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X-ray imaging has proved invaluable in medical diagnoses and non-destructive testing (NDT) in the past century. However, there remain two major limitations: radiation harm and inaccessibility to the sample. A recent imaging modality, X-ray induced acoustic computed tomography (XACT), allows a novel solution. In XACT, x-ray induced excitation causes localized heating (

Rationale: The epidemiology of hypoxemia in Sub-Saharan Africa is largely unknown., Objectives: Determine the prevalence, clinical care, and outcomes for hospitalized hypoxemic adults in Sub-Saharan Africa., Methods: We prospectively screened all adults admitted to five hospitals in Kenya, Malawi, and Rwanda over four months, and followed hypoxemic patients to discharge., Measurements and Main Results: Of the 24,724 adult patients admitted, 1,739 (7%) were hypoxemic on admission. Median imputed PaO 2 /FiO 2 was 168. Of all patients screened, 4,546 (18%) had complete oxygenation domains (SpO 2 , device, and quantity) documented in their charts on admission. Among hypoxemic patients, 44% of in-hospital days (6,890/15,553) had chart documentation of all three oxygenation domains. Of 1,508 unique hypoxemic patients, 770 (51%) had at least one day with subtherapeutic oxygen (SpO 2 <90%). Of patient-days with patients on oxygen therapy, 84% indicated supratherapeutic oxygen therapy (SpO 2 >94%). Of all hypoxemic adults, 35% died in-hospital. Sixteen percent of patients were mechanically ventilated during their stay, and in-hospital mortality in this subgroup was 49%., Conclusions: Hypoxemia is common and associated with high mortality in five referral hospitals in three countries in Sub-Saharan Africa. Monitoring and titration practices for oxygen therapy are inconsistent. Subtherapeutic and supratherapeutic oxygen therapy are common. Mortality is high among the few patients who receive mechanical ventilation. These findings suggest the urgent need to study interventions to improve survival for hypoxemic patients in Sub-Saharan Africa. Prioritized investigations include the development and implementation of oxygen monitoring and titration protocols, as well as studies of advanced oxygen therapies other than mechanical ventilation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background: In 2019, the World Health Organization called for operational research on all-oral shortened regimens for multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB). We report safety and effectiveness of three 9-month all-oral regimens containing bedaquiline (Bdq), linezolid (Lzd), and levofloxacin (Lfx) and reinforced with cycloserine (Cs) and clofazimine (Cfz), delamanid (Dlm) and pyrazinamide (Z), or Dlm and Cfz., Methods: We conducted a prospective cohort study of patients initiating treatment for pulmonary MDR/RR-TB under operational research conditions at public health facilities in Kazakhstan. Participants were screened monthly for adverse events. Participants with baseline resistance were excluded from the study and treated with a longer regimen. We analyzed clinically relevant adverse events of special interest in all participants and sputum culture conversion and end-of-treatment outcomes among individuals who were not excluded., Results: Of 510 participants, 41% were women, the median age was 37 years (25th-75th percentile: 28-49), 18% had a body mass index <18.5 kg/m2, and 51% had cavitary disease. A total of 399 (78%) initiated Bdq-Lzd-Lfx-Cs-Cfz, 83 (16%) started Bdq-Lzd-Lfx-Dlm-Z, and 28 (5%) initiated Bdq-Lzd-Lfx-Dlm-Cfz. Fifty-eight individuals (11%) were excluded from the study, most commonly due to identification of baseline drug resistance (n = 52; 90%). Among the remaining 452 participants, treatment success frequencies were 92% (95% CI: 89-95%), 89% (95% CI: 80-94%), and 100% (95% CI: 86-100%) for regimens with Cs/Cfz, Dlm/Z, and Dlm/Cfz, respectively. Clinically relevant adverse events of special interest were uncommon., Conclusions: All regimens demonstrated excellent safety and effectiveness, expanding the potential treatment options for patients, providers, and programs., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Loss to follow-up (LTFU) from tuberculosis (TB) treatment and care is a significant public health problem. It is important to understand what drives LTFU in children - a population whose treatment and management depend on an adult caregiver - to better provide support services to families affected by TB., Methods: We conducted a prospective cohort study of household contacts in Lima, Peru (2009-12). Using multilevel logistic regression analysis, we explored individual-level characteristics of children and their adult household members with TB disease to identify risk factors for LTFU among children initiated on treatment for TB., Results: A total of 154 child (0-14 years) household contacts were diagnosed with TB and initiated on treatment. While most (n = 133, 86.4%) had a successful outcome, 20 (13.0%) children were LTFU. Six (30.0%) children were LTFU within three months, nine (45.0%) between five to seven months, and three (15.0%) after seven months of treatment being initiated. In univariable analysis, children with index patients above 25 years of age had decreased odds of being LTFU (odds ratio = 0.26; 95% confidence interval = 0.08-0.84) compared to children with index patients 25 years or younger., Conclusions: In this cohort, more than 10% of children sick with TB who were exposed to the disease at home were LTFU. An integrated, family-centred TB prevention and management approach may reduce barriers to a child completing their course of TB treatment., Competing Interests: Disclosure of interest: The authors completed the ICMJE Disclosure of Interest Form (available upon request from the corresponding author) and disclose no relevant interests., (Copyright © 2024 by the Journal of Global Health. All rights reserved.)

