The human adenovirus PI3K-Akt activator E4orf1 is targeted by the tumor suppressor p53.

Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.

A randomized, double-blinded Phase 3 study to demonstrate lot-to-lot consistency and to confirm immunogenicity and safety of the live-attenuated chikungunya virus vaccine candidate VLA1553 in healthy adults†.

BACKGROUND: The global spread of the chikungunya virus (CHIKV) increases the exposure risk for individuals travelling to or living in endemic areas. This Phase 3 study was designed to demonstrate manufacturing consistency between three lots of the single shot live-attenuated CHIKV vaccine VLA1553, and to confirm the promising immunogenicity and safety data obtained in previous trials. METHODS: This randomized, double-blinded, lot-to-lot consistency, Phase 3 study, assessed immunogenicity and safety of VLA1553 in 408 healthy adults (18-45 years) in 12 sites across the USA. The primary endpoint was a comparison of the geometric mean titre (GMT) ratios of CHIKV-specific neutralizing antibodies between three VLA1553 lots at 28 days post-vaccination. Secondary endpoints included immunogenicity and safety over 6 months post-vaccination. RESULTS: GMTs were comparable between the lots meeting the acceptance criteria for equivalence. The average GMT (measured by 50% CHIKV micro plaque neutralization test; µPRNT50) peaked with 2643 at 28 days post-vaccination and decreased to 709 at 6 months post-vaccination. An excellent seroresponse rate (defined as µPRNT50 titre ≥ 150 considered protective) was achieved in 97.8% of participants at 28 days post-vaccination and still persisted in 96% at 6 months after vaccination. Upon VLA1553 immunization, 72.5% of participants experienced adverse events (AEs), without significant differences between lots (related solicited systemic AE: 53.9% of participants; related solicited local AE: 19.4%). Overall, AEs were mostly mild or moderate and resolved without sequela, usually within 3 days. With 3.9% of participants experiencing severe AEs, 2.7% were classified as related, whereas none of the six reported serious adverse events was related to the administration of VLA1553. CONCLUSIONS: All three lots of VLA1553 recapitulated the safety and immunogenicity profiles of a preceding Phase 3 study, fulfilling pre-defined consistency requirements. These results highlight the manufacturability of VLA1553, a promising vaccine for the prevention of CHIKV disease for those living in or travelling to endemic areas.

Safety and immunogenicity of a single-shot live-attenuated chikungunya vaccine: a double-blind, multicentre, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: VLA1553 is a live-attenuated vaccine candidate for active immunisation and prevention of disease caused by chikungunya virus. We report safety and immunogenicity data up to day 180 after vaccination with VLA1553. METHODS: This double-blind, multicentre, randomised, phase 3 trial was done in 43 professional vaccine trial sites in the USA. Eligible participants were healthy volunteers aged 18 years and older. Patients were excluded if they had history of chikungunya virus infection or immune-mediated or chronic arthritis or arthralgia, known or suspected defect of the immune system, any inactivated vaccine received within 2 weeks before vaccination with VLA1553, or any live vaccine received within 4 weeks before vaccination with VLA1553. Participants were randomised (3:1) to receive VLA1553 or placebo. The primary endpoint was the proportion of baseline negative participants with a seroprotective chikungunya virus antibody level defined as 50% plaque reduction in a micro plaque reduction neutralisation test (µPRNT) with a µPRNT50 titre of at least 150, 28 days after vaccination. The safety analysis included all individuals who received vaccination. Immunogenicity analyses were done in a subset of participants at 12 pre-selected study sites. These participants were required to have no major protocol deviations to be included in the per-protocol population for immunogenicity analyses. This trial is registered at ClinicalTrials.gov, NCT04546724. FINDINGS: Between Sept 17, 2020 and April 10, 2021, 6100 people were screened for eligibility. 1972 people were excluded and 4128 participants were enrolled and randomised (3093 to VLA1553 and 1035 to placebo). 358 participants in the VLA1553 group and 133 participants in the placebo group discontinued before trial end. The per-protocol population for immunogenicity analysis comprised 362 participants (266 in the VLA1553 group and 96 in the placebo group). After a single vaccination, VLA1553 induced seroprotective chikungunya virus neutralising antibody levels in 263 (98·9%) of 266 participants in the VLA1553 group (95% CI 96·7-99·8; p<0·0001) 28 days post-vaccination, independent of age. VLA1553 was generally safe with an adverse event profile similar to other licensed vaccines and equally well tolerated in younger and older adults. Serious adverse events were reported in 46 (1·5%) of 3082 participants exposed to VLA1553 and eight (0·8%) of 1033 participants in the placebo arm. Only two serious adverse events were considered related to VLA1553 treatment (one mild myalgia and one syndrome of inappropriate antidiuretic hormone secretion). Both participants recovered fully. INTERPRETATION: The strong immune response and the generation of seroprotective titres in almost all vaccinated participants suggests that VLA1553 is an excellent candidate for the prevention of disease caused by chikungunya virus. FUNDING: Valneva, Coalition for Epidemic Preparedness Innovation, and EU Horizon 2020.

