Pharmacokinetics of UC781-loaded intravaginal ring segments in rabbits: a comparison of polymer matrices

UC781 is a potent and poorly water-soluble nonnucleoside reverse transcriptase inhibitor being investi- gated as a potential microbicide for preventing sexual transmission of HIV-1. This study was designed to evaluate the in vivo release and pharmacokinetics of UC781 delivered from matrix-type intravaginal ring segments in rabbits. Three polymer matrices (polyurethane, ethylene vinyl acetate copolymer, and silicone elastomer) and two drug loadings (5 and 15 mg/segment) were evaluated in at least one of two independent studies for up to 28 days in vivo. Inter-study comparison of in vivo release, vaginal tissue, and plasma concentrations for similar formulations demonstrated good reproducibility of the animal model. Mean estimates for a 28-day in vivo release ranged from 0.35 to 3.17 mg UC781 per segment. Mean proximal vaginal tissue levels (adjacent to the IVR segment) were 8– 410 ng/g and did not change significantly with time for most formulations. Distal vaginal tissue levels of UC781 were 6- to 49-fold lower than proximal tissue levels. Mean UC781 plasma levels were low for all formulations, at 0.09–0.58 ng/mL. All formulations resulted in similar UC781 concentrations in vaginal tissue and plasma, except the low loading polyurethane group which provided significantly lower levels. Loading dependent release and pharmacokinetics were only clearly observed for the polyurethane matrix. Based on these results, intravaginal ring segments loaded with UC781 led to vaginal tissue concen- trations ranging from below to approximately two orders of magnitude higher than UC781’s EC50 under in vitro conditions (2.8 ng/mL), with little influence by polymer matrix or UC781 loading. Moreover, these findings support the use of rabbit vaginal pharmacokinetic studies in preclinical testing of microbicide intravaginal rings.

Tissue biomarker development in a multicentre trial context: a feasibility study on the PETACC3 stage II and III colon cancer adjuvant treatment trial.

Purpose: We evaluated the feasibility of biomarker development in the context of multicenter clinical trials.

Experimental Design: Formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from a prospective adjuvant colon cancer trial (PETACC3). DNA was isolated from tumor as well as normal tissue and used for analysis of microsatellite instability, KRAS and BRAF genotyping, UGT1A1 genotyping, and loss of heterozygosity of 18 q loci. Immunohistochemistry was used to test expression of TERT, SMAD4, p53, and TYMS. Messenger RNA was retrieved and tested for use in expression profiling experiments.

Results: Of the 3,278 patients entered in the study, FFPE blocks were obtained from 1,564 patients coming from 368 different centers in 31 countries. In over 95% of the samples, genomic DNA tests yielded a reliable result. Of the immmunohistochemical tests, p53 and SMAD4 staining did best with reliable results in over 85% of the cases. TERT was the most problematic test with 46% of failures, mostly due to insufficient tissue processing quality. Good quality mRNA was obtained, usable in expression profiling experiments.

Conclusions: Prospective clinical trials can be used as framework for biomarker development using routinely processed FFPE tissues. Our results support the notion that as a rule, translational studies based on FFPE should be included in prospective clinical trials.

Translating policy into practice: A case study in the secondary prevention of coronary heart disease

Background: This paper focuses on the relationships between health ‘policy’ as it is embodied in official documentation, and health ‘practice’ as reported and reflected on in the talk of policy makers, health professionals and patients. The specific context for the study involves a comparison of policies relating to the secondary prevention of coronary heart disease (CHD) in the two jurisdictions of Ireland – involving as they do a predominantly state funded (National Health Service) system in the north and a mixed healthcare economy in the south. The key question is to determine how the rhetoric of health policy as contained in policy documents connects to, and gets translated into practice and action.

Methods: The data sources for the study include relevant healthcare policy documents (N=5) and progress reports (N=6) in the two Irish jurisdictions, and semi-structured interviews with a range of policy-makers (N=28), practice nurses (14), general practitioners (12) and patients (13) to explore their awareness of the documents’ contents and how they saw the impact of ‘policy’ on primary care practice.

Results: The findings suggest that although strategic policy documents can be useful for highlighting and channelling attention to health issues that require concerted action, they have little impact on what either professionals or lay people do.

Conclusion: To influence the latter and to encourage a systematic approach to the delivery of health care it seems likely that contractual arrangements – specifying tasks to be undertaken and methods for monitoring and reporting on activity - are required.

Eyes open to stem cells: safety trial may pave the way for cell therapy to treat retinal disease in patients

A clinical trial using human embryonic stem cell (hESC) therapy for an inherited retinal degenerative disease is about to commence. The Advanced Cell Technology (ACT) trial will treat patients with Stargardt's macular dystrophy using transplanted retinal pigment epithelium derived from hESCs. Currently, no effective treatment is available for Stargardt's disease so a stem cell-based therapy that can slow progression of this blinding condition could represent a significant breakthrough. While there are some hurdles to clear, the ACT trial is a fine example of translational research that could eventually pave the way for a range of stem cell therapies for the retina and other tissues.

