Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

Optimization of crushing strength and disintegration time of a high-dose plant extract tablet by neural networks.

Optimization of crushing strength and disintegration time of a high-dose plant extract tablet was reached after extensive experimentation. Effects of the processing parameters, like compression force and tooling, and also of the excipients were found to be significant. Best results for both disintegration time and crushing strength were obtained with a plant extract that was granulated by roller compaction before compression. To gain more information about the different effects, artificial neural networks (ANNs) and a conventional multivariate method (partial least squares [PLS]) were used for data analysis. The topologies of the neural networks of the feed-forward type were optimized manually and by pruning methods. All methods were tested for contemplated parameters, crushing strength, and disintegration time. In general, ANNs were found to be more successful in characterizing the effects that influence crushing strength and disintegration time than the conventional multivariate methods.

[Arthroscopic therapy of patellar dislocation. Surgical technique and clinical results]. / Die arthroskopische Therapie der Patellaluxation. Operationstechnik und klinische Ergebnisse.

Between January 1986 and August 1995, we treated 86 patients suffering from lateral displacement of the patella with arthroscopic medial tightening and lateral release after a conservative functional treatment remained unsuccessful. 49 patients were available for follow-up studies. 29 patients were treated after primary dislocation of the patella, and 20 patients after recurrent dislocations (2-20). The lateral release was performed by arthroscopy in 28 patients and in 21 patients in an open procedure. The mean follow-up time was 47.3 months. The rate of reluxation was 8%. In the opinion of the patients, 44 (90% of the follow-up) operations were evaluated as good/very good, the average Lysholm score was 87.3 +/- 13.9. The clinical results were influenced by the point of time of the operation. Patients with monoluxation showed a lower rate of reluxation (3% vs. 15%) and superior functional and subjective results than those with recurrent dislocation. However, there were no significant differences between the open and closed performed lateral release. We recommend the technique presented here as a minimal-invasive method especially for patients with monoluxation of the patella.

Role of latency-associated transcript in herpes simplex virus infection.

Herpes simplex virus (HSV)-specified RNA transcript has been detected by in situ hybridization and by RNA blot analyses in latently infected peripheral nerve ganglia in experimentally infected mice and humans. Physical analysis of the nucleotide sequence, approximately 2,500, indicates possible splicing of the transcript into two overlapping open reading frames (ORF) and the presence of potential promoter elements. The latency-associated transcript, probably, is involved in the maintenance of latent herpes simplex virus infection; however, recent findings indicate that latent-phase transcription is involved in some in vivo reactivation.

Selection of L cell sublines resistant to (E)-5-(2-bromovinyl)-2'-deoxyuridine.

A mouse thymidine kinase (TK) deficient L cell subline and L cell sublines biochemically transformed by herpes simplex virus TK were cultured in the presence of increasing concentrations of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) which inhibited the growth of all sublines, and resistant sublines were isolated. Their growth properties were dependent on the medium condition used for selection. One subline had lost viral TK activity, while another retained viral TK activity with altered sensitivity of TK to BVDU in comparison with that of the original subline. Growth characteristics and TK activity of sublines are discussed.

Circulating human immunodeficiency virus (HIV) p24 antigen-positive lymphocytes: a flow cytometric measure of HIV infection.

Asymptomatic individuals seropositive for human immunodeficiency virus (HIV) progress in a heterogeneous fashion toward AIDS. To facilitate monitoring of disease progression and response to therapy, a rapid, new flow cytometric assay (FCA) lymphocyte p24-FCA, has been devised to quantify peripheral blood lymphocytes expressing cell-associated HIV-1 p24 antigen. Results from 55 asymptomatic, HIV-1-seropositive, serum p24 antigen-negative individuals ranged from undetectable (less than 0.1%) to 13.6% p24+ lymphocytes (mean, 2.0%). Mean values for three other groups studied were 0.1% for seronegative, viral culture-negative laboratory workers (n = 24); 4.2% for untreated patients with AIDS (n = 16); and 0.3% for AIDS patients receiving zidovudine (n = 11). Lymphocyte p24-FCA values were inversely related to the number of days to positive viral cultures and to levels of CD4+ lymphocytes. The ratio of p24+ lymphocytes to CD4+ lymphocytes may reflect the fraction of infected CD4+ lymphocytes. Lymphocyte p24-FCA determination may provide a method for monitoring response to antiretroviral therapy regardless of serum p24 antigen status.

Suppression of herpes simplex virus type 1 reactivation from latency by (+-)-9-([(Z)-2-(hydroxymethyl)cyclohexyl]methyl) guanine (L-653,180) in vitro.

