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A previous study showed failure of autologous engraftment in lethally irradiated dogs when marrow was treated before infusion with anti-class II antibody 7.2 and complement. The current study extended this observation to a second monoclonal antibody (HB10a) that identifies a different determinant on Ia-like molecules. These results suggest the presence of Ia-like antigens on pluripotent hematopoietic stem cells or on “accessory cells” needed for sustained engraftment to occur. To distinguish between these two possibilities, stem cell-depleted Ia- positive peripheral blood leukocytes obtained by discontinuous albumin density gradient were added as probable source of accessory cells to the marrow inoculum that was depleted of Ia-positive cells by treatment with antibody 7.2 and complement. Eight of ten dogs failed to show engraftment, providing further support for the hypothesis that pluripotent stem cells and not accessory cells were affected by cytolytic treatment. To provide direct evidence for the presence of Ia- like antigens on canine pluripotent hematopoietic stem cells, autologous transplants were performed using 0.7 to 13 X 10(6) Ia (7.2)- positive marrow cells per kg obtained with the help of fluorescence- activated cell sorter. Of three evaluable dogs, two showed sustained and complete engraftment, indicating that Ia-like antigens, as recognized by anti-class II antibody 7.2, are expressed at least on part of canine pluripotent hematopoietic stem cells. Concurrent in vitro studies revealed that canine CFU-GM also expressed Ia-like antigens as recognized by the class II-specific monoclonal antibodies 7.2 and HB10a.

Forty-six patients with severe aplastic anemia (median age, 23 years) were treated with high-dose cyclophosphamide followed by infusion of marrow from an HLA-identical family member. To evaluate postgrafting prophylaxis for graft-v-host disease (GVHD), they were entered into a prospective randomized trial comparing the effect of a combination of methotrexate and cyclosporine (n = 22) to that of methotrexate alone (n = 24). Forty-four of the forty-six patients had evidence of sustained marrow engraftment. Only one patient in each of the two study groups showed graft rejection. A significant reduction in the cumulative incidence of grades II to IV acute GVHD was seen in patients given methotrexate/cyclosporine (18%) compared with those given methotrexate alone (53%) (P = .012). In three patients given methotrexate alone, grade III developed, and in six, grade IV acute GVHD developed, compared with none given methotrexate/cyclosporine. Eighteen of the 22 patients given methotrexate/cyclosporine and 15 of the 24 given methotrexate alone are alive between 5.5 and 44.5 months (median, 18 months), with actuarial survival rates at 2 years of 82% and 60%, respectively (P = .062). The incidence of fatal infections was higher in patients given methotrexate alone, whereas there are as yet no significant differences in the incidence of chronic GVHD. We conclude that methotrexate/cyclosporine treatment resulted in a significant decrease in the incidence and severity of acute GVHD in patients who received transplants for severe aplastic anemia and thus an improvement in survival.

Eight patients with disseminated Hodgkin's disease resistant to MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) chemotherapy were treated with high-dose chemoradiotherapy and marrow transplantation from an HLA-identical sibling. Two patients remain alive in unmaintained complete remission (CR) at 38 and 39 months after transplant. In the other six patients, reasons for failure included relapse of lymphoma (two patients), or death due to complications of the transplant procedure, including Legionnaire's disease, disseminated zoster, graft-v-host disease, and aspiration pneumonia secondary to severe mucositis. These results demonstrate that some patients with MOPP-resistant Hodgkin's disease can obtain prolonged CR following intensive chemoradiotherapy and allogeneic marrow transplantation.

A murine monoclonal antibody (DT-2) is described which has been raised against canine thymocytes. DT-2 activates complement and is reactive with most canine thymocytes, peripheral blood T cells, thoracic duct lymphocytes, bronchoalveolar lymphocytes, and T chronic lymphatic leukemia cells. The antibody is nonreactive with surface immunoglobulin-positive blood lymphocytes, monocytes, bronchoalveolar macrophages, null acute lymphatic leukemia cells, granulocytes, erythrocytes, and platelets. In mixed lymphocyte cultures DT-2 and complement eliminate the responding but not the stimulating cell population. Mitogen stimulation (phytohemagglutinin, concanavalin A) experiments revealed that the responding cell affected by DT-2 is of lymphoid and not of monocyte/macrophage origin. All our data suggest that DT-2 is an antibody reacting specifically with canine T cells.

Four murine monoclonal antibodies were used in cytolytic assays to identify cell populations involved in canine T cell effector functions. Antibody DT-2, directed at T cells, and antibody DLy-6, a panlymphocyte antibody, inhibited mixed lymphocyte culture (MLC) responses and cytotoxic lymphocyte (CTL) activity only when lymphocytes were treated before culture (day 0), but they had no significant effect on these functions when cells were treated after 6 days in culture. Antibody DLy-1, reacting with canine lymphocytes and monocytes, abrogated MLC responses and CTL activity when responder cells were treated on day 0. When cells were treated after 6 days in culture, MLC responses were reduced to 47% of control, whereas CTL activity increased slightly. In contrast, the anti-Ia antibody 7.2 significantly reduced MLC responses and CTL activity of cells treated on either day 0 or day 6 of culture, suggesting that canine CTL express Ia antigens.

