Structure and Functional Characterization of a Humanized Anti-CCL20 Antibody following Exposure to Serum Reveals the Formation of Immune Complex That Leads to Toxicity.

mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.

The Effects of High-Load Slow-Velocity Resistance Exercise Training in Athletes With Tendinopathy: A Critically Appraised Topic.

CLINICAL SCENARIO: Tendinopathy is a musculoskeletal pathological condition experienced by athletes that can result in pain, impaired muscle performance, and loss of physical function and can hinder return to sports. Various types of resistance exercise training are effective for treating tendinopathy, including isometric, concentric, eccentric, and high-load slow-velocity resistance exercise. CLINICAL QUESTION: What are the effects of high-load slow-velocity resistance exercise training, compared with other forms of resistance exercise, on tendon morphology and patient-reported outcomes in athletes with tendinopathy? SUMMARY OF KEY FINDINGS: The findings of 4 randomized clinical trials were included. One study compared high-load slow-velocity resistance exercise with moderate-load slow-velocity resistance exercise. Two studies investigated the effects of high-load slow-velocity resistance exercise versus eccentric resistance exercise. The fourth study compared high-load slow-velocity resistance exercise with inertia-based resistance exercise. In all of the studies, high-load slow-velocity resistance exercise was as effective as the other forms of resistance exercise for improving patient-reported outcomes and pain. Three studies found no significant differences in changes in tendon morphology between patients who received high-load slow-velocity resistance exercise versus those who received the other forms of resistance exercise. One study showed that high-load slow-velocity resistance exercise was more effective than eccentric exercise for improving tendon morphology outcomes. CLINICAL BOTTOM LINE: Current evidence supports the use of high-load slow-velocity resistance exercise as a treatment option for patellar and Achilles tendinopathy in athletes. STRENGTH OF RECOMMENDATION: Results from level 2 studies suggest grade B evidence in support of high-load slow-velocity resistance exercise for treating athletes with tendinopathy.

The Role of Phosphodiesterase 12 (PDE12) as a Negative Regulator of the Innate Immune Response and the Discovery of Antiviral Inhibitors.

2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.

Reliability and Validity of the Patient-Specific Functional Scale in Community-Dwelling Older Adults.

BACKGROUND AND PURPOSE: Clinical measurement of physical function that is both specific to the individual and generates comparable outcome data is a fundamental need in physical therapy examination. The Patient-Specific Functional Scale (PSFS) has been found to be a reliable and valid measure of physical function in patients with musculoskeletal disorders and may have applications for other patient populations. However, the reliability and the validity of the PSFS have not been evaluated in older adults. The purpose of this study was to investigate the reliability and the validity of the PSFS in community-dwelling older adults. METHODS: Thirty-one community-dwelling older adults (11 males, 20 females), mean age = 81.1 (8.3) years, were included. Participants completed the PSFS, Lower Extremity Functional Scale (LEFS), Activity-specific Balance Confidence Scale (ABC), Short Physical Performance Battery (SPPB), Berg Balance Scale, and the Timed Up and Go on 2 separate days, 48 hours apart. Assessment scores were compared between testing days and reliability was analyzed using the intraclass correlation coefficient (ICC) and minimal detectable change (MDC). Validity of the PSFS was assessed by comparing initial scores with the other measures using the Pearson correlation coefficient, scatter plots, and Bland-Altman plots. RESULTS: The ICC and the MDC for the PSFS were 0.82 (95% confidence interval = 0.67-0.91) and 2.8, respectively. Significant correlations (P < .05) were found when the PSFS was compared with the ABC (r = 0.68), LEFS (r = 0.81), and SPPB (r = 0.37). Bland-Altman plots and 95% limits of agreement (LOA) using z scores indicated considerable agreement between the PSFS versus the ABC (LOA =-1.6 to 1.6), LEFS (LOA =-1.2 to 1.2), and SPPB (LOA =-2.1 to 2.1). CONCLUSION: The PSFS is a reliable and valid measure of physical function in community-dwelling older adults. A change of 2.8 or greater on the PSFS suggests a true change in physical function in this population.

BacMam production and crystal structure of nonglycosylated apo human furin at 1.89†Šresolution.

Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.

The impact of electronic mail versus print delivery of an exercise program on muscular strength and aerobic capacity in people with type 2 diabetes.

