Deciphering the Immune Microenvironment on A Single Archival Formalin-Fixed Paraffin-Embedded Tissue Section by An Immediately Implementable Multiplex Fluorescence Immunostaining Protocol

Technological breakthroughs have fundamentally changed our understanding on the complexity of the tumor microenvironment at the single-cell level. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunostaining on formalin-fixed paraffin-embedded (FFPE) tissue samples represents a widespread and simple procedure, allowing the visualization of cellular distribution and processes, on preserved tissue structure. Recent advances in microscopy and molecular biology have made multiplexing accessible, yet technically challenging. We herein describe a novel, simple and cost-effective method for a reproducible and highly flexible multiplex immunostaining on archived FFPE tissue samples, which we optimized for solid organs (e.g., liver, intestine, lung, kidney) from mice and humans. Our protocol requires limited specific equipment and reagents, making multiplexing (>12 antibodies) immediately implementable to any histology laboratory routinely performing immunostaining. Using this method on single sections and combining it with automated whole-slide image analysis, we characterize the hepatic immune microenvironment in preclinical mouse models of liver fibrosis, steatohepatitis and hepatocellular carcinoma (HCC) and on human-patient samples with chronic liver diseases. The data provide useful insights into tissue organization and immune-parenchymal cell-to-cell interactions. It also highlights the profound macrophage heterogeneity in liver across premalignant conditions and HCC

AN ESSENTIAL TOOL TO BE OPTIMIZED: SYNDROMIC MANAGEMENT OF VAGINAL DISCHARGE IN HAITI

Background: Haiti, like many low-income countries in crisis, has limited resources for etiologic diagnosis of vaginal discharge. As such, we sought to characterize variability in diagnoses of women presenting with vaginal discharge syndrome, with the goal to improve standardization of syndromic management. Materials and Methods: Participants aged 18 years and older endorsing vaginitis, or dysuria were recruited at Jerusalem Clinic over two, one-week periods in April 2018 and July 2019. We calculated Spearman rank correlations among history, exam findings, and diagnoses based on clinical presentation, to understand presentation groupings and their management. Results: Among 98 women, median age was 33.5 years, and most frequent symptoms were: vaginal discharge (97%), vaginal itch (73%), and/or suprapubic pain (68%). Most common physical exam findings were vaginal discharge (86%), suprapubic/lower quadrant tenderness (29%), cervical motion tenderness (24%), and cervical erythema (20%). Most symptoms and physical exam findings were weakly correlated with each other. Nearly one-third (31%) were diagnosed with normal physiologic vaginal discharge or no diagnosis, followed by Bacterial vaginosis (31%), vulvovaginal candidiasis (15%), cervicitis/PID (13%), and STI (7%). No reported symptoms strongly differentiated diagnostic categories. Diagnoses varied considerably by exam findings. Conclusions: The weak correlations between symptoms, exam findings, and diagnoses could represent variability in assessment. In the absence of reliable and accessible laboratory testing, the importance of standardizing syndromic management becomes increasingly relevant. Results from our study support the utility of speculum examination and more standardized documentation of physical exam findings. Next steps include the development of local algorithms to promote standardization of treatment of vaginal discharge syndrome

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Behçet’s disease physiopathology: a contemporary review

International audienceBehçet’s disease, also known as the Silk Road Disease, is a rare systemic vasculitis disorder of unknown etiology. Recurrent attacks of acute inflammation characterize Behçet’s disease. Frequent oral aphthous ulcers, genital ulcers, skin lesions and ocular lesions are the most common manifestations. Inflammation is typically self-limiting in time and relapsing episodes of clinical manifestations represent a hallmark of Behçet’s disease. Other less frequent yet severe manifestations that have a major prognostic impact involve the eyes, the central nervous system, the main large vessels and the gastrointestinal tract. Behçet’s disease has a heterogeneous onset and is associated with significant morbidity and premature mortality. This study presents a current immunological review of the disease and provides a synopsis of clinical aspects and treatment options

Identification of VKORC1 genotype leading to resistance to tecarfarin.

International audienceThe VKORC1 gene codes for the VKORC1 enzyme that is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). The use of VKA in human medicine is difficult because of inter-individual variability in VKA requirements. This variability is partly due to basic physiological parameters but also to genetic polymorphisms in CYP2C9 involved in VKA metabolism. Tecarfarin, a new VKA, was developed to avoid the CYP2C9-dependent metabolism. This molecule is metabolized by carboxylesterases. Nevertheless, genetic variations in the VKORC1 gene are also associated with inter-individual variability in VKA requirements. This study was performed to estimate the benefit of using tecarfarin versus traditional VKA in human patient carrier for hVKORC1 mutation. Wild type hVKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to tecarfarin was assessed by the in vitro determination of inhibition constants. Among the 14 mutated VKORC1 proteins analyzed in this study, 6 mutations (i.e., A26P, A41S, V54L, H68Y, I123N, and Y139H) led to resistant to traditional VKAs, while only 4 mutations (i.e., A26P, V54L, I123N, and Y139H) led to resistant to tecarfarin. Compared to other VKAs, a clear benefit of tecarfarin is observed for the mutants A26P, A41S, H68Y, and I123N and an absence of benefit for the mutant Y139H. Finally, tecarfarin is particularly inefficient on V54L-VKORC1

Identification of VKORC1 genotype leading to resistance to tecarfarin.

