Expression and frameshifting but extremely inefficient proteolytic processing of the HIV-1 gag and pol gene products in stably transfected rodent cell lines

Expression, ribosomal frameshifting, and proteolytic processing of HIV-1 GAG and POL proteins were investigated in heterologous mammalian cells in order to elucidate the influence of the cellular background on these events. DNA fragments encoded by the gag and pol region were expressed in two rodent cell lines, LTK- and BHK. Both stably transfected cell lines continuously produce recombinant proteins which react with HIV-specific antisera. The GAG precursor and a 39-kDa proteolytic fragment thereof were the major recombinant proteins detected. Expression of the gag-pol region leads to the production of the GAG-POL precursor. Ribosomal frameshifting at the HIV-1 shifty sequence to a typical extent could be positively demonstrated by an enzyme assay. Despite the presence of the viral protease within the GAG-POL precursors, proteolytic processing of the HIV-derived polyproteins was extremely inefficient. The efficiency could not be enhanced by overexpression of the HIV-1 protease encoding region.     

The contribution of RNA-binding motif (RBM) antibody to the histopathologic evaluation of testicular biopsies from infertile men

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.     

HepG2 cell binding activities of different hepatitis B virus isolates: inhibitory effect of anti-HBs and anti-preS1(21-47).

The antigenic relationships among different hepatitis B virus (HBV) isolates were investigated by using monoclonal antibodies (MAbs) specific for HBs, preS2 (pHSA binding site), and preS1 (hepatocyte receptor-binding site) epitopes in a double immunoradiometric assay. In order to define possible functional differences resulting from structural and antigenic differences in the HBV env protein, the HBV isolates were compared in an in vitro cell-binding assay based on the attachment of 125I-labeled HBV to human hepatoma HepG2 cells. We provided evidence for a variability of the expression of preS1 and preS2 specificities in the peplomer (glyco)protein of HBV depending on dly subtype of HBsAg, which could affect the viral infectivity. We showed that the integrity of the HBV envelope structure associated with a large expression of preS1(21-47) epitopes is an essential factor for effective binding to HepG2 cells. Interestingly, the HBs-specific MAbs directed to disulfide-bond-dependent epitopes were found to be the best inhibitors of the preS1-HepG2 cell interaction (greater than 50%, at the final concentration of 0.5 micrograms/ml). The MAb F35.25 directed to the preS1(21-47) sequence corresponding to the hepatocyte receptor recognition site was, however, also found to inhibit binding. Thus, our results demonstrate the abilities of both anti-HBs and anti-preS(21-41) to block the attachment of complete HBV particles to HepG2 cells, suggesting that these antibodies should be virus neutralizing and would be expected to confer protection against reinfection.     

Seasonal variations in pre- and post-thaw donor sperm quality

BACKGROUND: The present study was conducted to evaluate seasonal variability in the quality of pre- and post-thaw semen parameters among sperm bank donors. METHODS: The first two consecutive ejaculates during the months March (spring, 92 males), June (summer, 97 males), September (autumn, 81 males) and December (winter, 97 males) were analysed. A comparison was made between sperm parameters from the same sperm donor at different seasons. Only males who donated semen samples during at least two seasons were enrolled in the study group (n = 103). Sperm specimens were cryopreserved in aliquots with fixed range of 8-12 x 10(6)/ml of progressive motile sperm concentration after thawing. RESULTS: Differences between months were found in sperm concentration (P = 0.030) and normal morphology (P = 0.038); highest values were found in March and December, and the lowest in September. Mean specimen volume and percent of motile sperm cells did not vary throughout the seasons. The freezability of the donors' sperm dropped dramatically from March to September, as determined by the number of straws (fixed aliquots of 0.5 ml) and total thawed progressive motile sperm that were cryopreserved for each male (P = 0.017 and P = 0.002, respectively). CONCLUSIONS: Cryopreservation of donor sperm is more effective during winter and spring than during the rest of the year.     

