Multiplex immunohistochemical analysis of granulomatous inflammation in lung tissue sections using a mouse model of M. avium infection

INTRODUCTION: Investigating mechanisms of how intracellular bacterial pathogens such as Mycobacterium. avium (M. avium) evade the host immune response and replicate within macrophages is crucial to devising rational targets for host-directed therapies (HDT) against these associated diseases. This studied utilized the congenic mouse strain B6.Sst1S, which contains the super-susceptibility to tuberculosis (TB) allele. Among murine models of TB, this strain uniquely replicates human disease because mice develop granulomas with central caseous necrosis. Utilizing a susceptible model for M. avium infection, this study investigated the effect of mycobacterial pathogenesis on altering macrophage phenotypes and T cells distribution in areas of pulmonary granulomatous inflammation. METHODS:12 formalin fixed paraffin embedded (FFPE) lung sections from M. avium infected B6.Sst1S and B6 mice were examined microscopically (12 weeks post infection (wpi) n=5, 16 wpi=7). A targeted histology approach was initiated by using MRI coordinates to dictate the depths at which formalin fixed paraffin embedded (FFPE) lung samples were sectioned. Since interpretation of MRI images displayed no evidence of 2 discrete necrotizing granulomas, lungs were cut at sections representative of diffuse pathology at 2 mm into FFPE blocks. Using the Opal MethodTM (Akoya Biosciences), 6- plex immunohistochemical staining was performed with Arginase-1 (Arg1), inducible nitric oxide synthase (iNOS), CD68, CD3, M. tuberculosis antigen (cross-reacts with M. avium) and DAPI to segment nuclei. Slides were digitized by a Vectra PolarisTM fluorescent whole slide scanner. Autofluorescence was removed by InFormTM, and image analysis (IA) was conducted using HaloTM IA software. Statistical analysis was conducted using GraphPad PrismTM 8.0. RESULTS: Sst1 mediated susceptibility was statistically evident at 16 wpi but not at 12 wpi. B6.Sst1S mice showed a statistically significant (P <0.05) increase in M. avium+ cell expression in the non-inoculated lung lobes, but not the inoculated lung lobes. Pulmonary lesions within the inoculated and non-inoculated lung lobes contain different immune signatures. The predominately primary lesions of the inoculated lung lobes were associated with increased CD3+, M. avium+, and iNOS+ cell levels. When controlling for level of infection, there was lower levels of CD3+ cells within granulomatous lesions of B6.Sst1S mice, especially in the non-inoculated lung lobe. Controlling for level of infection also revealed elevated iNOS+ M. avium- cell expression in B6 mice. We observed elevated Arg1+ cell expression near iNOS+ M. avium+ cells, and, qualitatively, around larger lesions. T cell proximity analysis was contradictory and offers lessons for future the development of future IA modules. CONCLUSIONS: Sst1 mediated susceptibility was evident at 16 wpi and predominately mediated through secondary, metastatic lesions. Sst1 mediated susceptibility was also associated with fewer supportive cells (T cells and iNOS+ M. avium- cells) within granulomatous lesions. Future studies are necessary to evaluate to what degree granulomatous lesion Arg1+ cell expression and CD3+ proximity correlate to susceptibility

Emergency Care in the Occupied Palestinian Territory: A Scoping Review

The development of robust emergency care systems as a critical platform for addressing the global burden of disease has been increasingly recognized by global health policy makers over the past decade. A human rights-based approach to securing the right to quality emergency care is also essential to respond to the structural and political determinants of poor health outcomes. In the occupied Palestinian territory, human rights violations have contributed to significant deficiencies in health and quality of health care. In this scoping review, we identify deficiencies in the management of high-risk presentations to emergency departments in the Palestinian health care system for traumatic injury, acute myocardial infarction, and stroke. We subsequently apply a human rights-based analysis to demonstrate how structural racism in the administration of the occupation has contributed to deficiencies in emergency care. Specifically, deficiencies in resource and system organization within the Palestinian emergency care system arise due to occupation-related restrictions on freedom of movement, the procurement of essential drugs and medical equipment, and the development of a national Palestinian health care system. Further research and intervention are needed to understand gaps in emergency care for Palestinians and, in turn, to improve the management of emergency medical and traumatic conditions through capacity building of a Palestinian emergency care system. Importantly, deconstruction of the structural determinants of poor health for Palestinians in the occupied territory is needed to improve public health and ensure the protection of human rights

Family Physicians’ Attitudes and Practices Regarding Assessments of Medical Fitness to Drive in Older Persons

