ALBERT

Description: Leukemic blasts from 774 children with newly diagnosed acute lymphocytic leukemia (ALL) have been phenotyped by microcytotoxicity testing with a panel of monoclonal antibodies and heteroantisera as part of a Pediatric Oncology Group classification study of acute leukemia. One hundred twenty-two cases, or 16% were designated as T cell leukemia based on the reactivity of blast cells with previously well-characterized antisera (PT) against a T lymphocyte-associated antigen. Using this antisera-based definition as a standard, we looked for a monoclonal antibody combination that would be a suitable substitute. An algorithm calling for reactivity with either monoclonal antibody 3A1 or Leu-1 was a 92% sensitive and 97% specific predictor of PT reactivity. Only 27 of 755 cases of leukemia were incorrectly classified using this algorithm. Subsequently, Ficoll-Hypaque-separated bone marrow cells from 118 additional patients with ALL (21 of whom had T cell ALL) were stained by immunofluorescence using a combination of directly fluoresceinated 3A1 and Leu-1. Reactivity of 20% or more of the cells with this antibody combination was a 100% sensitive and 94% specific indicator of T cell ALL defined by PT positivity; with a higher cutoff value for positive values, or the use of supplemental tests, even this small number of false-positives could be eliminated. We conclude that this monoclonal antibody combination is a satisfactory replacement for our heteroantisera definition of T cell ALL.

Description: We have established two human cell lines which elaborate colony- stimulating activity (CSA) for at least four species: man, mouse, rabbit, and dog. One, GCT, was isolated from a lung metastasis of a fibrous histiocytoma; the other, RC4, from a monocyte-enriched fraction of normal blood. Medium conditioned by either GCT or RC4 cells was more potent in stimulating human marrow growth in vitro than was monocyte-conditioned medium or human leukocyte feeder layers. Fractionation of cell-line-conditioned medium by Sephacryl S-200 chromatography indicated that the maximum activity of the CSA for human marrow cells is eluted within the range of 30,000–40,000 daltons. These cells lines provide a continuous source of large quantities of conditioned medium for purification of CSA. Moreover, the invariable growth-supporting activity for all species tested and the high potency of cell-line CSA facilitates studies of its elaboration and biologic effects.

Description: The effects of administration of partially purified human urinary erythropoietin and rabbit thrombopoietin, and of endogenously produced erythropoietin and thrombopoietin on both red cell and platelet production were examined in mice. Partially purified thrombopoietin was prepared from rabbit plasma by sequential fractionation with ammonium sulfate precipitation, and DEAE and Sephadex G-100 chromatography. Preparations of thrombopoietin and partially purified human urinary erythropoietin (NIH No. H-11-TaLSL) were administered subcutaneously to normal mice, and the rate of incorporation of selenomethionine-75 Se into platelets was measured as an index of thrombopoietic activity of the infused material. Erythropoietin and thrombopoietin were assayed for erythropoietic activity by measuring the rate of appearance of 59Fe in the red cells of posthypoxic polycythemic mice. Preparations containing thrombopoietin had barely measurable erythropoietic activity, and 7 units of partially purified erythropoietin had little thrombopoietic activity. When endogenous levels of erythropoietin were increased by hypoxia, platelet production was not enhanced. Similarly, increased levels of thrombopoietin, induced in response to thrombocytopenia produced by platelet antiserum, did not alter red cell production. These data suggest that physiologically increased levels of thrombopoietin do not stimulate erythropoiesis, and that physiologically increased levels of erythropoietn do not stimulate thrombopoiesis. However, currently available, partially purified preparations of erythropoietin and thrombopoietin may be capable of stimulating both platelet and red cell production if used in sufficient quantities.

