High-Throughput CAMP Assay (HiTCA): A Novel Tool for Evaluating the Vitamin D-Dependent Antimicrobial Response.

Vitamin D is known to modulate human immune responses, and vitamin D deficiency is associated with increased susceptibility to infection. However, what constitutes sufficient levels or whether vitamin D is useful as an adjuvant therapeutic is debated, much in part because of inadequate elucidation of mechanisms underlying vitamin D's immune modulatory function. Cathelicidin antimicrobial peptide (CAMP) has potent broad-spectrum activity, and the CAMP gene is regulated in human innate immune cells by active 1,25(OH)2D3, a product of hydroxylation of inactive 25(OH)D3 by CYP27B1-hydroxylase. We developed a CRISPR/Cas9-edited human monocyte-macrophage cell line containing the mCherry fluorescent reporter gene at the 3' end of the endogenous CAMP gene. The High Throughput CAMP Assay (HiTCA) developed here is a novel tool for evaluating CAMP expression in a stable cell line that is scalable for a high-throughput workflow. Application of HiTCA to serum samples from a small number of human donors (n = 10) showed individual differences in CAMP induction that were not fully accounted for by the serum vitamin D metabolite status of the host. As such, HiTCA may be a useful tool that can advance our understanding of the human vitamin D-dependent antimicrobial response, which is being increasingly appreciated for its complexity.

Opioid and Naloxone Prescribing Following Insertion of Prompts in the Electronic Health Record to Encourage Compliance With California State Opioid Law.

Importance: Opioid addiction or dependency is a serious crisis in the US that affects public health as well as social and economic welfare. The State of California passed Assembly Bill (AB) 2760 in 2018 that mandates the coprescription of naloxone and opioids for patients with a high overdose risk. Objective: To assess whether the AB 2760-based electronic prompts were associated with increased naloxone orders for opioid users and reduced opioid prescribing when integrated into the practitioner workflow. Design, Setting, and Participants: This cohort study used interrupted time series mixed models to evaluate data obtained from the regional integrated health care system Kaiser Permanente Southern California (KPSC) from January 1, 2018, to December 31, 2019. Clinician participants were continuously employed at KPSC during the study period and ordered an opioid analgesic for eligible patients in 2018. Patient participants were KPSC members aged 18 years or older who received an opioid analgesic prescription during the study period. A series of AB 2760-based electronic prompts were integrated into the KPSC electronic health record system on December 27, 2018. The prompts are triggered or activated when 1 or more opioid prescribing conditions, defined in the AB 2760, are met at outpatient visits. Data were analyzed from January 8, 2021, to September 15, 2021. Exposures: Assembly Bill 2760-based electronic prompts for outpatient opioid prescriptions in the electronic health record system. Main Outcomes and Measures: Primary outcomes were changes in outpatient naloxone order rates among patients who were prescribed opioids and changes in outpatient opioid prescribing rates. Secondary outcomes were total morphine milligram equivalents (MMEs) ordered per prescriber-month, prompts-targeted objectives, and unintended consequences. Risk for opioid abuse among 3 types of patients was also assessed. Results: The 6515 eligible clinicians (mean [SD] age, 45.9 [9.43] years; 3604 men [55.3%]) included in the study served 500 711 unique patients in 1 903 289 outpatient encounters (mean [SD] age, 60.4 [15.67] years; 1 121 004 women [58.9%]) in which an opioid analgesic was prescribed. Naloxone order rate increased from 2.0% in December 2018 to 13.2% in January 2019 and then continued to increase to 27.1% in December 2019. Outpatient opioid prescribing rates decreased by 15.1% (rate ratio [RR], 0.85; 95% CI, 0.83-0.87) per prescriber-month when the electronic prompts were implemented. The postimplementation trend increased by 0.7% per prescriber-month (RR, 1.01; 95% CI, 1.01-1.01); the overall trend was still decreasing. The total MMEs per prescriber-month decreased by 7.8% (RR, 0.92; 95% CI, 0.89-0.96) after implementation of the prompts. The postimplementation trend tapered off. Other safe opioid prescribing measures also improved after implementation (decreases in concomitant muscle relaxants orders [RR, 0.94; 95% CI, 0.89-1.00], initial [RR, 0.86; 0.83-0.89] and renewal [RR, 0.65; 95% CI, 0.62-0.69] opioid orders, and long-term high-dose orders [RR, 0.96; 95% CI, 0.94-0.98]). Conclusions and Relevance: This study found an association between implementation of AB 2760-based prompts and increased naloxone order rate; improved opioid prescribing measures (ie, decreased concomitant muscle relaxants orders, initial and renewal opioid orders, and long-term high-dose orders), except monthly median MMEs; and reduced opioid prescribing. The findings suggest that opioid overdose risks can be mitigated by encouraging safe prescribing habits.

