Increased lipid peroxidation of erythrocytes in blood stored in polyvinyl chloride blood storage bags plasticized with di-[2-ethyl hexyl] phthalate and effect of antioxidants

We report two cases of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPD) after allogenic bone marrow transplantation which were conditioned with regimens including antithymocyte globulin (ATG). The first case was a 31 year-old man which severe aplastic anemia who was transplanted from HLA-matched unrelated donor conditioned with total lymphoid irradiation (TLI)/ cyclophosphamide/ATG and prophylactic administration of ganciclovir Grade I acute GVHD improved in response to cyclosporine (CsA). LPD as a polyclonal epipharyngeal mass developed at day +53 and spontaneously regressed along with the withdrawal of CsA. Second case was a 11 year-old boy with acute myelomonocytic leukemia (FAB:M4E). He was transplanted from HLA B locus mismatched mother conditioned with total body irradiation (TBI)/busulfan/L-PAM/ATG. He showed grade IV acute GVHD, which was controlled by steroids and FK-506. LPD as a monoclonal intestinal lymphoma was diagnosed at day +82, and he was unsuccessfully treated with ganciclovir, acyclovir, chemotherapy and transfusions of EBV-specific cytotoxic lymphocytes in addition to discontinuation of immunosuppressants, and died at day +18 due to sepsis and multiple cerebral infarction. Early detection and introduction of appropriate treatment for post bone marrow transplantation LPD is necessary.     

Impaired liver function tests in patients treated with antithymocyte globulin: implication for liver transplantation

Antithymocyte globulin (ATG) is traditionally used as a conventional immunosuppression agent in various pathological states including severe aplastic anaemia (SAA), graft versus host disease (GVHD), and for the prevention and treatment of graft rejection and GVHD post bone marrow and liver transplantation. We reviewed the liver functions of 16 haematological patients with no previous liver disorders who received ATG as part of their pre-bone marrow transplantation (BMT) conditioning regimen, and the liver function tests of five SAA patients who received ATG as part of their treatment. Liver functions were evaluated at day -1 pre-, and days +3 and +10 post-ATG treatment. All patients had normal liver functions before treatment. In the haematological patients, the mean serum lactic dehydrogenase (LDH) levels increased from 408.7 +/- 37.7 U/l pre-treatment to 1394.4 +/- 488.7 U/l 3 days post-treatment (n = 16; p < 0.029), and then declined to 561.4 +/- 61.3 U/l 10 days post-treatment (n = 16; p < 0.043). The mean alanine aminotransferase (ALT) levels increased from 51.9 +/- 11.3 U to 184.6 +/- 74.6 U (n = 16; p < 0.036), and then declined to 121.9 +/- 61.3 U (n = 16; NS). The mean aspartate amino transferase (AST) levels increased from 31.2 + 5.7 U to 152.0 +/- 67.0 U (n = 16; p < 0.44) and then declined to 46.0 +/- 14 (n = 16; p < 0.049). The mean tau-glutamyltransferase (GTP) levels increased from 93.0 +/- 34 to 188.0 +/- 36 (n = 16; p < 0.02), and were 168.0 +/- 37.0 at day +10 (n = 16; NS). The mean bilirubin levels increased from 18.0 +/- 1.9 microM l(-1) to 22.7 +/- 2.8 (n = 16); NS), at day +3 and to 31.9 +/- 6.9 at day +10 (n = 16; NS). In contrast, no significant changes in liver function tests were demonstrated in the SAA patients treated with ATG. The possible pathophysiologic mechanisms and the clinical implications for liver transplantation are discussed.     

