Complement-mediated regulation of Trichomonas vaginalis infection in mice.

Trichomonas vaginalis is a flagellated protozoan which causes trichomoniasis, a sexually transmitted disease of the human genitourinary tract. The importance of the alternative complement pathway in host defence against T. vaginalis was investigated in vitro. Kinetic studies utilising immunofixation following electrophoresis showed that both a strongly and weakly virulent strain of T. vaginalis activated murine serum C3. In vivo studies with congenic-resistant, C5-deficient, B10.D2/OSn- and C5-sufficient, B10.D2/nSn mice showed that the presence of C5 is a significant factor in the innate host resistance to primary infection with a strongly virulent, but not a weakly virulent trichomonad strain.

Involvement of Trichomonas vaginalis surface-associated glycoconjugates in the parasite/target cell interaction. A quantitative electron microscopy study.

Cytochemical labeling with gold particle-conjugated lectins in combination with transmission and scanning electron microscopy was used to localise specific sugar residues on the Trichomonas vaginalis cell surface. For investigation of the role played by the surface glycoconjugates of T. vaginalis in the process of parasite adhesion to the target cells, selected glycan moieties of parasite surface-bound molecules were removed by treatment with alpha-mannosidase and beta-N-acetylglucosaminidase. For observation of the parasite/epithelial cell interaction, human amnion membrane was employed as an in vitro model. Ultrastructure observations showed that T. vaginalis has distinct binding sites for concanavalin-A and wheat-germ agglutinin. This indicates the presence of mannose or mannose-like residues and N-acetyl-D-glucosamine-containing residues on the parasite membrane. The addition of inhibitory sugars to T. vaginalis incubation media diminished the subsequent labeling of the parasite cell coat with lectins. Enzyme treatment caused a significant reduction in the number of sugar residues on the cell surface of the parasite. The majority of the viable, motile, enzyme-treated T. vaginalis cells incubated with amnion membrane were incapable of adhering to the target cells. It was concluded that sugar residues, in particular alpha-D-mannose and N-acetyl-glucosamine, present in the parasite glycocalyx are involved in the process of T. vaginalis attachment to the host's epithelial cells. Removal of the T. vaginalis cell-surface sugars prevented the attachment to and damage of the epithelial cells.

Relationship of the virulence of Trichomonas vaginalis and the major histocompatibility complex in murine trichomonad infection.

A biological assay based upon the induction of abscesses in mice injected subcutaneously with Trichomonas vaginalis was shown to be a valid method for comparing the virulence of two isolates of T. vaginalis cultured from two patients, one suffering from severe vaginitis and the other exhibiting only mild disease. The data showed excellent correlation between the physical dimensions of abscesses in mice injected with each trichomonad isolate and the severity of vaginitis produced in the women from whom the isolates were obtained. The assay employed in our study incorporated measurement of the mean abscess volumes from day 1 to day 6 post-inoculation with T. vaginalis. We found that the abscess assay was clearly superior to a murine intraperitoneal assay for virulence evaluation of trichomonad isolates. We then used the murine abscess assay to determine the susceptibility of different strains of mice to infection with a virulent T. vaginalis isolate so as to test whether the genetic constitution of the host would influence the pathogenesis of the disease. BALB/c (H-2d) mice were susceptible to infection with T. vaginalis, but both CBA/CaH (H-2k) and BALB/c-H-2k mice were shown to be resistant. The quantitation of abscess formation in these inbred and congeneic resistant mouse strains demonstrates that the severity of infection with T. vaginalis is governed by genes mapping within the major histocompatibility complex.

An electron microscope study of the interaction between Trichomonas vaginalis and epithelial cells of the human amnion membrane.

The mechanism of cytopathogenicity of Trichomonas vaginalis is not well established. Adhesion of T. vaginalis to human epithelial cells is considered a prerequisite for parasitic infection and its pathogenic effect. To investigate cytopathological changes in the host caused by T. vaginalis infection, human amnion membrane was used as an in vitro model. T. vaginalis strain WAA38 from axenic culture was allowed to interact with the epithelial layer of the human amnion membrane for 6 and 9 h. Structural changes resulting from the interaction between parasite and host cells were studied with transmission (TEM) and scanning (SEM) electron microscopy. Analysis of the electron microscope data showed that T. vaginalis established contact with the host cells as early as after 6 h of incubation; however, a close attachment of parasites to the epithelial cells occurred only after 9 h. Amoeboid T. vaginalis formed numerous cytoplasmic extensions and adhered to the epithelial cells mostly through the portions of their body opposite the undulating membrane. A dense network of microfilaments was seen at the site of contact between T. vaginalis and epithelial cells. Damaged and desquamated epithelial cells were seen with TEM and SEM only in the areas where parasites were in direct contact with target cells.

Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water.

Cryptosporidium and Giardia are common causes of waterborne disease. The currently used methods of detecting these organisms in water rely on filtration capture, immunofluorescence labelling, and epifluorescence microscopy. These methods are inefficient, labour intensive, and require a highly skilled microscopist. We describe an alternative technique using flocculation concentration, followed by flow cytometry with fluorescence activated cell sorting. Environmental samples were analysed, and protozoan-like particles were sorted and collected before confirmation with epifluorescence microscopy. The technique was found to be significantly more sensitive and considerably faster than the conventional methods.

What's new in osteoclast ontogeny?

The osteoclast (OC) is a multinuclear bone-resorbing cell which shares several characteristics with cells of the mononuclear phagocyte system. Unlike terminally differentiated macrophages, OCs possess specialized characteristics such as tartrate resistant acid phosphatase activity and the presence of calcitonin receptors. It appears that myeloid progenitor cells, probably granulocyte-macrophage colony-forming units, generate OC precursors which then differentiate and fuse into OCs under the regulation of osteotropic hormones, cytokines and other local factors. Parathyroid hormone and 1,25 dihydroxy Vitamin D3 induce both the formation and fusion of OC precursors, while calcitonin inhibits fusion. Osteoblasts also produce factor(s) which regulate OC precursor differentiation and therefore bone resorption; the nature of these factor(s), however, is unknown. In addition, the OC surface interacts specifically with a range of cellular and extracellular matrix-associated ligands which influence OC differentiation. The precise regulation of OC formation, however, is complex and awaits further investigation.

Study of human epithelial cell detachment and damage: development of a model.

Detachment of epithelial structures from underlying basement membrane (BM) represents an important component of a number of human disease processes e.g. airway and alveolar diseases, gastrointestinal ulceration, and retinal diseases. This study describes a method of evaluating human epithelial cell detachment from BM that is simple, rapid, inexpensive, quantifiable and which, because it utilizes BM rather than tissue culture plastic, more closely mimics the in vivo situation than other methods. In this model human amnionic epithelial cells attached to their underlying BM are isolated from fresh placentae and mounted in a multi-well chemotaxis assembly. These membranes can be studied with the epithelial cell monolayer intact. Protease-induced detachment of the epithelial cells from the underlying BM was readily quantifiable using light microscopy and spectroscopy. Following removal of the native amnionic epithelial cells, immunoperoxidase staining for the BM attachment proteins laminin, fibronectin, and type IV collagen demonstrated that these molecules remain intact. The BM could also be used as an attachment surface to reconstitute other epithelial cell monolayers. Cultured human amnionic cells and human respiratory epithelial cells were both able to attach to the denuded BM in the absence of serum (% attachment = 85 +/- 15% and 92 +/- 8% respectively, P = 0.8). Natural BM was a better substrate for epithelial cell attachment than tissue culture plastic in that, in the absence of serum, cultured epithelial cell attachment to tissue culture plastic was 20 +/- 4% of the value for BM (P less than 0.05). Furthermore, cells attached to plastic adhered less effectively than to BM in that trypsin concentrations required to induce 50% cell detachment were 0.72 +/- 0.4 for plastic and 62 +/- 13 BAEE U/ml for BM (P less than 0.001). In view of the complex protein interactions known to be involved in the anchorage of human epithelial cells to BM, it is likely this model will be a useful tool for evaluating the mechanisms underlying human epithelial cell attachment and detachment in a variety of normal and disease situations.

Surface labelling of Strongyloides ratti: stage-specificity and cross-reactivity with S. stercoralis.

Various methods of radioiodination were compared for their efficacy in labelling the surface of Strongyloides ratti infective larvae and adult worms. The Iodogen method was chosen as the optimal technique for this parasite. The surface location of 125-iodine was confirmed with light microscope autoradiography of transverse sections of labelled worms. Stage-specific surface components were identified when the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles of infective larvae and adult worms were compared. Labelled surface molecules were solubilized with either the non-ionic detergent Triton-X-100, the anionic detergents sodium deoxycholate (DOC) or sodium dodecyl sulphate (SDS), or the cationic detergent cetyl trimethyl-ammonium bromide (CTAB). The CTAB extract yielded most labelled proteins that retained their antigenicity in an immunoprecipitation assay with hyperimmune mouse sera. Immunoprecipitation analysis with stage-specific mouse sera revealed that the surface of infective larvae is immunogenic and that there are no cross-reactions with adult worms. Adult worms resident in the intestine were not found to be immunogenic and showed a complete absence of reactivity. Antigenic determinants shared between S. ratti and S. stercoralis were identified. Patients infected with S. stercoralis precipitated bands with molecular weights 32 and 34 kD which were not reactive with normal sera. These reactions suggest the potential usefulness of the surface of S. ratti as a source of diagnostic antigens.