Pilot Trial of Vitamin D3 and Calcifediol in Healthy Vitamin D Deficient Adults: Does It Change the Fecal Microbiome?

CONTEXT: Experimental studies suggest that vitamin D receptor signaling may benefit the gut microbiome. In humans, whether vitamin D supplementation directly alters the gut microbiome is not well studied. OBJECTIVE: To determine whether correcting vitamin D deficiency with cholecalciferol (vitamin D3, D3) or calcifediol (25-hydroxyvitamin D3, 25(OH)D3) changes gut microbiome composition. METHODS: 18 adults with vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] <20 ng/mL) received 60 µg/day of D3 or 20 µg/day of 25(OH)D3 for 8 weeks. Changes in serum 25(OH)D, 1,25-diydroxyvitamin D (1,25(OH)2D), and 24,25-dihydroxyvitamin D (24,25(OH)2D) were assessed. We characterized composition of the fecal microbiota using 16S rRNA gene sequencing, and examined changes in α-diversity (Chao 1, Faith's Phylogenetic Diversity, Shannon Index), ß-diversity (DEICODE), and genus-level abundances (DESeq2). RESULTS: Vitamin D3 and 25(OH)D3 groups were similar. After 8 weeks of vitamin D3, mean 25(OH)D and 24,25(OH)2D increased significantly, but 1,25(OH)2D did not (25(OH)D: 17.8-30.1 ng/mL, P = .002; 24,25(OH)2D: 1.1 to 2.7 ng/mL, P =0.003; 1,25(OH)2D: 49.5-53.0 pg/mL, P = .9). After 8 weeks of 25(OH)D3, mean 25(OH)D, 24,25(OH)2D, and 1,25(OH)2D increased significantly (25(OH)D: 16.7-50.6 ng/mL, P < .0001; 24,25(OH)2D: 1.3-6.2 ng/mL, P = .0001; 1,25(OH)2D: 56.5-74.2 pg/mL, P = .05). Fecal microbial α-diversity and ß-diversity did not change with D3 or 25D3 supplementation. Mean relative abundance of Firmicutes increased and mean relative abundance of Bacterioidetes decreased from baseline to 4 weeks, but returned to baseline by study completion. DESeq2 analysis did not confirm any statistically significant taxonomic changes. CONCLUSION: In a small sample of healthy adults with vitamin D deficiency, restoration of vitamin D sufficiency with vitamin D3 or 25(OH)D3 did not lead to lasting changes in the fecal microbiota.

Association Between Removal of a Warning Against Cephalosporin Use in Patients With Penicillin Allergy and Antibiotic Prescribing.

Importance: Electronic health records (EHRs) often include default alerts that can influence physician selection of antibiotics, which in turn may be associated with a suboptimal choice of agents and increased antibiotic resistance. Objective: To examine whether removal of a default alert in the EHR to avoid cephalosporin use in patients with penicillin allergies is associated with changes in cephalosporin dispensing or administration in these patients. Design, Setting, and Participants: This retrospective cohort study of a natural experiment included data on patients who had received antibiotic treatment in the hospital or outpatient setting in 2 regions of a large, integrated health system in California from January 1, 2017, to December 31, 2018. Of 4 398 792 patients, 4 206 480 met the eligibility criteria: enrollment in the health system during antibiotic use, availability of complete demographic data, and use of antibiotics outside of the washout period. Interventions or Exposures: Oral or parenteral antibiotics dispensed or administered after removal of an EHR alert to avoid cephalosporin use in patients with a recorded penicillin allergy. Main Outcomes and Measures: Probability that an antibiotic course was a cephalosporin. A multinomial logistic regression model was used to examine the change in rates of cephalosporin use before and after an EHR penicillin allergy alert was removed in 1 of the study regions. Temporal changes in use rates were controlled for by comparing changes in cephalosporin use among patients with or without a penicillin allergy at the site that removed the warning and among patients at a comparison site that retained the warning. Regression models were used to examine adverse events. Results: Of the 4 206 480 patients who met all inclusion criteria, 2 465 849 (58.6%) were women; the mean (SD) age was 40.5 (23.2) years. A total of 10 652 014 antibiotic courses were administered or dispensed, divided approximately evenly between the period before and after removal of the warning. Before removal of an alert in the electronic health record system to avoid prescribing of cephalosporins to patients with a penicillin allergy at 1 of the 2 sites, 58 228 courses of cephalosporins (accounting for 17.9% of all antibiotic use at the site) were used among patients with a penicillin allergy; after removal of the alert, administration or dispensing of cephalosporins increased by 47% compared with cephalosporin administration or dispensing among patients without a penicillin allergy at the same site and patients at the comparison site that retained the warning (ratio of ratios of odds ratios [RROR], 1.47; 95% CI, 1.38-1.56) . No significant differences in anaphylaxis (9 total cases), new allergies (RROR, 1.02; 95% CI, 0.93-1.12), or treatment failures (RROR, 1.02; 95% CI, 0.99-1.05) were found at the course level. No significant differences were found in all-cause mortality (ratio of ratios of rate ratios [RRRR], 1.03; 95% CI, 0.94-1.13), hospital days (RRRR, 1.04; 95% CI, 0.99-1.10), and new infections (Clostridioides difficile: RRRR, 1.02; 95% CI, 0.84-1.22; methicillin-resistant Staphylococcus aureus: RRRR, 0.87; 95% CI, 0.75-1.00; and vancomycin-resistant Enterococcus: RRRR, 0.82; 95% CI, 0.55-1.22) at the patient level. Conclusions and Relevance: In this cohort study, removal of a warning in the electronic health record to avoid cephalosporin use in patients with penicillin allergies was associated with increased administration and dispensing of cephalosporin. This simple and rapidly implementable system-level intervention may be useful for improvement in antibiotic stewardship.