Rationale: The persistent burden of tuberculosis (TB) disease emphasizes the need to identify individuals with TB for treatment and those at a high risk of incident TB for prevention. Targeting interventions toward those at high risk of developing and transmitting TB is a public health priority. Objectives: We aimed to identify characteristics of individuals involved in TB transmission in a community setting, which may guide the prioritization of targeted interventions. Methods: We collected clinical and sociodemographic data from a cohort of patients with TB in Lima, Peru. We used whole-genome sequencing data to assess the genetic distance between all possible pairs of patients; we considered pairs to be the result of a direct transmission event if they differed by three or fewer SNPs, and we assumed that the first diagnosed patient in a pair was the transmitter and the second was the recipient. We used logistic regression to examine the association between host factors and the likelihood of direct TB transmission. Measurements and Main Results: Analyzing data from 2,518 index patients with TB, we identified 1,447 direct transmission pairs. Regardless of recipient attributes, individuals less than 34 years old, males, and those with a history of incarceration had a higher likelihood of being transmitters in direct transmission pairs. Direct transmission was more likely when both patients were drinkers or smokers. Conclusions: This study identifies men, young adults, former prisoners, alcohol consumers, and smokers as priority groups for targeted interventions. Innovative strategies are needed to extend TB screening to social groups such as young adults and prisoners with limited access to routine preventive care.

We previously showed that digitoxin inhibits angiogenesis and cancer cell proliferation and migration and these effects were associated to protein tyrosine kinase 2 (FAK) inhibition. Considering the interactions between FAK and Rho GTPases regulating cell cytoskeleton and movement, we investigated the involvement of RhoA and Rac1 in the antiangiogenic effect of digitoxin. Phalloidin staining of human umbilical vein endothelial cells (HUVECs) showed the formation of stress fibers in cells treated with 10 nM digitoxin. By Rhotekin- and Pak1- pull down assays, detecting the GTP-bound form of GTPases, we observed that digitoxin (10-25 nM) induced sustained (0.5-6 h) RhoA activation with no effect on Rac1. Furthermore, inhibition of HUVEC migration and capillary-like tube formation by digitoxin was counteracted by hindering RhoA-ROCK axis with RhoA silencing or Y-27632 treatment. Digitoxin did not decrease p190RhoGAP phosphorylation at Tyr1105 (a site targeted by FAK), suggesting that RhoA activation was independent from FAK inhibition. Because increasing evidence points to a redox regulation of RhoA, we measured intracellular ROS and found that digitoxin treatment enhanced ROS levels in a concentration-dependent manner (1-25 nM). Notably, the flavoprotein inhibitor DPI or the pan-NADPH oxidase (NOX) inhibitor VAS-2870 antagonized both ROS increase and RhoA activation by digitoxin. Our results provide evidence that inhibition of HUVEC migration and tube formation by digitoxin is dependent on ROS production by endothelial NOX, which leads to the activation of RhoA/ROCK pathway. Digitoxin effects on proteins regulating cytoskeletal organization and cell motility could have a wider impact on cancer progression, beyond the antiangiogenic activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rationale: Treatment outcomes may be compromised among patients with multidrug- or rifampicin-resistant tuberculosis with additional fluoroquinolone resistance. Evidence is needed to inform optimal treatment for these patients., Objectives: We compared the effectiveness of longer individualized regimens comprised of bedaquiline for 5 to 8 months, linezolid, and clofazimine to those reinforced with at least 1 third-tier drug and/or longer duration of bedaquiline., Methods: We emulated a target trial to compare the effectiveness of initiating and remaining on the core regimen to one of five regimens reinforced with (1) bedaquiline for ≥9 months, (2) bedaquiline for ≥9 months and delamanid, (3) imipenem, (4) a second-line injectable, or (5) delamanid and imipenem. We included patients in whom a fluoroquinolone was unlikely to be effective based on drug susceptibility testing and/or prior exposure. Our analysis consisted of cloning, censoring, and inverse-probability weighting to estimate the probability of successful treatment., Measurements and Main Results: Adjusted probabilities of successful treatment were high across regimens, ranging from 0.75 (95%CI:0.61, 0.89) to 0.84 (95%CI:0.76, 0.91). We found no substantial evidence that any of the reinforced regimens improved effectiveness of the core regimen, with ratios of treatment success ranging from 1.01 for regimens reinforced with bedaquiline ≥9 months (95%CI:0.79, 1.28) and bedaquiline ≥9 months plus delamanid (95%CI:0.81, 1.31) to 1.11 for regimens reinforced by a second-line injectable (95%CI:0.92, 1.39) and delamanid and imipenem (95%CI:0.90, 1.41)., Conclusions: High treatment success underscores the effectiveness of regimens comprised of bedaquiline, linezolid, and clofazimine, highlighting the need for expanded access to these drugs.

