Intensive lifestyle intervention is particularly advantageous in poorly controlled type 2 diabetes.

BACKGROUND AND AIMS: It is unknown whether lifestyle change is effective in people with type 2 diabetes with inadequate glucose control. The aim of this study was to asses, in a group of people with type 2 diabetes, the impact of baseline values of glycosylated haemoglobin (HbA1c) on the effects of an intensive lifestyle intervention on metabolic, clinical and strength parameters. METHODS AND RESULTS: 222 people with type 2 diabetes with mean ± standard deviation baseline HBA1c of 7.50% ± 1.27 (range 5.1-12.7%), were enrolled in a 3-month structured multidisciplinary lifestyle intervention. Anthropometric, biochemical, clinical and fitness measurements were collected at baseline, at the end of the lifestyle intervention program and at two-year follow-up visit. Significant improvements in glycometabolic control (HbA1c: p ≤ 0.0001); anthropometric parameters (BMI p ≤ 0.0001; waist circumference: p ≤ 0.0001); and systemic blood pressure (p ≤ 0.0001) were observed both at the end of the three month intensive lifestyle program and at the two-year follow up visit. In addition, defined daily doses of hypoglycaemic treatment significantly decreased (p = 0.001). Fitness measures exhibited significant increments in the whole sample at the end of the intensive intervention program (p ≤ 0.0001). When patients were divided into tertiles considering the baseline value of HbA1c, the most marked improvements in HbA1c, blood glucose and triglycerides were observed in the group with inadequate glucose control (Hba1c ≥ 7.71%), both at the three-month and two-year follow-ups. CONCLUSION: These results demonstrate that an intensive lifestyle intervention should be recommended for people with type 2 diabetes, particularly those with the most inadequate glycaemic control. REGISTRATION NUMBER: CURIAMO trial was registered in the Australian New Zealand Clinical Trials Registry, (ACTRN12611000255987).

Renal tuberculosis: a case report.

Tuberculosis or TB (tubercle bacillus) remains a major public health problem in developing countries. Over the last decades extrapulmonary locations of the disease have become more frequent due to the increased prevalence of acquired immune deficiency syndrome and the increase number of organ transplants. The urogenital localization represents about 27% of all extra-pulmonary localizations of TB and may be due either to a disseminated infection or to a primitive genitourinary localization. The majority of patients, has pyuria, sometimes with hematuria. The diagnosis of urinary tuberculosis is based on the finding of pyuria in the absence of infection by common bacteria. The initial medical treatment includes isoniazide, rifampicin, pyrazinamide, ethambutol and streptomycin. This disease should be suspected in patients with unexplained urinary tract infections, especially if immunocompromised and/or coming from endemic areas.

Adventitious Agent Risk Assessment Case Study: Evaluation of RotaTeq(R) for the Presence of Porcine Circovirus.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) In June of 2010, results of metagenomic and panmicrobial microarray analysis of a number of commercially available vaccine products were published, identifying the unexpected presence of porcine circovirus (PCV) in of one of the vaccine products tested. This testing did not detect any sequences of contaminating viruses in RotaTeq® (rotavirus vaccine, live, oral, pentavalent, RV5, Merck & Co., Inc., Whitehouse Station, NJ). To confirm this finding, Merck developed and applied a number of polymerase chain reaction-based analytical methods and a test algorithm to systematically demonstrate the absence of infectious PCV in RotaTeq®. This paper will describe the methodology and rationale developed to thoroughly assess key starting materials, product intermediates, and final product to demonstrate the absence of infectious PCV, and the continued quality of this product. This approach could be applied to assess the validity of other adventitious agent risks encountered in biological processes and products.

Separation and quantification of viral double-stranded RNA fragments by capillary electrophoresis in hydroxyethylcellulose polymer solutions.

