Intracellular parasitism by the human granulocytic ehrlichiosis bacterium through the P-selectin ligand, PSGL-1.

Human granulocytic ehrlichiosis (HGE) is a febrile tick-borne illness caused by a recently discovered intracellular bacterium remarkable for its tropism for professionally phagocytic neutrophils. Monoclonal antibodies against the P-selectin binding domain of the leukocyte P-selectin glycoprotein ligand, PSGL-1, prevented HGE cell binding and infection, as did enzymatic digestion of PSGL-1. Furthermore, simultaneous neoexpression in nonsusceptible cells of complementary DNAs for both PSGL-1 and its modifying alpha-(1,3) fucosyltransferase, Fuc-TVII, allowed binding and infection by HGE. Thus, the HGE bacterium specifically bound to fucosylated leukocyte PSGL-1. Selectin mimicry is likely central to the organism's unique ability to target and infect neutrophils.

Anti-inflammatory action of dapsone: inhibition of neutrophil adherence is associated with inhibition of chemoattractant-induced signal transduction.

Dapsone has clinical utility as an anti-inflammatory agent but the mechanism of this action remains unknown. We have previously reported that dapsone inhibits beta2 integrin (CD11b/CD18)-mediated adherence of human neutrophils in vitro and now describe studies designed to discover how dapsone-mediated inhibition of this neutrophil function occurs. Results indicate that dapsone interferes with the activation or function of the G-protein (Gi type) that initiates the signal transduction cascade common to chemotactic stimuli. They also show that dapsone-mediated suppression of this pathway inhibits the generation of second messengers essential to the activation of beta2 integrin molecules, as well as respiratory and secretory functions of neutrophils exposed to chemoattractants. We propose that the inhibition of chemoattractant-induced signal transduction by dapsone suppresses neutrophil recruitment and local production of toxic respiratory and secretory products in the affected skin of dermatitis herpetiformis and other neutrophilic dermatoses.

Receptors for the chemoattractants C5a and IL-8 are clustered on the surface of human neutrophils.

We have used high-resolution field emission scanning electron microscopy with backscatter electron imaging to detect immunogold-labeled C5a and interleukin-8 (IL-8) receptors on human blood neutrophils. The receptors were labeled with receptor-specific antibodies in combination with secondary antibody conjugated to immunogold. When neutrophils were isolated in a "nonactivated" state, both of these receptor populations were expressed primarily in clusters on nonprojecting domains of the cell membrane. When these cells were double labeled for C5a and IL-8 receptors, intermixing of these receptor species in a common cluster was not found. When neutrophils were isolated in an "activated" state, by mixing the blood with N-formylmethionyl-leucyl-phenylalanine, the cells were seen to be elongated and ruffled at their anterior pole, but the C5a receptors did not disperse or redistribute on the surface of the peptide-activated cells. Analysis of the distribution of human C5a receptors expressed by transfected mouse L-cell fibroblasts showed the C5a receptors to be clustered, but expressed on nonprojecting and projecting domains of the cell surface. These observations provide new information on the topographical expression of leukocyte receptors involved in directing cell migration.

The complexity of sexuality.

Recent genetic and environmental theories of the etiology of homosexuality are evaluated. Interactive theory is proposed as a better alternative for understanding the development of sexual identity and sexual preference.

Effects of detergents on proliferation and metabolism of human keratinocytes.

Effects of low concentrations of detergents on cultured human foreskin keratinocytes were assessed in vitro. The viability and activity of keratinocytes were assessed by measuring reduction of a tetrazolium dye as an indicator of mitochondrial metabolism. The keratinocyte proliferative responses after incubation with detergents were assessed by a spectrophotometric assay employing crystal violet dye and a fluorometric assay determining total DNA content. Both the cationic detergent cetyltrimethylammonium bromide [CTAB] and the anionic detergent sodium lauryl sulfate [SLS] showed toxic effects on keratinocytes at concentrations as low as 3 micrograms/mg, but SLS was less toxic. However, both SLS and CTAB activated keratinocytes at very low concentrations. Proliferative activity and mitochondrial metabolism increased. Serum partially protected keratinocytes against toxic and stimulatory activities of both detergents. We suggest that detergents may directly damage keratinocytes and thereby produce irritant contact dermatitis, but activation of keratinocytes by low concentrations may also produce dermatitis, perhaps by causing keratinocytes to release cytokines.

