Evaluation of a hand hygiene campaign in outpatient health care clinics.

OBJECTIVE: To improve hand hygiene in two outpatient health care clinics through the introduction of a gel sanitizer and an informational poster. METHODS: In this interventional study, health care workers at two outpatient clinics were observed for frequency of hand hygiene (attempts versus opportunities). Gel sanitizer and informational posters were introduced together as an intervention. Direct observation of the frequency of hand hygiene was performed during baseline, intervention, and follow-up. A poststudy survey of health care workers was also distributed and collected. RESULTS: In both clinics, the frequency of hand hygiene was poor at baseline (11% and 21%) but improved significantly after intervention (36% and 54%) and was maintained through the follow-up period (32% and 51%). Throughout the study, postcontact hand hygiene was observed significantly more often than precontact hand hygiene. In both clinics, health care workers reported a preference for soap and water; yet observations showed that when the intervention made gel sanitizer available, sanitizer use predominated. Fifty percent of the surveyed health care workers considered the introduction of gel sanitizer to be an effective motivating tool for improving hand hygiene. CONCLUSIONS: Hand hygiene performance by health care workers in outpatient clinics may be improved through promoting the use of gel sanitizer and using informational posters. Compared with surveys, direct observation by trained observers may provide more accurate information about worker preferences for hand hygiene tools.

Expression of pleiotrophin, an important regulator of cell migration, is inhibited in intestinal epithelial cells by treatment with non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs for the suppression of inflammation and pain. However, the analgesic properties of NSAIDs are also associated with significant negative side effects, most notably in the gastrointestinal (GI) tract. Increasingly, evidence indicates that the ulcerogenic properties of some NSAIDs are not exclusively the result of inhibition of cyclooxygenase isoforms in the GI tract, and other mechanisms, including inhibition of cell migration and epithelial restitution, are being explored. Recently, microarray analysis was used to identify potential novel targets of NSAID activity in intestinal epithelial cells. Treated cells exhibited significant reductions in the gene expression of pleiotrophin (PTN), a cytokine and growth factor known to participate in angiogenesis and bone growth. This report aimed to confirm the microarray results reported previously, and to measure protein expression of PTN in intestinal epithelial cells. Furthermore, we also examined the effects of exogenous PTN on cell migration in the presence and absence of either NSAIDs with variable ulcerogenic potential or PTN-specific siRNA. Our results demonstrated that indomethacin and NS-398, two NSAIDs with ulcerogenic potential significantly decrease both gene and protein expressions of PTN in IEC-6 cells and protein expression in IEC-6-Cdx2 cells. Additionally, cell migration experiments with PTN siRNA showed that PTN is an important mediator of IEC-6 cell migration, and addition of exogenous PTN partially restores the deficits in cell migration caused by treatment with indomethacin and NS-398. Finally, measurement of PTN protein expression in the GI tract of horses treated with phenylbutazone showed that PTN expression is reduced by NSAIDs in vivo. Our results show that PTN is an important mediator of cell migration in IEC-6 cells, and PTN is a potential target through which NSAIDs may inhibit cell migration, epithelial restitution, and wound healing in the GI tract.

Non-steroidal anti-inflammatory drugs inhibit calpain activity and membrane localization of calpain 2 protease.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used frequently worldwide for the alleviation of pain despite their capacity to cause adverse gastrointestinal (GI) side effects. GI toxicity, once thought to be the result of non-specific inhibition of cyclooxegenase (COX) enzymes, is now hypothesized to have multiple other causes that are COX independent. In particular, NSAIDs inhibit intestinal epithelial restitution, the process by which barrier function in intestinal mucosa is restored at sites of epithelial wounds within hours through cell spreading and migration. Accordingly, recent evidence indicates that the expression of calpain proteases, which play a key role in cell migration, is decreased by NSAIDs that inhibit cell migration in intestinal epithelial cells (IEC). Here, we examine the effect of NSAIDs on calpain activity and membrane expression in IEC-6 cells. Indomethacin, NS-398, and SC-560 inhibited calpain activity and decreased expression of calpain 2 in total membrane fractions and in plasma membranes involved in cell attachment to the substrate. Additionally, we demonstrated that inhibition of calpain activity by NSAIDs or ALLM, a calpain inhibitor, limits cell migration and in vitro wound healing of IEC-6 cells. Our results indicate that NSAIDs may inhibit cell migration by decreasing calpain activity and membrane-associated expression of calpain 2. Our results provide valuable insight into the mechanisms behind NSAID-induced GI toxicity and provide a potential pathway through which these negative side effects can be avoided in future members of the NSAID class.

NC-1059: a channel-forming peptide that modulates drug delivery across in vitro corneal epithelium.

