Independent data collectors decrease bias in the measurement of adherence to anterior cruciate ligament injury prevention programs.

OBJECTIVES: Studies on adherence to neuromuscular training (NMT) for anterior cruciate ligament (ACL) injury prevention are frequently biased due to the use of self-reporting by coaches or the athletes themselves. Few NMT studies use data collectors (aside from the athletes or the individuals administering the NMT program) to decrease bias when assessing the adherence of coaches and sports teams. We hypothesized that the use of a data collector who is independent of the team to evaluate adherence to NMT programs would be reliable. METHODS: In a prior a cluster-randomized controlled trial evaluating adherence to NMT training trial, twelve boys' and nine girls' high school athletic teams in a variety of sports were enrolled. Eight data collectors (unaffiliated with the NMT program) were hired specifically to record adherence of the athletes to the NMT exercises at each team's warm-ups 2-3 times a week, prior to practices and games. In addition to the data collectors, a control group of independent observers made visits throughout the season to also record adherence (solely for the purpose of this study, alongside the data collectors and in the same fashion) in order to evaluate the data collectors' performance and determine inter-observer reliability. The inter-observer reliability between data collectors and independent observers was measured using the Kappa statistic. RESULTS: A total of 399 warm-ups for practices or games were observed by data collectors to obtain adherence data. Independent observers also measured adherence at 58 practices or games for inter-observer reliability. Exercise instruction and alignment cues for 29 different exercises were analysed. The Kappa values ranged from 0.63 to 1.0, indicating substantial to perfect agreement. The overall Kappa values of 0.89 and 0.90 for exercise instruction and alignment cues, respectively, indicated almost perfect agreement. CONCLUSION: The use of a data collector who is independent of the team to evaluate adherence to NMT programs (rather than athlete or coach self-reporting), was shown to be a reliable method for measurement of adherence in studies of NMT for injury prevention. Avoiding self-reporting in adherence research to NMT training may decrease bias. LEVEL OF EVIDENCE: I.

Graft choices for paediatric anterior cruciate ligament reconstruction: State of the art.

The paediatric population is at particularly high risk for anterior cruciate ligament (ACL) injuries due to high rates of sports participation. Other risk factors for ACL injuries in children include but are not limited to being female, generalised ligamentous laxity, a high body mass index (BMI), and poor neuromuscular control. ACL reconstruction (ACLR) is commonly done to treat ACL injuries and allow for return to sports and daily activities. ACL repair is another option with ongoing techniques being developed. The high rates of graft failure in children reported in recent publications on ACL repair are very concerning. Special consideration must be taken in ACLR in the skeletally immature patient due to the risk of growth-related complications, such as limb deformity or growth arrest, that can arise from drilling across or disrupting the physis. Graft choices for paediatric ACLR include iliotibial band (ITB) over the top and over the front, hamstring autograft, bone patellar tendon bone (BTB) autograft, quadriceps tendon autograft, and allograft. Factors for each graft choice to consider include graft size, graft failure rates, donor site morbidity, requirement for bony tunnels, the post-op rehabilitation process, and return to sport outcomes. Each graft has its benefits and disadvantages for the individual patient, depending on age, skeletal maturity, and goals for recovery. Lateral extra-articular tenodesis (LET) is another option to consider with paediatric ACLR because LET has been shown to decrease the re-rupture rate in adult ACLR. After surgery, patient follow-up until at least the growth plates are closed is important. This article aims to provide an overview and comparison of the various graft types to aid in the graft choice decision making process for paediatric ACLR.

Effects of Church-Based Parent-Child Abstinence-Only Interventions on Adolescents' Sexual Behaviors.