Computerized microassay of keratinocyte cell-plastic attachment and proliferation for assessing net stimulatory, inhibitory and toxic effects of compounds on nonimmortalized cell lines.

Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA plate reader. Optical density readings are directly converted into cell counts by computer software. Counts obtained by this method strongly correlate with the results obtained using the [3H]thymidine uptake assay and direct cell counts. The assay standardizes measurements of nonimmortalized EK lines with different innate proliferative properties and allows accurate quantitation of EK numbers in the range of 2,500-500,000 EK/well.

Binding and uptake of Trichophyton rubrum mannan by human epidermal keratinocytes: a time-course study.

Trichophyton rubrum infects skin. This fungus or its products might affect the function of epidermal cells. We previously reported that T. rubrum mannan (TRM) exhibits a suppressive effect on proliferation of human lymphocytes. The goal of the present study was to investigate the possibility of direct interaction of TRM with cultured normal human epidermal keratinocytes (EK). Mannan, a cell wall glycoprotein, was extracted from T. rubrum by precipitation with cetyltrimethylammonium bromide and conjugated with fluorescein isothiocyanate (FITC-TRM). After incubation of EK with 50 micrograms/ml FITC-TRM for 30 min, the surface of EK showed bright fluorescent staining. EK cultures pretreated with non-labelled TRM remained unstained. The fate of TRM bound to EK surface was determined in a time-course study. After pulse exposure to FITC-TRM, EK cultures were washed and incubated for various time periods. The EK moved surface mannan to the one area of cell membrane, so that at 4-6 h, the homogeneous staining of the entire cell surface was replaced by staining in a "cap" pattern. By 12 h, FITC-TRM was taken up into the cell, brought to the nuclear area and concentrated in the EK nucleoli. During the next 3 days nucleolar and cytoplasmic staining of the cells was observed. The intensity of fluorescence gradually diminished. On the 4th day, the sharp staining of organelles disappeared; instead, a large number of small fluorescent granules were seen intra- and extracellularly. By the 6th day after exposure, no EK staining remained. Thus, EK specifically bound, internalized and apparently catabolized TRM.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of Trichophyton rubrum mannan to human monocytes in vitro.

We recently reported that the mannan component of Trichophyton rubrum cell wall (TRM) has an inhibitory influence on cell-mediated immune function in vitro. We now describe experiments designed to identify the target cell for this effect of TRM. T. rubrum mannan labeled with fluorescein (FITC-TRM) was incubated with peripheral blood mononuclear leukocytes, monocytes, or lymphocytes. Binding and uptake of the FITC-TRM were monitored by fluorescence microscopy and flow cytometry. Approximately 10% of mononuclear leukocytes were stained with this reagent and the fluorescent cells appeared to be monocytes by morphology. Virtually all purified monocytes and no purified lymphocytes stained with FITC-TRM. Flow cytometry to analyze FITC-TRM monocyte-specific binding of FITC-TRM involved the use of a phycoerythrin-labeled anti-CD14 antibody to identify monocytes. The only cells stained with FITC-TRM were those stained with the monocyte-specific antibody. The ability of monocytes to endocytose mannan was assessed by fluorescence microscopy. Cells were exposed to FITC-TRM and washed, and the staining pattern recorded periodically over a 48-h incubation period. After 15 min, staining was homogeneous and involved the entire cell surface; by 30 min, "patching" was observed; by 90 min, bright granules had formed along the cell border and a large number of small granules were present in the cytoplasm; by 8-12 h, the fluorescent granules were enlarged in size and reduced in number; by 24-36 h, the intensity of cytoplasmic fluorescence began to diminish; and, after 48 h, all fluorescent staining had disappeared. An additional feature of staining during the 8-12-h period was the appearance of a large round bright spot in the nuclear region of each cell, which may represent nucleolar staining. A role for "mannan receptors" is suggested by observations that FITC-TRM binding was prevented by unlabeled TRM or pretreatment of the monocytes with trypsin. Our finding that monocytes selectively and specifically bind TRM appears to identify the monocyte rather than the lymphocyte as the target cell for the inhibitory effect of mannan on cell-mediated immune function.