Quantitation of concanavalin A and wheat germ agglutinin binding by two strains of Trichomonas vaginalis of differing pathogenicity using gold particle-conjugated lectins.

Two strains of Trichomonas vaginalis, which differed in their pathogenicity for both women and experimental animals hosts, were compared for the presence and number of concanavalin A (ConA)- and wheat germ agglutinin (WGA)-binding sites on their surface using gold lectin conjugates. Both strains showed a high affinity for ConA and WGA and a similar pattern of gold particle distribution on the surface coat. The gold marker was distributed over the cytoplasmic membrane sparsely as single particles but more often in groups, suggesting the presence of single and clustered sugar residues on the parasite surface. Statistical analysis of the level of lectin binding, expressed as the number of gold particles attached per 1 micron plasmalemma, by pathogenic and non-pathogenic strains of T. vaginalis show that these two strains do not differ in the number of ConA receptors on their surfaces. However, WGA-binding receptors were more numerous on the surface of the pathogenic than on the non-pathogenic strain. This suggests that these two strains differ in the number of N-acetyl-D-glucosamine residues on their surfaces. The lectin-gold particle conjugate technique therefore appears more sensitive than agglutination assays or the horseradish peroxidase-3,3'-diaminobenzidine method for the assessment of lectin-binding sites on the surface of T. vaginalis.

Electron microscopical studies of Strongyloides ratti infective larvae: loss of the surface coat during skin penetration.

Previous indications using radiolabelled larvae that Strongyloides ratti free-living infective larvae lose a surface coat during penetration of the skin were further investigated by transmission electron microscopy of the cuticle of S. ratti infective larvae in the free-living stage, after penetration of mouse skin, and after migration to the lungs. These studies demonstrated the presence of a faint electron-dense surface coat external to the epicuticle on free-living worms which was absent from larvae recovered from the skin and lungs. When free-living infective larvae were incubated in 10% CO2 at 37 C and then examined with phase-contrast microscopy, worms were observed in the process of losing this coat. These observations confirm the hypothesis that S. ratti infective larvae lose a surface coat during penetration of the skin.

An ultrastructural study of mast cells in the alveolar wall of normal and asthmatic lung.

Specimens of normal and asthmatic lungs were studied at the electron microscopic level and the frequency and ultrastructural features of mast cells and their granules within the alveolar wall were assessed with morphometric techniques. The numerical density of mast cells per square millimetre of alveolar wall was 299 (SD = 258) in normal and 366 (SD = 260) in asthmatic lung. The mean area of the mast cell nucleated profile was 25.7 microns2 (SD = 6.3) in normal lung and 29.8 (SD = 6.2) in asthmatic. The average number of secretory granules per single mast cell nucleated profile was 55 (SD = 13) in normal lung and 60 (SD = 12) in asthmatic lung. The diameter of the individual secretory granule was 338.9 nm (SD = 42.6) in normal and 345.6 (SD = 47.7) in asthmatic lung. The volume density of secretory granules in normal and asthmatic lung was 6.31 microns3 and 5.81 microns3, respectively. The mean diameter of the individual subunit ('scroll') inside the secretory granule was 88.8 nm for both normal and asthmatic lung. In normal lung 64.2% of granules were of 'scroll' and 'combined' type, and 35.8% of granules were 'particulate' or 'empty'. In specimens from asthmatic patients 40.3% of granules had 'scroll' or 'combined' structures and 59.7% were 'particulate' or 'empty'. Our data suggest that there is no difference between the number of mast cells in normal and asthmatic lung. However, in pulmonary mast cells from asthmatic lung, degranulation is more common than in normal lung.

Ultrastructural and cytophotometric studies of lectin binding by human pulmonary macrophages from asthmatic and normal subjects.

Pulmonary macrophages from normal subjects and asthmatic patients were examined for the presence of sugar residues on their surface. The technique of bronchoalveolar lavage was employed to obtain cell samples. Ultrastructural and cytophotometric methods were used for studying the patterns of lectin binding by these two groups of macrophages. Three lectins, Concanavalin A (Con A), Wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA), were used in this investigation. Pulmonary macrophages from both normal and asthmatic persons revealed a high level of Con A, WGA and RCA binding. The distribution of the electron dense reaction product on the macrophage surfaces was relatively uniform. Quantitative cytophotometric studies showed that the level of binding of Con A by macrophages from both groups was approximately the same. Similar results were obtained with WGA--the difference between macrophages from normal and asthmatic persons was not statistically significant. In the case of RCA, macrophages from asthmatic patients showed a higher level of lectin binding than macrophages from normal persons. The conclusion is made that macrophages from asthmatic persons have more D-galactose residues on their surface.

