Challenges to Achieving HCV Micro-Elimination in People With HIV in the United States: Provider Perspectives and the Role of Implicit Bias.

The prevalence of HIV/HCV (hepatitis C virus) co-infection is high particularly in persons who inject drugs (PWID) and is increasing because of the evolving opioid epidemic in the United States. The introduction of effective antiviral medications for HCV has raised the strategic goal of HCV micro-elimination, and efforts to understand the barriers to treatment are critical. In this study, we explored the provider perspective of factors that inhibit HCV micro-elimination efforts in people with HIV (PWH), including the role of implicit bias and related stigma in providers' health care decision making. We used the mixed-methods approach of nominal group technique (NGT) with 14 participants from 11 different clinics engaged in two virtual focus group sessions (n = 5 and n = 9). Responses from the NGTs were rank ordered during the sessions to identify providers' perspectives of major barriers and facilitators, then identified possible implicit bias after the NGTs concluded. There were 12 responses given for micro-elimination barriers with the three most prioritized being housing instability, medication nonadherence concerns, and inability to motivate patients. Of these, eight were categorized as potential implicit biases. Among the 14 responses given for facilitators of treatment, the three major solutions included distributive models of care, improved provider knowledge, and increased patient engagement. Although the solutions offered were insightful, there was consensus that the individual lives of patients were the root cause of most barriers to care. We recommend further research on behavioral design interventions that promote patients' involvement in decision making and focus on patients' eligibility criteria for HCV treatment as opposed to providers' perceived barriers to treatment.

Serum Amylase and Lipase Estimation in Diabetic Ketoacidosis.

Diabetic ketoacidosis is one of the hyperglycemic emergencies, there is insulin deficiency coupled with concomitant elevation of counter regulatory hormones. This hormonal imbalance promotes gluconeogenesis, glycolysis, glycogenolysis, protein breakdown and lipolysis.The symptoms of DKA like nausea, vomiting, epigastric pain can be present in acute pancreatitis also. From various studies it has been identified that in DKA, non specific elevation of serum amylase and lipase levels occurs in 16-25% of cases. Elevation of serum amylase, and lipase levels in association with severe abdominal pain often trigger the initial diagnosis of acute pancreatitis. So this study was carried out to study the elevation of serum amylase and lipase levels in patients with DKA. MATERIAL: This cross sectional study was conducted in department of medicine KR Hospital,Mysore medical college and research institute, mysore during the study period of six months from June 2021 to november 2021. A total of 50 patients were included in the study after fulfilling the inclusion and exclusion criteria. OBSERVATION: Among 50 cases studied, 9 cases (18%) with DKA are showing elevation of serum amylase levels and 13 cases(26%) of cases are showing elevation of serum lipase,34 cases(68%) were males and 16 cases(32%) were female. Among the 50 cases studied,infection is the most precipitating factor seen in 34cases (68%),followed by omission of insulin in 12 cases(24%), unidentified cause in 4 cases(8%). CONCLUSION: significant elevation of serum amylase and serum lipase which are more specific for diagnosis of acute pancreatitis can also be seen in patients with diabetic ketoacidosis. Elevated serum amylase and lipase can occur in patients with DKA probably due to metabolic derangements,decreased clearance of enzymes and not due to acute pancreatitis The clinician must take these data into account when evaluating abdominal symptoms in DKA patients.

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Use of Pre-exposure Prophylaxis Among People Who Inject Drugs: Exploratory Findings of the Interaction Between Race, Homelessness, and Trust.

Scale-up of pre-exposure prophylaxis (PrEP) in people who inject drugs (PWID) remains suboptimal. Patient-level factors are often complex and may contribute to scale-up. Using cross-sectional data from 234 opioid-dependent patients on methadone who met eligibility criteria for PrEP, we conducted logistic regression analyses to assess potential moderators associated with being on PrEP (n = 60). Mean provider trust was significantly higher among Blacks vs Whites (39.4 vs 34.9; p = 0.002) and non-homeless vs homeless participants (37.5 vs 34.8; p = 0.008). Though race/ethnicity was not a significant moderator on provider trust and PrEP use, increased provider trust was marginally associated with increased PrEP use among Blacks (p = 0.058). Additionally, homelessness significantly moderated provider trust and PrEP use (p = 0.024). Provider trust among non-homeless participants was positively correlated with PrEP use (p = 0.013) but not among homeless participants. Strategies that promote provider trust in Blacks and non-homeless PWID on methadone may improve PrEP scale-up.

