The effects of thrombopoietic activity of rabbit plasma fractions on megakaryocytopoiesis in agar cultures.

A plasma fraction that stimulates platelet production in vivo also stimulates megakaryocytopoiesis in vitro. The plasma activity is attributed to a humoral regulator, thrombopoietin. Addition of the thrombocytopenic plasma (TP) fraction to an agar system supporting megakaryocyte colonies increased the frequency of colony formation significantly over that stimulated by spleen cell-conditioned medium (SCM). TP had no effect on the size of the colonies and, in the absence of SCM, TP did not stimulate colony formation. In studies of single megakaryocytes, the numbers of small megakaryocytes, specifically those 5-10 microns in diameter, increased significantly after 3 days of incubation with TP alone. SCM supported not an increase in the numbers, but an increase in the proportion of larger (30- to 40-microns) megakaryocytes. A normal plasma fraction contained similar but consistently less activity than fractions containing TP. The findings indicated that TP stimulates differentiation of megakaryocyte precursors from unidentifiable to identifiable cells but does not alone support colony formation. Thus, TP appears to be a potentiator of megakaryocytopoiesis. However, the augmentation of colony frequency by TP further suggests that TP may also play a role in early colony development, either by enhancing progenitor responsiveness to a megakaryocyte colony-stimulating factor or by recruiting additional colony progenitors from a noncycling progenitor population. These studies establish a link between the stimulation of platelet production observed after TP administration in vivo and the effects of TP on early events in megakaryocytopoiesis.

Antibody to human immunodeficiency virus in factor-deficient plasma. Effect of heat treatment on lyophilized plasma.

The prevalence of antibody to human immunodeficiency virus (HIV) was determined in various commercial substrate plasmas used in clotting factor assays, and viral isolation was attempted from both seropositive and seronegative samples. Antibody to HIV was detected in 13 of 13 plasma substrates used for Factor VIII assays and in 2 of 3 plasma substrates used for Factor IX assays. Antibodies were not detected in any of the other factor-specific substrates. Virus could not be isolated from any of the seropositive samples after 28 days in culture. Heat treatment of the samples under conditions known to inactivate HIV in plasma products indicated that heating the lyophilized substrate plasmas at 60 degrees C for up to eight hours had little effect on factor substrates and factor assays. Progressive loss of Factor V in the deficient plasmas was the most serious effect produced by heat treatment.

A longitudinal study of patients with hemophilia: immunologic correlates of infection with HTLV-III/LAV and other viruses.

A comprehensive study was initiated to examine the immunologic status of a sample (n = 47) of the asymptomatic hemophilia A and B populations of metropolitan Atlanta and to determine if any of the abnormalities changed with time or correlated with infection by human T cell leukemia virus type III and lymphadenopathy-associated virus (HTLV-III/LAV) or other viruses either alone or in combination. Patients with hemophilia A (Hem-A) showed a defect in cellular immunity evidenced by a depressed T cell helper/suppressor ratio (P less than .0001), an increased absolute T suppressor cell number (P less than .0001), and a diminished number of T helper cells (P = .003) when compared with health professionals. Lymphocytes from these patients also showed a reduced ability to transform in response to phytohemagglutinin and pokeweed mitogen. No deterioration in immune status was seen during a median ten-month period of follow-up. Sixty-four percent of Hem-A patients had antibodies to HTLV-III/LAV and those who were seropositive had a significantly decreased helper/suppressor cell ratio (P = .018) and a diminished T helper cell number (P = .002); they were also more likely to have had exposure to cytomegalovirus than HTLV-III/LAV-negative Hem-A patients (P = .016). Heavy use of factor VIII concentrate was associated with a decreased number of T helper cells (P = .037) and seropositivity for HTLV-III/LAV (P = .011 in 1982). Hemophilia patients had higher IgG, immune complex, and beta 2-microglobulin levels than health professionals (P less than .0001). Although the most prominent abnormality observed in T cell subsets of patients with hemophilia is an increase in T suppressor cells, a finding likely to be associated with immune augmentation in response to multiple stimuli, the T cell abnormality that was predictive of exposure to HTLV-III/LAV, the putative acquired immunodeficiency syndrome agent, was a diminished number of T helper cells.

