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The capacity of busulfan and total body irradiation to ablate hematopoietic stem cells as preparation for the allogeneic bone marrow transplantation of patients with congenital bone marrow disorders was studied. Fourteen patients received 18 transplants; busulfan was used in the preparatory regimen of eight transplants and total body irradiation in the regimens of six transplants. Sustained hematopoietic ablation was achieved in six of eight patients prepared with busulfan and in all six patients prepared with total body irradiation. Three patients prepared with total body irradiation died with idiopathic interstitial pneumonitis, whereas no patients receiving busulfan developed interstitial pneumonitis. The optimal antihematopoietic stem cell agent to be used for the preparation of patients with congenital bone marrow disorder for bone marrow transplantation is not certain.

Prompted by previous reports that in certain patients with aplastic anemia, cell-mediated autoimmune suppression of myeloid stem cell proliferation may be demonstrable in vitro, we studied the effects of bone marrow lymphocytes from 18 patients with myeloid aplasia on the proliferation of committed granulocytic-monocytic progenitor cells (CFU- C). When assayed in soft agar cultures, marrow suspensions from 10 patients with aplastic anemia contained significantly fewer viable CFU- C than similar cell preparations from control subjects. To deplete marrow cell suspensions of lymphocytes, we employed rabbit anti-human thymocyte serum (ATS), which after multiple adsorptions exhibited marked cytotoxicity for human B and T lymphocytes but had negligible effect on normal CFU-C proliferation. Preincubation of marrow samples from 12 patients with ATS and complement resulted in no inhibition or enhancement of CFU-C growth. In further experiments, marrow cells from 8 patients were incubated with marrow from control subjects prior to CFU-C culture. No suppression of donor CFU-C proliferation was observed in any of these studies, and in 4 cocultures, mixture of the 2 marrow suspensions resulted in stimulation of CFU-C growth. Using these assays, we detected no evidence of cell-mediated inhibition of CFU-C proliferation in any of the 18 patients that we evaluated. Our data support the conclusion that in the majority of patients with aplastic anemia, an absolute deficiency of hemopoietic stem cells is present within the marrow that does not appear to be effected or sustained by suppressor lymphocytes. Whether the reduction of viable stem cells is the cause or the consequence of the process that leads to marrow failure remains unknown.

Isolated thrombocytopenia after bone marrow transplantation was investigated in 65 fully engrafted patients surviving at least 60 days posttransplant. Twenty-four patients (37%) developed this complication, which occurred most frequently in patients receiving pretransplant preparation with total body irradiation or busulfan. Two distinct thrombocytopenic syndromes were identified: (1) transient thrombocytopenia (nine patients), in which a normal platelet count (greater than 100,000/microL) was initially established by day +40 but then diminished to less than 10,000 to 45,000/microL on day +40 to +70, with subsequent resolution of the thrombocytopenia by day +90; (2) chronic thrombocytopenia (15 patients), in which a platelet count greater than 100,000/microL was not achieved at any time during the first four months posttransplant, despite the simultaneous presence of normal granulocyte and reticulocyte counts. Although the transient syndrome did not adversely affect prognosis, the chronic syndrome carried a high mortality (21% actuarial survival at 1,000 days posttransplant compared with 67% survival for all patients, P less than .01) and had a high association with both severe (grades 3 to 4) acute graft-versus-host disease (GVHD) and chronic GVHD. In three of nine patients with transient thrombocytopenia, a temporal association with trimethoprim-sulfamethoxazole administration was observed, whereas in all other patients, no drug association could be found. Bone marrow biopsies in those patients with drug-associated thrombocytopenia showed decreased numbers of megakaryocytes, whereas biopsies in the remainder of the transiently thrombocytopenic patients demonstrated adequate numbers of platelet precursors, suggesting peripheral platelet destruction or ineffective thrombopoiesis. Biopsies in the chronic thrombocytopenic patients included those with and without adequate numbers of platelet precursors, although the association with chronic GVHD was strongest in patients demonstrating normal numbers of megakaryocytes. We conclude that isolated thrombocytopenia represents a significant complication of bone marrow transplantation, particularly in patients receiving hematopoietic ablative preparatory regimens, and that it is the chronic, not the transient, thrombocytopenic syndrome that is associated with an adverse patient prognosis.