Latent herpes simplex virus type 1 (HSV-1) infection was induced in human embryonic lung cells in vitro by using a combination of viral replication inhibitors and elevated temperature. Under reactivating conditions (superinfection by human cytomegalovirus or temperature manipulation), a nonantiviral thymidine kinase inhibitor (L-653,180) was found to suppress or delay reactivation of HSV-1 from latently infected human embryonic lung cells. L-653,180 alone or in combination with interferon was ineffective as a primary or acute viral replication inhibitor and was unable to induce latent HSV-1 infection in cell culture. These data suggest that initial or acute virus replication and replication resulting from reactivation from latency are separate events.

Viral oncogenesis and the immune system.

Oncogenic transformation of normal cells and the establishment of transformed cells to form malignant tumors is a complex, multistep process influenced by viruses in multiple ways. The relationship between viruses and the immune system manifests itself, in part, through various roles of viruses in transformation of host cells, including cells of the immune system. A large number of viruses participate in oncogenic transformation of cells in many animal species. Candidates for oncogenic transformation in man are human T lymphotropic viruses I and II, certain human papillomavirus types, hepatitis B virus, and Epstein-Barr virus. Various mechanisms, which may overlap with one another, have been proposed to account for viral oncogenesis. These include introduction of a directly transforming viral gene, retroviral transduction of protooncogenes, mutagenesis, uncoupling of cellular protooncogene expression from normal regulatory controls, overexpression of normal cellular genes resulting from effects of viral cis- or trans-acting factors, and inactivation of tumor suppressor genes. A second critical area of interaction between viruses and the immune system is in the selection of transformed cells. When cell transformation is accompanied by expression of tumor antigens, the immune system may influence tumor cell establishment and selection of transformed cells for metastatic outgrowth. Finally, host well-being may be severely compromised when viruses infect cells of the immune system, leading to an inability to mount immunological responses specific for opportunistic microorganisms and for cells transformed by viruses or nonviral agents. Human immunodeficiency virus infection exemplifies this phenomenon, although other viruses also negatively affect the immune system. The role of normal immune responses in limiting tumor cell growth is evident from the increased incidence of malignancies in immunocompromised hosts.

Effects of cellular fixatives on human immunodeficiency virus production.

Effects of cell fixation procedures appropriate for flow cytometric analysis on the infectivity of human T lymphoblastoid H9 cells infected with human immunodeficiency virus-1 (HIV-1) were evaluated to provide guidelines for choosing cell treatments for potentially infectious samples. H9 cells experimentally infected with HIV-1 were treated by the test fixation procedure, washed, and cocultured with equal numbers of live, uninfected H9 cells. To estimate the reduction in infectivity due to the fixation procedure, dilution series of live infected H9 cells in uninfected H9 cells were simultaneously established in culture. Cell cultures were incubated 8-10 d, harvested, and evaluated for evidence of HIV-1 infection by the presence of cell-associated HIV-1 antigens and/or by the presence of particle-associated reverse transcriptase activity in cell culture supernatants. Thirty-minute fixation with formaldehyde (1.85%), methanol (absolute), methanol:acetone (1:1), or paraformaldehyde (0.5%) reduced the infectivity of HIV-1-infected H9 cells by greater than 99.99%. To the same degree, a multi-step fixation procedure utilizing formaldehyde and ethanol was effective in reducing HIV-1 infectivity. Conversely, the erythrocyte fixative dimethylsuberimidate at 3 micrograms/ml was ineffective in reducing HIV-1 infectivity.

Tropism of human immunodeficiency virus 1 isolates for H9 cells and U937 cells.

Human immunodeficiency virus 1 (HIV-1) produced in the human T lymphoblastoid H9 cell line infected cells of that line more readily than cells of the human monocytoid U937 line. While both cell lines expressed detectable levels of the CD4 molecule on their surfaces, the H9 and U937 cell lines differed in expression of major histocompatibility complex class I and class II antigens. Both H9 and U937 cells were infected initially with HIV-1 derived from H9 cells. Cell-free culture supernatants were harvested after the cells had been infected for at least 1 month. Culture supernatant from HIV-infected H9 cells was used to infect H9 and U937 cells. Conversely, culture supernatant from HIV-infected U937 cells was used to infect H9 and U937 cells. The percentages of cells infected at each of several time points during the first few days after infection were determined by flow cytometric analysis of cell-associated HIV-1 major core protein p24. Infection of each cell line was more efficient when the cell type infected was identical to that in which the infecting supernatant was produced. However, this difference in tropism was not generated early after infection of each cell line, as might have been expected if this effect were mediated by cell surface molecules acquired during the process of budding through the cell membrane.

Anti-interferon antibody inhibits natural killer cell activity in HSV-2 genital infection in C57BL/6J mice.