Thirty patients with advanced preleukemic syndromes were treated with marrow transplantation. Most cases were diagnosed by the presence of peripheral pancytopenia and a diagnostic marrow examination but in 6 of the 30 patients pretransplant chromosome studies were instrumental in establishing the diagnosis. Three patients prepared for transplantation with cyclophosphamide alone recurred with their disease within 6 months of transplantation. The other 27 patients were treated with cyclophosphamide and total body irradiation. Twenty of these 27 patients had preleukemia not associated with prior therapy or severe marrow fibrosis. Thirteen of these 20 are alive and well 9 to 56 months from transplant and 7 died, 4 of interstitial pneumonia, 2 of candida septicemia, and 1 of disseminated zoster. There have been no disease recurrences in this group. The remaining preleukemic patients, which include 3 patients transplanted for preleukemia secondary to prior therapy and 4 patients transplanted for preleukemia associated with severe marrow fibrosis, have all died. Major problems in these patients included disease recurrence (2 cases) and, in those with severe marrow fibrosis, graft failure (2 cases). These results suggest that for patients with life-threatening pancytopenia due to spontaneous preleukemia without severe marrow fibrosis, marrow transplantation can prolong disease-free survival and may result in cure of the disease.

Twenty-three of 37 anti-Ia McAb reactive with human B cells, as determined by indirect immunocytofluorescence, were shown to be reactive with canine peripheral blood mononuclear cells (PBMC). Using a panel of human B cell lines that differ in their expression of HLA-DR, -DP, and -DQ molecules, it was shown that 15 of these antibodies identify HLA-DR and DP molecules (i.e., broadly reactive), while 22 identify only HLA-DR molecules. Fourteen of the 15 broadly reactive McAb were reactive with canine PBMC while only 9 of the 22 HLA-DR-specific McAb reacted with canine PBMC, suggesting that broadly reactive anti-Ia McAb are much more likely to react with canine cells than narrowly reactive McAb. Ten of the canine reactive McAb that were shown to identify typical Ia bimolecular structures on canine cells using immune precipitation analysis were tested for blocking activity in the canine mixed lymphocyte culture (MLC). All four of the broadly reactive McAb (B1F6, J-70, 9-49, and HB10a) plus two of the six narrowly reactive McAbs (H81.98.21 and H40.164.3) blocked the canine MLC when added to culture wells on day 0, suggesting that inhibition may be related to the specificity of the anti-Ia McAb employed. Since the MLC may reflect cellular interactions occurring during graft-versus-host disease, this assay may be useful for screening functionally relevant broadly reactive McAb in experimental canine bone marrow transplantation studies. These data suggest that the dog may be a useful model to study anti-Ia immunotherapy.

Severe hemolytic in Basenji dogs secondary to pyruvate kinase deficiency was corrected by marrow transplantation from hematologically normal littermates. These dogs have now been followed for more than 5.5 yr. Essentially normal hematopoiesis has persisted, and the dogs remain in good health without cirrhosis or osteosclerosis. Furthermore, hepatic iron overload present before transplantation has gradually decreased. These results in dogs suggest that marrow transplantation could prevent the morbidity and mortality of severe hemolytic anemia and associated iron overload in man.

Previous work has shown failure of engraftment in lethally irradiated dogs when autologous marrow was depleted of Ia-positive cells with an anti-Ia antibody and complement before infusion. In the current study, we have utilized an avidin-biotin immunoadsorption procedure to obtain a population of highly enriched Ia-positive cells for autologous bone marrow transplantation in dogs given lethal irradiation. Dog marrow cells (2.4 to 7.0 X 10(9) cells) that contained 8.6% to 19.9% Ia- positive cells were treated successively with monoclonal antibody 7.2, which reacts with a framework determinant of Ia-antigen, and biotin- conjugated goat antimouse immunoglobulin. These treated cells were passed over a column of avidin-Biogel (polyacrylamide) and the adherent cells removed by mechanical agitation. Seven lethally irradiated dogs were transplanted with 5.9 to 33.4 X 10(6) recovered adherent cells per kilogram of which 69.0% to 88.0% were Ia-positive. All dogs had hematologic recovery; six are alive and well with durable engraftment and one died on day 15 posttransplant. They are immunologically normal as determined by lymph node and bone marrow biopsies, lymphocyte function, and immunophenotyping of peripheral blood and bone marrow cells. These data provide further evidence that canine hematopoietic stem cells express Ia-like antigens and that these cells are capable of complete hematopoietic and immunologic reconstitution in an autologous model.

Based on studies in multiply transfused dogs showing that cyclosporine (CsA) pregrafting reduced the incidence of rejection, we treated 11 multiply transfused patients with severe aplastic anemia with CsA, 12.5-20 mg/kg/day i.m. on days -6 to -1, and cyclophosphamide, 50 mg/kg/day i.v. on days -5 to -2, followed on day 0 by a marrow graft from an HLA-identical sibling donor. Patients were not given additional infusions of buffy coat cells from their marrow donors. As postgrafting prophylaxis for graft-versus-host disease 10 patients were given CsA, 12.5 mg/kg/day orally, and one patient was given a standard regimen of intermittent methotrexate. Among 10 evaluable patients, 2 rejected their marrow grafts. Eight patients had sustained engraftment. Two of these developed severe veno-occlusive disease of the liver, a complication previously observed only in patients conditioned with total-body irradiation but not in more than 200 patients with aplastic anemia conditioned with cyclophosphamide without the addition of CsA. These data indicate that conditioning of patients with cyclophosphamide and concurrent CsA does not uniformly prevent graft rejection and can result in severe hepatic toxicity.