Previous research indicates that the Internet, electronic mail (e-mail), and printed materials can be used to deliver interventions to improve physical activity in people with type 2 diabetes. However, no studies have been conducted investigating the effect of e-mail or print delivery of an exercise program on muscular strength and aerobic capacity in people with type 2 diabetes. The purpose of this clinical trial was to investigate the impact of e-mail vs. print delivery of an exercise program on muscular strength and aerobic capacity in people with type 2 diabetes. Nineteen participants with type 2 diabetes were allocated to either a group that was delivered a prescribed exercise program using e-mail (e-mail group, n = 10) or a group that was delivered the same prescribed exercise program in print form (print group, n = 9). Chest press and leg press estimated one-repetition maximum (1-RM) scores as well as estimated peak oxygen uptake ([latin capital V with dot above]O2peak) were measured at baseline and follow-up. Intention-to-treat analysis indicated significant improvements in chest press (mean = 7.00 kg, p = 0.001, effect size = 2.22) and leg press (mean = 19.32 kg, p = 0.002, effect size = 1.98) 1-RM scores and [latin capital V with dot above]O2peak (mean = 9.38 mL of oxygen uptake per kilogram of body mass per minute, p = 0.01, effect size = 1.45) within the e-mail group. Within the print group, significant improvements in chest press (mean = 9.13 kg, p = 0.01, effect size = 1.49) and leg press (mean = 16.68 kg, p = 0.01, effect size = 1.31) 1-RM scores and [latin capital V with dot above]O2peak (mean = 5.14 ml of oxygen uptake per kilogram of body mass per minute, p = 0.03, effect size = 1.14) were found. No significant between-group differences in improvements were found. Clinicians can deliver a prescribed exercise program, either by e-mail or in print form, to significantly improve muscular strength and aerobic capacity in people with type 2 diabetes, and expect similar outcomes.

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

Effects of moderate- versus high-intensity exercise training on physical fitness and physical function in people with type 2 diabetes: a randomized clinical trial.

BACKGROUND: Exercise training is effective for improving physical fitness and physical function in people with type 2 diabetes. However, limited research has been conducted on the optimal exercise training intensity for this population. OBJECTIVE: The primary study objective was to investigate the effects of moderate- versus high-intensity exercise training on physical fitness and physical function in people with type 2 diabetes. DESIGN: This was a randomized clinical trial. SETTING: The setting was a university campus. PARTICIPANTS: Twenty-one people with type 2 diabetes were randomly allocated to receive either moderate-intensity training (MOD group) or high-intensity training (HIGH group). INTERVENTION: The MOD group performed resistance training at an intensity of 75% of the 8-repetition maximum (8-RM) and aerobic training at an intensity of 30% to 45% of the heart rate reserve (HRR). The HIGH group performed resistance training at an intensity of 100% of the 8-RM and aerobic training at an intensity of 50% to 65% of the HRR. MEASUREMENTS: Muscle strength (peak torque [newton-meters]), exercise capacity (graded exercise test duration [minutes]), and physical function (Patient-Specific Functional Scale questionnaire) were measured at baseline and 3 months later. Acute exercise-induced changes in glucose levels were assessed immediately before exercise, immediately after exercise, and 1 hour after exercise during the first exercise training session. RESULTS: Although both groups showed improvements in physical fitness and physical function, the between-group effect sizes were not statistically significant (exercise capacity estimated marginal mean [EMM] difference=2.1, 95% confidence interval [95% CI]=-0.2, 4.5; muscle strength EMM difference=20.8, 95% CI=-23.3, 65.0; and physical function EMM difference=0.1, 95% CI=-0.6, 0.9). Mean percent changes in glucose levels measured immediately before exercise and immediately after exercise, immediately after exercise and 1 hour after exercise, and immediately before exercise and 1 hour after exercise for the MOD group were -11.4%, -5.0%, and -15.8%, respectively; those for the HIGH group were -21.5%, 7.9%, and -15.3%, respectively. LIMITATIONS: Sample size, lack of outcome assessor masking, and physical function measurement subjectivity were limitations. CONCLUSIONS: Moderate- and high-intensity exercise training, as defined in this study, may lead to similar improvements in physical fitness and physical function in people with type 2 diabetes.

Correlation between the 8-repetition maximum test and isokinetic dynamometry in the measurement of muscle strength of the knee extensors: A concurrent validity study.