Dapsone suppresses integrin-mediated neutrophil adherence function.

The anti-inflammatory influence of dapsone may involve suppression of neutrophil chemotaxis to selected attractants, but other actions of the drug are likely also involved. We have discovered that dapsone may suppress migration of neutrophils to extravascular sites through inhibition of adherence functions required for neutrophil recruitment. Neutrophil adherence mediated by integrins (CD11/CD18 or Mac-1 family receptors) was measured in vitro in terms of binding of stimulated cells to albumin-coated wells of microtiter plates, using phorbol myristate acetate (PMA) and N-formylmethionyl-leucyl-phenylalanine (FMLP) as stimuli. Adherence was assessed by staining attached cells with crystal violet dye and measuring the dye concentration at OD590 using an automated plate reader. The role of integrins in this assay was confirmed by the ability of anti-integrin antibody to suppress stimulated neutrophil adherence. The OD590 value for cells adhering to albumin in the absence of stimulus and dapsone averaged 0.2 +/- 0.04 (SEM) over five experiments. In the presence of 0.1 microM PMA or 10(-6) M FMLP, the OD590 values averaged 0.88 +/- 0.1 and 0.75 +/- 0.12, respectively. Dapsone did not affect unstimulated neutrophil adherence but, when present with stimulus, produced a dose-related inhibitory effect on adherence. Fifty percent inhibitory doses were approximately 150 micrograms/ml dapsone for both stimuli. Sulfapyridine reproduced the inhibitory effect of dapsone, but two structurally related compounds, hydrochlorothiazide and furosamide, did not. The observed ability of dapsone to inhibit neutrophil chemotaxis under agarose to FMLP and interleukin-8 may also be explained by interference with integrin-mediated adherence required for motility in this assay system. To consider if dapsone might have a similar inhibitory influence on neutrophil adherence in vivo, we tested the stimulated adherence function of neutrophils isolated from three individuals on dapsone therapy for dermatitis herpetiformis. Stimulated adherence of patients' cells averaged less than 40 percent of the control value. Suppression of leukocyte integrin function may therefore also contribute to the ability of dapsone to inhibit neutrophil infiltration in neutrophilic dermatoses.

Experimental investigation of a free-space optical switching network by using symmetric self-electro-optic-effect devices.

A prototype digital free-space photonic switching fabric is demonstrated. It consists of three cascaded 16 x 8 arrays of symmetric self-electro-optic-effect devices that are used as logic gates that implement part of a multistage interconnection network. We discuss architecture, device tolerancing, optical system design, and optomechanical design. This optical circuit is successfully configured as a fully operational array of 32 independent 2 x 2 nodes and operates at 100 kHz.

An immunoinhibitory cell wall glycoprotein (mannan) from Trichophyton rubrum.

Trichophyton rubrum causes 90% of chronic dermatophyte infections. Most patients with widespread chronic T. rubrum infection fail to express a delayed hypersensitivity reaction to intradermally injected trichophytin. We propose that cell-mediated immunity to T. rubrum may be suppressed in chronic infections by the mannan cell wall component of the fungus. The proposed suppressive effect of T. rubrum mannan on cell-mediated immunity was tested by measuring the ability of extracted mannan to inhibit lymphoproliferative responses of human mononuclear leukocytes to antigens, mitogens, and an anti-T-cell receptor antibody (anti-CD3) in vitro. Mannan was found to be highly antigenic in two of five donors and weakly antigenic in the other three. Despite its antigenic property, mannan exhibited a dose-related ability to inhibit lymphoproliferation stimulated by other agents including 1) antigens from Candida albicans, T. rubrum, and tetanus toxoid (ID50 = 250 micrograms/ml); 2) anti-CD3 antibody (ID50 = 250 micrograms/ml); and 3) Phaseolus limensis mitogenic lectin (ID50 = 64 micrograms/ml). Mannan added to cultures later than 24 h after initiation had no inhibitory influence, but culture of cells with mannan for a period of 24 h prior to the addition of stimulus enhanced the inhibitory effect of the glycoprotein. Lymphoproliferation in response to recombinant interleukin-2 (IL-2) was not inhibited. The influence of time of addition of mannan and the failure of mannan to inhibit IL-2-stimulated lymphoproliferation demonstrate that the suppressive effect of mannan must be pharmacologic rather than cytotoxic. The observed ability of T. rubrum cell wall mannan to suppress cell-mediated immune function in vitro may provide an important clue to a mechanism enabling the fungus to avoid elimination in chronically infected patients.