Leukoencephalopathy probably caused by tacrolimus hydrate after stem cell transplantation in a girl with MDS 7 monosomy

Leukoencephalopathy probably caused by tacrolimus hydrate after stem cell transplantation in a girl with MDS 7 monosomy is reported. The conditioning regimen consisted of thiotepa (150 mg/m2 x 4), melphalan (70 mg/m2 x 2) and 12 Gy total body irradiation. She received peripheral blood CD34 positive cells (4.17 x 10(6)/kg) from her HLA-mismatched father and tacrolimus hydrate was used for GVHD prophylaxis. Engraftment was rapid and grade 1 acute GVHD of the skin responded well to pulse therapy. From day 27 she became irritable and sleepless, and right facial convulsions developed on day 37. No abnormality was found in the cerebrospinal fluid. Cranial CT findings showed no abnormalities except for low density lesions around the bilateral ventricle. Leukoencephalopathy was suspected and tacrolimus hydrate was discontinued. Thereafter psychosomatic symptoms improved, temporarily however, similar symptoms again developed following cyclosporine administration. Therefore we had to halt the administration of both tacrolimus and cyclosporine. She died on day 104 because of GVHD and fungal infection without recovering from leukoencephalopathy.     

Comparison of supercritical fluid extraction and conventional liquid-solid extraction for the determination of benzo[a]pyrene in water-soluble smoke

In patients undergoing bone marrow transplantation cryptococcosis is rarely encountered. We report a fatal case of Cryptococcus meningitis in a 12-year-old girl with acute lymphoblastic leukemia (ALL) in second remission who had a transplant from a human leukocyte antigen (HLA)-identical unrelated bone marrow donor. The conditioning regimen was thiotepa, cyclophosphamide, and total body irradiation (TBI); graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A, methotrexate, and antilymphocyte globulin (ALG). The patient experienced stage III GVHD responsive to high-dose corticosteroids. On day +54 a thrombotic microangiopathy occurred. On day +64 neurological status worsened; a brain computed tomographic (CT) scan showed hyperdense lesions suggesting fungal infection. Detection of cryptococcal antigen by latex agglutination was positive but India ink stain and culture were negative. Despite treatment with amphotericin B, 5-flucytosine, and granulocyte-macrophage colony-stimulating factor, the patient died 13 days after the diagnosis.     

A case of prostatic ductal adenocarcinoma with endometrioid features, associated with rectal invasion and multiple liver metastases

A 66-year-old man was admitted with destructive arthropathy, and calcium pyrophosphate dihydrate was demonstrated in the synovial fluid specimen. He was found to have a hyponatremia. The serum sodium concentration was 121 mmol/l, plasma arginine vasopressin (AVP) 6.6 pmol/l, and serum interleukin (IL)-6 96 pg/l. The clinical findings suggest the diagnosis of syndrome of inappropriate secretion of antidiuretic hormone (SIADH). However, destructive arthropathy with increased values of C-reactive protein and IL-6 is the only background of SIADH in this patient. We suggest the possibility that IL-6 produced at inflammatory lesions may have stimulated an excessive release of AVP resulting in the hyponatremia and hypochloremia of SIADH.     

Alprazolam as an alternative to low-dose haloperidol in older, cognitively impaired nursing facility patients

BACKGROUND: Using unrelated bone marrow, there is an increased risk of graft-versus-host disease (GVHD). METHODS: HLA-A-, HLA-B-, and HLA-DR-compatible unrelated bone marrow was given to 132 patients. The diagnoses included chronic myeloid leukemia (n=43), acute lymphoblastic leukemia (n=29), acute myeloid leukemia (n=27), myelodysplastic syndrome (n=4), lymphoma (n=3), myeloma (n=1), myelofibrosis (n=1), severe aplastic anemia (n=12), and metabolic disorders (n=12). The median age was 25 years (range 1-55 years). HLA class I was typed serologically, and class II was typed by polymerase chain reaction using sequence-specific primer pairs. Immunosuppression consisted of antithymocyte globulin or OKT3 for 5 days before transplantation and methotrexate combined with cyclosporine. RESULTS: Engraftment was seen in 127 of 132 patients (96%). Bacteremia occurred in 47%, cytomegalovirus (CMV) infection in 49%, and CMV disease in 8%. The cumulative incidences of acute GVHD > or = grade II and of chronic GVHD were 23% and 50%, respectively. The 5-year transplant-related mortality rate was 39%. The overall 5-year patient survival rate was 49%; in patients with metabolic disorders and severe aplastic anemia, it was 61% and 48%, respectively. The disease-free survival rate was 47% in patients with hematological malignancies in first remission or first chronic phase and 38% in patients with more advanced disease (P=0.04). Acute GVHD was associated with early engraftment of white blood count (P=0.02). Poor outcome in multivariate analysis was associated with acute myeloid leukemia (P=0.01) and CMV disease (P=0.04). CONCLUSION: Using HLA-A-, HLA-B-, and HLA-DR-compatible unrelated bone marrow and immunosuppression with antithymocyte globulin, methotrexate, and cyclosporine, the probability of GVHD was low and survival was favorable.     

