Long-term outcome of childhood aplastic anemia patients who underwent allogeneic hematopoietic SCT from an HLA-matched sibling donor in Japan.

We report long-term outcomes of 329 childhood severe aplastic anemia (SAA) patients who underwent hematopoietic SCT (HSCT) from an HLA-matched sibling donor in the Japanese Hematopoietic Cell Transplantation Registry. OS and EFS at 10 years were as high as 89.7+/-1.7% and 85.5+/-2.0%, respectively. Five cases of late malignancies (LM) were identified (malignant peripheral nerve sheath tumor, thyroid carcinoma, colon carcinoma, MDS and hepatoblastoma). Cumulative incidence of LM was 0.8% at 10 years and 2.5% at 20 years, respectively, which was lower than that in previous reports. This low incidence is in keeping with the low occurrence of skin cancer in Japanese population and of acute GVHD in our study group. Radiation-containing conditioning was not significantly associated with the incidence of LM after HSCT probably because of absolute low patient number who developed LM in our series. In terms of LM development after HSCT, low-dose TBI in HSCT for SAA to avoid graft rejection, which is commonly used in Japan, might be tolerable in the Japanese population because of its low incidence.

Second allogeneic hematopoietic SCT for relapsed ALL in children.

A second SCT is generally accepted as the only potentially curative approach for ALL patients that relapse after SCT, but the role of second SCT for pediatric ALL is not fully understood. We performed a retrospective analysis of 171 pediatric patients who received a second allo-SCT for relapsed ALL after allo-SCT. OS at 2 years was 29.4 ± 3.7%, the cumulative incidence of relapse was 44.1 ± 4.0% and non-relapse mortality was 18.8 ± 3.5%. Relapse occurred faster after the second SCT than after the first SCT (117 days vs 164 days, P=0.04). Younger age (9 years or less), late relapse (180 days or more after first SCT), CR at the second SCT, and myeloablative conditioning were found to be related to longer survival. Neither acute GVHD nor the type of donor influenced the outcome of second SCT. Multivariate analysis showed that younger age and late relapse were associated with better outcomes. Our analysis suggests that second SCT for relapsed pediatric ALL is an appropriate treatment option for patients that have achieved CR, which is associated with late relapse after the first SCT.

Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations.

We have produced a novel monoclonal antibody (mAb) directed against Wiskott-Aldrich syndrome protein (WASP) by immunizing mice with the recombinant protein. The mAb designated 5A5 is highly specific to WASP and suitable for Western blot analysis and immunoprecipitation. A flow cytometric assay using the 5A5 mAb identifies expression of intracytoplasmic WASP in lymphocytes from normal individuals. Double staining analysis with cell surface CD3, CD19, and CD56, and intracytoplasmic molecules revealed WASP expression in each subpopulation. With regard to WASP expression in patients with Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), peripheral blood mononuclear cells (PBMCs) from nine patients and Epstein-Barr virus-transformed B-lymphoblastoid cell lines from seven patients examined did not show WASP expression by flow cytometric analysis. These results were confirmed by Western blot analysis. We conclude that WASP expression in lymphocyte subpopulations from patients with WAS and XLT can be more precisely evaluated by flow cytometry as compared with Western blot analysis. This flow cytometry method is important as a supplement to Western blots, but even more important as an alternative and powerful assay that can contribute to research on WASP as well as diagnosis in a clinical setting.

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults.

BACKGROUND: X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Bruton's tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood. METHODS: Flow cytometric analysis of the peripheral monocytes using the anti-BTK antibody was used to characterize a 27 year old male patient with mild hypogammaglobulinemia (IgG, 635 mg/dl; IgM, 11 mg/dl; IgA, <5 mg/dl). He had suffered from frequent pneumonia since age 25 but had no history of frequent infections in his childhood or in adolescence. Sequencing of the BTK cDNA obtained from an Epstein-Barr virus-transformed B lymphoblastoid cell line derived from the bone marrow of the patient was performed to confirm a genetic defect. RESULTS: Flow cytometric analysis of cytoplasmic BTK protein in peripheral monocytes indicated that the patient presents a rare case of adult-onset XLA and that his mother is an XLA carrier. Sequencing of the BTK gene revealed a deletion of AG in the codon for Glu605 (AGT), resulting in an aberrant stop codon that truncates the BTK protein in its kinase domain. CONCLUSIONS: This case suggests that some XLA cases may remain undiagnosed because they only show mild hypogammaglobulinemia and they lack repeated infections in childhood. Flow cytometric analysis is a powerful method to screen these patients.

