Langerhans Cell Histiocytosis With Unusual Hexagonal Crystals in Addition to Usual Charcot-Leyden Crystals. Report of a Patient With Possible Process of Crystal Formation and Clinical Significance of a "Necrotic" Change.

Langerhans cell histiocytosis is a rare neoplastic disorder characterized by the proliferation of Langerhans cells and often accompanied by eosinophil infiltration. Charcot-Leyden crystals, composed of galectin 10, are occasionally observed in Langerhans cell histiocytosis; however, histological images are rarely reported. We herein present a patient with Langerhans cell histiocytosis with Charcot-Leyden crystals and hexagonal crystals by describing the histologic and immunohistochemical features of a lymph node. A unique distribution of Charcot-Leyden crystals and hexagonal crystals was observed in this patient, shedding light on their possible formation process of the latter. We discuss the biological significance of eosinophilic abscesses in Langerhans cell histiocytosis and propose that these crystals may be linked to extracellular trap-cell death (ETosis). This example challenges the conventional characterization of "necrosis" in Langerhans cell histiocytosis and underscores the importance of recognizing ETosis as a potential mechanism involved in the pathogenesis of Langerhans cell histiocytosis. Further studies are underway to validate significance of these findings in a larger cohort of Langerhans cell histiocytosis patients.

[A case of gastric cancer accompanied by disseminated carcinomatosis of bone marrow wherein long-term chemotherapy was enabled by early supportive palliative care].

Here we report gastric cancer accompanied by bone marrow carcinomatosis in a patient for whom long-term chemotherapy was enabled by early pain-relief therapy. A 45-year-old man was admitted to our hospital because of back pain associated with multiple spinal tumors in June 2011. Blood tests showed a trend toward disseminated intravascular coagulation(DIC) and gastric cancer was suspected as the primary lesion. Because pain was severe, emergency pain relief was provided by flurbiprofen axetil and a continuous subcutaneous infusion of fentanyl citrate. After bone marrow examination gave a diagnosis of poorly differentiated adenocarcinoma, we performed sequential methotrexate(MTX)and 5-fluorouracil(5-FU)therapy. The therapy successfully decreased tumor marker levels, and alkaline phosphatase and lactate dehydrogenase levels normalized. Finally, gastric cancer accompanied by bone marrow carcinomatosis was diagnosed. Because the patient had anxiety and spiritual pain from the time of admission, psychiatric care was also required. In November 2011, the tumor recurred, and we switched therapy to a combination of S-1 and cisplatin. The patient's pain was controlled by combined treatment with a fentanyl patch and etodolac, and he was discharged in December 2011. However, severe pain recurred and pain therapy was continued. DIC developed in February 2012 and transiently resolved after resuming combination therapy with MTX and 5-FU; however, it subsequently recurred, leading to the patient's death in May 2012.

[Palliative care for patients with hematological malignancies in the Japan Baptist Medical Foundation].

The Japan Baptist Medical Foundation has established a "hospice triangle" system consisting of the hospice ward, general ward, and home hospice. Palliative care is provided for patients with various types of cancer, including hematological malignancies, in the place where they desire to receive care. From December 2010 to December 2013, 37 patients with hematological malignancies received palliative care and died at our foundation. Eleven (30%) patients died in the hospice ward, 24 (65%) in the general ward, and 2 (5%) at home. The median interval between the final dose of chemotherapy and death was 12 (1- 88) days. Twenty (54%) patients received transfusions during the last 2 weeks prior to death. Quick response to patient situations and early introduction of palliative care are essential to support end-of-life decision-making processes, because the clinical course of hematological malignancies generally differs from that of other cancers.

Interferon-α and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells.

Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio-defence systems through the production of cytokines such as interferon-α (IFN-α) and interleukin-12 (IL-12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti-microbial innate immune responses but also in the pathogenesis of IFN-related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA-mediated innate immunity by possessing DNA-sensing toll-like receptor 9 (TLR9). We here found that double-stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte-derived DCs (moDCs), leading to the preferential production of IFN-α but not IL-12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA-mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co-localization of endosomal LAMP1 staining, and the dsDNA-mediated IFN-α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single-stranded RNA (ssRNA) stimulated moDCs to secrete IL-12 but not IFN-α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA-mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN-α production and acquired immunity via IL-12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA-mediated activation of protective or pathogenic immunity.