Aims: Estrogen-regulated pathways are involved in the etiology and progression of epithelial ovarian cancer (EOC), but the relative contribution of estrogen receptor isoforms is unclear. Only a subset of patients responds to antiestrogens including tamoxifen. Based on our previous evidence that miR-206 behaves as an oncosuppressor in EOC, we hypothesized that miR-206 would interfere with G protein-coupled estrogen receptor (GPER)-mediated signaling and cell motility., Main Methods: PFKFB3 and FAK proteins from OC cells challenged with selective estrogen receptor agonist and antagonist were measured by Western blotting. Cell proliferation and motility were analyzed by MTT and Boyden chamber, respectively. Estrogen-dependent cells were transfected with miR-206 mimic or control using Lipofectamine., Key Findings: The migration of SKOV3 and OVCAR5 cells significantly increased following treatment with 17β-estradiol (E2) and the selective GPER agonist G1. However, tamoxifen failed to inhibit E2 effect and even promoted SKOV3 cell migration. Estrogen receptor ligands did not affect SKOV3 proliferation. The GPER antagonist G15 significantly prevented E2-mediated upregulation of PFKFB3 expression, while G1 concentration-dependently upregulated PFKFB3 levels. Consistent with the functional link between PFKFB3 and FAK activation, E2 and G1 increased FAK phosphorylation at Tyr397. Transfection with miR-206 abolished estrogen-induced EOC migration and down-regulated PFKFB3 protein levels. Notably, miR-206 transfection reduced ERα protein abundance, whereas GPER amount was unchanged., Significance: By blocking estrogen signaling and G1-induced EOC cell invasiveness with no direct interference with GPER levels, miR-206 mimics have the potential to act as pathway-selective antagonists and deserve further testing as RNA therapeutics in estrogen-dependent EOC., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Current recommendations for the treatment of rifampicin- and multidrug-resistant tuberculosis include bedaquiline (BDQ) used for 6 months or longer. Evidence is needed to inform the optimal duration of BDQ. Objectives: We emulated a target trial to estimate the effect of three BDQ duration treatment strategies (6, 7-11, and ⩾12 mo) on the probability of successful treatment among patients receiving a longer individualized regimen for multidrug-resistant tuberculosis. Methods: To estimate the probability of successful treatment, we implemented a three-step approach comprising cloning, censoring, and inverse probability weighting. Measurements and Main Results: The 1,468 eligible individuals received a median of 4 (interquartile range, 4-5) likely effective drugs. In 87.1% and 77.7% of participants, this included linezolid and clofazimine, respectively. The adjusted probability of successful treatment was 0.85 (95% confidence interval [CI], 0.81-0.88) for 6 months of BDQ, 0.77 (95% CI, 0.73-0.81) for 7-11 months, and 0.86 (95% CI, 0.83-0.88) for ⩾12 months. Compared with 6 months of BDQ, the ratio of treatment success was 0.91 (95% CI, 0.85-0.96) for 7-11 months and 1.01 (95% CI, 0.96-1.06) for ⩾12 months. Naive analyses that did not account for bias revealed a higher probability of successful treatment with ⩾12 months (ratio, 1.09 [95% CI, 1.05-1.14]). Conclusions: BDQ use beyond 6 months did not increase the probability of successful treatment among patients receiving longer regimens that commonly included new and repurposed drugs. When not properly accounted for, immortal person-time bias can influence estimates of the effects of treatment duration. Future analyses should explore the effect of treatment duration of BDQ and other drugs in subgroups with advanced disease and/or receiving less potent regimens.

Programmed cell death 1 ligand 1 (PD-L1) expressed in non-immune cells is involved in immune-mediated tissue damage in the context of inflammatory conditions and tumor immune escape. Emerging evidence suggests soluble (s)PD-L1 as a marker of inflammation. Based on well-established sex-specific differences in immunity, we tested the novel hypotheses that (i) endothelial cell PD-L1 is modulated by inflammatory cytokines and vascular endothelial growth factor (VEGF) in a sex-specific fashion, and (ii) the endothelium is a source of sPD-L1. After exposure of human umbilical vein endothelial cells (HUVECs) to lipopolysaccharide, interleukin (IL)1β or VEGF for 24 h, total PD-L1 levels were upregulated solely in cells from female donors, while being unchanged in those from male donors. Accordingly, exposure to synovial fluids from patients with inflammatory arthritis upregulated PD-L1 levels in HUVECs from female donors only. Membrane PD-L1 expression as measured by flow cytometry was unchanged in response to inflammatory stimuli. However, exposure to 2 ng/mL IL-1β or 50 ng/mL VEGF time-dependently increased sPD-L1 release by HUVECs from female donors. Treatment with the metalloproteinase (MMP) inhibitor GM6001 (10 μM) prevented IL-1β-induced sPD-L1 release and enhanced membrane PD-L1 levels. The anti-VEGF agents bevacizumab and sunitinib reduced both VEGF-induced PD-L1 accumulation and sPD-L1 secretion. Thus, inflammatory agents and VEGF rapidly increased endothelial PD-L1 levels in a sex-specific fashion. Furthermore, the vascular endothelium may be a sPD-L1 source, whose production is MMP-dependent and modulated by anti-VEGF agents. These findings may have implications for sex-specific immunity, vascular inflammation and response to anti-angiogenic therapy., Competing Interests: Conflict of interest statement None., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Ethinylestradiol (EE) and estetrol (E4) are the two main estrogenic agents used in combined oral contraceptives. These compounds have different binding affinity to and efficacy on estrogen receptors (ER) subtypes. We previously reported that treatment with estrogenic agents enhances angiogenesis via nongenomic, G protein-coupled estrogen receptor (GPER)-dependent mechanisms. However, the impact of EE and E4 on human endothelial function has been little investigated. EE and E4 (10 -9 - 10 -7  M) significantly enhanced migration of human umbilical vein endothelial cells (HUVECs) using scratch and Boyden chamber assays. Mechanistically, both agents increased accumulation of phosphorylated protein tyrosine kinase 2 on tyrosine 397 (FAK Y397), a key player in endothelial cell motility, after 30-min treatment. Treatment with increasing concentrations of EE, but not E4, enhanced accumulation of the glycolysis activator PFKFB3. Of note, effects of EE and E4 on endothelial migration and signalling proteins were abolished by addition of the GPER antagonist G36 (10 -6  M). Thus, EE and E4 induced comparable endothelial responses in vitro, suggesting no apparent alterations of vascular remodelling and regeneration capacity by oral contraceptives containing these agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Spatially targeted interventions may be effective alternatives to individual or population-based prevention strategies against tuberculosis (TB). However, their efficacy may depend on the mechanisms that lead to geographically constrained hotspots. Local TB incidence may reflect high levels of local transmission; conversely, they may point to frequent travel of community members to high-risk areas. We used whole-genome sequencing to explore patterns of TB incidence and transmission in Lima, Peru. Between 2009 and 2012, we recruited incident pulmonary TB patients and their household contacts, whom we followed for the occurrence of TB disease. We used whole-genome sequences of 2,712 Mycobacterial tuberculosis isolates from 2,440 patients to estimate pariwise genomic distances and compared these to the spatial distance between patients' residences. Genomic distances increased rapidly as spatial distances increased and remained high beyond 2 km of separation. Next, we divided the study catchment area into 1 × 1 km grid-cell surface units and used household spatial coordinates to locate each TB patient to a specific cell. We estimated cell-specific transmission by calculating the proportion of patients in each cell with a pairwise genomic distance of 10 or fewer single-nucleotide polymorphisms. We found that cell-specific TB incidence and local transmission varied widely but that cell-specific TB incidence did not correlate closely with our estimates of local transmission (Cohen's k = 0.27). These findings indicate that an understanding of the spatial heterogeneity in the relative proportion of TB due to local transmission may help guide the implementation of spatially targeted interventions.