Lymphocytes from 18 patients with the Wiskott-Aldrich Syndrome (WAS) were examined by scanning electron microscopy (SEM). Most peripheral blood lymphocytes from normal individuals are covered with slender microvillus projections, but a large proportion of lymphocytes from WAS patients were found to be relatively devoid of microvilli. A lymphocyte morphology scoring system was developed to quantify the density of microvilli: Grade 4 classified those lymphocytes with greater than 75% of the surface covered with microvilli with progressive decrements to grade 1, which were those without microvilli. The mean lymphocyte morphology score of eight normal individuals was 3.62 +/- .22. The mean lymphocyte score of WAS patients was substantially lower (2.89 +/- .27, P less than .001). In addition, WAS lymphocytes often were qualitatively abnormal, with short, blunted microvilli. These morphological criteria were used to diagnose WAS from the cord blood lymphocytes of one “at-risk” patient. Thus, WAS is the first primary immunodeficiency in which morphological abnormalities have been identified that can aid in diagnosis.

We performed a prospective randomized trial of antithoracic duct lymphocyte globulin (ATDLG), HLA-haploidentical marrow, and androgen (regimen ABA) versus androgen alone (concurrent STANDARD care controls) in 42 newly diagnosed individuals with severe aplastic anemia. ABA patients also were matched with patients from our preceding study (historical STANDARD care controls). Supportive care and pretreatment patient characteristics were the same in all groups. By life table analysis, 76% of patients receiving ABA are alive at 2 yr compared to 31% of the concurrent control group (p less than 0.002 versus ABA) and 19% of the historical controls (p less than 0.0001 versus ABA) given STANDARD care. ABA patients had greater hematologic improvement than either control group (p less than 0.001). However, improvement with ABA was often incomplete. Toxicity of ATDLG was considerable but manageable. Further studies to determine the mechanism of action and active component(s) of ABA are indicated.

Of 23 patients with severe aplastic anemia, 17 were sensitized to histocompatibility antigens of HLA-A, -B, and -D loci-identical potential sibling donors as determined by cell-mediated lysis (CML) assays in vitro. Antibody-dependent sensitization was detected in 3 patients, antibody-independent cellular sensitization in 11, and both in 3. Fourteen sensitized patients were transplanted after initial multiagent immunosuppression consisting of rabbit anti-human thymocyte serum, procarbazine, and cyclophosphamide, eleven with a CML-positive donor and three with a CML-negative donor. Engraftment was achieved in each of 13 patients who were evaluable, and only 2 ultimately rejected their marrow grafts, 1 with subsequent return of his own marrow function. Five patients without evidence in vitro of sensitization were transplanted after immunosuppression with cyclophosphamide alone; none of these rejected their grafts. These studies show that sensitized bone marrow transplant recipients can be successfully transplanted after optimal donor selection and multiagent immunosuppression.

The capacity of peripheral blood mononuclear cells from bone marrow transplant recipients to proliferate and produce interferon in response to mitogens and specific antigens was tested. Proliferation in response to phytohemagglutinin or pokeweed mitogen occurred in cells from more than 90% of the recipients, and interferon was present in 60 to 70% of the supernatants from these cultures, even when tested as soon as 8 weeks after transplantation. Proliferation in response to bacterial antigens was infrequent, and interferon release was not detected. In the early post-transplantation period (less than 13 weeks), cells from only two of four cytomegalovirus (CMV) antibody-positive patients proliferated normally in response to CMV antigen and interferon release was detected only once. In the late post-transplantation period (more than 13 weeks), in only two of five instances did cells proliferating in response to CMV antigen release interferon. The response to CMV antigen of mononuclear cells from many transplant recipients differs from that of cells from normal controls.

Despite androgens and intensive supportive care, satisfactory survival in severe aplastic anemia remains at 20% or less. Histocompatible bone marrow transplantation can restore normal hematopoiesis in approximately 40% of similarly severe individuals. Delay of transplantation for 3 wk after diagnosis allows time for proper evaluation and for many spontaneous recoveries. Further delay increases risks of fatal complications and decreases chances for successful transplantation while the incidence of spontaneous remission declines. When available, early histocompatible bone marrow transplantation may be the treatment of choice for severe aplastic anemia.

