Tissue compatibility and pharmacokinetics of three potential subcutaneous injectables for low‐pH drug solutions

Objectives The aim of the study was to investigate the tissue tolerance and bioavailability of four formulations containing 5% ricobendazole solubilised at low pH, following subcutaneous injection in sheep. Formulations were: a water‐in‐oil emulsion, a microemulsion, a hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD, 20%) drug solution, and a low‐pH drug solution (reference). Methods In‐vitro cytotoxicity of the formulations was investigated in L929 fibroblasts using MTS viability and lactate dehydrogenase leakage assays. Each formulation and respective vehicle was injected into either side of the back of a sheep to investigate the tissue tolerance and pharmacokinetics. Key findings In‐vitro studies suggested that both the emulsion and the microemulsion are unlikely to give a burst release of the low‐pH drug solution in aqueous media. The microemulsion showed the greatest in‐vitro cytotoxic effect but no significant difference was observed between the other formulations. In sheep, the three new formulations and vehicles caused little or no injection‐site reactions compared with a marked response to the reference formulation. Bioavailabilities of HP‐β‐CD formulation, emulsion and microemulsion formulations, relative to the reference formulation, were 194, 155 and 115%, respectively. Conclusions The three new subcutaneous injectables showed promise for reducing irritation of low‐pH solubilised ricobendazole. HP‐β‐CD significantly enhanced the drug absorption. Controlling the burst release of the low‐pH drug solution may improve tissue tolerance and minimise post‐injection precipitation, and hence increase drug bioavailability. The in‐vitro cytotoxicity studies did not predict the in‐vivo irritation effects.     

Virulence Genes and Genotypic Associations in Nasal Carriage,Community-Associated Methicillin-Susceptible and Methicillin-Resistant USA400 Staphylococcus aureus Isolates