Engagement in Harm Reduction Strategies After Suspected Fentanyl Contamination Among Opioid-Dependent Individuals.

The evolving opioid epidemic in the United States has increased drug-related overdose rates exponentially (Centers for Disease Control and Prevention in Opioid overdose, 2020c, https://www.cdc.gov/drugoverdose/data/otherdrugs.html#:~:text=Polysubstance%20drug%20use%20occurs%20with,or%20other%20non%2Dopioid%20substances ). Fentanyl, a synthetic opioid, has recently fueled the epidemic, increasing overdose death rates (Centers for Disease Control and Prevention in Drug overdose deaths involving fentanyl, 2011-2016, 2019a, https://www.cdc.gov/nchs/data/nvsr/nvsr68/nvsr68_03-508.pdf ). Harm reduction strategies (drug checking, naloxone administration, etc.) are at the forefront of preventing opioid-related overdoses in high-risk populations (Kennedy et al. in Drug Alcohol Depend 185:248-252, 2018, https://doi.org/10.1016/j.drugalcdep.2017.12.026 ; Laing et al. in Int J Drug Policy 62:59-66, 2018, https://doi.org/10.1016/j.drugpo.2018.10.001 ). Little is known, however, about how people who inject drugs (PWID) may modify their drug use behaviors after suspected fentanyl contamination in their drugs. We conducted a cross-sectional survey among 105 opioid-dependent PWID enrolled in a methadone maintenance program. We assessed their willingness to engage in various harm reduction methods (i.e., slowing down drug use, not using drugs, carrying naloxone, using with someone who has naloxone) after suspected fentanyl contamination of their drugs. In a multivariable analysis, participants who were white, low-income, polysubstance users, and had previously experienced an overdose or had previously administered naloxone were more likely to report a willingness to engage in harm reduction measures. These findings provide an evidence-based understanding of PWID's engagement in harm reduction behaviors after suspecting potential fentanyl exposure as well as a basis for tailoring intervention strategies in the context of fentanyl-adulterated markets.

Purposeful Fentanyl Use and Associated Factors among Opioid-Dependent People Who Inject Drugs.

Introduction: In recent years, opioid-related mortality has increased steadily in the United States. Fentanyl, a synthetic opioid, has been a primary driver of the current wave of overdose-related deaths. Little is known, however, about fentanyl use among opioid-dependent people who inject drugs (PWID). We, therefore, sought to characterize purposeful fentanyl use among PWID. Methods: A cross-sectional survey was administrated to 104 opioid-dependent PWID on methadone who self-reported drug- or sex-related risk behaviors. Participants were recruited between July 2018 and October 2019 from a methadone clinic in New Haven, Connecticut. Multivariable logistic regressions were used to identify independent correlates of purposeful fentanyl use. Results: Overall, 43.2% and 22.1% of the participants reported any (lifetime) or recent (past 30 days) purposeful fentanyl use, respectively. Independent correlates of any purposeful fentanyl use were younger age (aOR= 0.94; p = 0.021), recent daily injection (aOR= 3.52; p = 0.047), recent cocaine use (aOR= 3.54; p = 0.041), and moderate/severe depression (aOR= 3.82; p = 0.028). Independent correlates of recent purposeful fentanyl use were moderate/severe depression (aOR= 10.94; p = 0.031), recently sharing injection equipment (aOR= 2.96; p = 0.044), and recently engaging in transactional sex (aOR= 3.60; p = 0.026). Conclusions: These findings indicate that opioid-dependent PWID on methadone remain at increased risk for overdose given the high prevalence of ongoing purposeful fentanyl exposure. Interventions to reduce the harms associated with fentanyl use should target young PWID and active fentanyl users, with an emphasis on achieving adequate methadone dosage and screening and treating depression in methadone-maintained patients who preferentially seek fentanyl.

