Protein arginine deiminase 4 inhibition is sufficient for the amelioration of collagen-induced arthritis.

Citrullination of joint proteins by the protein arginine deiminase (PAD) family of enzymes is recognized increasingly as a key process in the pathogenesis of rheumatoid arthritis. This present study was undertaken to explore the efficacy of a novel PAD4-selective inhibitor, GSK199, in the murine collagen-induced arthritis model of rheumatoid arthritis. Mice were dosed daily from the time of collagen immunization with GSK199. Efficacy was assessed against a wide range of end-points, including clinical disease scores, joint histology and immunohistochemistry, serum and joint citrulline levels and quantification of synovial autoantibodies using a proteomic array containing joint peptides. Administration of GSK199 at 30 mg/kg led to significant effects on arthritis, assessed both by global clinical disease activity and by histological analyses of synovial inflammation, pannus formation and damage to cartilage and bone. In addition, significant decreases in complement C3 deposition in both synovium and cartilage were observed robustly with GSK199 at 10 mg/kg. Neither the total levels of citrulline measurable in joint and serum, nor levels of circulating collagen antibodies, were affected significantly by treatment with GSK199 at any dose level. In contrast, a subset of serum antibodies reactive against citrullinated and non-citrullinated joint peptides were reduced with GSK199 treatment. These data extend our previous demonstration of efficacy with the pan-PAD inhibitor Cl-amidine and demonstrate robustly that PAD4 inhibition alone is sufficient to block murine arthritis clinical and histopathological end-points.

Clinically significant human papilloma virus in squamous cell carcinoma of the head and neck in UK practice.

AIMS: The association between squamous cell carcinoma of the head and neck (HNSCC) and infection with human papilloma viruses (HPV) has created considerable interest. Rates of primary oropharyngeal cancers have shown increasing incidence and declining age at presentation over the last decade, believed to relate to infection with oncogenic or high-risk subtypes of HPV (HR-HPV). HR-HPV-associated tumours have reportedly improved outcomes when compared with HPV-negative cancers. Within the UK, rates of HR-HPV in HNSCC have not yet been reported. MATERIALS AND METHODS: We analysed consecutive retrospective cases of oropharyngeal cancer presenting between 2004 and 2007. RESULTS: Thirty-seven per cent of 83 oropharyngeal tumours stained positively for p16(INK4A), a marker of HPV infection (73% tonsillar cancers being p16 (INK4A) positive, 30% tongue and 43% floor of mouth tumours). HPV16 DNA was demonstrated in 75% p16 (INK4A) cases. Despite being more advanced with higher T-stage and nodal burden at presentation, HR-HPV-associated HNSCC showed significantly improved rates of disease-free and overall survival, in particular with improved rates of response to radical radiotherapy. CONCLUSION: HPV16 infection seems to be a clinically significant cause of oropharyngeal HNSCC in the UK and the collection of national data should be supported.

A new scaleable freeze-thaw technology for bulk protein solutions.

A new method of freeze-thaw is described using experimental data obtained from freezing of purified rhGH (recombinant human growth hormone). The method is based on freezing protein solutions in rectangular rather than cylindrical containers. It is hypothesized that the change in container geometry allows for linear scale-up of the freeze-thaw operation based on equivalency of temperature-time profile. The hypothesis is tested using freeze-thaw data from a miniature (30 ml) and a 2.4 litre container. Computational fluid dynamics techniques are used to simulate the freeze process and the simulations are compared with experimental results. Protein quality is assessed as a function of freeze conditions using dynamic light scattering, circular CD, size-exclusion and reverse-phase HPLC measurements. The results demonstrate the applicability of the new approach. Freezing of rhGH solution at concentrations of approx. 30 mg/ml is shown to be possible with no damage to the molecule for up to five cycles of freeze-thaw. A nitrogen blast chest-freezer is designed and evaluated as part of the process. The refrigeration system and the freeze-thaw method can be used to freeze-thaw bulk protein solutions for development work and has the potential for transfer to manufacturing.

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection.

Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human alpha1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galalpha1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.

CD4+ T-cell testing practices as implications for training.

As new diseases and new testing methods emerge, clinical laboratories are faced with updating the skills of their personnel. Complex techniques, such as flow cytometry, require both education and experience to achieve a high level of proficiency. One of the ways to determine areas in which training is needed is to assess laboratory practices and compare them with practices recommended in guidelines or by panels of experts. In this paper we describe practices reported in a written survey of 206 laboratories that perform CD4+ T-cell counts (CD4). We provided a list of alternate practices for each of the key steps in the testing process and asked participants to select the practices they use in their laboratories. Published guidelines and interviews with knowledgeable "key informants" and focus groups of people who perform CD4 testing were used to formulate the questions. We interpreted variations from recommended practices as indicators of training needs. Other factors that can affect performance, such as workload, supervision, and resources, were satisfactory to the respondents. A response rate of 73% (247 of 337 laboratories) revealed that laboratories followed most of the recommended practices. Notable exceptions included some areas of quality control and quality assurance and safety. This paper also describes flow cytometry testing as it was practiced in 1993 shortly after release of some of the testing guidelines and provides a baseline of practices for that time frame.

Design considerations for mental health facilities.

The design environment of mental health facilities can facilitate the human interactions essential to treatment and can help in meeting clients' basic needs for safety and security, for self-esteem, and for the development of interpersonal and social skills. To determine the factors in the design of interior spaces that optimize clients' response to therapy, the author made a study of six Indiana community mental health centers. Drawing on that study and other sources, she presents design recommendations for mental health facilities for such areas as reception and admission areas, corridors and stairwells, therapists' offices, inpatient rooms, and dayrooms. Other discussions cover the relation of color, visual patterning, and light, and the selection of materials and finishes.