Orthogonal IMiD-Degron Pairs Induce Selective Protein Degradation in Cells.

Immunomodulatory imide drugs (IMiDs) including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short zinc finger (ZF) degron motif. These IMiDs, however, also induce degradation of endogenous neosubstrates, including IKZF1 and IKZF3. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogs against 8380 ZF mutants. This yielded a bumped IMiD analog that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1 and IKZF3. In proof-of-concept studies, this system was applied to induce efficient degradation of TRIM28, a disease-relevant protein with no known small molecule binders. We anticipate that this system will make a valuable addition to the current arsenal of degron systems for use in target validation.

TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Pla2g5 contributes to viral-like-induced lung inflammation through macrophage proliferation and LA/Ffar1 lung cell recruitment.

Macrophages expressing group V phospholipase A2 (Pla2g5) release the free fatty acid (FFA) linoleic acid (LA), potentiating lung type 2 inflammation. Although Pla2g5 and LA increase in viral infections, their role remains obscure. We generated Pla2g5flox/flox mice, deleted Pla2g5 by using the Cx3cr1cre transgene, and activated bone marrow-derived macrophages (BM-Macs) with poly:IC, a synthetic double-stranded RNA that triggers a viral-like immune response, known Pla2g5-dependent stimuli (IL-4, LPS + IFNγ, IL-33 + IL-4 + GM-CSF) and poly:IC + LA followed by lipidomic and transcriptomic analysis. Poly:IC-activated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs had downregulation of major bioactive lipids and critical enzymes producing those bioactive lipids. In addition, AKT phosphorylation was lower in poly:IC-stimulated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs, which was not restored by adding LA to poly:IC-stimulated BM-Macs. Consistently, Pla2g5flox/flox;Cx3cr1cre/+ mice had diminished poly:IC-induced lung inflammation, including inflammatory macrophage proliferation, while challenging Pla2g5flox/flox;Cx3cr1cre/+ mice with poly:IC + LA partially restored lung inflammation and inflammatory macrophage proliferation. Finally, mice lacking FFA receptor-1 (Ffar1)-null mice had reduced poly:IC-induced lung cell recruitment and tissue macrophage proliferation, not corrected by LA. Thus, Pla2g5 contributes to poly:IC-induced lung inflammation by regulating inflammatory macrophage proliferation and LA/Ffar1-mediated lung cell recruitment and tissue macrophage proliferation.

PPARγ agonist treatment reduces fibroadipose tissue in secondary lymphedema by exhausting fibroadipogenic PDGFRα+ mesenchymal cells.

Secondary lymphedema occurs in up to 20% of patients after lymphadenectomy performed for the surgical management of tumors involving the breast, prostate, uterus, and skin. Patients develop progressive edema of the affected extremity due to retention of protein-rich lymphatic fluid. Despite compression therapy, patients progress to chronic lymphedema in which noncompressible fibrosis and adipose tissue are deposited within the extremity. The presence of fibrosis led to our hypothesis that rosiglitazone, a PPARγ agonist that inhibits fibrosis, would reduce fibrosis in a mouse model of secondary lymphedema after hind limb lymphadenectomy. In vivo, rosiglitazone reduced fibrosis in the hind limb after lymphadenectomy. Our findings verified that rosiglitazone reestablished the adipogenic features of TGF-ß1-treated mesenchymal cells in vitro. Despite this, rosiglitazone led to a reduction in adipose tissue deposition. Single-cell RNA-Seq data obtained from human tissues and flow cytometric and histological evaluation of mouse tissues demonstrated increased presence of PDGFRα+ cells in lymphedema; human tissue analysis verified these cells have the capacity for adipogenic and fibrogenic differentiation. Upon treatment with rosiglitazone, we noted a reduction in the overall quantity of PDGFRα+ cells and LipidTOX+ cells. Our findings provide a framework for treating secondary lymphedema as a condition of fibrosis and adipose tissue deposition, both of which, paradoxically, can be prevented with a pro-adipogenic agent.

COVID-19 vaccine uptake before and after a vaccine mandate at a major academic hospital: Trends by race/ethnicity and level of patient contact.

