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S M Crowe

Publications and source records attributed to S M Crowe.

At least 19 recordsLinked to original sources
PubMed1997

Constitutive expression of p50 homodimer in freshly isolated human monocytes decreases with in vitro and in vivo differentiation: a possible mechanism influencing human immunodeficiency virus replication in monocytes and mature macrophages.

Human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in vitro in differentiated macrophages than in freshly isolated monocytes. We investigated whether this may be partly explained by changes in expression of NF-kappaB with monocyte differentiation. We demonstrated that constitutive expression of NF-kappaB in primary human monocytes changed significantly with differentiation in vitro to monocyte-derived macrophages (MDMs) and differentiation in vivo to alveolar macrophages (AMs). Freshly isolated monocytes constitutively expressed high levels of transcriptionally inactive p50 homodimer which decreased with time in culture in favor of the transcriptionally active p50/p65 and p50/RelB heterodimers. As in MDMs, AMs constitutively expressed p50/p65 and p50/RelB although at lower levels. HIV infection of fresh monocytes failed to induce p50/p65 as seen in MDMs. The replacement of p50 homodimers with transcriptionally active heterodimers following time in culture may partially explain the progressive increase in susceptibility of monocytes to HIV infection during in vitro culture. The change in NF-kappaB components with monocyte differentiation in vivo may also explain the different transcriptional activities of these cell populations in HIV-infected individuals.

Cell Differentiation↗
PubMed1996

Surface CD4 is critical to in vitro HIV infection of human alveolar macrophages.

The CD4 glycoprotein is the major cellular receptor for HIV. CD4 surface expression of monocytes decreases with time in culture while their susceptibility to HIV-1 increases. Our aim was to investigate whether this phenomenon occurs in macrophages that have differentiated in vivo by investigating CD4 expression and HIV-1 infection of human alveolar macrophages (AMs). Using flow cytometry to detect CD4 expression by Leu-3a labeled indirectly with fluorescein isothiocyanate or allophycocyanin, we found that CD4 was expressed at low but detectable levels, despite the high background autofluorescence well described in AMs. This finding was supported by the detection of CD4 mRNA in AMs using RT-PCR. T cell contamination of mRNA extracts of AMs was excluded by amplifying in parallel with primers to the constant region of the T cell receptor. Despite this low level of surface CD4, recombinant soluble CD4 and anti-CD4 antibody completely inhibited HIV-1 infection of AMs. We conclude that CD4, although expressed at low levels on the surface of AMs, appears to be critical to HIV-1 infection of these cells.

Acquired Immunodeficiency Syndrome↗
PubMed1996

The interaction of macrophage and non-macrophage tropic isolates of HIV-1 with thymic and tonsillar dendritic cells in vitro.

Dendritic cells isolated from thymus and tonsil were tested for susceptibility to HIV-1 strains that are tropic for macrophages or for T cell lines. DCs were purified by cell sorting and before infection expressed high levels of CD4 and HLA-DR and lacked markers for T, B, NK cells, or macrophages. Viral entry and reverse transcription was found after pulsing with strains of HIV-1 that could infect macrophages. During the first 36 h the PCR signals for gag sequences increased in DCs and macrophages. In contrast little if any viral DNA was found after pulsing macrophages or DCs with HIV-1 that was able to infect T cell lines. DCs pulsed with HIV-1 were able to transmit infection to responding T cells during an allogeneic or superantigen response. Selection for virus able to infect lymphoid DCs and other DCs expressing CD4 and its transfer to T cells during subsequent immune responses may provide a mechanism for the observed predominance of macrophage-tropic HIV-1 after in vivo transmission.

CD4 Antigens↗
PubMed1996

Quantifying phagocytosis of Mycobacterium avium complex by human monocytes in whole blood.

Studies of phagocytic efficiency in cells of the macrophage lineage have assumed additional importance since the discovery that HIV infection of these cells impairs their immune function. A rapid method has been developed for measuring phagocytosis of the opportunistic pathogen Mycobacterium avium complex by human monocytes. Fluoresceinated M. avium complex (F-MAC) was incubated with whole blood at 37 degrees C and the fluorescence of extracellular F-MAC was quenched using a vital blue stain. Monocytes were then stained with a monoclonal antibody (mAb) to human CD14 conjugated to phycoerythrin (PE) red cells were lysed, and the percentage of monocytes which had phagocytosed F-MAC was measured by flow cytometry. The results were reproducible in samples of blood taken from individual donors over a period of 1 or 2 weeks, and optimum F-MAC concentrations and an optimum incubation time were determined by experiment. This method has the advantages of requiring only a small volume of blood, not necessitating manipulation of cells before testing, and using a phagocytic target relevant to the pathogenesis of HIV infection.