Isolation and characterization of a human T cell leukemia virus type II from a hemophilia-A patient with pancytopenia.

Human T cell leukemia virus (HTLV) type I has been isolated from the cultured T cells of several patients with adult T cell leukemia (ATL) and has been etiologically linked to ATL. However, HTLV-type II has been isolated only once, from the T cells of a patient with a T cell variant of hairy-cell leukemia. We report here the isolation of HTLV-II-related virus from the cultured T cells of a hemophilia-A patient with pancytopenia. The T cell line (CM) grows in the absence of T cell growth factor. Cord blood T cells were rapidly transformed when co-cultivated with irradiated CM cells. Heterologous competition radioimmunoassays using purified HTLV-I p24 showed the expression of HTLV-IIMO-related protein in these cells. Electron microscopy of the CM cells showed the presence of intracellular and extracellular type C viral particles. Comparison of the proviral genome in the CM cell line and the prototype HTLV-IIMO-containing cell line (MO) by molecular hybridization with probes specific for HTLV-IIMO indicated that restriction cleavage sites were identical. The fresh peripheral blood leukocytes of the patient contained two complete copies of the proviral genome, despite the lack of HTLV-II p24 expression. The virus from the cell line CM is designated as HTLV-IICM to distinguish it from the original HTLV-IIMO isolate.

HTLV-I antibody status in hemophilia patients treated with factor concentrates prepared from U.S. plasma sources and in hemophilia patients with AIDS.

Serum samples from 85 Austrian hemophilia patients treated with lyophilized factor concentrates prepared from U.S. plasma sources, 24 hemophilia patients from Georgia on a home therapy program with factor concentrates, and 10 U.S. hemophilia patients with acquired immunodeficiency syndrome (AIDS) were analyzed by two different methods for the presence of antibodies to the major internal antigen of human T-cell leukemia virus I (HTLV-I) p24. All but one, a Georgia sample, were negative. The absence of antibody to HTLV-I p24 in the serum of European hemophilia patients, of U.S. hemophilia patients with no symptoms of AIDS, and of U.S. hemophilia patients with AIDS is interpreted as an indication of the lack of ready transmissibility of HTLV-I in lyophilized factor concentrates.

Coincidental appearance of LAV/HTLV-III antibodies in hemophiliacs and the onset of the AIDS epidemic.

PIP: To determine when antibody to lymphadenopathy-associated virus (LAV) emerged in the US hemophiliac population, serum bank samples that had been collected from hemophiliacs from California, Georgia, New York, and Texas in 1968 and in 1978-84 were tested for antibody to viral proteins p25 and p41 of LAV. Antibody to LAV was not detected in the serum samples collected in 1968-69 from 15 patients with mild hemophilia A. Before 1980, it was present in only 1 of 21 California patients with hemophilia A. Among the latter group, seroconversion tended not to occur until 1982, rapidly increased in 1983, and had reached a rate of over 85% by 1984. Among Georgian hemophiliacs, 8 of 14 (57%) were seropositive in 1981 and 8 of 8 (100%) were positive in 1984. The extent of seroconversion was not as great among patients with hemophilia B as among those with hemophilia A. None of the patients with antibody to LAV has contracted acquired immunodeficiency syndrome (AIDS). Of the 22 patients who received factor VIII concentrates (21 with hemophilia A and 1 with von Willebrand's disease), 7(32%) have mild lymphadenopathy. Of the 7 patients who received factor IX concentrates (6 with hemophilia B and 1 with a factor VII deficiency), only 1 had lymphadenopathy. These findings show that LAV exposure is widespread among patients receiving factor VIII concentrate and suggest that the concentrate may have contained LAV during 1980-83. Of interest was the observation that seroconversion in hemophiliacs began shortly after the outbreak of the AIDS epidemic among homosexual men and intravenous drug users (1979-80), and the timing of seroconversion correlates with the 1st cases of AIDS in hemophiliacs in late 1981-early 1982. The temporal relationship between seroconversion and the epidemic among hemophiliacs supports the hypothesis that LAV is the etiologic agent of AIDS.^ieng

Human T-cell leukemia virus in lymphocytes of two hemophiliacs with the acquired immunodeficiency syndrome.

