Social Network Organization, Structure, and Patterns of Influence Within a Community of Transgender Women in Lima, Peru: Implications for Biomedical HIV Prevention.

Understanding social network structures can contribute to the introduction of new HIV prevention strategies with socially marginalized populations like transgender women (TW). We conducted 20 semi-structured interviews and four focus groups (n = 32) with TW from selected social networks in Lima, Peru between May and July, 2015. Participants described layers of social influence from diverse actors in their social networks. The majority identified a close relative as their primary social support, with whom they confided secrets but avoided issues of transgender identity, sexuality, and sex work. Participants described close circles of TW friends with whom they shared information about gender identity, body modification, and sexual partners, but avoided issues like HIV. Community leadership included political leaders (who advocated for transgender rights) as well as social leaders (who introduced TW to hormone therapy, body modification, and commercial sex). Detailed analysis of TW social networks can contribute to implementation and acceptability of new HIV prevention technologies.

Responses of bovine T cells to fractionated lysate and culture filtrate proteins of Mycobacterium bovis BCG.

Bacterial cell lysates and culture filtrate proteins of Mycobacterium bovis BCG were each separated in a two-dimensional system that yields soluble protein fractions immediately available for probing with T cells. The fractions were used in lymphocyte proliferation assays using blood lymphocytes from cattle immunized with either viable or gamma-irradiated BCG. Cattle immunized with either form of BCG responded similarly to fractionated lysate proteins. Cattle immunized with viable BCG responded to culture filtrate proteins that were not recognized by cattle immunized with dead BCG. Marked heterogeneity of the responses to the culture filtrate proteins was seen.

Resistance against Taenia hydatigena in sheep after passive transfer of serum or colostrum.

The role of antibody in the resistance of sheep to infection with Taenia hydatigena metacestodes was examined using passive transfer of immunoglobulin. The immunoglobulin either was experimentally transferred in serum, or was transferred from immune ewes to their new-born lambs in colostrum. Pooled serum from donor lambs which had received one, light, oral infection did not protect recipients although the donors themselves were immune. However, transfer of pooled serum from donors which had either received three oral infections, or three immunizations with solubilized T. hydatigena oncospheres in a water-in-oil adjuvant, resulted in 70-80% fewer cysts in the recipients. Colostrum from ewes infected with three high or low doses of T. hydatigena eggs was transferred to their lambs. A short acting protection (one to three weeks) was observed in the lambs. Comparisons by ELISA and Western blot, of the anti-T. hydatigena oncosphere antibody content of the donor sera, the sera of the recipients collected 24 h and seven days after transfer, the sera of the lambs and ewes, and the colostrum of the ewes, indicated that resistance to the challenge infection depends upon a critical level of antibody.

Rapid electroelution of two-dimensionally separated protein mixtures: its use in in vitro assays of T cell activities.

A recently developed electroelution method for separated mixtures of proteins and its application in vaccine research were investigated. The method combines the high resolution power of two-dimensional gel electrophoresis with the advantage of direct probing of separated proteins with viable cells. An electroelution time of only 30 min was sufficient for complete protein transfer, as shown by Coomassie Brilliant Blue and silver staining. Inclusion of sodium dodecyl sulfate (SDS) into the electrophoresis buffer for the second dimension considerably improved the separation capacity. Furthermore, because of the low concentration of SDS (0.03%) no deleterious effects on the cells were seen. It was shown that T lymphocytes from cattle vaccinated with dead M. bovis BCG responded to numerous mycobacterial protein antigens, whereas unvaccinated control animals showed no, or very weak, responses. A comparison of T cell proliferation profiles obtained with different protein separations demonstrated the reproducibility of the method.

Protein antigens secreted by Mycobacterium paratuberculosis.