gpL115 is a lymphocyte surface component that is deficient in patients with the X-chromosome-linked immune deficiency Wiskott-Aldrich syndrome (6). The glycoprotein nature of gpL115 is demonstrated through labeling in carbohydrate moieties by [3H]NaBH4 and its synthesis by lymphocytes through labeling with [35S]methionine. Native gpL115 adheres to wheat germ lectin-Sepharose and sialidase-treated gpL115 does not adhere, indicating that native gpL115 adheres via clusters of sialic acid residues. When tested on peanut lectin, which shows specificity for the disaccharide Gal beta 1-3GalNAc, gpL115 is nonadherent and sialidase-treated gpL115 is adherent, indicating the presence of the sequence sialic acid-Gal beta 1-3GalNAc, which is characteristic for O-linked (mucin-type, acidic-type) carbohydrates. A surface glycoprotein with all the above characteristics was found on the lymphoblastoid cell line CEM. CEM cells were used as immunogen to generate the monoclonal antibody L10, an IgG1, which binds native and sialidase-treated gpL115 . Sialidase-treatment of gpL115 significantly alters its physical properties, reducing its electrophoretic mobility and changing its behavior on isoelectrofocusing. Cumulatively, these findings indicate that gpL115 , like glycophorin of erythrocytes and GPIb of platelets, is a sialoglyco protein with significant quantities of O-linked carbohydrate. On treatment with limiting sialidase concentrations, gpL115 of normal lymphocytes is transformed into a series of partially desialylated species of decreasing electrophoretic mobility. This finding resembles the situation with lymphocytes of some Wiskott-Aldrich syndrome patients. Lymphocytes of eight Wiskott-Aldrich syndrome patients were found to be deficient in 125I-labeled gpL115 . Lymphocytes from three of these patients displayed an abnormal 125I-component of apparent mol wt 135,000.

A naturally occurring subpopulation of human peripheral blood lymphocytes is cytotoxic to autologous and/or allogeneic fibroblasts. The autocytotoxic lymphocytes have a receptor for the third component of complement and for aggregated gamma globulin, do not form rosettes with sheep red blood cells, and are not removed by passage through nylon. The autocytotoxic subpopulation is not present in the thymus and tonsils of normal children or in the peripheral blood of individuals with X-linked agammaglobulinemia. Fibroblast absorption experiments demonstrate that the autocytotoxic cells are "sensitized" to antigens expressed on allogeneic fibroblasts in addition to the antigens expressed on autologous cells. Some normal individuals have a second subpopulation of lymphocytes that may "regulate" the autocytotoxic cells. The relevance of these observations to the murine autocytotoxic cells is discussed.

Objective: The purpose of this study was to determine whether thymectomy in the newborn has a negative effect on immune function. Methods: Twenty-five neonates (<30 days) who had thymectomy at congenital heart repair were prospectively studied to determine immune function. The percentage of T-cell subtypes including CD3 (all T cells), CD4 (helper T cells), and CD8 (suppressor T cells) was determined. In six patients, further testing of CD4 cells was done to determine whether they were newly formed, recent thymic emigrants (CD4, CD45, and RA), or older educated lymphocytes (CD4, CD45, and RO). Response to the mitogen phytohemagglutinin and to tetanus toxoid were determined, as were antibody titers to tetanus. Samples were drawn before the thymectomy, at approximately 3 months after immunization and at 1 year. Ten age-matched control patients were tested. At follow-up, parents were asked about infections. Results: Prethymectomy T-cell subsets were all normal and comparable to controls. At 12 months, the percent of CD3 was significantly less than in the control group (48% +/- 3% versus 64% +/- 2% [mean +/- standard error of the mean]; p < 0.01) as was CD4 (31% +/- 2% versus 46% + 2% [mean +/- standard error of the mean]; p = < 0.01). CD8 did not drop. Surprisingly, the percent of CD4 that were recent thymic emigrants did not decrease significantly (50% +/- 8% versus 60% +/- 6% [mean +/- standard error of the mean]; p = not significant). Lymphocyte blastogenesis to phytohemagglutinin and tetanus toxoid and antibody to tetanus were all normal at 12 months. No patient required readmission for infection, and there were the expected number of minor infectious events (median 3; 95% confidence interval 1,4). Conclusion: Thymectomy in neonates results in a modest but significant decrease in T-lymphocyte levels, but there is no compromise in immune function. (J Thorac Cardiavasc Surg 1998;115:1041-6)