PURPOSE: The aim of the study was to evaluate the efficacy of faith-based and nonfaith-based parent-child abstinence-only interventions in reducing sexual risk behavior among young African American adolescents. METHODS: Randomized controlled trial recruiting 613 African American parent-adolescent dyads from urban African American Baptist churches and randomizing them to one of three parent-child interventions: faith-based abstinence-only intervention emphasizing delaying or reducing sexual intercourse drawing on Biblical scriptures; nonfaith-based abstinence-only emphasizing intervention delaying or reducing sexual intercourse without referencing scriptures; or attention-matched control intervention targeting health issues unrelated to sexual behavior. Primary outcome was the self-reported frequency of condomless sexual intercourse in the past 3 months assessed periodically through 18 months postintervention. Secondary outcomes were frequency of sexual intercourse, number of sexual partners, consistent condom use and, among sexually inexperienced adolescents, sexual debut. RESULTS: Generalized estimating equations analyses revealed that nonfaith-based abstinence-only intervention reduced the frequency of condomless sexual intercourse, frequency of sexual intercourse, and number of sexual partners compared with the attention-matched control intervention, whereas faith-based abstinence-only intervention did not. Neither intervention affected consistent condom use or sexual debut. CONCLUSIONS: Parent-child abstinence-only interventions can reduce condomless sexual intercourse among young African American adolescents in church settings. Linking the abstinence message to Biblical scriptures may not be efficacious.

Co-coating of receptor-targeted drug nanocarriers with anti-phagocytic moieties enhances specific tissue uptake versus non-specific phagocytic clearance.

Nanocarriers (NCs) help improve the performance of therapeutics, but their removal by phagocytes in the liver, spleen, tissues, etc. diminishes this potential. Although NC functionalization with polyethylene glycol (PEG) lowers interaction with phagocytes, it also reduces interactions with tissue cells. Coating NCs with CD47, a protein expressed by body cells to avoid phagocytic removal, offers an alternative. Previous studies showed that coating CD47 on non-targeted NCs reduces phagocytosis, but whether this alters binding and endocytosis of actively-targeted NCs remains unknown. To evaluate this, we used polymer NCs targeted to ICAM-1, a receptor overexpressed in many diseases. Co-coating of CD47 on anti-ICAM NCs reduced macrophage phagocytosis by ∼50% for up to 24 h, while increasing endothelial-cell targeting by ∼87% over control anti-ICAM/IgG NCs. Anti-ICAM/CD47 NCs were endocytosed via the CAM-mediated pathway with efficiency similar (0.99-fold) to anti-ICAM/IgG NCs. Comparable outcomes were observed for NCs targeted to PECAM-1 or transferrin receptor, suggesting broad applicability. When injected in mice, anti-ICAM/CD47 NCs reduced liver and spleen uptake by ∼30-50% and increased lung targeting by ∼2-fold (∼10-fold over IgG NCs). Therefore, co-coating NCs with CD47 and targeting moieties reduces macrophage phagocytosis and improves targeted uptake. This strategy may significantly improve the efficacy of targeted drug NCs.

Risky Trade: Individual and Neighborhood-Level Socio-Demographics Associated with Transactional Sex among Urban African American MSM.

There is a clear, persistent association between poverty and HIV risk and HIV infection. Low educational attainment, neighborhood disadvantage, and residential instability are ways in which poverty is instrumentally experienced in urban America. We investigated the role of lived poverty at both the individual and neighborhood levels in transactional sex behavior among African American men who have sex with men (MSM) residing in urban neighborhoods. Using population-averaged models estimated by generalized estimating equation (GEE) models, we identified individual-level and neighborhood-level factors that are associated with exchanging sex for drugs and/or money. We tested the association between neighborhood and individual-level socioeconomic status and HIV risk behavior by combining area-based measures of neighborhood quality from the US Census with individual survey data from 542 low-income African American MSM. The primary outcome measure was self-reported transactional sex defined as exchanging sex for drugs or money. Individual-level covariates included high school non-completion, income, and problem drug use. Neighborhood-level covariates were high school non-completion and poverty rates. The findings suggested that educational attainment is associated with both the individual level and neighborhood level. Participants were more likely to engage in transactional sex if they did not complete high school (OR = 1.78), and similarly if their neighbors did not complete high school (OR = 7.70). These findings suggest potential leverage points for both community-level interventions and advocacy for this population, particularly related to transactional sex and education, and will aid HIV prevention efforts that seek to address the contextual constraints on individual risk behavior.