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice.

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8alpha(+) conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8(+) T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3(+) DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8(+) T cells through XCR1.

Dic (17;20) (p11;q11) preceded MLL gene amplification in a patient with de novo mixed-lineage leukemia.

We report a case of acute mixed-lineage leukemia, as seen in a 65 year-old female with MLL gene amplification and biallelic loss of wild type p53 gene. The diagnosis was based on the findings that her bone marrow (BM) blasts expressed cytoplasmic CD3 (cyCD3), B-lineage antigens and myeloid antigens accompanied by clonal rearrangements of IgH gene. The BM blasts consisted of small-sized peroxidase-negative blasts (97%) and large-sized peroxidase-positive blasts (3%). The BM blasts showed a complex "karyotype," including dic(17;20) (p11;q11), -5 and add (11q23). Add (11q23) abnormality was found in sideline karyotypes as well as the stemline abnormality of dic(17;20) (p11;q11). For the p53 gene, which is located at 17p13, fluorescence in situ hybridization analysis showed the loss of one of two p53 alleles. Furthermore, polymerase chain reaction-single-strand conformation polymorphism and following nucleotide sequencing showed that the p53 gene was mutated at codon 215, leading to an amino acid substitution from Ser to Arg. For the MLL gene, southern blot analysis showed that the MLL gene locus was amplified but not rearranged at its breakpoint cluster region, which is usually rearranged in balanced translocations with many partner genes. These findings suggest that MLL gene amplification may in this case be based on the genetic instability caused by the preceding biallelic loss of the wild type p53 gene.

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, function as inhibitors of cellular and molecular components involved in type I interferon production.

OBJECTIVE: Statins, which are used as cholesterol-lowering agents, have pleiotropic immunomodulatory properties. Although beneficial effects of statins have been reported in autoimmune diseases, the mechanisms of these immunomodulatory effects are still poorly understood. Type I interferons (IFNs) and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Therefore, PDCs may be a specific target of statins in therapeutic strategies against SLE. This study was undertaken to investigate the immunomodulatory mechanisms of statins that target the IFN response in PDCs. METHODS: We isolated human blood PDCs by flow cytometry and examined the effects of simvastatin and pitavastatin on PDC activation, IFNalpha production, and intracellular signaling. RESULTS: Statins inhibited IFNalpha production profoundly and tumor necrosis factor alpha production modestly in human PDCs in response to Toll-like receptor ligands. The inhibitory effect on IFNalpha production was reversed by geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert their inhibitory actions through geranylgeranylated Rho inactivation. Statins inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory effect on the IFN response was through the prevention of nuclear translocation of IFN regulatory factor 7. In addition, statins had an inhibitory effect on both IFNalpha production by PDCs from SLE patients and SLE serum-induced IFNalpha production. CONCLUSION: Our findings suggest a specific role of statins in controlling type I IFN production and a therapeutic potential in IFN-related autoimmune diseases such as SLE.

Thymic stromal lymphopoietin plays an adjuvant role in BCG-mediated CD8(+) cytotoxic T cell responses through dendritic cell activation.

Although Bacillus Calmette-Guérin (BCG) has historically emerged as a potent adjuvant in cancer immunization through dendritic cell (DC) activation, the efficacy of its antitumor effect has been limited. Therefore, the strategy of adjuvant therapy using BCG needs to be improved by adding enhancers. Here we found that thymic stromal lymphopoietin (TSLP) acts as an enhancer for the BCG-mediated antitumor effect. While BCG-stimulated DCs induced CD8(+) T cell production of IFN-gamma without strong cell expansion, TSLP-stimulated DCs induced robust CD8(+) T cell expansion without high quantities of IFN-gamma production. Notably, DCs stimulated with both BCG and TSLP induced robust expansion of CD8(+) T cells that produced a large amount of IFN-gamma with a potent cytolytic activity related to granzyme B expression. Our data suggest that TSLP is a good adjuvant to enhance the BCG-mediated cytotoxic T cell effect through DC activation, and provide a functional basis for a novel strategy for antitumor immune-based therapy.