The objectives of this study were to characterize canine peripheral blood dendritic cells (DC), to compare their functional role in lectin (Con-A) induced and oxidative (NaIO4-induced) mitogenesis, and to investigate the effect of ultraviolet (UV) irradiation on DC function. We used, sequentially, Ficoll-Hypaque fractionation, discontinuous percoll gradients, rosette, sedimentation with human red blood cells, adherence to plastic and cytofluorometric sorting after labeling with an anti-Ia monoclonal antibody (7.2) to obtain cells of low buoyant density that were rosette-negative, plastic-non-adherent, Ia-positive, nonspecific-esterase-negative, and nonphagocytic. By transmission and scanning electron microscopy these cells had large irregular nuclei, numerous spherical mitochondria, elongated cytoplasmatic processes, and lacked phagosomes. These features clearly separate these cells from canine monocytes and characterize them as DC. These cells were capable of reconstituting the proliferative responses of accessory-cell-depleted autologous lymphocytes to both Con-A and NaIO4. The responses to both mitogens showed a similar time course: an initial cluster formation of lymphocytes with DC was followed by lymphocyte blastogenesis (48-72 hr) and proliferation (72-96 hr). UV irradiation of DC before culture completely abrogated accessory activity for the response to both Con-A and NaIO4, and neither cluster formation nor blast transformation or proliferation occurred. These data provide evidence that a UV-sensitive step of direct interaction of lymphocytes and DC is involved in the mechanism of T cell activation and proliferation.

Two patients with Fanconi's anemia treated for 5 years with oxymetholone developed hepatic function abnormalities in association with hepatic tumors demonstrated by isotope liver-spleen scan or abdominal echogram. The lesions resolved over a period of 26 months after allogeneic marrow transplantation, and the patients are alive and well 3 and 4 years following transplantation. The course of these patients indicates that marrow transplantation for Fanconi's anemia allows the withdrawal of androgens and subsequent regression of androgen-related hepatic tumors in patients who might otherwise have a fatal outcome.

We examined the correlation between cyclosporine (CsA) levels and in vitro assays of immune function and hematopoiesis. Mixed lymphocyte reaction (MLR), mitogen responses, suppressor cell (SC), cell-mediated lympholysis (CML), and erythroid colony (EC) assays were studied in dogs, and in vitro megakaryocytopoiesis was studied in mice. Serum CsA concentrations were measured by radioimmunoassay. After oral or intramuscular CsA dosing, lymphocyte proliferation, as measured by MLR, inversely correlated with in vivo serum CsA concentration. MLR responses decreased rapidly, and nearly complete inhibition coincided with peak CsA levels. While CsA concentration-related suppression of lymphocyte stimulation was also observed in mitogen-stimulated cultures, results were less predictable and similar to results with in vitro CsA addition, and higher serum CsA levels were required to achieve comparable suppression. In vivo serum or in vitro CsA levels greater than 300 ng/ml completely inhibited the development of cytotoxic effector cells but had no measureable effect on the expression of suppressor cells in the same cultures. Furthermore, CsA serum also caused concentration-related inhibition of EC growth. The addition of human embryonic kidney-conditioned medium, however, abrogated CsA-related inhibition of EC growth, which suggested that CsA indirectly inhibited EC growth, presumably by interfering with CsA-sensitive accessory cells. This was supported by studies in an in vitro model of murine megakaryocytopoiesis. In normal conditioned medium, megakaryocyte colonies were unaffected by the presence of CsA. However, when cells were cultured in conditioned medium prepared in the presence of CsA, profound inhibition of megakaryocyte growth was observed. These studies show that biologic assays can be used reliably to measure concentration-related changes in immunosuppressive activity of CsA. Further clinical studies are needed to evaluate the usefulness of pharmacodynamic monitoring of CsA therapy.