Several mediators including cytokines, growth factors and metalloproteinases (MMP) modulate pathological angiogenesis associated with inflammatory arthritis. The biological factors underlying sex disparities in the incidence and severity of rheumatic musculoskeletal diseases are only partially understood. We hypothesized that synovial fluids (SFs) from rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients would impact on endothelial biology in a sexually dimorphic fashion. Immune cell counts and levels of pro-angiogenic cytokines found in SFs from RA and PsA patients (n = 17) were higher than in osteoarthritis patients (n = 6). Synovial VEGF concentration was significantly higher in male than in female RA patients. Zymography revealed that SFs comprised solely MMP-9 and MMP-2, with significantly higher MMP-9 levels in male than female RA patients. Using in vitro approaches that mimic the major steps of the angiogenic process, SFs from RA and PsA patients induced endothelial migration and formation of capillary-like structures compared to control. Notably, endothelial cells from female donors displayed enhanced angiogenic response to SFs with respect to males. Treatment with the established anti-angiogenic agent digitoxin prevented activation of focal adhesion kinase and SF-induced in vitro angiogenesis. Thus, despite higher synovial VEGF and MMP-9 levels in male patients, the responsiveness of vascular endothelium to SF priming was higher in females, suggesting that gender differences in angiogenic responses were mainly related to the endothelial genotype. These findings may have implications for pathogenesis and targeted therapies of inflammatory arthritis., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Vasoactive molecules, such as vascular endothelial growth factor (VEGF) and endothelins, share cytokine-like activities and regulate endothelial cell (EC) growth, migration, and inflammation. Some endothelial mediators and their receptors are targets for currently approved angiogenesis inhibitors, drugs that are either monoclonal antibodies raised towards VEGF, or inhibitors of vascular receptor protein kinases and signalling pathways. Pharmacological interference with the protective functions of ECs results in a similar spectrum of adverse effects. Clinically, the most common side effects of VEGF signalling pathway inhibition include an increase in arterial pressure, left ventricular dysfunction facilitating the development of heart failure, thromboembolic events including pulmonary embolism and stroke, and myocardial infarction. Sex steroids, such as androgens, progestins, and oestrogens and their receptors (ERα, ERβ, GPER; PR-A, PR-B; AR) have been identified as important modifiers of angiogenesis, and sex differences have been reported for anti-angiogenic drugs. This review article discusses the current challenges clinicians are facing with regard to angiogenesis inhibitor therapy, including the need to consider sex differences affecting clinical efficacy and safety. We also propose areas for future research taking into account the role of sex hormone receptors and sex chromosomes. Development of new sex-specific drugs with improved target- and cell-type selectivity likely will open the way to personalized medicine in men and women requiring anti-angiogenic therapy to reduce adverse effects and to improve therapeutic efficacy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Few studies explored the role of microRNAs (miRNAs) in the post-transcriptional regulation of glycolytic proteins and downstream effectors in ovarian cancer cells. We recently showed that the functional activation of the cytoskeletal regulator FAK in endothelial cells is fostered by the glycolytic enhancer 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We tested the hypothesis that miR-206 and mir-26b, emerging onco-suppressors targeting PFKFB3 in estrogen-dependent tumors, would regulate proliferation and migration of serous epithelial ovarian cancer (EOC) cells via common glycolytic proteins, i.e., GLUT1 and PFKFB3, and downstream FAK. PFKFB3 was overexpressed in SKOV3, and its pharmacological inhibition with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) significantly reduced cell proliferation and motility. Both miR-206 and miR-26b directly targeted PFKFB3 as evaluated by a luciferase reporter assay. However, endogenous levels of miR-26b were higher than those of miR-206, which was barely detectable in SKOV3 as well as OVCAR5 and CAOV3 cells. Accordingly, only the anti-miR-26b inhibitor concentration-dependently increased PFKFB3 levels. While miR-206 overexpression impaired proliferation and migration by downregulating PFKFB3 levels, the decreased PFKFB3 protein levels related to miR-26 overexpression had no functional consequences in all EOC cell lines. Finally, consistent with the migration outcome, exogenous miR-206 and miR-26b induced opposite effects on the levels of total FAK and of its phosphorylated form at Tyr576/577. 3PO did not prevent miR-26b-induced SKOV3 migration. Overall, these results support the inverse relation between endogenous miRNA levels and their tumor-suppressive effects and suggest that restoring miR-206 expression represents a potential dual anti-PFKFB3/FAK strategy to control ovarian cancer progression., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Acute kidney injury (AKI) in Covid-19 patients admitted to the intensive care unit (ICU) is common, and its severity may be associated with unfavorable outcomes. Severe Covid-19 fulfills the diagnostic criteria for acute respiratory distress syndrome (ARDS); however, it is unclear whether there is any relationship between ventilatory management and AKI development in Covid-19 ICU patients., Purpose: To describe the clinical course and outcomes of Covid-19 ICU patients, focusing on ventilatory management and factors associated with AKI development., Methods: Single-center, retrospective observational study, which assessed AKI incidence in Covid-19 ICU patients divided by positive end expiratory pressure (PEEP) tertiles, with median levels of 9.6 (low), 12.0 (medium), and 14.7 cmH 2 O (high-PEEP)., Results: Overall mortality was 51.5%. AKI (KDIGO stage 2 or 3) occurred in 38% of 101 patients. Among the AKI patients, 19 (53%) required continuous renal replacement therapy (CRRT). In AKI patients, mortality was significantly higher versus non-AKI (81% vs. 33%, p < 0.0001). The incidence of AKI in low-, medium-, or high-PEEP patients were 16%, 38%, and 59%, respectively (p = 0.002). In a multivariate analysis, high-PEEP patients showed a higher risk of developing AKI than low-PEEP patients (OR = 4.96 [1.1-21.9] 95% CI p < 0.05). ICU mortality rate was higher in high-PEEP patients, compared to medium-PEEP or low-PEEP patients (69% vs. 44% and 42%, respectively; p = 0.057)., Conclusion: The use of high PEEP in Covid-19 ICU patients is associated with a fivefold higher risk of AKI, leading to higher mortality. The cause and effect relationship needs further analysis., (© 2021. The Author(s).)

