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Bestatin, an inhibitor of leucine aminopeptidase (LAPase), significantly decreased HIV infection as reflected by a reduced number of positive immunofluorescent cells, p24 levels, reverse transcriptase activity and the number of proviral copies found in Bestatin-treated cells. Cellular and extracellular LAPase activity in infected cells was higher than the LAPase activity found in uninfected cells. However, cellular and extracellular LAPase activity as well as total protein kinase C activity was lower in Bestatin-treated cells. Conversely, the incubation of human lymphocytic HUT78 cells with LAPase promotes HIV infectivity. The possible role of LAPase in the pathophysiology of HIV was assessed by determining LAPase serum levels in HIV infected patients. LAPase activity levels were three orders of magnitude greater in sera obtained from HIV patients than those detected in sera of uninfected individuals. Although Bestatin reduced HIV infection, a moderate decrease in the reverse transcriptase activity of chronically-infected H9 human T-lymphocytic cells was observed. Based on the higher levels of LAPase present in the serum of HIV patients and on the combined inhibitory effect of Bestatin on LAPase and on protein kinase C activities, we suggest that LAPase may play an important role in the early events of HIV infection such as viral entry.

In blood, the CD4+ T cells of patients with human immunodeficiency virus type 1 (HIV-1) harbor HIV-1; however, whether the CD4+ blood monocytes carry the virus is controversial. Tissue macrophages are known to be infected. To determine in blood monocytes from HIV-1-seropositive patients contain HIV-1, we separated monocytes and T-cell subsets by using monoclonal antibodies bound to magnetic beads and by monocyte adherence to glass. Monocytes were cultured with macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3. After 14 days in culture, cells were analyzed for the presence of HIV-1 antigen and multinucleated giant cells (MGCs). Freshly isolated cell subsets were analyzed for HIV-1 proviral DNA by PCR with modified env (SK68i and SK69i2) and gag (SK145i and SK150) primers. We found that (i) monocytes cultured without depletion of CD4+ T cells (11 of 11 patients) were HIV-1 antigen positive and showed dramatically increased spontaneous formation of MGCs (ii) monocytes cultured after depletion of CD4+ T cells (three experiments) were HIV-1 antigen negative and showed markedly decreased MGC formation, and (iii) in specimens from 14 patients subsequently analyzed by PCR, purified CD4+ T cells were positive for HIV-1 proviral DNA in all patients. In 11 of 14 patients (79%), the monocyte fractions were HIV-1 proviral DNA negative, while in the remaining 3 patients, the monocytes were positive for HIV-1 proviral DNA. In conclusion, the major reservoir for HIV-1 infection in human peripheral blood is the CD4+ T cell (14 of 14 cases).(ABSTRACT TRUNCATED AT 250 WORDS)

Members of the anthracycline family of drugs such as daunorubicin (DNR) are currently being employed in cancer therapy, treatment of Kaposi's sarcoma and lymphomas in HIV-1 infected patients. These drugs may have an anti-viral activity against HIV-1. Experiments conducted on U937 (a promonocytic cell line), peripheral blood monocytes (without T cells) and infected with HIV-1 showed anti-viral activity at nanogram/ml concentrations, without affecting cell proliferation and cell viability. Treatment of Hut 78 cells (a T cell line) with DNR was not as effective in inhibiting HIV-1 replication when compared to the anti-HIV activity in cells of the monocyte lineage.

To determine the effect of HIV infection on the accessory cell function of monocytes we measured the ability of HIV-infected monocytes to restore PHA-induced and soluble anti-CD3-induced T cell blastogenesis. These T cells were highly purified and depleted of monocytes (< 0.5%) and activated T cells. Monocytes were isolated using gelatin-fibronectin-coated flasks (< 1% T cells) and after 4 days in culture with granulocyte/macrophage-colony stimulating factor, they were infected with HIV. Accessory cell (AC) function was tested 2 and 7 days later, employing autologous cryopreserved T lymphocytes. Monocytes infected with HIV for 2 days lacked the ability to permit phytohemagglutinin (PHA) and anti-CD3-induced T cell blastogenesis. Noninfected monocytes restored the proliferative response of purified T cells. Interleukin 1 (IL-1) and interleukin 6 (IL-6) levels in culture supernatants were low when compared to cultures with noninfected AC. Preincubation of monocytes with human anti-HIV neutralizing antibodies did not restore either of the responses. AC treated with heat-inactivated HIV had normal accessory cell function. The addition of IL-1 and/or IL-6 partially restored the AC function for PHA stimulation, but not for anti-CD3 stimulation. We conclude that HIV infection of monocytes suppressed their accessory cell function in the T cell blastogenesis assay. The response was partially restored with IL-1 and/or IL-6, suggesting that HIV infection down-regulated the monocyte production of both cytokines.