Validation of natural language processing to determine the presence and size of abdominal aortic aneurysms in a large integrated health system.

OBJECTIVE: Previous studies of the natural history of abdominal aortic aneurysms (AAAs) have been limited by small cohort sizes or heterogeneous analyses of pooled data. By quickly and efficiently extracting imaging data from the health records, natural language processing (NLP) has the potential to substantially improve how we study and care for patients with AAAs. The aim of the present study was to test the ability of an NLP tool to accurately identify the presence or absence of AAAs and detect the maximal abdominal aortic diameter in a large dataset of imaging study reports. METHODS: Relevant imaging study reports (n = 230,660) from 2003 to 2017 were obtained for 32,778 patients followed up in a prospective aneurysm surveillance registry within a large, diverse, integrated healthcare system. A commercially available NLP algorithm was used to assess the presence of AAAs, confirm the absence of AAAs, and extract the maximal diameter of the abdominal aorta, if stated. A blinded expert manual review of 18,000 randomly selected imaging reports was used as the reference standard. The positive predictive value (PPV or precision), sensitivity (recall), and the kappa statistics were calculated. RESULTS: Of the randomly selected 18,000 studies that underwent expert manual review, 48.7% were positive for AAAs. In confirming the presence of an AAA, the interrater reliability of the NLP compared with the expert review showed a kappa value of 0.84 (95% confidence interval [CI], 0.83-0.85), with a PPV of 95% and sensitivity of 88.5%. The NLP algorithm showed similar results for confirming the absence of an AAA, with a kappa of 0.79 (95% CI, 0.799-0.80), PPV of 77.7%, and sensitivity of 91.9%. The kappa, PPV, and sensitivity of the NLP for correctly identifying the maximal aortic diameter was 0.88 (95% CI, 0.87-0.89), 88.8%, and 88.2% respectively. CONCLUSIONS: The use of NLP software can accurately analyze large volumes of radiology report data to detect AAA disease and assemble a contemporary aortic diameter-based cohort of patients for longitudinal analysis to guide surveillance, medical management, and operative decision making. It can also potentially be used to identify from the electronic medical records pre- and postoperative AAA patients "lost to follow-up," leverage human resources engaged in the ongoing surveillance of patients with AAAs, and facilitate the construction and implementation of AAA screening programs.

Comparison of Direct Oral Anticoagulants Versus Warfarin in Patients With Atrial Fibrillation and Bioprosthetic Heart Valves.