PURPOSE: The goal of this study was to determine whether a synthetic peptide, NC-1059, can modulate the corneal epithelium to increase the permeation of therapeutic agents across this barrier. METHODS: An in vitro system employing transformed human corneal epithelial (THCE) cells was optimized for this study. Culture conditions were identified to promote formation of a confluent monolayer that rapidly develops a substantial transepithelial electrical resistance. Electrical parameters were measured with a modified Ussing flux chamber, and solute flux was quantified with fluorescently labeled compounds. RESULTS: NC-1059 causes a concentration-dependent increase in short-circuit current and an increase in transepithelial electrical conductance when assessed in modified Ussing chambers. The effect of NC-1059 on transepithelial electrical resistance was reversible. To test for paracellular permeability and size exclusion, FITC-labeled dextran ranging in size from 10 to 70 kDa was used. Dextran permeated the corneal cell monolayer in the presence, but not the absence, of NC-1059. Fluorescein sodium and carboxyfluorescein were then used as low molecular weight markers with similar NC-1059-modulated kinetics being observed. Maximum permeation for the fluorescein derivatives occurred 30 to 90 minutes after a 5-minute NC-1059 exposure. A prototypical drug, methotrexate, also exhibited increased permeation in the presence of NC-1059. CONCLUSIONS: NC-1059 enhances drug permeation across cultured corneal epithelial cell monolayers by transiently affecting the paracellular pathway. Thus, NC-1059 is a lead compound for development of cotherapeutic agents to enhance access and effectiveness of ophthalmic compounds.

Public-health education at Kansas State University.

What are veterinary medical and public-health professionals doing to remedy the immediate and impending shortages of veterinarians in population health and public practice? This question was addressed at the joint symposium of the Association of American Veterinary Medical Colleges and the Association of Schools of Public Health, held in April 2007. Thinking locally, faculty and students at Kansas State University (KSU) asked similar questions after attending the symposium: What are we doing within the College of Veterinary Medicine to tackle this problem? What can we do better with new collaborators? Both the professional veterinary curriculum and the Master of Public Health (MPH) at KSU provide exceptional opportunities to address these questions. Students are exposed to public health as a possible career choice early in veterinary school, and this exposure is repeated several times in different venues throughout their professional education. Students also have opportunities to pursue interests in population medicine and public health through certificate programs, summer research programs, study abroad, and collaborations with contributing organizations unique to KSU, such as its Food Science Institute, National Agricultural Biosecurity Center, and Biosecurity Research Institute. Moreover, students may take advantage of the interdisciplinary nature of public-health education at KSU, where collaborations with several different colleges and departments within the university have been established. We are pleased to be able to offer these opportunities to our students and hope that our experience may be instructive for the development of similar programs at other institutions, to the eventual benefit of the profession at large.

R(X) for recruitment and retention of veterinarian scientists: money, marketing, mentoring.

Veterinary medicine is failing both to sustain its academic base and to meet national needs for research in the fields of comparative medicine (translational research), public health, and food production. The basis for the shortage of veterinarians with research expertise is multi-factorial and related to the substantial commitment of time and money required to obtain both a DVM and advanced training, as well as the lack of motivation among veterinary students to engage in biomedical science. Effective strategies for increasing the number of veterinarian scientists must address these issues using a balanced combination of money, marketing, and mentoring. Success will require not only that we increase and improve opportunities for research training, but also that we create and sustain veterinary college environments that attract, foster, and reward dedication to research. The ''research pipeline'' needs to be transformed into a ''research manifold'' with multiple portals for entry and re-entry of trainees. Age-appropriate educational and mentoring programs should be implemented at K-14, baccalaureate, veterinary college, post-graduate, and junior faculty levels to promote recruitment, training, and retention of veterinarian scientists. New initiatives are especially needed to attract students with primary interests in science and biomedical research to the veterinary profession and to facilitate transition of motivated veterinary graduates from private practice to research careers. Specific examples of such programs are presented and future directions are discussed.

Structural and functional basis for the long QT syndrome: relevance to veterinary patients.

Long QT syndrome (LQTS) is a condition characterized by prolongation of ventricular repolarization and is manifested clinically by lengthening of the QT interval on the surface ECG. Whereas inherited forms of LQTS associated with mutations in the genes that encode ion channel proteins are identified only in humans, the acquired form of LQTS occurs in humans and companion animal species. Often, acquired LQTS is associated with drug-induced block of the cardiac K+ current designated I(Kr). However, not all drugs that induce potentially fatal ventricular arrhythmias antagonize I(Kr), and not all drugs that block I(Kr), are associated with ventricular arrhythmias. In clinical practice, the extent of QT interval prolongation and risk of ventricular arrhythmia associated with antagonism of I(Kr) are modulated by pharmacokinetic and pharmacodynamic variables. Veterinarians can influence some of the potential risk factors (eg, drug dosage, route of drug administration, presence or absence of concurrent drug therapy, and patient electrolyte status) but not all (eg, patient gender/genetic background). Veterinarians need to be aware of the potential for acquired LQTS during therapy with drugs identified as blockers of HERG channels and I(Kr).