Deficient activity of von Willebrand factor-cleaving protease in patients with Upshaw-Schulman syndrome.

We identified unusually large von Willebrand factor (vWF) multimers caused by deficient activity of vWF-cleaving protease in 2 patients with Upshaw-Schulman syndrome. The autoantibodies that inhibited the protease activity were not detected in the plasma of either patient. Periodic fresh-frozen plasma transfusion was effective for management of the hemolysis and thrombocytopenia. We detected enriched enzyme activity in a particular plasma fraction, although molecular cloning of this specific protease is needed to determine a more detailed pathogenesis and to develop new therapeutic approaches.

Successful umbilical cord blood transplantation from an unrelated donor for a patient with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis.

We report a case of a 5-year-old girl with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) who underwent cord blood (CB) stem cell transplantation (CBSCT) from an unrelated donor. The patient presented with persistent high-grade fever and hepatosplenomegaly. Because the disease was refractory to immunochemotherapy according to the HLH94 protocol, she received 2.0 x 10(7) CB nucleated cells/kg body weight (BW) after conditioning with BU/CY/etoposide. No acute GVHD developed, using FK506 for prophylaxis. The neutrophil count reached >0.5 x 10(9)/l by day 21 and the platelet count reached >50 x 10(9)/l by day 84. The patient recovered well with sequelae of neurological deficits more than 10 months after receiving CBSCT, without showing evidence of HLH or chronic GVHD. Real-time PCR proved applicable for estimation of the EBV load in PBMC of the patient. We conclude that CBSCT may be indicated for some cases of refractory EBV-HLH, who have no HLA-matched siblings and are therefore dependent on unrelated marrow donors.

Epstein-Barr virus-associated hodgkin's disease in a patient with Wiskott-Aldrich syndrome.

UNLABELLED: Wiskott-Aldrich syndrome is a primary immunodeficiency syndrome in which the majority of malignant complications are non-Hodgkin's lymphoma. We report here a Wiskott-Aldrich syndrome patient who developed Epstein-Barr virus-positive Hodgkin's disease in the bilateral pulmonary hilar lymph nodes. The treatment was successful as the patient achieved a complete response and event-free survival for more than 4 y. CONCLUSION: This case is very rare, but highly suggestive of the immune-mediated mechanisms in the pathogenesis of Epstein-Barr virus-associated Hodgkin's disease in an immunodeficiency patient.

Columnar epidermal necrosis: a unique manifestation of transfusion-associated cutaneous graft-vs-host disease.

BACKGROUND: In 1978, the first case of columnar epidermal necrosis was reported in a 6-year-old boy. There were scaly, partially vesicular or crusty, erythematous lesions mainly involving the extremities that histopathologically showed peculiar features of focal, total epidermal necrosis accompanied by a lichenoid tissue reaction. He developed the skin eruption after receiving a blood transfusion from his mother when he showed debility induced by vaccination with an alternated live measles virus vaccine. The lesions rapidly regressed after sun exposure. To our knowledge, there has been no report of a similar case despite such unique features. OBSERVATION: We encountered a similar case of columnar epidermal necrosis in a 15-year-old Japanese girl with chronic graft-vs-host disease; the lesions occurred 3 months after the transfusion of peripheral blood stem cells from her HLA antigen-matched brother. However, there was no exacerbation of liver dysfunction, diarrhea, or bone marrow aplasia. The peculiar cutaneous lesions responded well to topical phototherapy. CONCLUSION: These 2 patients shared a similarity in their lesions and circumstances under which the blood transfusion was performed to a debilitated patient from a close family member. We believe that focal epidermal necrosis observed in patients with this condition represents a variant of blood transfusion-associated lichenoid graft-vs-host disease that occurs uniquely in a skin-targeted fashion.