Female hormones and sex-specific factors are established determinants of endothelial function, yet their relative contribution to human endothelium phenotypes has not been defined. Using human umbilical vein endothelial cells (HUVECs) genotyped by donor's sex, we investigated the influence of sex and estrogenic agents on the main steps of the angiogenic process and on key proteins governing HUVEC metabolism and migratory properties. HUVECs from female donors (fHUVECs) showed increased viability ( p < 0.01) and growth rate ( p < 0.01) compared with those from males (mHUVECs). Despite higher levels of G-protein coupled estrogen receptor (GPER) in fHUVECs ( p < 0.001), treatment with 17β-estradiol (E2) and the selective GPER agonist G1 (both 1-100 nM) did not affect HUVEC viability. Migration and tubularization in vitro under physiological conditions were higher in fHUVECs than in mHUVECs ( p < 0.05). E2 treatment (1-100 nM) upregulated the glycolytic activator PFKFB3 with higher potency in fHUVECs than in mHUVECs, despite comparable baseline levels. Moreover, Y576/577 phosphorylation of focal adhesion kinase (FAK) was markedly enhanced in fHUVECs ( p < 0.001), despite comparable Src activation levels. While the PI3K inhibitor LY294002 (25 µM) inhibited HUVEC migration ( p < 0.05), Akt phosphorylation levels in fHUVECs and mHUVECs were comparable. Finally, digitoxin treatment, which inhibits Y576/577 FAK phosphorylation, abolished sexual dimorphism in HUVEC migration. These findings unravel complementary modulation of HUVEC functional phenotypes and signaling molecules involved in angiogenesis by hormone microenvironment and sex-specific factors, and highlight the need for sex-oriented pharmacological targeting of endothelial function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Boscaro, Trenti, Scapin, Baggio, Trevisi, Cignarella and Bolego.)

Background: There is limited research to guide TB treatment specifically in pregnant women and few studies have described the presentation of TB in pregnant women. We aimed to understand TB presentation and treatment outcomes in pregnant women in a low HIV burden setting. We describe a cohort of women of childbearing age treated for TB disease in Lima, Peru, and compare clinical presentation and treatment outcomes among pregnant and non-pregnant women between 2009 and 2012, including 36 pregnant women., Methods: This is a prospective cohort study. Subjects were recruited from across 106 public health centers in Lima, Peru. Baseline demographic, medical history, and drug-susceptibility test results were collected. We used descriptive statistics to describe demographic and clinical characteristics of the women using Pearson chi-squared, Fisher's exact tests, or Kruskal-Wallis., Results: Among 4500 individuals with pulmonary TB disease, 1334 women were included in analysis with 36 (2.69%) pregnant women. Pregnant women had similar demographics, past medical histories, and clinical presentation to non-pregnant women, except being more likely to be married (p = 0.01) and have cardiac disease (p = 0.04) and less likely to have weight loss (p = 0.05). Twenty (71.4%) pregnant women had pan-susceptible TB compared with 616 (63.1%) non-pregnant women; four (14.3%) pregnant women had mono-resistant TB compared with 154 (15.8%) non-pregnant women; and four (14.3%) pregnant women had multi-drug-resistant TB compared with 140 (14.3%) of non-pregnant women (p = 0.53). Twenty-eight (96.6%) pregnant women had a successful outcome (cure, completed treatment, treatment ended early by clinical team) while one (3.4%) had an unsuccessful outcome (treatment failed) and 1074 (97.3%) non-pregnant women had a successful outcome while 30 (2.7%) had an unsuccessful outcome (p = 0.56)., Conclusion: In this cohort with low HIV co-infection, we found high TB treatment success rates in both pregnant and non-pregnant women, irrespective of drug-susceptibility profiles. If treated appropriately, pregnant women with TB disease can have successful outcomes.