Inhibitor of IkappaB kinase activity, BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells.

INTRODUCTION: Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IkappaB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs. METHODS: We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-alpha production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-alpha production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U. RESULTS: Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-alpha production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-alpha production, more strongly inhibited IFN-alpha production than TNF-alpha production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-alpha production by human PBMCs induced by the SLE serum and the in vivo serum IFN-alpha level induced by injecting mice with poly U. CONCLUSIONS: These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.

Parotid mucosa-associated lymphoid tissue lymphoma regression after Helicobacter pylori eradication.

A 60-year-old woman with Sjögren's syndrome showed recurrence of parotid mucosa-associated lymphoid tissue (MALT) lymphoma with a simultaneous increase of serum sIL-2R antigen levels 10 years after surgical treatment. Helicobacter pylori infection had been detected in the stomach since the beginning of the lymphoma. Although H. pylori was not detected in the recurrent parotid lymphoma, antibiotic therapy contributed not only to successful eradication of gastric H. pylori but also to disappearance of the recurrent lymphoma. Further studies on the mechanisms of occurrence of extragastric MALT lymphomas are needed to establish the treatment of extragastric MALT lymphomas.

Imidazoquinoline acts as immune adjuvant for functional alteration of thymic stromal lymphopoietin-mediated allergic T cell response.

Atopic dermatitis is a major allergic disease that develops through dysregulation of Th2-mediated inflammation. Although dendritic cells (DCs) have been thought to play a critical role in the upstream phase of the allergic cascade, conventional drugs such as steroids and chemical mediator antagonists target the effector cells or factors in allergic inflammation. Recently, it has been demonstrated that interaction between thymic stromal lymphopoietin (TSLP) and human DCs plays an essential role in evoking inflammatory Th2 responses in allergy through OX40 ligand expression on DCs. In this study, we provide evidence that R848, an imidazoquinoline compound, which is a TLR ligand and a strong Th1 response-inducing reagent, is a potent adjuvant for the alteration of the Th2-inducing potency of human DCs activated by TSLP (TSLP-DCs). R848 inhibited the inflammatory Th2-inducing capacity of TSLP-DCs and redirected them to possessing an IL-10 and IFN-gamma-producing regulatory Th1-inducing capacity. This functional alteration depended on both repression of OX40 ligand expression and induction of IL-12 production from DCs by the addition of R848. Additionally, R848 had the ability to inhibit the TSLP-mediated expansion and maintenance of the Th2 memory response. These findings suggest that imidazoquinoline may be a useful in the treatment of allergic diseases that are triggered by TSLP.

Mycobacterium bovis Bacillus Calmette-Guérin suppresses inflammatory Th2 responses by inducing functional alteration of TSLP-activated dendritic cells.

Allergic diseases such as atopic dermatitis and asthma develop as a consequence of dysregulated T(h)2 responses. Recently, it has been demonstrated that interaction between dendritic cells (DCs) and thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, is essential for evoking T(h)2 responses in allergy. In this study, we investigated whether Mycobacterium bovis Bacillus Calmette-Guérin (BCG), a strong T(h)1 response-inducing adjuvant, can alter the function of DCs activated by TSLP (TSLP-DCs). We demonstrated that BCG redirects TSLP-DCs away from inducing inflammatory T(h)2 cells that produce IL-4, IL-5, IL-13 and tumor necrosis factor (TNF)-alpha and toward regulatory T(h)1 cells that produce IFN-gamma and IL-10. We also demonstrated that this functional alteration of TSLP-DCs by BCG depended on both production of IL-12 from DCs and down-regulation of OX40 ligand, a member of the TNF family, on DCs. These findings suggest that BCG might be a useful adjuvant for the treatment of allergic diseases that are triggered by TSLP.

[Elevation of Epstein-Barr virus (EBV)-DNA in the peripheral blood of a patient with age-related EBV-associated B-cell lymphoproliferative disorder].

