Alpha 1-acid glycoprotein is upregulated in severe COVID-19 patients and decreases neutrophil NETs in SARS-CoV-2 infection.

Orosomucoid, or alpha-1 acid glycoprotein (AGP), is a major acute-phase protein expressed in response to systemic injury and inflammation. AGP has been described as an inhibitor of neutrophil migration on sepsis, particularly its immunomodulation effects. AGP's biological functions in coronavirus disease 2019 (COVID-19) are not understood. We sought to investigate the role of AGP in severe COVID-19 infection patients and neutrophils infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Epidemiological data, AGP levels, and other laboratory parameters were measured in blood samples from 56 subjects hospitalized in the ICU with SARS-CoV-2 infection. To evaluate the role of AGP in NETosis in neutrophils, blood samples from health patients were collected, and neutrophils were separated and infected with SARS-CoV-2. Those neutrophils were treated with AGP or vehicle, and NETosis was analyzed by flow cytometry. AGP was upregulated in severe COVID-19 patients (p<0.05). AGP level was positively correlated with IL-6 and C-reactive protein (respectively, p=0.005, p=0.002) and negatively correlated with lactate (p=0.004). AGP treatment downregulated early and late NETosis (respectively, 35.7% and 43.5%) in neutrophils infected with SARS-CoV-2 and up-regulated IL-6 supernatant culture expression (p<0.0001). Our data showed increased AGP in COVID-19 infection and contributed to NETosis regulation and increased IL-6 production, possibly related to the Cytokine storm in COVID-19.

Isolation and primary culture of human abdominal aorta smooth muscle cells from brain-dead donors: an experimental model for vascular diseases.

Primary cell cultures are essential tools for elucidating the physiopathological mechanisms of the cardiovascular system. Therefore, a primary culture growth protocol of cardiovascular smooth muscle cells (VSMCs) obtained from human abdominal aortas was standardized. Ten abdominal aorta samples were obtained from patients diagnosed with brain death who were organ and tissue donors with family consent. After surgical ablation to capture the aorta, the aortic tissue was removed, immersed in a Custodiol® solution, and kept between 2 and 8 °C. In the laboratory, in a sterile environment, the tissue was fragmented and incubated in culture plates containing an enriched culture medium (DMEM/G/10% fetal bovine serum, L-glutamine, antibiotics and antifungals) and kept in an oven at 37 °C and 5% CO2. The aorta was removed after 24 h of incubation, and the culture medium was changed every six days for twenty days. Cell growth was confirmed through morphological analysis using an inverted optical microscope (Nikon®) and immunofluorescence for smooth muscle alpha-actin and nuclei. The development of the VSMCs was observed, and from the twelfth day, differentiation, long cytoplasmic projections, and adjacent cell connections occurred. On the twentieth day, the morphology of the VSMCs was confirmed by actin fiber immunofluorescence, which is a typical characteristic of VSMCs. The standardization allowed VSMC growth and the replicability of the in vitro test, providing a protocol that mimics natural physiological environments for a better understanding of the cardiovascular system. Its use is intended for investigation, tissue bioengineering, and pharmacological treatments.

Cytokine storm in individuals with severe COVID-19 decreases endothelial cell antioxidant defense via downregulation of the Nrf2 transcriptional factor.

The cytokine storm in SARS-CoV-2 infection contributes to the onset of inflammation and target-organ damage. The endothelium is a key player in COVID-19 pathophysiology and it is an important target for cytokines. Considering that cytokines trigger oxidative stress and negatively impact endothelial cell function, we sought to determine whether serum from individuals with severe COVID-19 decreases endothelial cells' main antioxidant defense, i.e., the antioxidant transcriptional factor Nrf2. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from patients with severe COVID-19 at different time points and the effects on redox balance and Nrf2 activity were determined. Serum from individuals with COVID-19 increased oxidant species, as indicated by higher DHE (dihydroethydine) oxidation, increased protein carbonylation, and induced mitochondrial reactive oxygen species (ROS) generation and dysfunction. Serum from patients with COVID-19, but not serum from healthy individuals, induced cell death and diminished nitric oxide (NO) bioavailability. In parallel, Nrf2 nuclear accumulation and the expression of Nrf2-targeted genes were decreased in endothelial cells exposed to serum from individuals with COVID-19. In addition, these cells exhibited higher expression of Bach-1, a negative regulator of Nrf2 that competes for DNA binding. All events were prevented by tocilizumab, an IL-6 receptor blocker, indicating that IL-6 is key to the impairment of endothelial antioxidant defense. In conclusion, endothelial dysfunction related to SARS-CoV-2 infection is linked to decreased endothelial antioxidant defense via IL-6-dependent mechanisms. Pharmacological activation of Nrf2 may decrease endothelial cell damage in individuals with severe COVID-19.NEW & NOTEWORTHY We demonstrate that endothelial cell dysfunction in SARS-CoV-2-infected individuals is linked to decreased activity of the major antioxidant system regulator, the Nrf2 transcription factor. We provide evidence that this phenomenon relies on IL-6, an important cytokine involved in the pathophysiology of COVID-19. Our data support the view that Nrf2 activation is a potential therapeutical strategy to prevent oxidative stress and vascular inflammation in severe cases of COVID-19.