Retention of South African Adolescents in a 54-Month Longitudinal HIV Risk Reduction Trial.

Retention of participants in clinical trials is a central concern of HIV/STI behavioral researchers and research sponsors. This article describes the strategies used for addressing the challenges in retaining South African adolescents for a 54-month longitudinal study. The objective of the South African adolescent health promotion long-term follow-up trial was to test the sustainability of the effects of an HIV/STI risk reduction intervention, "Let Us Protect Our Future," on young adolescents as they aged into middle and late adolescence. Inaccurate contact information, invalid mobile telephone numbers, lack of transportation, transitory family addresses, and family relocation were among the challenges to retaining participants. Despite a significant gap in time of 36 months between the main trial and the long-term follow-up study, 99.2% of 1057 participants were retained. Solutions used for retaining the adolescents are discussed with suggestions offered for retaining adolescents in longitudinal HIV/STI prevention clinical trials in low resource countries.

Efficacy and Mediation of a Theory-Based Physical Activity Intervention for African American Men Who Have Sex with Men: A Randomized Controlled Trial.

BACKGROUND: Few trials have tested physical-activity interventions among sexual minorities, including African American men who have sex with men (MSM). PURPOSE: We examined the efficacy and mediation of the Being Responsible for Ourselves (BRO) physical-activity intervention among African American MSM. METHOD: African American MSM were randomized to the physical-activity intervention consisting of three 90-min one-on-one sessions or an attention-matched control intervention and completed pre-intervention, immediately post-intervention, and 6- and 12-month post-intervention audio computer-based surveys. RESULTS: Of the 595 participants, 503 completed the 12-month follow-up. Generalized estimating equation models revealed that the intervention increased self-reported physical activity compared with the control intervention, adjusted for pre-intervention physical activity. Mediation analyses suggested that the intervention increased reasoned action approach variables, subjective norm and self-efficacy, increasing intention immediately post-intervention, which increased physical activity during the follow-up period. CONCLUSIONS: Interventions targeting reasoned action approach variables may contribute to efforts to increase African American MSM's physical activity. CLINICAL TRIAL REGISTRATION: The trial was registered with the ClinicalTrials.gov Identifier NCT02561286 .

On the Efficacy and Mediation of a One-on-One HIV Risk-Reduction Intervention for African American Men Who Have Sex with Men: A Randomized Controlled Trial.

We examined the efficacy and mediation of Being Responsible for Ourselves (BRO), an HIV/STI risk-reduction intervention for African American men who have sex with men (MSM), the population with the highest HIV-diagnosis rate in the US. We randomized African American MSM to one of two interventions: BRO HIV/STI risk-reduction, targeting condom use; or attention-matched control, targeting physical activity and healthy diet. The interventions were based on social cognitive theory, the reasoned-action approach, and qualitative research. Men reporting anal intercourse with other men in the past 90 days were eligible and completed pre-intervention, immediately post-intervention, and 6 and 12 months post-intervention surveys. Of 595 participants, 503 (85 %) completed the 12-month follow-up. Generalized-estimating-equations analysis indicated that, compared with the attention-matched control intervention, the BRO intervention did not increase consistent condom use averaged over the 6- and 12-month follow-ups, which was the primary outcome. Although BRO did not affect the proportion of condom-protected intercourse acts, unprotected sexual intercourse, multiple partners, or insertive anal intercourse, it did reduce receptive anal intercourse compared with the control, a behavior linked to incident HIV infection. Mediation analysis using the product-of-coefficients approach revealed that although BRO increased seven of nine theoretical constructs it was designed to affect, it increased only one of three theoretical constructs that predicted consistent condom use: condom-use impulse-control self-efficacy. Thus, BRO indirectly increased consistent condom use through condom-use impulse-control self-efficacy. In conclusion, although BRO increased several theoretical constructs, most of those constructs did not predict consistent condom use; hence, the intervention did not increase it. Theoretical constructs that interventions should target to increase African American MSM's condom use are discussed.

