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.

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.

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.

Polyneuropathy complicating bone marrow and solid organ transplantation.

We report a generalized polyneuropathy coincident with the occurrence of graft-versus-host disease in four patients undergoing bone marrow transplantation and accompanying solid organ rejection (heart and kidney) in two patients. The neuropathy affected proximal and distal muscles, demonstrated hyporeflexia or areflexia, and usually had elevated CSF protein. Electrophysiologic studies did not meet strict criteria for demyelination. The signs of neuropathy improved after immunosuppressive treatment or simultaneous to the resolution of graft-versus-host disease or tissue rejection. Polyneuropathy must be considered as a potential complication of tissue transplantation.

Bone marrow transplant in adrenoleukodystrophy.

An allogeneic bone marrow transplant (BMT) from a normal HLA identical sibling donor was performed in a 13-year-old boy with rapidly progressive adrenoleukodystrophy (ALD). Engraftment and complete hematologic recovery occurred within 4 weeks, but neurologic deterioration continued. The patient died of an adenovirus infection 141 days after BMT. ALD is characterized by abnormally high plasma levels of very long chain fatty acids (VLCFA) as a result of impaired capacity to degrade them. Ten days after BMT, the white blood cell VLCFA levels and enzyme activity became normal; after 3 months, there was progressive reduction of plasma VLCFA to levels only slightly above normal.

Bone marrow transplantation in the busulfan-treated rat. II. Effect of cyclophosphamide and antithymic serum on the presensitized state.

The purpose of the present study was to develop a model in the busulfan (BU)-treated aplastic rat for investigating sensitization to donor marrow in various donor and recipient combinations differing in their degree of histocompatibility. In addition, cyclophosphamide (CY) and rabbit antirat thymocyte serum (RARTS) were studied as potential agents for reversing the presensitized state. Marrow transplantation was studied in the following donor-host combinations: ACI-Lewis (Ag-B-histoincompatible), F344-Lewis (Ag-B-histocompatible), and F344-LBNF1 ("very histocompatible"). The administration of 10(7) donor-type spleen cells i.v. 9 days before marrow infusion sensitized (prevented marrow takes) in all three systems. BN cells were able to sensitize Lewis recipients to F344 marrow because of minor antigens shared by the BN and F344 rat strains. RARTS given as six consecutive daily doses (1.5 ml each) beginning 1 day after immunization and ending 1 day before marrow grafting failed to abolish the presensitized states in all three systems studied. A single dose of CY (150 mg/kg), given alone or combined with RARTS, was able to reverse the measurable effects of presensitization in the histocompatible and "very histocompatible" situations, but was without effect on the histoincompatible system. The present studies were performed in a limited number of rat strain combinations, and the general and specific effects of CY on presensitization to minor histocompatibility antigens in other situations such as outbred mammalian systems remain to be determined.

Bone marrow transplantation in the busulfan-treated rat. I. Effect of cyclophosphamide and rabbit antirat thymocyte serum as immunosuppression.

It was previously shown that lethal doses of busulfan (BU) were not immunosuppressive in the rat. In the present studies the effect of rabbit antirat thymocyte serum (RARTS) and cyclophosphamide (CY) as immunosuppressive preparation for marrow allografts in the rat given supralethal doses of BU was investigated. Lewis or LBNF rats were given RARTS for 4-12 consecutive daily doses. They were given a supralethal dose of BU in addition on the last day of RARTS administration. Ag-B-incompatible (ACI). Ag-B-compatible (F344), or syngeneic marrow was infused 24 hr later. In general, RARTS was not toxic to the marrow graft and permitted engraftment of Ag-B-incompatible and compatible marrow. All animals given Ag-B-incompatible marrow and Ag-B-compatible marrow that survived to day 21 were shown to be chimeric, but all those with incompatible marrow subsequently died from severe graft-versus-host disease (GVHD). In other experiments Lewis or LBNF1 rats were given Ag-B-incompatible (ACI) and compatible (F344) marrow after a supralethal dose of BU and single graded doses of CY. Doses of CY as low as 25-50 mg/kg provided sufficient immunosuppression to allow allogeneic engraftment. Transient chimerism was seen up to doses of 100 mg/kg of CY. Permanent chimerism was seen with doses of CY from 150 to 250 mg/kg. The incidence of lethal GVHD increased in parallel with the percentage of animals that were demonstrated to be chimeric. Mild but transient GVHD was seen with Ag-B-compatible marrow given to Lewis rats.

