Active surveillance for scrapie by third eyelid biopsy and genetic susceptibility testing of flocks of sheep in Wyoming

Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrP(Sc), the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrP(Sc) assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrP(Sc)-positive sheep were identified through the passive surveillance program, and an additional three PrP(Sc)-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrP(Sc)-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program.     

Neuromuscular electrical stimulation and volitional exercise for individuals with rheumatoid arthritis: a multiple-patient case report

BACKGROUND AND PURPOSE: Muscle atrophy is common in patients with rheumatoid arthritis (RA). Although neuromuscular electrical stimulation (NMES) is a viable treatment for muscle atrophy, there is no evidence about the use of NMES in patients with RA. The purposes of this multiple-patient case report are: (1) to describe the use of NMES applied to the quadriceps femoris muscles in conjunction with an exercise program in patients with RA; (2) to report on patient tolerance and changes in lean muscle mass, quadriceps femoris muscle strength (force-producing capacity), and physical function; and (3) to explore how changes in muscle mass relate to changes in quadriceps femoris muscle strength, measures of physical function, and patient adherence. CASE DESCRIPTION: Seven patients with RA (median age=61 years, range=39-80 years) underwent 16 weeks of NMES and volitional exercises. Lean muscle mass and strength of the quadriceps femoris muscle and physical function were measured before and after treatment. OUTCOMES: One patient did not tolerate the NMES treatment, and 2 patients did not complete at least half of the proposed treatment. Patients who completed the NMES and volitional exercise program increased their lean muscle mass, muscle strength, and physical function. DISCUSSION: Because of the small sample, whether NMES combined with exercises is better than exercise alone or NMES alone could not be determined. However, the outcomes from this multiple-patient case report indicate that NMES is a viable treatment option to address muscle atrophy and weakness in patients with RA. Strategies to increase tolerance and adherence to NMES are warranted.     

Telavancin Disrupts the Functional Integrity of the Bacterial Membrane through Targeted Interaction with the Cell Wall Precursor Lipid II

Telavancin is an investigational lipoglycopeptide antibiotic currently being developed for the treatment of serious infections caused by gram-positive bacteria. The bactericidal action of telavancin results from a mechanism that combines the inhibition of cell wall synthesis and the disruption of membrane barrier function. The purpose of the present study was to further elucidate the mechanism by which telavancin interacts with the bacterial membrane. A flow cytometry assay with the diethyloxacarbocyanine dye DiOC₂(3) was used to probe the membrane potential of actively growing Staphylococcus aureus cultures. Telavancin caused pronounced membrane depolarization that was both time and concentration dependent. Membrane depolarization was demonstrated against a reference S. aureus strain as well as phenotypically diverse isolates expressing clinically important methicillin-resistant (MRSA), vancomycin-intermediate (VISA), and heterogeneous VISA (hVISA) phenotypes. The cell wall precursor lipid II was shown to play an essential role in telavancin-induced depolarization. This was demonstrated both in competition binding experiments with exogenous d-Ala-d-Ala-containing ligand and in experiments with cells expressing altered levels of lipid II. Finally, monitoring of the optical density of S. aureus cultures exposed to telavancin showed that cell lysis does not occur during the time course in which membrane depolarization and bactericidal activity are observed. Taken together, these data indicate that telavancin''s membrane mechanism requires interaction with lipid II, a high-affinity target that mediates binding to the bacterial membrane. The targeted interaction with lipid II and the consequent disruption of both peptidoglycan synthesis and membrane barrier function provide a mechanistic basis for the improved antibacterial properties of telavancin relative to those of vancomycin.The increasing prevalence of serious infections caused by gram-positive bacteria, including those caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), highlights the need for new agents with enhanced antimicrobial properties (2, 10, 21, 26, 35). One promising approach has been the development of lipoglycopeptide antibiotics, semisynthetic derivatives of glycopeptides that contain hydrophobic substituents and that possess improved antimicrobial properties (1, 4, 13, 32, 39). Telavancin, a lipoglycopeptide derivative of vancomycin, exhibits enhanced potency in vitro, concentration-dependent bactericidal activity, and activity both in vitro and in vivo against organisms that display reduced susceptibility to vancomycin (17, 18, 23, 24, 28, 31, 33, 36, 42). Telavancin has been evaluated in phase III clinical trials for the treatment of complicated skin and skin structure infections and hospital-acquired pneumonia (46, 53).The bactericidal action of telavancin results from a mechanism that includes the inhibition of cell wall synthesis and the disruption of essential membrane barrier functions (25). Telavancin possesses the glycopeptide core of vancomycin, which binds with a high affinity to the acyl-d-alanyl-d-alanine (d-Ala-d-Ala) terminus of cell wall precursors through a network of hydrogen bonds and hydrophobic packing interactions (3, 45). Inhibition of cell wall synthesis by telavancin therefore involves binding to late-stage peptidoglycan precursors, including membrane-embedded lipid II. These interactions prevent both the polymerization of the precursor into peptidoglycan and subsequent cross-linking events. Telavancin also binds to bacterial membranes and causes membrane depolarization and increased membrane permeability. The mechanism by which telavancin binds to and disrupts the function of the bacterial membrane has not been determined.The present study was undertaken to further explore the interaction of telavancin with the bacterial membrane. Using a flow cytometry assay optimized for the accurate measurement of membrane potential in bacteria, we demonstrate that telavancin causes pronounced, concentration-dependent depolarization in S. aureus cells. Isolates of S. aureus expressing important and emerging resistance phenotypes, such as MRSA, heterogeneous vancomycin-intermediate S. aureus (hVISA), vancomycin-intermediate S. aureus (VISA), and daptomycin-nonsusceptibile MRSA, are equally susceptible to depolarization by telavancin. We provide evidence, through multiple lines of investigation, that membrane disruption by telavancin requires binding to the bacterial specific target, lipid II. Finally, we demonstrate that telavancin does not lyse bacteria during the time course that membrane effects are assayed. Importantly, the latter observation indicates that telavancin-induced membrane depolarization is not a consequence of a weakened cell wall. The findings of the studies reported here enhance our understanding of telavancin''s mechanism of action and, in assays designed to be representative of physiological conditions, demonstrate that therapeutically relevant concentrations of telavancin inhibit essential functions of the bacterial membrane.     