Few studies have explored the mechanisms coupling estrogen signals to metabolic demand in endothelial cells. We recently showed that 17β-estradiol (E2) triggers angiogenesis via the membrane G-protein coupled estrogen receptor (GPER) and the key glycolytic protein PFKFB3 as a downstream effector. We herein investigated whether estrogenic agents regulate the stability and/or degradation of glycolytic proteins in human umbilical vein endothelial cells (HUVECs). Similarly to E2, the GPER selective agonist G1 rapidly increased PFKFB3 protein amounts, without affecting mRNA levels. In the presence of cycloheximide, E2 and G1 treatment counteracted PFKFB3 degradation over time, whereas E2-induced PFKFB3 stabilization was abolished by the GPER antagonist G15. Inhibitors of selective SCF E3 ubiquitin ligase (SMER-3) and proteasome (MG132) rapidly increased PFKFB3 protein levels. Accordingly, ubiquitin-bound PFKFB3 was lower in E2- or G1-treated HUVECs. Both agents increased deubiquitinase USP19 levels through GPER signaling. Notably, USP 19 siRNA decreased PFKFB3 levels and abolished E2- and G1-mediated HUVEC tubularization. Finally, E2 and G1 treatments rapidly enhanced glucose transporter GLUT1 levels via GPER independent of transcriptional activation. These findings provide new evidence on mechanisms coupling estrogen signals with the glycolytic program in endothelium and unravel the role of USP19 as a target of the pro-angiogenic effect of estrogenic agents., (© 2020 Federation of American Societies for Experimental Biology.)

Introduction: The Community Outreach and Patient Empowerment (COPE) intervention provides integrated outreach through community health representatives (CHRs) to people living with diabetes in Navajo Nation. The aim of this study was to identify groups for whom the intervention had the greatest effect on glycated hemoglobin A 1c (HbA 1c )., Methods: We analyzed de-identified data extracted from routine health records dated from December 1, 2010, through August 31, 2014, to compare net change in HbA 1c among COPE patients and non-COPE patients. We used linear mixed models to assess whether the intervention was modified by age, sex, preferred language, having a primary care provider, baseline HbA 1c , or having a mental health condition., Results: Age, having a primary care provider, and baseline HbA 1c significantly modified HbA 1c levels. Among patients aged 64 or younger, COPE participation was associated with a net decrease in HbA 1c of 0.77%; among patients aged 65 or older, the net decrease was 0.49% (P = .03). COPE participation was associated with a steeper decrease in HbA 1c among patients without a primary care physician (net decrease, 0.99%) than among patients with a primary care provider (net decrease, 0.57%) (P = .03). COPE patients with a baseline HbA 1c >9% had a net decrease of 0.70%, while those with a baseline HbA 1c ≤9% had a net decrease of 0.34% (P = .01). We found no significant differences based on sex, preferred language, or having a mental health condition., Conclusion: Findings suggest that the COPE intervention was robust and equitable, benefiting all groups living with diabetes in Navajo Nation, but conferring the greatest benefit on the most vulnerable.

Background: Navajo community members face high rates of diabetes mellitus and other chronic diseases. The Navajo Community Health Representative Outreach Program collaborated with healthcare providers and academic partners to implement structured and coordinated outreach to patients living with diabetes. The intervention, called Community Outreach and Patient Empowerment or COPE, provides home-based health coaching and community-clinic linkages to promote self-management and engagement in healthcare services among patients living with diabetes. The purpose of this study was to evaluate how outreach by Navajo Community Health Representatives ("COPE Program") affected utilization of health care services among patients living with diabetes., Methods: De-identified data from 2010 to 2014 were abstracted from electronic health records at participating health facilities. In this observational cohort study, 173 cases were matched to 2880 controls. Healthcare utilization was measured as the number of times per quarter services were accessed by the patient. Changes in utilization over 4 years were modeled using a difference-in-differences approach, comparing the trajectory of COPE patients' utilization before versus after enrollment with that of the control group. The model was estimated using generalized linear mixed models for count outcomes, controlling for clustering at the patient level and the service unit level., Results: COPE enrollees showed a 2.5% per patient per quarter (pppq) greater increase in total utilization (p = 0.001) of healthcare services than non-COPE enrollees; a 3.2% greater increase in primary care visits (p = 0.024); a 6.3% greater increase in utilization of counseling and behavioral health services (p = 0.013); and a 9.0% greater increase in pharmacy visits (p <  0.001). We found no statistically significant differences in utilization trends of inpatient, emergency room, specialty outpatient, dental, laboratory, radiology, or community encounter services among COPE participants versus control., Conclusions: A structured intervention consisting of Community Health Representative outreach and coordination with clinic-based providers was associated with a modest increase in health care utilization, including primary care and counseling services, among Navajo patients living with diabetes. Community health workers may provide an important linkage to enable patients to access and engage in clinic-based health care., Trial Registration: NCT03326206, registered 10/31/2017, retrospectively registered.

