Ubiquitin-dependent proteolysis of trihydrophobin 1 (TH1) by the human papilloma virus E6-associated protein (E6-AP).

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

THE CARE NEEDS OF WOMEN INFECTED WITH THE HUMAN PAPILLOMA VIRUS: A COMPREHENSIVE APPROACH

This study is founded on the phenomenology of Martin Heidegger, with the objective to understand the care needs of women infected with the human papilloma virus. Participants were fourteen women who had been diagnosed with this infection. The guiding questions were: What is it like to have this diagnosis? Tell me your experience, from when you received your diagnosis until today. What has your health care been like? The questions revealed the theme - seeking care as solicitude - which showed the importance of the support of family and friends. The presence of the infection as the cause of marital conflicts and separation was another highlighted aspect. The statements showed that there was a sense of resignation after an unsuccessful attempt to find accurate and clear information in order to make assertive decisions. Health interventions for infected women must overcome the traditional models of care, including interventions for health promotion and prevention, with trained professionals who are sensitive to the subjective dimension.

The human papilloma virus 16E6 gene sensitizes human mammary epithelial cells to apoptosis induced by DNA damage.

Programmed cell death (apoptosis) is a normal physiological process, which could in principle be manipulated to play an important role in cancer therapy. The key importance of p53 expression in the apoptotic response to DNA-damaging agents has been stressed because mutant or deleted p53 is so common in most kinds of cancer. An important strategy, therefore, is to find ways to induce apoptosis in the absence of wild-type p53. In this paper, we compare apoptosis in normal human mammary epithelial cells, in cells immortalized with human papilloma virus (HPV), and in mammary carcinoma cell lines expressing wild-type p53, mutant p53, or no p53 protein. Apoptosis was induced with mitomycin C (MMC), a DNA cross-linking and damaging agent, or with staurosporine (SSP), a protein kinase inhibitor. The normal and HPV-transfected cells responded more strongly to SSP than did the tumor cells. After exposure to MMC, cells expressing wild-type p53 underwent extensive apoptosis, whereas cells carrying mutated p53 responded weakly. Primary breast cancer cell lines null for p53 protein were resistant to MMC. In contrast, two HPV immortalized cell lines in which p53 protein was destroyed by E6-modulated ubiquitinylation were highly sensitive to apoptosis induced by MMC. Neither p53 mRNA nor protein was induced in the HPV immortalized cells after MMC treatment, although p53 protein was elevated by MMC in cells with wild-type p53. Importantly, MMC induced p21 mRNA but not p21 protein expression in the HPV immortalized cells. Thus, HPV 16E6 can sensitize mammary epithelial cells to MMC-induced apoptosis via a p53- and p21-independent pathway. We propose that the HPV 16E6 protein modulates ubiquitin-mediated degradation not only of p53 but also of p21 and perhaps other proteins involved in apoptosis.

Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7.

Multiple mammary epithelial cell (MEC) types are observed both in mammary ducts in vivo and in primary cultures in vitro; however, the oncogenic potential of different cell types remains unknown. Here, we used human papilloma virus 16 E6 and E7 oncogenes, which target p53 and Rb tumor suppressor proteins, respectively, to immortalize MECs present in early or late passages of human mammary tissue-derived cultures or in milk. One MEC subtype was exclusively immortalized by E6; such cells predominated in late-passage cultures but were rare at early passages and apparently absent in milk. Surprisingly, a second cell type, present only in early-passage tissue-derived cultures, was fully immortalized by E7 alone. A third cell type, observed in tissue-derived cultures and in milk, showed a substantial extension of life span with E7 but eventually senesced. Finally, both E6 and E7 were required to fully immortalize milk-derived MECs and a large proportion of MECs in early-passage tissue-derived cultures, suggesting the presence of another discrete subpopulation. Identification of MECs with distinct susceptibilities to p53- and Rb-targeting human papillomavirus oncogenes raises the possibility that these cells may serve as precursors for different forms of breast cancer.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

Low expression of human histocompatibility soluble leukocyte antigen-G (HLA-G5) in invasive cervical cancer with and without metastasis, associated with Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class Ib molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICCW)] and absence [ICC without metastasis (ICCWT)] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICCWT (n=52 patients) and ICCW (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice

Human immunodeficiency virus type 1 (HIV-1) subtype C is the predominant HIV in southern Africa, and is the target of a number of recent vaccine candidates. It has been proposed that a heterologous prime/boost vaccination strategy may result in stronger, broader and more prolonged immune responses. Since HIV-1 Gag Pr55 polyprotein can assemble into virus-like particles (VLPs) which have been shown to induce a strong cellular immune response in animals, we showed that a typical southern African subtype C Pr55 protein expressed in insect cells via recombinant baculovirus could form VLPs. We then used the baculovirus-produced VLPs as a boost to a subtype C HIV-1 gag DNA prime vaccination in mice. This study shows that a low dose of HIV-1 subtype C Gag VLPs can significantly boost the immune response to a single subtype C gag DNA inoculation in mice. These results suggest a possible vaccination regimen for humans. © 2004 SGM.