Capillary electrophoresis (CE) is an analytical technique widely utilized to resolve complex mixtures of nucleic acids. CE uses a variety of polymers in solution that act as a molecular sieve to separate nucleic acid fragments according to size. It has been shown previously that purified dsDNA can be resolved efficiently by solutions of hydroxyethylcellulose (HEC) polymer, providing a rapid and high resolution method of separation. We have applied this separation technique to viral double-stranded (ds) RNA segments derived from rotavirus process samples. HEC polymers of various molecular masses and concentrations were identified and compared for their ability to separate dsRNA based on the extent of expected polymer network formation. The HEC polymer exhibiting the most desirable separation characteristics was then used for subsequent optimization of various method parameters, such as, injection time, electric field strength, dye concentration and capillary equilibration. The optimized method was then applied to the quantification of genome concentration based on a representative segment of the rotavirus genome. This study demonstrated that purified viral dsRNA material of known concentration could be used to generate an external standard curve relating concentration to peak area. This standard curve was used to determine the concentration of unknown samples by interpolation. This novel RNA quantification assay is likely to be applicable to other types of virus, including those containing dsDNA.

Substrate microtopography can enhance cell adhesive and migratory responsiveness to matrix ligand density.

The regulation of cell motility by ligand density on substrates with variable microtopography is not well understood. In this report, we studied the adhesion and motility behavior of HepG2 cells on microtextured poly(glycolic-co-lactic)acid (PGLA) copolymer substrates, whose surface bioactivity was differentially modified through the adsorption of 0-5.5 ng/cm(2) collagen. Microtextured PGLA substrates were fabricated as thin films with a uniform surface distribution of micropores of median size of 3.1 +/- 1.5 microm and three-dimensional root mean squared roughness of 0.253 microm. Even in the absence of collagen, cells on microtextured substrates responded to substrate topography by exhibiting a 200% increase in adhesion strength compared with untextured controls and ventral localization of the intracellular adhesion protein vinculin. Further enhancement in adhesion strength (420% over untextured, untreated substrates) was demonstrated with bioactivated, microtextured surfaces, indicating that cell adhesion responses to topography and surface ligand density were cooperative. Our motility studies of cells on untextured substrates adsorbed with different levels of collagen demonstrated that a classical biphasic relationship between the cell population averaged migration rate, mu, and the collagen ligand density was preserved. However, comparison of cell motility responses between untextured and microtextured substrates indicates that the motility versus ligand density curve shifted, such that equivalent levels of cell motility were achieved at lower ligand density on microtextured surfaces. Furthermore, the maximum mu values achieved on the microtextured substrates exceeded those on untextured substrates by twofold. Taken together, we show that the magnitude of subcellular scale microtexture of a polymer substrate can sensitize the cell motility responsiveness to substrate ligand concentration; we suggest that the underlying mechanisms involve alteration in the degree of cell-substrate adhesivity as well as changes in the nature of ligand-induced cell activation processes.

Mechanochemical manipulation of hepatocyte aggregation can selectively induce or repress liver-specific function.

Controlled activation of hepatocyte aggregation is critical to three-dimensional (3D) multicellular morphogenesis during native regeneration of liver as well as tissue reconstruction therapies. In this work, we quantify the stimulatory effects of two model hepatotrophic activators, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), on the aggregation kinetics and liver-specific function of hepatocytes cultured on organotypic substrates with differing mechanical resistivity. Substrate-specific morphogenesis of cultured hepatocytes is induced on a tissue basement membrane extract, Matrigel, formulated at two distinct levels of mechanical compliance (storage modulus G', at oscillatory shear rate 1 rad/s, was 34 Pa for basal Matrigel and 118 Pa for crosslinked Matrigel). Overall, we report that growth factor stimulation selectively promotes the kinetics of aggregation in the form of two-dimensional corded aggregates on basal Matrigel and three-dimensional spheroidal aggregates on crosslinked Matrigel. Our analysis also indicates that costimulation with EGF and HGF (20 ng/mL each) cooperatively maximizes the kinetics of aggregation in a substrate-specific manner. In addition, we show that the role of growth factor stimulation on hepatocyte function is sensitively governed by the mechanical compliance of the substrate. In particular, on matrices with high compliance, costimulatory aggregation is shown to elicit a marked increase in albumin secretion rate, whereas on matrices with low compliance aggregation results in effective functional repression to basal, unstimulated levels. Thus, our studies highlight a novel interplay of physicochemical parameters of the culture microenvironment, leading to selective enhancement or repression of differentiated functions of hepatocytes, in concert with the activation of cellular morphogenesis.

Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis.