Candida mannan: chemistry, suppression of cell-mediated immunity, and possible mechanisms of action.

The ability of Candida albicans to establish an infection involves multiple components of this fungal pathogen, but its ability to persist in host tissue may involve primarily the immunosuppressive property of a major cell wall glycoprotein, mannan. Mannan and oligosaccharide fragments of mannan are potent inhibitors of cell-mediated immunity and appear to reproduce the immune deficit of patients with the mucocutaneous form of candidiasis. However, neither the exact structures of these inhibitory species nor their mechanisms of action have yet been clearly defined. Different investigators have proposed that mannan or mannan catabolites act upon monocytes or suppressor T lymphocytes, but research from unrelated areas has provided still other possibilities for consideration. These include interference with cytokine activities, lymphocyte-monocyte interactions, and leukocyte homing. To stimulate further research of the immunosuppressive property of C. albicans mannan, we have reviewed (i) the relationship of mannan to other antigens and virulence factors of the fungus; (ii) the chemistry of mannan, together with methods for preparation of mannan and mannan fragments; and (iii) the historical evidence for immunosuppression by Candida mannan and the mechanisms currently proposed for this property; and (iv) we have speculated upon still other mechanisms by which mannan might influence host defense functions. It is possible that understanding the immunosuppressive effects of mannan will provide clues to novel therapies for candidiasis that will enhance the efficacy of both available and future anti-Candida agents.

Pathogenesis of candidiasis. Immunosuppression by cell wall mannan catabolites.

Candida albicans cell wall mannan polysaccharide has an ability to negatively influence cell-mediated immune function. We have attempted to identify the mechanism of this phenomenon by testing the modulatory effects of isolated mannan and the chemical catabolites of mannan on cell-mediated immune function in vitro. We have determined that mannan isolated by complexation with cetyltrimethylammonium bromide (CTAB) is more antigenic than mannan isolated by precipitation with copper and that CTAB mannan does not inhibit lymphoproliferation stimulated by another antigen. We have also determined that oligosaccharides of three sizes, derived by chemical catabolism of CTAB mannan, are not antigenic, but instead are immunoinhibitory. Immunoinhibition does not involve interference with the mitogenic activity of interleukin 2. A similar occurrence of oligosaccharides may be produced by catabolism of mannan in vivo as evidenced by the presence of oligosaccharides of similar size in cell-free supernatant fluids derived from mononuclear leukocytes incubated with tritiated mannan. We propose that catabolites of fungal mannan may contribute significantly to suppression of cell-mediated immunity in candidiasis.

Homosexual and heterosexual sex-role orientation on six sex-role scales.

A comparison was made of the sex roles of homosexual and heterosexual men and women on the Bem Sex Role Inventory, Personality Attributes Questionnaire, Personality Research Form Androgyny Scale, Adjective Checklist Masculinity and Femininity Scales, Extended Personality Attributes Questionnaire and Undesirable Characteristics Scale. The results indicated that homosexuals and heterosexuals differ in their response to different aspects of sex roles. The most consistent difference was the greater femininity of male homosexuals in respect to male heterosexuals. Other differences were scale-specific and the low interscale comparability indicated such scales should not be used interchangeably. Differences between results of studies comparing sex roles of the homosexuals and heterosexuals appear attributable to sample heterogeneity and distinctions between sex-role scales.