Fluorescein-5-isothiocyanate↗
PubMed1996

Human immunodeficiency virus grown in CD4-expressing cells is associated with CD4.

Using a CD4-capture immunoassay for gp120, several strains of human immunodeficiency virus type 1 (HIV-1) grown in CD4-expressing T lymphoblastoid cells were found to contain little CD4-reactive gp120 (0.3-1.0 ng/ml) relative to virus titre (10(3.2)-10(5.0) TCID50/ml) and p24 antigen (80-1000 ng/ml). The measured CD4-reactive gp120 concentrations of HIV-1 suspensions grown in CD4-negative human neuroblastoma cells were 100- to 10,000-fold greater than those of HIV-1 grown in CD4-positive lymphoblastoid cells, even though both virus suspensions contained abundant viral gp120 as shown by immunoblot assay. It was postulated that CD4 derived from host cells might be associated with virions, concealing the binding domains of gp120. CD4 association with HIV-1 virions grown in CD4-positive cells was demonstrated directly by immunoblot assay of sucrose gradient-purified virus suspensions and by specific co-sedimentation of 125I-labelled OKT4 with virions propagated in CD4-expressing cells. CD4 coating of primary HIV-1 isolates grown in peripheral blood mononuclear cells was also observed. The biological significance of CD4 coating of HIV particles remains to be determined.

CD4 Antigens↗
PubMed1995

HIV-1 infection of human macrophages impairs phagocytosis and killing of Toxoplasma gondii.

The susceptibility of patients with AIDS to certain opportunistic infections is due to defective cell-mediated immunity. The contribution of direct infection of macrophages with HIV-1 to this defect is unknown. To address this issue, we infected normal human monocyte-derived macrophages with a monocytotropic strain of HIV-1 and examined their ability to phagocytose and kill the opportunistic pathogen, Toxoplasma gondii. Phagocytosis of heat-killed T. gondii was reduced in HIV-infected macrophages compared with mock-infected controls. Opsonization of heat-killed T. gondii increased phagocytosis by both mock- and HIV-infected macrophages, but phagocytosis in HIV-infected cultures remained lower than in controls. Internalization of live T. gondii by macrophages was unaffected by HIV infection. Intracellular replication of live T. gondii was enhanced by HIV infection, as shown in four experiments, each using monocyte-derived macrophages from a different donor. Treatment of HIV-infected macrophages with IFN-gamma decreased parasite replication but not to control levels. These findings suggest that infection of macrophages by HIV may be a contributing factor to the reactivation of T. gondii infection in patients with AIDS.

Animals↗
PubMed1995

The kinetics of surface expression of CD11/CD18 integrins and CD54 on monocytes and macrophages.

Cells of the macrophage lineage mediate extremely important normal functions of the immune system. Such functions are in part related to interactions between cell-bound LeuCAMs and their ligands. MoAb staining and flow cytometric analysis were used to follow changes in surface expression of LeuCAMs and the LFA-1 ligand CD54 during maturation of peripheral blood monocytes (BM) in vitro. Surface expression of these molecules increased on BM following isolation, the greatest increase being in CD54 and CD11c. Following an initial increase, there was a reduction in CD11a expression after 2 weeks in culture, this being greater on adherent compared with suspension-maintained cells. Expression of CD11b remained high throughout the culture period. LeuCAM and CD54 expression was further compared on freshly isolated alveolar macrophages (AM) and BM paired donors. A reciprocal relationship was observed between CD11c and CD11b on AM and BM, in that BM expressed higher levels of CD11b than CD11c, whilst the converse was true for AM. CD54 expression was also higher on AM than on BM, whilst there was no significant difference in expression of CD11a on these cells. These data suggest that consistent changes occur in the surface expression of the LeuCAMs and CD54 as monocytes mature into macrophages, which may reflect the specific functions of these cells.

CD11 Antigens↗
PubMed1995

The role of bronchoscopy in the diagnosis and treatment of pulmonary disease in HIV-infected patients.