It is not well understood why strains of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), a major cause of skin and soft tissue infections, became successful so quickly, overtaking the place of methicillin-sensitive S. aureus (MSSA) in many communities. To evaluate the genetic basis of differences in their virulence traits, 293 S. aureus isolates consisting of three cohorts, genotypically defined clinical CA-MRSA (n = 77), clinical MSSA (n = 103), and nasal carriage MSSA (n = 113), collected over a 19-year period in two Midwestern states in the United States, were (i) extensively genotyped and (ii) screened for 40 known virulence genes which included those for enterotoxins, leukocidins, hemolysins, and surface proteins and several newly identified putative toxin genes from the USA400 lineage of CA-MRSA. Genotypically, nasal carriage and clinical MSSA isolates were much more diverse than was the CA-MRSA group, which was found to be of USA400 lineage only. Virulence gene profiles of the three groups showed that CA-MRSA strains harbored significantly higher percentages (≥95%; P value, <0.05) of the sea, sec, sec4, seg2, seh, sek, sel, sel2, ear, ssl1, lpl10, lukSF-PV, lukD, lukE, and clfA genes than did the carriage and the clinical MSSA group (range, 0% to 58%). Genes of the enterotoxin gene cluster, seg, sei, sem, sen, and seo, were present in the clinical and carriage isolates but not in the CA-MRSA group. These results suggest that the presence of additional virulence factors in USA400 CA-MRSA strains compared to the nasal carriage and clinical MSSA strains probably contributed to their enhanced virulence.Staphylococcus aureus both is a benign commensal and common pathogen in humans and is responsible for a variety of infections, ranging from superficial skin and soft tissue infections to bacteremia, endocarditis, and osteomyelitis (33). Based on its susceptibility to beta-lactams, S. aureus is commonly described as methicillin-susceptible S. aureus (MSSA) or methicillin-resistant S. aureus (MRSA) (15). Infections due to health care-associated MRSA (HA-MRSA) have been a problem since the 1970s; however, starting in the 1990s, new strains of MRSA, referred to as community-associated MRSA (CA-MRSA), appeared in community dwellers and were genetically different from HA-MRSA strains (7). Until recently, individuals who presented with infections due to CA-MRSA typically have had none of the established risk factors associated with HA-MRSA, such as recent hospitalization, surgery, dialysis, long-term care residence, or indwelling medical devices. Lately however, CA-MRSA strains have been reported from both the community and the health care settings (25, 45). Genotyping tools such as pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa typing have helped in distinguishing the genotypes of CA-MRSA strains from those of other S. aureus strains (2, 14, 26). In PFGE, SmaI-restricted S. aureus genomes are compared to determine their genetic relatedness and also compared against the reference USA genotypes (USA100, USA200, etc., up to USA1200) as described by the Centers for Disease Control and Prevention (CDC) (34, 53). Of these, CA-MRSA isolates mostly belong to USA300 and USA400 clones and in some cases USA1000 and USA1100 clones as well. HA-MRSA isolates generally belong to USA100, USA200, and USA500 (34, 35). One of the two major clones of CA-MRSA, USA400, recognized in the early 1990s and initially referred to as the MW2 clone, was the predominant CA-MRSA clone that initially circulated in the midwestern United States in the 1990s (8, 17, 40, 52). The second and more recent CA-MRSA clone, USA300, was first recognized in 2000 and has since spread throughout the world (54). More than a thousand MLST allelic profiles for S. aureus have been identified so far, of which CA-MRSA strains are primarily represented by sequence type 1 (ST1) (USA400) and ST8 (encompasses USA300 and USA500). Of the several thousand spa types in the Ridom database (http://spaserver.ridom.de/), the most predominant CA-MRSA spa types are t008 and t128.CA-MRSA strains, besides their distinct PFGE, ST, and spa profiles, almost ubiquitously possess the Panton-Valentine leukocidin (PVL or lukSF-PV) genes, in contrast to only 1% to 5% of S. aureus strains overall having these genes (33, 50, 57). The PVL toxin has been implicated in many skin and soft tissue infections and lethal necrotizing pneumonia, but the exact role of PVL during S. aureus infections remains controversial (10, 28, 31, 58). The genome sequence of the CA-MRSA strain MW2 of the USA400 lineage showed the presence of several additional putative toxin genes (e.g., ear, sec4, sel2, seg2, ssl1 [set16], and lpl10) compared with other S. aureus strains that had been sequenced (1). Some of these toxin genes share homology with classic staphylococcal enterotoxin genes that encode pyrogenic exotoxins (49) typically produced during the post-exponential phase of growth, and the genes encoding these exotoxins are most often carried on plasmids, bacteriophages, or pathogenicity islands. The classic staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, and seo) are commonly found in strains of S. aureus (12, 23, 39, 42, 43, 46, 62). Pyrogenic exotoxin genes are common in S. aureus, and as many as 73% of S. aureus isolates carry at least one of the genes encoding a classic pyrogenic exotoxin; however, the distribution among various clonal types differs (3). Strains of USA400 CA-MRSA typically have been shown to possess the sea, sec, seh, and sek enterotoxin genes, whereas HA-MRSA strains usually carry the sed, seg, sei, sej, sem, sen, and seo enterotoxin genes (40). Currently, the distribution of the newly identified putative toxin genes (seg2, sel2, sec4, ssl1, and lpl10) (1) from the MW2 strain has not been reported from among CA-MRSA strains in general or from clinical and nasal carriage MSSA strains. Since CA-MRSA isolates are able to cause disease in humans without predisposing risk factors and have spread rapidly in communities, these strains may possess a greater number of toxin genes than do the other strains of S. aureus.The aim of this study was to compare the genotypes of clinical CA-MRSA USA400, clinical MSSA, and colonizing nasal carriage MSSA isolates and determine the frequency and distribution of the classic enterotoxin genes as well as the new putative toxin genes in them. Our results showed that MSSA strains were much more diverse in their genotypes than were CA-MRSA USA400 strains. In addition, CA-MRSA USA400 strains possessed a distinct array of toxin genes compared to MSSA strains. These data may provide insight into the success of CA-MRSA USA400 and its ability to cause severe disease in previously healthy people.     

The potential of pulsed low intensity ultrasound to stimulate chondrocytes matrix synthesis in agarose and monolayer cultures

Pulsed low intensity ultrasound (PLIUS) has been used successfully for bone fracture repair and has therefore been suggested for cartilage regeneration. However, previous in vitro studies with chondrocytes show conflicting results as to the effect of PLIUS on the elaboration of extracellular matrix. This study tests the hypothesis that PLIUS, applied for 20 min/day, stimulates the synthesis of sulphated glycosaminoglycan (sGAG) by adult bovine articular chondrocytes cultured in either monolayer or agarose constructs. For both culture models, PLIUS at either 30 or 100 mW/cm² intensity had no net effect on the total sGAG content. Although PLIUS at 100 mW/cm² did induce a 20% increase in sGAG content at day 2 of culture in agarose, this response was lost by day 5. Intensities of 200 and 300 mW/cm² resulted in cell death probably due to heating from the ultrasound transducers. The lack of a sustained up-regulation of sGAG synthesis may reflect the suggestion that PLIUS only induces a stimulatory effect in the presence of a tissue injury response. These results suggest that PLIUS has a limited potential to provide an effective method of stimulating matrix production as part of a tissue engineering strategy for cartilage repair.     