OBJECTIVES: To assess vaccine coverage rates before and after implementation of a COVID-19 vaccine mandate among Health care Personnel (HCP) and demographic characteristics associated with vaccine uptake Design, Setting, and Participants: Cohort study conducted among 10,889 hospital employees followed from Dec 16, 2020 - October 31, 2021, at a large academic hospital in Philadelphia. MAIN OUTCOME AND MEASURES: Time to COVID-19 vaccination and vaccine series completion rates before and after implementation of a COVID-19 vaccine mandate based on age, gender, race/ethnicity, and level of patient contact/occupational group. RESULTS: The vaccination series completion rate was 86.0% prior to mandate announcement, and increased to 98.7% after mandate implementation. Rates before mandate announcement were highest among Asians (96.2%), Whites (94.0%), males (89.7%), employees ≥ 65 years of age (95.2%), and employees with direct patient care (physicians, 99.0%, and nurses, 93.3%). Hospital educational initiatives (including Town Halls and discussions with Black and Hispanic employees with the lowest vaccination rates) appeared to improve uptake. The largest increase in series completion after mandate announcement occurred among Blacks, those of other/multiracial backgrounds, and Hispanics (35.6%, 22.4%, and 10.8%, respectively) as well as those with some or no direct patient contact (24.5% and 18.3%, respectively). Medical or religious exemptions were approved for 64 (<0.6%) employees and 38 (<0.4%) left their positions (8 voluntary, 30 involuntary) specifically due to the COVID-19 vaccine mandate. No clinically meaningful differences by age, gender, or race/ethnicity for those who were vaccinated under the mandate versus those who left their positions were noted. CONCLUSIONS AND RELEVANCE: These results suggest that while mandates may be challenging to institutions and enforcement unpopular, they play an important role in reducing hesitancy and securing high vaccination rates among HCP, a group at high risk of COVID-19 given their employment and who can be a source of disease transmission to patients.

Early-life fecal metabolomics of food allergy.

BACKGROUND: Intestinal microenvironmental perturbations may increase food allergy risk. We hypothesize that children with clinical food allergy, those with food sensitization, and healthy children can be differentiated by intestinal metabolites in the first years of life. METHODS: In this ancillary analysis of the Vitamin D Antenatal Asthma Reduction Trial (VDAART), we performed untargeted metabolomic profiling in 824 stool samples collected at ages 3-6 months, 1 year and 3 years. Subjects included 23 with clinical food allergy at age 3 and/or 6 years, 151 with food sensitization but no clinical food allergy, and 220 controls. We identified modules of correlated, functionally related metabolites and sought associations of metabolite modules and individual metabolites with food allergy/sensitization using regression models. RESULTS: Several modules of functionally related intestinal metabolites were reduced among subjects with food allergy, including bile acids at ages 3-6 months and 1 year, amino acids at age 3-6 months, steroid hormones at 1 year, and sphingolipids at age 3 years. One module primarily containing diacylglycerols was increased in those with food allergy at age 3-6 months. Fecal caffeine metabolites at age 3-6 months, likely derived from breast milk, were increased in those with food allergy and/or sensitization (beta = 5.9, 95% CI 1.0-10.8, p = .02) and were inversely correlated with fecal bile acids and bilirubin metabolites, though maternal plasma caffeine levels were not associated with food allergy and/or sensitization. CONCLUSIONS: Several classes of bioactive fecal metabolites are associated with food allergy and/or sensitization including bile acids, steroid hormones, sphingolipids, and caffeine metabolites.

Patient Characteristics Associated With Reactions to Mrgprx2-Activating Drugs in an Electronic Health Record-Linked Biobank.

BACKGROUND: Mas-related G protein-couple receptor x2 (Mrgprx2) activation underlies many common non-IgE-mediated adverse drug reactions (ADRs), yet the features of patients with reactions to Mrgprx2-activating drugs are unknown. OBJECTIVE: To characterize the patient-specific comorbidities and laboratory characteristics associated with listed reactions to Mrgprx2-activating drugs, including fluoroquinolones, morphine, neuromuscular blockade agents, vancomycin, and leuprolide. METHODS: We used a retrospective, observational cohort study design using electronic health record data from adults with an Mrgprx2-activating drug exposure recorded within a hospital system clinical Biobank. Odds ratios (ORs) and incidence rate ratios for clinical characteristics associated with ADRs, including immediate hypersensitivity reactions, were calculated using multivariable logistic regression. RESULTS: Among 59,763 patients exposed to Mrgprx2-activating drugs, 4846 had a listed ADR. Female sex, White race, asthma (OR: 1.81, 95% confidence interval [CI]: 1.68-1.94), chronic urticaria (OR: 1.73, 95% CI: 1.46-2.05), and mastocytosis (OR: 12.79, 95% CI: 5.98-27.02) were associated with increased odds of a reaction. Overall, patients with allergic disease had 1.21 times the rate of an ADR compared with patients without allergic disease. Elevated absolute eosinophil count was inversely associated with reactions, and there was no association with elevated total IgE. Observed associations were similar in a patient subgroup with immediate-type hypersensitivity reactions. CONCLUSION: Specific allergic diseases and common allergic biomarkers are differentially associated with ADRs to Mrgprx2-activating drugs. These findings from a large, "real world" drug-exposed population highlight clinical factors that may contribute to non-IgE-mediated drug allergy.