BACKGROUND: Pulmonary disease is the most common reason for presentation and the major cause of death in HIV-infected patients. There has been an evolution in the optimal approach to the investigation of a pulmonary infiltrate in HIV-infected patients since the introduction of induced sputum for the diagnosis of Pneumocystis carinii pneumonia (PCP). AIMS: To evaluate the usefulness of flexible fibreoptic bronchoscopy (FFB), bronchoalveolar lavage (BAL), transbronchial biopsy (TBB) and bronchial brushings (BB) in the diagnosis of pulmonary disease in HIV-infected patients and to examine the effect of FFB on changes in therapy and survival. METHODS: The histories of all HIV-infected patients referred to Fairfield Hospital for FFB between January 1990 and June 1993 were examined retrospectively. RESULTS: Forty-two FFB were performed on 41 patients (40 male and one female). Definitive diagnoses made at FFB included Kaposi's sarcoma (KS) (n = 9), invasive aspergillosis (n = 5), PCP (n = 4), Mycobacterium avium complex (MAC) pneumonia (n = 2), cytomegalovirus (CMV) pneumonia (n = 1), Cryptococcus neoformans pneumonia (n = 1), microsporidium (n = 1) and Pseudomonas aeruginosa pneumonia (n = 1). TBB and BB did not provide a diagnosis for diseases not seen macroscopically at FFB or diagnosed by BAL. FFB findings altered diagnosis in 21/42 (50%) presentations and changed therapy in 26/42 (62%) cases. CONCLUSIONS: FFB together with BAL altered the working diagnosis and changed therapy in a significant number of patients. TBB and BB should not be routinely performed in all patients as these procedures are of limited value in this setting.

AIDS-Related Opportunistic Infections↗
PubMed1995

Role of macrophages in the pathogenesis of human immunodeficiency virus (HIV) infection.

There are a number of machanisms by which HIV-infected macrophages contribute to the pathogenesis of the Acquired Immunodeficiency Syndrome (AIDS). Macrophage-tropic strains of HIV are present at the time of infection, and persist throughout the course of infection, despite the emergence of T cell tropic quasispecies. As HIV causes chronic infection of macrophages with only minimal cytopathology, these cells can provide an important viral reservoir in HIV-infected persons. Macrophages are more susceptible to HIV infection than freshly isolated monocytes. HIV-infected macrophages can contribute to CD4 T lymphocyte depletion through a gp120-CD4 dependent fusion process with uninfected CD4-expressing T cells. Increasing data support the role of HIV-infected macrophages and microglia in the pathogenesis of HIV-related encephalopathy and AIDS-related dementia through the production of neurotoxins. HIV infection of macrophages in vitro results in impairment of many aspects of their function. Reduced phagocytic capacity for certain opportunistic pathogens, including Toxoplasma gondii and Candida albicans, may be responsible for reactivation of these pathogens in persons with advanced HIV infection, although the mechanisms underlying reactivation of infections and susceptibility to disease from new infections are likely to be multifactorial. Our studies showing defective phagocytosis and killing provide additional information that contribute to our understanding of the pathogenesis of AIDS. Studies of in vitro efficacy of potential antiretroviral therapies should be performed in both primary lymphocyte and monocyte cultures, given the importance of both of these cell populations to HIV pathogenesis and their differing biology.

AIDS Dementia Complex↗
PubMed1995

A simple method for the propagation of cervical lymphocytes.

Local immune function is most likely a key influence in the establishment of human papillomavirus infections and its subsequent disease. Unfortunately, little information is known about local cervical immunity, and even less is known about human papillomavirus immunoreactivity. In addition, studies of local immunoreactivity have been hampered by the technical difficulty in obtaining cervical lymphocytes. The objective of the present study was to develop a simple method for the propagation of cervical lymphocytes from biopsy-size specimens. Cervical tissue was obtained from women undergoing a hysterectomy. Cervical samples measuring approximately 3 by 5 by 2 mm were minced and divided into two portions. One portion was digested by standard digestion methods and density gradient lymphocyte separation. The sample was then immunocharacterized for CD4 and CD8 cells by flow cytometry. The other portion was minced into 1-mm3 sections, and each section was placed into a separate well with tissue culture medium and interleukin-2. Lymphocyte counts and immunophenotypic analysis were performed after 18 to 20 days in culture. After 18 to 20 days in culture, the analysis demonstrated that this method of direct lymphocyte culture from a biopsy specimen yielded approximately 1 x 10(6) to 5 x 10(6) lymphocytes. Immunophenotypic studies of the digested sample at day 0 revealed CD4-to-CD8 ratios of between 0.7:1 and 3.5:1, and at days 18 to 20 they revealed ratios of between 2.3:1 and 98:1. In summary, we developed a simple technique for propagating cervical lymphocytes from small tissue samples for the study of the local immune response. Studies are under way to optimize lymphocyte growth and to preserve CD8 populations.