There are limited data regarding direct oral anticoagulants (DOACs) for stroke prevention in patients with bioprosthetic heart valves (BHVs) and atrial fibrillation (AF). The objectives of this study were to evaluate the ambulatory utilization of DOACs and to compare the effectiveness and safety of DOACs versus warfarin in patients with AF and BHVs. We conducted a retrospective cohort study at a large integrated health care delivery system in California. Patients with BHVs and AF treated with warfarin, dabigatran, rivaroxaban, or apixaban between September 12, 2011 and June 18, 2020 were identified. Inverse probability of treatment-weighted comparative effectiveness and safety of DOACs compared with warfarin were determined. Use of DOACs gradually increased since 2011, with a significant upward in trend after a stay-at-home order related to COVID-19. Among 2,672 adults with BHVs and AF who met the inclusion criteria, 439 were exposed to a DOAC and 2233 were exposed to warfarin. For the primary effectiveness outcome of ischemic stroke, systemic embolism and transient ischemic attack, no significant association was observed between use of DOACs compared with warfarin (HR 1.19, 95% CI 0.96 to 1.48, p = 0.11). Use of DOACs was associated with lower risk of the primary safety outcome of intracranial hemorrhage, gastrointestinal bleeding, and other bleed (HR 0.69, 95% CI 0.56 to 0.85, p < 0.001). Results were consistent across multiple subgroups in the sensitivity analyses. These findings support the use of DOACs for AF in patients with BHVs.

Low fundamental and formant frequencies predict fighting ability among male mixed martial arts fighters.

Human voice pitch is highly sexually dimorphic and eminently quantifiable, making it an ideal phenotype for studying the influence of sexual selection. In both traditional and industrial populations, lower pitch in men predicts mating success, reproductive success, and social status and shapes social perceptions, especially those related to physical formidability. Due to practical and ethical constraints however, scant evidence tests the central question of whether male voice pitch and other acoustic measures indicate actual fighting ability in humans. To address this, we examined pitch, pitch variability, and formant position of 475 mixed martial arts (MMA) fighters from an elite fighting league, with each fighter's acoustic measures assessed from multiple voice recordings extracted from audio or video interviews available online (YouTube, Google Video, podcasts), totaling 1312 voice recording samples. In four regression models each predicting a separate measure of fighting ability (win percentages, number of fights, Elo ratings, and retirement status), no acoustic measure significantly predicted fighting ability above and beyond covariates. However, after fight statistics, fight history, height, weight, and age were used to extract underlying dimensions of fighting ability via factor analysis, pitch and formant position negatively predicted "Fighting Experience" and "Size" factor scores in a multivariate regression model, explaining 3-8% of the variance. Our findings suggest that lower male pitch and formants may be valid cues of some components of fighting ability in men.

The Society for Vascular Surgery Alternative Payment Model Task Force report on opportunities for value-based reimbursement in care for patients with peripheral artery disease.

The Society for Vascular Surgery Alternative Payment Model (APM) Taskforce document explores the drivers and implications for developing objective value-based reimbursement plans for the care of patients with peripheral arterial disease (PAD). The APM is a payment approach that highlights high-quality and cost-efficient care and is a financially incentivized pathway for participation in the Quality Payment Program, which aims to replace the traditional fee-for-service payment method. At present, the participation of vascular specialists in APMs is hampered owing to the absence of dedicated models. The increasing prevalence of PAD diagnosis, technological advances in therapeutic devices, and the increasing cost of care of the affected patients have financial consequences on care delivery models and population health. The document summarizes the existing measurement methods of cost, care processes, and outcomes using payor data, patient-reported outcomes, and registry participation. The document also evaluates the existing challenges in the evaluation of PAD care, including intervention overuse, treatment disparities, varied clinical presentations, and the effects of multiple comorbid conditions on the cost potentially attributable to the vascular interventionalist. Medicare reimbursement data analysis also confirmed the prolonged need for additional healthcare services after vascular interventions. The Society for Vascular Surgery proposes that a PAD APM should provide patients with comprehensive care using a longitudinal approach with integration of multiple key medical and surgical services. It should maintain appropriate access to diagnostic and therapeutic advancements and eliminate unnecessary interventions. It should also decrease the variability in care but must also consider the varying complexity of the presenting PAD conditions. Enhanced quality of care and physician innovation should be rewarded. In addition, provisions should be present within an APM for high-risk patients who carry the risk of exclusion from care because of the naturally associated high costs. Although the document demonstrates clear opportunities for quality improvement and cost savings in PAD care, continued PAD APM development requires the assessment of more granular data for accurate risk adjustment, in addition to largescale testing before public release. Collaboration between payors and physician specialty societies remains key.

Predictors of Hyperkalemia and Hypokalemia in Individuals with Diabetes: a Classification and Regression Tree Analysis.