Intestinal Shedding of SARS-CoV-2 in Children: No Evidence for Infectious Potential.

The clinical courses of COVID-19 in children are often mild and may remain undiagnosed, but prolonged intestinal virus shedding has been documented, thus potentially enabling fecal-oral transmission. However, the infectious potential of SARS-CoV-2 viruses excreted with feces has remained unclear. Here, we investigated 247 stool specimens from 213 pediatric patients to assess the prevalence of intestinal SARS-CoV-2 shedding in hospitalized children without or with COVID-19 and determined the infectious capacity of stool-borne viruses. Upon RT-qPCR screening, the infectivity of virus-positive samples was tested in cell culture using the Vero-E6 permissive cell line. SARS-CoV-2 RNA was detected by RT-qPCR in 32 (13%) stool specimens, but the analysis of virus-positive samples in cell culture revealed no cytopathic effects attributable to SARS-CoV-2-related cell damage. Our findings do not support the notion of potential fecal-oral SARS-CoV-2 spreading, thus questioning the role of hygienic measures designed to prevent this mode of viral transmission.

Polylactic acid as a suitable material for 3D printing of protective masks in times of COVID-19 pandemic.

A critical lack of personal protective equipment has occurred during the COVID-19 pandemic. Polylactic acid (PLA), a polyester made from renewable natural resources, can be exploited for 3D printing of protective face masks using the Fused Deposition Modelling technique. Since the possible high porosity of this material raised questions regarding its suitability for protection against viruses, we have investigated its microstructure using scanning electron microscopy and aerosol generator and photometer certified as the test system according to the standards EN 143 and EN 149. Moreover, the efficiency of decontaminating PLA surfaces by conventional chemical disinfectants including 96% ethanol, 70% isopropanol, and a commercial disinfectant containing 0.85% sodium hypochlorite has been determined. We confirmed that the structure of PLA protective masks is compact and can be considered a sufficient barrier protection against particles of a size corresponding to microorganisms including viruses. Complete decontamination of PLA surfaces from externally applied Staphylococcus epidermidis, Escherichia coli, Candida albicans and SARS-CoV-2 was achieved using all disinfectants tested, and human adenovirus was completely inactivated by sodium hypochlorite-containing disinfectant. Natural contamination of PLA masks worn by test persons was decontaminated easily and efficiently by ethanol. No disinfectant caused major changes to the PLA surface properties, and the pore size did not change despite severe mechanical damage of the surface. Therefore, PLA may be regarded as a suitable material for 3D printing of protective masks during the current or future pandemic crises.

Intestinal HAdV Infection: Tissue Specificity, Persistence, and Implications for Antiviral Therapy.

