Effect of Pseudomonas aeruginosa cytotoxin on thymidine incorporation by murine splenocytes.

The interaction of highly purified Pseudomonas aeruginosa cytotoxin (PAC) with murine splenocytes was examined. Added at culture initiation, PAC (0.1 to 0.5 microgram/ml) inhibited subsequent [3H]deoxythymidine incorporation measured between 42 to 48 h. Incorporation of [3H]deoxythymidine was inhibited 50% in lipopolysaccharide-, phytohemagglutinin-, and concanavalin A-stimulated cultures by 0.20, 0.32, and 0.39 microgram of PAC per ml, respectively. It is concluded that PAC exhibits a narrow inhibitory concentration response range of 0.1 to 0.5 microgram/ml which, secondarily, is affected by the presence of mitogens. Antitoxin added at splenocyte culture initiation, directly after PAC, yielded greater than or equal to 86% protection against PAC inhibition of [3H]deoxythymidine incorporation. Addition of antitoxin to cultures at different times after PAC demonstrated a time-dependent loss of antitoxin protective effect over a 12-h period, indicating that PAC became cell associated and refractory to antitoxin within this time period. PAC preincubated with splenocytes at 4 degrees C for less than or equal to 1 h could not be removed by washing of cells and was fully inhibitory to [3H]deoxythymidine incorporation when these cells were cultured at 37 degrees C. This finding was confirmed by demonstrating that 125I-labeled PAC bound immediately to cells. It is concluded that PAC action on splenocytes is dose- and time-dependent and consists of a two-phase process: (i) a very rapid binding of PAC to the cell surface available to antitoxin, and (ii) a slower toxicity development phase of ca. 12 h, during which PAC becomes refractory to antitoxin.     

Review of Technology: Planning for the Development of Telesonography

Teleradiology allows contemporaneous interpretation of imaging exams performed at some distance from the interpreting radiologist. The transmitted images are usually static. However, there is benefit to real-time review of full-motion ultrasound (US) exams as they are performed. Telesonography is transmission of full-motion sonographic data to a remote site. We hypothesize that US exams, read after having been compressed utilizing Motion Picture Experts Group version 4 (MPEG-4) compression scheme, transmitted over the Internet as streaming multimedia, decompressed, and displayed, are equivalent in diagnostic accuracy to reading the examinations locally. MPEG-4 uses variable compression on each image frame to achieve a constant output bit rate. With less compression, the bit rate rises, and the only way the encoder can contain bit rate within the set bandwidth is by lowering frame rate or reducing image quality. We review the relevant technologies and industry standard components that will enable low-cost telesonography.     

Interaction of lipopolysaccharides of Helicobacter pylori with basement membrane protein laminin.

The ability of hemagglutinating and poorly hemagglutinating strains of the gastroduodenal pathogen Helicobacter pylori to bind 125I-radiolabelled laminin was quantitated in a liquid phase assay. Although all strains bound laminin, some hemagglutinating strains were good binders of laminin (maximum of 31% binding), whereas poorly hemagglutinating strains bound intermediate to small amounts of laminin (minimum of 6% binding). Since a hydrophobic component of the bacterium has been reported to be involved in binding of laminin (T. J. Trust, P. Doig, L. Emödy, Z. Kienle, T. Wadström, and P. O'Toole, Infect. Immun. 59:4398-4404, 1991), we investigated the role of lipopolysaccharide (LPS) in the interaction of both types of strains with laminin. Although the extent of inhibition varied among strains, laminin binding to hemagglutinating and poorly hemagglutinating strains was inhibited with homologous and heterologous smooth-form LPS. The ability of heterologous rough-form LPS to produce inhibition comparable to that shown by smooth-form LPS indicated that the O side chain of H. pylori LPS was not involved in the interaction. Further inhibition experiments with dephosphorylated LPS, isolated core oligosaccharide, and free lipid A suggested that a phosphorylated structure in the core oligosaccharide mediates the interaction of a hemagglutinating strain of H. pylori with laminin, whereas a conserved nonphosphorylated structure in the core oligosaccharide mediates the interaction of a poorly hemagglutinating strain. Furthermore, we showed that the interaction of H. pylori LPS with 125I-radiolabelled laminin in a solid phase assay was saturable, specific, and inhibitable with unlabelled laminin. It was postulated that the initial recognition and binding of laminin by H. pylori may occur through LPS and that subsequently a more specific interaction with a lectin-like adhesin on the bacterial surface occurs.     