Highland games: A benchmarking exercise in predicting biophysical and drug properties of monoclonal antibodies from amino acid sequences.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Design of affinity peptides from natural protein ligands: A study of the cardiac troponin complex.

We describe a general strategy for the design and discovery of affinity peptides for a protein from its natural ligands. Our approach is guided by protein-protein interactions in natural systems and focuses on the hetero-trimeric complex of cardiac troponin I (cTnI), C (cTnC) and T (cTnT). A key premise of this work is that cTnC and cTnT, owing to their innate ability to bind cTnI, are potential templates for the design and discovery of cTnI-binding peptides. Relying only on the knowledge of primary sequences of cTnC and cTnT, we designed a library of short overlapping peptides that span the entirety of cTnC and cTnT and tested them for binding to cTnI. We were successful in identifying several peptides that display high affinity (1-100 nM) for cTnI. The specific implication of this work is that mimicking natural protein-protein interactions is an excellent starting point for the discovery and rational design of peptide ligands. The knowledge of secondary or tertiary structures of the proteins involved is not a necessary precondition for this approach. Nevertheless, we show that structural information can be used to validate the results of a fragment-based peptide design, and can be potentially beneficial for refining the lead candidates. Our approach is broadly applicable to any protein with at least one natural binding ligand with known primary sequence. For protein targets with multiple natural ligands, this approach can potentially yield several distinct affinity peptides capable of simultaneously binding the target protein via orthogonal modes or at complementary interfaces.

Design of peptide affinity ligands for S-protein: a comparison of combinatorial and de novo design strategies.

Design of peptide affinity ligands against biological targets is important for a broad range of applications. Here, we report on de novo and combinatorial strategies for the design of high-affinity and high-specificity peptides against S-protein as a target. The peptide libraries employed in this study contain (1) consensus motif (CM) sequences identified from high-throughput phage combinatorial screening, (2) point mutations of CM sequences, and (3) de novo sequences rationally designed based on stereo-chemical information of the complex between S-protein and its natural ligand, S-peptide. In general, point mutations to CM allowed for modulating peptide affinity and specificity over a broad range. This is particularly useful in designing peptides with varying affinities and specificities for the target. De novo sequences, especially those based on the S-protein binding pocket, on average bound with higher affinities within a narrow range (10-100 nM) as compared to point mutations to CM (1 nM-2 µM). As such, the approaches described here serve as a general guide for optimizing the design of peptide affinity ligands for a wide range of target proteins or applications.

Structural barriers to implementing recommended tuberculosis preventive treatment in primary care clinics in rural South Africa.

The World Health Organization (WHO) recommends tuberculosis preventive treatment (TPT) in people with HIV (PWH), yet implementation remains poor, especially in rural communities. We examined factors influencing TPT initiation in PWH on antiretroviral therapy (ART) in rural South Africa using the Promoting Action on Research Implementation in Health Services (PARiHS) framework to identify contextual factors and facilitation strategies to successfully implement TPT. Patient and clinical factors were extracted from medical records at two primary healthcare clinics (PHCs). Among 455 TPT eligible indivdiuals, only 263 (57.8%) initiated TPT. Patient-level characteristics (older age and symptoms of fever or weight loss) were significantly associated with TPT initiation in bivariate analysis, but PHC was the only independent correlate of TPT initiation (aOR: 2.24; 95% CI: 1.49-3.38). Clinic-level factors are crucial targets for implementing TPT to reduce the burden of HIV-associated TB. Gaps in knowledge of HCW, staff shortages, and non-integrated HIV/TB services were identified barriers to TPT implementation. Evidence-based strategies for facilitating TPT implementation that might be under-prioritized include ongoing reprioritization, expanding training for primary care providers, and quality improvement strategies (organisational changes, multidisciplinary teams, and monitoring and feedback). Addressing contextual barriers through these facilitation strategies may improve future TPT implementation in this setting.

Purification of proteins using peptide-ELP based affinity precipitation.

