Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

The H8 protein is a surface-exposed antigen that is found, among members of the Neisseria genus, primarily on pathogenic species. In this study, the surface exposure of H8 was reassessed by four techniques. Results of slide agglutination, indirect fluorescent antibody binding, absorption of sera with whole gonococci, and immune electron microscopy all confirmed the presence of H8 in the outer membrane. The degree to which protein A-gold-labeled monoclonal antibodies bound to H8 was marked, and suggested that this antigen was present in abundant amounts in the outer membrane. Also in this study, the electrophoretic heterogeneity of this common surface antigen was examined. Because H8 stains poorly, electrophoretic mobility was assessed using polyclonal antibodies and a monoclonal antibody that recognizes a common H8 epitope. H8 was analyzed with respect to protein I, lipopolysaccharide (LPS), and pilus and opacity phenotypic variation; results confirmed that heterogeneity of Mr was the rule among strains (21 were examined), however, the variability in Mr was independent of protein I or LPS Mr. In one strain (FA1090), the heterogeneity of H8 was examined among 10 piliation/opacity variants; the H8 (and LPS) Mr was identical in all variants; similar data were generated in strains JS3 and JS1. The electrophoretic mobility of H8 was altered in serum-resistant and neutrophil enzyme-resistant gonococci compared to the sensitive gonococci. Some of the unusual electrophoretic migration characteristics of the antigen were also examined. H8 formed a unique mushroom-shaped band in one-dimensional gels; in a two-dimensional electrophoresis system, the antigen migrated aberrantly, very similarly to LPS. Also seen in the two-dimensional electrophoresis profile were multimers of the H8 antigen; in strain JS3 (Mr 23,500), these migrated at 43,600, 86,000, and greater than 150,000. In other strains, the Mr of the multimers differed depending upon the Mr of the monomer. The two-dimensional migration characteristics (as measured by antigenicity) were completely destroyed by proteinase K digestion. Activity of H8 polyclonal antibodies to the antigens in two-dimensional gels was completely removed by adsorption of formalin-fixed whole cells, but was not affected by adsorption with LPS. These electrophoretic characteristics may reflect the close association of some nonprotein constituent, perhaps lipid or carbohydrate or both.

Variants selected by treatment of human immunodeficiency virus-infected cells with an immunotoxin.

An immunotoxin has been made by coupling anti-human immunodeficiency virus (HIV) envelope antibody 907 to ricin A chain (907-RAC). 907 recognizes an epitope within the immunodominant PB-1 loop of gp120. Variant cells were selected by cloning persistently infected H9/human T lymphocyte virus IIIB cells in the presence of the immunotoxin. Clones resistant to 907-RAC arose at a frequency of 0.1-1.0%. Seven clones were selected for intensive analysis. When studied, these clones fell into two distinct groups, members of which appeared to be identical, suggesting that the variation arose before the selection process. In contrast to the parent cells, none of the cloned variants produced infectious HIV. The first set of clones, designated the "E" variants, expressed decreased levels of the HIV envelope on the cell surface. However, levels of intracellular HIV antigens and reverse transcriptase were equal to or greater than that of the parental cell line. Radioimmunoprecipitation demonstrated that the gp160 was truncated to 145 kD (gp120 was normal length), capable of binding to CD4, and, unlike normal gp160, was released in its unprocessed form into the cellular supernatant. Sequence analysis demonstrated that a deletion at codon 687 of the envelope gene resulted in the production of this truncated protein. Ultrastructural analysis of E variants demonstrated some budding forms of virus, but also large numbers of HIV within intracellular vesicles. The second set of variants, the "F" series, produced no HIV antigens, reverse transcriptase, nor was there ultrastructural evidence of virus. However, proviral DNA was present. Virus could not be induced with agents known to activate latent HIV. These cells also lacked cell surface CD4 and could not be infected with HIV. These studies demonstrate that variation in HIV can affect the phenotype of the cells carrying the altered virus, allowing for escape from immunologic destruction. The E variants may serve as prototypes for attenuated HIV, which could be used as a vaccine. We have reconstructed the mutation found in the E variants within the infectious HIV clone HXB-2 and demonstrated that the resulting virus retains its noninfectious phenotype.