Human adenovirus (HAdV) causes infections predominantly in early childhood and the tissue tropism of specific HAdV species determines the clinical manifestation, including infections of the gastrointestinal tract, respiratory tract, and keratoconjunctivitis. Why HAdV shows such a tropism has not yet been fully elucidated, but in the intestine different mechanisms for virus entry or resistence to immune modulatory factors have been described. Recently identified antiviral strategies by interferons provide evidence about the repression of E1A and maybe even promote HAdV persistence. The presence of HAdV in a persistent status in the gut is of importance in the setting of pediatric stem cell transplant recipients where HAdV detection in stool usually preceds clinical signs and severe infections are related to mortality. The reactivation of persistent intestinal HAdV infections in these patients needs further investigation also with regard to successful therapy options. In addition, several newly identified recombinant HAdV types have been isolated from stool samples, thus raising the question of possible recombination events in the gut. In this review, intestinal HAdV infections are discussed in relation to the tissue tropism, persistence, recombination, and new in-vitro models to enhance the knowledge about virus-host interactions and support the development of new treatment approaches.

Diagnostic Parameters of Adenoviremia in Pediatric Stem Cell Transplant Recipients.

Despite recent progress in the diagnostic risk assessment of human adenovirus (HAdV) infections in immunocompromised patients, clinical complications mediated by these viruses continue contributing to significant morbidity and mortality, particularly in the pediatric hematopoietic allogeneic stem cell transplant (HSCT) setting. Current data highlight the importance of monitoring stool samples to assess the risk of invasive HAdV infections in children undergoing HSCT. The advent of novel, more effective antiviral treatment options might permit successful virus control even at the stage of systemic infection, thus increasing the interest in optimized HAdV monitoring in peripheral blood (PB). We have screened over 300 pediatric HCST recipients by serial monitoring of stool and PB specimens, and identified 31 cases of invasive HAdV infection by quantitative pan-adenovirus RQ-PCR analysis of consecutive PB specimens. The diagnostic parameters assessed included HAdV peak levels (PL) and the time-averaged area under the curve (AAUC) of virus copy numbers. The predictive value for patient outcome reflected by non-relapse and HAdV-related mortality was determined. The patients were assigned to quartiles based on their PL and AAUC, and the readouts were highly correlated (p < 0.0001). Non-relapse mortality in patients by AAUC quartile (lowest to highest) was 26, 50, 75, and 86%, respectively, and AAUC was strongly correlated with non-relapse mortality (p < 0.0001), while the association between PL and non-relapse mortality was less pronounced (p = 0.013). HAdV-related mortality was absent or very low in patients within the two lower quartiles of both PL and AAUC, and increased to ≥70% in the upper two quartiles. Despite the significant correlation of PL and AAUC with patient outcome, it is necessary to consider that the risk of non-relapse mortality even within the lowest quartile was still relatively high, and it might be difficult therefore to translate the results into differential treatment approaches. By contrast, the correlation with HAdV-related mortality might permit the identification of a low-risk patient subset. Nevertheless, the well-established correlation of HAdV shedding into the stool and intestinal expansion of the virus with the risk of invasive infection will expectedly remain an essential diagnostic parameter in the pediatric HSCT setting.

Post-transplant Replication of Torque Teno Virus in Granulocytes.