Durability of Severe Acute Respiratory Syndrome Coronavirus 2 Messenger RNA Booster Vaccine Protection Against Omicron Among Healthcare Workers With a Vaccine Mandate.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread rapidly throughout the world since being identified in South Africa in November 2021. Few studies have assessed primary series and booster vaccine effectiveness against Omicron among US healthcare workers. METHODS: We conducted a test-negative case-control design to estimate BNT162b2 and mRNA1273 primary vaccination and booster effectiveness against SARS-CoV-2 infection and symptomatic coronavirus disease 2019 during an Omicron surge among employees of the University of Pennsylvania Health System. The study period was between 1 July 2021 and 5 April 2022. We defined the Delta period as 1 July to 12 December 2021 and the Omicron period as beginning 12 December 21. RESULTS: Our sample included 14 520 tests (2776 [19%] positive)-7422 (506 [7%] positive) during Delta and 7098 (2270 [32%] positive) during Omicron. Benchmarked against Delta, the vaccine effectiveness of 2 vaccine doses was lower during Omicron, with no significant protection against infection. Booster doses added significant protection, although they also showed reduced effectiveness during Omicron. Compared with findings in employees who had received 2 vaccine doses, 3 doses of BNT162b2 had a relative effectiveness of 50% (95% confidence interval, 42%-56%) during Omicron, relative to 78% (63%-87%) during Delta; 3 doses of mRNA1273 had a relative effectiveness of 56% (45%-65%) during Omicron, relative to 96% (82%-99%) during Delta. Restricting the sample to symptomatic tests yielded similar results to our primary analysis. After initial waning in BNT162b2 booster protection against infection, it remained largely stable for ≥16 weeks after vaccination. CONCLUSIONS: Our findings provide a strong rationale for boosters among healthcare workers in the Omicron era.

Providing Remote Aid During a Humanitarian Crisis.

Humanitarian crises create opportunities for both in-person and remote aid. Durable, complex, and team-based care may leverage a telemedicine approach for comprehensive support within a conflict zone. Barriers and enablers are detailed, as is the need for mission expansion due to initial program success. Adapting a telemedicine program initially designed for critical care during the severe acute respiratory syndrome coronavirus 2 pandemic offers a solution to data transfer and data analysis issues. Staffing efforts and grouped elements of patient care detail the kinds of remote aid that are achievable. A multiprofessional team-based approach (clinical, administrative, nongovernmental organization, government) can provide comprehensive consultation addressing surgical planning, critical care management, infection and infection control management, and patient transfer for complex care. Operational and network security create parallel concerns relevant to avoid geolocation and network intrusion during consultation. Deliberate approaches to address cultural differences that influence relational dynamics are also essential for mission success.

Community-Based COVID-19 Vaccine Clinics in Medically Underserved Neighborhoods to Improve Access and Equity, Philadelphia, 2021-2022.

Vaccination remains key to reducing the risk of COVID-19-related severe illness and death. Because of historic medical exclusion and barriers to access, Black communities have had lower rates of COVID-19 vaccination than White communities. We describe the efforts of an academic medical institution to implement community-based COVID-19 vaccine clinics in medically underserved neighborhoods in Philadelphia, Pennsylvania. Over a 13-month period (April 2021-April 2022), the initiative delivered 9038 vaccine doses to community members, a majority of whom (57%) identified as Black. (Am J Public Health. 2022;112(12):1721-1725. https://doi.org/10.2105/AJPH.2022.307030).

Understanding Contributors to Racial/Ethnic Disparities in Emergency Department Throughput Times: a Sequential Mixed Methods Analysis.