Fresh and cultured peripheral blood cells from two patients with hemophilia A and the acquired immunodeficiency syndrome were examined for markers of infection with human T-cell leukemia virus (HTLV) type 1. Neither patient had antibody to membrane antigens of HTLV-infected cells at the time of culture. Electron microscopy of peripheral blood cells from Patient 1 and cultured cells from Patient 2 showed type C retrovirus-like particles. Examination of peripheral blood lymphocytes showed other smaller virus-like particles in circulating mononuclear cells from both patients. Indirect immunofluorescence of peripheral mononuclear cells from both patients and of cultured cells from Patient 2 showed staining with antibodies to purified HTLV and to HTLV core proteins p24 and p19.

The acquired immunodeficiency syndrome in patients with hemophilia.

Since mid-1981 the Centers for Disease Control (CDC) has received reports of more than 1900 cases of the acquired immunodeficiency syndrome. These cases had either Kaposi's sarcoma confirmed by biopsy or a life-threatening opportunistic infection confirmed by biopsy or culture. In January 1982 a hemophiliac with Pneumocystis carinii pneumonia was reported to the CDC, and by July 1982 two other hemophiliacs had developed P. carinii pneumonia. During the next 12 months a total of 22 confirmed cases of the acquired immunodeficiency syndrome occurred in hemophiliacs, 17 in the United States and 5 outside the United States. We report the nature of the epidemic of the acquired immunodeficiency syndrome in hemophiliacs and summarize pertinent clinical aspects.

Unusual urinary cholesterol metabolites following intracerebral injection of [4-14C]cholesterol into rats: I. The minor 14C-metabolite.

[4-14C]Cholesterol injected intracerebrally into 10-12-day-old rats becomes localized largely in central nervous system myelin. If sufficient 14C is injected, myelin cholesterol remains labeled for the rest of the rats' lives. In the course of the slow myelin cholesterol turnover that ensues, a unique series of cholesterol metabolites is excreted exclusively in the rats' urine. There is reason to believe that the metabolites are formed in the central nervous system before entering the urine. This manuscript describes separation of the 2 urinary 14C-labeled metabolic types and isolation and identification of the minor 14C-labeled material which consists of cholesterol and 2 other sterols bound covalently to short-chain peptides. The minor sterols have been tentatively identified as 24- and 26-hydroxycholesterol. The sterol-peptide combinations have been isolated from human male urine, also.

Subcellular localization of indium-111 in indium-111-labeled platelets.

Human platelets labeled with [111In]oxine have been suggested for use in platelet kinetic studies, thrombus localization, detection of atherosclerotic lesions, and other scintigraphic techniques. To demonstrate the subcellular localization of indium-111 in labeled platelets, we did subcellular fractionation and measured the amount of indium-111 localized in major platelet components. Human platelets were labeled with [111In]oxine and disrupted by nitrogen cavitation. The cellular material was fractionated on sucrose density gradients. When the subcellular fractions were analyzed, more than 70% of the indium-111 was located in the cytoplasmic pool of the platelets. Chromatography on Sephadex G-100 of aliquots of the labeled cytosol material showed that most of the indium-111 was associated with components of approximately 46,000 daltons. This demonstrated that indium-111 in human platelets labeled with [111In]oxine is located predominantly in platelet cytosol and is associated with cytoplasmic components.

Effects of lipid A and liposomes containing lipid A on platelet and fibrinogen production in rabbits.