BACKGROUND: Both hyperkalemia and hypokalemia can lead to cardiac arrhythmias and are associated with increased mortality. Information on the predictors of potassium in individuals with diabetes in routine clinical practice is lacking. OBJECTIVE: To identify predictors of hyperkalemia and hypokalemia in adults with diabetes. DESIGN: Retrospective cohort study, with classification and regression tree (CART) analysis. PARTICIPANTS: 321,856 individuals with diabetes enrolled in four large integrated health care systems from 2012 to 2013. MAIN MEASURES: We used a single serum potassium result collected in 2012 or 2013. Hyperkalemia was defined as a serum potassium ≥ 5.5 mEq/L and hypokalemia as < 3.5 mEq/L. Predictors included demographic factors, laboratory measurements, comorbidities, medication use, and health care utilization. KEY RESULTS: There were 2556 hypokalemia events (0.8%) and 1517 hyperkalemia events (0.5%). In univariate analyses, we identified concordant predictors (associated with increased probability of both hyperkalemia and hypokalemia), discordant predictors, and predictors of only hyperkalemia or hypokalemia. In CART models, the hyperkalemia "tree" had 5 nodes and a c-statistic of 0.76. The nodes were defined by prior potassium results and eGFRs, and the 5 terminal "leaves" had hyperkalemia probabilities of 0.2 to 7.2%. The hypokalemia tree had 4 nodes and a c-statistic of 0.76. The hypokalemia tree included nodes defined by prior potassium results, and the 4 terminal leaves had hypokalemia probabilities of 0.3 to 17.6%. Individuals with a recent potassium between 4.0 and 5.0 mEq/L, eGFR ≥ 45 mL/min/1.73m2, and no hypokalemia in the previous year had a < 1% rate of either hypokalemia or hyperkalemia. CONCLUSIONS: The yield of routine serum potassium testing may be low in individuals with a recent serum potassium between 4.0 and 5.0 mEq/L, eGFR ≥ 45 mL/min/1.73m2, and no recent history of hypokalemia. We did not examine the effect of recent changes in clinical condition or medications on acute potassium changes.

Comparative Morpho-Anatomical and HPTLC Profiling of Tinospora Species and Dietary Supplements.

Overlapping geographical occurrence, history of traditional use, confusion in species identification, and morphological resemblances among various species are some considerations that necessitate the importance of qualitative analysis for efficient quality control and safer botanical products. This paper provides detailed morpho-anatomies of the leaves and stems of Tinospora cordifolia, Tinospora crispa, and Tinospora sinensis, and stems of Tinospora baenzigeri. Microscopy studies of the selected Tinospora species revealed key diagnostic features that can help distinguish the closely related species of Tinospora as well as to detect any adulteration or substitution in the raw materials. HPTLC profiles of the authenticated plant materials, as well as commercial products claiming to contain Tinospora, were compared to distinguish T. crispa from other closely related species and to establish an efficient method to assess the identity and quality of the products using qualified chemical markers. HPTLC chromatograms of both plant samples and dietary supplements were compared with six reference marker compounds. The analysis revealed that borapetoside B and C were useful to identify T. crispa while tinosineside A was found to be characteristic to authenticate the T. sinensis products.

Impact of Social Needs Navigation on Utilization Among High Utilizers in a Large Integrated Health System: a Quasi-experimental Study.

BACKGROUND: Programs addressing social determinants of health for high-utilizing patients are gaining interest among health systems as an avenue to promote health and decrease utilization. OBJECTIVE: To evaluate impacts of a social needs screening and navigation program for adult predicted high utilizers on total medical visit utilization. DESIGN: A prospective, quasi-experimental study using an intent-to-treat propensity-weighted difference-in-differences approach. Stratified analyses assessed intervention effects among three low-socioeconomic status sub-samples: patients in low-income areas, in low-education areas, and with Medicaid insurance. PARTICIPANTS: Predicted high utilizers-patients predicted to be in the highest 1% for total utilization in a large integrated health system. INTERVENTION: A telephonic social needs screening and navigation program. MAIN MEASURES: Primary difference-in-difference analyses compared total visit count utilization, including outpatient, emergency department (ED), and inpatient utilization, between the intervention and control groups at both in-network and out-of-network facilities. Prevalence of social needs among sample patients and their connection rates to social needs resources are also described. KEY RESULTS: The study included 34,225 patients (7107 intervention, 27,118 control). Most (53%) patients screened reported social needs, but only a minority (10%) of those with a need were able to connect with resources to address these needs. Primary analysis found total utilization visits decreased 2.2% (95% CI - 4.5%, 0.1%; p = 0.058) in the intervention group. Stratified analyses showed decreases in total utilization for all low-socioeconomic status subgroups receiving the intervention compared with controls: - 7.0% (95% CI - 11.9%, - 1.9%; p = 0.008) in the low-income area group, - 11.5% (- 17.6%, 5.0%; p < 0.001) in the low-education area group, and - 12.1% (- 18.1%, - 5.6%; p < 0.001) in the Medicaid group. CONCLUSIONS: Social needs navigation programs for high-utilizing patients may have modest effects on utilization for the population overall. However, significant decreases in utilization were found among low-socioeconomic status patients more likely to experience social needs.

