Thomas M. Folks

Infection and replication of HIV-1 in purified progenitor cells of normal human bone marrow.

Myeloid progenitor cells were highly purified from normal human bone marrow by positive immunoselection with high-affinity monoclonal antibodies linked to magnetic beads and were successfully infected in vitro with the human immunodeficiency virus type 1 (HIV-1). From 99 to 100 percent pure bone marrow cells expressing the CD34 phenotypic marker were obtained. These cells were devoid of mature myeloid or T cell surface and intracellular markers as analyzed by immunohistochemical staining and flow cytometry. HIV-1 particles were detected by supernatant reverse transcriptase activity and transmission electron microscopy 40 to 60 days after infection. Viral particles were predominantly observed assembling and accumulating from within intracellular membranes, while phenotypically the cells were observed to have differentiated into CD4+ monocytes. These studies have important implications in understanding the pathogenesis of HIV-1 as well as the possible cause of certain of the observed hematologic abnormalities in HIV-1 infection. They also indicate that the bone marrow may serve as a potentially important reservoir of HIV-1 in the body.

Flow-cytometric detection of circulating immune complexes

In an effort to develop an assay which would rapidly detect and analyze circulating immune complexes, we have adapted the Raji cell radioimmunoassay (RIA) to a flow cytometric (FCM) analysis. The advantages of immune complex analysis by FCM are many. Foremost is the effectiveness and efficiency of the FCM method relative to the radioimmunoassay (RIA) method. The data demonstrated that FCM detection is three times more sensitive than RIA detection. Only populations of viable Raji cells bearing immune complexes are analyzed because parameters of the FCM analysis permitted the elimination (gating out) of dead cells. The determinations are rapid and the data are immediately available for several additional analyses. Because of the availability of many fluorescent monoclonal antibodies to complement components, viral antigens, light chains and immunoglobulin isotypes, it is possible to detect many components that might be present in the Raji cell bound complexes. Finally, the Raji cells can be characterized in different stages of their cell cycle to generate information about the state of the cells and the density of the receptors involved in binding the complexes.

Unusual isolates of Thermoascus crustaceus from three monocyte cultures of aids patients

lished on twigs of U. alata, Fraxinus caroliniana Miller, Alnus serrulata (Aiton) Willd., and Platanus occidentalis L. The inability of spores and hyphal fragments of Padgett and Heitmans test fungi to survive exposure to 25%o NaCl suggests, however, that survival of hyphal fragments in the present study may not represent a widespread phenomenon within the Saprolegniaceae. As is often the case with ecological investigations the present study poses more questions than it answers. One must wonder, for instance, why water molds are not routinely recovered from euryhaline water samples if hyphal fragments or infested detritus are contained therein? How long can these fungi tolerate exposure to seawater? What is the mechanism of their resistance, and, more importantly, what are the ecological implications of this phenomenon relative to fish diseases, decomposition, or fungal distribution? It is hoped that these preliminary findings will invite additional research in this area. I am indebted to Drs. Armando A. De La Cruz (Mississippi State Univ.) and Courtney T. Hackney (UNC-Wilmington) for their critical comments on prelim? inary drafts of this manuscript.

Detection of helper and suppressor T cell lines to soluble antigens using a modified ELISA system.

Helper and suppressor activity of T cell lines were investigated using an assay system which is based on measurement by the enzyme-linked immunosorbent assay (ELISA) of the antibody produced in vitro. T cell lines were established from spleens of BALB/c mice immunized with ovalbumin and human serum albumin. The T cells were expanded in culture with irradiated spleen cells and antigen or concanavalin A supernatant. The culture system used for assay of T cell activities contained antigen (Ag)-primed unfractionated spleen cells or Ag-primed B cells. Ag-primed cells and cultured T cells were incubated together with Ag (0.05 ng/ml-100 micrograms/ml) for various times, washed at varying intervals, and supernatants assayed for specific antibody activity by an ELISA adapted for this purpose. A total incubation time of 9 days was required for significant antibody production. Complete reconstitution of the antibody response was observed with Ag-primed B cells and Ag-primed T cells were combined. In one experiment, a helper cell line was shown to restore specific antibody production to approximately 50% of the normal response while a suppressor cell line suppressed antibody production by 90%. A linear response of between 0.2 and 1.4 OD units was observed in the ELISA allowing easy detection of help or suppression. As little as 80 ng of specific antibody could be detected. This technique allows quantitative determination of antibody production for a large number of individual microcultures.

Increased number of Leu-2-bearing non-T cells with natural killer activity in chimpanzees.

Peripheral blood lymphocytes from adult and adolescent chimpanzees, as well as adult humans, were studied for phenotypic surface markers by flow cytometry. Lymphocytes from chimpanzees were found to have increased numbers of Leu-1-, Leu-2+ cells as compared to humans. These cells, following preparative electronic cell sorting, were shown to possess natural killer function. Further analysis of this subpopulation indicated that they lacked responsiveness to a number of T-cell mitogens. The differences in lymphocyte subpopulations between chimpanzees and humans can almost be totally accounted for by the Leu-1-, Leu-2+ cells. Phylogenetic disparity between these two species may also be found within this population.

An autoreactive T hybridoma expressing nonspecific helper activity

Two functional T hybridomas were prepared by fusing BW5147 with ovalbumin (OVA)-primed splenic T blast cells. One was OVA specific for helper function requiring concanavalin A supernatant (CAS) for activity while the other, termed autoreactive, was nonspecific for helper-augmenting activity. Both required H-2d presenter cells for interleukin 2 (IL-2) production. The autoreactive clone showed helper activity only in the presence of suboptimal numbers of antigen (Ag)-primed T cells. Both T hybridomas were Lyt 1+2+ and Thy 1+. Cells produced from such fusions should provide a useful instrument not only in dissecting the T-cell regulatory mechanism, but also in isolating and characterizing self-recognition structures.

Lipopolysaccharide-induced suppression of antibody-directed cell-mediated cytotoxicity to chicken red blood cells

Abstract Prior treatment with commercially prepared and acetone-extracted lipopolysaccharide (LPS) was found to suppress the expression of antibody-directed, cell-mediated cytotoxicity (ADCC) to chicken red blood cells (CRBC) by spleen cells from C57/BL10, C3H, and BALB/c mice. The in vitro incubation with commercial LPS suppressed ADCC-CRBC activity of spleen cells from both C3H/HeJ and C3H/HeN mice. Only the C3H/HeN strain was suppressed when treated with purified LPS. ADCC-CRBC activity of neonatal spleen cells could be suppressed after a 3-hr in vivo incubation with LPS while adult spleen cells required a minimum of 15 hr preincubation.