The induction of graft versus host disease in mice treated with cyclophosphamide.

In these studies adult mice treated with cyclophosphamide and foreign immunologically competent cells developed a graft versus host disease which outwardly resembled that encountered in other experimental systems. Progressively larger doses of cyclophosphamide produced an increasingly severe disease whereas comparable doses of mechlorethamine were ineffective. Increasingly larger cell inocula from parental, allogeneic, and xenogeneic donors resulted in a correspondingly more severe disease. Nucleated cells obtained from the peripheral blood were found to be the most potent inducers of this syndrome, while cells from the spleen, bone marrow, and thymus displayed lesser degrees of reactivity in that order. No such graft versus host disease occurred in mice given saline, lysed, or heat-killed cells in place of viable foreign cells. Neither did the disorder develop when comparable inocula of isogeneic cells were used.

Graft-versus-host disease in cyclosporin A-treated rats after syngeneic and autologous bone marrow reconstitution.

Lethally irradiated rats treated with cyclosporin A (CsA) for 20-40 d develop classic graft-versus-host disease (GVHD) when reconstituted with syngeneic or autologous bone marrow, upon discontinuation of CsA, whereas normal rats do not. Syngeneic GVHD may be transferred to irradiated but not normal syngeneic recipients. Normal spleen cells fail to prevent the development or adoptive transfer of syngeneic GVHD.

Development of graft-vs.-host disease-like syndrome in cyclosporine-treated rats after syngeneic bone marrow transplantation. I. Development of cytotoxic T lymphocytes with apparent polyclonal anti-Ia specificity, including autoreactivity.

Lethally irradiated rats reconstituted with syngeneic bone marrow and treated with cyclosporine (CsA) for 40 d develop a graft-vs.-host disease-like syndrome (GVHD) after CsA therapy. We attempted to assess the development of autoreactivity in these animals. Results revealed that a majority of the animals with syngeneic GVHD develop autocytotoxic T lymphocytes of the OX8 phenotype. In addition to reactivity with self, these cells were capable of lysing appropriate target cells from a variety of different rat strains. The target antigens appeared to be class II major histocompatibility antigens, because lysis could be effectively blocked by an anti-Ia monoclonal antibody. Cold target inhibition studies indicated that one effector cell was capable of lysing various target cells, and provided evidence against a polyclonal activation of multiple anti-Ia-reactive cells. These results suggested that the anti-class II autoreactive cell associated with syngeneic GVHD either recognizes a common class II determinant ("public" epitope) shared by multiple strains of rats, or was polyspecific with respect to "private" class II determinants.

Prolonged survival and remyelination after hematopoietic cell transplantation in the twitcher mouse.

The twitcher mouse is an animal model of galactosylceramidase deficiency (Krabbe's disease), a human sphingolipidosis. The effects of hematopoietic cell transplantation as potential enzyme replacement therapy were examined in the twitcher mouse. Survival in twitcher mice with transplants was significantly prolonged and was associated with gradual repair of demyelination in peripheral nerves. In contrast, there was no improvement in the neurodegenerative process in the central nervous system after transplantation. These observations indicate that cellular transplantation may effectively provide in vivo enzyme replacement for the peripheral manifestations of genetic storage diseases. Strategies to perturb the blood-brain barrier may be necessary for enzyme replacement to be therapeutic in diseases with central nervous system manifestations.

Theta-sensitive cell and erythropoiesis: identification of a defect in W/Wv anemic mice.

Nonirradiated mice of the W/WV genotype were injected with normal (+/+) bone marrow cells that had been treated with antiserum to Thy 1.2 and complement (C'). Such bone marrow cells had no effect on the number of macroscopic colonies formed in the spleens of these mice, but did not cure the anemia. The addition of +/+ thymocytes to these bone marrow cells restored their ability to cure the anemia in W/WV mice. These data suggest that a theat-sensitive cell is required in the promotion of differentiation of murine hematopoietic stem cells into erythrocytes, and that there is a deficiency of such a cell in the W/WV mouse.

Viral hemorrhagic encephalopathy of rats.