Novel mutations, no detectable mRNA and familial genetic analysis of the Wiskott-Aldrich syndrome protein gene in six Japanese patients with Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a primary X-linked immunodeficiency disease caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene. The present molecular studies of six Japanese WAS patients identified five different mutations of WASP, including two novel mutations (45delG, 395insGGAGAT), the latter appearing to have occurred de novo. Familial carriers were detected by polymerase chain reaction-single strand conformational polymorphism analysis, restriction enzyme digestion and direct sequencing of PCR products. Neither mRNA nor the protein product were detectable in any of the patients, while various amounts of WASP protein were expressed in carriers, normal controls, haematopoietic cell lines of all lineages and in one patient after receiving allogeneic bone marrow transplantation. Conclusion Genetic and protein analysis is useful in the definite diagnosis and follow up of Wiskott-Aldrich syndrome patients and in carrier detection, especially of atypical or sporadic patients.

Immunological reconstitution by allogeneic bone marrow transplantation in a child with the X-linked hyper-IgM syndrome.

UNLABELLED: A successful transplantation of sibling marrow in a patient with the X-linked hyper-IgM syndrome is reported. Engraftment of HLA-identical marrow cells was obtained, although complicated by grade I acute graft-versus-host disease. Expression of the CD40 ligand (CD40L, CD154) by activated T-cells from the recipient remained at low levels until 10 months after the transplantation, but then normalized. The patient is now fully competent in immune function without any episodes of severe infection 24 months later. CONCLUSION: Allogeneic bone marrow transplantation is a reasonable therapeutic option for X-linked hyper-IgM syndrome if HLA-matched family donors are available. Whether dysregulation of CD40L expression causes post-transplant immunological abnormalities remains to be clarified.

Prolonged secretion of IL-15 in patients with severe forms of acute graft-versus-host disease after allogeneic bone marrow transplantation in children.

Graft-versus-host disease (GVHD) is one of the most common and fatal complications that follows allogeneic bone marrow transplantation (BMT). Donor origin T cells are responsible for the initiation of GVHD. In this report, we demonstrate that conditioning regimens for BMT resulted in elevated serum levels of interleukin-15 (IL-15), which reached maximum levels within 15 days and returned to basal levels within 25 days after allogeneic BMT, in all patients examined. Thereafter, circulating IL-15 was detected only in patients with grade III or IV acute GVHD with gut involvement. In contrast, IL-2 was not detected at any time in these patients. Since IL-15 is able to activate antigen-stimulated T cells and natural killer (NK) cells, IL-15 may play an important role in the development of severe forms of acute GVHD.

Epstein-Barr virus-associated lymphoproliferative disorder after unrelated bone marrow transplantation in a young child with Wiskott-Aldrich syndrome.

We report a case of a 16-month-old Wiskott-Aldrich syndrome (WAS) patient with a WASP gene mutation who received human leukocyte antigen (HLA)-matched, unrelated allogeneic bone marrow transplantation (BMT) followed by an Epstein-Barr virus-associated lymphoproliferative disorder (EB-LPD), diagnosed by clinical findings, polymerase chain reaction detection of the EB virus genome, and spontaneous lymphocyte proliferation of donor cell origin. EB-LPD is one of frequent lethal complications in HLA-mismatched or unrelated BMT in this syndrome. Adoptive immunotherapy with donor leukocyte transfusion, including appropriate numbers of CD3-positive T cells, was effective for the EB-LPD, achieving almost complete recovery 1 year later without any findings of graft-versus-host disease.

Detection of the PGP9.5 and tyrosine hydroxylase mRNAs for minimal residual neuroblastoma cells in bone marrow and peripheral blood.