We describe a method for simultaneous analysis of CD3, CD4, and CD8 positive cells from whole blood utilizing single laser flow cytometers. All three T cell values are attained from a single test tube. CD4 and CD8 positive cells are identified only if they are CD3 positive. Thus the values obtained by this method for T helper/inducer and T cytotoxic/suppressor cells can be reported directly as a percentage of T lymphocytes. Analysis for CD4 and CD8 positive cells is accomplished, by first gating on CD3 positive T lymphocytes, hence the approach is referred to as a T gating method. As the third dye, conjugated to anti-CD3 monoclonal antibodies (MAbs), we utilized peridinin chlorophyll protein (PerCP), a new red fluorochrome. The proposed method may prove to be practical for monitoring disease progression in AIDS, where longitudinal T helper/inducer and T cytotoxic/suppressor cell enumeration must be performed unambiguously by a simple, reproducible, and fast method.

The hemopoietic inductive microenvironment (HIM) of the bone marrow is responsible for secretion of growth factors that regulate hemopoiesis. It is composed of an extracellular matrix and a complex variety of cell types with a range of functions related to blood cell development. In order to understand how such a complex system operates, it will first be necessary to determine the role(s) of the integral parts. Several of the stromal cell types have been identified morphologically in various culture systems, and some of their functions have been elucidated. We have identified a new stromal cell type in mouse bone marrow that appears similar if not identical to its human counterpart. When bone marrow cells are placed in methylcellulose/plasma clot culture with phytohemagglutinin-stimulated human leukocyte-conditioned medium in the presence of bovine calf serum (BCS), mercaptoethanol, and hydrocortisone, extensive branching colonies develop within 14 days. These "reticulo-fibroblastoid" (RF) colonies arise from a putative reticulo-fibroblastoid colony-forming unit (CFU-RF) stem cell, and many become adipocytic by day 14; the addition of fresh medium, methylcellulose, and BCS on day 7 inhibits this change. The batch of human citrated plasma used in the culture system and the type and source of stimulating factor all influence the number of RF colonies that develop as well as the percent of colonies that become adipocytic. Whether this adipogenesis represents functional maturity or terminal differentiation is not yet known. Information gained on the role of these RF cells in normal and impaired hemopoiesis should contribute to the elucidation of the complicated interactive role of the microenvironment in the support and modulation of hemopoiesis.

Using long-term culture techniques, it has been shown that stromal cells in the marrow microenvironment are essential for the continued production and self-renewal of hematopoietic stem cells. We previously reported the development of a methylcellulose colony assay for a population of marrow stromal progenitors called CFU-RF. In this paper, a method is described for subculturing cells from individual CFU-RF-derived colonies to allow conditioned medium production (StCM). StCM, prepared in this way, was found to possess an erythroid lineage-specific activity that stimulated the formation of macroscopic erythroid colonies in cultures containing erythropoietin (epo). Using dose-response curves, the KG1 colony assay, and antibody neutralization, it was shown that the activity could not be attributed to interleukin 3 (IL3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). However, it was further shown that a monolayer of stromal cells, which had earlier been producing the erythroid activity, could be stimulated by IL1 to produce granulocytic colony-stimulating activity, but only as long as IL1 was present in the culture medium. These findings indicate a mechanism whereby the same stromal population could be modulated to promote growth and differentiation of different hematopoietic lineages.