Torque Teno virus (TTV) in humans is characterized by ubiquitous occurrence in peripheral blood (PB), without any related disease described to date. Several studies reported a significant increase of TTV plasma DNA levels in allogeneic transplant recipients, and suggested a correlation of elevated virus titers with immunosuppression and transplant-related complications. However, the site of viral replication in this setting has remained unclear. We have studied TTV in serial plasma specimens derived from 43 pediatric allogeneic hematopoietic stem cell transplantation (HSCT) recipients by RQ-PCR, and found increasing TTV-DNA levels in all patients post-transplant, with a peak around day +100 and maximum virus copy numbers reaching 4 × 10E9/ml. To assess whether the virus replicates in PB-cells, leukocyte subsets including granulocytes, monocytes, NK-cells, T- and B-lymphocytes were serially isolated by flow-sorting for TTV analysis in 19 patients. The virus was undetectable in most cell types, but was identified in granulocytes in all instances, revealing a median DNA copy number increase of 1.8 logs between days +30-100 post-transplant. Our data therefore provide evidence for TTV replication in granulocytes in this setting. In a control cohort of immunocompetent children and in HSCT recipients before day +30, TTV positivity in granulocytes was less common (33%), and the copy numbers were considerably lower. However, rising TTV replication about 2 weeks after granulocyte engraftment (>500 cells/µl) was observed suggesting that granulocyte recovery might be required for TTV expansion in severely immunosuppressed transplant recipients.

Management of adenovirus infection in patients after haematopoietic stem cell transplantation: State-of-the-art and real-life current approach: A position statement on behalf of the Infectious Diseases Working Party of the European Society of Blood and Marrow Transplantation.

The important insights gained over the past years in diagnosis and treatment of invasive adenoviral infections provide new paradigms for the monitoring and clinical management of these life-threatening complications. A meeting was held to discuss and subsequently disseminate the current advances in our understanding of the aetiology/pathogenesis and future treatment options facilitating effective control or prevention of adenovirus-related diseases in the allogeneic haematopoietic stem cell transplant setting. Invited experts in the field discussed recent progress with leading members of the Infectious Diseases Working Party of the European Society of Blood and Marrow Transplantation at the "State-of-the-art" Meeting in Poznan, Poland, in October 2017. In this review article, the panel of experts presents a concise summary of the current evidence based on published data from the last 15 years and on recent achievements resulting from real-life practice. The present position statement reflects an expert opinion on current approaches to clinical management of adenovirus infections in patients undergoing allogeneic haematopoietic stem cell transplant and provides graded recommendations of the panel for diagnostic approaches and preemptive therapy reflecting the present state of knowledge.

Broad-spectrum antiviral activity of the deubiquitinase inhibitor HBX against human adenoviruses

BACKGROUND: Human adenoviral (HAdV) infections are usually mild and self-limited, however, some infections from species A, B, C, D and E, can cause severe illnesses, which have raised public health concerns over the past few years. Current available antiviral therapies have limited efficacy and severe toxicity; therefore, finding new targets for specific anti-adenoviral drug design is urgently needed. Our previous work showed that the small molecule compound, HBX, inhibits HAdV type 5 (species C, HAdV-C5) replication and oncogenic transformation through inhibition of the cellular pro-viral factor ubiquitin-specific protease 7 (USP7). Here, we have tested the ability of HBX to inhibit other HAdV species, as well as different clinical isolates that are the cause of severe infections. METHODS: We treated HAdV-infected A549 cells with different concentrations of HBX and analysed the antiviral efficacy of the drug by determining the half maximal inhibitory concentration (IC50) necessary to decrease both viral genome copies and virus progeny production at different time points after infection. RESULTS: In addition to its effect on HAdV-C5, HBX was able to significantly inhibit virus genome replication and progeny release of all adenovirus types tested, with the exception of types 12 and 31, from species A. Of note, clinical isolates were more sensitive to HBX treatment than their prototype strains. CONCLUSIONS: These results point to HBX as a promising broad-spectrum anti-adenoviral drug, opening new opportunities to prevent severe adenoviral infections and to improve their treatment.

Intestinal Adenovirus Shedding Before Allogeneic Stem Cell Transplantation Is a Risk Factor for Invasive Infection Post-transplant.

