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Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte–macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.

The graft-versus-host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell-mediated attack on host lymphohaematopoietic populations, resulting in the reconstitution of the host with donor cells. We examined Fas-Fas ligand (FasL) interactions in donor and host haematopoietic cells over a prolonged period of parental-induced GVHD. Fas expression on bone marrow cells of both donor and host origin increased at 2 weeks. Host cell incubation with anti-Fas antibody induced apoptosis, and the number of haematopoietic progenitor cells decreased. Fas-induced apoptosis by the repopulating donor cells, however, did not increase until 12 weeks, when more than 90% of the cells were donor cells. The expression of various cytokines, such as interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and FasL gene expression in the bone marrow increased concomitantly. To examine directly whether FasL has a major role in the development of donor cell engraftment, FasL-deficient (gld) mice were used as donors. Injection of B6/gld spleen cells induced significantly less host lymphohaematopoietic depletion, resulting in a failure of donor cell engraftment. Furthermore, injection of IFN-gamma gene knockout (gko) B6 spleen cells failed to augment Fas and FasL expression in recipient mice, resulting in a failure of donor cell engraftment. This suggests that the induction of apoptosis by Fas-FasL interactions in host cells may contribute to a reconstitution of the host with donor cells and that donor-derived IFN-gamma plays a significant role for Fas-FasL interactions in host cells during parental-induced GVHD.

Patients with trilineage myelodysplasia (TMDS) in de novo acute myeloid leukaemia (AML) at diagnosis and remission were clinically evaluated between 1983 and 1996. AML with TMDS (AML/TMDS) was observed in 20 (12%) of 162 patients with de novo AML at diagnosis. Complete remission (CR) was achieved with combination chemotherapy in 12 (67%) of 18 AML/TMDS cases. This CR rate was relatively worse than the rate of 78% (106/136 cases) of AML without TMDS, but this difference was not significant. Disease-free survival curves also showed no difference between AML/TMDS and AML without TMDS. During remission, eight (67%) of 12 AML/TMDS cases had myelodysplastic remission marrow (AML/MRM). AML/MRM was also seen in seven (7%) of 106 AML cases without TMDS. The actuarial disease-free survival was significantly lower in AML/MRM than in AML without MRM (P = 0.0003). All of the AML/MRM cases exhibited early leukaemic relapse; median remission duration was only 9 months. Clonal changes occurred in two cases of AML/TMDS and five cases of AML/MRM at the time of relapse. These findings suggest that TMDS during remission predicts a poorer prognosis and early leukaemic relapse when compared with the absence of TMDS.

A 43-year-old man with chronic myeloid leukaemia underwent a second transplant with CD34 + bone marrow cells selected from his two-loci HLA-mismatched sibling after rejection of the first graft from an HLA-matched unrelated donor. By immunomagnetic positive selection, CD34 + marrow cells at 0.95 x 10 6 /kg with 97% purity and CD3 + T lymphocytes at 1.3 x 10 4 /kg were collected and transplanted. Engraftment was confirmed to be of CD34 + cell-donor origin. The patient developed only grade I acute graft-versus-host disease (GVHD) and no chronic GVHD to date. These observations suggest that allogeneic CD34 + bone marrow cells are capable of reconstituting haemopoiesis and that CD34 + selection could be applicable to T-cell depletion.

Fas/APO-1 (CD95) is a cell surface molecule that can transduce apoptotic signals into cells. We examined the expression of Fas antigen on CD4+ and CD8+ T cells of patients who received total body irradiation (TBI) as a preparative regimen for allogeneic bone marrow transplantation. Numbers of peripheral blood lymphocytes were significantly reduced after TBI. Cytofluorometric analysis revealed a significantly higher expression of Fas on CD4+ and CD8+ T cells after TBI. Serum soluble Fas concentrations were significantly elevated after TBI. Changes in the Fas system were therefore accompanied by TBI-induced lymphocytopenia, suggesting that Fas plays a role in irradiation-induced apoptosis in vivo.

It is known that the topoisomerase II inhibitors, MST-16 (sobuzoxane) and VP-16 (etoposide), are effective for the treatment of lymphoma. Five patients with refractory or relapsed non-Hodgkin’s lymphoma (NHL) were treated with a combination of oral MST-16 and VP-16 over a long period. Two patients had severely refractory NHL. The remaining 3 patients could not be treated with intensive chemotherapy because of severe organ dysfunction or a poor hematopoietic reserve. All 5 are alive and well after MST-16 and VP-16 treatment. MST-16 and VP-16 are effective for NHL when intensive chemotherapy is ineffective or contraindicated.