4-aminopyridine decreases progesterone production by porcine granulosa cells.

BACKGROUND: Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels. METHODS: Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17beta, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size. RESULTS: 4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17beta to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production. CONCLUSION: 4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Expression of the ether-a-go-go (ERG) potassium channel in smooth muscle of the equine gastrointestinal tract and influence on activity of jejunal smooth muscle.

OBJECTIVE: To determine whether ether-a-go-go (ERG) potassium channels are expressed in equine gastrointestinal smooth muscle, whether ERG channel antagonists affect jejunal muscle contraction in vitro, and whether plasma cisapride concentrations in horses administered treatment for postoperative ileus (POI) are consistent with ERG channels as drug targets. SAMPLE POPULATION: Samples of intestinal smooth muscle obtained from 8 horses free of gastrointestinal tract disease and plasma samples obtained from 3 horses administered cisapride for treatment of POI. PROCEDURE: Membranes were prepared from the seromuscular layer of the duodenum, jejunum, ileum, cecum, large colon, and small colon. Immunoblotting was used to identify the ERG channel protein. Isolated jejunal muscle strips were used for isometric stress response to ERG channel blockers that included E-4031, MK-499, clofilium, and cisapride. Plasma concentrations of cisapride were determined in 3 horses administered cisapride for treatment of POI after small intestinal surgery. RESULTS: Immunoblotting identified ERG protein in all analyzed segments of the intestinal tract in all horses. The selective ERG antagonist E-4031 caused a concentration-dependent increase in jejunal contraction. Clofilium, MK-499, and cisapride also increased jejunal contraction at concentrations consistent with ERG channel block; effects of E-4031 and cisapride were not additive. Peak plasma cisapride concentrations in treated horses were consistent with ERG block as a mechanism of drug action. CONCLUSIONS AND CLINICAL RELEVANCE: The ERG potassium channels modulate motility of intestinal muscles in horses and may be a target for drugs. This finding may influence development of new prokinetic agents and impact treatment of horses with POI.

Expression and coassociation of ERG1, KCNQ1, and KCNE1 potassium channel proteins in horse heart.

In dogs and in humans, potassium channels formed by ether-a-go-go-related gene 1 protein ERG1 (KCNH2) and KCNQ1 alpha-subunits, in association with KCNE beta-subunits, play a role in normal repolarization and may contribute to abnormal repolarization associated with long QT syndrome (LQTS). The molecular basis of repolarization in horse heart is unknown, although horses exhibit common cardiac arrhythmias and may receive drugs that induce LQTS. In horse heart, we have used immunoblotting and immunostaining to demonstrate the expression of ERG1, KCNQ1, KCNE1, and KCNE3 proteins and RT-PCR to detect KCNE2 message. Peptide N-glycosidase F-sensitive forms of horse ERG1 (145 kDa) and KCNQ1 (75 kDa) were detected. Both ERG1 and KCNQ1 coimmunoprecipitated with KCNE1. Cardiac action potential duration was prolonged by antagonists of either ERG1 (MK-499, cisapride) or KCNQ1/KCNE1 (chromanol 293B). Patch-clamp analysis confirmed the presence of a slow delayed rectifier current. These data suggest that repolarizing currents in horses are similar to those of other species, and that horses are therefore at risk for acquired LQTS. The data also provide unique evidence for coassociation between ERG1 and KCNE1 in cardiac tissue.

Potassium channel antagonists influence porcine granulosa cell proliferation, differentiation, and apoptosis.