CD4 on blood monocytes is generally regarded as being found on a subset of blood monocytes. However, our results show that all monocytes are CD4+ but the number of molecules per cell is lower than T cells. We have performed immunofluorescent (flow cytometry, microscopy) analysis of monocytes from normal donors and HIV-1-infected patients using anti-CD4 (Leu-3a) and the monocyte-specific marker anti-CD14 (Leu-M3) monoclonal antibodies. Specifically: (1) 91% of monocytes from normal individuals show dual positivity for CD14 and CD4 (n = 14) as measured by flow cytometry; (2) 76% of monocytes expressed surface bound CD4 and CD14 when an enhanced two colour immunofluorescence microscopic technique was employed; (3) all CD14+ monocytes stained with an intensity of 3+(-)4+ for cytoplasmic CD4. Few monocytes were CD14- and CD4+. Cell surface CD4 expression was blocked with unconjugated anti-CD4 prior to staining; (4) the staining intensity for cytoplasmic CD4 in T cells was negligible; (5) CD4 expression on monocytes obtained from patients was as observed with normal individuals. The conclusion drawn is that all monocytes are CD4+ and the CD4 expression in monocytes is mainly cytoplasmic. Thus, all monocytes are potentially infectable with HIV-1.

A patient with longstanding Mollaret's meningitis had an increased number of circulating natural killer cells during an acute attack, as evidenced by 73% large granular lymphocytes and 80% Leu-11a-positive peripheral mononuclear cells. Numbers of large granular lymphocytes and Leu-11a-positive cells returned to normal by three months after the attack. Similarity of the morphological features of the Mollaret's cell to those of large granular lymphocytes is noted.

We have developed an assay for specific genes in DNA based on transient expression. DNA prepared from patients with acute myeloblastic or acute lymphoblastic leukemia or from the continuous leukemic cell line HL60 was transferred to LTA cells; 48-72 hr later, these recipients expressed hemopoietic differentiation markers as detected by monoclonal antibodies against My-1 (granulocyte specific) and OK-T3 (T-cell specific) using immunofluorescence. The efficiency of transfer was dose and time dependent. We found that genes not expressed in the original cells were expressed after transfer by using this assay. Restriction enzyme analysis showed that My-1 was not expressed with DNA that had been incubated before transfer with either HindIII or Sal I but was present after digestion with either EcoRI or BamHI; after digestion of DNA with the same enzymes, OK-T3 expression was observed only in the HindIII-treated DNA. These studies indicate that DNA was transferred; this approach may provide an efficient method for detecting the activity of specific genes in the absence of selection.

Evidence is presented for an inhibitory action of the bone marrow adherent cell layer on erythropoiesis in a case of pure red cell aplasia. Cultures of the patient's bone marrow with autologous and normal plasma showed normal numbers of CFU-C, but total absence of BFU-E. There was no inhibition of BFU-E when normal bone marrow was cultured with the patient's plasma. T-cell depletion of the patient's bone marrow did not affect these results, but after removal of the adherent cells, normal numbers of BFU-E were cultured. These observations suggest that the adherent cells, which are predominantly macrophages, were responsible for the inhibitory action on erythropoiesis.

The phytohemagglutinin (PHA) blastogenic response of normal healthy individuals was studied before and after vaccination with hepatitis B surface antigen. The PHA response was suppressed 2 d after the first dose of vaccine but was not affected by the second and the third doses of vaccine. The suppressed PHA blastogenic response on day 7 was not enhanced by the addition of interleukin-2 or indomethacin even though an increase in cell number expressing CD25 was observed. The removal of CD4+ or CD8+ cells enhanced the PHA response but only on days 2 or 4 and not at other sampling times, which suggests that the suppression is mediated by CD4+ or CD8+ cells. The addition of interleukin-2 alone or with PHA did not reverse the suppression at any time tested. In vitro induction of suppressor cells was performed and was blocked by the addition of indomethacin at the time of culture initiation.