Nucleoid condensation in Escherichia coli that express a chlamydial histone homolog.

Chlamydial cell types are adapted for either extracellular survival or intracellular growth. In the transcriptionally inert elementary bodies, the chromosome is densely compacted; in metabolically active reticulate bodies, the chromatin is loosely organized. Condensation of the chlamydial nucleoid occurs concomitant with expression of proteins homologous to eukaryotic histone H1. When the Chlamydia trachomatis 18-kilodalton histone homolog Hc1 is expressed in Escherichia coli, a condensed nucleoid structure similar to that of chlamydiae is observed with both light and electron microscopy. These results support a role for Hc1 in condensation of the chlamydial nucleoid.

Isolation of a spirochete from the soft tick, Ornithodoros coriaceus: a possible agent of epizootic bovine abortion.

A Borrelia-like spirochete was detected in all three parasitic stages of Ornithodoros coriaceus, the soft tick implicated in the epizoology of epizootic bovine abortion. After the spirochete had been isolated, its distinctness from other North American tick-borne borreliae as well as from Spirochaeta aurantia, Treponema pallidum, and Leptospira interrogans serovar pomona was established on the basis of its morphology, protein components, and inability to infect mice. The spirochete is passed trans-stadially and via eggs by ticks, and it is also excreted in coxal fluid after ticks have fed and detached. Circumstantial evidence suggests that the spirochete may be causally related to epizootic bovine abortion.

Lyme disease-a tick-borne spirochetosis?

A treponema-like spirochete was detected in and isolated from adult Ixodes dammini, the incriminated tick vector of Lyme disease. Causally related to the spirochetes may be long-lasting cutaneous lesions that appeared on New Zealand White rabbits 10 to 12 weeks after infected ticks fed on them. Samples of serum from patients with Lyme disease were shown by indirect immunofluorescence to contain antibodies to this agent. It is suggested that the newly discovered spirochete is involved in the etiology of Lyme disease.

Murine retrovirus-induced spongiform encephalopathy: productive infection of microglia and cerebellar neurons in accelerated CNS disease.

We have examined the pathological lesions and sites of infection in mice inoculated with a highly neurovirulent recombinant wild mouse ecotropic retrovirus (FrCasE). The spongiform lesions appeared initially as swollen postsynaptic neuronal processes, progressing to swelling in neuronal cell bodies, all in the absence of detectable gliosis. Infection of neurons in regions of vacuolation was not detected. However, high level infection of cerebellar granule neurons was observed in the absence of cytopathology, wherein viral protein was found associated with both axons and dendrites. Infection of ramified and amoeboid microglial cells was associated with cytopathology in the brain stem, and endothelial cell-pericyte infection was found throughout the CNS. No evidence of defective retroviral expression was observed. These results are consistent with an indirect mechanism of retrovirus-induced neuropathology.

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand.

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne illness caused by an intracellular bacterium that infects neutrophils. Cells susceptible to HGE express sialylated Lewis x (CD15s), a ligand for cell selectins. We demonstrate that adhesion of HGE to both HL60 cells and normal bone marrow cells directly correlates with their CD15s expression. HGE infection of HL60 cells, bone marrow progenitors, granulocytes, and monocytes was blocked by monoclonal antibodies against CD15s. However, these antibodies did not inhibit HGE binding, and anti-CD15s was capable of inhibiting the growth of HGE after its entry into the target cell. In contrast, neuraminidase treatment of HL60 cells prevented both HGE binding and infection. A cloned cell line (HL60-A2), derived from HL60 cells and resistant to HGE, was deficient in the expression of alpha-(1, 3)fucosyltransferase (Fuc-TVII), an enzyme known to be required for CD15s biosynthesis. Less than 1% of HL60-A2 cells expressed CD15s, and only these rare CD15s-expressing cells bound HGE and became infected. After transfection with Fuc-TVII, cells regained CD15s expression, as well as their ability to bind HGE and become infected. Thus, CD15s expression is highly correlated with susceptibility to HGE, and it, and/or a closely related sialylated and alpha-(1,3) fucosylated molecule, plays a key role in HGE infection, an observation that may help explain the organism's tropism for leukocytes.