Increasing age has been reported to be a poor prognostic factor for survival after bone marrow transplantation. We evaluated causes of death and frequency and type of complications after marrow grafting in 24 syngeneic and 39 allogeneic recipients who were 45 to 68 years old at the time of transplant. Most patients were in an advanced stage of hematologic malignancy. Among patients given syngeneic transplants, actuarial disease-free survival at 7 years is 20%. The major causes of death were relapse of leukemia and idiopathic interstitial pneumonia. Among allogeneic recipients, 9 (23%) are currently alive, and actuarial disease-free survival at 7 years is 11%. Cytomegalovirus pneumonia and septicemia were the most frequent causes of death. Patients over 50 years of age had the poorest survival rate (1/13), but many of these were transplanted in an advanced stage of their disease. However, among 12 patients transplanted while in remission or at an early stage of their disease, 5 are surviving 65 to 1,160 days after transplantation, with an actuarial survival rate of 22% at 3 years. This is in contrast to those who received their transplant in relapse: 2 out of 20 patients (10%) became long-term survivors, with a probability of survival of 15% at 3 years. The actuarial incidence of grade II through IV acute graft- v-host disease (GVHD) was 30% for allogeneic recipients 45 to 50 years of age. This was not significantly different from the incidence in younger patients. In patients 51 to 62 years of age, the actuarial incidence of acute GVHD was 79%; however, this group included three partially HLA-mismatched transplants. Ten of 15 patients surviving at least 3 months developed chronic GVHD. These results suggest that marrow transplantation is feasible and should be considered in patients over 45 years, especially if recipients are in good clinical condition and are at an early stage of their disease, such as the chronic phase of chronic myelogenous leukemia and preleukemia. For patients more than 50 years of age, allogeneic marrow grafting cannot presently be considered first-line therapy.

The Third International Workshop on Canine Immunogenetics involved 80 potentially DLA-D homozygous typing cells obtained from dogs of various breeds and submitted from five laboratories in Europe and the United States. Mutual reactivity of all cells was studied in mixed leukocyte cultures, and stabilized relative responses were used for analysis. Intralaboratory and interlaboratory comparisons of results suggest that a stabilized relative response of 30% represents an acceptable parameter for "typing responses" indicating phenotypic DLA-D identity of stimulator and responder cells. Using this criterion, 10 clusters of homozygous typing cells were defined and accepted on an international level, and they were assigned the specificities Dw1 to Dw10. At least six additional (provisional) specificities were recognized that were well characterized within individual laboratories but require additional testing before workshop specificities can be assigned. These data show that DLA-D typing is feasible and represents a useful tool in the genetic analysis of the canine major histocompatibility complex. Much work is needed to confirm the present results in family studies, to determine gene frequencies, and to analyze at a molecular level the antigens responsible for mixed leukocyte culture reactivity.

Subsegmental bronchoalveolar lavages were performed in 18 healthy beagles. The average yield per lavage was 45 X 10(6) cells consisting on the average of 24% lymphocytes, 71% macrophages, and 4% granulocytes. Cells were further examined in cytofluorometric studies using monoclonal (anti-Ia, antilymphocyte, anti-T) and polyspecific (anti-Ig) antibodies. Sixty to 90% of lymphocytes were T cells as determined by the T cell antibody DT-2. No surface immunoglobulin-positive cells (B cells) could be detected. All macrophages expressed Ia antigens (p 29/34) whereas lymphocytes did not. In assays of concanavalin A-induced blastogenesis of thymocytes, alveolar macrophages functioned as accessory cells. The anti-Ia antibody 7.2 interfered with this function, indicating that Ia antigens on canine alveolar macrophages are involved in interaction with T cells resulting in T cell proliferation.

We have previously shown that grafts with low doses of bone marrow (≤ 4 × 108 cells/kg) after 1200 R total-body irradiation (TBI) are successful in DLA-identical. but not DLA-nonidentical littermate or unrelated dogs, suggesting a locus of resistance associated with the major canine histocompatibility complex (DLA). Addition of viable donor blood leukocytes to the marrow inoculum has consistently overcome resistance, either by administration of an increased number of stem cells from the peripheral blood or through an effect mediated by lymphocytes. In-vitro-irradiated leukocytes have been ineffective. In the current study 8 dogs were given 1200 R TBI followed by 2.5 ± 1.4 × 108 marrow cells/kg and 7.7 ± 3.6 × 108 thoracic duct lymphocytes/kg from unrelated DLA-nonidentical donors. All 8 dogs showed prompt and sustained hemopoietic engraftment, and none rejected. Since canine thoracic duct lymphocytes have previously been shown not to contain hemopoietic stem cells, abrogation of resistance to canine marrow grafts appears to be related either to active suppression of residual host immunity or to enhancement of hemopoiesis by cell-cell interaction of thoracic duct lymphocytes with marrow cells. The latter mechanism is suggested by in vitro studies showing that dog thoracic duct lymphocytes cocultured with autologous marrow significantly increase the number of erythroid colonies.

Cytotoxic lymphocytes (CTL) were generated in bulk mixed leukocyte culture (MLC) using peripheral blood mononuclear cells from homozygous dogs identical to each other for DLA-A and -B but different for DLA-D loci. These CTL were tested against 51Cr-labeled targets (Con-A-stimulated PBMC) derived from the MLC sensitizing dogs and from other dogs homozygous for various DLA-D locus alleles. Indirect cell-mediated lympholysis (CML) assays, with and without cold target blocking and pretreatment of 51Cr-labeled targets with DLA alloantisera were used to study the target cell determinants recognized by these CTL. The resultant target cell lysis and cold target blocking of specific target cell lysis indicated target determinants associated with DLA-D-locus-encoded specificities, and different from those encoded by DLA-A and -B loci. The pattern of shared target determinants among different DLA-D HTC may be explained by: (A) two separate previously unrecognized MHC associated loci, each with 2 alleles defined so far; or (B) epitopes shared among certain DLA-D alleles but not present in others. This study demonstrates that DLA-D associated determinants different from previously described DLA-D specificities may serve as CML target cell determinants when there are no apparent DLA-A and -B loci encoded antigen differences between responder and stimulator cells used to generated MLC-derived CTL.