Immunogenicity in guinea pigs and tolerance in grass pollen-sensitive volunteers of enteric-coated grass pollen allergens

An acid-insoluble, methacrylic acid, methyl methacrylate copolymer (Eudragit L-100) was used to give an enteric coating to a grass pollen extract in order to protect it against gastric degradation. Substantial protection against the degradative effects of simulated gastric secretion was demonstrated using this preparation which was well tolerated by grass pollen-allergic volunteers. The enteric-coated allergen induced a greater secondary antibody response than did an aqueous presentation when administered orally to guinea pigs which had been primed previously by subcutaneous injection. This result indicates that an effective hyposensitisation regimen could consist of a short series of initial parenteral injections, followed by an oral course of the protected allergen.     

Serological analysis of the heat-stable antigens involved in serotyping Campylobacter jejuni and Campylobacter coli

Analysis with serotyping antisera showed that carbohydrate determinants were the dominant heat-stable antigens of Campylobacter jenuni/coli involved, whereas proteins did not contribute to the serological reactions. Lipopolysaccharide (LPS) along with a polysaccharide extract from whole bacteria (PS(WB] conferred strain serospecificity. In general, analysis with monoclonal antibodies in passive haemagglutination and co-agglutination tests showed the existence of similar antigenic determinants in LPS and PS(WB) of the same strain. However, in some strains determinants were detectable in LPS but not in PS(WB) using monoclonal antibodies, in other strains the situation was reversed. All of these monoclonal antibodies reacted with LPS in the more sensitive immunoblotting technique. The presence of 2-keto-3-deoxyoctonic acid in PS(WB) preparations, in the absence of endotoxin, supported the conclusion that PS(WB) was derived from LPS during extraction. The lack of detection of a reaction by monoclonal antibodies with LPS in passive haemagglutination, in contrast to immunoblotting, was suggested due to the presence of low concentrations of the relevant epitopes because of the procedure used to prepare the LPS tested.     

Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro

The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM(2) and GM(3) gangliosides, with minor amounts of structures mimicking GD(1b) and GD(2). Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM(2) to GM(3) ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM(3) ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.     

Methods for the selection and growth of antigen-specific cytolytic T lines and clones bearing a defined T cell receptor beta chain marker

Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed.     

Neuroendocrine carcinomas (carcinoid tumor) of the testis. A clinicopathologic and immunohistochemical study of ten cases

We studied 10 cases of primary pure testicular neuroendocrine carcinoma. Patients were between 16 and 48 years old and had testicular swelling with pain or a painless testicular mass and no history of neuroendocrine carcinoma or other malignant neoplasm. All underwent orchiectomy. The tumors were low (n = 9) and intermediate (n = 1) grades with a variegated histologic appearance characterized by a nesting pattern, cords of neoplastic cells with rosettes, or sheets of neoplastic cells. Mitotic activity was lacking in 9 cases. In 1 case, mitotic figures ranged from 7 to 8 per 10 high-power fields, and cellular atypia and comedo-like necrosis were present. Immunohistochemical studies using a keratin cocktail, chromogranin, synaptophysin, epidermal growth factor, p53, placental-like alkaline phosphatase, and CD117 (c-kit) were performed in all cases. Keratin, chromogranin, and synaptophysin were positive in all tumors. Clinical follow-up information was obtained for 6 patients (range, 12-60 months): 5 with low-grade tumors were alive 24 to 60 months after diagnosis; 1 with an intermediate-grade tumor died of tumor 12 months after initial diagnosis. The behavior of these tumors, while in the testicular region, correlates well with the histologic grade. We propose replacing the term testicular carcinoid with neuroendocrine carcinoma, which better reflects the nature of these neoplasms.