BACKGROUND: Ensuring equitable care remains a critical issue for healthcare systems. Nationwide evidence highlights the persistence of healthcare disparities and the need for research-informed approaches for reducing them at the local level. OBJECTIVE: To characterize key contributors in racial/ethnic disparities in emergency department (ED) throughput times. DESIGN: We conducted a sequential mixed methods analysis to understand variations in ED care throughput times for patients eventually admitted to an emergency department at a single academic medical center from November 2017 to May 2018 (n=3152). We detailed patient progression from ED arrival to decision to admit and compared racial/ethnic differences in time intervals from electronic medical record time-stamp data. We then estimated the relationships between race/ethnicity and ED throughput times, adjusting for several patient-level variables and ED-level covariates. These quantitative analyses informed our qualitative study design, which included observations and semi-structured interviews with patients and physicians. KEY RESULTS: Non-Hispanic Black as compared to non-Hispanic White patients waited significantly longer during the time interval from arrival to the physician's decision to admit, even after adjustment for several ED-level and patient demographic, clinical, and socioeconomic variables (Beta (average minutes) (SE): 16.35 (5.8); p value=.005). Qualitative findings suggest that the manner in which providers communicate, advocate, and prioritize patients may contribute to such disparities. When the race/ethnicity of provider and patient differed, providers were more likely to interrupt patients, ignore their requests, and make less eye contact. Conversely, if the race/ethnicity of provider and patient were similar, providers exhibited a greater level of advocacy, such as tracking down patient labs or consultants. Physicians with no significant ED throughput disparities articulated objective criteria such as triage scores for prioritizing patients. CONCLUSIONS: Our findings suggest the importance of (1) understanding how our communication style and care may differ by race/ethnicity; and (2) taking advantage of structured processes designed to equalize care.

Transparency, Public Reporting, and a Culture of Change to Quality and Safety in Cardiac Surgery.

Academic medical centers have a duty to serve as hospitals of last resort for advanced cardiac surgical care and therefore manage patients at elevated risk of postoperative morbidity and mortality. They must also meet state and professional quality targets devised to protect the public. The tension between these imperatives can be managed by a multidimensional quality improvement program that aims to manage risk, optimize outcomes, and exclude futile operations. We here share our approach to this process, its impact on our institution, and discuss pertinent issues relevant to institutions in a similar situation.

Factors Associated With COVID-19 Vaccine Receipt by Health Care Personnel at a Major Academic Hospital During the First Months of Vaccine Availability.

Importance: Several COVID-19 vaccines have been authorized in the US, yet preliminary evidence suggests high levels of vaccine hesitancy and wide racial, ethnic, and socioeconomic disparities in uptake. Objective: To assess COVID-19 vaccine acceptance among health care personnel (HCP) during the first 4 months of availability in a large academic hospital, compare acceptance with previously measured vaccine hesitancy, and describe racial, ethnic, and socioeconomic disparities in vaccine uptake. Design, Setting, and Participants: This cross-sectional study included 12 610 HCP who were offered COVID-19 vaccination at a major academic hospital in Philadelphia between December 16, 2020, and April 16, 2021. Exposures: For each HCP, data were collected on occupational category, age, sex, race and ethnicity (Asian or Pacific Islander, Black or African American [Black], Hispanic, White, and multiracial), and social vulnerability index (SVI) at the zip code of residence. Main Outcomes and Measures: Vaccine uptake by HCP at the employee vaccination clinic. Results: The study population included 4173 men (34.8%) and 7814 women (65.2%) (623 without data). A total of 1480 were Asian or Pacific Islander (12.4%); 2563 (21.6%), Black; 452 (3.8%), Hispanic; 7086 (59.6%), White; and 192 (1.6%), multiracial; 717 had no data for race and ethnicity. The mean (SD) age was 40.9 (12.4) years, and 9573 (76.0%) received at least 1 vaccine dose during the first 4 months of vaccine availability. Adjusted for age, sex, job position, and SVI, Black (relative risk [RR], 0.69; 95% CI, 0.66-0.72) and multiracial (RR, 0.80; 95% CI, 0.73-0.89) HCP were less likely to receive vaccine compared with White HCP. When stratified by job position, Black nurses (n = 189; 62.8%), Black HCP with some patient contact (n = 466; 49.9%), and Black HCP with no patient contact (n = 636; 56.3%) all had lower vaccine uptake compared with their White and Asian or Pacific Islander counterparts. Similarly, multiracial HCP with some (n = 26; 52.0%) or no (n = 48; 58.5%) patient contact had lower vaccine uptake. In contrast, Black physicians were just as likely to receive the vaccine as physicians of other racial and ethnic groups. Conclusions and Relevance: In this cross-sectional study, more than two-thirds of HCP at a large academic hospital in Philadelphia received a COVID-19 vaccine within 4 months of vaccine availability. Although racial, ethnic, and socioeconomic disparities were seen in vaccine uptake, no such disparities were found among physicians. Better understanding of factors driving these disparities may help improve uptake.