Chronic graft-versus-host disease in the rat radiation chimera. III. Immunology and immunopathology in rapidly induced models.

Although chronic graft-versus-host disease (GVHD) frequently develops in the long-term rat radiation chimera, we present three additional models in which a histologically similar disease is rapidly induced. These include adoptive transfer of spleen and bone marrow from rats with spontaneous chronic GVHD into lethally irradiated rats of the primary host strain; sublethal irradiation of stable chimeras followed by a booster transplant; and transfer of spleen cells of chimeras recovering from acute GVHD into second-party (primary recipient strain) or third-party hosts. Some immunopathologic and immune abnormalities associated with spontaneous chronic GVHD were not observed in one or more of the induced models. Thus, IgM deposition in the skin, antinuclear antibodies, and vasculitis appear to be paraphenomena. On the other hand, lymphoid hypocellularity of the thymic medulla, immaturity of splenic follicles, and nonspecific suppressor cells were consistently present in the long term chimeras, and in all models. These abnormalities therefore may be pathogenetically important, or closely related to the development of chronic GVHD.

Bone marrow transplantation without total-body irradiation in patients aged 40 and older.

We evaluated relapse-free survival and the incidence and type of complications in 17 patients aged 40 or older with chronic myelogenous leukemia, acute myelogenous leukemia, or lymphoma who underwent allogeneic marrow transplantation following busulfan 16 mg/kg and cyclophosphamide 120 mg/kg. Nine patients are disease-free survivors 5-38 months (median 26 months) following transplantation. The incidence of grades II-IV acute graft-versus-host disease was 35%. No significant difference was detected in the incidence of GVHD or interstitial pneumonia between patients aged 40 and older and a group of younger patients transplanted over the same time period. These observations should encourage consideration of allogeneic marrow transplantation in older patients and suggest that this busulfan-cyclophosphamide regimen is a promising alternative to regimens containing total-body irradiation in older individuals.

Suppressor cells in transplantation tolerance. II. maturation of suppressor cells in the bone marrow chimera.

Histoincompatible bone marrow allografts were established in lethally irradiated rats. At various times after transplantation, the spleen cells were harvested, subjected to mixed lymphocyte cultures, and assayed for suppressor cells in vitro and in vivo by adoptive transfer studies. Alloantigen-nonspecific suppressor cells appeared in the chimera at 40 days after grafting, coinciding with the resolution of graft-versus-host disease (GVHD). At 250 days the nonspecific suppressor cells were replaced by suppressor cells specifically suppressing donor-versus-host alloantigen responses. At 720 days suppressor cells could no longer be identified by in vitro methods but were identified by in vivo adoptive transfer of transplantation tolerance. After injection of host-type antigen into chimeras, the suppressor cells could be again demonstrated by in vitro methods.

Suppressor cells in transplantation tolerance. I. Suppressor cells in the mechanism of tolerance in radiation chimeras.

Histoincompatible-complete radiation chimeras, after resolving acute graft-versus-host disease (GVHD), establish specific tolerance to host and donor alloantigens. This tolerance can be perturbed with immunosuppressive agents and infusions of small numbers of donor-type cells, with infusions of massive numbers of donor-type cells, or with infusions of a small number of donor-type cells, that were sensitized against host antigens prior to transfer. These chimeras possess T lymphocytes in the spleen that specifically suppress donor to host mixed lymphocyte reactions and adoptively transfer suppression of GVHD to secondary hosts. Nylon-wool fractionation of chimeric spleen cells restores the response of chimeric lymphocytes to host alloantigens, suggesting that transplantation tolerance is not attributable to clonal deletion but the activity of nylon-wool-adherent T suppressor spleen cells.

Suppressor cells in transplantation tolerance. III. The role of antigen in the maintenance of transplantation tolerance.

Suppressor cells, which in an alloantigen-specific manner inhibit proliferation of donor cells to host antigens in a mixed lymphocyte culture and adoptively transfer the suppression of graft-versus-host disease (GVHD), undergo a gradual clonal reduction in long-term, allogeneic, histoincompatible rat radiation chimeras until they can no longer be measured in an in vitro suppressor cell assay. When lymphohematopoietic cells from these chimeras are transferred into lethally irradiated secondary recipients of original donor strain, the suppressor cells, now in a target antigen-free environment, undergo a further clonal reduction. After "parking" for 120 days, the chimeric cells are specifically tolerant to original host antigens, but cannot adoptively transfer suppression of GVHD. When chimeric cells, parked for 120 days in secondary recipients of original donor strain, are stimulated with original host-type antigen repeatedly during or once at the end of the parking period, the suppressor cell clone is expanded, suppressor cells can be identified in vitro, and suppression of GVHD can adoptively be transferred to tertiary recipients.