In this work, we employed fusions of affinity peptides and elastin-like polypeptide (ELP) to carry out a proof-of-concept study for the single-step purification of model, tag-free proteins. Three known peptide-protein binding pairs of varied binding strengths were evaluated. The peptide-protein binding was first characterized through in-solution binding techniques such as fluorescence spectroscopy. Peptide-ELP constructs were then produced in E. coli and purified by phase transition. The binding of the peptide-ELP constructs to the products were then evaluated using competitive fluorescence spectroscopy. Affinity capture, precipitation and recovery experiments were then conducted using the three constructs to evaluate the efficacy of these peptide-ELP based affinity precipitation processes. Two out of the three systems tested were successful in capturing the product and yielding pure protein in a single affinity precipitation step. These results indicated that peptide-protein affinity played an important role in both the effective capture of the protein, and also in the required binding molar ratio for the affinity precipitation process. In addition, for intermediate affinity systems, constructs containing two copies of the peptide at the N-terminus of the ELP were beneficial towards achieving both high purity and yield, likely due to increased affinity from avidity effects. This proof-of-concept study lays the foundations for the development of new peptide-ELP-based affinity purification processes for industrially relevant proteins and other classes of biologics.

Retention in clinical trials after prison release: results from a clinical trial with incarcerated men with HIV and opioid dependence in Malaysia.

Background: Study retention is a major challenge in HIV clinical trials conducted with persons recruited from correctional facilities. Objective: To examine study retention in a trial of within-prison methadone initiation and a behavioral intervention among incarcerated men with HIV and opioid dependence in Malaysia. Methods: In this 2x2 factorial trial, 296 incarcerated men with HIV and opioid dependence were allocated to (1) an HIV risk reduction intervention, the Holistic Health Recovery Program for Malaysia (HHRP-M), (2) pre-release methadone initiation, (3) both interventions, or (4) standard care (NCT02396979). Here we estimate effects of these interventions on linkage to the study after prison release and completion of post-release study visits. Results: Most participants (68.9%) completed at least one post-release study visit but few (18.6%) completed all 12. HHRP-M was associated with a 13.5% (95% confidence interval (CI): 3.8%, 23.2%) increased probability of completing at least one post-release study visit. Although not associated with initial linkage, methadone treatment was associated with an 11% (95% CI: 2.0%, 20.6%) increased probability of completing all twelve post-release study visits. Being subject to forced relocation outside Kuala Lumpur after prison release decreased retention by 43.3% (95% CI: -51.9%, -34.8%). Conclusion: Retaining study participants in HIV clinical trials following prison release is challenging and potentially related to the broader challenges that participants experience during community reentry. Researchers conducting clinical trials with this population may want to consider methadone and HHRP as means to improve post-release retention, even in clinical trials where these interventions are not being directly evaluated.

All-cause mortality among people with HIV released from an integrated system of jails and prisons in Connecticut, USA, 2007-14: a retrospective observational cohort study.