Between July 1970 and January 1985, 100 patients with malignant lymphoma were treated with high-dose chemoradiotherapy and bone marrow transplantation. Twenty-eight of the 100 are alive and the actuarial probability of disease-free survival 5 years from transplantation is 22%. The most common reason for treatment failure was disease recurrence, with an actuarial probability of 60%. A proportional hazards regression analysis showed that the likelihood of disease-free survival was less in those patients transplanted in resistant relapse and in those previously treated with chest radiotherapy. Neither disease histology (Hodgkin's disease, high-grade lymphoma or intermediate-grade lymphoma), nor source of marrow (syngeneic, allogeneic, or autologous) significantly influenced either disease-free survival or probability of relapse. The use of high-dose chemoradiotherapy and marrow transplantation appears to offer a better chance for long-term survival than any other form of therapy for young patients with disseminated malignant lymphoma whose disease has progressed after initial combination chemotherapy. The best results with marrow transplantation were obtained in patients transplanted in early relapse or second remission who had not received prior chest radiotherapy.

Ten dogs were given 9.2 Gy of total body irradiation and autologous bone marrow infusion followed by ten daily transfusions of leukocytes for a total of 11.5 to 36.2 (median, 18.8) x 10(8)/kg obtained via leukapheresis from histoincompatible unrelated donors. Four dogs were given unirradiated leukocytes, and all developed graft-versus-host disease (GVHD). In contrast, only two of three dogs given leukocytes irradiated with 20 mJ/cm2 of ultraviolet (UV) light (200 to 300 nm), and none of three dogs given leukocytes irradiated with 1,000 mJ/cm2 developed GVHD. These data indicate that UV irradiation abrogates the alloreactive potential of transfused leukocytes, and suggest that UV irradiation can be used to prevent the development of transfusion- induced GVHD.

Past studies in dogs have suggested that marrow graft rejection was mediated by major histocompatibility complex (MHC) class II antigen-positive non-T cells that survived standard doses of total-body irradiation (TBI). We have now raised 4 monoclonal antibodies (mAbs) against marrow cells harvested 6 days after TBI. The mAbs are highly reactive (greater than 70%) with marrow cells surviving radiation and also bind strongly (greater than 50%) to normal marrow cells, lymphocytes, monocytes, and granulocytes. One of the mAbs (34-S3) reacted strongly with NK-like cells. In vitro treatment of marrow with mAb and rabbit complement (C') did not affect erythroid colony-forming unit (CFU-E) growth, whereas 2 of the 4 mAbs inhibited granulocyte-macrophage colony-forming unit (CFU-GM) growth, and all 3 mAbs tested suppressed autologous marrow engraftment. One of the mAbs, 69-S5 (IgG1), bound to a 95,000 dalton antigen. It crossreacted with human cells, but not with cells from Rhesus monkeys, baboons, and cats. We administered this mAb intravenously at 0.2 mg/kg/day on days -5 to 0 to dogs given 9.2 Gy TBI on day 0 followed by marrow grafts (less than or equal to 4 x 10(8) cells/kg) from DLA-nonidentical unrelated donors. Three of five dogs had sustained grafts. Increasing the dose of mAb ten-fold (2 mg/kg/day) resulted in graft failure (2 of 2 dogs). Treatment with a dose of 0.2 mg/kg/day from day -7 to -2 showed sustained engraftment in 7 of 10 dogs. This result is in contrast to sustained grafts in 3 of 36 dogs not given mAb, and in 1 of 7 dogs treated with an irrelevant mAb (P = 0.0002 and 0.04, respectively). We conclude that treatment of recipients with a mAb raised against marrow cells surviving radiation and not directed at major histocompatibility complex (MHC) class II antigens and NK-like cells can also facilitate engraftment of DLA-nonidentical canine marrow. These results may be relevant for the transplantation of HLA-incompatible marrow in man, particularly after in vivo T cell depletion, where graft failure is frequent.

Summary An 11 · 5-year-old girl developed acute lymphoblastic leukaemia 7 months after bone marrow transplantation for severe aplastic anaemia. Before transplantation there were neither morphologic nor cytogenetic abnormalities to suggest preleukaemia. The conditioning regimen consisted only of cyclophosphamide. At the time of development of acute lymphoblastic leukaemia, chromosome analysis showed the blasts to be of host origin with clonal abnormalities including monosomy 7. Such a preleukaemic syndrome presenting as severe aplastic anaemia is a very rare event (the case reported here is the only one of 436 patients in Seattle) and cannot be reliably excluded before transplantation.

Total body irradiation is being used with increasing frequency for the treatment of lymphopoietic malignancies and in preparation for marrow transplantation. Acute toxicities include reversible gastroenteritis, mucositis, myelosuppression and alopecia. As the success of treatment improves and more patients become long-term survivors, manifestations of delayed and chronic toxicity become evident. These include impairment of growth and development, gonadal failure and sterility, cataract formation and possibly secondary malignancies. The contribution of total body irradition to the development of pneumonitis and pulmonary fibrosis is still poorly understood. Some of these changes are reversible or correctable, whereas others are permanent. Nevertheless, until equally effective but less toxic regimens become available, total body irradiation appears to be the treatment of choice to prepare patients with leukemia for marrow transplantation.