A virus has been isolated and serially passed in suckling rats; it causes an acute fatal paralysis associated with hemorrhage and necrosis in the brain and spinal cord. The agent is relatively resistant to heat and ether, is about 20 millimicrons in diameter, and is antigenically closely related to rat virus. Its isolation resulted from the study of occasional cases of paralysis in adult rats after administration of cyclophosphamide.

Cytotoxic antibody in normal human serums reactive with tumor cells from acute lymphocytic leukemia.

Serums showing complement-dependent cytotoxic reactions to acute lymphocytic leukemia cells were detected in three normal unimmunized subjects. These serums were reactive with tumor cells from 514 (514 tested) acute lymphocytic leukemia patients, and three (12 tested) patients with acute myelocytic leukemia; they did not react with tumor cells from patients with acute monocytic leukemia (two tested), with chronic lymphocytic leukemia (two tested) or with leukolymphosarcoma (two tested); nor did they react with normal lymphocytes from 52 different donors. These reactive serums appear to recognize antigens primarily associated with acute lymphocytic leukemia.

Nutritional support of bone marrow transplant recipients: a prospective, randomized clinical trial comparing total parenteral nutrition to an enteral feeding program.

Although standard supportive care for bone marrow transplant (BMT) recipients includes total parenteral nutrition (TPN), it has not been shown that this is the most appropriate method of nutritional support. To determine whether current BMT recipients require TPN during the early recovery period, we conducted a prospective, randomized clinical trial comparing TPN and an individualized enteral feeding program (counseling, high protein snacks and/or tube feeding). Nutritional assessment included measurement of serum proteins, anthropometry, and body composition analysis. For the latter, total body water and extracellular fluid were measured by standard radioisotope dilution techniques and used to quantitate body cell mass and body fat plus extracellular solids (FAT + ECS). In 27 TPN patients, body composition 28 days after BMT, expressed as a percentage of baseline, was body cell mass, 100%, extracellular fluid, 108%, FAT + ECS, 108%, and in 30 enteral feeding program patients, was body cell mass, 93%, extracellular fluid, 104%, and FAT + ECS, 94%. Only the difference in FAT + ECS was statistically significant (p less than 0.01). Compared to the enteral feeding program, TPN was associated with more days of diuretic use, more frequent hyperglycemia, and more frequent catheter removal (prompted by catheter-related complications), but less frequent hypomagnesemia. There were no significant differences in the rate of hematopoietic recovery, length of hospitalization, or survival, but nutrition-related costs were 2.3 times greater in the TPN group. We conclude that TPN is not clearly superior to individualized enteral feeding and recommend that TPN be reserved for BMT patients who demonstrate intolerance to enteral feeding.

Phase I study of combination drug purging for autologous bone marrow transplantation.

In a phase I clinical trial of autologous bone marrow transplantation, we determined the feasibility of ex vivo purging with high concentrations of pharmacologics in combination. Light-density cells isolated from the grafts of 26 patients with acute leukemia or lymphoblastic lymphoma were treated with 4-hydroperoxycyclophosphamide (4-HC; 30 to 60 micrograms/mL), vincristine (Vcr; 1.5 to 3.0 micrograms/mL), and methylprednisolone sodium succinate (MP; 5 mg/mL). All patients received marrow-lethal induction therapy followed by infusion of the treated grafts. Three patients died of transplant-related complications before achieving peripheral blood recovery of greater than 0.5 x 10(9) granulocytes per liter. All other patients achieved this parameter of engraftment at a median of 35 days after marrow infusion. The median time to last platelet transfusion was 45 days. These durations of aplasia were similar to those experienced by other patients receiving density-gradient separated grafts treated with 60 micrograms/mL of 4-HC as a single agent. No patient required infusion of untreated reserve marrow because of engraftment failure. The colony-forming unit-granulocyte macrophage (CFU-GM) content of the grafts after purging predicted these parameters of engraftment. Colony-forming unit-leukemia (CFU-L) cultured from the grafts of 12 of the patients treated for acute lymphoblastic leukemia (ALL) were considerably more sensitive to the combination regimen than to 4-HC alone; the sensitivity of CFU-GM from these patients did not differ between the two regimens. The results of this trial indicate the feasibility of treating autologous bone marrow grafts with high concentrations of pharmacologics in combination. The use of combinations may increase the efficacy of ex vivo purging without increasing the toxicity to normal hematopoietic cells.