The 8-repetition maximum test has the potential to be a feasible, cost-effective method of measuring muscle strength for clinicians. The purpose of this study was to investigate the concurrent validity of the 8-repetition maximum test in the measurement of muscle strength by comparing the 8-repetition maximum test to the gold standard of isokinetic dynamometry. Thirty participants (15 males and 15 females, mean age = 23.2 years [standard deviation = 1.0]) underwent 8-repetition maximum testing and isokinetic dynamometry testing of the knee extensors (at 60, 120, and 240 degrees per second) on two separate sessions with 2-3 days between each mode of testing. Linear regression was used to assess the validity by comparing the findings between 8-repetition maximum testing and isokinetic dynamometry testing. Significant correlations were found between the 8-repetition maximum and isokinetic dynamometry peak torque at each testing velocity (r = 0.71-0.85). The highest correlations were between the 8-repetition maximum and isokinetic dynamometry peak torques at 60 (r = 0.85) and 120 (r = 0.85) degrees per second. The findings of this study provide supportive evidence for the use of 8-repetition maximum testing as a valid, alternative method for measuring muscle strength.

Reliability of the 8-repetition maximum test in men and women.

OBJECTIVE: The 8-repetition maximum test has been recommended as a method of prescribing an intensity for resistance training in healthy adults, athletes, and patients with health conditions. Yet, limited research related to the reliability of 8-repetition maximum testing has been conducted. The purpose of this study was to determine the reliability of the 8-repetition maximum test in men and women. DESIGN: Test-retest reliability study. METHODS: Twenty-eight people (14 males, 14 females, mean age=23.0 years [standard deviation=1.3]) with no exercise contraindications participated in this study. After familiarization, each participant underwent 8-repetition maximum testing using 4 different exercises. For all participants, the 8-repetition maximum test was performed during 2 sessions with 2-3 days between sessions. The intraclass correlation coefficient (ICC([1,2])), typical error as the coefficient of variation (TE(CV)), and the Bland-Altman plot were used to assess reliability. Unpaired t-test was used to determine the influence of gender on differences between initial test and retest values. RESULTS: Excellent reliability of the 8-repetition maximum test was found for all exercises (ICC([1,2])>0.9). The range of TE(CV) values was 3.4-10.4%. The Bland-Altman plot illustrated that 27 out of 28 data points for total 8-repetition maximum scores were within the 95% limits of agreement. Unpaired t-test indicated no significant difference between men and women in variations between initial test and retest 8-repetition maximum scores. CONCLUSION: The findings of this study suggest that an 8-repetition maximum test protocol that includes familiarization is reliable in men and women.

Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.

Impact of physical therapist-directed exercise counseling combined with fitness center-based exercise training on muscular strength and exercise capacity in people with type 2 diabetes: a randomized clinical trial.

BACKGROUND: Assessing muscular strength (force-generating capacity) and exercise capacity in response to an intervention for people with type 2 diabetes is clinically important in the prevention of type 2 diabetes-related complications. OBJECTIVE: The purpose of this study was to investigate the impact of physical therapist-directed exercise counseling combined with fitness center-based exercise training on muscular strength and exercise capacity in people with type 2 diabetes. DESIGN: This study was a randomized clinical trial. SETTING: The study was conducted on a university campus, with patient recruitment from the local community. PATIENTS: Twenty-four people with type 2 diabetes were randomly allocated to either a group that received physical therapist-directed exercise counseling plus fitness center-based exercise training (experimental group) or a group that received laboratory-based, supervised exercise (comparison group). INTERVENTION: The experimental group received physical therapist-directed exercise counseling on an exercise program and was provided access to a fitness center. The comparison group received the same exercise program as the experimental group while under supervision. MEASUREMENTS: For all participants, chest press, row, and leg press muscular strength (1-repetition maximum [in kilograms]) and exercise capacity (graded exercise test duration [in minutes]) testing were conducted at baseline and 2 months later. RESULTS: No significant differences in improvements in muscular strength were found for the chest press (adjusted mean difference=1.2; 95% confidence interval [CI]=-5.5 to 7.8), row (adjusted mean difference=0.1; 95% CI=-9.0 to 9.1), or leg press (adjusted mean difference=2.7; 95% CI=-9.1 to 14.6) between the groups. No significant difference in improvement in exercise capacity (adjusted mean difference=0.2; 95% CI=-0.9 to 1.2) was found between the groups. LIMITATIONS: Lack of group allocation blinding and the small sample size were limitations of this study. CONCLUSIONS: The results suggest that physical therapist-directed exercise counseling combined with fitness center-based exercise training can improve muscular strength and exercise capacity in people with type 2 diabetes, with outcomes comparable to those of supervised exercise.