In order to detect chimerism, fluorescence in situ hybridization (FISH) and cytogenetic analyses were performed on bone marrow cells from 47 patients with hematological malignancies following allogeneic hematopoietic cell transplant (HCT). The dual-color XY, major Bcr-Abl (M-Bcr-Abl), and specific alpha-satellite probes were used for sex-mismatched HCT, chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS) cases with karyotypic abnormalities before HCT, respectively. Donor cells were found using FISH analysis in all 32 cases examined within 2 months following HCT, confirming engraftment. In six cases, however, cytogenetic analysis failed to detect donor cells due to lack of metaphases. Relapse occurred in four of the six cases in which mixed chimerism was detected using FISH analysis after 6 months of HCT. In contrast, after 12 months of HCT, no relapse was found in 24 patients without host cells. For two patients with mixed chimerism, gradual reduction of immunosuppressants or donor lymphocyte infusion resulted in the disappearance of host cells as analyzed using FISH analysis. In three extramedullary relapse cases, however, cytogenetic relapse preceded morphological and FISH relapse. These findings suggest that FISH analysis is more useful for detecting residual host cells after HCT, and the combination of FISH and cytogenetic analyses provide a more detailed evaluation for HCT patients. The results also indicate that monitoring of mixed chimerism using FISH analysis after 6 months of HCT is important for allowing the early detection of hematological relapse.

We evaluated the relationship between CMV and human herpes virus-6 (HHV-6) reactivation and the incidence of grades 2 to 4 acute GVHD post BMT. Bronchoalveolar lavage fluid (BALF) samples extracted from 54 BMT recipients on post-BMT day 35 were analyzed by PCR for detection of CMV DNA, HHV-6 DNA and CMV plus HHV-6 DNA. CMV DNA was detected in 26 patients and 13 (50%) developed grades 2 to 4 acute GVHD. Of the 28 who were CMV negative, only six (21.4%) developed grades 2 to 4 acute GVHD. HHV-6 was detected in 18 patients, and 11 (61.1%) developed grades 2 to 4 acute GVHD. Of the 36 who were HHV-6 negative, only eight (22.2%) developed grades 2 to 4 acute GVHD. CMV and HHV-6 were detected in 13 patients, and eight (61.5%) developed grades 2 to 4 acute GVHD. Of the 23 who were negative for both CMV and HHV-6, only three (13%) developed grades 2 to 4 acute GVHD. In all experiments, the difference between the groups was significant (P

The acute graft-versus-host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell-mediated attack on host lymphohematopoietic populations, resulting in the reconstitution of the host with donor hematopoietic stem cells. We examined the effect of GVHD on the donor and host hematopoiesis in parental-induced acute GVHD. The bone marrow was hypoplastic and the number of hematopoietic progenitor cells significantly decreased at 4 weeks after GVHD induction. However, extramedullary splenic hematopoiesis was present and the number of hematopoietic progenitor cells in the spleen significantly increased at this time. Fas expression on the host spleen cells and bone marrow cells significantly increased during weeks 2 to 8 of GVHD. Host cell incubation with anti-Fas Ab induced apoptosis, and the number of hematopoietic progenitor cells decreased during these weeks. A significant correlation between the augmented Fas expression on host bone marrow cells and the decreased number of host bone marrow cells by acute GVHD was observed. Furthermore, the injection of Fas ligand (FasL)-deficient B6/gld spleen cells failed to affect host bone marrow cells. Although Fas expression on repopulating donor cells also increased, Fas-induced apoptosis by the repopulating donor cells was not remarkable until 12 weeks, when more than 90% of the cells were donor cells. The number of hematopoietic progenitor cells in the bone marrow and the spleen by the repopulating donor cells, however, decreased over an extended time during acute GVHD. This suggests that Fas–FasL interactions may regulate suppression of host hematopoietic cells but not of donor hematopoietic cells. Hematopoietic dysfunctions caused by the reconstituted donor cells are independent to Fas–FasL interactions and persisted for a long time during parental-induced acute GVHD.

Although all-trans retinoic acid (ATRA) has been shown to improve the outcome of patients with acute promyelocytic leukemia (APL) compared with chemotherapy alone, various adverse effects have been reported. We report here the development of scrotal ulcer in four patients with APL during ATRA treatment. ATRA 45 mg/m 2 was administered orally to four patients with newly diagnosed APL, two of whom also received chemotherapy. Scrotal ulcers appeared in all four patients after a median of 22 days (range: 17-29 days) of ATRA treatment ATRA was discontinued in only one patient, who was then treated with methylprednisolone pulse therapy. The fever resolved, and scrotal ulcer improved after this treatment. The other three patients were treated with steroid or antibiotic ointment for scrotal ulcers. ATRA was re-administered 2 years later in one patient who had relapse of APL. Ulcers appeared again on the scrotal and lower abdominal skin on day 27 of ATRA treatment. These findings strongly suggest that scrotal ulcer is a potential adverse effect of ATRA.

