Biphenylsulfonacetic acid inhibitors of the human papillomavirus type 6 E1 helicase inhibit ATP hydrolysis by an allosteric mechanism involving tyrosine 486

Human papillomaviruses (HPVs) are the causative agents of benign and malignant lesions of the epithelium. Despite their high prevalence, there is currently no antiviral drug for the treatment of HPV-induced lesions. The ATPase and helicase activities of the highly conserved E1 protein of HPV are essential for viral DNA replication and pathogenesis and hence are considered valid antiviral targets. We recently described novel biphenylsulfonacetic acid inhibitors of the ATPase activity of E1 from HPV type 6 (HPV6). Based on kinetics and mutagenesis studies, we now report that these compounds act by an allosteric mechanism. They are hyperbolic competitive inhibitors of the ATPase activity of HPV6 E1 and also inhibit its helicase activity. Compounds in this series can also inhibit the ATPase activity of the closely related enzyme from HPV11; however, the most potent inhibitors of HPV6 E1 are significantly less active against the type 11 protein. We identified a single critical residue in HPV6 E1, Tyr-486, substituted by a cysteine in HPV11, which is primarily responsible for this difference in inhibitor potency. Interestingly, HPV18 E1, which also has a tyrosine at this position, could be inhibited by biphenylsulfonacetic acid derivatives, thereby raising the possibility that this class of inhibitors could be optimized as antiviral agents against multiple HPV types. These studies implicate Tyr-486 as a key residue for inhibitor binding and define an allosteric pocket on HPV E1 that can be exploited for future drug discovery efforts.     

Methicillin-resistant Staphylococcus aureus: A public health issue with economic consequences

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become endemic worldwide in hospitals, and community-associated MRSA is spreading into the community at large. OBJECTIVES: To estimate the current cost of MRSA in Canada and to assess the magnitude of this public health issue. METHODS: An extensive review of the literature was conducted to gather epidemiology, health care resource utilization and cost data for MRSA in Canadian settings. The current MRSA burden was estimated using available cost data and the most recent epidemiology data. RESULTS: The rate of MRSA in Canadian hospitals increased from 0.46 to 5.90 per 1000 admissions between 1995 and 2004, while community-associated MRSA continued to spread into the community. Patients harbouring MRSA required prolonged hospitalization (average 26 days of isolation per patient), special control measures, expensive treatments and extensive surveillance. Total cost per infected MRSA patient averaged $12,216, with hospitalization being the major cost driver (81%), followed by barrier precautions (13%), antimicrobial therapy (4%) and laboratory investigations (2%). The most recent epidemiological data, combined with available cost data, suggest that direct health care cost attributable to MRSA in Canada, including cost for management of MRSA-infected and-colonized patients and MRSA infrastructure, averaged $82 million in 2004 and could reach $129 million in 2010. CONCLUSION: MRSA is a costly public health issue that needs to be tackled if the growing burden of this disease in Canadian hospitals and in the community is to be limited.     

Reduced encephalopathy in pigs with ischemia-induced acute hepatic failure treated with a bioartificial liver containing alginate-entrapped hepatocytes