Proteins secreted by Mycobacterium paratuberculosis (M.ptb) during short-term cultivations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western(Immuno) blotting. Cultivation in a defined medium containing 35S methionine allowed autoradiographic detection of proteins which had been secreted or passively released by actively metabolizing M.ptb organisms. After the first 3 days of cultivation, 4 proteins with molecular weights of approximately 38, 50, 65 and 110 kilodaltons (kd) were detected on SDS gels. Longer incubation up to 12 days resulted in an increased concentration of these proteins as well as in appearance of additional proteins ranging from 14 to over 90 kd. In long-term (8-10 weeks) culture filtrates only two prominent proteins with molecular weights of 30 and 65 kd proteins could be detected. Immunoblot analysis showed that some of the proteins secreted during short-term cultivations were recognized by sera from M.ptb-infected sheep and more significantly by sera from animals which had been immunized with a M.ptb live vaccine strain. The study indicates that during short incubation times M.ptb may secrete immunoreactive proteins which are not dominant in long-term cultures.

Expression of Escherichia coli beta-galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses.

A promoter sequence, PAN, was isolated from Mycobacterium paratuberculosis and characterized. This promoter lies adjacent to, and outside, the 3' end of an IS900 insertion element. IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence. A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3. Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited beta-galactosidase activity. However, lacZ was only expressed in Escherichia coli under the control of PAN, when ORF2 was deleted. Immunization of mice with the recombinant M. bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against beta-galactosidase. The PAN-ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.

Mycobacterium paratuberculosis binds fibronectin.

Fibronectin, an adhesive glycoprotein which is present in plasma and on many host cell surfaces of many host organisms, binds to certain bacterial pathogens. This study demonstrates the ability of Mycobacterium paratuberculosis (M.ptb) to interact with 125I-labelled fibronectin purified from bovine and ovine plasma. Two M.ptb strains were tested: a clinical isolate and a commercially available vaccine strain. Both strains showed significant fibronectin-binding activities of 22 and 41%, respectively, whereas non-pathogenic M.phlei had almost no affinity for fibronectin. Binding activities were similar for ovine and bovine fibronectin. We found that fibronectin binding by M.ptb was (1) time-dependent, reaching saturation within 90 min, (2) specific, since it was inhibited by an excess of unlabelled fibronectin but not by albumin, (3) saturable, with an apparent dissociation constant of 1.25 x 10(-9) M and a maximal number of 1,600 binding sites per bacterium, and (4) sensitive to detergents, proteases and heat treatments, indicating the protein nature of the responsible binding component(s). Scatchard plot analysis gave a straight line suggesting the presence of a single type of fibronectin receptor on M.ptb.

Amplification and differentiation of the DNA of an abortigenic (type 1) and a respiratory (type 4) strain of equine herpesvirus by the polymerase chain reaction.

Unpurified DNA derived from cultures of equine fetal kidney cells infected with either equine herpesvirus type 1 or equine herpesvirus type 4 was amplified by the polymerase chain reaction using one pair of oligonucleotide primers. Restriction endonuclease digestion of the amplified segments with PvuII, followed by electrophoresis, revealed restriction fragment length polymorphisms which enabled the two virus types to be differentiated.

Antibody reactivities of Mycobacterium paratuberculosis infected sheep as analyzed by enzyme-linked immunosorbent assay and western blotting.

Antibody reactivities in sera from Mycobacterium paratuberculosis (M. ptb) infected and vaccinated sheep were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western (immuno)blotting using a sonicate antigen from M. ptb. Both methods allowed good differentiation between infected/vaccinated animals and noninfected controls. Removal of nonspecific crossreactive antibodies by absorption with a M. phlei sonicate antigen coupled to Sepharose reduced ELISA reactivities of positive sera by 50% and those of noninfected serum by 85%. Immunoblotting analysis revealed that reduction by M. phlei absorption was due to lower reactivities of M. ptb antigens in the range of 30 to 45 kDa. However, one protein with a molecular mass of approx. 27 kDa seemed to be specific for M. ptb since it reacted similarly with nonabsorbed and absorbed serum but not with antibodies which were eluted from M. phlei-Sepharose after absorption. Our findings indicate that M. ptb and M. phlei share a number of common antigens of potential pathogenic importance and that only a smaller part of proteins (i.e. the 27 kDa protein) might be specific for M. ptb.