A female patient with ALL received a bone marrow transplant (BMT) from an unrelated donor with a one locus HLA mismatch. The donor was heterozygous at the HLA-A locus, while the patient was homozygous at this locus. The patient had cytomegalovirus (CMV) antigenemia on day 42 following an intensive preparative regimen that included anti-thymocyte globulin and BU+CY+TBI to prevent graft rejection. Ganciclovir was given initially for the treatment of CMV antigenemia, although the patient soon developed severe myelosuppression. The patient's hematopoietic recovery was poor, and CMV and human herpesvirus-6 (HHV-6) were detectable in the peripheral blood. Severe CMV retinitis was treated with foscarnet and the intraocular injection of ganciclovir. The CMV retinitis improved and the marrow gradually recovered. CMV and HHV-6 were no longer detectable in the peripheral blood. Foscarnet and intraocular injection of ganciclovir appeared to be an effective treatment for CMV retinitis in this myelosuppressed BMT patient.

A female patient received a renal transplantation from an unrelated cadaver donor, and five years later developed myelodysplastic syndrome (MDS) with immunological abnormalities. Chromosomal analysis showed trisomy 8 in bone marrow cells. FISH analysis indicated that trisomy 8 was present in 4% of CD19 + cells, suggesting that the MDS clone involved B lymphocytes. Polymerase chain reaction of the human androgen receptor gene indicated B cell clonality (54%) and granulocyte clonality (63%). There have been no previous reports of secondary MDS following renal transplantation in which the MDS clone involved the B cell lineage. The abnormal MDS B cell clone may have caused the immunological abnormalities.

Recombinant human erythropoietin (rHu EPO) has been reported to accelerate early erythroid reconstitution after bone marrow transplantation (BMT). We conducted a pilot study on rHu EPO for late-onset anemia in 9 patients after allogeneic BMT. The patients achieved initial erythropoietic recovery (hemoglobin (Hb) range 9.1-13.4, mean 10.8 g/dl), but then developed transplant-related anemia (Hb range 6.3-9.7, mean 8.2 g/dl) more than 50 days after BMT. This type of anemia was related to graft-versus-host disease (GVHD), cytomegalovirus infection, and/or impaired EPO secretion. The patients received 3,000 or 12,000 U of rHu EPO subcutaneously three or seven times weekly. Hb levels increased by more than 2 g/dl in 6 of the 9 patients, but were unchanged in the 3 patients with severe GVHD. These findings suggest that in some cases rHu EPO is effective for the treatment of late-onset anemia after BMT.

Immunological abnormalities (IA) are frequently observed in patients with myelodysplastic syndromes (MDS). Although there have been a number of analyses of the prognostic factors, there have been few studies, if any, to determine whether IA affects prognosis. We investigated the prognosis of 153 MDS patients with or without IA who were treated at a single Japanese institute for 10 years. Nineteen of 153 patients (12%) developed autoimmune disorders. One hundred of 153 patients (63%) had an abnormality in at least one immunological laboratory test. Hypergammaglobulinemia was found in 50 of 128 (39%) patients tested, hypogammaglobulinemia was observed in 10 of 128 (8%), positivities of antinuclear antibody, RA factor. DNA antibody, and direct antiglobulin test were observed in 30%, 14%, 7% and 12%, respectively, and a CD4/CD8 ratio 1 was also significantly superior to the survival of those with an inverted CD4/CD8 ratio. Patients with IA tended to die of infection or leukemic progression in comparison with those without IA, suggesting that IA may be associated with susceptibility to opportunistic infection and disease progression.

Three patients with severe aplastic anemia, acute promyelocytic leukemia (M3) and chronic myelogenous leukemia, developed myelodysplastic changes with trilineage morphological abnormalities in a few months following allogenic bone marrow transplantation (BMT). Morphologically dysplastic changes associated with moderate-severe anemia, leukopenia and/or thrombocytopenia appeared on day 40, day 62 and day 68 after BMT. A ferrokinetics study clearly showed ineffective erythropoiesis in one patient. Hematopoietic cells were shown to be of donor-origin in all three cases. The levels of vitamin B12 and folic acid were normal. Laboratory tests showed no signs of hemolysis or fragmentation of red blood cells. Although the cause of aberrant hematopoiesis compatible with MDS within 100 days post-BMT remains to be determined, cytomegalovirus infection, ganciclovir and/or graft-versus host disease (GVHD) might be associated with this myelodysplasia following allogeneic BMT.