Human adenoviruses (HAdV) are a major cause of morbidity and mortality in pediatric human stem cell transplant (HSCT) recipients. Our previous studies identified the gastrointestinal tract as a site of HAdV persistence, but the role of intestinal virus shedding pre-transplant for the risk of ensuing invasive infection has not been entirely elucidated. Molecular HAdV monitoring of serial stool samples using RQ-PCR was performed in 304 children undergoing allogeneic HSCT. Analysis of stool and peripheral blood specimens was performed pre-transplant and at short intervals until day 100 post-HSCT. The virus was detected in the stool of 129 patients (42%), and 42 tested positive already before HSCT. The patients displaying HAdV shedding pre-transplant showed a significantly earlier increase of intestinal HAdV levels above the critical threshold associated with high risk of invasive infection (p<0.01). In this subset of patients, the occurrence of invasive infection characterized by viremia was significantly higher than in patients without HAdV shedding before HSCT (33% vs 7%; p<0.0001). The data demonstrate that intestinal HAdV shedding before HSCT confers a greatly increased risk for invasive infection and disseminated disease post-transplant, and highlights the need for timely HAdV monitoring and pre-emptive therapeutic considerations in HSCT recipients.

Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Viruses shape a diversity of ecosystems by modulating their microbial, eukaryotic, or plant host metabolism. The complexity of virus-host interaction networks is progressively fathomed by novel metagenomic approaches. By using a novel metagenomic method, we explored the virome in mammalian cell cultures and clinical samples to identify an extensive pool of mobile genetic elements in all of these ecosystems. Despite aseptic treatment, cell cultures harbored extensive and diverse phage populations with a high abundance of as yet unknown and uncharacterized viruses (viral dark matter). Unknown phages also predominated in the oropharynx and urine of healthy individuals and patients infected with cytomegalovirus despite demonstration of active cytomegalovirus replication. The novelty of viral sequences correlated primarily with the individual evaluated, whereas relative abundance of encoded protein functions was associated with the ecologic niches probed. Together, these observations demonstrate the extensive presence of viral dark matter in human and artificial ecosystems.-Thannesberger, J., Hellinger, H.-J., Klymiuk, I., Kastner, M.-T., Rieder, F. J. J., Schneider, M., Fister, S., Lion, T., Kosulin, K., Laengle, J., Bergmann, M., Rattei, T., Steininger, C. Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Human cytomegalovirus infection downregulates vitamin-D receptor in mammalian cells.

Vitamin D (VD) is essential for the human body and involved in a wide variety of critical physiological processes including bone, muscle, and cardiovascular health, as well as innate immunity and antimicrobial responses. Here, we elucidated the significance of the VD system in cytomegalovirus (CMV) infection, which is one of the most common opportunistic infections in immunocompromised or -suppressed patients. We found that expression of vitamin D receptor (VDR) was downregulated in CMV-infected cells within 12h [hrs] post infection [p.i.] to 12% relative to VDR expression in mock-infected fibroblasts and did not recover during the CMV replication cycle of 96h. None of the biologically active metabolites of VD, cholecalciferol, calcidiol, or calcitriol, inhibit CMV replication significantly in human fibroblasts. In a feedback loop, expression of CYP24A1 dropped to 3% by 12h p.i. and expression of CYP27B1 increased gradually during the replication cycle of CMV to 970% probably as a consequence of VDR inhibition. VDR expression was not downregulated during influenza virus or adenovirus replication. The potent synthetic vitamin D analog EB-1089 was not able to inhibit CMV replication or antagonize its effect on VDR expression. Only CMV replication, and none of the other viral pathogens evaluated, inhibited the vitamin D system in vitro. In view of the pleiotropism of VDR, CMV-mediated downregulation may have far-reaching virological, immunological, and clinical implications and thus warrant further evaluations in vitro and in vivo.

Screening for adenoviruses in haematological neoplasia: High prevalence in mantle cell lymphoma.