Associations Between Change in Total and Free 25-Hydroxyvitamin D With 24,25-Dihydroxyvitamin D and Parathyroid Hormone.

Context: The physiologic role of free 25-hydroxyvitamin D [25(OH)D] in humans is unclear. Objective: To assess whether rise in total vs free 25(OH)D is associated with change in downstream biomarkers of 25(OH)D entry into target cells in kidney and parathyroid: 24,25-dihyroxyvitamin D [24,25(OH)2D] and PTH, respectively. Design: 16-week randomized controlled trial. Intervention: 60 µg (2400 IU)/d of D3 or 20 µg/d of 25(OH)D3. Setting: Academic medical center. Participants: 35 adults age ≥18 years with 25(OH)D levels < 20 ng/mL. Main Outcome Measures: 24,25(OH)2D, 1,25-dihyroxyvitamin D [1,25(OH)2D] and PTH. Results: At baseline, participants [D3 and 25(OH)D3 groups combined] were 35.1 ± 10.6 years. Mean total 25(OH)D, free 25(OH)D, 24,25(OH)2D, and PTH were 16.6 ng/mL, 4.6 pg/mL, 1.3 ng/mL, and 37.2 pg/mL, respectively. From 0 to 4 weeks, rise in only free 25(OH)D was associated with a concurrent 24,25(OH)2D increase [P = 0.03, adjusted for change in 1,25(OH)2D and supplementation regimen] and PTH decrease (P = 0.01, adjusted for change in calcium and supplementation regimen). Between 4 and 8 weeks, and again from 8 to 16 weeks, rises in free and total 25(OH)D were associated with 24,25(OH)2D increase; in contrast, rise in neither total nor free 25(OH)D was associated with PTH decrease during these time periods. Conclusions: Early rise in free 25(OH)D during treatment of vitamin D deficiency was more strongly associated with changes in biomarkers of 25(OH)D entry into target kidney and parathyroid cells, suggesting a physiologic role of free 25(OH)D in humans.

Bisphosphonate Drug Holiday and Fracture Risk: A Population-Based Cohort Study.

Holidays from bisphosphonates (BPs) may help to prevent rare adverse events such as atypical femoral fractures, but may be appropriate only if risk of osteoporosis-related fractures does not increase. Our objective was to compare the incidence of osteoporosis-related fractures among women who had a BP holiday to those who continued to use BPs. This retrospective cohort study, conducted within four Kaiser Permanente integrated health system regions, included 39,502 women aged ≥45 years with ≥3 years exposure to BP. Participants with a BP holiday (≥12 months with no use) were compared to persistent (use with ≥50% adherence) and nonpersistent (use with <50% adherence) users for incident osteoporosis-related fractures. The BP holiday (n = 11,497), nonpersistent user (n = 10,882), and persistent user groups (n = 17,123) were observed for 156,657 person-years. A total of 5199 osteoporosis-related fractures (including 1515 hip fractures and 2147 vertebral fractures) were observed. Compared to the persistent use group, there was a slight difference in overall osteoporosis-related fracture risk (HR 0.92; 95% CI, 0.84 to 0.99)and no difference in hip fracture risk (HR 0.95; 95% CI, 0.83 to 1.10) for the BP holiday group. A slight reduction in risk of vertebral fracture was observed (HR 0.83; 95% CI, 0.74 to 0.95). Compared to the nonpersistent user group, the BP holiday group was at decreased risk for osteoporosis-related fractures (HR 0.71; 95% CI, 0.65 to 0.79), vertebral fractures (HR 0.68; 95% CI, 0.59 to 0.78), and hip fractures (HR 0.59; 95% CI, 0.50 to 0.70). Women who undertake a BP holiday from BP of ≥12 months duration for any reason after ≥3 years of BP use do not appear to be at greater risk of osteoporosis-related fragility fracture, hip, or vertebral fractures compared to ongoing BP users. In our cohort, BP holiday remains a viable strategy for balancing the benefits and potential harms associated with long-term BP use. © 2018 American Society for Bone and Mineral Research.

Norepinephrine in Combination with Antibiotic Therapy Increases both the Bacterial Replication Rate and Bactericidal Activity.