Background: Stool is a promising specimen option to diagnose pediatric tuberculosis (TB), but studies have reported a wide range of test sensitivities. We conducted a meta-analysis to assess the accuracy of Xpert MTB/RIF or 'in-house' molecular tests on stool samples against culture or Xpert MTB/RIF on respiratory samples or clinically-diagnosed unconfirmed TB and aimed to identify factors that contribute to the heterogeneity of reported sensitivity., Methods: We searched EMBASE and Pubmed databases and conference abstract books for studies reporting molecular stool testing against a clinical or microbiological reference standard among children., Results: We identified 16 studies that included 2,481 children in stool test analyses. Pooled specificity was 98% [95%CI: 96-99], pooled sensitivity was 57% [95%CI: 40-72] against culture and 3% [95%CI: 2-6] among children with clinically-diagnosed, unconfirmed TB. There was much heterogeneity. Sensitivity was higher among children with a smear-positive sputum test. Rifampin resistance in stool was reported in two studies and detected in 5/14 children (36%)., Conclusion: Our results suggest molecular stool tests have potential as diagnostic rule-in tests, but it is challenging to optimize sensitivity due to between-study variation in methodology and test procedures. Therefore, we recommend future research with rigorous study design and standardized results reporting., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: We studied the impact of Community Outreach and Patient Empowerment (COPE) intervention to support Community Health Representatives (CHR) on the clinical outcomes of patients living with diabetes in the Navajo Nation extending into the States of Arizona, Utah, and New Mexico. The COPE intervention integrated CHRs into healthcare teams by providing a structured approach to referrals and home visits., Methods: We abstracted routine clinical data from the Indian Health Service's information system on individuals with diabetes mellitus seen at participating clinical sites from 2010 to 2014. We matched 173 COPE participants to 2880 patients with similar demographic and clinical characteristics who had not participated in COPE. We compared the changes in clinical outcomes between the two groups using linear mixed models., Results: Over the four years of the study, COPE patients had greater improvements in glycosylated hemoglobin (- 0.56%) than non-COPE participants (+ 0.07%) for a difference in differences of 0.63% (95% confidence interval (CI): 0.50, 0.76). Low-density lipoprotein fell more steeply in the COPE group (- 10.58 mg/dl) compared to the non-COPE group (- 3.18 mg/dl) for a difference in differences of 7.40 mg/dl (95%CI: 2.00, 12.80). Systolic blood pressure increased slightly more among COPE (2.06 mmHg) than non-COPE patients (0.61 mmHg). We noted no significant change for body mass index in either group., Conclusion: Structured outreach by Community Health Representatives as part of an integrated care team was associated with improved glycemic and lipid levels in the target Navajo population., Trial Registration: Trial registration: NCT03326206. Registered 31 October 2017 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/study/NCT03326206.

Birthweight is associated with health outcomes across the life course, DNA methylation may be an underlying mechanism. In this meta-analysis of epigenome-wide association studies of 8,825 neonates from 24 birth cohorts in the Pregnancy And Childhood Epigenetics Consortium, we find that DNA methylation in neonatal blood is associated with birthweight at 914 sites, with a difference in birthweight ranging from -183 to 178 grams per 10% increase in methylation (P Bonferroni  < 1.06 x 10 -7 ). In additional analyses in 7,278 participants, <1.3% of birthweight-associated differential methylation is also observed in childhood and adolescence, but not adulthood. Birthweight-related CpGs overlap with some Bonferroni-significant CpGs that were previously reported to be related to maternal smoking (55/914, p = 6.12 x 10 -74 ) and BMI in pregnancy (3/914, p = 1.13x10 -3 ), but not with those related to folate levels in pregnancy. Whether the associations that we observe are causal or explained by confounding or fetal growth influencing DNA methylation (i.e. reverse causality) requires further research.