STIMULATION OF PHOSPHOLIPASE C-BETA(2) BY RECOMBINANT GUANINE-NUCLEOTIDE-BINDING PROTEIN BETA-GAMMA DIMERS PRODUCED IN A BACULOVIRUS/INSECT CELL EXPRESSION SYSTEM - REQUIREMENT OF GAMMA-SUBUNIT ISOPRENYLATION FOR STIMULATION OF PHOSPHOLIPASE-C

Recombinant wild-type beta(1) gamma(1) dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and beta(1) gamma 1 dimers carrying a mutation known to block gamma-subunit isoprenylation (beta(1) gamma(1)C71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant beta(1) gamma(1) dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble beta(1) gamma(1) preparation contained approximately equal amounts of non-isoprenylated and isoprenylated beta(1) gamma(1) dimers. Soluble wild-type and mutant beta(1) gamma(1) dimers and native beta(1) gamma(1) dimers purified from bovine retina were reconstituted with recombinant phospholipase C-beta(2). Only isoprenylated beta(1) gamma(1) dimers were capable of stimulating phospholipase C-beta(2). The results show that gamma-subunit isoprenylation and/or additional post-translational processing of the protein are required for beta gamma subunit stimulation of phospholipase C.

Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C

We have previously shown that isoprenylation and/or additional pest-translational processing of the G protein gamma(1) subunit carboxyl terminus is required for beta(1) gamma(1) subunit stimulation of phospholipase C-beta(2) (PLC beta(2)) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma(1) subunit alone is sufficient for beta(1) gamma(1)-mediated PLC beta(2) stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta(1) gamma(1) dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta(1) gamma(1) dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma(1) subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta(1) gamma(1) dimers with a recombinant PLC beta(2) isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta(1) gamma(1) dimers capable of stimulating PLC beta(2) and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta(1) gamma(1) dimers than for fully modified native beta(1) gamma(1) purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms.

ELECTRON IMMUNOGOLD LABELING OF REGULATORY PEPTIDE IMMUNOREACTIVITY IN THE NERVOUS-SYSTEM OF MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

An electron immunogold-labeling technique was used in conjunction with a post-embedding procedure to demonstrate for the first time the ultrastructural distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF), in the nervous system of the cestode Moniezia expansa. Two axon types, distinguished by their populations of different-sized electron-dense vesicles, were identified. Immunogold labeling demonstrated an apparent homogeneity of PP, FMRFamide and NPF (M. expansa) antigenic sites throughout the larger dense-cored vesicles within the central nervous system. Triple labeling clearly demonstrated the co-localisation of immunoreactivities (IR) for NPF, PP and FMRFamide within the same dense-cored vesicles. The presence of NPF-IR within the vesicles occupying the perikaryon of the neuronal cell body indicated that the peptides had undergone post-translational C-terminal amidation prior to entering the axon. Antigen pre-absorption experiments using NPF prevented labeling with either PP or FMRFamide antisera, and the failure of these antisera to block NPF-IR supports the view that some, if not all, of the PP/FMRFamide-IR is due to NPF-like peptides.

PREDISSOCIATION OF H-3 N = 2 RYDBERG STATES - PRODUCT BRANCHING AND ISOTOPE EFFECTS

A translational spectroscopy technique is used to obtain predissociation kinetic energy release spectra from the lowest bound states of H-3, 2s 2A1' and 2p 2A

Turning the tide: A critique of Natural Semantic Metalanguage from a translation studies perspective

Starting from the premise that human communication is predicated on translational phenomena, this paper applies theoretical insights and practical findings from Translation Studies to a critique of Natural Semantic Metalanguage (NSM), a theory of semantic analysis developed by Anna Wierzbicka. Key tenets of NSM, i.e. (1) culture-specificity of complex concepts; (2) the existence of a small set of universal semantic primes; and (3) definition by reductive paraphrase, are discussed critically with reference to the notions of untranslatability, equivalence, and intra-lingual translation, respectively. It is argued that a broad spectrum of research and theoretical reflection in Translation Studies may successfully feed into the study of cognition, meaning, language, and communication. The interdisciplinary exchange between Translation Studies and linguistics may be properly balanced, with the former not only being informed by but also informing and interrogating the latter.

Conceptualizing a transformative research agenda

Transformative consumer researchers seek to enhance consumer well-being by tackling some of the more difficult and intractable social problems and getting the results into the hands of stakeholders who can apply the research findings. Although traditional research approaches offer a viable path for doing research seeking positive social change, alternative research approaches exist that are uniquely suited to the challenges of Transformative Consumer Research. This article examines the standard research process from the perspective of a transformative research agenda to highlight steps in the process that can be better adapted and suggests innovations inspired from other translational research programs.

Slower postnatal growth is associated with delayed cerebral cortical maturation in preterm newborns

Slower postnatal growth is an important predictor of adverse neurodevelopmental outcomes in infants born preterm. However, the relationship between postnatal growth and cortical development remains largely unknown. Therefore, we examined the association between neonatal growth and diffusion tensor imaging measures of microstructural cortical development in infants born very preterm. Participants were 95 neonates born between 24 and 32 weeks gestational age studied twice with diffusion tensor imaging: scan 1 at a median of 32.1 weeks (interquartile range, 30.4 to 33.6) and scan 2 at a median of 40.3 weeks (interquartile range, 38.7 to 42.7). Fractional anisotropy and eigenvalues were recorded from 15 anatomically defined cortical regions. Weight, head circumference, and length were recorded at birth and at the time of each scan. Growth between scans was examined in relation to diffusion tensor imaging measures at scans 1 and 2, accounting for gestational age, birth weight, sex, postmenstrual age, known brain injury (white matter injury, intraventricular hemorrhage, and cerebellar hemorrhage), and neonatal illness (patent ductus arteriosus, days intubated, infection, and necrotizing enterocolitis). Impaired weight, length, and head growth were associated with delayed microstructural development of the cortical gray matter (fractional anisotropy: P <0.001), but not white matter (fractional anisotropy: P = 0.529), after accounting for prenatal growth, neonatal illness, and brain injury. Avoiding growth impairment during neonatal care may allow cortical development to proceed optimally and, ultimately, may provide an opportunity to reduce neurological disabilities related to preterm birth.