Human adenoviruses possess oncogenic capacity which is well documented in mammalian animal models, but their possible implication in human malignancy has remained enigmatic. Following primary infection, adenoviruses can persist in a latent state in lymphocytes where the virus is apparently able to evade immune surveillance. In the present study, we have employed a broad-spectrum adenovirus polymerase chain reaction (PCR) assay to systematically screen more than 200 diagnostic specimens of different lymphoid malignancies including acute lymphocytic leukaemia (n=50), chronic lymphocytic leukaemia (n=50), various types of malignant lymphoma (n=100) and multiple myeloma (n=11) for the presence of adenoviral sequences. While most entities analysed revealed negative findings in virtually all specimens tested, adenoviral DNA was detected in 15/36 (42%) mantle cell lymphomas investigated. The most prevalent adenoviral species detected was C, and less commonly B. Adenovirus-positive findings in patients with mantle cell lymphoma were made at different sites including bone marrow (n=7), intestine (n=5), lymph nodes (n=2) and tonsillar tissue (n=1). The presence of adenoviral sequences identified by PCR was confirmed in individual cells by fluorescence in-situ hybridisation (FISH). The frequent observation of adenoviruses in mantle cell lymphoma is intriguings, and raises questions about their possible involvement in the pathogenesis of this lymphoid malignancy.

Assessment of drug candidates for broad-spectrum antiviral therapy targeting cellular pyrimidine biosynthesis.

Currently available antiviral drugs frequently induce side-effects or selection of drug-resistant viruses. We describe a novel antiviral principle based on targeting the cellular enzyme dihydroorotate dehydrogenase (DHODH). In silico drug design and biochemical evaluation identified Compound 1 (Cmp1) as a selective inhibitor of human DHODH in vitro (IC50 1.5±0.2nM). Crystallization data specified the mode of drug-target interaction. Importantly, Cmp1 displayed a very potent antiviral activity that could be reversed by co-application of uridine or other pyrimidine precursors, underlining the postulated DHODH-directed mode of activity. Human and animal cytomegaloviruses as well as adenoviruses showed strong sensitivity towards Cmp1 in cell culture-based infection systems with IC50 values in the low micromolar to nanomolar range. Particularly, broad inhibitory activity was demonstrated for various types of laboratory and clinically relevant adenoviruses. For replication of human cytomegalovirus in primary fibroblasts, antiviral mode of activity was attributed to the early stage of gene expression. A mouse in vivo model proved reduced replication of murine cytomegalovirus in various organs upon Cmp1 treatment. These findings suggested Cmp1 as drug candidate and validated DHODH as a promising cellular target for antiviral therapy.

Presence of adenovirus species C in infiltrating lymphocytes of human sarcoma.

Human adenoviruses are known to persist in T-lymphocytes of tonsils, adenoids and intestinal tract. The oncogenic potential of different adenovirus types has been widely studied in rodents, in which adenovirus inoculation can induce multiple tumors such as undifferentiated sarcomas, adenocarcinomas and neuroectodermal tumors. However, the oncogenic potential of this virus has never been proven in human subjects. Using a highly sensitive broad-spectrum qRT-PCR, we have screened a set of different human sarcomas including leiomyosarcoma, liposarcoma and gastro intestinal stroma tumors. Primers binding the viral oncogene E1A and the capsid-coding gene Hexon were used to detect the presence of adenovirus DNA in tumor samples. We found that 18% of the tested leiomyosarcomas and 35% of the liposarcomas were positive for the presence of adenovirus DNA, being species C types the most frequently detected adenoviruses. However, only in one sample of the gastro intestinal stroma tumors the virus DNA could be detected. The occurrence of adenovirus in the tumor sections was confirmed by subsequent fluorescence in-situ-hybridization analysis and co-staining with the transcription factor Bcl11b gives evidence for the presence of the virus in infiltrating T-lymphocytes within the tumors. Together these data underline, for the first time, the persistence of adenovirus in T-lymphocytes infiltrated in muscular and fatty tissue tumor samples. If an impaired immune system leads to the viral persistence and reactivation of the virus is involved in additional diseases needs further investigation.

