The rare alpha-thalassemia-1 of blacks is a zeta alpha-thalassemia-1 associated with deletion of all alpha- and zeta-globin genes

Restriction endonuclease mapping with alpha and zeta-globin gene probes showed differences between the alpha-thalassemia-1 (alpha-thal-1) condition in two patients with HbH disease. One patient had the rare black type of alpha-thal-1 together with alpha-thal-2 and HbS heterozygosities. The second patient was a Laotian child with HbE, Hb Constant Spring (alpha-thal-2), and alpha-thal-1 heterozygosities. The diagnoses were based on clinical, hematologic, and biochemical data. Whereas DNA fragments hybridizing to a zeta-probe were obtained from the Laotian type of alpha-thal-1, neither alpha nor zeta-gene fragments could be identified deriving from the black type of alpha-thal-1. Therefore, the black type of alpha-thal-1 is associated with a deletion of the entire zeta 2-psi zeta-psi alpha-alpha 2-alpha 1 gene complex and can be considered a zeta alpha-thal-1. It is likely that homozygosity for such a condition will lead to embryonic wastage, explaining the absence of hydrops fetalis in blacks.     

Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro

Two anti-nerve growth factor receptor (LNGFR or p75NGFR) antibodies, Me20.4 and Me8211, label stromal cells with dendritic features in fresh smears and in formalin-fixed, paraffin-embedded human bone marrow (BM). The LNGFR+ cells have an oval nucleus, a scanty cytoplasm with long dendrites that intermingle with the hematopoietic cells, line the abluminal side of sinus endothelial cells, and provide the scaffold for the hematopoietic marrow. At the electron microscopy level, the immunogold tag labels the body and the long branching dendrites of fibroblast-like cells with scanty cytoplasm containing mitochondria, endoplasmic reticulum, and dense bodies. The LNGFR+ cells are positive for alkaline phosphatase, reticulin, collagen III, vimentin, TE-7, and CD13 but negative for endothelial (vWF, CD34, Pal-E), neural (CD56, neurofilament) and leukocyte markers (CD45, CD68). The LNGFR+ stromal cells appear in the fetal BM before the hematopoietic activity begins, originate from the vessel adventitia, and radiate in the Bm cavity. Long-term BM culture (LTBMC) in vitro contain LNGFR+ stromal cells. We document the presence of RNA message for the low- (LNGFR) and the high- affinity NGF receptor (NTRK1) by using RT-PCR on fresh BM aspirate and on LTBMC. BM biopsies from patients with hematologic fibrogenic diseases and in cytokine-treated cancer patients are evaluated for LNGFR+ cells: the amount of stained cells is correlated with the traditional reticulin stain in cases of myelofibrosis, therapy-related myelodysplasia, leukemia, and detected an increase of stromal cells in cytokine-treated patients. The anti-LNGFR antibodies represent a specific membrane marker for the adventitial reticular cells (ARC) of the human marrow and allow precise evaluation and quantitation of this important BM microenvironment component in vivo and in vitro.     

Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum

Patients who undergo transplantation with haploidentical "three-loci" mismatched T-cell-depleted bone marrow (BM) are at high risk for graft failure. To overcome the host-versus-graft barrier, we increased the size of the graft inoculum, which has been shown to be a major factor in controlling both immune rejection and stem cell competition in murine models. Seventeen patients (mean age, 23.2 years; range, 6 to 51 years) with end-stage chemoresistant leukemia were received transplants of a combination of BM with recombinant human granulocyte colony- stimulating factor-mobilized peripheral blood progenitor cells from HLA- haploidentical "three-loci" incompatible family members. The average concentration of colony-forming unit-granulocyte-macrophage in the final inoculum was sevenfold to 10-fold greater than that found in BM alone. The sole graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion of the graft by the soybean agglutination and E- rosetting technique. The conditioning regimen included total body irradiation in a single fraction at a fast dose rate, antithymocyte globulin, cyclophosphamide and thiotepa to provide both immunosuppression and myeloablation. One patient rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient who received a much greater quantity of T lymphocytes than any other patient died from grade IV acute GVHD. There were no other cases of GVHD > or = grade II. Nine patients died from transplant-related toxicity, 2 relapsed, and 6 patients are alive and event-free at a median follow-up of 230 days (range, 100 to 485 days). Our results show that a highly immunosuppressive and myeloablative conditioning followed by transplantation of a large number of stem cells depleted of T lymphocytes by soybean agglutination and E-rosetting technique has made transplantation of three HLA-antigen disparate grafts possible, with only rare cases of GVHD.     