The "touchdown" polymerase chain reaction (PCR) technique has been applied to analyze expression of the neuron-specific protein, PGP9.5, and tyrosine hydroxylase (TH) genes for detection of minimal residual neuroblastoma cells in bone marrow and peripheral blood. PGP9.5 and TH gene products were not detected in any normal samples (n = 72) examined. However, in patients more than 1 year of age with stage III and IV neuroblastoma PGP9.5 mRNA was detected in six of seven bone marrow samples and in four of eight peripheral blood samples, and TH mRNA in four of seven and three of eight, respectively. The detection sensitivity was up to 10(-6) to 10(-7) micrograms of total cellular RNA for PGP9.5 and 10(-4) micrograms for TH. Among forty bone marrow specimens from nineteen patients with neuroblastoma both PGP9.5 and TH mRNAs were detected in six, and only PGP9.5 mRNA was detected in ten. Since detection of PGP9.5 and TH gene transcripts by the "touchdown" PCR was highly specific and sensitive, it might be most informative at present to carry out both PGP9.5 and TH mRNA assays for minimal residual neuroblastoma cells in blood and bone marrow.

Okadaic acid suppresses neural differentiation-dependent expression of the neurofilament-L gene in P19 embryonal carcinoma cells by post-transcriptional modification.

Mouse P19 embryonal carcinoma cells in aggregation culture in the presence of 10(-6) M retinoic acid followed by monolayer culture differentiate into nerve and glial cells. In this study, we demonstrated that the neurofilament-L (NF-L) mRNA and protein levels of these cells were enhanced in accordance with their retinoic acid-induced neural differentiation. Okadaic acid (OA) treatment of the cells markedly suppressed this differentiation-dependent NF-L gene expression increase and neurite outgrowth of the cells. Similar results were obtained when tautomycin was used instead of OA, suggesting that inhibition of protein phosphatase(s) is involved in the suppression of neural differentiation. OA treatment did not affect the NF-L gene transcription level, determined by the nuclear run-on transcription assay, but it did reduce the stability of both the 3.5- and 2.3-kilobase NF-L mRNAs. The expression and activity levels of protein phosphatase 2A (PP2A) and 2B (PP2B) but not protein phosphatase 1 (PP1) in P19 cells increased in accordance with the enhanced NF-L gene expression. The presence of OA in the culture medium during the course of the neural differentiation caused a reduced PP2A activity but not PP1 and PP2B activities of the cell extracts. On the other hand, both PP1 and PP2B activities but not PP2A activity of cell extracts were suppressed by the addition of cyclosporin A or FK506 in the culture medium. However, both cyclosporin A and FK506 treatments affected neither NF-L gene expression nor neurite outgrowth. These results demonstrate that the OA treatment inhibits the differentiation-dependent increase in NF-L gene expression by destabilizing its mRNAs and suggest that PP2A plays key roles in the differentiation-dependent enhanced expression of the NF-L gene and is the point of the action of OA.

Enhanced UV sensitivity of yeast cells induced by overexpression of Mg(2+)-dependent protein phosphatase alpha (type 2C alpha).

The UV sensitivity of wild-type Saccharomyces cerevisiae cells was increased 2-fold when rat Mg(2+)-dependent protein phosphatase alpha (protein phosphatase type 2C alpha) was overexpressed in the cells. The overexpression of this enzyme rendered the rad 18 mutant (defective in postreplication repair) more UV-sensitive than was observed in the wild-type cells. However, this increase in UV sensitivity disappeared when the host cells had a rad 1 mutation (defective in excision repair). These results suggest that the Mg(2+)-dependent protein phosphatase overexpressed in the yeast cells inhibited their excision repair system.

Localization of the mouse protein serine/threonine phosphatase 2C beta gene to chromosome 17E 4-5.

Protein phosphatase 2C (PP2C) is one of four major classes of protein serine/threonine phosphatase and is considered to have a role in signal transduction of stress responses. It has two isotypes, alpha and beta, encoded by different genes. In this study, the mouse PP2C beta gene was mapped by in situ hybridization to chromosome 17E 4-5.