Graft-versus-host disease and sialodacryoadenitis viral infection in bone marrow transplanted rats.

The effect of a localized viral infection on the occurrence of graft-vs.-host disease (GVHD) was examined in allogeneic rat bone marrow chimeras (ACI/LEW). Animals without clinical evidence of GVHD, 62 days after bone marrow transplant, were infected in salivary and lacrimal glands with sialodacryoadenitis virus (SDAV), and sacrificed 8-25 days postinfection. Using established histologic criteria, GVHD was found more frequently in salivary and lacrimal glands of SDAV-infected chimeras than uninfected chimeras. Skin and oral mucosa, tissues not infected by the virus, showed no differences in occurrence of GVHD, suggesting that the viral infection induced only local and not systemic GVHD. GVHD and SDAV infection, which are histologically similar, were differentiated by examining tissues for SDAV antigen using immunoperoxidase technique. Histologic changes were present for at least 1 week longer than viral antigen, suggesting they represented GVHD rather than viral infection. GVHD and SDAV infection were also differentiated by looking for a histologic feature characteristic of GVHD and not found in SDAV infection (periductal lymphocytic infiltrate). This was found in SDAV-infected chimeras more frequently than uninfected chimeras, suggesting that the viral infection somehow induced GVHD. Results showed a localized increase in the occurrence of GVHD subsequent to localized viral infection.

Cyclosporine-associated seizures in bone marrow transplant recipients given busulfan and cyclophosphamide preparative therapy.

Five of 182 recipients of allogeneic bone marrow transplants performed between 2/84 and 6/90 developed seizures while receiving cyclosporine and methylprednisolone to prevent acute graft-versus-host disease. All received a radiation-free regimen of busulfan and cyclophosphamide as preparative therapy. Two patients received HLA-mismatched allografts; and three patients received marrow from HLA-identical sibling donors. Two patients had received extensive intrathecal therapy prior to transplantation. All patients were receiving standard prophylactic doses of CsA and MP at the time of onset (median 31 days posttransplantation) of seizures. Three patients had mild-to-moderate hypertension and varying degrees of morphologic evidence of microangiopathic hemolytic anemia. None had unusually low magnesium levels. Cyclosporine levels were not in the toxic range. Cranial magnetic resonance imaging and computed tomography (CT) showed bilateral abnormalities primarily in the posterior temporal, occipital, and parietal lobes. These abnormalities were shown to be transient on sequential MRI exams in two patients. Seizures as well as radiologic abnormalities resolved on stopping CsA and did not recur in 2 patients who subsequently received CsA in lower doses. These findings confirm and expand previous observations of CsA-associated seizures and demonstrate that they occur in allogeneic bone marrow transplant recipients following a radiation-free preparative regimen of busulfan and cyclophosphamide.

Functional asplenia in patients with chronic graft-versus-host disease: concise communication.

Liver/spleen images were performed with technetium-99m sulfur colloid in 53 patients who had undergone bone-marrow transplantation. The spleen was not seen in the images in five out of the ten patients with chronic graft-versus-host disease (GVHD). None of the five had a history of splenectomy. In two of these patients, anatomical presence of the spleen had been documented earlier by scintigram. The spleen was visible in all seven patients with acute and in all 36 patients without GVHD. Neither the differences in methods of treating the patients before bone-marrow transplantation nor the time lapse between transplantation and the liver/spleen image correlated with the observed effect among these three groups of transplant patients. We conclude that there is a high association between chronic GVHD and functional asplenia.

Analysis of airflow obstruction by bronchoalveolar lavage following bone marrow transplantation. Implications for pathogenesis and treatment.

The development of airflow obstruction, most often due to bronchiolitis, is a significant cause of morbidity and mortality in recipients of allogeneic BMT. Current consensus holds that this airways disease is the result of chronic GVHD and/or CMV infection. However, recent studies of idiopathic forms of BRO have demonstrated a striking influx of neutrophils into the lungs of affected individuals. Reasoning that the immune cell populations involved in tissue injury associated with either CGVHD or CMV infection would consist predominantly of lymphocytes, we tested this hypothesis by performing BAL in 12 adults with minimal or absent smoking histories who developed significant airflow obstruction (FEV1/FVC = 80.7 +/- 1 percent preBMT and 56.8 +/- 2.4 percent postBMT; p less than 0.001) following allogeneic BMT. Eleven of 12 patients had evidence of chronic, stable GVHD at the time of the study. In contrast to non-BMT patients with BRO, BAL defined two distinct patterns of lung inflammation in the BMT patients with airflow obstruction: (a) neutrophil predominance (five patients; neutrophil percentage = 20.2 +/- 6.6 percent); and (b) lymphocyte predominance (three patients; lymphocyte percentage = 35.9 +/- 12.1 percent). These data suggest that the pattern of inflammation in the lungs of BMT patients with BRO is not uniform and is not associated with active microbial infection. From these results, it is inferred that the airways injury in BMT patients may reflect diverse pathogenetic mechanisms initiated in the context of CGVHD and cytotoxic drug therapy.