Acute myeloblastic leukemia, like certain other hematologic disorders, originates in pluripotent stem cells. Two general biologic processes underlie development of the disease. Over long times, clonal progression leads from normal polyclonal hemopoiesis through clonal preleukemia to leukemia. Overt leukemia is characterized by the emergence of blast cell populations. Over shorter times, clonal expansion yields cellular diversity based upon randomizing events. The analysis indicates that that blast population is of crucial importance. Characteristics of a colony assay for blast cell progenitors are presented.

Cell populations depleted of sheep erythrocytes (E) rosette-forming T cells (E-cells) contain cells capable of giving rise to T cell colonies. We have characterized the T cell colony-forming cell from human bone marrow, blood and tonsil E- cells using a T-cell colony assay. Depletion of CD2+, CD3+ or CD4+ cells from E- cells reduced colony formation by 70-100%. Removal of CD8+ cells did not reduce, but rather enhanced colony formation by 50% or more. The most effective reduction (100%) in colony formation was obtained with anti-CD4, indicating that CD4 is a marker of all colony-forming T cells. The CD4+ lymphocytes generated two types of colonies, types I and II, in the presence or absence, respectively, of CD8+ lymphocytes. Type I were small and compact, reached a peak on days 5-7, and contained CD4+ and CD8+ cells. Type II were large and diffuse, reached a peak on days 9-10 and contained CD4+ cells. In continuous culture of single type II colony cells, we observed a consistent increase of CD8+ cells. In one colony the combined percentage of CD4+ and CD8+ cells exceeded 100% (averaging 83% CD4+ and 72% CD8+), indicating the presence of dual markers on some cells. We suggest that colony forming T cells are CD2+ CD3+ CD4+, the CD4+ antigen being the most consistent marker of such precursor cells.

Anti-endothelial cell antibodies (AECA) have been detected in autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma (PSS) but their role in pathogenesis is unknown. Immunofluorescence, immunohistochemistry, complement-dependent antibody lysis, and radioimmunoassay have been used in the past to detect AECA. We have developed a rapid, sensitive, and quantitative cellular enzyme-linked immunosorbent assay (ELISA) to detect and characterize AECA. Sera were obtained from 28 normal volunteers, 28 patients with SLE, and 14 patients with PSS. We also performed studies in 47 patients with various monoclonal gammopathies. Endothelial cells (EC) were obtained from human umbilical veins by standard methods and subcultured on 96-well tissue culture plates without fixation. EC were then sequentially incubated with sera, peroxidase-conjugated goat anti-human Ig (IgG, IgM, or IgA), and substrate. Optical density readings were converted to arbitrary units by developing a standard curve. Heavy-chain specific antibodies were used to determine the class of AECA binding to EC. IgG was purified by using protein A columns and digested with pepsin to obtain F(ab')2 fragments. The mean units of AECA from normals were 19.3 for IgG and 12.5 for IgM. SLE sera showed significant levels of IgM AECA (37 units, P less than 0.001) but not IgG (29 units, P less than 0.1). PSS sera showed significant levels of both IgM AECA (38 units, P = 0.001) and IgG AECA (42.7 units, P less than 0.005). IgA AECA were not detected in normal, SLE, or PSS sera. Blocking Fc receptors with rabbit IgG did not affect the titer of IgG or IgM AECA.(ABSTRACT TRUNCATED AT 250 WORDS)

Several techniques are available to study cell membrane glycoproteins in pathological conditions but these either lack sensitivity or require radiolabelled material or expensive apparatus. We have, therefore, developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) to study patients with the Bernard-Soulier syndrome (BSS), a hereditary platelet disorder characterized primarily, at the molecular level, by glycoprotein Ib (GpIb) deficiency. We have used the lectin wheat germ agglutinin to capture GpIb in the microtiter wells. Following incubation with monoclonal antibody AN51 which recognizes an epitope on GpIb, the immune complex was detected using the streptavidin-peroxidase-biotin complex. Platelet samples from 24 normal controls gave a mean value of 106% whereas in the six BSS patients the mean value was 14% and in the eight obligatory heterozygotes it was 78%. The technique is simple, inexpensive and sensitive and does not require the use of radioactive material. The assay method could be applied to quantitate other cellular glycoproteins where specific lectins and monoclonal antibodies are available.