Inhibition of calcineurin phosphatase activity in adult bone marrow transplant patients treated with cyclosporine A

In vitro studies have demonstrated that cyclosporine A (CsA) acts by inhibiting the phosphatase activity of calcineurin, an important mediator of T-cell activation. The relationship of CsA administration in vivo, calcineurin activity, and graft-versus-host disease (GVHD) has yet to be studied. The calcineurin activities of mononuclear cells isolated from 62 bone marrow transplant recipients and 12 normal volunteers were determined and analyzed with respect to administration of CsA, presence or absence of CsA in plasma, and presence or absence of GVHD. Of 62 patients, 33 were taking CsA and 29 were not. Early posttransplant (< 100 days), the calcineurin activity of patients on CsA was significantly lower than that of patients not on CsA (P = .0004) and than that of normal volunteers (P < .0001). Similarly, late posttransplant (> 100 days), the calcineurin activity of patients taking CsA was inhibited compared with normal volunteers (P < .05). The calcineurin activity of patients with acute GVHD who were taking CsA was lower than that of patients on CsA without acute GVHD matched for time posttransplant (P = .02). Calcineurin activity in patients on CsA with chronic GVHD was similar to those without chronic GVHD on drug. In conclusion, calcineurin activity is significantly suppressed by in vivo administration of CsA. The lower calcineurin activity of patients on CsA with acute GVHD suggests that CsA-resistant GVHD is not the result of inadequate suppression of calcineurin activity. These data suggest that if inhibition of calcineurin is the only physiologic target of CsA administration, simply increasing doses of CsA or treatment with other inhibitors of calcineurin, such as FK506, would not be expected to ameliorate GVHD.     

Murine graft-versus-host disease in an F1-hybrid model using IFN-gamma gene knockout donors

These experiments were performed to determine whether the absence of donor-derived IFN-gamma would influence the outcome of acute graft-vs-host disease (GVHD). Graft-vs-host reactions were induced in B6D2F1 hybrids using grafts from either IFN-gamma gene knockout (gko) or wild-type, C57BL/6J, parental strain donors. GVHD was equally lethal in both groups, but IFN-gamma gko graft recipients developed a more protracted form of the disease. These mice developed early wasting that persisted until death. IFN-gamma was present in spleen cell cultures from wild-type graft recipients, but was absent in cultures from IFN-gamma gko graft recipients. Both recipient groups showed macrophage priming for LPS-induced TNF-alpha release. Engraftment of donor-derived CD4+ and CD8+ cells was greater in IFN-gamma gko graft recipients. Pathologic changes in IFN-gamma gko graft recipients were different from those typically seen in acute GVHD. The syndrome developing in IFN-gamma gko recipients consisted of patchy alopecia, corneal dryness and clouding, and lymphocytic infiltration of the liver, pancreas, salivary gland, lung, and kidney. Lymphocytic infiltrates were also present in the epidermis and the epithelium of both bile and salivary gland ducts. Some of the lesions closely resembled those seen in the "sicca"/Sjogren's-like syndrome associated with chronic GVHD; however, there was no evidence of immune complex deposition in the kidney. These results indicate that GVHD in IFN-gamma gko graft recipients shares many features with acute GVHD, but both the duration of the disease and its pathologic manifestations are different. Our results suggest that IFN-gamma plays a significant role in the pathogenesis of acute GVHD by increasing the rate at which mortality develops.     