A 69-year-old man was admitted to our hospital with fever and right neck lymphadenopathy. A neck lymph node biopsy was performed. In the specimens, immunoblasts were present with an admixture of small T lymphocytes and macrophagocytes. Immunohistochemcal staining of immunoblasts was positive for CD20, CD30 and EBER. Epstein-Barr virus (EBV) serology showed elevated IgG antibody to VCA, and EBV DNA was detected in the peripheral blood. Since he showed latency II without immunodeficiency disease, we diagnosed age-related EBV-associated B-cell lymphoproliferative disorder (EBV-LPD) as a result of aging and EBV infection. He was treated with 6 courses of the CHOP regimen plus rituximab, and achieved complete remission. EBV-DNA became undetectable at remission. In age-related EBV-LPD, there is some possibility that EBV-DNA in the peripheral blood is valuable as a tumor biomarker.

Hodgkin's reed-sternberg cell line (KM-H2) promotes a bidirectional differentiation of CD4+CD25+Foxp3+ T cells and CD4+ cytotoxic T lymphocytes from CD4+ naive T cells.

A recent report revealed that a large population of Hodgkin's lymphoma-infiltrating lymphocytes (HLILs) consisted of regulatory T cells. In this study, we cocultured CD4+ naive T cells with KM-H2, which was established as a Hodgkin's Reed-Sternberg cell line, to clarify their ability to induce CD25+ Forkhead box P3+ (Foxp3+) T cells. The characteristic analyses of T cells cocultured with KM-H2 revealed the presence of CD4+CD25+ T cells. They expressed CTLA-4, glucocorticoid-induced TNFR family-related gene, and Foxp3 and could produce large amounts of IL-10. Conversely, KM-H2 also generated CD4+ CTLs, which expressed Granzyme B and T cell intracellular antigen-1 in addition to Foxp3+ T cells. They exhibit a strong cytotoxic effect against the parental KM-H2. In conclusion, KM-H2 promotes a bidirectional differentiation of CD4+ naive T cells toward Foxp3+ T cells and CD4+ CTLs. In addition to KM-H2, several cell lines that exhibit the APC function were able to generate Foxp3+ T cells and CD4+ CTLs. Conversely, the APC nonfunctioning cell lines examined did not induce both types of cells. Our findings suggest that the APC function of tumor cells is essential for the differentiation of CD4+ naive T cells into CD25+Foxp3+ T cells and CD4+ CTLs and at least partly explains the predominance of CD25+Foxp3+ T cells in HLILs and their contribution to a better prognosis. Therefore, in APC-functioning tumors, including classical Hodgkin lymphomas, which generate Foxp3+ T cells and CD4+ CTLs, these T cell repertories play a beneficial role synergistically in disease stability.

Prostaglandin E2 is a negative regulator on human plasmacytoid dendritic cells.

Prostaglandin E2 (PGE2), a major lipid derived from the metabolism of arachidonic acid, is an environmentally bioactive substance produced by inflammatory processes and acts as a cAMP up-regulator that plays an important role in immune responses. It has been reported that PGE2 has the ability to inhibit the production of interleukin-12 by myeloid dendritic cells (MDCs) and macrophages, and then induce preferential T helper type 2 (Th2) cell responses. However, little is known of the function of PGE2 for plasmacytoid dendritic cells (PDCs), which may contribute to the innate and adaptive immune response to viral infection, allergy and autoimmune diseases. In the present study, we compared the biological effect of PGE2 on human PDCs and MDCs. PGE2 caused the death of PDCs but MDCs survived. Furthermore, we found that, whereas PGE2 inhibited interferon-alpha production by PDCs in response to virus or cytosine-phosphate-guanosine, it inhibited interleukin-12 production by MDCs in response to lipopolysaccharide (LPS) or poly(I:C). Although both virus-stimulated PDCs and LPS-stimulated MDCs preferentially induced the development of interferon-gamma-producing Th1 cells, pretreatment with PGE2 led both DC subsets to attenuate their Th1-inducing capacity. These findings suggest that PGE2 represents a negative regulator on not only MDCs but also PDCs.

Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis.