Description: Hereditary elliptocytosis is a heterogeneous disorder resulting from defects in the erythrocyte membrane skeleton. Although some cases of elliptocytosis result from defects in spectrin, the specific structural abnormality has yet to be identified in the majority of cases. Protein 4.1 plays an essential role in erythrocyte membrane physiology, and deficiencies have been implicated in only a few rare cases of elliptocytosis. By using 4.1 immunoblots and a 4.1 radioimmunoassay we identified distinct variants of protein 4.1 in 15 elliptocytic members of three US white families with the Rh-linked form of elliptocytosis. Elliptocytic members of family G were heterozygotes for a low-molecular weight (mol wt) 4.1 variant (65,000 to 68,000 daltons; normal, 80,000) inherited in linkage with the Rz phenotype. Elliptocytic members of family C expressed a simple partial deficiency of protein 4.1 (63% of the normal level) that was inherited in linkage with the r phenotype. Elliptocytic members of family N were heterozygotes for a high-mol wt 4.1 variant (100,000 daltons) also inherited in linkage with the r phenotype. These studies indicate that mutant forms of protein 4.1 are not uncommon in elliptocytosis among whites and that different kindreds probably express different mutations. The observed linkage of elliptocytosis and Rh blood type most likely results from the close proximities of the 4.1 gene (site of the mutation) and the Rh gene, which is located nearby on the short arm of chromosome 1.

Description: A murine model of bone marrow (BM) transplantation in which donor (B10.D2) and recipient (BALB/c) mice were major histocompatibility complex (MHC) (H-2d) and Mls-1 identical, but incompatible at multiple non-MHC minor histocompatibility (H) antigens, and at Mls-2,3 was used to examine regeneration of B-cell development during the minor H antigen graft-versus-host reaction (GVHR). Mice that received T-cell- depleted allogeneic BM regained significant pre-B cells (sIg- 14.8+) in their BM. Mice undergoing GVHR after transplantation with allogeneic BM + T cells had less than 2% pre-B cells in their BM at day 7 and only 12% to 14% pre-B cells at days 21 and 28 compared with greater than 20% pre-B cells in the allogeneic controls. After partial recovery, the pre- B cells in the BM of GVH mice again decreased to less than 3% by day 42. This abnormal pattern of pre-B cell development in mice undergoing GVHR was associated with a reduced response to interleukin-7 (IL-7) in vitro. The delay in B-lineage cell reconstitution in mice with GVHR correlated with the expansion of donor V beta 3+ T cells in both the spleen and BM. BM T cells from mice with GVHR as well as isolated V beta 3+ T cells inhibited IL-7 colony-forming units from normal BM in co-culture assays. This inhibition could be reversed with anti- interferon gamma (IFN gamma) antibody. These data suggest that the delay in appearance and the reduction in proportion and number of pre-B cells observed early during the GVH reaction in this model is caused, in part, by the inhibitory actions of IFN gamma derived from donor V beta 3+ T cells on B-lineage cell development.

Description: The human T-cell lymphotropic virus type I (HTLV-I) is causally associated with adult T-cell leukemia, but its role in mycosis fungoides (MF) has remained enigmatic. The virus is suspect because a small percentage of patients with MF have antibodies to it, the cells of others harbor deleted HTLV-I proviral sequences, and particles resembling HTLV-I emerge in cultured blood lymphocytes obtained from most patients. An alternative possibility is that disparate lymphotropic retroviruses may infect or affect a population of epidermotropic lymphocytes, leading to the same outcome, ie, MF. In studies designed to identify the particles detected in lymphocyte cultures of nine patients with a diagnosis of skin involvement characteristic of MF, this concept has gained support. While the cells of four patients provided evidence of HTLV-I infection, molecular hybridization with HTLV-II-specific pol probes showed HTLV-II in the cells of another patient. The 103-bp fragment amplified by the HTLV-II- specific probe was sequenced and proved to have greater than 90% homology with the same fragment amplified from cells known to be infected with HTLV-II. A role for HTLV-II in MF has not been suggested heretofore. Therefore, HTLV-I, HTLV-II, and their incomplete forms may be found in cells of MF patients, suggesting new theories regarding the pathogenesis of this disease.