PMN heterogeneity: long-term stability of fluorescent membrane potential responses to the chemoattractant N-formyl-methionyl-leucyl- phenylalanine in healthy adults and correlation with respiratory burst activity

Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N- formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3'-di-pentyl-oxacarbocyanine [di-O- C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women.     

Cutaneous lymphoblastic lymphoma with pre-B markers

Two children with cutaneous convoluted lymphoblastic lymphoma are reported. Malignant cells from both patients contained cytoplasmic Mu heavy chains characteristic of pre-B-cells and expressed CALLA and la antigens as well. Most cases of convoluted lymphoblastic lymphoma are T- cell-derived neoplasms. The non-T, non-B phenotype found in these two children demonstrates that histology does not necessarily predict immunophenotype. The association of the pre-B phenotype with cutaneous lymphoma has not been previously reported, but may represent a unique clinical-histopathologic-immunologic entity that occurs in young children.     

Improved results of treatment of adult acute lymphoblastic leukemia

We designed a treatment program to improve the outcome for adults with acute lymphoblastic leukemia (ALL). Treatment included a remission- induction phase followed by intensive alternating cycles of non-cross- resistant chemotherapy and prolonged oral maintenance therapy. Eighty- one consecutive previously untreated patients were entered on this study. Ninety-four percent of patients entered complete remission. A Kaplan-Meier analysis predicts that 53% +/- 9% (SEM) of patients in remission will remain free of disease at 3 years. Neither age, sex, WBC count, nor immunophenotype had a significant effect on remission duration. This program of intensive cyclical chemotherapy has improved the disease-free survival of patients with adult ALL.     

Basophils (Bsp-1+) derive from the leukemic clone in human myeloid leukemias involving the chromosome breakpoint 9q34

The monoclonal antibody (MoAb) Bsp-1 was used to purify basophilic cells from leukemic blood of five patients with Philadelphia chromosome (Ph') positive chronic myeloid leukemia (CML) and two patients with acute myeloid leukemia (AML) characterized by the chromosomal translocation t(6;9)(p23;q34). When cultured, Bsp-1 positive cells from all CML and AML patients showed the same clonal karyotype changes observed in diagnostic buffy coat preparations, indicating that the basophilic cells were of leukemic origin. In contrast, T lymphocytes from four of five CML patients cultured in the presence of interleukin- 2 (IL-2) showed a normal karyotype and were therefore not derived from the leukemic clone. Bsp-1 staining correlated with toluidine blue- positive basophils in chronic phase CML and with toluidine blue- negative blast cells expressing an immature myeloid phenotype in blast crisis CML and AML. Chromosome in situ hybridization showed that the ABL oncogene was translocated from chromosome 9 to chromosome 22 in the CML patients but remained on chromosome 9 in the AML patients. These results indicate that the breakpoint at 9q34 in CML is 5' of ABL, whereas the breakpoint at 9q34 in AML is 3' of ABL. Field inversion gel electrophoresis showed that the 9q34 breakpoint was not within 200 kb 3' of ABL in one of the AML patients, nor was there any rearrangement of the PIM oncogene locus at 6p21.     

Human CD34+ fetal liver stem cells differentiate to T cells in a mouse thymic microenvironment

Hematopoietic stem cells differentiate in the thymus to T cells along precisely defined intermediates. This process is thymic epithelium dependent and involves cytokines and cell-cell interactions between thymic stroma and T-cell precursors. Here we report that highly purified human CD34++ fetal liver stem cells differentiate to mature T cells, when seeded into isolated fetal thymic lobes of severe combined immunodeficient mice, and subsequently cultured in vitro. The human stem cells differentiate sequentially into CD4+CD8-CD3-, CD4+CD8+CD3-, CD4+CD8+CD3+, and finally, CD4+CD8-CD3+4 and CD4-CD8+CD3++ cells. Phenotypic analysis for additional maturation markers showed that these CD4 and CD8 single-positive thymocytes are fully maturate cells. By immunochemistry, human HLA-DR+ cells with a dendritic morphology could be detected. This novel chimeric human-mouse fetal thymus organ culture offers a tool to study human T-cell ontogeny in vitro and is a rapid and reliable test method for T-cell precursor activity of cultured or transfected human stem cells.