Extraction of immune and inflammatory cells from human lung parenchyma: evaluation of an enzymatic digestion procedure.

The inflammatory and immune cell populations of the human lung parenchyma have not been characterized in detail. This report describes a novel and efficient procedure for their extraction. Histologically normal human lung tissue samples from pneumonectomy specimens were sliced to 0.5 mm, and digested in collagenase/DNAse. Viable mononuclear cell yields ranged from 15-48 X 10(6)/g, and were markedly in excess of reported methods employing mechanical tissue disruption, which normally yield populations containing almost exclusively macrophages. The lung digest population was examined by flow cytometry using monoclonal antibodies against cell surface receptors, and found to comprise up to 40% T lymphocytes, 10% B lymphocytes and 30% macrophages, contaminated by less than 1% peripheral blood cells. Based upon these figures, the recoverable lung parenchymal lymphoid cell pool appears considerably larger than previously recognized, being of the same order as the peripheral blood pool. Initial functional studies suggest that such cellular activities as antigen-specific T cell proliferation, antigen-presentation, interleukin 1 production and natural killer cell activity survive the extraction process, and controlled enzymatic digestion experiments with peripheral blood cells indicate that the degree of enzyme-mediated damage to these functions and to cell-surface structures, was minimal. The extraction method thus appears suitable for studying the types and functions of human parenchymal lung cells in health and disease.

Pharmacology of the erectile tissue of the canine penis.

To assess the role of different neuroeffector systems in penile erection, pharmacological agents were tested in vitro using strips of corpus cavernosum tissue from the dog. Alpha-adrenoceptor agonists, K+ or histamine caused concentration-dependent contraction. Tyramine caused a poorly sustained contraction, as did nicotine in some cases. In precontracted preparations, relaxation was caused by beta2-adrenoceptor or muscarinic cholinoceptor agonists, or by vasoactive intestinal peptide. The results suggest that a noradrenergic alpha-adrenoceptor system maintains penile flaccidity in the dog. Penile erection may result from the activation of inhibitory beta2-adrenergic, muscarinic and/or peptidergic systems.

Binding of concanavalin A and wheat germ agglutinin by murine peritoneal macrophages: ultrastructural and cytophotometric studies.

Concanavalin A and wheat germ agglutinin were employed in conjunction with the horseradish peroxidase-diaminobenzidine method for the detection of sugar residues on the surface coat of exudate and resident murine peritoneal macrophages. Electron microscopical and cytophotometric techniques were used for the visualization and quantification of the final reaction product on the surface of cells. After incubation with concanavalin A and wheat germ agglutinin, both exudate and resident macrophages showed readily detectable final reaction product indicating the presence of numerous, easily accessible, alpha-methyl-D-mannosyl and N-acetyl-D-glucosaminyl residues on their surface. The binding of concanavalin A was higher with resident than with exudate macrophages. With wheat germ agglutinin, a different pattern of lectin binding was observed: more electron-dense product was deposited on exudate than on resident macrophage surfaces. The binding of concanavalin A and wheat germ agglutinin to macrophages was inhibited by the competing sugars alpha-methyl-D-mannoside and N-acetyl-D-glucosamine, respectively.

Pathogenicity of Trichomonas vaginalis: cytopathologic and histopathologic changes of the cervical epithelium.

Virulence of 52 Trichomonas vaginalis isolates was estimated by the subcutaneous mouse assay. A positive linear relationship was found between the mean volumes of subcutaneous abscesses caused by the parasites in mice and severity of cervical epithelial abnormalities observed in the patients from whom these strains had been isolated. This relationship implies that virulence of the human urogenital trichomonad, as measured by the mouse assay, may be related to some factors associated with dysplastic changes in the cervical epithelium. No relationships appeared to exist between the results of the mouse assay and inflammation of the vagina and cervix as evaluated clinically or pathologically, although these data were not analyzed statistically; likewise, no relationships were found between the mouse assay and nonprotozoal microorganisms identified in donors of the trichomonad strains.

Effects of adrenaline and carbachol on the morphology of alveolar contractile cells in the lung of the toad (Bufo marinus).

Smooth muscle-like interstitial cells in toad (Bufo marinus) lung strips were examined by light and electron microscopy and subjected to morphometric analysis in an attempt to assess the effect of spasmogenic and relaxant agonists on contractile cells in peripheral airways. Carbachol (12.8 microM) caused a 43.6% reduction in contractile cell length compared to control, while adrenaline (100 microM) caused a 21.3% increase. The mean transverse and longitudinal-sectional area and perimeter of cell profiles were significantly greater in carbachol treated preparations than in control. In tissue that had relaxed after exposure to adrenaline, the mean area of longitudinally sectioned cells and the perimeter of cell profiles were significantly reduced.