients who underwent allogeneic hematopoietic cell transplantation and those who did not in the overall series of relapsed patients, we found that transplantation significantly improved survival among patients who had t(8;21) (54% versus 26% at 3 years, P=0.002). In addition, among patients with t(8;21), those who had different cytogenetics at relapse had a significantly improved survival after transplantation, while those who had same cytogenetics did not. We showed that the prognosis differs significantly and optimal treatment strategies may vary between groups of patients with core binding factor acute myeloid leukemia with different cytogenetic profiles at relapse. These findings may help to guide therapeutic decisions after first relapse.

Differential effects of interleukin-12 and interleukin-15 on expansion of NK cell receptor-expressing CD8+ T cells

The cytolytic activity of cells expressing natural killer cell receptors (NKRs) depends on the balance between stimulatory and inhibitory signals. We investigated both inhibitory NK receptor (CD94/NKG2A) expression and stimulatory NKR (NKG2D) expression on T cells after stimulation with cytokines (IL-12 or IL-15). Cytolytic NKR-expressing CD8+ T cells were expanded from normal adult peripheral blood mononuclear cells using anti-CD3 monoclonal antibody and cytokines (IL-12 or IL-15). The proportion and absolute number of CD94/NKG2A-expressing T cells expanded by IL-12 were significantly larger than those of the cells expanded by IL-15. On the other hand, the proportion and absolute number of NKG2D-expressing T cells expanded by IL-15 were significantly larger than those of the cells expanded by IL-12. The proportions of NKG2D and intracellular granzyme A expression in CD94-expressing cells were much more increased in PBMCs cultured with IL-15 than those of cells cultured with IL-12. A real-time polymerase chain reaction assay showed that there was a 1.68-fold increase in NKG2D mRNA expression level and a 1.37-fold increase in DAP10 mRNA expression level in CD94-expressing cells expanded by IL-15 compared with those of the cells expanded by IL-12. The cytolytic activity levels of purified CD94-expressing cells from 8-day culture with IL-15 tested against 51Cr-labeled K562 cells by standard 4-h 51Cr release assays without prior sensitization were much higher than those of cells from 8-day culture with IL-12. IL-15 appears to be able to enhance the cytolytic activity of CD94/NKG2A-expressing cells through induction of NKG2D and intracellular granzyme expression much more efficiently than does IL-12.

A fatal case of cytomegalovirus ventriculoencephalitis in a mycosis fungoides patient who received multiple umbilical cord blood cell transplantations

Cytomegalovirus (CMV) infection is latent in the majority of adult humans. The reactivation of CMV causes pneumonia and gastrointestinal disease in severely immunosuppressed patients, who consequently suffer very high mortality due to CMV central nervous system disease. We report here a case involving a 28-year-old female patient with mycosis fungoides who underwent umbilical cord blood transplantation three times and developed CMV ventriculoencephalitis. The patient’s CMV viremia was successfully preempted with ganciclovir (GCV) as indicated by undetectable CMV antigenemia; despite this successful treatment, the patient developed CMV ventriculoencephalitis. Foscarnet (FCV) therapy led to a temporary recovery, after which CMV ventriculoencephalitis recurred, and the patient died after receiving combination GCV and FCV therapy. Autopsy samples revealed CMV ventriculoencephalitis, as indicated by numerous inclusion-bearing cells (Owl’s eye). It is likely that this patient harbored a GCV-resistant CMV strain; however, it was not possible to obtain nucleic acids suitable for use in assessing this possibility.

Immune response against serial infusion of factor VIII antigen through an implantable venous-access device system in haemophilia A mice.

Summary.  Haemophilia A is a life long bleeding disorder caused by an inherited deficiency of factor VIII (FVIII). About 30% of haemophilia A patients develop neutralizing antibodies as a consequence of treatment with FVIII concentrates. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII. We evaluated the immune responses to serial intravenous administration of FVIII in preimmunized haemophilia A mice. We introduced an implantable venous‐access device (iVAD) system into haemophilia A mice to facilitate sequential infusion of FVIII. After preimmunization with FVIII, the haemophilia A mice were subjected to serial intravenous administration of FVIII through the iVAD system. In all mice with serial infusion of FVIII, high titers of anti‐FVIII inhibitory antibodies developed at 10 exposure days (EDs). However, the anti‐FVIII IgG titers were decreased after 150 EDs of sequential low‐dose infusion of FVIII [0.05 U g−1 body weight (BW) five times per week]. Proliferative response to ex vivo FVIII stimulation was significantly suppressed in splenic CD4+ T cells from mice with serial low‐dose FVIII infusion compared with those from mice with high‐dose FVIII infusion (0.5 U g−1 BW five times per week) or preimmunized mice. Moreover, splenic CD4+ T cells from mice with serial low‐dose infusion of FVIII failed to produce interleukin‐2 and interferon‐γ. These data suggest that serial infusion of FVIII could induce T‐cell anergy in haemophilia A mice with inhibitor antibodies.

Sequential chemotherapy and myeloablative allogeneic hematopoietic stem cell transplantation for refractory acute lymphoblastic leukemia

The prognosis of patients receiving allogeneic hematopoietic stem cell transplantation (HSCT) for refractory acute lymphoblastic leukemia (ALL) is very poor. To improve survival rates, we attempted to intensify the conditioning regimen with daunorubicin, vincristine, prednisolone, medium-dose etoposide, cyclophosphamide, and total body irradiation (DNR/VCR/PSL plus medium-dose VP/CY/TBI). Four patients in relapse or induction failure of B-precursor ALL without other complications underwent allogeneic HSCT. Initially, chemotherapy comprising DNR 60 mg/m2 for 3 days, VCR 1.4 mg/m2 for 1 day, and PSL 60 mg/m2 for 3 days was administered, which was followed by medium-dose VP/CY/TBI; some modifications were made for individual patients. All patients achieved engraftment and complete remission after HSCT. Regimen-related toxicities were tolerable and no patient died within 100 days. Two patients were alive without disease on days 563 and 1,055. The third patient relapsed on day 951, while the fourth died on day 179 without disease. Our results indicate that intensified myeloablative HSCT should be considered for patients with refractory ALL.