Research issues in cancer survivorship: report of a workshop sponsored by the Office of Cancer Survivorship, National Cancer Institute

We present the results of a prospective, randomised study comparing PBPC and BM focusing on engraftment, acute and chronic GVHD and survival. Forty patients with haematological malignancies received HLA-identical sibling BM (group A) or PBPC (group B). Evaluable patients were 19 (A) and 18 (B). Median age was 35 (17-56) in A and 29.5 (9-51) in B. Conditioning was mainly Bu-Cy2; GVHD prophylaxis was CSA-MTX. PBPC were harvested after 5 days of G-CSF 10 microg/kg/day. Median days for an ANC >0.5 x 10(9)/l was 18 (13-30) in A and 16 (11-25) in B (P = 0.10). Platelets >20 x 10(9)/l occurred at +17 (10-40) in A and +12 (9-36) in B (P = 0.01). The probability of > or =2 grade a-GVHD was 19% (A) and 27% (B) (P = 0.53). The probability of all grade c-GVHD was 70% with BM. In spite of the small number of patients in group B (PBPC), our data suggest the great majority of them will have c-GVHD (P = 0.08); extensive disease was present in 50 and 100%, respectively (P = 0.05). The estimates of overall survival for A and B at 1000 days are 51 and 47%, respectively (P = 0.67); DFS at 1000 days are 52 and 58%, respectively (P = 0.50). PBPC resulted in faster platelet engraftment. The incidence of acute and chronic GVHD was similar in both groups, but the severity of c-GVHD was higher with PBPC. No differences in survival and DFS have been observed to date.     

A phase II study of BTI-322, a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graft-versus-host disease

BTI-322, a rat monoclonal IgG2b directed against the CD2 antigen on T cells and natural killer (NK) cells, blocks primary and memory alloantigen proliferative responses in vitro. We have evaluated the pharmacokinetics and safety of BTI-322 during treatment of 20 transplant recipients with steroid-refractory acute graft-versus-host disease (GVHD). Treatment consisted of BTI-322 by intravenous (IV) bolus or 30-minute infusion at approximately 0.1 mg/kg/d for 10 days in addition to continuing high-dose steroids and tacrolimus or cyclosporine. Pharmacokinetic sampling was performed in 10 patients; the t1/2 +/- SE was 9.1 +/- 1.3 hours, the Cmax was 2,549 +/- 291 ng/mL, the Vd was 3.97 +/- 0.95 L, and the Vd/kg was 0. 05 +/- 0.01 L/kg. Ten patients experienced transient dyspnea sometimes accompanied by nausea, vomiting, diarrhea, and tachycardia shortly after the initial bolus dose of drug, but serious drug-related adverse events were not seen during the remainder of the infusions. At the end of treatment (day 11), there were six patients with complete responses and five with a reduction in grade of GVHD for a total response rate of 55% (95% confidence interval [CI], 32% to 77%). Antibodies targeting CD2 may be active in the treatment of acute GVHD, and evaluation of a humanized form of BTI-322 is warranted.     

Unusual case of disseminated sarcoidosis with prominent gastrointestinal symptoms

HISTORY AND ADMISSION FINDINGS: A 63-year-old man had for 10 months suffered from marked weight loss, night sweats, diffuse abdominal pain and increased stool frequency. He was admitted to evaluate an ultrasonically abnormal focus in the liver parenchyma and elevated liver function parameters. His sclerae were obviously icteric and he looked under-weight. INVESTIGATIONS: He had a hypochromic microcytic anemia and abnormal liver and pancreatic function tests: total bilirubin 3.11 mg/dl, direct bilirubin 2.21 mg/dl, GOT21U/l, gamma-GT 422 U/l, alkaline phosphatase 1449 U/l, alpha-amylase 481 U/l, lipase 2827 U/l. The serum creatinine level was elevated to 1.47 mg/dl. Computed tomography revealed enlarged liver and spleen as well as an enlargement of intraabdominal lymph nodes, chest radiogram and endoscopic cholangio-pancreatography were unremarkable. Biopsies from the lower duodenum, large intestine, bone marrow and liver showed inflammatory changes with Langhans-type mononuclear granulomas. Together with these findings an increased activity of the angiotensin-converting-enzyme (ACE) indicated sarcoidosis, other causes having been excluded. TREATMENT AND COURSE: All signs and symptoms rapidly improved under prednisolone, and 4 weeks after begin of treatment the biochemical abnormalities had clearly regressed. The raised serum levels of soluble IL-2 receptors and of neopterin, measures of sarcoidosis activity, had decreased. Activity of ACE had fallen. CONCLUSION: Sarcoidosis can present with diverse clinical signs and symptoms. In a case of multi-system disease that cannot be readily classified, sarcoidosis should be included in the differential diagnosis.     