OBJECTIVE: To analyze the effects of an extracorporeal bioartificial liver containing alginate bead-entrapped hepatocytes on pigs with ischemia-induced acute hepatic failure. DESIGN: Prospective animal study. SETTING: University and INSERM laboratory. SUBJECTS: Fifteen Large White/Pietrin female pigs weighing 20-30 kg. INTERVENTIONS: Acute hepatic failure was induced by end-to-side portocaval shunt and ligature of the whole porta hepatitis. The bioartificial liver was in a thermostabilized column, containing a fluidized bed of alginate beads that embedded porcine hepatocytes, connected to a plasmapheresis system. Intracranial pressure; survival; ammonia, total bilirubin, aminotransferases, alkaline phosphatase, and lactate concentrations; and clotting factors were studied. The groups were pigs with acute hepatic failure (group 1, n = 4), pigs with acute hepatic failure treated with bioartificial liver containing empty beads (group 2, n = 4), or porcine hepatocytes (group 3, n = 5). MEASUREMENTS AND MAIN RESULTS: In group 1, survival of pigs averaged 10.9 +/- 1.0 hrs; intracranial pressure reached 32.3 +/- 3.8 mm Hg and was associated with coma and cerebral edema. After connection to the bioartificial liver, the survival of acute hepatic failure pigs was 12.1 +/- 1.4 hrs in group 2 and 14.8 +/- 2.5 hrs in group 3. In group 3, intracranial pressure and bilirubin concentrations were reduced significantly compared with both group 1 and group 2. Neither signs of encephalopathy nor cerebral edema was observed in any animal of group 3. In all animals, plasma ammonium, aminotransferases, alkaline phosphatase, and lactate concentrations increased and clotting factors decreased with no significant differences between the three groups. Autopsy revealed a total necrosis of the liver, which was histologically confirmed. CONCLUSIONS: The ischemia-induced model of acute hepatic failure in pigs is reproducible and provides measurable clinical and biological features. A bioartificial liver containing alginate bead-entrapped hepatocytes improves the signs of encephalopathy in pigs with ischemia-induced acute hepatic failure, suggesting that the bioartificial liver can clear out toxic compounds that are released from necrotic livers.     

Antigen‐specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy

Tumor infiltrating lymphocyte (TIL) therapy has shown objective clinical response rates of 50% in stage IV melanoma patients in a number of clinical trials. Nevertheless, the majority of patients progress either directly upon therapy or after an initial period of tumor control. Recent data have shown that most TIL products that are used for therapy contain only low frequencies of T cells reactive against known melanoma‐associated epitopes. Because of this, the development of a technology to create T‐cell products that are enriched for reactivity against defined melanoma‐associated antigens would seem valuable, both to evaluate the tumoricidal potential of T cells directed against different antigen classes and to potentially increase response rates. Here, we developed and validated a conditional MHC streptamer‐based platform for the creation of TIL products with defined antigen reactivities. We have used this platform to successfully enrich both high‐frequency (≥1%) and low‐frequency (<1%) tumor‐specific CD8⁺ T‐cell populations, and thereby created T‐cell products with enhanced tumor recognition potential. Collectively, these data demonstrate that selection of antigen‐specific T‐cell populations can be used to create defined T‐cell products for clinical use. This strategy thus forms a highly flexible platform for the development of antigen‐specific cell products for personalized cancer immunotherapy.     

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.     

Evidence for involvement of a tumor suppressor gene on 1p in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNST) are rare soft-tissue malignancies. The genetic basis of these tumors is still poorly understood. Cytogenetic analyses predominantly revealed complex karyotypes, precluding the identification of recurrent chromosomal changes. We report loss of 1p material in a near-diploid karyotype with few or no additional structural chromosome changes in two sporadic cases of MPNST, indicating an important role of 1p loss in MPNST development. In one of these two tumors, a distal 1p deletion (1p31.2 approximately pter) was detected suggesting involvement of a tumor suppressor gene located within this distal region of 1p. Further evidence for recurrent 1p loss in MPNST was obtained by interphase fluorescence in situ hybridization, which showed loss of 1p material in 3 out of 13 tumors. These findings together with data from the literature suggest that loss of a tumor suppressor gene located within distal 1p is implicated in the pathogenesis of MPNST.     

PiggyMac,a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia

Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase.     

Cholera Vaccination in Urban Haiti

Successful and sustained efforts have been made to curtail the major cholera epidemic that occurred in Haiti in 2010 with the promotion of hygiene and sanitation measures, training of health personnel and establishment of treatment centers nationwide. Oral cholera vaccine (OCV) was introduced by the Haitian Ministry of Health as a pilot project in urban and rural areas. This paper reports the successful OCV pilot project led by GHESKIO Centers in the urban slums of Port-au-Prince where 52,357 persons received dose 1 and 90.8% received dose 2; estimated coverage of the at-risk community was 75%. This pilot study demonstrated the effort, community mobilization, and organizational capacity necessary to achieve these results in a challenging setting. The OCV intervention paved the way for the recent launching of a national cholera vaccination program integrated in a long-term ambitious and comprehensive plan to address Haiti''s critical need in water security and sanitation.