Two mechanisms of inhibition of human lymphocyte proliferation by soluble yeast mannan polysaccharide.

The literature on chronic mucocutaneous candidiasis contains multiple reports which suggest that loss of cell-mediated immunity in this disease may be related in part to the presence of an inhibitory factor(s) present in patient plasma. One such inhibitory factor has been suggested to be mannan polysaccharide released from the cell wall of the pathogen. The present report describes results of experiments to consider mechanisms by which yeast mannan influences proliferative responses of human lymphocytes. Mannan for these experiments was isolated from Saccharomyces cerevisiae. We observed that mannan-mediated inhibition of proliferative responses to a battery of stimuli (phytohemagglutinin, pokeweed mitogen, and Candida, mumps, streptococcus, cytomegalovirus, and herpes simplex virus antigens) was related in part to an effect of copper associated with the mannan and possibly to the superoxide dismutase activity of the mannan-copper complex. Mannan made deficient in copper by use of a copper-chelating resin appeared to inhibit only lymphoproliferation stimulated by the Candida antigen. These results suggest that inhibitory effects of yeast mannans on lymphoproliferative responses may involve at least two mechanisms, one related to hydrogen peroxide production augmented by mannan-copper complexes and another related to still unknown effects independent of the metal ligand. We propose that our results represent a significant novel observation which may be useful in understanding mechanisms of immunoinhibitory effects of C. albicans mannan.

Chemotactic requirements of bovine leukocytes.

A system was described for studying bovine leukotaxis. Chemotaxis was readily observed toward bovine serum activated by zymosan of agarose. However, bacterial culture filtrates and peptides, which were potent chemotaxins for leukocytes from other species, failed to affect bovine leukotaxis. Using conditions suitable for studying binding to leukocytes from other species, specific binding of a radiolabeled chemotactic peptide to bovine leukocytes was not apparent. These data indicate that bovine leukocytes have chemotactic requirements distinct from those of other species.

Chemotactic deactivation of human neutrophils: protective influence of phenylbutazone.

The antiinflammatory drug, phenylbutazone (PBZ), has been studied in terms of its influence on chemotactic deactivation of human neutrophils. When PBZ was present during the time of preincubation of cells with N-formyl-methionyl-phenylalanine (F-Met-Phe), loss of subsequent spontaneous mobility and chemotactic responsivity to F-Met-Phe did not occur. The action of PBZ to protect neutrophils from peptide-mediated chemotactic deactivation was found to involve in part its inhibitory influence on hexose monophosphate shunt activity and in part its antagonistic effect on interaction of peptide receptors with N-formyl peptide. Phenylbutazone interfered with binding of N-formyl-methionyl-leucyl-[3H]phenylalanine but not [125I]C5a to the neutrophil, displaced labeled tripeptide bound in the absence of PBZ, increased the dissociation constant (KD) for labeled tripeptide, and limited down regulation of peptide receptor function. These results provide an example of drug-mediated modulation of the interaction of neutrophils with N-formyl peptide and strongly support the possibility that PBZ interacts directly and specifically with the human neutrophil peptide receptor as a competitive antagonist. They also provide an additional example of a compound outside of th N-formyl peptide series that interacts with the peptide receptor.

Influence of yeast mannan on human neutrophil functions: inhibition of release of myeloperoxidase related to carbohydrate-binding property of the enzyme.

We investigated the possibility that dissolved mannan polysaccharide from Candida may have an inhibitory influence on the host defense mechanisms associated with neutrophils and thereby contribute to the pathogenesis of candidoses. We created a model for this hypothesis by using mannan derived from bakers' yeast (Saccharomyces cerevisiae) and mannan isolated from the serum of a patient with chronic mucocutaneous candidosis. We observed that these mannans inhibited the respiratory burst and release of myeloperoxidase stimulated by phagocytosis of serum-opsonized zymosan in vitro. Mannan did not inhibit release of three other lysosomal enzymes. The selective inhibition of release of myeloperoxidase was attributable to a carbohydrate-binding property of the enzyme, with the mannan causing co-sedimentation of the enzyme with the cellular fraction.