Geographical differences in the proportion of human group A rotavirus strains within New Zealand during one epidemic season

The prospect of future rotavirus vaccine programs means it is important to understand rotavirus strain diversity within New Zealand, especially if this was to influence vaccine effectiveness. The G‐genotype of 359 group A rotavirus strains isolated from 416 stool samples collected from June 2005 to May 2006 (inclusive) from children less than 5 years of age in multiple centers throughout New Zealand was determined. G1 was the dominant circulating strain (55.8%) followed by G4 (21.4%), G3 (3.4%), G9 (3.4%) G2 (1.0%), and mixed infection (1.0%). Two less common strains, G6 and G8, were identified for the first time in New Zealand. P genotypes were determined for a random 10% of samples containing the common G‐type strains, and all samples with an unusual G‐type. All samples able to be tested contained P[8] bearing strains, except for G1P[4], G2P[4], and G8P[14] strains. Importantly, significant differences in strain frequency were found between samples collected from the North and South Islands of New Zealand. G1 was the most commonly identified strain in the North Island (81.9%); whereas G4 predominated in the South Island (39.6%). Of note, no significant differences in the relative frequency of rotavirus strains were observed between samples collected from children treated in hospital compared to samples collected from children seen by their primary healthcare provider in the community. Regional strain variation highlights the importance of ensuring multi‐center surveillance to help monitor program effectiveness when rotavirus vaccines are introduced into New Zealand's national childhood immunization schedule. J. Med. Virol. 82: 897–902, 2010. © 2010 Wiley‐Liss, Inc.     

Behavioural and electrophysiological measures of task switching during single and mixed-task conditions

In order to understand how the brain prepares for and executes a switch in task demand, we measured reaction time (RT), accuracy, and event-related brain potentials associated with performance in single and mixed-task blocks using a cued design. Our results show that trials which repeat in a mixed-task block (repeat trials) were more demanding than trials which repeated in a single-task block, as reflected by the presence of a RT mixing cost and by the presence of a smaller target-locked positivity (P3b) on repeat trials. Within a mixed-task block, repeat and switch trials also differed, where repeat trials showed evidence of greater preparation (larger cue-locked negativity), more efficient target processing (larger target-locked P3b), and shorter RTs. In addition, the cue-locked negativity difference remained despite equating repeat and switch trials on RT, suggesting that this negativity difference is specific to the switching process. Our results are discussed in light of existing models of task switching.     

Effect of co-solvents on the characteristics of enkephalin microcapsules

The objective of this project was to study the effect of the presence of co-solvents with dichloromethane on the properties of leu-enkephalin microcapsules. Six co-solvents, including ethyl acetate, methanol, ethanol, acetone, isopropanol and acetonitrile, at three concentrations of 5%, 10% and 20% (v/v), respectively, of dichloromethane were selected for this study. The resulting microcapsules were evaluated for morphology and particle size, surface area, thermal characteristics and efficiency of encapsulation. The analysis of particle size distribution showed bi- and tri-modal distribution of the microcapsules. The median particle size of the microcapsules was between 17 microm and 57 microm. All microcapsules were smaller than 90 microm. Approximately 10% of the microcapsules were smaller than 500 nm. In general, the microcapsules prepared with co-solvents showed relatively smaller median size. The microcapsules were spherical in shape. DSC analysis of the microcapsules showed that there were no significant differences in the glass transition temperatures. There were significant changes in the efficiency of encapsulation due to the addition of co-solvents. Substitution with 20% methanol resulted in 40% increase in the efficiency of encapsulation (12% vs. 17%). Furthermore, substitution with 20% ethyl acetate, isopropanol, or acetonitrile, reduced the efficiency of encapsulation to as low as 6%.     

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.     

Exit interview-consultation for research validation and dissemination

Dissemination of research findings to practice and maintenance of rigor and validity in qualitative research are continuing challenges for nurse researchers. Using three nursing home case studies as examples, this article describes how exit interview-consultation was used as (a) a validation strategy and (b) a rapid research dissemination tool that is particularly useful for nursing systems research. Through an exit interview-consultation method, researchers validated inferences made from qualitative and quantitative data collected in three comprehensive nursing home case studies that examined nursing management practices. This exit interview-consultation strategy extends the traditional member-check approach by providing confirmation at the individual and organizational level. The study examined how using the exit interview-consultation strategy can potentially assist nursing home organizations to increase their capacity for improving operations. Benefits from research participation are often indirect; this study's results suggest that exit interview-consultation can provide direct and immediate benefits to organizations and individuals.     

Genital findings of women after consensual and nonconsensual intercourse

After a sexual assault, forensic nurses, nurse practitioners, and physicians are called on to collect evidence, document any genital injuries, and testify about the significance of injuries. Recently, the scientific rigor of the research has been challenged in the courts.