Subchronic infusion of clenbuterol, a beta-adrenergic agonist, decreases the cerebellar beta receptor

Subchronic, peripheral infusion of clenbuterol, a beta-adrenergic agonist, markedly reduced beta receptor density and isoproterenol-sensitive adenylate cyclase activity in the cerebellum of the rat. In contrast, infusion of salbutamol, isoproterenol or desipramine did not alter the beta receptor. The result of clenbuterol administration demonstrates, for the first time, a significant alteration of the cerebellar beta 2 receptor to a pharmacologic manipulation. This alteration may influence physiological and behavioral processes regulated by the cerebellum.     

Impact of Strain Heterogeneity on Lyme Disease Serology in Europe: Comparison of Enzyme-Linked Immunosorbent Assays Using Different Species of Borrelia burgdorferi Sensu Lato

For the standardization of serological tests for Lyme borreliosis (LB) in Europe, the influence of the heterogeneity of Borrelia burgdorferi sensu lato must be assessed in detail. For this study four immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISAs) with octyl-β-d-glucopyranoside extracts of strains PKo (Borrelia afzelii), PBi (Borrelia garinii), and PKa2 and B31 (both B. burgdorferi sensu stricto) were compared. Strains PKo, PBi, and PKa2 at the passages used for antigen preparations abundantly expressed outer surface protein C (OspC), whereas strain B31 at the passage used for antigen preparation did not express OspC. Sera (all from Germany) from 222 patients with clinically defined LB of all stages, 133 blood donors, and 458 forest workers were tested. None of the forest workers had symptoms consistent with LB at the time that the samples were collected. For IgM tests, receiver operating characteristic curves demonstrated that discrimination between sera from patients and blood donors was best with strain PKo and worst with strain B31. The discriminatory abilities of the four IgG ELISAs were similar in a diagnostically reasonable specificity range (90 to 100%). More than 20% of the sera from forest workers reacted strongly in the PKo IgG ELISA (optical density value, >1.5; other assays, less than 8%). Western blots of the sera with the most discrepant ELISA results revealed almost exclusive reactivity with p17. This highly immunogenic antigen is only expressed by strain PKo. This observation might be important for the development of assays enabling discrimination between asymptomatic or previous infection and active disease.Lyme borreliosis (LB) is a global tick-associated disease caused by infection with Borrelia burgdorferi sensu lato. The disorder develops in stages and has different manifestations. In Europe, three species pathogenic for humans (2) and at least eight serotypes of B. burgdorferi sensu lato (40, 44) demonstrating both inter- and intraspecies heterogeneity are known. Different species seem to show a preferential association with different clinical manifestations (1, 35, 44). The most frequent disorders in Eurasia are erythema migrans (EM) localized around the tick bite lesion, neuroborreliosis (NB), acrodermatitis chronica atrophicans (ACA), and arthritis (19, 32). In Europe, all three pathogenic species have been isolated from human biopsy specimens and cerebrospinal fluid (CSF) as well as from ticks (Ixodes ricinus), but the predominant species seem to be Borrelia afzelii and Borrelia garinii. B. afzelii has been found to be associated more frequently with skin lesions, whereas B. garinii is the predominant species found in patients with neurological disorders (8, 35, 40, 44). In North America only B. burgdorferi sensu stricto occurs (30, 44); arthritis occurs frequently, whereas ACA is almost unknown, and multiple EM lesions are more common in North America than in Europe (33).The diagnosis of Lyme disease is based on the recognition of typical clinical signs and is supported by laboratory tests, especially if the clinical picture is not clear. Since culture is laborious and insensitive and PCR assays are still considered controversial, routine testing comprises mostly serological methods. However, serology also harbors several problems: The occurrence of cross-reacting antibodies may result in false-positive findings (5). Furthermore, patients may still be seronegative in the early stages of the infection and the humoral immune response can be diminished after the early onset of antibiotic treatment (37). Several strategies for increasing both sensitivity and specificity (i.e., the discriminatory ability of the test) have been developed, for instance, preabsorption of cross-reactive antibodies with Treponema phagedenis (49), the use of detergent extracts of B. burgdorferi sensu lato (3), and the use of purified flagella (16) or various recombinant antigens (7, 31, 41, 42).Serological tests for Lyme disease have not been standardized so far, leading to considerable variations in test results among different laboratories. The heterogeneity of Lyme disease borreliae as well as different methods of antigen preparation and test performance may contribute to the problem.In Europe, the extent of variation resulting from the use of different strains for antigenic preparations is still widely discussed (1, 23, 46, 48). Differences in the regional distributions of borrelial species may further influence the preferential reactivities of sera from patients with LB (6, 26).In this study, enzyme-linked immunosorbent assays (ELISAs) with detergent extracts of different strains representing the three species pathogenic for humans were compared. Strains PKo (B. afzelii), PBi (B. garinii), and PKa2 (B. burgdorferi sensu stricto) at the passage used to obtain coating antigens abundantly expressed outer surface protein C (OspC), whereas strain B31 (B. burgdorferi sensu stricto) at the passage used to obtain coating antigen did not express OspC. In Western blot studies OspC has been shown to be one of the most immunodominant antigens for the early immune response (12, 13, 17, 48). The influence of the expression of this lipoprotein on the results of ELISAs was especially monitored by comparison of the test results for the otherwise closely related B. burgdorferi sensu stricto strains PKa2 and B31 (20, 29, 38, 43). To analyze the sensitivities and specificities of the four ELISAs, we probed sera from German patients with different clinical manifestations as well as sera from healthy blood donors and forest workers (as an example of a group highly exposed to ticks and at high risk of contracting infections with B. burgdorferi sensu lato).     