Targeting, endocytosis, and lysosomal delivery of active enzymes to model human neurons by ICAM-1-targeted nanocarriers.

PURPOSE: Delivery of therapeutics to neurons is paramount to treat neurological conditions, including many lysosomal storage disorders. However, key aspects of drug-carrier behavior in neurons are relatively unknown: the occurrence of non-canonical endocytic pathways (present in other cells); whether carriers that traverse the blood-brain barrier are, contrarily, retained within neurons; if neuron-surface receptors are accessible to bulky carriers compared to small ligands; or if there are differences regarding neuronal compartments (neuron body vs. neurites) pertaining said parameters. We have explored these questions using model polymer nanocarriers targeting intercellular adhesion molecule-1 (ICAM-1). METHODS: Differentiated human neuroblastoma cells were incubated with anti-ICAM-coated polystyrene nanocarriers and analyzed by fluorescence microscopy. RESULTS: ICAM-1 expression and nanocarrier binding was enhanced in altered (TNFα) vs. control conditions. While small ICAM-1 ligands (anti-ICAM) preferentially accessed the cell body, anti-ICAM nanocarriers bound with faster kinetics to neurites, yet reached similar saturation over time. Anti-ICAM nanocarriers were also endocytosed with faster kinetics and lower saturation levels in neurites. Non-classical cell adhesion molecule (CAM) endocytosis ruled uptake, and neurite-to-cell body transport was inferred. Nanocarriers trafficked to lysosomes, delivering active enzymes (dextranase) with substrate reduction in a lysosomal-storage disease model. CONCLUSION: ICAM-1-targeting holds potential for intracellular delivery of therapeutics to neurons.

Multiple partners and condom use among students at a South African University.

The prevalence of HIV in sub-Saharan Africa is the highest in the world. Young people, including university students, are at risk. Many sexually active young people have multiple partners, but little is known about how university students who have multiple partners differ from those who do not. This study examined such differences among randomly selected first-year students at a university in the Eastern Cape Province, South Africa, who completed a confidential questionnaire via audio computer-assisted self-interviewing. Of 201 participants, 93 (46.3%) reported sexual intercourse in the previous 3 months. Of those, 52 (55.91%) reported sexual intercourse with more than one partner in the past 3 months. Controlling for gender, students who reported multiple partners were younger at first coitus, had a greater number of lifetime coital partners, and reported more frequent coitus and unprotected coitus but a lower proportion of condom-protected coital acts in the past 3 months than did those reporting only one partner. However, those reporting multiple partners and one partner did not differ in religiosity, drinking problems, or victimization by childhood sexual abuse. HIV/sexually transmitted disease risk reduction interventions must address unprotected coitus and failure to use condoms among university students reporting multiple partners.

Combination-targeting to multiple endothelial cell adhesion molecules modulates binding, endocytosis, and in vivo biodistribution of drug nanocarriers and their therapeutic cargoes.