Medical and nursing students perceived knowledge, attitudes, and practices concerning human immunodeficiency virus

Objective. To assess medical and nursing students’ knowledge, attitudes, and practices (KAP) regarding human immunodeficiency virus (HIV) in Fiji. Methods. A cross-sectional study of 275 medical and 252 nursing students that participated in a questionnaire survey on HIV KAP. Data was analysed according to their gender, program of study, and academic year. Results. The mean HIV knowledge (HK) and attitude scores were 16.0 and 41.3, respectively. Mean HK score was significantly higher in males compared to females. Significant positive correlations were found between HK and academic year for medical () and nursing () students and between HK and attitude scores (). The majority of students indicated fear in contracting HIV through clinical practice and felt that health care workers have the right to know a patients HIV status for their own safety. The majority would wear gloves to touch a patient if suspected of HIV. Conclusions. The study found a high level of HIV knowledge and positive attitude towards HIV patients. However, respondents also displayed negative attitudes and unacceptable practices probably due to fear. Training institutions need to ensure that students gain accurate knowledge on HIV especially on transmission routes to allay the fear of caring for HIV-infected patients.

Analysis of human immunodeficiency virus type 1 drug resistance in children receiving nucleoside analogue reverse-transcriptase inhibitors plus nevirapine, nelfinavir, or ritonavir (Pediatric AIDS Clinical Trials Group 377)

In Pediatric AIDS Clinical Trials Group 377, antiretroviral therapy-experienced children were randomized to 4 treatment arms that included different combinations of stavudine, lamivudine (3TC), nevirapine (Nvp), nelfinavir (Nfv), and ritonavir (Rtv). Previous treatment with zidovudine (Zdv), didanosine (ddI), or zalcitabine (ddC) was acceptable. Drug resistance ((R)) mutations were assessed before study treatment (baseline) and at virologic failure. Zdv(R), ddI(R), and ddC(R) mutations were detected frequently at baseline but were not associated with virologic failure. Children with drug resistance mutations at baseline had greater reductions in virus load over time than did children who did not. Nvp(R) and 3TC(R) mutations were detected frequently at virologic failure, and Nvp(R) mutations were more common among children receiving 3-drug versus 4-drug Nvp-containing regimens. Children who were maintained on their study regimen after virologic failure accumulated additional Nvp(R) and 3TC(R) mutations plus Rtv(R) and Nfv(R) mutations. However, Rtv(R) and Nfv(R) mutations were detected at unexpectedly low rates.

Undertreatment of human immunodeficiency virus in psychiatric inpatients: a cross-sectional study of seroprevalence and associated factors

Background: The aims of this study were to evaluate the prevalence of HIV and its associated demographic and clinical factors among psychiatric inpatients of a general hospital. Methods: This was a single-center, observational, cross-sectional study that included patients consecutively admitted to our unit aged 16 years or older and with no relevant cognitive problems. The patients were evaluated using a semistructured interview and an appropriate test for HIV infection. Results: Of the 637 patients who were screened, 546 (86%) who consented to participate were included in the analyses. Twenty-five (4.6%, 95% confidence interval [CI] 3.0-6.8) patients were HIV-positive. The prevalence was higher among patients with substance misuse (17.4%, 95% CI 9.7-28.8). All except one of the 25 patients knew of their seropositive condition prior to participation in the study. Only 14 (56%) of the 25 seropositive patients had previously received pharmacological treatment for their infection. According to the multiple logistic regression analysis, the likelihood of HIV infection was lower in patients with higher levels of education and higher among patients who were single, had history of intravenous drug use, and had an HIV-positive partner, particularly if they did not use condoms. Among the patients with HIV infection, 18 (72%) had a history of suicide attempts compared with 181 (34.7%) of the patients without HIV infection (relative risk 2.1, 95% CI 1.6-2.7; P<0.001). Conclusion: HIV infection is highly prevalent in patients admitted to a psychiatric unit, especially those with a diagnosis of substance misuse. Seropositive patients show very poor treatment adherence. The risk of suicide seems to be very high in this population. Implementing interventions to reduce the suicide risk and improve adherence to antiretroviral therapy and psychotropic medications seems crucial.

Genotyping of TRIM5 locus in northern pig-tailed macaques (Macaca leonina), a primate species susceptible to Human Immunodeficiency Virus type 1 infection

Background: The pig-tailed macaques are the only Old World monkeys known to be susceptible to human immunodeficiency virus type 1 (HIV-1) infection. We have previously reported that the TRIM5-Cyclophilin A (TRIMCyp) fusion in pig-tailed macaques (Macaca n