Separation of human bone marrow cell populations by density gradient electrophoresis: differential mobilities of myeloid (CFU-C), monocytoid, and lymphoid cells.
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Biomedical subjects
Publications and source records attributed to N Kapoor.
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Marrow cells from 16 patients with severe combined immunodeficiency diseases (SCID) were examined for the presence of T cell precursors which could be induced to express surface markers and functions of T lymphocytes after exposure in vitro to thymic extracts or peptides of thymic origin (thymopoietin and thymopoietin32-36 (TP-5). Marrow cells from 14 patients studied before transplantation revealed three patterns of response. In five patients, inducible T cell precursors were not detected. In six patients, precursors were detected which could be induced to express a human T lymphocyte antigen (HTLA) but acquired little or no ability to rosette with sheep erythrocytes (SRBCs). Induction of HTLA-positive and E rosette-positive lymphocytes was normal in only two patients, both of whom were engrafted with maternal lymphocytes as an apparent result of an intrauterine infusion. Induction of mixed leukocyte reaction (MLR) responses following exposure to thymic hormones was also observed in one of these two patients. In no case could significant responses to mitogens be elicited. Following transplantation, marrow cells from fully engrafted, immunologically reconstituted patients could be induced to bear HTLA, to form E rosettes, and to respond to mitogens and allogeneic cells following exposure to thymic extracts, thymopoietin, or TP-5. Thus, most patients with SCID manifest differentiative abnormalities intrinsic to lymphoid precursors which are corrected following engraftment of functioning allogeneic lymphoid precursors from normal donors.
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Three patients with Wiskott-Aldrich syndrome received transplants of marrow from their HLA-A, B, C, D identical siblings after myeloablation with busulfan, 2 mg/kg/day x 4 days, followed by immunosuppression with cyclophosphamide, 50 mg/kg/day x 4. Sustained engraftment of lymphoid and hematopoietic elements was documented in each case. Platelet counts in excess of 100,000/cu mm were restored 20--50 days posttransplant and remain in the normal range 6--12 mo later. Platelets exhibit normal size and in vitro aggregation. The patients produce isoagglutinins and antibodies to other polysaccharides. The use of busulfan in moderate dosages as a myeloablative agent, coupled with cyclophosphamide, may offer an improved alternative to the use of lethal total body irradiation as a preparative regimen for complete correction of Wiskott-Aldrich syndrome by marrow transplantation.
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A 2 1/2-year-old male infant with severe combined immunodeficiency was documented by HLA typing and the presence of a female karyotype in the few spontaneously dividing cells to have an intrauterine-derived maternal lymphocyte graft. The presence of DR antigens on the engrafted maternal E rosette-forming T cells was demonstrated by both cytotoxicity and immunofluorescence techniques using both conventional and monoclonal antihuman DR antisera. These DR antigens were of the same allospecificity, DR4, as the mother's peripheral blood B cells. The patient's B cells and monocytes expressed DR alloantigens DR4 and DR3, corresponding to his genotype. Although fresh maternal lymphocytes react strongly in vitro against patient non-T cells, the engrafted maternal T lymphocytes no longer show this activity. Furthermore, clinical evidence suggesting mild graft-versus-host disease was completely resolved by the end of his first year. The presence of the DR-positive maternal cells may reflect the survival of a group of activated suppressor cells mediating graft tolerance of host tissue.
Bone marrow and peripheral blood cells of patients with non-leukemic neutropenia contain and elaborate a granulocyte-progenitor cell inhibitory activity. The inhibitory activity is common to the neutropenias of the various etiologies studied, which included congenital, idiopathic, autoimmune, cyclical, common variable immuno-deficiency with hypogammaglobulinemia and drug induced states. It derives from non-adherent, low density, slowly sedimenting and non-E-rosetting cells and appears to require RNA and protein synthesis, but not cell division, for its production. The material is not species specific, inhibits autologous and allogeneic normal CFUgm and leukemic CFUgm, is not cell-cycle specific in action and is most effective against granulocyte colony forming cells (CFUg), less effective against mixed granulocyte-macrophage colony forming cells (CFUgm) and least or non-effective against macrophage colony forming cells (CFUm). This inhibitory activity has no influence on cells which generate CFUc in suspension culture or on the erythroid colony forming (CFUe) and burst forming (BFUe) units. It is different from other known inhibitory activities such as lactoferrin, leukemia inhibitory activity, E type prostaglandins, interferon and immunoglobulins. This inhibitory activity, while at present an in vitro phenomenon, may be produced as a secondary response within a compromised host.
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A patient with aplastic anemia who was found to be homozygous for an HLA-D determinant shared by her unrelated parents achieved sustained engraftment and full restoration of hematopoietic and lymphoid function following a transplant from an HLA-A and -B nonidentical, ABO incompatible sibling who was heterozygous for the shared HLA-D specificity. Transplantation was complicated by transient graft-versus-host disease of moderate severity, which resolved completely following treatment with antithymocyte globulin and prednisone. The case indicates that patients found to be HLA-D-homozygous may be successfully transplanted from HLA-D-heterozygous sibling donors despite HLA-A and HLA-B incompatibilities, and thus further demonstrates the importance of the HLA-D region as a marker of donor-host histocompatibility.
A procedure was developed for the isolation from human bone marrow of a cell fraction enriched for haematopoietic precursors and depleted of T lymphocytes. T cells are eliminated from bone marrow by rosetting with sheep red blood cells, followed by differential agglutination of residual T lymphocytes in the non-rosetting population by the lectin, soybean agglutinin. The fraction unagglutinated by the lectin contains a high proportion of colony-forming cells and non detectable T cell alloreactivity in vitro. Similar results were obtained with monkey bone-marrow cells, suggesting that monkeys can be used for evaluation of this fractionation technique for bone-marrow transplantation across histocompatibility barriers.
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