The marrow microenvironment is composed of an extracellular matrix as well as a heterogeneous population of cells. Isolation of the various cell types and analysis of their function is necessary for a better understanding of their roles in hemopoiesis. We have recently reported a colony assay for a cellular component of the marrow microenvironment. The assay consists of a cellular component of the marrow microenvironment. The assay consists of a plasma clot-methylcellulose marrow culture. The stimulator is PHA-stimulated leukocyte conditioned medium (PHA-LCM) and hydrocortisone (5 x 10(-5)M). The fibrin strands appear to act as a substrate for the growth of Reticulo-Fibroblastoid colonies derived from the CFU-RF precursor. RF colonies can be subcultured forming adherent layers when transferred to liquid cultures. Confluent adherent layers can be maintained for long periods of time by changing medium every 3 to 5 days. Supernatants derived from unstimulated RF cultures (RF-CM) were tested for growth promotion of hemopoietic precursors. We found: (1) RF-CM by itself does not induce colony formation. (2) In the presence of erythropoietin, RF-CM enhances the growth of BFU-E. (3) Recombinant IL 4 also enhances BFU-E formation, but in our assays IL 4 induced fewer colonies than RF-CM and the colonies were smaller. (4) Because neither IL 4 nor RF-CM, by themselves, can stimulate colony formation, we compared the effect of RF-CM on assays that are known to show other IL 4 functions. RF-CM did not induce proliferation of PHA induced blast T cells, a known property of IL 4.(ABSTRACT TRUNCATED AT 250 WORDS)

A 29-year-old woman developed acute promyelocytic leukemia during pregnancy. At diagnosis, immediately postpartum, she was found to have IgG kappa immunoglobulin on the surface of the leukemic cells as well as a monoclonal protein of IgG kappa specificity in her serum. These resolved with chemotherapy which induced a complete remission. Immunoglobulin gene rearrangement was not found in the leukemic cells, thus indicating that the blasts were not secreting the monoclonal protein. The authors believe that the patient had an autoantibody directed at myeloid cells which was amplified by the development of the leukemic process.

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.

Diseaes originating in pluripotent stem cells, then developing through clonal expansion and clonal progression may properly be grouped together because of their common features. Among these, AML appears to be unique by reason of the presence within the clone of a blast cell population. Studies of cellular composition and regulation in AML clones require assays that measure not only myelopoiesis but also blast cell proliferation. The relation of the blast population to other components of AML clones remains uncertain. Resolution of the uncertainty is important in considering therapeutic strategies.

A 67-year-old male with a prolonged activated partial thromboplastin time (APTT) of 43 seconds (normal, 25-40 seconds) was found to have laboratory features of von Willebrand's disease and IgA myeloma but had a normal bleeding time and no bleeding tendency. Plasma Factor VIII coagulant activity (F.VIII:C) was 80 U/L (0.08 U/mL), Factor VIII antigen (F.VIII:Ag) 70 U/L (0.07 U/mL), and von Willebrand's factor antigen (vWF:Ag) 50 U/L (0.05 U/mL) and ristocetin cofactor (vWF:RiCoF) 10 U/L (0.10 U/mL). The platelet vWF:Ag level was normal, whereas both platelet lysate and plasma vWF high molecular weight multimers were decreased. Patient plasma had no inhibitory effect on either F.VIII:C or vWF:RiCoF. However, when patient plasma was incubated with normal plasma, crossed immunoelectrophoresis for vWF:Ag demonstrated the presence of immune complexes. Infusion of 1-desamino-8-D-arginine vasopressin led to a transient correction of the plasma vWF:Ag multimer pattern. The survival of all components of vWF/F.VIII was decreased, as also occurred after cryoprecipitate. The levels of plasma F.VIII/vWF increased as the IgA values decreased after chemotherapy, whereas the platelet high molecular weight multimers remained decreased. The data suggest that the plasma vWF/F.VIII deficiency results from complexing of the IgA myeloma protein with vWF, resulting in premature clearance of the vWF/F.VIII complex. The absence of clinical bleeding likely results from the combination of a normal platelet vWF:Ag level and persistence of intermediate molecular weight vWF multimers.