We reported previously that the administration of anti-alpha/beta murine interferon antibody (anti-IFN-Ab) to C57BL/6J mice genitally infected with herpes simplex virus type 2 (HSV-2) dramatically increases morbidity and mortality. The effect of anti-IFN-Ab is visible early postinfection and it is more evident when low virus doses are used. This report describes our study of the mechanism of action of anti-IFN-Ab. We found that anti-IFN-Ab did not affect peripheral multiplication of the virus at the site of infection. The T-cell cytotoxic activity of spleen and lymph node cells of HSV-2 genitally infected C57BL/6J mice was dependent on the virus dose and peaked 7 days postinfection and, therefore, was excluded as a possible mechanism for the effect of anti-IFN-Ab. Natural killer (NK) cell activity paralleled the effect of anti-IFN-Ab closely; the activity peaked early postinfection, and there was no correlation between NK cell activity and virus dose. We have now observed that in mice treated with anti-IFN-Ab, the NK cell activity was significantly lower than in mice infected and not treated with anti-IFN-Ab. The lack of effect of anti-IFN-Ab on local multiplication of HSV-2 coupled with the decrease in NK cell activity indirectly point to a non-antiviral mechanism of action of endogenous IFN in vivo.

Protein analysis of herpes simplex virus latency in vitro established with cycloheximide.

Herpes simplex virus (HSV)-specific protein synthesis was examined during establishment of HSV latency and reactivation of virus in human embryonic lung cells treated with cycloheximide and incubated at 40.5 degrees. Eight viral proteins, identified during the first two days of establishment of latency at 40.5 degrees, were undetectable by Day 3. At least two synthesized proteins were present during the maintenance phase of latency. Reactivation of HSV (viral protein 135K) was first detected in latently infected cultures between 2 and 3 hr after superinfection with human cytomegalovirus (HCMV). During this period an 82K protein with the same molecular weight as one of the HCMV immediate-early proteins (82 and 75K) was detected in the immunoprecipitates of latently infected cultures with anti-HSV serum. Thus, this HSV latency system can be used to analyze protein synthesis and clarify reactivation of HSV by HCMV superinfection.

Analysis of viral proteins in human cytomegalovirus-infected cells during impaired lytic replication of herpes simplex virus.

Herpes simplex virus (HSV) latency can be established in vitro following arrest of virus replication and survival of infected cells in culture. Human cytomegalovirus (HCMV) has been shown to interact with HSV, resulting in reactivation of latent HSV. In addition, impaired replication of superinfecting HSV occurs in HCMV-infected human cells. HCMV-infected human embryonic lung cells inhibit production of infectious HSV despite replication of HSV DNA at levels comparable to those in control cultures infected only with HSV. Using radioimmunoprecipitation techniques, we found that the synthesis of HSV type 1 proteins of the alpha, beta/gamma, and gamma kinetic classes was impaired during the restricted replication of HSV in HCMV-infected HEL cells. However, synthesis of the HSV beta protein ICP-8 and HCMV alpha and beta proteins was not significantly affected in superinfected cell cultures.

Sexually transmitted viruses.

Human viruses known to be spread by sexual contact include herpes simplex viruses (HSV), papillomaviruses (HPV), human immunodeficiency virus (HIV), hepatitis B virus, and cytomegalovirus. Infections with the first three (HSV, HPV, and HIV) have reached epidemic proportions and pose global health concerns. Most of what we know about these human pathogens has been learned only recently, owing to the advent of DNA technologies and advances in culture techniques. In fact, our awareness of one virally transmitted venereal disease, acquired immunodeficiency syndrome, dates to the early 1980s. This paper touches on various aspects of the biology, pathogenesis, clinical manifestations, and, where applicable, oncogenicity of these agents, as well as current treatments and vaccine initiatives.

Kinetics of infected cell appearance as a determinant of number of human immunodeficiency virus-1 infectious units.

In order to optimize detection of human immunodeficiency virus-1 (HIV-1)-infected cells, the temporal appearance of virus antigens in newly infected H9 cell cultures was examined. Analyses were accomplished by indirect immunofluorescence labeling with each of 10 monoclonal antibodies and evaluation by flow cytometry. Of the antibodies examined, those specific for HIV-1 capsid protein p24, matrix protein p17, or their precursor molecule p55 allowed the earliest and most sensitive detection in infected cells fixed to allow detection of intracellular antigen. Discrimination of infected cells from uninfected cells was much less sensitive when three antibodies specific for HIV-1 glycoproteins were used to detect intracellular or cell surface antigen. In several experiments involving the time course of infection, we observed no differences in cell numbers between infected and uninfected H9 cultures initiated at identical cell concentrations. We hypothesized that it might be possible to quantitate infectious HIV-1 virions from the kinetics of infected cell appearance. Straight-line relationships between the log p24-positive cells and the time after infection were observed. These quantitative observations were employed to calculate the number of infectious units originally added to the culture that were capable of infecting H9 cells. The production of infectious virus, but not of cytopathic effects, was required. The results of this novel approach to the titration of infectious HIV-1 particles agreed well with those from median cell culture infective dose determination. This method could be employed with other infectious agents for which detection of cell-associated antigens is possible in cell cultures not destroyed by infection.