Serum intact parathyroid hormone and ionised calcium concentration in children with renal insufficiency

We report our experience of the use of an immunoradiometric assay for intact parathyroid hormone (i-PTH) and the measurement of plasma ionised calcium concentration (PCa2+) in 73 children with chronic renal insufficiency (CRI); plasma creatinine concentration (PCr) 52-856 mumol/l. There was a poor correlation between i-PTH and PCr (r = 0.10, n = 552) compared with that for C-terminal PTH and PCr (r = 0.60, n = 248), suggesting that the i-PTH assay is independent of renal function in this group of treated children. A clear response of i-PTH to a low total plasma Ca (tPCa) and PCa2+ was observed. There was a significant positive correlation between both tPCa and PCa2+ (r = 0.50, n = 389) and the fraction of Ca2+ (the fraction of tCa which was ionised) and PCa2+ (r = 0.50, n = 389). The finding of a low or normal PCa2+ with a low calculated fraction of Ca2+ was frequently observed, i.e. the measured tPCa was unexpectedly high, suggesting complexing of Ca2+ by accumulated anions in CRI. There was a poor relationship between the plasma albumin concentration and both bound plus complexed Ca (tPCa minus PCa2+) and the fraction of Ca2+ (r = 0.15 and -0.17, respectively). The positive predictive value for a raised i-PTH of a tubular reabsorption of phosphate of less than 80% was 0.87, and of an alkaline phosphatase greater than 800 U/l was 0.37.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linkage map integration

Cyclosporin A (CsA) has been shown to be useful in the prophylaxis of acute graft-versus-host-disease (GVHD). However, this immunosuppressive agent produces multiple side-effects including nephrotoxicity, hypertension, hypertricosis, gum hyperplasia, infections, and neurotoxicity. We report a retrospective analysis of neurotoxicity in 625 recipients transplanted for thalassemia and given CsA as part of GVHD prophylaxis. Neurotoxicity consisted in mental status changes, tremor, headache (grade 1), visual disturbance and cortical blindness (grade 2) and seizures and coma (grade 3). The overall toxicity was 28.8% and the incidence of convulsions was 10.1%. Neurological findings were reversible after temporary reduction or discontinuation of CsA. Class 3 patients, when prepared with protocol 6 (Bu 14 + Cy 200 and CsA for GVHD) or when they developed acute GVHD, had the highest risk of convulsions. Age, sex, different conditioning regimens, different anticonvulsive prophylaxis, liver damage due to iron-overload and/or to chronic inflammation did not influence the occurrence of CsA-related CNS toxicity. The occurrence of acute GVHD with concomitant use of high-dose corticosteroids is the single significant predisposing factor in the occurrence of convulsions. Grades 1 and 2 of neurotoxicity occurred earlier and were not influenced even by acute GVHD.     

Immune suppression by lysosomotropic amines and cyclosporine on T-cell responses to minor and major histocompatibility antigens: does synergy exist?