Description: Decreases in protein S levels have recently been reported in some human immunodeficiency virus (HIV)-infected patients. To examine predisposing factors, 25 men randomly selected from a long-term study of HIV- infected patients were studied. The minimum mean duration of HIV seropositivity in this group was 106.6 months (range 15 to 143 months). No patients were anticoagulated at the time of the study. Three of the 25 randomly selected patients gave a history of thrombosis, in each instance occurring after the onset of HIV positivity. Two of the 3 patients with thrombosis had more than one episode. Coagulation studies showed that 3 of 3 (100%) of the patients with thrombosis and 16 of 22 (72.7%) of those without previous thrombosis had decreased free protein S. Mean-free and total protein S levels were statistically lower for HIV-infected patients with and without previous thrombosis compared with healthy male controls. C4b-binding protein was not increased in study patients with decreased protein S levels. Decreases in protein S levels did not correlate with CD4+ cell levels, CDC class, p24 antigen positivity, zidovudine (AZT) use, or Pneumocystis carinii prophylaxis. The duration of disease statistically correlated with decreases in protein S levels (r = .37, P 〈 .05). A linear correlation existed between increasing IgG anticardiolipin antibody levels and decreasing free protein S antigen (r = .67, P 〈 .005). This study shows that protein S deficiency is common in long-term HIV-infected patients and is caused by a decrease in the free protein, rather than by changes in the bound complex. The data suggest that protein S deficiency is not correlated with HIV disease severity but may predispose patients to thromboembolic complications.

Description: A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity 〉 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

Description: Mast cells accumulate at sites of angiogenesis. The factor(s) that control mast-cell recruitment at these sites have yet to be defined. We sought to determine if angiogenic factors result in mast-cell chemotaxis. In this study, we observed that platelet-derived growth factor-AB (PDGF-AB), vascular endothelial cell growth factor (VEGF), and basic fibroblast growth factor (bFGF) each cause directed migration of murine mast cells at picomolar concentrations, with a typical bell-shaped dose-response curve. Another potent angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), appears to promote chemokinesis of mast cells, whereas tumor necrosis factor-alpha, a weak angiogenic factor, is less robust but still functions as a mast cell chemotactic factor. Epidermal growth factor (EGF), a growth factor with minimal angiogenic properties, was ineffective as a mast cell chemotactic factor. A checkerboard analysis confirmed the directional chemotactic response of PDGF-AB, VEGF, and bFGF, while indicating the chemokinetic response induced by PD-ECGF. Cross-desensitization of growth-factor-induced directed migration was observed between PDGF-AB and bFGF, and also between PDGF-AB and PD-ECGF. Tyrosine kinase-inhibitor genistein effectively dampened the chemotactic responses, whereas pertussis toxin had no effect. In summary, our findings suggest that factors known to act on endothelial cells and stimulate neovascularization may simultaneously serve to recruit mast cells to these sites. The local accumulation of mast cells is believed to facilitate new vessel formation through complex cell:cell interactions.

Description: Despite significant progress in elucidating the biochemistry of the hemostatic mechanism, the process of blood coagulation in vivo remains poorly understood. Factor IX is a vitamin K-dependent glycoprotein that can be activated by factor XIa or the factor VII-tissue factor complex in vitro. To investigate the role of these two pathways in factor IX activation in humans, we have developed a sensitive procedure for quantifying the peptide that is liberated with the generation of factor IXa. The antibody population used for the immunoassay was raised in rabbits and chromatographed on a factor IX-agarose immunoadsorbent to obtain antibody populations with minimal intrinsic reactivity toward factor IX. We determined that the mean level of the factor IX activation peptide (FIXP) in normal individuals under the age of 40 years was 203 pmol/L and that levels increased significantly with advancing age. The mean concentration of FIXP was markedly reduced to 22.7 pmol/L in nine patients with hereditary factor VII deficiency (factor VII coagulant activity less than 7%) but was not significantly different from normal controls in nine subjects with factor XI deficiency (factor XI coagulant activity less than 8%). These data indicate that factor IXa generation in vivo results mainly from the activity of the tissue factor mechanism rather than the contact system (factor XII, prekallikrein, high molecular-weight kininogen, factor XI). Our results may also help to explain the absence of a bleeding diathesis in many patients with deficiencies of the contact factors of coagulation.