This investigation determined the effects of K(+) channel antagonists on proliferation, differentiation, and apoptosis of porcine granulosa cells. The drugs screened for functional effects included the class III antiarrhythmic agents MK-499 and clofilium, the chromanol I(Ks) antagonist 293B, the benzodiazepine I(Ks) antagonists L-735,821 and L-768,673, and the peptidyl toxins charybdotoxin (CTX) and margatoxin (MTX). Granulosa cell proliferation and differentiation were assessed by serial measurements of cell number and progesterone accumulation in the culture media, respectively. Granulosa cell apoptosis was evaluated using flow cytometry. Additional information about drug effects was obtained by immunoblotting to detect expression of proliferating cell nuclear antigen, p27(kip1) and the caspase-3 substrate poly(ADP-ribose) polymerase. The ERG channel antagonist MK-499 had no functional effects on cultured granulosa cells. However, the broad spectrum K(+) channel antagonist clofilium decreased, in a concentration-dependent fashion, the number of viable granulosa cells cultured, and these effects were associated with induction of apoptosis. All three I(Ks) antagonists (293B, L-735,821, and L-768,673) increased basal, but not FSH-enhanced progesterone accumulation on Day 1 after treatment without affecting the number of viable cells in culture, an effect that was blocked by pimozide. In contrast, CTX and MTX increased the number of viable cells in FSH-stimulated cultures on Day 3 after treatment without affecting progesterone output per cell. These data demonstrate that selective antagonism of granulosa cell K(+) channels with distinct molecular correlates, electrophysiological properties, and expression patterns can influence differential granulosa cell proliferation, steroidogenic capability, and apoptosis. Thus, K(+) channels may represent pharmacological targets for affecting Granulosa cell function and oocyte maturation, in vivo or in vitro.

Hypercalcemia due to latrogenic secondary hypoadrenocorticism and diabetes mellitus in a cat.

A 9-year-old, spayed female domestic shorthair cat presented for polyphagia, polydipsia, and polyuria following chronic methylprednisolone acetate therapy for pruritus. Initial diagnostics were consistent with uncomplicated diabetes mellitus. Serum calcium was within reference range. Within 12 hours the cat developed depression, anorexia, vomiting, and severe dehydration. Laboratory analysis indicated marked hypercalcemia as measured by both ionized and total calcium concentration. No underlying neoplastic or inflammatory process was identified. An adrenocorticotropic hormone stimulation test was indicative of adrenocortical insufficiency. The hypercalcemia resolved with glucocorticoid supplementation and correction of the dehydration. The diabetes mellitus and adrenal insufficiency both resolved within 9 weeks.

Concurrent administration of water-soluble vitamin E can increase the oral bioavailability of cyclosporine a in healthy dogs.

Randomized crossover studies were performed to determine the effect of coadministration of d-alpha-tocopheryl polyethylene glycol 1,000 succinate (vitamin E TPGS) on the oral bioavailability of cyclosporine A (CsA) formulated as either Sandimmune (Novartis Pharmaceuticals) or Neoral (Novartis Pharmaceuticals). Healthy dogs were given a single oral dose of each cyclosporine formulation with and without vitamin E TPGS. Blood samples were collected from each dog before and at various intervals for 24 hours after drug administration. Whole-blood CsA concentrations were determined by high-pressure liquid chromatography. Noncompartmental pharmacokinetic analysis showed that coadministration of vitamin E TPGS increased the oral bioavailability of Sandimmune. The bioavailability of Neoral was greater than that of cyclosporine. Concurrent administration of vitamin E TPGS had no consistent effect on the bioavailability of Neoral.

Molecular basis of voltage-dependent potassium currents in porcine granulosa cells.

The major objective of this study was to elucidate the molecular bases for K(+) current diversity in porcine granulosa cells (GC). Two delayed rectifier K(+) currents with distinct electrophysiological and pharmacological properties were recorded from porcine GC by using whole-cell patch clamp: 1) a slowly activating, noninactivating current (I(Ks)) antagonized by clofilium, 293B, L-735,821, and L-768,673; and 2) an ultrarapidly activating, slowly inactivating current (I(Kur)) antagonized completely by clofilium and 4-aminopyridine and partially by tetraethylammonium, charybdotoxin, dendrotoxin, and kaliotoxin. The molecular identity of the K(+) channel genes underlying I(Ks) and I(Kur) was examined using reverse transcription-polymerase chain reaction and immunoblotting to detect K(+) channel transcripts and proteins. We found that GC could express multiple voltage-dependent K(+) (Kv) channel subunits, including KCNQ1, KCNE1, Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kvbeta1.3, and Kvbeta2. Coimmunoprecipitation was used to establish the hetero-oligomeric nature of granulosa cell Kv channels. KCNE1 and KCNQ1 were coassociated in GC, and their expression coincided with the expression of I(Ks). Extensive coassociation of the various Kv alpha- and beta-subunits was also documented, suggesting that the diverse electrophysiological and pharmacological properties of I(Kur) currents may reflect variation in the composition and stoichiometry of the channel assemblies, as well as differences in post-translational modification of contributing Kv channel subunits. Our findings provide an essential background for experimental definition of granulosa K(+) channel function(s). It will be critical to define the functional roles of specific GC K(+) channels, because these proteins may represent either novel targets for assisted reproduction or potential sites of drug toxicity.