BACKGROUND: Using murine models, we have shown that the lysosomotropic amine, chloroquine, is effective in the prevention of graft-versus-host disease (GVHD) mediated by donor T cells reactive with recipient minor histocompatibility antigens (MiHCs). Because lysosomotropic amines can suppress major histocompatibility complex (MHC) class II antigen presentation, their mechanism of action is potentially different from current immune suppressant drugs used to control GVHD such as cyclosporine. METHODS: We investigated the use of cyclosporine and the lysosomotropic amines chloroquine and hydroxychloroquine in combination for additive or synergistic immunosuppression on T-cell responses in vitro to MiHC and MHC in mice. RESULTS: We found that similar concentrations of chloroquine and hydroxychloroquine suppress the T-cell response to MiHC in mice (C57BL/6 anti-BALB.B) and that lysosomotropic amines in combination with cyclosporine result in synergistic suppression of a proliferative response to MiHC. Similar suppression and synergy appear to be present in an alloreactive response (C57BL/6 anti-BALB/c). Direct inhibition by chloroquine of T-cell proliferative responses induced by anti-CD3epsilon in the absence of antigen-presenting cells is present at higher concentrations than that required to suppress responses to MiHC or MHC. Chloroquine appears to induce decreased T-cell viability at high concentrations. This effect does not appear to be due to decreased T-cell production of interleukin-2 or interferon-gamma. At lower concentrations (<25 microg/ml), chloroquine can also decrease the ability of antigen-presenting cells to stimulate an a C57BL/6 anti-BALB/c T-cell response and can inhibit MHC class II expression after activation with lipopolysaccharide. CONCLUSIONS: Lysosomotropic amines in combination with cyclosporine appear to be synergistic in the suppression of T-cell proliferation to MiHC and MHC. Use of chloroquine in combination with cyclosporine may result in improved control of GVHD.     

Rapid engraftment without significant graft-versus-host disease after allogeneic transplantation of CD34+ selected cells from peripheral blood

We have prospectively evaluated the feasibility and results of the biotin-avidin immunoadsorption method (Ceprate SC system) for a phase I/II study of T-cell depletion of granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cells (PBPC) for allogeneic transplantation. Twenty consecutive patients, median age, 40 years (21 to 54) and diagnoses of chronic myeloid leukemia in chronic phase (n = 5), acute myeloblastic leukemia (n = 7), acute lymphoblastic leukemia (n = 2), chronic myelomonocytic leukemia (n = 1), refractory anemia with excess of blasts in transformation (n = 3), histiocytosis X (n = 1), and chronic lymphocytic leukemia (n = 1), were conditioned with cyclophosphamide (120 mg/kg) and total body irradiation (13 Gy; 4 fractions). HLA identical sibling donors received G-CSF at 10 microg/kg/d subcutaneously (SC); on days 5 and 6 (19 cases) and days 5 to 8 (1 case) donors underwent 10 L leukapheresis. PBPC were purified by positive selection of CD34+ cells using immunoadsorption biotin-avidin method (Ceprate SC) and were infused in the patients as the sole source of progenitor cells. No growth factors were administered posttransplant. The median recovery of CD34+ cells after the procedure was of 65%. The median number of CD34+ cells infused in the patients was 2.9 (range, 1.5 to 8.6) x 10(6)/kg. The median number of CD3+ cells administered was 0.42 x 10(6)/kg (range, 0.1 to 2). All patients engrafted. Neutrophil counts >500 and >1,000/microL were achieved at a median of 14 days (range, 10 to 18) and 15 days (range, 11 to 27), respectively. Likewise, platelet counts >20,000 and >50,000/microL were observed at a median of 10 days (range, 6 to 23) and 17 days (range, 12 to 130), respectively. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine plus methylprednisolone. No patient developed either grade II to IV acute or extensive chronic GVHD. After a median follow-up of 7.5 months (range, 2 to 22) three patients have relapsed, and one of them is again in hematologic and cytogenetic remission after infusion of the donor lymphocytes. Two patients died in remission: one on day +109 of pulmonary aspergillosis and the other on day +251 of metastasic relapse of a previous breast cancer. Sixteen of the 20 patients are alive in remission after a median follow-up of 7.5 months (range, 2 to 22). In conclusion, despite the small number of patients and limited follow-up, it appears that this method allows a high CD34+ cell recovery from G-CSF mobilized PBPC and is associated with rapid engraftment without significant GVHD, and with low transplant related mortality.     