Adolescent↗
PubMed1995

In vitro growth of the microsporidian Septata intestinalis from an AIDS patient with disseminated illness.

A new species of microsporidian, Septata intestinalis, was recently recognized as an opportunistic pathogen of AIDS patients. In this study, it was cultured from the nasopharyngeal aspirate of a human immunodeficiency virus type 1-infected patient with disseminated microsporidiosis. In human embryonic lung cells exposed to S. intestinalis, a cytopathic effect appeared within 28 days as foci of rounded up cells. Thin-section electron microscopy showed a variety of developmental stages of the microsporidium within parasitophorous vacuoles. In monocyte-derived macrophages, evidence of infection and development of the parasite was demonstrated by light and electron microscopy. In both infected human embryonic lung cells and monocyte-derived macrophages, a network of septa separated individual spores. Partial sequencing of the RNA small-subunit gene (16S rDNA gene) confirmed the identity of this parasite as S. intestinalis. This is the first report of the isolation of S. intestinalis in vitro and provides evidence that the parasite can be disseminated by macrophages.

AIDS-Related Opportunistic Infections↗
PubMed1994

Lymphocyte subset analysis by Boolean algebra: a phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence.

Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre-mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conjugated monoclonal antibodies. We describe a rapid method whereby total CD3+ T-cells, CD4+ T-cells (CD3+ CD4+), CD8+ T-cells (CD3+ CD8+), putative gamma delta-receptor-T-cells (CD3+ CD4- CD8-), and T-cells that are CD3+ CD4+ CD8+ as well as B-lymphocytes and NK-cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC-conjugated antibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time.

Antibodies, Monoclonal↗
PubMed1994

Susceptibility of dendritic cells to HIV-1 infection in vitro.

We review recent work on the extent of HIV-1 infection of dendritic cells (DCs) and the consequences of exposure to virus. The reported levels of infection of DCs from blood have varied from "explosive" to "undetectable." The only study that used sorted DCs demonstrated little if any infectability, which may not be surprising given the very low levels of CD4 on the populations that were studied. HIV-1-pulsed, highly purified DCs function as potent antigen-presenting cells during the mixed leukocyte reaction and responses to superantigens. At the same time that the HIV-1-pulsed DCs stimulate CD4+ T cells in DC-T clusters, the virus is transferred to the responding lymphocytes and a vigorous productive infection of the T cells takes place. This pool of transferable HIV-1 is short lived in cultured human blood DCs and likely reflects the capacity of these cells to internalize and recycle vesicles in the endocytic pathway, as revealed with experiments using 0.1-micron fluorescent latex beads. Current efforts are directed to analyzing the interaction of HIV-1 with several populations of DCs that express higher levels of CD4. These include DCs studied in fresh, uncultured blood, as well as skin, thymus, and tonsil DCs. In each case, entry and reverse transcription of HIV-1 are seen, but again, coculture with T cells is required for a productive infection to take place. We conclude that DCs could play a critical role in the pathogenesis of HIV-1 infection, but that the interaction with CD4+ T cells is a critical variable in analyzing the extent of productive infection and its consequences.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Accumulation of unintegrated circular viral DNA in monocytes and growth-arrested T cells following infection with HIV-1.

Cytocidal retrovirus infection is characterized by rapid accumulation of unintegrated viral DNA forms. These are thought to be generated by multiple rounds of reinfection and have been suggested to play a central role in cytopathogenesis. Here we have reviewed the work done in this area with HIV-1, mostly using acutely and chronically infected T cell and monocytic cell lines and in some cases T cells blocked at S phase of the cell cycle by aphidicolin treatment. To these studies, we have compared our findings with HIV-1 infected primary peripheral blood monocyte-derived macrophages and untreated and growth-arrested MT-2 cells, two biologically disparate cell populations. Using 1- and 2-long terminal repeat (LTR) circular forms as indicators of unintegrated viral DNA, we found similar rapid accumulation in both untreated and growth-arrested MT-2 cells. In contrast, we found much lower levels in monocyte/macrophages. Our findings suggest that accumulation of unintegrated viral DNA does not require virus production and reinfection in growth-arrested T cells. The significantly lower levels found in monocyte/macrophages may reflect superinfection resistance, allowing the maintenance of a persistent infection.

Acquired Immunodeficiency Syndrome↗