In vitro and in vivo interaction between sporocysts of Sarcocystis muris and mouse peritoneal macrophages.

The interaction between the sporocysts of Sarcocystis muris and mouse peritoneal macrophages was studied both in vitro and in vivo in an attempt to determine whether or not resident peritoneal macrophages might effect the excystation of S. muris sporozoites from sporocysts injected intraperitoneally. Sporocysts of S. muris were phagocytosed by peritoneal macrophages both in vitro and in vivo. The addition of either unheated mouse serum or fetal calf serum did not significantly alter the level of phagocytosis. The percentage of phagocytosis in vivo and by thioglycolate-, proteose peptone- and BCG-elicited macrophages in vitro was greater than that shown by unstimulated macrophages in vitro. After 8 h incubation in vivo and in vitro a small proportion of sporocysts (less than 5%) was seen to have collapsed walls and up to 5% to have stained sporozoites, suggesting increased permeability of the sporocyst wall. The significance of increased permeability of the cyst wall in the process of sporozoite excystation is discussed.

A cloned DNA probe for the detection of Mycobacterium paratuberculosis.

DNA extracted from Mycobacterium paratuberculosis, which had been isolated from a cow with clinical Johne's disease, was used to make a gene library in the Escherichia Coli expression vector phage lambda gt11. Plaque-lifts were made from the library onto nitrocellulose membranes. These were screened by differential hybridization using radiolabelled chromosomal DNA from M. paratuberculosis and Mycobacterium phlei. By this method six recombinants that hybridized to M. paratuberculosis but not to M. phlei were identified. Three of these, designated lambda gt-R3, lambda gt-R4 and lambda gt-RS, containing DNA inserts of 2.5,1.5 and 3.7 kilobases (kb), respectively, were chosen for further analysis of their insert specificities. Following restriction with the endonucleases EcoRI and BamHI, the digestion fragments from the three recombinants were transferred to nitrocellulose membranes and probed with radiolabelled DNA from M. paratuberculosis and M. phlei. As expected, M. paratuberculosis DNA hybridized to all the fragments. M. phlei DNA hybridized to both the fragments that were generated from lambda gt-R3, to the single fragment from lambda gt-R4 and to two of the three fragments generated from lambda gt-RS. The fragment with which M. phlei DNA failed to hybridize was 0.45 kb in length. Multiple copies of this fragment were made in the plasmid pGEM-2; the plasmid DNA was then harvested and radiolabelled. Designated PAM-1, the radiolabelled material hybridized to a 3.7 kb fragment of EcoRI-digested M. paratuberculosis and to 2.2 kb fragments of similarly digested M. avium serovars 2 and 3. PAM-1 did not hybridize to DNA from the other four mycobacterial species examined or from Nocardia asteroides. The restriction fragment length polymorphism thus demonstrated distinguishes M. paratuberculosis from M. avium serovars 2 and 3.

Cellular changes in the spleens of mice infected with Sarcocystis muris.

Cellular changes in the spleens of mice infected with Sarcocystis muris have been studied. Immunofluorescent staining for B and T cells and alpha-naphthyl acetate esterase (ANAE) staining for macrophages combined with histological studies revealed marked changes in the populations and distributions of all three cell types. Infection was accompanied by a marked splenomegaly, attributable mainly to widespread hyperplasia of the white pulp. Following infection there was an increase in the relative proportions of B cells (i.e. surface immunoglobulin+) and ANAE+ cells and a decrease in the proportion of T cells (i.e. Thy 1.2+). There was also a progressive accumulation of immunoglobulin-containing cells in the periarteriolar lymphocytic sheaths. Splenomegaly was most pronounced 20 days after infection. At this time there were 9.3 times as many B cells, 3.7 times as many T cells and 16.6 times as many ANAE+ cells as in uninfected mice.