BACKGROUND: Higher crash rates per mile driven in older drivers have focused attention on the assessment of older drivers. OBJECTIVE: To examine the attitudes and practices of family physicians regarding fitness-to-drive issues in older persons. DESIGN: Survey questionnaire. PARTICIPANTS: The questionnaire was sent to 1,000 randomly selected Canadian family physicians. Four hundred sixty eligible physicians returned completed questionnaires. MEASUREMENTS: Self-reported attitudes and practices towards driving assessments and the reporting of medically unsafe drivers. RESULTS: Over 45% of physicians are not confident in assessing driving fitness and do not consider themselves to be the most qualified professionals to do so. The majority (88.6%) feel that they would benefit from further education in this area. About 75% feel that reporting a patient as an unsafe driver places them in a conflict of interest and negatively impacts on the patient and the physician–patient relationship. Nevertheless, most (72.4%) agree that physicians should be legally responsible for reporting unsafe drivers to the licensing authorities. Physicians from provinces with mandatory versus discretionary reporting requirements are more likely to report unsafe drivers (odds ratio [OR], 2.78; 95% confidence interval [CI], 1.58 to 4.91), but less likely to perform driving assessments (OR, 0.58; 95% CI, 0.39 to 0.85). Most driving assessments take between 10 and 30 minutes, with much variability in the components included. CONCLUSIONS: Family physicians lack confidence in performing driving assessments and note many negative consequences of reporting unsafe drivers. Education about assessing driving fitness and approaches that protect the physician–patient relationship when reporting occurs are needed

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Progression and Dissemination of Pulmonary Mycobacterium Avium Infection in a Susceptible Immunocompetent Mouse Model

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are a growing public health concern with incidence and mortality steadily increasing globally. Granulomatous inflammation is the hallmark of MAC lung infection, yet reliable correlates of disease progression, susceptibility, and resolution are poorly defined. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. We characterized pulmonary temporospatial outcomes of intranasal and left intrabronchial M. avium spp. hominissuis (M.av) induced pneumonia in B6.Sst1S mice, which carries the sst1 mutant allele. We utilized traditional semi-quantitative histomorphological evaluation, in combination with fluorescent multiplex immunohistochemistry (fmIHC), whole slide imaging, and quantitative digital image analysis. Followingintrabronchiolar infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12&ndash;16 weeks post infection (wpi), with plateauing and/or resolving disease by 21 wpi. Caseating granulomas were not observed during the study. Disease progression from 12&ndash;16 wpi was associated with increased acid-fast bacilli, area of secondary granulomatous pneumonia lesions, and Arg1+ and double positive iNOS+/Arg1+ macrophages. Compared to B6 WT, at 16 wpi, B6.Sst1S lungs exhibited an increased area of acid-fast bacilli, larger secondary lesions with greater Arg1+ and double positive iNOS+/Arg1+ macrophages, and reduced T cell density. This morphomolecular analysis of histologic correlates of disease progression in B6.Sst1S could serve as a platform for assessment of medical countermeasures against NTM infection

Epidemiology of asylum seekers and refugees at the Mexico-US border: a cross-sectional analysis from the migrant settlement camp in Matamoros, Mexico

Abstract Background The number of migrants and asylum seekers at the Mexico-US border has increased to historic levels. Our objective was to determine the medical diagnoses and treatments of migrating people seeking care in humanitarian clinics in Matamoros, Mexico. Methods We conducted a cross-sectional study of patient encounters by migrating people through a humanitarian clinic in Matamoros, Mexico, from November 22, 2019, to March 18, 2021. The clinics were operated by Global Response Medicine in concert with local non-governmental organizations. Clinical encounters were each coded to the appropriate ICD-10/CPT code and categorized according to organ system. We categorized medications using the WHO List of Essential Medicines and used multivariable logistic regression to determine associations between demographic variables and condition frequency. Results We found a total of 8,156 clinical encounters, which included 9,744 diagnoses encompassing 132 conditions (median age 26.8 years, female sex 58.2%). People originated from 24 countries, with the majority from Central America (n = 5598, 68.6%). The most common conditions were respiratory (n = 1466, 15.0%), musculoskeletal (n = 1081, 11.1%), and skin diseases (n = 473, 4.8%). Children were at higher risk for respiratory disease (aOR = 1.84, 95% CI: 1.61–2.10), while older adults had greater risk for joint disorders (aOR = 3.35, 95% CI: 1.73–6.02). Women had decreased risk for injury (aOR = 0.50, 95% CI: 0.40–0.63) and higher risk for genitourinary diseases (aOR = 4.99, 95% CI: 3.72–6.85) compared with men. Among 10,405 medications administered, analgesics were the most common (n = 3190, 30.7%) followed by anti-infectives (n = 2175, 21.1%). Conclusions In this large study of a migrating population at the Mexico-US border, we found a variety of clinical conditions, with respiratory, musculoskeletal, and skin illnesses the most common in this study period which encompassed a period of restrictive immigration policy and the first year of the COVID-19 pandemic