Corrigendum: Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

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Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

Effective Reduction in Opioid Prescriptions for Ambulatory Lesion Excisions in Pediatric Patients.

Childhood opioid consumption is potentially deleterious to cognitive development and may predispose children to later addiction. Opioids are frequently prescribed for outpatient surgery but may not be necessary for adequate pain control. We aimed to reduce opioid prescriptions for outpatient pediatric skin and soft tissue lesion excisions using quality improvement (QI) methods. METHODS: A multidisciplinary team identified drivers for opioid prescriptions. Interventions were provider education, improving computer order set defaults, and promoting non-narcotic pain control strategies and patient-family education. Outcomes included percentage of patients receiving opioid prescriptions and patient-satisfaction scores. Data were retrospectively collected for 3 years before the QI project and prospectively tracked over the 8-month QI period and the following 18 months. RESULTS: The percentage of patients receiving an opioid prescription after outpatient skin or soft tissue excision dropped significantly from 18% before intervention to 6% at the end of the intervention period. Patient-reported satisfaction with pain control improved following the QI intervention. Satisfaction with postoperative pain control was independent of closure size or receipt of a postoperative opioid prescription. Intraoperative use of lidocaine or bupivacaine significantly decreased the incidence of postoperative opioid prescription in both bivariate and multivariate analyses. Results were maintained at 18 months after the conclusion of the QI project. CONCLUSION: Raising provider awareness, educating patients on expected postoperative pain management options, and prioritizing non-narcotic medications postoperatively successfully reduced opioid prescription rates in children undergoing skin and soft tissue lesion excisions and simultaneously improved patient-satisfaction scores.

Characteristics, Outcomes, and Trends of Patients With COVID-19-Related Critical Illness at a Learning Health System in the United States.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic continues to surge in the United States and globally. OBJECTIVE: To describe the epidemiology of COVID-19-related critical illness, including trends in outcomes and care delivery. DESIGN: Single-health system, multihospital retrospective cohort study. SETTING: 5 hospitals within the University of Pennsylvania Health System. PATIENTS: Adults with COVID-19-related critical illness who were admitted to an intensive care unit (ICU) with acute respiratory failure or shock during the initial surge of the pandemic. MEASUREMENTS: The primary exposure for outcomes and care delivery trend analyses was longitudinal time during the pandemic. The primary outcome was all-cause 28-day in-hospital mortality. Secondary outcomes were all-cause death at any time, receipt of mechanical ventilation (MV), and readmissions. RESULTS: Among 468 patients with COVID-19-related critical illness, 319 (68.2%) were treated with MV and 121 (25.9%) with vasopressors. Outcomes were notable for an all-cause 28-day in-hospital mortality rate of 29.9%, a median ICU stay of 8 days (interquartile range [IQR], 3 to 17 days), a median hospital stay of 13 days (IQR, 7 to 25 days), and an all-cause 30-day readmission rate (among nonhospice survivors) of 10.8%. Mortality decreased over time, from 43.5% (95% CI, 31.3% to 53.8%) to 19.2% (CI, 11.6% to 26.7%) between the first and last 15-day periods in the core adjusted model, whereas patient acuity and other factors did not change. LIMITATIONS: Single-health system study; use of, or highly dynamic trends in, other clinical interventions were not evaluated, nor were complications. CONCLUSION: Among patients with COVID-19-related critical illness admitted to ICUs of a learning health system in the United States, mortality seemed to decrease over time despite stable patient characteristics. Further studies are necessary to confirm this result and to investigate causal mechanisms. PRIMARY FUNDING SOURCE: Agency for Healthcare Research and Quality.

Reductive power generated by Mycobacterium leprae through cholesterol oxidation contributes to lipid and ATP synthesis

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.