Designing of drug nanocarriers to aid delivery of therapeutics is an expanding field that can improve medical treatments. Nanocarriers are often functionalized with elements that recognize cell-surface molecules involved in subcellular transport to improve targeting and endocytosis of therapeutics. Combination-targeting using several affinity elements further modulates this outcome. The most studied example is endothelial targeting via multiple cell adhesion molecules (CAMs), which mimics the strategy of leukocytes to adhere and traverse the vascular endothelium. Yet, the implications of this strategy on intracellular transport and in vivo biodistribution remain uncharacterized. We examined this using nanocarriers functionalized for dual- or triple-targeting to intercellular, platelet-endothelial, and/or vascular CAMs (ICAM-1, PECAM-1, VCAM-1). These molecules differ in expression level, location, pathological stimulation, and/or endocytic pathway. In endothelial cells, binding of PECAM-1/VCAM-1-targeted nanocarriers was intermediate to single-targeted counterparts and enhanced in disease-like conditions. ICAM-1/PECAM-1-targeted nanocarriers surpassed PECAM-1/VCAM-1 in control, but showed lower selectivity toward disease-like conditions. Triple-targeting resulted in binding similar to ICAM-1/PECAM-1 combination and displayed the highest selectivity in disease-like conditions. All combinations were effectively internalized by the cells, with slightly better performance when targeting receptors of different endocytic pathways. In vivo, ICAM-1/PECAM-1-targeted nanocarriers outperformed PECAM-1/VCAM-1 in control and disease-like conditions, and triple-targeted counterparts slightly enhanced this outcome in some organs. As a result, delivery of a model therapeutic cargo (acid sphingomyelinase, deficient in Niemann-Pick disease A-B) was enhanced to all affected organs by triple-targeted nanocarriers, particularly in disease-like conditions. Therefore, multi-CAM targeting may aid the optimization of some therapeutic nanocarriers, where the combination and multiplicity of the affinity moieties utilized allow modulation of targeting performance.

Enhancing biodistribution of therapeutic enzymes in vivo by modulating surface coating and concentration of ICAM-1-targeted nanocarriers.

Coupling therapeutic proteins to targeted nanocarriers can enhance their biodistribution. This is the case for enzyme replacement therapies where intravenously injected enzymes must avoid prolonged blood exposure while reaching body organs. We have shown enhanced tissue targeting of various lysosomal enzymes by coupling to nanocarriers targeted to intercellular adhesion molecule-1 (ICAM-1). Here, we varied design parameters to modify tissue enzyme levels without affecting specific targeting and relative biodistribution. We coupled a-galactosidase (aGal; affected in Fabry disease) to model polymer nanocarriers and varied enzyme load (50 vs. 500 molecules/particle), anti-ICAM surface density (80 vs. 180 molecules/particle), and nanocarrier concentration (1.6 x 1013 vs. 2.4 x 1013 carriers/kg) to render three formulations (45, 449, 555 microg alphaGal/kg). Naked alpha Gal preferentially distributed in blood vs. organs, while nanocarriers shifted biodistribution from blood to tissues. Accumulation in brain, kidneys, heart, liver, lungs, and spleen did not vary among nanocarrier formulations, with enhanced specific tissue accumulation compared to naked aGal. The highest specificity was associated with lowest antibody density and nanocarrier concentration, but highest enzyme load; possibly because of synergistic enzyme affinity toward cell-surface markers. Variation of these parameters significantly increased absolute enzyme accumulation. This strategy may help optimize delivery of lysosomal enzyme replacement and, likely, other protein delivery approaches.

Specific binding, uptake, and transport of ICAM-1-targeted nanocarriers across endothelial and subendothelial cell components of the blood-brain barrier.

PURPOSE: The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transport drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule -1 (ICAM-1), to transport drug carriers into and across BBB models. METHODS: Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. RESULTS: ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. CONCLUSIONS: CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier.

Enhanced delivery of α-glucosidase for Pompe disease by ICAM-1-targeted nanocarriers: comparative performance of a strategy for three distinct lysosomal storage disorders.

Enzyme replacement therapies for lysosomal storage disorders are often hindered by suboptimal biodistribution of recombinant enzymes after systemic injection. This is the case for Pompe disease caused by acid α-glucosidase (GAA) deficiency, leading to excess glycogen storage throughout the body, mainly the liver and striated muscle. Targeting intercellular adhesion molecule-1 (ICAM-1), a protein involved in inflammation and overexpressed on most cells under pathological conditions, provides broad biodistribution and lysosomal transport of therapeutic cargoes. To improve its delivery, we coupled GAA to polymer nanocarriers (NCs; ∼180 nm) coated with an antibody specific to ICAM-1. Fluorescence microscopy showed specific targeting of anti-ICAM/GAA NCs to cells, with efficient internalization and lysosomal transport, enhancing glycogen degradation over nontargeted GAA. Radioisotope tracing in mice demonstrated enhanced GAA accumulation in all organs, including Pompe targets. Along with improved delivery of Niemann-Pick and Fabry enzymes, previously described, these results indicate that ICAM-1 targeting holds promise as a broad platform for lysosomal enzyme delivery. FROM THE CLINICAL EDITOR: In this study, ICAM-1 targeted nanocarriers were used to deliver GAA (acid alpha glucosidase) into cells to address the specific enzyme deficiency in Pompe's disease. The results unequivocally demonstrate enhanced enzyme delivery over nontargeted GAA in a mice model.