We examined the effect of methotrexate (MTX) and cyclosporin A (Cy A) on engraftment, graft-versus-host disease (GVHD), and the induction of tolerance in dogs prepared for marrow transplantation by 9 Gy of total body irradiation and grafted with bone marrow and buffy coat cells. Nineteen dogs were given grafts from DLA-identical littermates followed by immunosuppression with Cy A for 25 or 100 days. All had sustained engraftment, and 12 became healthy long-term chimeras. Sixty dogs were given grafts from DLA-nonidentical unrelated donors. Among nine given MTX only postgrafting, one rejected the graft nd eight died with GVHD. Among 18 dogs given Cy A only postgrafting, eight failed to achieve engraftment, seven died of various causes, and three died with GVHD. Thirty-four dogs were given both MTX and Cy A in various regimens postgrafting. The only long-term survivors were 4 of 10 dogs given MTX on days 1, 3, 6, and 11 and Cy A from days 0 through 100. Two have chronic GVHD. We conclude that Cy A can induce graft-host tolerance across minor, but not major, histocompatibility differences. The combination of MTX early after transplantation with Cy A prevents failure of engraftment of histoincompatible marrow and some recipients become long-term survivors.

Forty-eight patients with chronic myelocytic leukemia, aged 11 to 47, were treated with high-dose cyclophosphamide and fractionated total body irradiation, followed by infusion of marrow from HLA-identical siblings. They were randomized to receive either methotrexate (MTX) (n = 23) or cyclosporine (CSP) (n = 25) as postgrafting prophylaxis for graft-v-host disease (GVHD). All patients had evidence of sustained hematopoietic engraftment. Seventeen of the 25 patients receiving CSP and 17 of the 23 patients receiving MTX are alive between one and almost four (median, 1.7) years, with an actuarial survival rate at three years of 62% and 66%, respectively (P = .60). Also, with respect to most other parameters studied, the two drugs were identical. The probability of acute GVHD was .42 and .46, respectively (P = .70), that of chronic GVHD, .50 and .63 (P = .44), and that of death from transplant-related causes, .30 and .24 (P = .51). There were no differences in the speed of granulocyte and platelet engraftment (P = .82 and .94, respectively), and the duration of hospitalization was comparable (P = .58). Patients receiving MTX required red cell transfusions for a shorter period of time (P = .02), but had a slightly increased morbidity from early oral mucositis. The leukemia recurrence rates were comparable (P = .60). With the regimens used in this study, we conclude that CSP failed to reduce the incidence of GVHD and improve the survival of patients with chronic myelocytic leukemia when compared to results with standard MTX.

We reviewed the records and marrow biopsy specimens of 75 patients with leukemic, myelodysplastic, or myeloproliferative disorders to determine whether the presence of marrow fibrosis affected engraftment after allogeneic marrow transplantation. While 28 control patients without fibrosis achieved prompt engraftment, two of 32 patients (6%) with mild and five of 15 patients (33%) with severe fibrosis failed. The rate of myeloid recovery was significantly slower and the dependence on platelet and red blood cell transfusions was significantly longer in patients with severe fibrosis than in patients with no fibrosis. A finding of severe marrow fibrosis should therefore be taken into account when evaluating the risks and benefits of marrow transplantation.

Les transfusions prealables, surtout si le donneur est le futur donneur de moelle, reduisent les chances de reussite de la greffe de moelle. Elles semblent sensibiliser le receveur vis-a-vis d'antigenes d'histocompatibilite exprimes sur les cellules sanguines et medullaires, ce qui amene une reaction au moment de la greffe de moelle. Cette reaction secondaire, due apparemment aux lymphocytes T, est plus difficile a maitriser qu'une reaction immune primaire

We retrospectively analyzed the relationship of serum cyclosporine concentration to renal dysfunction in 63 marrow transplant recipients who received cyclosporine for prophylaxis of acute graft-versus-host disease. Patients were divided into three groups according to their mean trough cyclosporine concentration for the first 28 days of therapy: less than 150, 150-250, and greater than 250 ng/ml. Baseline renal function and exposure to nephrotoxic antibiotics was comparable in the three groups. Renal dysfunction was defined as doubling of baseline serum creatinine. The likelihood of developing renal dysfunction was analyzed with Kaplan-Meier product limit estimates. The log-rank test was used to compare the three groups. Fifty-four (86%) of the patients developed renal dysfunction. The incidence of renal dysfunction was 73%, 95%, and 100%, and it developed at a median of 46, 29, and 20 days in patients with a mean trough concentration of less than 150, 150-250, and greater than 250 ng/ml, respectively (P less than 0.001). Eight of the nine patients who did not develop renal dysfunction had a mean trough concentration of less than 150 ng/ml. These data indicate that the incidence and the rate of development of renal dysfunction are related to serum cyclosporine concentration.