IL-2 reduces graft-versus-host disease and preserves a graft-versus-leukemia effect by selectively inhibiting CD4+ T cell activity

We have recently demonstrated, in a fully MHC-mismatched murine bone marrow transplantation model, that administration of a short course of high dose IL-2 markedly diminishes graft-vs-host disease (GVHD) without compromising alloengraftment or the graft-vs-leukemia (GVL) effect of allogeneic T cells. We have now evaluated the mechanism of the dissociation of GVL and GVHD observed in this model. We demonstrate that CD4+ T cells were required to produce severe, acute GVHD in the fully MHC-mismatched plus minor histocompatibility Ag-mismatched A/J-->B10 strain combination. The GVHD-producing activity of A/J CD4+ T cells administered without CD8+ T cells was inhibited by IL-2 treatment. In contrast, CD8+ T cells alone mediated the GVL effect observed in the EL4 leukemia/lymphoma model, and CD4+ cells did not contribute to this effect. This CD8-mediated GVL activity was not inhibited by IL-2 treatment. Because naive A/J CD8+ T cells administered without CD4+ T cells did not produce acute GVHD, we were unable to evaluate the effect of IL-2 in this model. However, when A/J donors were presensitized with B10 skin grafts, CD4-depleted A/J spleen cells were capable of causing acute GVHD in B10 recipients. This CD8-mediated GVHD was not inhibited by treatment with IL-2. However, IL-2 did partially inhibit the GVHD produced by nondepleted presensitized A/J spleen cells, probably due to selective inhibition of the function of presensitized A/J CD4+ T cells. The dissociation of GVHD and GVL against the EL4 leukemia/lymphoma in IL-2-treated mice can therefore be explained by selective inhibition by IL-2 of CD4 activity.     

Busulfan/cyclophosphamide in volunteer unrelated donor (VUD) BMT: excellent feasibility and low incidence of treatment-related toxicity

An increasing number of volunteer unrelated donor bone marrow transplantations (VUD-BMT) are performed every year for hematological malignancies due to the availability of a large donor pool. Here we show the results of 36 VUD transplants from our institution using a chemotherapy-only conditioning regimen comprising busulfan 4 x 4 mg/kg and cyclophosphamide 2 x 60 mg/kg. All patients received heparin 200 IU/kg bw continuous i.v. infusion starting the day before conditioning until day +30. Thirty-four of 36 patients (94%) engrafted and no secondary graft failure was observed. The two non-engraftments occurred in patients with CML in blast crisis with extensive myelofibrosis. All 34 engrafted patients (100%) were in complete remission on day +30 as shown by bone marrow biopsy and cytogenetic examinations. No life-threatening treatment-related morbidity or mortality (TRM) were observed, in particular, no severe veno-occlusive disease (VOD) of the liver and no fatal pulmonary complication. Use of G-CSF significantly shortened the time of neutropenia by 5 days. GVHD prophylaxis consisted of CsA/methylprednisolone with or without MTX. Acute GVHD grade II-IV was observed in 18/34 patients (53%) and cGVHD in 12/27 patients (45%), who survived to day +100. In seven patients (four with HLA class I or II mismatch) anti-T-lymphocyte globulin (ATG) was added for acute GVHD prophylaxis. One of seven had aGVHD grade II and none developed grade III to IV GVHD or graft failure. We conclude that Bu/CY is a feasible, save and sufficiently immunosuppressive regimen for VUD transplantation. Severe acute GVHD might be avoided by additional use of ATG in GVHD prophylaxis.     

Digoxin in heart failure: results of the recent Digoxin Investigation Group trial in the context of other treatments for heart failure

BACKGROUND: This case report is the first documentation of the occurrence and potential source of lethal graft-versus-host disease (GVHD) after simultaneous kidney-pancreas transplantation. The patient was a 27-year-old African-American male who received an ABO-compatible, five HLA antigen-mismatched kidney-pancreas transplant from a 17-year-old African-American female donor, who died after childbirth. METHODS: Preoperative crossmatches using lymphocytotoxicity and flow cytometry were negative. The patient received four blood transfusions within 10 days of transplantation. Immunosuppression consisted of OKT3 induction, and then cyclosporine, azathioprine, and corticosteroids. RESULTS: On postoperative day (POD) 9, the patient became febrile, and leukocytopenia and pancytopenia developed. Immunosuppression was reduced and granulocyte colony-stimulating factor was begun. Cultures were negative, interleukin 6 and interleukin 8 levels were elevated, and a cutaneous rash appeared on POD 18. A skin biopsy demonstrated dermatitis with focal epidermal necrosis consistent with GVHD. In an attempt to identify the source of GVHD, variable-number tandem repeat analysis fingerprinting was performed with DNA from donor splenocytes, from the skin biopsy, as well as from the patient's buccal mucosa. The skin biopsy showed a mixed variable-number tandem repeat analysis type containing DNA fragments matching the recipient and donor. Blood donors were excluded as a source because they were serologically different from the organ donor. The patient developed liver abnormalities and died from multiorgan failure on POD 22. CONCLUSIONS: We speculate that carryover of passenger donor lymphocytes within the transplanted organ were responsible for GVHD. Furthermore, donor traits such as sexual mismatching, African-American race, and alloimmune status may be important potential risk factors for GVHD.     