Low-efficiency (macro-)pinocytic internalization of non-pathogenic Escherichia coli into HEp-2 cells.

HEp-2 cells internalize non-pathogenic Escherichia coli bacteria by a low-efficiency internalization mechanism which is upregulated in Pho-derepressed strains (as shown by Sinai and Bavoil in 1993), and is independent of microfilament integrity but requires functional microtubules. Here, we further characterize the microtubule requirement of this pathway using various effectors of microtubule integrity and function. Furthermore, we show that internalization is enhanced upon treatment with monodansylcadaverine, a specific inhibitor of receptor mediated endocytosis, and is insensitive to brefeldin A, which promotes the microtubule-dependent reorganization of the endosome. An assay system is also described to directly evaluate the contribution of pinocytosis to this pathway based on the ability of the bacteria to cointernalize and consequently colocalize with the fluid-phase marker, Texas-red-conjugated dextran (TRD). Using this assay, Hoescht-stained bacteria were observed in TRD-containing vesicles in numbers that are consistent with their observed internalization rate. Overall, these data are strongly supportive of the existence of a low-efficiency macropinocytic mechanism of entry for these non-pathogenic bacteria. Moreover, the observed requirements for host tyrosine kinase and protein kinase C activities suggest that it is inducible.

Identification of an isolate of Rickettsia canada from California.

A strain of Rickettsia canada was recovered in 1980 an adult rabbit tick, Haemaphysalis leporispalustris, taken from a black-tailed jack rabbit, Lepus californicus, in Mendocino County, California. In all examined biologic characteristics, this isolate, CA410, is indistinguishable from the prototype, strain 2678, isolated in Ontario, Canada, in 1963. These similarities include serologic and immunologic reactivity in laboratory mice and guinea pigs, cultural characteristics in Vero cells, chick embryo cells and embryonated eggs, low pathogenicity for mice, meadow voles and guinea pigs, unusual resistance to streptomycin, morphology by electron microscopy, and molar percentages of guanine plus cytosine of the deoxyribonucleic acids. Recovery of this second strain in the same species of tick, but far removed in time and place from the origin of the prototype, provides evidence that R. canada is an established, ecologically stable, rickettsia in North America.

Production of monoclonal antibodies reactive with a denatured form of the Friend murine leukemia virus gp70 envelope protein: use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting.

Four monoclonal antibodies were selected for their ability to recognize the envelope protein of Friend murine leukemia virus (F-MuLV) in methanol-fixed tissue culture cells. Each of these monoclonal antibodies was found to react only with F-MuLV. By using recombinant retroviruses, it was determined that each of the monoclonal antibodies recognized the C-terminal one-third of the F-MuLV gp70 envelope protein. The monoclonal antibodies were effective in radioimmunoprecipitation of F-MuLV proteins, and one of the antibodies, 720, was also effective in Western blotting. The ability of antibody 720 to react with F-MuLV in methanol-fixed cells facilitated the use of a sensitive immunoperoxidase method with a focal virus infectivity assay. In immunohistochemical studies using light microscopy, antibody 720 could specifically label F-MuLV-infected cells in acetone-fixed tissue sections from F-MuLV-infected animals. Finally, in immuno-gold labelling studies using electron microscopy, antibody 720 could be used to distinguish F-MuLV from amphotropic MuLV.

Ixodes ricinus: vector of a hitherto undescribed spotted fever group agent in Switzerland.