We previously demonstrated that the rate and extent of an antimicrobial agent's bactericidal effects were coupled to the bacterial replication rate, the latter of which was modulated with the sodium chloride concentration. Herein, we describe the results from a 24-h one-compartment in vitro infection model study that was designed to demonstrate that an antimicrobial agent's bactericidal effects could be amplified when it is administered with a pharmaceutical agent that increases the bacterial replication rate. The antimicrobial and growth-promoting agents selected were levofloxacin and norepinephrine, respectively. The challenge isolate was Escherichia coli JMI 21711R (levofloxacin MIC, 8 mg/liter). Within the in vitro infection model, a human levofloxacin concentration-time profile (half-life, 7 h) was simulated and the challenge isolate was subjected to an ineffective monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC] ratio of 6) with and without norepinephrine as a continuous infusion (275 mg/liter). Samples were collected from the model during the course of the study for bacterial density determinations and drug concentration assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS). As expected, the norepinephrine and no-treatment control arms failed immediately, followed by the levofloxacin monotherapy arm, which failed slowly over time. The levofloxacin-epinephrine regimen resulted in a 2-log10 CFU reduction in bacterial density over the first 6 to 8 h of the study, which was followed by regrowth of a highly levofloxacin-resistant subpopulation (MIC, 64 mg/liter). These data demonstrate that increasing the rate of bacterial replication with a pharmaceutical product in combination with antimicrobial therapy represents an opportunity to increase the rate and magnitude of bactericidal effect.

Single-Dose, Preoperative Vitamin-D Supplementation Decreases Infection in a Mouse Model of Periprosthetic Joint Infection.

BACKGROUND: Despite recent advances, infection remains the most common etiology of arthroplasty failure. Recent work suggests that 25-hydroxyvitamin D (25D) deficiency correlates with the frequency of periprosthetic joint infection (PJI). We endeavored to examine whether 25D3 deficiency leads to increased bacterial burden in vivo in an established mouse model of PJI and, if so, whether this effect can be reversed by preoperative 25D3 supplementation. METHODS: Mice (lys-EGFP) possessing fluorescent neutrophils were fed a vitamin D3-sufficient (n = 20) or deficient (n = 40) diet for 6 weeks. A group of 25D3-deficient mice (n = 20) were "rescued" with 1 intraperitoneal dose of 25D3 at 3 days before surgery. A stainless steel implant was inserted into the knee joint and the joint space was inoculated with bioluminescent Staphylococcus aureus (1 × 10 colony forming units [CFUs]). In vivo imaging was used to monitor bacterial burden and neutrophil infiltration. Blood was drawn to confirm 25D3 levels 3 days before surgery and on postoperative days (PODs) 0 and 14. Mice were killed at POD 21, and CFUs were quantified after culture. Myeloperoxidase (MPO) and ß-N-acetylglucosaminidase (NAG) were assayed to look at neutrophil infiltration and activated tissue macrophage recruitment, respectively. RESULTS: Serum values confirmed 25D3 deficiency and repletion of the 25D3-rescued group. Bacterial bioluminescence and neutrophil fluorescence were significantly greater (p < 0.05) in the 25D3-deficient group. CFU counts from the joint tissue and implant were also significantly greater in this group (p < 0.05). Rescue treatment significantly decreased bacterial burden and neutrophil infiltration (p < 0.05). Compared with the 25D3-sufficient and 25D3-rescued groups, MPO activity was higher (p < 0.02) and NAG activity was lower (p < 0.03) in the 25D3-deficient group. CONCLUSIONS: This study demonstrated in vivo in a mouse model of PJI that (1) 25D3 deficiency results in increased bacterial burden and neutrophil infiltration, and (2) this effect can be reversed with preoperative repletion of 25D3. CLINICAL RELEVANCE: Considering that >65% of patients undergoing arthroplasty have insufficient or low levels of total 25D and that 25D levels can be replenished with ease using a U.S. Food and Drug Administration (FDA)-approved, oral 25D3 product, 25D deficiency may be an important modifiable risk factor in humans undergoing joint replacement.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Effects of High-Dose Vitamin D2 Versus D3 on Total and Free 25-Hydroxyvitamin D and Markers of Calcium Balance.