Age-related functional changes in the follicle-associated epithelium of the bursa of Fabricius in Shaver cockerels.

A study of the age-related functions of immunologically important components of the bursa of Fabricius in Shaver cockerels showed that endocytosis of carbon particles by the specialised follicle-associated epithelium was at a high level from hatching until 5 weeks of age and thereafter declined until at 18 weeks it could no longer be detected. The follicle-associated epithelium had marked non-specific esterase activity during the first 15 weeks of life as determined by a standard acid alpha-naphthyl acetate esterase method. The absolute weight of the bursa was at a maximum at 9 to 10 weeks. Involution began before 14 weeks and was complete by 22 weeks. The results indicate that the critical period for the bursa in regard to acquiring immunity from either local vaccination or environmental challenge is likely to be within the first five weeks of life.

Immunosuppression in Sarcocystis muris-infected mice: evidence for suppression of antibody and cell-mediated responses to a heterologous antigen.

Mice infected with Sarcocystis muris showed a significant reduction in plaque-forming cells (PFC) and delayed-type hypersensitivity (DHS) responses to an unrelated protein antigen, bovine gamma-globulin, when compared with uninfected controls. This immunosuppression was observed only when infection preceded immunization or when mice were immunized concurrently with infection, suggesting that suppression induced by murine sarcocystosis affected the induction and/or the differentiation of antigen-sensitive immunocytes. The immunosuppression lasted for 5 weeks, the period of this study, and affected cell-mediated responses more than antibody responses. The secondary PFC and DHS responses of mice immunized 14 days after infection and re-immunized 21 days later were also significantly lower than those of uninfected controls, whereas the secondary PFC and DHS responses of mice primed before infection were unimpaired. This indicated that S. muris infection affects only the induction but not the expression of immune memory.

Absence of lymphokine-enhanced macrophage migration in vitro in the Australian brush-tailed opossum, Trichosurus vulpecula.

The supernatants from cultures of either opossum or guinea pig splenic lymphocytes, stimulated with phytohaemagglutinin, significantly enhanced the in vitro migration of guinea pig peritoneal macrophages (P greater than 0.001) but not that of opossum peritoneal macrophages. Failure of opossum macrophages to respond to a putative macrophage chemotactic factor might account for the observed paucity of immune granulomas in these animals and help explain the species' susceptibility to tuberculosis.

Experimental trials on the use of radioimmunoassay for the detection of leptospiral antigens in urine.

Trials were conducted on the use of the solid phase radioimmunoassay (RIA) to detect leptospires or their antigens in simulated urine samples. The procedure was relatively simple to perform and appeared to be specific in detecting certain numbers of leptospiral organisms or their antigens in experimentally prepared samples. With this technique, it was possible to examine individual or pooled urine samples for the presence of leptospires within half a day. This technique may be of value for the detection of leptospiruric animals if the sensitivity of the technique could be further increased. Suggestions for the improvement of the procedure are discussed.

The acute psychiatric diagnostic interview.

The general outline of a psychiatric diagnostic interview given in Table 1 includes some broad suggestions for the amount of time to spend on each section. As a structured interview based on a symptom checklist questionnaire yields higher frequency of reports of symptoms, it is advisable to follow this type of format rather than a totally unstructured interview technique. Sim recommends a structured format that lends itself to computerization. Griest and colleagues suggest a computer interview, and there are data supporting the diagnostic accuracy of such a system. Within the framework of any diagnostic interview, a thorough exploration of the 10 critical elements listed in Table 5 is essential for accurate diagnosis. This information, which is usually obtainable in about 30 minutes, will enable the clinician to make a preliminary diagnosis, decide upon pharmacotherapy, and determine if hospitalization is warranted. A more intensive but lengthy and time-consuming structured diagnostic interview is the Schedule for Affective Disorders (SADS), which is more appropriate for inpatients or patients being considered for a research protocol.