While microporous biopolymer matrices are being widely tested as cell culture substrates in hepatic tissue engineering, the microstructural basis for their control of cell differentiation is not well understood. In this paper, we studied the role of void size of collagen foams in directing the induction of liver-specific differentiated morphology and secretory activities of cultured rat hepatocytes. Hepatocytes cultured on collagen foams with subcellular sized pore diameters of 10 microm assumed a compact, cuboidal cell morphology, rapidly achieving monolayer coverage, and secreted albumin at the rate of 40 +/- 8 pg/cell/d. Increasing the pore size to 18 microm elicited a distinctly spread cellular phenotype, with discontinuous surface coverage, and albumin secretion rates declined precipitiously to 3.6 +/- 0.8 pg/cell/d. However, when collagen foams with an even higher average void size of 82 microm were used, hepatocytes exhibited high degree of spreading within an extensive three-dimensional cellular network, and exhibited high albumin secretory activity (26 +/- 0.6 pg/cell/d). The effect of void geometry on cellular ultrastructral polarity was further analyzed for the three void size configurations employed. The distribution of the cell-cell adhesion protein, E-cadherin, was primarily restricted to cell-cell contacts on the 10 microm foams, but was found to be depolarized to all membrane regions in cells cultured on the 18 and 82 microm foams. Vinculin-enriched focal adhesions were found to be peripherally clustered on cells cultured on 10 microm foams, but were found to redistribute to the entire ventral surface of cells cultured on the 18 and 82 microm foams. Overall, we demonstrate the significance of designing pore sizes of highly adhesive substrates like collagen toward selective cell morphogenesis in 2-D or 3-D. Subcellular and supercellular ranges of pore size promote hepatocellular differentiation by limiting 2-D cell spreading or effecting 3-D intercellular contacts, while intermediate range of pore sizes repress differentiation by promoting 2-D cell spreading.

Polymer substrate topography actively regulates the multicellular organization and liver-specific functions of cultured hepatocytes.

This study examines the role of topography of porous synthetic polymer substrates in regulating the tissue-specific morphogenesis of cultured hepatocytes. Porous foams of amorphous 50/50 poly(D,L glycolic-co-lactic acid) (PGLA) with a wide range of controlled pore-size distributions ( approximately 1 to 100 microm) were used as culture model surfaces. We found that the induction of microporosity in PGLA substrates significantly improved cell attachment and viability in comparison to those observed on non-porous PGLA films. A detailed evaluation of cellular morphogenesis on the microporous matrices showed that hepatocellular organization was sensitively dependent on the topographical feature size of the foam surfaces. Foams with subcellular size voids ( approximately 3 microm) induced kinetics of two-dimensional hepatocyte reorganization, yet limited the extent of three-dimensional aggregation. In contrast, foams with supercellular size voids ( approximately 67-microm) restricted hepatocyte motility, thereby promoting the kinetics of 3D aggregation. At intermediate void sizes ( approximately 17 microm), both 2D and 3D reorganization kinetics were promoted. Albumin secretory kinetics progressively increased on all void size configurations, the most rapid and sustained kinetics observed in supercellular sized voids, which may serve to minimize cell-polymer contacts and maximize cell-cell contacts in 3D. Overall, these studies demonstrate that void topography of porous polymer substrates is a critical textural feature to induce short-term cell adhesion and viability, and to also selectively regulate the kinetics and extent of multicellular spreading versus 3D aggregation. By virtue of its effects on cell adhesion and morphogenesis, the void topography of nonphysiological polymer scaffolds also is a powerful variable to microengineer hepatospecific activity of tissue analogs.

Monoclonal antibody process development using medium concentrates.

A fed-batch process using concentrated medium was evaluated for its ability to improve cell culture longevity and final monoclonal antibody (MAb) titers for two monoclonal antibody producing cell lines. It was found to result in up to 7-fold increases in final antibody titers compared to batch culture controls. Although the development cell line specific fed-batch protocols is critical to the development of cost-efficient large-scale production processes, the use of complete medium concentrates provided us with a quick and simple method for producing large quantities of antibodies in the early stages of process development, thus accelerating early work on purification process development, analytical development, biochemical characterization, and safety studies. Insights gained from the concentrated medium fed-batch approach were valuable for the development of refined, cell line specific feeding strategies yielding final MAb titers on the order of 1-2 g/L. Process development data on the effects of inhibitory growth byproducts, medium osmolarity, and the mode of nutrient feed addition on culture longevity and MAb production and information on culture metabolic behavior were successfully incorporated in the development of the optimized fed-batch protocols.