The effect of the lipid A moiety of endotoxin on platelet and fibrinogen production was studied in rabbits. Lipid A was infused intravenously in doses ranging from 1 to 100 micrograms/kg body mass; 18 hr later, selenomethionine-75Se was injected intravenously and its incorporation into fibrinogen and platelets determined. Lipid A in saline stimulated fibrinogen and platelet production, but the dose required was 50--100 times that required for an intact endotoxin. Although lipid A solubilized in triethylamine (TEA) was at least 60 times more active in the Limulus amebocyte lysate assay than was lipid A suspended in saline, the sensitivity of platelet and fibrinogen production to solubilized lipid A was increased only twofold. Incorporation of lipid A into liposomes had no effect on its Limulus activity. Lipid A in liposomes continued to stimulate platelet, but not fibrinogen, production. Leukopenia that was induced by lipid A in TEA did not occur when rabbits received the same dose of lipid A in liposomes. Lipid A, like intact endotoxin, can stimulate platelet and fibrinogen production and induce leukopenia but the doses required are high. The low solubility of lipid A in aqueous solutions may be only one factor that determines its biologic activity.

Hydrolytic enzyme activities of the nervous system.

The activities of five hydrolytic enzymes (acid and alkaline phosphatase, hexosaminidase [N-acetyl-beta-D-glucosaminidase], beta-galactosidase, and beta-glucorinidase) were measured in reconstituted homogenates of lyophilized human brain tissue and primary and metastatic tumors. The linearity of reaction, with respect to incubation time, and optimal pH of each enzyme and in tumor tissues were comparable to those in normal brain tissue. Total enzyme activities of hexosaminidase, beta-glucuronidase, and beta-galactosidase were significantly higher in tumors than in normal cerebral white matter. The ratio of hexosaminidase activity to beta-glucuronidase activity was significantly lower for metastatic than for primary tumors or normal white matter. When histological observations do not clearly establish if a brain tumor is primary or metastatic, this ratio may help. Alteration of hydrolytic enzyme activities as demonstrated here may be indicative of "ket enzymes" that are essential for maintaining the metabolic advantages of tumors.

The biochemical and morphological response of hydrolytic enzymes in the developing brain to hypocholesterolemic agents.

Administration of hypocholesterolemic agents to developing rats has been found to selectively induce brain hydrolases. Certain regimes also caused an appreciable increase in total brain protein content. The hypocholesterolemic agents AY-9944 and zuclomiphene were tested individually and in combination. A fourth type of treatment utilized the above drugs in combination with Triparanol. Whenever AY-9944 was used, singly or in combination with other compounds, the beta-glucuronidase activity of developing brain was increased. Acid phosphatase and total brain protein were increased in animals treated with AY-9944 plus zuclomiphene or AY-9944 plus zuclomiphene and Triparanol. Neither AY-9944 nor zuclomiphene alone significantly affected brain total protein or acid phosphatase. Electron microscopic examination of tissue specifically reacted for acid phsophatase demonstrated that the increased enzyme activity was localized in cells in the perivascular spaces. Alkaline phosphatase and N-acetyl-beta-glucosaminidase, two other hydrolytic enzymes assayed, seemed to be much less influenced by the drug treatments.

Hydrolytic enzymes in meningiomal subtypes.

Estimation of activity of five hydrolytic enzymes was made in foru histologically different types of human meningiomas derived from surgery. The hydrolytic enzymes examined in 13 tumors included four lysosomal enzymes: beta-glucuronidase, N-acetyl-beta-D-glucosaminidase (hexosaminidase), beta-galactosidase, and acid phosphatase. The fifth enzyme studied was alkaline phosphatase. The one papillary-type meningioma examined appeared to contain generally greater activities of the lysosomal enzymes than the other tumor types. Alkaline phosphatase was decidedly greater in transitional type meningiomas. The correlation of histological types with alkaline phosphatase activity is discussed with regard to previous observations.