Introduction: Lead (Pb) crosses the placenta and can cause oxidative stress, reduced fetal growth and neurological problems. The principal source of oxidative stress in human cells is mitochondria. Therefore, disruption of normal mitochondrial function during pregnancy may represent a primary mechanism behind the adverse effects of lead. We sought to assess the association of Pb exposure during pregnancy with mitochondrial DNA (mtDNA) content, a sensitive marker of mitochondrial function, in cord blood., Materials and Methods: This study comprised mother-infant pairs from the Programming Research in Obesity, Growth, Environment and Social Stressors (PROGRESS) study, a prospective birth-cohort that enrolled 1050 pregnant women from Mexico City who were receiving prenatal care between December 2007 and July 2011. Quantitative PCR was used to calculate relative MtDNA content (mitochondrial-to-nuclear DNA ratio (mtDNA/nDNA)) in cord blood. Lead concentrations in both maternal blood (2nd and 3rd trimester and at delivery day) and in cord blood were measured by ICP-MS. Multivariable regression models adjusting for multiple confounders were fitted with 410 mother-infant pairs for whom complete data for mtDNA content, lead levels, and covariates were available., Results: Maternal blood Pb measured in the second (mean 3.79 μg/dL, SD 2.63; β = 0.059, 95% CI 0.008, 0.111) and third trimester (mean 3.90 μg/dL; SD 2.84; β = 0.054, 95% CI 0.002, 0.107) during pregnancy and PB in cord blood (mean 3.50 μg/dL, SD 2.59; β = 0.050, 95% CI 0.004; 0.096) were associated with increased cord blood mtDNA content (mean 1.46, SD 0.44). In two-way interaction analyses, cord blood Pb marginally interacted with gestational age leading to an increase in mtDNA content for pre-term births (Benjamini-Hochberg False Discovery Rate correction; BH-FDR = 0.08)., Conclusion: This study shows that lead exposure in pregnancy alters mtDNA content in cord blood; therefore, alteration of mtDNA content might be a mechanism underlying the toxicity of lead., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Clinical and experimental evidence supports a role for cardiac glycosides (CGs) as potential novel anticancer drugs. However, there are no studies reporting the effect of CGs on the inflammatory tumor microenvironment (TME), which plays a central role in tumor progression and invasiveness. We investigated whether digitoxin affects a) specific pathways involved in motility and/or activation of different cell types shaping TME, and b) cancer cell growth and invasiveness in response to TME-associated factors. To test our hypothesis, conditioned media (CM) from polarized macrophages, and apoptotic or non-apoptotic ovarian cancer cells (SKOV3) were tested as chemoattractants for endothelial cells, monocytes and cancer cells. We demonstrated that CM from M1 (LPS/IFNγ) and M2 (IL-4/IL-13) polarized macrophages, which mimic inflammatory TME, increased both HUVEC migration and tubularization. Treatment of HUVECs with digitoxin at concentrations within its plasma therapeutic range counteracted these effects. Digitoxin affected the expression of neither M1 (CD80/CD68) nor M2 (CD206/CD163) activation markers, nor the amount of cell-bound IL-1β and CCL22. Accordingly, HUVEC migration in response to CM from digitoxin-treated activated macrophages was unchanged. These data point to a direct effect of digitoxin on HUVEC signaling rather than on the modulation of the cytokine profile released from activated macrophages. At variance with what observed for HUVECs, digitoxin did not prevent monocyte migration induced by SKOV3 CM. In addition, digitoxin significantly impaired SKOV3 growth and migration in response to M1 or M2 macrophage CM. Finally, we showed that digitoxin inhibited FAK phosphorylation in SKOV3 but not PYK2 phosphorylation in monocytes, thus providing a molecular mechanism accounting for the observed differential anti-migratory effect. Overall, digitoxin counteracted salient features of the inflammatory ovarian cancer microenvironment, laying the ground for potential digitoxin repositioning as an anticancer drug., (Copyright © 2018. Published by Elsevier Inc.)

Estrogen plays an important role in the regulation of cardiovascular physiology and the immune system by inducing direct effects on multiple cell types including immune and vascular cells. Sex steroid hormones are implicated in cardiovascular protection, including endothelial healing in case of arterial injury and collateral vessel formation in ischemic tissue. Estrogen can exert potent modulation effects at all levels of the innate and adaptive immune systems. Their action is mediated by interaction with classical estrogen receptors (ERs), ERα and ERβ, as well as the more recently identified G-protein coupled receptor 30/G-protein estrogen receptor 1 (GPER1), via both genomic and non-genomic mechanisms. Emerging data from the literature suggest that estrogen deficiency in menopause is associated with an increased potential for an unresolved inflammatory status. In this review, we provide an overview through the puzzle pieces of how 17β-estradiol can influence the cardiovascular and immune systems., Competing Interests: The authors declare no conflict of interest.

Human peripheral-blood monocytes are used as an established in vitro system for generating macrophages. For several reasons, monocytic cell lines such as THP-1 have been considered as a possible alternative. In view of their distinct developmental origins and phenotypic attributes, we set out to assess the extent to which human monocyte-derived macrophages (MDMs) and phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells were overlapping across a variety of responses to activating stimuli. Resting (M0) macrophages were polarized toward M1 or M2 phenotypes by 48-h incubation with LPS (1 μg/ml) and IFN-γ (10 ng/ml) or with IL-4 (20 ng/ml) and IL-13 (5 ng/ml), respectively. At the end of stimulation, MDMs displayed more pronounced changes in marker gene expression than THP-1. Upon assaying an array of 41 cytokines, chemokines and growth factors in conditioned media (CM) using the Luminex technology, secretion of 29 out of the 41 proteins was affected by polarized activation. While in 12 of them THP-1 and MDM showed comparable trends, for the remaining 17 proteins their responses to activating stimuli did markedly differ. Quantitative comparison for selected analytes confirmed this pattern. In terms of phenotypic activation markers, measured by flow cytometry, M1 response was similar but the established MDM M2 marker CD163 was undetectable in THP-1 cells. In a beads-based assay, MDM activation did not induce significant changes, whereas M2 activation of THP-1 decreased phagocytic activity compared to M0 and M1. In further biological activity tests, both MDM and THP-1 CM failed to affect proliferation of mouse myogenic progenitors, whereas they both reduced adipogenic differentiation of mouse fibro-adipogenic progenitor cells (M2 to a lesser extent than M1 and M0). Finally, migration of human umbilical vein endothelial cells was enhanced by CM irrespective of cell type and activation state except for M0 CM from MDMs. In summary, PMA-differentiated THP-1 macrophages did not entirely reproduce the response spectrum of primary MDMs to activating stimuli. We suggest that THP-1 be regarded as a simplified model of human macrophages when investigating relatively straightforward biological processes, such as polarization and its functional implications, but not as an alternative source in more comprehensive immunopharmacology and drug screening programs.