Babesia bovis : identification of immunodominant merozoite surface proteins in soluble culture-derived exoantigen

Babesia bovis merozoite proteins presenting as exoantigens in in vitro culture supernatants have been characterized. Bovine antisera to B. bovis exoantigens were used to immunoprecipitate [³⁵S]-methionine metabolically labeled or lactoperoxidase-catalyzed radioiodinated B. bovis merozoite proteins. A total of 24 metabolically labeled proteins ranging in molecular weight from 24,000 to 225,000 Da and 9 radioiodinated proteins with molecular weights varying between 24,000 and 225,000 Da were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies to B. bovis merozoite surface proteins were also used to immunoprecipitate metabolically labeled exoantigens directly from in vitro culture supernatants. These results demonstrate epitopes from at least nine merozoite surface proteins present in the exoantigen fraction, among which are the recently characterized major surface antigens 1 and 2, rhoptry-associated protein 1, and spherical body protein 2. Received: 1 April 1997 / Accepted: 6 May 1997     

Variation of the Mycobacterium tuberculosis PE_PGRS 33 gene among clinical isolates

PE_PGRS33, one of about 60 PE_PGRS genes in the Mycobacterium tuberculosis genome, encodes a surface-expressed protein that may be involved in the antigenic variation of M. tuberculosis strains and evasion of the host immune system. While genetic differences between the PE_PGRS33 genes of H37Rv and CDC1551 have been noted, genetic variation in this gene among clinical isolates has not been evaluated. In order to gain a better understanding of the genetic basis for the role of PE_PGRS in antigenic variation and evasion of the host immune system, we investigated the genetic diversity of the PE_PGRS33 gene among 123 clinical M. tuberculosis isolates from a population-based study, using PCR and DNA sequencing. The 123 isolates belonged to principal genetic groups 1, 2, and 3 and had IS6110 copy numbers ranging from 1 to 22. Eighty-four (68.3%) of the 123 isolates were found to have at least one sequence variation in the PE_PGRS33 gene, relative to that of H37Rv. Twenty-five different sequence variations were observed and included three insertions (ranging from 9 to 87 bp), nine deletions (ranging from 1 to 273 bp), one insertion-and-deletion event, and 12 single-nucleotide polymorphisms (six synonymous and six nonsynonymous). Analysis of the relationships among the different PE_PGRS33 gene sequence variations suggests that polymorphisms in the gene are shifting along evolutionary lineages. The observed genetic diversity of the PE_PGRS33 gene supports its role in antigenic variation and can serve as a basis for future investigations of the function of the PE_PGRS33 gene among clinical isolates.     

Evaluation of Previously Assigned Antibody Concentrations in Pneumococcal Polysaccharide Reference Serum 89SF by the Method of Cross-Standardization

An enzyme-linked immunosorbent assay (ELISA) and the antibody concentrations assigned to different pneumococcal capsular polysaccharide types were used to estimate concentrations of antibody to additional pneumococcal types in reference serum 89SF and to confirm assigned antibody values. This was possible because the slopes of curves of antibody binding to all polysaccharide types evaluated (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) were similar. The point estimates for total anti-pneumococcal antibody and immunoglobulin G (IgG) antibody determined by cross-standardization by an ELISA based on use of methylated human serum albumin (mHSA) to improve the efficiency of polysaccharide binding to the ELISA plate differed by less than 40% from those reported by Quataert et al. (Clin. Diagn. Lab. Immunol. 2:590–597, 1995) for types 1, 4, 6B, 7F, 9V, 14, 18C, and 23F. However, large differences were found between the assigned values and those obtained by our mHSA ELISA for types 3 and 19F. The mHSA ELISA and the direct polysaccharide coat ELISA may not measure antibodies to the same epitopes on polysaccharides of types 3 and 19F. The functional importance of these different antibody specificities is being investigated. We have thus confirmed the assigned IgG antibody values for most types by a different method and have extended antibody assignments to several additional types.     

Electron microscopic demonstration of horseradish peroxidase-tetramethylbenzidine reaction product as a method for identifying sensory nerve fibers in the rat tooth pulp

The purpose of the present investigation was to determine if the horseradish peroxidase could be used as a method for labeling sensory nerve fibers (specifically, tooth pulp afferents) for detailed ultrastructural analyses. HRP injected into the trigeminal ganglion of adult rats was taken up by ganglion cell bodies and transported anterogradely to their peripheral endings in the dental tissues. Following perfusion-fixation, the teeth were decalcified in EDTA, sectioned, reacted for HRP activity according to the tetramethylbenzidine (TMB) technique, and processed for electron microscopy. The HRP-TMB reaction product was clearly visible within most of the axons in the dental pulp, appearing as conspicuous, rectangular shaped aggregates of fine rods or needles.     

Rapid colormetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines

A rapid colormetric microtiter assay has been developed to detect cytotoxic lymphokines produced by human lymphocytes activated with lectins or tumor cells. The viability of lymphotoxin-treated target cells was detected using a tetrazolium dye that is reduced to a blue formazan by living but not dead cells. The amount of dye formed was quantitated using a microplate spectrophotometer (ELISA plate reader) and visual observations confirmed the amount of formazan dye produced was directly proportional to the number of viable target cells. The advantages of using this colormetric method are that it requires no washing steps or radioisotopes and its precision and rapidity. Optimal conditions were established using the murine L929 and human ESH-5L cell lines as target cells for detecting lymphotoxins produced by human lymphocytes. The data indicate that the L929 cell line was 10–50-fold more sensitive than the ESH-5L line to the lytic activity of cytotoxins produced by human phytohemagglutinin-P-activated T lymphocytes, or the cytotoxins produced by peripheral blood lymphocytes stimulated with various tumor cell lines. This assay system was also useful in detecting antibodies capable of neutralizing lymphotoxin activity and thus should be a suitable method to aid in the molecular characterization of these lymphokines.     