International audienceThe VKORC1 gene codes for the VKORC1 enzyme that is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). The use of VKA in human medicine is difficult because of inter-individual variability in VKA requirements. This variability is partly due to basic physiological parameters but also to genetic polymorphisms in CYP2C9 involved in VKA metabolism. Tecarfarin, a new VKA, was developed to avoid the CYP2C9-dependent metabolism. This molecule is metabolized by carboxylesterases. Nevertheless, genetic variations in the VKORC1 gene are also associated with inter-individual variability in VKA requirements. This study was performed to estimate the benefit of using tecarfarin versus traditional VKA in human patient carrier for hVKORC1 mutation. Wild type hVKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to tecarfarin was assessed by the in vitro determination of inhibition constants. Among the 14 mutated VKORC1 proteins analyzed in this study, 6 mutations (i.e., A26P, A41S, V54L, H68Y, I123N, and Y139H) led to resistant to traditional VKAs, while only 4 mutations (i.e., A26P, V54L, I123N, and Y139H) led to resistant to tecarfarin. Compared to other VKAs, a clear benefit of tecarfarin is observed for the mutants A26P, A41S, H68Y, and I123N and an absence of benefit for the mutant Y139H. Finally, tecarfarin is particularly inefficient on V54L-VKORC1

Brain-Targeting Form of Docosahexaenoic Acid for Experimental Stroke Treatment: MRI Evaluation and Anti-Oxidant Impact

Epidemiologic studies report cardiovascular protection conferred by omega-3 fatty acids, in particular docosahexaenoic acid (DHA). However, few experimental studies have addressed its potential in acute stroke treatment. The present study used multimodal MRI to assess in vivo the neuroprotection conferred by DHA and by a brain-targeting form of DHA-containing lysophosphatidylcholine (AceDoPC) in experimental stroke. Rats underwent intraluminal middle cerebral artery occlusion (MCAO) and were treated at reperfusion by intravenous injection of i) saline, ii) plasma from donor rats, iii) DHA or iv) AceDoPC, both solubilized in plasma. Twenty-four hours after reperfusion, animals underwent behavioral tests and were sacrificed. Multiparametric MRI (MRA, DWI, PWI, T2-WI) was performed at H0, during occlusion, and at H24, before sacrifice. Brain tissue was used for assay of F2-isoprostanes as lipid peroxidation markers. Initial lesion size and PWI/DWI mismatch were comparable in the four groups. Between H0 and H24, lesion size increased in the saline group (mean +/- s.d.: +18%+/- 20%), was stable in the plasma group (-3%+/- 29%), and decreased in the DHA (-17%+/- 15%, P=0.001 compared to saline) and AceDoPC (-34%+/- 29%, P=0.001 compared to saline) groups. Neuroscores in the AceDoPC group tended to be lower than in the other groups (P=0.07). Treatments (pooled DHA and AceDoPC groups) significantly decreased lipid peroxidation as compared to controls (pooled saline and vehicle) (P=0.03). MRI-based assessment demonstrated the neuroprotective effect of DHA in the MCAO model. Results further highlighted the therapeutic potential of engineered brain-targeting forms of omega-3 fatty acids for acute stroke treatment

Brain-Targeting Form of Docosahexaenoic Acid for Experimental Stroke Treatment: MRI Evaluation and Anti-Oxidant Impact

Epidemiologic studies report cardiovascular protection conferred by omega-3 fatty acids, in particular docosahexaenoic acid (DHA). However, few experimental studies have addressed its potential in acute stroke treatment. The present study used multimodal MRI to assess in vivo the neuroprotection conferred by DHA and by a brain-targeting form of DHA-containing lysophosphatidylcholine (AceDoPC) in experimental stroke. Rats underwent intraluminal middle cerebral artery occlusion (MCAO) and were treated at reperfusion by intravenous injection of i) saline, ii) plasma from donor rats, iii) DHA or iv) AceDoPC, both solubilized in plasma. Twenty-four hours after reperfusion, animals underwent behavioral tests and were sacrificed. Multiparametric MRI (MRA, DWI, PWI, T2-WI) was performed at H0, during occlusion, and at H24, before sacrifice. Brain tissue was used for assay of F2-isoprostanes as lipid peroxidation markers. Initial lesion size and PWI/DWI mismatch were comparable in the four groups. Between H0 and H24, lesion size increased in the saline group (mean +/- s.d.: +18%+/- 20%), was stable in the plasma group (-3%+/- 29%), and decreased in the DHA (-17%+/- 15%, P=0.001 compared to saline) and AceDoPC (-34%+/- 29%, P=0.001 compared to saline) groups. Neuroscores in the AceDoPC group tended to be lower than in the other groups (P=0.07). Treatments (pooled DHA and AceDoPC groups) significantly decreased lipid peroxidation as compared to controls (pooled saline and vehicle) (P=0.03). MRI-based assessment demonstrated the neuroprotective effect of DHA in the MCAO model. Results further highlighted the therapeutic potential of engineered brain-targeting forms of omega-3 fatty acids for acute stroke treatment