Infections and immunodeficiency in bone marrow transplantation.

After allogeneic bone marrow transplantation certain patterns of infectious complications emerge that follow the clinical course, are correlated to the immunobiology of transplantation and are almost predictable in their character and expression. The preparative regimen, designed to generate complete aplasia, will be associated with severe and sometimes life-threatening bacterial infections, predominantly with Gram-negative organisms derived from bowel flora, but also Gram-positive skin saprophytes. In this early aplastic phase, life-threatening viral infections are less common, consisting mainly of herpes simplex and possibly Epstein-Barr stomatitis and BK papovavirus cystitis. Systemic infections with invasive filamentous fungi are rare and are seen only when the induced aplasia is markedly prolonged. Once early marrow recovery has been achieved, systemic infections will generally disappear unless acute graft-vs.-host disease develops. This complication, which will lead to the breakdown of natural barriers such as skin and gastrointestinal epithelium and the marked impairment of all systemic defense mechanisms, can cause polymicrobial infections as well as set the stage for life-threatening viral infections. Such opportunistic viral infections, leading to either interstitial pneumonia or hemorrhagic gastroenteritis, are the major threat in the early recovery phase after engraftment has taken place. Usually caused by cytomegalovirus and rotavirus, respectively, these infections are the primary expression of the severe combined immunodeficiency post transplant, statistically associated with the presence of acute graft-vs.-host disease and amenable to immunologic manipulations. With the recovery of cellular and humoral immune function derived from transplanted donor lymphoid cells, the third phase of infectious complications is reached, covering 3 months to 2 years post grafting.(ABSTRACT TRUNCATED AT 250 WORDS)

Marrow transplantation following busulfan and cyclophosphamide in multiple myeloma.

We report the use of busulfan and cyclophosphamide as conditioning before allogeneic bone marrow transplantation for a 38-year-old man with multiple myeloma. Three months post-transplant the monoclonal IgA band previously present was no longer detectable in the serum and plasma cells were no longer visible in the marrow. The patient died on day 93 with fungal infection. We conclude that the combination of busulfan and cyclophosphamide could be valuable before transplant for other patients with multiple myeloma.

Autologous peripheral blood progenitor cell transplantation for non-Hodgkin's lymphoma with extensive bone marrow necrosis.

A patient with malignant lymphoma, large cell type and extensive bone marrow necrosis at presentation is reported. Bone marrow necrosis persisted even after a complete remission was induced with standard chemotherapy. Because of this adverse prognostic factor, circulating stem cells were collected and reinfused following a myeloablative regimen consisting of busulfan, etoposide and cyclophosphamide. The patient engrafted rapidly and a subsequent bone marrow examination was free of both lymphoma and necrosis. To the best of our knowledge, this is the first reported patient with extensive bone marrow necrosis in whom circulating progenitor cells were harvested and utilized successfully.

Allogeneic marrow transplantation in non-Hodgkin's lymphoma.

Nine individuals between 15 and 43 years of age with non-Hodgkin's lymphoma underwent allogeneic marrow transplantation following busulfan 16 mg/kg and cyclophosphamide 120 mg/kg. These individuals were not considered optimal candidates for autologous transplantation chiefly because of marrow involvement or resistance to chemotherapy. All patients engrafted and eight achieved complete remission. Three patients relapsed; one patient died of transplant-related complications. Five individuals are disease-free survivors between 103 and 1169 days following transplantation. Of three individuals with relapsed Burkitt's lymphoma none experienced a sustained disease-free interval following transplantation. Three of four individuals with large cell or lymphoblastic lymphoma are surviving 585 to 1169 days following transplantation. Allogeneic marrow transplantation following busulfan and cyclophosphamide appears reasonably safe and is effective in selected patients with non-Hodgkin's lymphoma who are not good candidates for autologous marrow transplantation.