The impact of a supervised strength and aerobic training program on muscular strength and aerobic capacity in individuals with type 2 diabetes.

An intervention in the clinical management of individuals with type 2 diabetes is strength and aerobic training. Limited research has been conducted that investigates the effect of a supervised strength and aerobic training program on muscular strength and aerobic capacity in people with type 2 diabetes. The purpose of this 1-group repeated-measures-designed study was to investigate the impact of a supervised strength and aerobic training program on muscular strength and aerobic capacity in subjects with type 2 diabetes. Thirteen subjects with type 2 diabetes completed the training program. Subjects met the American Diabetes Association diagnostic criteria for type 2 diabetes. For each subject, muscular strength (estimated 1 repetition maximum) and aerobic capacity (estimated maximal oxygen uptake) were measured before and after a supervised strength and aerobic training program as well as during a 6-week follow-up. Repeated-measures analysis of variance was used to compare muscular strength and aerobic capacity between pretesting, posttesting, and follow-up testing periods. Significant improvements in muscular strength (p < 0.01) and aerobic capacity (p < 0.01) were found during posttesting and follow-up testing, as compared to pretesting measures. Yet a significant loss in muscular strength (p < 0.01) and no significant change in aerobic capacity (p > 0.05) were found during follow-up testing, as compared to posttesting measures. This study indicates that a supervised strength and aerobic training program can significantly improve muscular strength and aerobic capacity in people with type 2 diabetes. Yet improvements in muscular strength due to training will not be maintained if individuals with type 2 diabetes do not adhere to a continuous training program. In addition, aerobic capacity can be improved with training, but aerobic capacity will not continue to improve if people with type 2 diabetes are not compliant with a continuous training program.

Intrarater reliability of 1 repetition maximum estimation in determining shoulder internal rotation muscle strength performance.

The purpose of this study was to determine intrarater reliability of the 1 repetition maximum (1RM) estimation for shoulder internal rotation. The accuracy of the estimated 1RM was determined by establishing the actual 1RM. Fifteen subjects were positioned supine with the shoulder in 0 degrees abduction (position 1) and prone with the shoulder in 90 degrees abduction (position 2). Subjects were placed in both testing positions and performed resisted shoulder internal rotation. A 1RM estimation equation was used to estimate shoulder internal rotation strength. After 1 week, procedures were repeated and intrarater reliability was calculated. One week after 1RM estimation procedures were completed, the accuracy of an estimated 1RM was determined by establishing an actual 1RM. The results indicated excellent intrarater reliability for position 1 (intraclass correlation coefficient [ICC] = 0.99) and position 2 (ICC = 0.96). The correlation coefficients for accuracy indicated excellent concurrent validation was attained for position 1 (ICC = 0.99) and position 2 (ICC = 0.97). Shoulder internal rotation 1RM estimation appears to be reliable and accurate. Clinicians may use submaximal loads to estimate the 1RM and decrease the possibility of injury during actual 1RM strength testing.

Intrarater reliability of the KT1000 arthrometer in determining anterior translation of the glenohumeral joint.

OBJECTIVES: To establish intrarater reliability of the KT1000 arthrometer in determining glenohumeral anterior translation and to determine if a difference existed between measurements of glenohumeral anterior translation in 2 testing positions. DESIGN: Intrarater reliability study. SETTING: Academic laboratory. PARTICIPANTS: Convenience sample of 15 unimpaired volunteers (mean age +/- standard deviation, 25+/-4 y). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Subjects were positioned supine with the shoulder in 20 degrees of abduction and 0 degrees of external rotation (position 1) and 90 degrees of abduction and 90 degrees of external rotation (position 2). The KT1000 was placed on the shoulder with the tibia sensor pad near the joint line and the patella sensor pad over the coracoid process. Testing involved an anteriorly directed force of 67N. Testing procedures were repeated after 1 week. RESULTS: The intraclass correlation coefficients (ICCs) for intrarater reliability for position 1 (ICC=.93; 95% confidence interval [CI], .81-.98) and for position 2 (ICC=.93; 95% CI, .80-.97) were excellent. The degree of anterior translation measured in position 1 was significantly greater than in position 2 ( t =4.79, P <.01). CONCLUSIONS: Use of the KT1000 to measure glenohumeral anterior translation in the 2 testing positions appears to be a relatively simple, reliable method. Because testing position 1 allowed significantly greater anterior translation than testing position 2, the 2 positions should not be used interchangeably.