A tick/rickettsial survey in various parts of Switzerland revealed the presence of a new, hitherto undescribed spotted fever group rickettsia ("Swiss agent") in up to 11.7% of I. ricinus collected off vegetation. Infection in ticks was found to be generalized with rickettsiae developing intracellularly and occasionally also intranuclearly. As a result of massive growth in ovarial tissues, including the germinative cells, the rate of transovarial and filial infection was 100%. The "Swiss agent" appears to be nonpathogenic for guinea pigs, domestic rabbits, and Swiss mice, but in male meadow voles (Microtus pennsylvanicus) it produces a microscopically detectable infection in the tunica vaginalis. The rickettsia grows well in tissue culture systems including chick embryo fibroblast, Vero, and vole tissue cells, when inoculated via yolk sac into 5-day-old hens' eggs, it kills 100% of the embryos after 5 to 7 days. Antigenic relatedness of the "Swiss agent" to rickettsiae of the spotted fever group was indicated by indirect and direct fluorescent antibody staining. Preliminary serologic typing by microimmunofluorescence and by microagglutination indicated that the "Swiss agent" differs from all prototype strains of spotted fever group rickettsiae studied so far.

Challenging heterosexism in college health service delivery.

The empowerment and affirmation of lesbian, bisexual, and gay students is long overdue. This article explores how human immunodeficiency virus and acquired immune deficiency syndrome (HIV/AIDS), substance abuse, violence and hate-related crimes, suicide, and heterosexism all adversely affect the physical and emotional health of nonheterosexual college students. College health services must expand their current scope and practice and assume a leadership role in combating all forms of oppression by actively incorporating and addressing the unique health issues and needs of the lesbian, bisexual, and gay population. This article provides a brief overview of the relevant healthcare issues for lesbians, bisexuals, and gays; examples of heterosexism in college health services; and recommendations for institutional and personal and professional change.

Reactivation of Rickettsia rickettsii in Dermacentor andersoni ticks: an ultrastructural analysis.

Virulent Rickettsia in Dermacentor andersoni lose their pathogenicity and virulence for guinea pigs when subjected to physiological stresses, such as starvation (overwintering), of its tick vector. However, incubation of infected ticks at an elevated temperature (37 degrees C) for 24 to 48 h or feeding for a time (usually greater than 10 h) induces R. rickettsii to revert to a virulent state, a phenomenon defined as "reactivation." Electron microscopy reveals that the microcapsular and slime layers of R. rickettsii undergo changes dependent upon the physiological conditions within the tick vector. In engorged ticks, the microcapsular layer is readily identified as a discrete layer, approximately 16 nm thick, composed of globular subunits that have a periodicity of approximately 10 nm. The slime layer external to the microcapsular layer forms a discrete electron-lucent zone around the rickettsia. In starved ticks, neither the microcapsular layer nor slime layer remains a discrete entity. Instead, they are shed and form stringy, shredded, and somewhat flocculent strands of low electron density without periodicity. Incubation at 37 degrees C or feeding of starved infected ticks results in the restoration of a discrete microcapsular and slime layer. These reversible structural modifications are linked to physiological changes in the tick host and correlate with reactivation, i.e., restoration of pathogenicity and virulence of R. rickettsii.

Ultrastructure of Rickettsia rhipicephali, a new member of the spotted fever group rickettsiae in tissues of the host vector Rhipicephalus sanguineus.

Rickettsia rhipicephali is similar in ultrastructure to R. rickettsii while differing from other rickettsiae of the typhus group and of Q fever and others by its lack of a prominently reticulated cytoplasmic matrix and in the thickness of the inner osmophilic layer of the cell wall. In tissues of the tick vector Rhipicephalus sanguineus, R. rhipicephali had a mean length and width of 1.2 and 0.46 micrometer, respectively. It possessed a trilaminar cell wall with an adhering capsule-like layer. The trilaminar cell wall was approximately 12 to 18 nm thick; its inner osmophilic layer was thicker than that previously reported for other rickettsiae. The capsule-like layer varied from 7 to 18 nm thick. The plasma membrane was similar in structure, measurement, and appearance to that of other reported rickettsiae. The cytoplasm appeared to be composed of a finely granular, amorphous, ground substance and randomly dispersed ribosomes and lacked a reticular matrix or nuclear fibrils. In massively infected salivary glands and ovarial tissues of its tick vector, R. rhipicephali produced a low degree of histopathology which does not appear to affect the engorgement and egg-laying process of the ticks.