Epidermal lipid in several cetacean species: ultrastructural observations

The ultrastructure of the skin of four cetacean species, bottlenose dolphin (Tursiops truncatus) long-finned pilot whale (Globicephala melaena), humpback whale (Megaptera novaeangliae), and fin whale (Balaenoptera physalus) was investigated with particular reference to epidermal lipid. It has already been established that massive lipid reservoirs exist in whales, that the biochemical structures of cetacean lipids are unique, and that unusual intracellular lipid droplets appear in the epidermis. We report here some novel findings on scanning electron microscopic morphology of epidermal lipid, and on its ultrastructural morphology in general and specialized integumentary sites, including species not previously investigated. The intracellular epidermal lipid droplets were more extensive than lamellar body-derived intercellular lipid which is within the interstices of stratum externum cells. The intracellular droplets were spherical, highly variable in size ranging from 0.24 m to 3.0 m in diameter, appeared singly or were aggregated in cytoplasmic cavitations, and often were closely associated with epidermal cell nuclei. Evidence for exocytosis of the intracellular droplets was not observed. Significant numbers of intracellular lipid droplets are not observed in the epidermis of terrestrial mammals, so their presence is one of several aquatic specializations of the cetacean integument. Its full significance remains obscure, but it is more probably associated with epidermal cell metabolism than with secretion of lipid.     

Physiological response and childhood anxiety: association with symptoms of anxiety disorders and cognitive bias.

This study examined the physiological response (skin conductance and heart rate [HR]) of youth exposed to a mildly phobic stimulus (video of a large dog) and its relation to child- and parent-reported anxiety symptoms and cognitive bias in a community-recruited sample of youth (n = 49). The results of this study indicated that HR and skin-conductance response were associated with youth report but not parent report of their child's symptoms of anxiety disorders and that HR response was more strongly associated with anxiety symptoms than skin-conductance response. Physiological response was uniquely associated with youth-reported symptoms of anxiety rather than youth-reported depression. Finally, HR response interacted with cognitive bias in predicting childhood anxiety disorder symptoms in a manner consistent with theories of the etiology of anxiety disorders.     

Glial Fibrillary Acidic Protein Expression in Pleomorphic Adenoma of Salivary Gland: An Immunoelectron Microscopic Study

Previous immunocytochemical studies of pleomorphic adenomas have demonstrated consistent labeling with glial fibrillary acidic protein (GFAP). Cross-reactivity with other intermediate filaments of similar structure and chemical composition has been suggested to account for this seemingly inappropriate pattern of immunoreactivity. To investigate further this phenomenon, we examined five pleomorphic adenomas by immunoelectron microscopy. Ultrastructural features were similar to those described by other investigators, with ductal epithelium being surrounded by myoepithelial cells and modified cells becoming detached to form the isolated stellate and spindle cells of the stroma. As part of this process, many neoplastic myoepithelial cells appeared to lose their specialized ultrastructural features, assuming a rather undifferentiated appearance. Single and double immunoelectron microscopic labeling showed vimentin filaments in all these neoplastic myoepithelial cells. In contrast, GFAP filaments were identified only in the most undifferentiated cells. Such restriction of GFAP filaments to an ultrastructurally definable subset of neoplastic cells provides strong evidence against nonspecific staining due to cross-reactivity. Given the previously described coexpression of vimentin and GFAP by neoplastic cartilage, it appears likely that this immunophenotype in neoplastic myoepithelial cells reflects early chondroid differentiation.     

Distress and DNA repair in human lymphocytes

This research assessed differences in DNA repair in lymphocytes from high-and low-distressed individuals. A median split on Minnesota Multiphasic Personality Inventory (MMPI) Scale 2 divided 28 newly admitted nonpsychotic psychiatric inpatients into high- and low-distress subgroups. The high-distress subgroup had significantly poorer DNA repair in lymphocytes exposed to X-irradiation than low-distress subjects. We also found that lymphocytes obtained from this psychiatric sample had significantly poorer DNA repair than lymphocytes from nonpsychiatric control subjects when compared 5 hr after X-irradiation. A high level of distress therefore appears to be associated with significant dysfunctional differences at the molecular level which may have important implications for health. These data provide evidence for a direct pathway through which distress could influence the incidence of cancer.This research was funded in part by General Molecular Applications, Inc., the Bremer Foundation, the Samuel J. Roessler Fund, and Comprehensive Cancer Center Core Grant CA-16068-09.     

Molecular identification of T cell-specific antigens on human T lymphocytes and thymocytes

We have identified membrane glycoproteins which carry T cell-specific antigens on human T lymphocytes and thymocytes. Purified cells were surface-labeled with NaB³H₄ after treatment with neuraminidase and galactose oxidase. Immunoprecipitations were performed with rabbit anti-human T cell-specific antibodies using co precipitation with protein A-containing staphylococci strain Cowan I. The labeled membrane glycoproteins and the precipitates were subjected to polyacrylamide slab gel electrophoresis and visualized by fluorography. The antibodies specifically precipitated 4 proteins called GP200, GP180, GP165 and GP160 (mol. wts. = 200000, 180000, 165000 and 160000) from surface-labeled T lymphocytes and low-density (medullary) thymocytes. The GP200 and GP180 were not labeled on high-density (cortical) thymocytes. A protein with a mol. wt. of 45000 was precipitated from thymocytes. Another glycoprotein on T lymphocytes and thymocytes with a mol. wt. similar to that of mouse and rat Thy-1 or Θ antigen (mol. wt. 25000) reacted with the antibodies.