Successful marrow transplantation for acute myelocytic leukemia following therapy for Hodgkin's disease.

Five patients with acute myelocytic leukemia (AML) after combined modality therapy for Hodgkin's disease (HD) were treated with cyclophosphamide and busulfan followed by bone marrow transplantation (BMT). Four patients received allogeneic transplants from histocompatibility locus antigen (HLA)-compatible siblings and the fifth patient received an autologous marrow treated with 4-hydroperoxycyclophosphamide. Two patients died of complications of acute graft-v-host disease (GVHD) despite prophylaxis with either low-dose cyclophosphamide or cyclosporine. The remaining three patients were alive and disease-free 382, 617, and 620 days after transplant. These initial results are encouraging and more patients with treatment-related AML need to be evaluated with both allogeneic and autologous BMT to fully elucidate the potentially curative role of this intensive therapy in an otherwise fatal hematologic malignancy.

Allogeneic bone marrow transplantation for patients with high-risk acute lymphoblastic leukemia.

Seventy-four consecutive patients with high-risk acute lymphoblastic leukemia (ALL) were given cyclophosphamide (CY; 50 mg/kg on each of 4 days) plus total body irradiation (TBI; 300 rad on each of 4 days) followed by a human leukocyte antigen (HLA)-identical allogeneic bone marrow transplant (BMT). Eighteen patients in first complete remission (CR1), 36 in CR2, 16 in CR3, and four in CR4 were transplanted. Patients in CR1 were transplanted 1 to 8 months (median, 3 months) after attaining CR. All 18 patients in CR1 had one or more poor risk factors: age more than 18 (N = 17), initial leukocyte count greater than or equal to 20,000 (N = 11), Ph 1 chromosome (N = 2), delay in attaining CR more than 6 weeks (N = 8), or extramedullary disease (N = 1). Of those transplanted in CR2, 72% had relapsed on therapy. The 5-year event-free survival (EFS) rates for patients transplanted in CR1, CR2, and CR3 are 42%, 43%, and 25%, respectively, at median follow-up times of 57, 54, and 72 months, respectively. Children aged less than 18 years transplanted in CR2 have a 5-year EFS rate of 54%. All CR4 patients died early after transplant. The actuarial probability of relapse is 20%, 26%, and 48% for those transplanted in CR1, CR2, and CR3, respectively. Although there was substantial transplant-associated mortality, it decreased over the decade of the study (P = .01). This study indicates that BMT offers an attractive alternative to postremission chemotherapy in patients in CR1 with poor prognostic factors and in patients in second remission.

Cure of acute myelocytic leukemia in adults: a reality.

Adult acute myelocytic leukemia (AML) is a curable disease in responsive patients with aggressive treatment in remission. Over the past decade at the Johns Hopkins Oncology Center, AML has been treated with either allogeneic bone marrow transplantation or with intensive timed sequential treatment using high dose cytarabine in remission. With either treatment modality comparable cure rates were obtained. The role, if any, of randomized trials to adequately determine the preferred treatment for appropriate patients has yet to be defined.

Characterization of the natural suppressor cell population in adult rat bone marrow.