Variation in a major surface protein of Lyme disease spirochetes.

Two monoclonal antibodies (H6831 and H5TS) differed in their indirect immunofluorescence reactivity when tested against 14 strains of Lyme disease spirochetes. Strains were bound by both antibodies, by H6831 or H5TS alone, or by neither. Western blot and immunoprecipitation studies revealed that the determinants of both antibodies were associated with abundant proteins with apparent subunit molecular weights of ca. 34,000 (34K-range proteins). The following results indicated that the 34K-range proteins were exposed on the surface of the spirochetes. (i) Antibody H6831 agglutinated the spirochetes; (ii) immune electron microscopy showed that the H6831 determinant was associated with the outer membrane; (iii) radiolabeled H6831 bound to live organisms; and (iv) proteases effectively removed the 34K-range proteins from intact cells. With their demonstrated variability and exposure on the surface, the 34K-range proteins may contribute to the serotype specificity of Lyme disease spirochetes.

Sexual transmission of spotted fever group rickettsiae by infected male ticks: detection of rickettsiae in immature spermatozoa of Ixodes ricinus.

Electron microscopic evidence is provided showing that the newly described "Swiss agent," a spotted fever group rickettsia, is incorporated into the reproductive cells of its male tick vector, Ixodes ricinus. Rickettsiae were found in spermatogonia, spermatocytes, and maturing spermatids. The potential significance of these observations is briefly discussed in relation to published data on sexual transmission of rickettsiae by ticks.

Monocytic differentiation inhibits infection and granulocytic differentiation potentiates infection by the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne infection with a specific tropism for granulocytes. We previously isolated and cultivated the HGE agent in the promyelocytic leukemia cell line HL-60 and have also demonstrated the susceptibility of both granulocytic and monocytic human marrow progenitors. Circulating monocytes have not been observed to be infected, suggesting that cell susceptibility may be differentiation specific. To evaluate this hypothesis, HL-60 cells were differentiated towards granulocytes (with dimethyl sulfoxide or all-trans retinoic acid) or toward monocytes-macrophages (with 12-O-tetradecanoylphorbol-13-acetate [TPA], gamma interferon, or 1, 25-dihydroxyvitamin D3) and then challenged with HGE. HGE binding, internalization, and proliferation were compared in differentiated and untreated control HL-60 cells by immunofluorescence, electron microscopy, and Giemsa staining. Granulocytic differentiation resulted in a doubling of HGE binding and enhanced infection consistent with the agent's clinical tropism for neutrophils. Granulocytic cells were unable to kill internalized ehrlichiae even after activation induced by N-formyl-Met-Leu-Phe alone or together with tumor necrosis factor alpha. In contrast, monocyte-macrophage differentiation with TPA resulted in complete resistance to infection through at least two distinct mechanisms: (i) reduction in binding and uptake and (ii) killing of any internalized organisms. Diminished binding in TPA-treated cells correlated with their reduced expression of sialyl Lewis x (CD15s), a putative cellular receptor component for HGE. The degree of monocytic differentiation and activation induced (i.e., TPA > gamma interferon > vitamin D3) correlated with resistance to HGE. Thus, HL-60 cells exhibit a striking differentiation-specific susceptibility to HGE. Differentiation-induced changes in bacterial adhesion and killing capacity underlie the tropism of HGE for granulocytic HL-60 cells and, conversely, the resistance of activated macrophages to infection.