Disseminated skeletal lesions associated with pathogenic actinomycetes in a Pygmy hog (Sus salvanius)

The clinical and pathological features of an unusual crippling bone disease in an adult male Pygmy Hog, Sus salvanius, born the smallest in a litter of five and representing the rarest of the known living Suidae, are described. Radiological studies revealed severe spondylosis deformans and focal sharply demarcated radiolucencies in virtually the whole skeleton, but particularly in the bones of the skull, the processes of multiple vertebrae, the ribs, scapulae and parts of the humeri and femora. The clinical chemistry measurements were indicative of pathological lytic processes in the skeleton. The focal bone lesions consisted of caseous necrosis, dystrophic calcification and peripheral fibroblastic demarcation. They contained colonies of filamentous bacteria identified as members of the Order Actinomycetales. Case history evidence suggests that the infection may have resulted from repeated skin trauma inflicted by litter-mates.     

Autoimmunity following viral infection: demonstration of monoclonal antibodies against normal tissue following infection of mice with reovirus and demonstration of shared antigenicity between virus and lymphocytes

Splenic lymphocytes from adult C57BL/6 mice infected with purified reovirus type 1 or 3 particles were fused with NS1 myeloma cells. Approximately 300 clones were obtained from each fusion (type 1 or type 3) and the supernatants from these clones were screened by radioimmunoassay for their ability to bind virus, T lymphocytes, brain, liver, lung tissues and isolated oligodendrocytes and ependymal cells. Approximately 10% of clones (33 and 26 clones, respectively) were positive for each fusion. For reovirus type 1:21% of positive clones bound only virus, 64% bound one of the normal tissues but not virus, and 15% bound both virus and one or more of the normal tissues. For reovirus type 3: 19% of positive clones bound only virus, 73% bound normal tissue only, and 8% bound both virus and normal tissue. Only 3 positive clones were obtained from uninfected control animals. These experiments demonstrate that (a) during the course of an immune response to a virus, autoantibodies are generated which react with a large variety of normal tissues and that (b) there are shared antigenic structures between viral determinants and normal tissue that can be identified by monoclonal antibodies. Although these results suggest two mechanisms by which an autoimmune response may develop following viral infection, the biological significance of these autoreactive monoclonal antibodies remains to be elucidated.     

Life skill training: psychoeducational training as mental health treatment

Fifty-four patients of a Veterans Administration Medical Center were assigned to either a life-skill training program that emphasized psychoeducational instruction and skill building or to a group counseling control condition. Subjects assigned to life-skill training were provided with 28 hours of instruction in interpersonal communication, purpose in life problem solving, and physical fitness/health maintenance. Control subjects received equal time engaged in psychiatric treatment that emphasized the analysis and exploration of personal problems, but with no direct coping skill training. Significant differences between the two groups were found on measures of interpersonal communication and meaningful purpose in life. Both groups received lower staff ratings on psychopathological behavior and demonstrated improvement on ratings of health and physical fitness upon completion of treatment. Twelve- and 24-month follow-up data that include rehospitalization rates are presented for each group.