CONTEXT: Controversy persists over: 1) how best to restore low serum 25-hydroxyvitamin D (25D) levels (vitamin D2 [D2] vs vitamin D3 [D3]); 2) how best to define vitamin D status (total [protein-bound + free] vs free 25D); and 3) how best to assess the bioactivity of free 25D. OBJECTIVE: To assess: 1) the effects of D2 vs D3 on serum total and free 25D; and 2) whether change in intact PTH (iPTH) is more strongly associated with change in total vs free 25D. DESIGN: Participants previously enrolled in a D2 vs D3 trial were matched for age, body mass index, and race/ethnicity. Participants received 50 000 IU of D2 or D3 twice weekly for 5 weeks, followed by a 5-week equilibration period. Biochemical assessment was performed at baseline and at 10 weeks. SETTING AND PARTICIPANTS: Thirty-eight adults (19 D2 and 19 D3) ≥18 years of age with baseline 25D levels <30 ng/mL were recruited from an academic ambulatory osteoporosis clinic. OUTCOME MEASURES: Serum measures were total 25D, free 25D (directly measured), 1,25-dihydroxyvitamin D, calcium, and iPTH. Urine measure was fasting calcium:creatinine ratio. RESULTS: Baseline total (22.2 ± 3.3 vs 23.3 ± 7.2 ng/mL; P = .5) and free (5.4 ± 0.8 vs 5.3 ± 1.7 pg/mL; P = .8) 25D levels were similar between D2 and D3 groups. Increases in total (+27.6 vs +12.2 ng/mL; P = .001) and free (+3.6 vs +6.2 pg/mL; P = .02) 25D levels were greater with D3 vs D2. Percentage change in iPTH was significantly associated with change in free (but not total) 25D, without and with adjustment for supplementation regimen, change in 1,25-dihydroxyvitamin D, and change in calcium. CONCLUSIONS: D3 increased total and free 25D levels to a greater extent than D2. Free 25D may be superior to total 25D as a marker of vitamin D bioactivity.

Success of Standard Dose Vitamin D Supplementation in Treated Human Immunodeficiency Virus Infection.

Background. Vitamin D insufficiency is prevalent in human immunodeficiency virus-positive (HIV+) persons. Human immunodeficiency virus and antiretroviral therapy (ART) may create unique risk factors, and the optimal vitamin D repletion and maintenance regimen in HIV+ persons remains unclear. Methods. Human immunodeficiency virus-positive adults on suppressive ART underwent routine serum 25-hydroxyvitamin D (25OHD) screening. Persons with vitamin D insufficiency (25OHD <30 ng/mL) received open-label, oral vitamin D3 50 000 international units (IU) twice weekly for 5 weeks, then 2000 IU daily to complete 12 weeks. We predicted 70% (95% confidence interval, 60%-80%) repletion to 25OHD ≥30 ng/mL compared with 85% among historical HIV-negative controls. Eighty participants provided 91% power to detect this difference. Ability to maintain 25OHD ≥30 ng/mL after 24 weeks was also assessed. Results. Baseline characteristics were similar between the 82 vitamin D insufficient and 40 sufficient persons enrolled: 95% male, 60% white, 88% nonsmokers, median age 49 years, body mass index 26 kg/m(2), and CD4(+) T lymphocyte count 520 cells/mm(3). After 12 weeks, 81% (66 of 82) of insufficient persons achieved 25OHD ≥30 ng/mL (P = .32 vs historical controls), with only older age (odds ratio [OR] = 1.06; P = .06), higher baseline 25OHD (OR = 1.14; P < .01), white race (OR = 3.39; P = .04), and current smoking (OR = 0.25; P = .06) associated with successful repletion. After 24 weeks, 73% (48 of 66) maintained 25OHD ≥30 ng/mL, with tenofovir (OR = 5.00; P = .01) and abacavir use (OR = 0.23; P = .02) associated with success and failure, respectively, to maintain 25OHD levels. Conclusions. The 25OHD repletion rates were comparable between HIV+ adults on suppressive ART and historical HIV-negative controls, indicating that successful oral repletion can be achieved in this population.

NELL-1 in the treatment of osteoporotic bone loss.

NELL-1 is a secreted, osteoinductive protein whose expression rheostatically controls skeletal ossification. Overexpression of NELL-1 results in craniosynostosis in humans and mice, whereas lack of Nell-1 expression is associated with skeletal undermineralization. Here we show that Nell-1-haploinsufficient mice have normal skeletal development but undergo age-related osteoporosis, characterized by a reduction in osteoblast:osteoclast (OB:OC) ratio and increased bone fragility. Recombinant NELL-1 binds to integrin ß1 and consequently induces Wnt/ß-catenin signalling, associated with increased OB differentiation and inhibition of OC-directed bone resorption. Systemic delivery of NELL-1 to mice with gonadectomy-induced osteoporosis results in improved bone mineral density. When extended to a large animal model, local delivery of NELL-1 to osteoporotic sheep spine leads to significant increase in bone formation. Altogether, these findings suggest that NELL-1 deficiency plays a role in osteoporosis and demonstrate the potential utility of NELL-1 as a combination anabolic/antiosteoclastic therapeutic for bone loss.