A flow cytometry-based method for detecting antibody responses to murine cytomegalovirus infection

An assay based on target cells infected with green fluorescent protein labeled murine cytomegalovirus (GFP-MCMV) and dual color flow cytometry for detecting antibody to MCMV is described. After optimizing conditions for this technique, kinetics of anti-MCMV IgG antibody response was tested in susceptible (BALB/c) and resistant (C57BL/6) mouse strains following primary MCMV infection. Previously published antibody kinssetics were confirmed in susceptible mice, with peak IgG response seen approximately 8 weeks after primary infection, decreasing by 20 weeks after infection. In contrast, MCMV resistant C57BL/6 mice showed significantly lower IgG antibody responses than susceptible mice. Although several techniques have been previously described to detect murine antibody responses to MCMV, including nuclear anti-complement immunofluorescence, viral immunoblotting, complement fixation, indirect immunofluorescence, indirect hemagglutination, and enzyme-liked immunosorbent assay techniques, these techniques are all time consuming and laborious. The technique presented is a simple time efficient alternative to detect previous MCMV antibody responses in experimentally infected mice.     

Isokinetic Peak Torque Ratios and Shoulder Injury History in Adolescent Female Volleyball Athletes

Context:

Few researchers have examined shoulder strength in adolescent volleyball athletes despite increasing levels of participation in this age group.

Objective:

To compare medial and lateral isokinetic peak torque of the rotator cuff among skill levels and between athletes with and without a history of shoulder injury.

Design:

Cross-sectional design.

Setting:

The Human Performance Lab and Athletic Training Lab.

Patients or Other Participants:

Thirty-eight female adolescent club volleyball athletes from 10 to 15 years of age (mean  =  13.02 ± 1.60 years).

Main Outcome Measure(s):

We measured concentric and eccentric peak torque of the medial and lateral rotators of the shoulder and calculated resultant cocking and spiking ratios based on peak torque values.

Results:

Athletes at higher skill levels had higher peak torque measurements in concentric and eccentric medial and lateral rotation compared with the athletes at lower skill levels. No differences in peak torque existed between participants with or without an injury history 6 months before the study. Strength ratios did not differ across skill levels, but previously injured participants produced lower eccentric medial rotation to concentric lateral rotation ratios compared with participants without a history of injury (P  =  .02). At the highest skill level, previously injured participants produced lower eccentric lateral rotation to concentric medial rotation ratios compared with participants without an injury history (P  =  .04).

Conclusions:

Differences in medial and lateral shoulder rotator strength ratios appear to be related more to injury prevalence than to absolute strength. Shoulder dysfunction related to strength ratio deficits also may exist in adolescent female volleyball athletes. Preventive shoulder strengthening programs focused on improving eccentric strength and correcting imbalances between medial and lateral rotators may be warranted for all female adolescent volleyball athletes.