BACKGROUND: People transitioning from prisons or jails have high mortality, but data are scarce for people with HIV and no studies have integrated data from both criminal justice and community settings. We aimed to assess all-cause mortality in people with HIV released from an integrated system of prisons and jails in Connecticut, USA. METHODS: We linked pharmacy, custodial, death, case management, and HIV surveillance data from Connecticut Departments of Correction and Public Health to create a retrospective cohort of all adults with HIV released from jails and prisons in Connecticut between 2007 and 2014. We compared the mortality rate of adults with HIV released from incarceration with the general US and Connecticut populations, and modelled time-to-death from any cause after prison release with Cox proportional hazard models. FINDINGS: We identified 1350 people with HIV who were released after 24 h or more of incarceration between 2007 and 2014, of whom 184 (14%) died after index release; median age was 45 years (IQR 39-50) and median follow-up was 5·2 years (IQR 3·0-6·7) after index release. The crude mortality rate for people with HIV released from incarceration was 2868 deaths per 100 000 person-years, and the standardised mortality ratio showed that mortality was higher for this cohort than the general US population (6·97, 95% CI 5·96-7·97) and population of Connecticut (8·47, 7·25-9·69). Primary cause of death was reported for 170 individuals; the most common causes were HIV/AIDS (78 [46%]), drug overdose (26 [15%]), liver disease (17 [10%]), cardiovascular disease (16 [9%]), and accidental injury or suicide (13 [8%]). Black race (adjusted hazard ratio [HR] 0·52, 95% CI 0·34-0·80), having health insurance (0·09, 0·05-0·17), being re-incarcerated at least once for 365 days or longer (0·41, 0·22-0·76), and having a high percentage of re-incarcerations in which antiretroviral therapy was prescribed (0·08, 0·03-0·21) were protective against mortality. Positive predictors of time-to-death were age (≥50 years; adjusted HR 3·65, 95% CI 1·21-11·08), lower CD4 count (200-499 cells per µL, 2·54, 1·50-4·31; <200 cells per µL, 3·44, 1·90-6·20), a high number of comorbidities (1·86, 95% CI 1·23-2·82), virological failure (2·76, 1·94-3·92), and unmonitored viral load (2·13, 1·09-4·18). INTERPRETATION: To reduce mortality after release from incarceration in people with HIV, resources are needed to identify and treat HIV, in addition to medical comorbidities, psychiatric disorders, and substance use disorders, during and following incarceration. Policies that reduce incarceration and support integrated systems of care between prisons and communities could have a substantial effect on the survival of people with HIV. FUNDING: US National Institutes of Health.

Nanofluidic device for continuous multiparameter quality assurance of biologics.

Process analytical technology (PAT) is critical for the manufacture of high-quality biologics as it enables continuous, real-time and on-line/at-line monitoring during biomanufacturing processes. The conventional analytical tools currently used have many restrictions to realizing the PAT of current and future biomanufacturing. Here we describe a nanofluidic device for the continuous monitoring of biologics' purity and bioactivity with high sensitivity, resolution and speed. Periodic and angled nanofilter arrays served as the molecular sieve structures to conduct a continuous size-based analysis of biologics. A multiparameter quality monitoring of three separate commercial biologic samples within 50 minutes has been demonstrated, with 20 µl of sample consumption, inclusive of dead volume in the reservoirs. Additionally, a proof-of-concept prototype system, which integrates an on-line sample-preparation system and the nanofluidic device, was demonstrated for at-line monitoring. Thus, the system is ideal for on-site monitoring, and the real-time quality assurance of biologics throughout the biomanufacturing processes.

Increased expression of activating factors in large osteoclasts could explain their excessive activity in osteolytic diseases.

Large osteoclasts (>or=10 nuclei) predominate at sites of pathological bone resorption. We hypothesized this was related to increased resorptive activity of large osteoclasts and have demonstrated previously that larger osteoclasts are 8-fold more likely to be resorbing than small osteoclasts (2-5 nuclei). Here we ask whether these differences in resorptive activity can be explained by differences in expression of factors involved in osteoclast signaling, fusion, attachment, and matrix degradation. Authentic rabbit osteoclasts and osteoclasts derived from RAW264.7 cells showed similar increases in c-fms expression (1.7- to 1.8-fold) in large osteoclasts suggesting that RAW cells are a viable system for further analysis. We found 2- to 4.5-fold increases in the expression of the integrins alpha(v) and beta(3), the proteases proMMP9, matMMP9 and pro-cathepsinK, and in activating receptors RANK, IL-1R1, and TNFR1 in large osteoclasts. In contrast, small osteoclasts had higher expression of the fusion protein SIRPalpha1 and the decoy receptor IL-1R2. The higher expression of activation receptors and lower expression of IL-1R2 in large osteoclasts suggest they are hyperresponsive to extracellular factors. This is supported by the observation that the resorptive activity in large osteoclasts was more responsive to IL-1beta, and that this increased activity was inhibited by the IL-1 receptor antagonist, IL-1ra. This increased responsiveness of large osteoclasts to IL-1 may, in part, explain the pathological bone loss noted in inflammatory diseases. The heterogeneity in receptor expression and the differential response to cytokines and their antagonists could prove useful for selective inhibition of large osteoclasts actively engaged in pathological bone loss.