R-(+)-alpha-lipoic acid inhibits endothelial cell apoptosis and proliferation: involvement of Akt and retinoblastoma protein/E2F-1.

Lipoic acid was recently demonstrated to improve endothelial dysfunction or retinopathy not only in rats but also in diabetic patients. We tested the hypothesis that R-(+)-alpha-lipoic acid (LA) directly affects human endothelial cell (EC) function (e.g., apoptosis, proliferation, and protein expression), independent of the cells' vascular origin. Macrovascular EC (macEC), isolated from umbilical (HUVEC) and adult saphenous veins and from aortae, as well as microvascular EC (micEC) from retinae, skin, and uterus, were exposed to LA (1 mumol/l-1 mmol/l) with/without different stimuli (high glucose, TNF-alpha, VEGF, wortmannin, LY-294002). Apoptosis, proliferation, cell cycle distribution, and protein expression were determined by DNA fragmentation assays, [(3)H]thymidine incorporation, FACS, and Western blot analyses, respectively. In macro- and microvascular EC, LA (1 mmol/l) reduced (P < 0.05) basal (macEC, -36 +/- 4%; micEC, -46 +/- 6%) and stimulus-induced (TNF-alpha: macEC, -75 +/- 11%; micEC, -68 +/- 13%) apoptosis. In HUVEC, inhibition of apoptosis by LA (500 mumol/l) was paralleled by reduction of NF-kappaB. LA's antiapoptotic activity was reduced by PI 3-kinase inhibitors (wortmannin, LY-294002), being in line with LA-induced Akt phosphorylation (Ser(437), +159 +/- 43%; Thr(308), +98 +/- 25%; P < 0.01). LA (500 mumol/l) inhibited (P < 0.001) proliferation of macEC (-29 +/- 3%) and micEC (-29 +/- 3%) by arresting the cells at the G(1)/S transition due to an increased ratio of cyclin E/p27(Kip) (4.2-fold), upregulation of p21(WAF-1/Cip1) (+104 +/- 21%), and reduction of cyclin A (-32 +/- 11%), of hyperphosphorylated retinoblastoma protein (macEC: -51 +/- 7%; micEC: -50 +/- 15%), and of E2F-1 (macEC: -48 +/- 3%; micEC: -31 +/- 10%). LA's ability to inhibit apoptosis and proliferation of ECs could beneficially affect endothelial dysfunction, which precedes manifestation of late diabetic vascular complications.

Investigation of adenovirus occurrence in pediatric tumor entities.

Adenoviruses (AdVs) contain genes coding for proteins with transforming potential, and certain AdV serotypes have been shown to induce tumors in rodents. However, data on the possible oncogenicity of AdVs in humans are scarce. We have therefore employed a real-time quantitative PCR (RQ-PCR) assay permitting highly sensitive detection of all 51 currently known human AdV serotypes to screen more than 500 tumor specimens derived from 17 different childhood cancer entities including leukemias, lymphomas, and solid tumors. Most tumor entities analyzed showed no evidence for the presence of AdV sequences, but AdV DNA was detected by RQ-PCR in different brain tumors including 25/30 glioblastomas, 22/30 oligodendrogliomas, and 20/30 ependymomas. Nonmalignant counterparts of AdV-positive brain tumors, including specimens of ependymal cells, plexus choroideus, and periventricular white matter, were screened for control purposes and revealed the presence of AdV DNA in most specimens tested. Identification of the AdV types present in positive malignant and nonmalignant brain tissue specimens revealed predominantly representatives of species B and D and, less commonly, C. To exclude contamination as a possible cause of false-positive results, specimens with AdV sequences detectable by PCR were subsequently analyzed by in situ hybridization, which confirmed the PCR findings in all instances. The central nervous system appears to represent a common site of AdV infection with virus persistence, thus providing the first evidence for the possible contribution of AdVs to the multistep process of tumor pathogenesis in brain tissue.