Frequent platelet support is an essential part of the management of patients with severe aplastic anaemia and platelet transfusions from random donors are usually given as initial therapy. To evaluate those parameters that might correlate with the development of refractoriness to platelets from random donors, we performed a retrospective multivariate analysis in 264 patients with severe aplastic anaemia who presented for allogeneic bone marrow transplantation. Two hundred and ten (79.5%) of these patients had received multiple platelet and red cell transfusions, and 71 (34%) were refractory to random donor platelets. The strongest factor correlating with refractoriness was the presence of lymphocytotoxic antibodies, followed by the number of platelet units previously transfused. However, the latter variable attained significance only when the number of platelet units transfused exceeded 40. When given HLA-compatible platelet transfusions, only five (7%) of the refractory patients did not show a reasonable post-transfusion platelet increment. Measures which would delay or prevent platelet alloimmunization might include a policy of therapeutic rather than prophylactic platelet transfusions, and referring patients early in the course of their disease for marrow grafting if a suitable donor is available.

Marrow transplants were carried out between unrelated DLA-nonidentical dogs. Recipients were conditioned for transplantation by total body irradiation (TBI) given eigher as a single dose of 9 Gy (900 rad) or fractionated in three increments of 6 Gy (600 rad) each at intervals of 48 hr. All recipients received marrow, less than or equal to 4 x 10(8) cells/kg, and no buffy coat cells. No immunosuppression was given after grafting. All 10 dogs given single dose total body irradiation failed to show engraftment and died with marrow aplasia and infectious complications (median survival 12 days). In contrast, all 10 dogs given fractionated TBI had sustained engraftment and died with graft-versus-host disease (GVHD) and infectious complications (median survival 12.5 days). None of the dogs died from radiation-induced gastroenteritis. In conclusion, resistance to DLA-nonidentical unrelated marrow grafts can be abrogated by high-dose TBI. This technique may allow hemopoietic engraftment even after i vitro manipulation of the marrow such as lymphocyte depletion by cell separation or treatment with anti-T cell antisera.

Canine lymphocytes alloantigen-primed in mixed leukocyte culture (MLC) were tested for their ability to suppress MLC reactivity. These cells were added to fresh responder cells in MLC at ratios of 1:50 and 1:20 and suppressed reactivity 90 +/- 7% (mean +/- 1 SD) and 91 +/- 3% respectively. At lower ratios no suppression was observed. Monoclonal antibody E11 (IgG3) recognized a subset of canine Thy1+ T cells, and with complement treatment was capable of depleting suppressor cells from bulk MLC (11 +/- 6% of control suppression), but DT-2 (IgG2a), another canine T-cell antibody, had no such effect (84 +/- 11%) (P = 0.028). Antibody A5 (IgG2b), reactive with all T and most non-T cells, also eliminated suppression. Positively selected E11+ bulk-MLC cells suppressed (91%) and E11- bulk-MLC cells did not (4%). At the ratios described, suppression was specific for the DLA-D phenotype of the stimulator cells. In tests with the original stimulators, suppression was 91 +/- 7%; in tests with DLA-D identical stimulators it was 92 +/- 5%; and for DLA-D nonidentical stimulators it was 38 +/- 25% (P = 0.005). Nonspecific suppression increased with increasing numbers of suppressor cells. At ratios of 1:10 and 1:5 nonspecific suppression was 72 +/- 13% and 92 +/- 8%, respectively. These studies show that under appropriate conditions, Thy1+, E11+, A5+, DT-2- alloantigen-activated canine T lymphocytes function as DLA-D-specific suppressor cells and DT-2+ cells do not.

We investigated the frequency of hypertension (sustained diastolic blood pressure greater than or equal to 90 mmHg) in 112 patients given HLA-identical marrow grafts. Patients were conditioned with 2 X 60 mg/kg of cyclophosphamide and 6 X 2 Gy of total body irradiation and randomized to receive as graft-versus-host disease prophylaxis either the standard methotrexate regimen (n = 61) or cyclosporine (n = 51), starting on day -1 as 12.5 mg/kg/d orally or as 3 mg/kg/d i.v. and later converting to p.o. when oral intake was tolerated. Kaplan-Meier estimates indicate a 60% incidence of hypertension in the first 120 d in patients given cyclosporine (median time to onset: 4 d post transplant) compared to 20% in patients given methotrexate (P less than 0.0001). Multifactorial analysis using a Cox regression model showed that cyclosporine was was the most significant risk factor for developing hypertension (relative risk: 32.1, P less than 0.0001). In addition, glucocorticoids, used for treatment of GVHD, were associated with an increased risk for hypertension (relative risk 7.2, P less than 0.0001). Age, sex, underlying disease, cyclosporine trough levels, and renal function had no significant association with hypertension. Early therapy of hypertension in cyclosporine-treated patients appears to be indicated.