Engagement of the Fas receptor (CD95) initiates multiple signaling pathways that lead to apoptosis, such as the formation of death-inducing signaling complex (DISC), activation of caspase cascades, and the generation of the lipid messenger, ceramide. Sphingomyelin (SM) is a major component of lipid rafts, which are specialized structures that enhance the efficiency of membrane receptor signaling and are a main source of ceramide. However, the functions of SM in Fas-mediated apoptosis have yet to be clearly defined, as the responsible genes have not been identified. After cloning a gene responsible for SM synthesis, SMS1, we established SM synthase-defective WR19L cells transfected with the human Fas gene (WR/Fas-SM(-)), and cells that have been functionally restored by transfection with SMS1 (WR/Fas-SMS1). We show that expression of membrane SM enhances Fas-mediated apoptosis through increasing DISC formation, activation of caspases, efficient translocation of Fas into lipid rafts, and subsequent Fas clustering. Furthermore, WR/Fas-SMS1 cells, but not WR/Fas-SM(-) cells, showed a considerable increase in ceramide generation within lipid rafts upon Fas stimulation. These data suggest that a membrane SM is important for Fas clustering through aggregation of lipid rafts, leading to Fas-mediated apoptosis.

[Splenic marginal zone lymphoma associated with antiphospholipid antibodies].

A 61-year-old woman experienced a high fever with anemia and APTT prolongation after suffering a herpes zoster virus infection. Physical examination revealed a large splenomegaly without lymphadenopathy. Laboratory evaluations were positive for lupus anticoagulant (LA) and monoclonal IgM-kappa protein. LA was associated with the presence of anti-beta2GPI antibody, anti-cardiolipin antibody, and anti-prothrombin antibody. Moreover, the results of factors IX, XI, and XII assays and CRP and FDP-E were disturbed. A splenectomy was performed, and a splenic marginal zone lymphoma (SMZL) was diagnosed. All hematological findings rapidly recovered after the splenectomy. No thrombotic events occurred after the splenectomy even though thrombosis prophylaxis was not performed. The clinical course suggested that the SMZL-producing antibody induced immunological abnormalities in the labolatory tests. Since the patient suffered disease progression soon after the splenectomy, an autologous peripheral stem cell transplantation with rituximab administration was performed.

Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs.

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-alpha in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-gamma-producing Th cells depending on their capacity to residually produce IFN-alpha. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-alpha-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.

Membranous nephropathy associated with donor lymphocyte infusion following allogeneic bone marrow transplantation.

Nephrotic syndrome after hematopoietic stem cell transplantation (HSCT) followed by donor lymphocyte infusion (DLI) has never been described. We report the case of a myelodysplastic syndrome (MDS) patient who developed nephrotic syndrome with membranous nephropathy 18 months after allogeneic HSCT and 4 months after DLI. A 50-year-old woman with MDS underwent allogeneic bone marrow transplantation from her HLA-matched brother. MDS relapsed 55 days after transplantation, donor lymphocytes were infused as adoptive immunotherapy, and complete remission was achieved. Four months after the third DLI, the patient developed nephrotic syndrome with proteinuria up to 9 g/day. Renal biopsy revealed granular deposits of immunoglobulin G along the glomerular basement membrane, and subepithelial electron-dense deposits. A diagnosis of membranous nephropathy was made. For maintenance of the immunotherapeutic effect of DLI, minimum doses of immunosuppressive therapy for decreasing proteinuria were administered, and improvement of nephrotic syndrome and persistent complete remission of MDS were achieved.

[Unrelated HLA-mismatched cord blood transplantation using nonmyeloablative conditioning in an adult patient with very severe aplastic anemia].

An adult patient suffering from G-CSF-resistant very severe aplastic anemia received a cord blood transplantation from a three-loci HLA-mismatched unrelated donor after nonmyeloablative conditioning. Cord blood was infused after conditioning with fludarabine (180 mg/m2) and cyclophosphamide (100 mg/kg). Cyclosporin A and short-term methotrexate were used for prophylaxis against acute GVHD. Engraftment was achieved on day 23, and there was no serious GVHD. A full-donor type T-cell chimerism was obtained by day 30. Normal hematopoiesis and complete chimerism have been maintained 14 months after transplantation.