Enhanced endothelial delivery and biochemical effects of α-galactosidase by ICAM-1-targeted nanocarriers for Fabry disease.

Fabry disease, due to the deficiency of α-galactosidase A (α-Gal), causes lysosomal accumulation of globotriaosylceramide (Gb3) in multiple tissues and prominently in the vascular endothelium. Although enzyme replacement therapy (ERT) by injection of recombinant α-Gal improves the disease outcome, the effects on the vasculopathy associated with life-threatening cerebrovascular, cardiac and renal complications are still limited. We designed a strategy to enhance the delivery of α-Gal to organs and endothelial cells (ECs). We targeted α-Gal to intercellular adhesion molecule 1 (ICAM-1), a protein expressed on ECs throughout the vasculature, by loading this enzyme on nanocarriers coated with anti-ICAM (anti-ICAM/α-Gal NCs). In vitro radioisotope tracing showed efficient loading of α-Gal on anti-ICAM NCs, stability of this formulation under storage and in model physiological fluids, and enzyme release in response to lysosome environmental conditions. In mice, the delivery of (125)I-α-Gal was markedly enhanced by anti-ICAM/(125)I-α-Gal NCs in brain, kidney, heart, liver, lung, and spleen, and transmission electron microscopy showed anti-ICAM/α-Gal NCs attached to and internalized into the vascular endothelium. Fluorescence microscopy proved targeting, endocytosis and lysosomal transport of anti-ICAM/α-Gal NCs in macro- and micro-vascular ECs and a marked enhancement of Gb3 degradation. Therefore, this ICAM-1-targeting strategy may help improve the efficacy of therapeutic enzymes for Fabry disease.

NO signaling and S-nitrosylation regulate PTEN inhibition in neurodegeneration.

BACKGROUND: The phosphatase PTEN governs the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway which is arguably the most important pro-survival pathway in neurons. Recently, PTEN has also been implicated in multiple important CNS functions such as neuronal differentiation, plasticity, injury and drug addiction. It has been reported that loss of PTEN protein, accompanied by Akt activation, occurs under excitotoxic conditions (stroke) as well as in Alzheimer's (AD) brains. However the molecular signals and mechanism underlying PTEN loss are unknown. RESULTS: In this study, we investigated redox regulation of PTEN, namely S-nitrosylation, a covalent modification of cysteine residues by nitric oxide (NO), and H2O2-mediated oxidation. We found that S-nitrosylation of PTEN was markedly elevated in brains in the early stages of AD (MCI). Surprisingly, there was no increase in the H2O2-mediated oxidation of PTEN, a modification common in cancer cell types, in the MCI/AD brains as compared to normal aged control. Using several cultured neuronal models, we further demonstrate that S-nitrosylation, in conjunction with NO-mediated enhanced ubiquitination, regulates both the lipid phosphatase activity and protein stability of PTEN. S-nitrosylation and oxidation occur on overlapping and distinct Cys residues of PTEN. The NO signal induces PTEN protein degradation via the ubiquitin-proteasome system (UPS) through NEDD4-1-mediated ubiquitination. CONCLUSION: This study demonstrates for the first time that NO-mediated redox regulation is the mechanism of PTEN protein degradation, which is distinguished from the H2O2-mediated PTEN oxidation, known to only inactivate the enzyme. This novel regulatory mechanism likely accounts for the PTEN loss observed in neurodegeneration such as in AD, in which NO plays a critical pathophysiological role.