Natural suppressor cell activity (NSCA) has been ascribed to a subset of cells present in human and murine hematopoietic tissues which can suppress a variety of lymphocyte responses without MHC restriction. We investigated NSCA in lymphocyte-depleted rat bone marrow (BM) which is used as a model for prevention of graft vs host disease (GVHD) following allogeneic BM transplantation (BMT). The T-cell depleted fraction obtained after elutriation contained higher levels of NSCA than the unseparated BM. Further separation of this graft fraction by discontinuous Percoll gradient centrifugation revealed high levels of radiosensitive NSCA in the low density (less than 1.070) fraction which represented 0.5% of the original BM population. These cells were of blast morphology, stained intensely with a dansylated derivative of cyclosporine A (dans CsA) and weakly expressed macrophage/granulocyte antigens and non-specific esterase (NSE). These cells were initially non-adherent but proliferated in culture to produce intensely NSE positive, adherent, phagocytic cells of macrophage morphology. We conclude that the highly suppressive, radiosensitive cell present in rat BM may be of early progenitor or monocyte lineage. The grafting of natural suppressor (NS) cells and progenitor cells may affect graft/host immunoregulation and their characterization may provide insight into GVH biology and graft rejection.

Mitigation of Graft-versus-host disease in mice with xenogeneic antithymocyte serum and complement.

In vitro treatment of parental C57BL/6 lymphohematopoietic cell grafts with unabsorbed guinea pig anti-mouse thymocyte serum (ATS) and guinea pig complement (GPC), prior to inoculation into lethally irradiated B6D2F hybrid hosts, has proven to be of value in terms of mitigating graft-versus-host disease (GvHD). However, the beneficial effect of such a pregrafting procedure is limited to the prevention of acute GvHD. The late GvHD remains a continuing problem, and is probably due to the graft-versus-host activity (GvHA) of newly produced nontolerant lymphocytes from lymphoid precursors resistant to ATS. Possible ways to render these precursors sensitive to ATS and complement are discussed. The potential significance of thymic hormones and cyclic AMP in achieving this is emphasized.

In vitro activation of human suppressor cells: relative quantitation and specificity of suppressor cells generated in primary and secondary mixed lymphocyte cultures.

Human suppressor cells activated in primary and secondary mixed lymphocyte cultures (MLC) were quantitatively assessed for their ability to suppress the proliferative response of fresh responding lymphocytes to the original sensitizing (specific suppression) and third party, unrelated (non-specific suppression) alloantigens. Maximal levels of both specific and non-specific suppressor cell activities generated in primary MLC were found in the population of cells harvested on day 7 which corresponded to the peak of proliferation in bulk culture and decayed upon further culture reaching a nadir concomitant with the end of proliferation in primary bulk MLC. Maximal reactivation of the suppressor cells was accomplished by restimulation in secondary MLC with the original sensitizing alloantigens. Assessment of suppressor cell activity revealed that specific rechallenge with the original stimulating alloantigens in secondary MLC resulted in significant increases in both the levels of suppressor cell activity and in the specificity of action of the suppressor cell population as compared to suppressor cells harvested from primary MLC on day 7. Both primary and secondary MLC activated suppressor cells were capable of suppressing the induction of cytolytic lymphocytes in primary MLC. Suppression was not simply the result of altered kinetics of the fresh micro MLC response.

Recovery of hematologic competence without engraftment following attempted bone marrow transplantation for aplastic anemia: Report of a case with diffusion chamber studies.

An 18-year-old male with severe aplastic anemia associated with hepatitis was prepared for bone marrow transplantation with cyclophosphamide (50 mg/kg/day) for 4 consecutive days. He then received 3.47 X 10(8) nucleated bone marrow cells per kilogram of body weight from his HL-A identical sister by intravenous infusion. Although subsequent karyotyping showed that engraftment was not achieved, prompt hematologic recovery ensued by the third week post-transplant. Diffusion chamber culture of the patient's bone marrow stroma, obtained prior to cyclophosphamide therapy, was carried out. A prompt increase in the number of cells in the chambers was observed. A differential cell count of the contents was similar to that seen when normal bone marrow is cultured in a similar manner.

Separation of rat bone marrow cells by counterflow centrifugal elutriation: a model for studying the effects of lymphocyte depletion.