Allogeneic hematopoietic cell transplantation (HCT) offers curative therapy for many patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). However, post-HCT relapse remains a major problem, particularly in patients with high-risk cytogenetics. In this prospective phase II trial, we assessed the efficacy and toxicity of treosulfan, fludarabine, and 2 Gy total body irradiation (TBI) as conditioning for allogeneic HCT in patients with MDS or AML. Ninety-six patients with MDS (n = 36: 15 refractory cytopenia with multilineage dysplasia, 10 refractory anemia with excess blasts type 1, 10 refractory anemia with excess blasts type 2, 1 chronic myelomonocytic leukemia type 1) or AML (n = 60: 35 first complete remission [CR], 18 second CR, 3 advanced CR, 4 refractory relapse) were enrolled; median age was 51 (range, 1 to 60) years. Twelve patients had undergone a prior HCT with high-intensity conditioning. Patients received 14 g/m2/day treosulfan i.v. on days −6 to −4, 30 mg/m2/day fludarabine i.v. on days −6 to −2, and 2 Gy TBI on day 0, followed by infusion of hematopoietic cells from related (n = 27) or unrelated (n = 69) donors. Graft-versus-host disease prophylaxis consisted of tacrolimus and methotrexate. With a median follow-up of 30 months, the 2-year overall survival (OS), relapse incidence, and nonrelapse mortality were 73%, 27%, and 8%, respectively. The incidences of grades II to IV (III to IV) acute and chronic graft-versus-host disease were 59% (10%) and 47%, respectively. Two-year OS was not significantly different between MDS patients with poor-risk and good/intermediate-risk cytogenetics (69% and 85%, respectively) or between AML patients with unfavorable and favorable/intermediate-risk cytogenetics (64% and 76%, respectively). In AML patients, minimal residual disease (MRD; n = 10) at the time of HCT predicted higher relapse incidence (70% versus 18%) and lower OS (41% versus 79%) at 2 years, when compared with patients without MRD. In conclusion, treosulfan, fludarabine, and low-dose TBI provided effective conditioning for allogeneic HCT in patients with MDS or AML and resulted in low relapse incidence, regardless of cytogenetic risk. In patients with AML, MRD at the time of HCT remained a risk factor for post-HCT relapse.

Previous studies had shown that marrow graft rejection generally did not occur in untransfused dogs given 900-rad total body irradiation and hemopoietic grafts from DLA identical littermates (only 1 of 59 rejected), but was seen in all instances after three preceding transfusions of whole blood from the marrow donor on days -24, -17, and -10 before transplantation (19 of 19 rejected). The present study was undertaken to investigate whether immunization by 3 preceding transfusions of whole blood from the DLA-identical littermate marrow donor could be abrogated by administration of the immunosuppressive agent cyclo-sporin-A, 20 mg/kg/day intramuscularly on days -5 to 0. Seven of 10 dogs showed sustained marrow engraftment. 2 failed to engraft, and 1 dog died to early to be evaluated. It was concluded that immunization to non-DLA antigens by preceding whole blood transfusions could be abrogated in most cases by a short-course of cyclosporin-A before total body irradiation and marrow transplantation, resulting in successful and sustained marrow engraftment.

One hundred thirty-seven patients had gonadal function evaluated 1-11 years after marrow transplantation. All 15 women less than age 26 and three of nine older than age 26 who were treated with 200 mg/kg cyclophosphamide recovered normal gonadotropin levels and menstruation. Five have had five pregnancies resulting in three live births, one spontaneous abortion, and one elective abortion. Three of 38 women who were prepared with 120 mg/kg cyclophosphamide and 920-1200 rad total-body irradiation had normal gonadotropin levels and menstruation. Two had pregnancies resulting in one spontaneous and one elective abortion. Of 31 men prepared with 200 mg/kg cyclophosphamide, 30 had normal luteinizing hormone levels, 20 had normal follicle-stimulating hormone levels, and 10 of 15 had spermatogenesis. Four have fathered five normal children. Thirty-six of 41 men prepared with 120 mg/kg cyclophosphamide and 920-1750 rad total-body irradiation had normal luteinizing hormone levels, ten had normal follicle-stimulating hormone levels, and 2 of 32 studied had spermatogenesis. One has fathered two normal children. It was concluded that cyclophosphamide does not prevent return of normal gonadal function in younger women and in most men. Total-body irradiation prevents return of normal gonadal function in the majority of patients.

Bone marrow cells from ten normal donors were exposed to ultraviolet (UV)C or UVB light for total exposures of 0.1 to 100 mJ/cm2, and assayed for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and phytohemagglutinin (PHA)- stimulated proliferative responses. After exposure to UVC CFU-GM, BFU-E and PHA responses showed a UV dose-dependent sharp decrease to levels less than 1% of controls with 0.5, 2.0, and 10 mJ/cm2, respectively. With UVB, PHA responses were most sensitive, declining to less than 1% at 5 mJ/cm2. BFU-E decreased to less than 1% of control with 15 mJ/cm2 UVB. CFU-GM, at UVB doses of 0.1 to 2.0 mJ/cm2, increased to 125% to 130% of control and decreased to less than 1% only at exposures greater than 20 mJ/cm2. Thus, these studies show that UVB, but not UVC light, can be used to inactivate bone marrow T lymphocytes selectively while sparing hematopoietic precursor cells. The data suggest that UVB irradiation can be used for T-lymphocyte purging for allogeneic marrow transplantation.