Allogeneic peripheral blood stem cell transplantation in 30 patients with hematologic disorders

Thirty patients (median age of 32 years; range, 6-61) with hematologic disorders received unmanipulated peripheral blood stem cell transplants from HLA-matched or one-antigen-mismatched related donors following myeloablative therapy for acute lymphoblastic leukemia (7), acute myelogenous leukemia (6), chronic myelogenous leukemia (8), myelodysplastic syndrome (3), or other disorders (6). Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells were collected from donors in 1 to 3 aphereses. The apheresis products contained mean counts of 11.3 x 10(8) (range, 3.8-17.2) nucleated cells/kg and 6.7 x 10(6) (range, 1.3-16.7) CD34+ cells/kg. Graft-versus-host-disease (GVHD) prophylaxis consisted of cyclosporin A plus methotrexate, or FK506 plus methotrexate. All patients received G-CSF following their transplant. Although 1 patient died of pneumonia 6 days after transplantation, the others demonstrated rapid engraftment. Median days to recovery to 500/microliter neutrophils and 20,000/microliter platelets were 13 (range, 8-21) and 14 (range, 1-23) days, respectively. The incidence of acute GVHD grade II-IV was 33%; chronic GVHD developed in 57% of the assessable patients. There were no episodes of graft failure or rejection. Nineteen patients (63%) were alive and in complete remission from 147 to 839 days following their transplant (median follow-up of 560 days). Further follow-up study will be required to assess the incidence of chronic GVHD and graft-versus-leukemia (GVL) effects.     

Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graft-versus-host disease

Allogeneic CD8+ T cells mediate both a graft-vs-leukemia (GVL) effect and graft-vs-host disease (GVHD). To evaluate whether CD8 cells of defined cytokine phenotype differentially mediate these processes, alloreactive donor CD8+ T cells preferentially secreting type I or type II cytokines were generated by alloantigenic priming in vitro in the presence of IL-12 or IL-4, respectively. Both cytokine-secreting subsets lysed allogeneic tumor targets in vitro ("Tc1" and "Tc2" subsets). A transplantation model was established (B6 into B6C3F1, 1050 cGy host irradiation) using the 32Dp210 myeloid line (bcr/abl transfected, H-2k; 1 x 10(4) tumor cells/recipient). Compared with leukemia controls (death at 12.9 days post-bone marrow transplantation), both Tc1 and Tc2 recipients were conferred a survival advantage. At cell doses of 2 to 2.5 x 10(7), the Tc1-mediated GVL effect (mean survival of 34.2 days) was more potent than the Tc2-mediated GVL effect (mean survival of 20.5 days; Tc1 > Tc2, p = 0.009). On day 15, histologic examination showed that Tc1 recipients had undetectable tumor burdens, whereas Tc2 recipients had extensive leukemic infiltrates. However, Tc2 recipients had essentially no histologic evidence of GVHD, whereas Tc1 recipients had mild to moderate GVHD (average GVHD scores of 1/40 and 9.3/40, respectively). In contrast, recipients of uncultured CD8+ donor T cells developed severe GVHD (average GVHD score of 26.7/40). Because in vitro-generated, alloreactive Tc1 and Tc2 populations mediated GVL with reduced GVHD, we conclude that both subsets may improve the therapeutic outcome of allogeneic T cell transfers in patients with leukemia.