We have developed a simple three flow-rate, fixed rotor speed, counterflow centrifugal elutriation (CCE) procedure that permits the isolation of an engraftable lymphocyte-depleted (greater than 98%) fraction from ACI rat bone marrow. The different cell fractions were characterized by morphology, alloreactivity in mixed lymphocyte culture and limiting dilution analysis, colony-forming capacity, and their capacity to reconstitute hematopoiesis and effect a graft-versus-host reaction in lethally irradiated allogeneic hosts. After CCE fractionation of ACI rat marrow, transplantation of the lymphocyte-depleted marrow fraction resulted in sustained engraftment without evidence of clinical or histologic acute graft-versus-host disease (GVHD). CCE fractionation of rat bone marrow may be a useful preclinical model for studying lympho-hematopoietic and immune reconstitution after transplantation with lymphocyte-depleted donor marrow, as well as for studying the role of lymphocyte subpopulations on engraftment, acute GVHD, and leukemia relapse in syngeneic and allogeneic bone marrow transplantation.

Autologous bone marrow transplantation in the treatment of selected human malignancies: The Johns Hopkins Oncology Center Program.

Preliminary clinical trials using cryopreserved autologous bone marrow reinfusion have now been carried out at our institution in 5 children and 2 adults with advanced stages of neuroblastoma, rhabdomyosarcoma, non-Hodgkin's lymphoma and small cell carcinoma of the lung. Normal numbers of in vitro colony forming cells (CFU-C) were obtained from these patients despite prior courses of combination chemotherapy. The dose of marrow cells cryopreserved ranged from 1-6 X 10(8) cells/kg and recovery of CFU-C after thawing averaged 50%. Partial or complete hematologic reconstitution was achieved in all patients. The time for recovery ranged from 10-43 days for leukocytes (greater than 1000 cells/mm3) and 23-45 days for platelets (greater than 50,000/mm3). Two patients have died of interstitial pneumonitis due to cytomegalovirus. Three patients have died of recurrent tumor at 40, 48 and 156 days post-transplant. Two patients have had significant therapeutic benefit. One of these had a stable partial response permitting the use of further post-transplant therapy and is alive and well 16+ months post-transplant. The other patient had a complete response and remains tumor-free 25+ months following therapy. We conclude: 1) Autologous bone marrow reinfusion permits hematologic reconstitution following marrow-ablative therapy. 2) A quantity of marrow sufficient for this purpose remains viable following cryopreservation even when obtained from patients previously exposed to chemotherapy. 3) Autologous bone marrow reinfusion now allows the exploration of more intensive cytoreductive therapy in selected malignancies.

Cutaneous graft-versus-host reaction lacks evidence of cutaneous cytomegalovirus by the immunoperoxidase technique.

Sixty skin biopsy specimens from 21 bone-marrow transplant patients were evaluated for the presence of cytomegalovirus (CMV) using two monoclonal antibodies to early and late antigens. Each patient had at least one biopsy showing an acute graft-versus-host reaction (GVHR), grade 2, and one positive culture for CMV from blood, bone marrow or urine. In no case could CMV antigens be identified in biopsies showing an acute or chronic cutaneous GVHR or in any other of the skin biopsies obtained from these patients. While CMV may play a role in immunologic events culminating in graft-versus-host disease (GVHD), this immunoperoxidase study did not reveal evidence of viral antigens in tissue displaying features of cutaneous GVHR.

Acute graft-versus-host disease: clinical characteristics in the cyclosporine era.

Graft-versus-host disease (GVHD) remains the major problem in allogeneic bone marrow transplantation. GVHD has limited the use of this technique to HLA-matched donor recipient pairs. Thus, only a quarter of patients who ultimately may have benefited from bone marrow transplantation are currently eligible. Even in matched patient recipient pairs, GVHD accounts for approximately 40% of the deaths following allogeneic bone marrow transplants. One of the major challenges for transplantation is to derive better strategies to prevent and treat GVHD while retaining the allogeneic benefit of graft-versus-leukemia. Current pharmacologic approaches have used cyclosporine, usually in combination with other drugs. More experimental approaches have removed lymphocytes from the marrow grafts. With either approach, maintaining the anti-leukemic benefit of an allogeneic transplant (i.e., immunologic attack of the leukemia resulting in a lower relapse rate), will need to be maintained if that approach will ultimately prove to be useful.