Inhibition of IL-6 signaling prevents serum-induced umbilical cord artery dysfunction from patients with severe COVID-19.

Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.

Implementation and certification of ISO 9001:2015 seal in human tissue bank HCFMRP-USP.

The Quality Management System has been a management technology applied to guarantee the quality of processes and products of a given organization. Thus, ISO 9001:2015 certification assists organizations that want to develop, implement, maintain, and improve a quality management system to enable process improvements and assessments to meet the needs of its customers. This experience report addresses the implementation and certification of the ISO 9001:2015 quality management seal at the Human Tissue Bank of the Ribeirão Preto Clinical Hospital (HCRP) at the University of São Paulo (USP) at Ribeirão Preto Medical School (FMRP)-Brazil. In 2018, the Human Tissue Bank, in partnership with HCRP, received consultancies from PRIMEMODE. The consulting consisted of visits that elucidated the processes to the employees of the Tissue Bank, enabling them to clarify the applicability of all items of the standard. In March 2019, the Tissue Bank received the audit visit and was certified with the ISO 9001:2015 Seal by the Carlos Alberto Vanzolini Foundation. The Tissue Bank aims to provide human tissues for transplantation and research from organ donors. It has a complex physical structure within the required health and legal standards. Also, now it has a quality management seal. The certification guarantees the process organization and the high quality standard of the supplied tissues.

The Role of Interleukins and Inflammatory Markers in the Early Restenosis of Covered Stents in the Femoropopliteal Arterial Segment.

BACKGROUND: The objective of this study was to evaluate the relationship between inflammatory markers, such as interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor α (TNF-α), transforming growth factor ß (TGF-ß), and highly sensitive C-reactive protein, and the development of arterial restenosis 6 months after femoropopliteal percutaneous transluminal angioplasty (PTA) with covered stent implantation. METHODS: We recruited 27 patients of a tertiary hospital in Brazil who were treated with covered stents for atherosclerotic peripheral arterial disease. Serum samples were collected before stent implantation, then 24 hr later, and 6 months after the procedure. RESULTS: At 6-month follow-up, 4 patients (15%) presented restenosis. IL1- ß, IL-6, IL-8, and TNF-α levels showed a statistically significant reduction after both 24 hr and 6 months compared with pretreatment levels (P < 0.01). There were increased levels of IL-10 and TGF-ß both 24 hr and 6 months after PTA and stenting compared with pretreatment levels (P < 0.01). None of the cytokines studied were correlated with restenosis. CONCLUSIONS: This study demonstrated a significant increase in anti-inflammatory TGF-ß and IL-10 and a decrease in proinflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α 6 months after the procedure, but no inflammatory marker was independently identified as a risk factor for in-stent restenosis.

Moderate-to-severe obstructive sleep apnea is associated with telomere lengthening.

Obstructive sleep apnea (OSA) is associated with cardiometabolic diseases. Telomere shortening is linked to hypertension, diabetes mellitus, and cardiovascular diseases. Because these conditions are highly prevalent in OSA, we hypothesized that telomere length (TL) would be reduced in OSA patients. We identified 106 OSA and 104 non-OSA subjects who underwent polysomnography evaluation. Quantitative PCR was used to measure telomere length in genomic DNA isolated from peripheral blood samples. The association between OSA and TL was determined using unadjusted and adjusted linear models. There was no difference in TL between the OSA and non-OSA (control) group. However, we observed a J-shaped relationship between TL and OSA severity: the longest TL in moderate-to-severe OSA [4,918 ± 230 (SD) bp] and the shortest TL in mild OSA (4,735 ± 145 bp). Mean TL in moderate-to-severe OSA was significantly longer than in the control group after adjustment for age, sex, body mass index, hypertension, dyslipidemia, and depression (ß = 96.0, 95% confidence interval: 15.4-176.6, P = 0.020). In conclusion, moderate-to-severe OSA is associated with telomere lengthening. Our findings support the idea that changes in TL are not unidirectional processes, such that telomere shortening occurs with age and disease but may be prolonged in moderate-to-severe OSA.NEW & NOTEWORTHY Here, we show that moderate-to-severe obstructive sleep apnea is associated with longer telomeres, independent of age and cardiovascular risk factors, challenging the hypothesis that telomere shortening is a unidirectional process related to age/disease. A better understanding of the mechanisms underlying telomere dynamics may identify targets for therapeutic intervention in cardiovascular aging/other chronic diseases.

The role of the kallikrein-kinin system, matrix metalloproteinases, and tissue inhibitors of metalloproteinases in the early restenosis of covered stents in the femoropopliteal arterial segment.

OBJECTIVE: The purpose of this study was to investigate the roles of the kallikrein-kinin system and matrix metalloproteinases (MMPs) in the development of arterial restenosis attributable to intimal hyperplasia in the femoropopliteal arteries. METHODS: This report describes a single-center prospective study of 27 patients with peripheral artery disease who required percutaneous transluminal angioplasty and stenting of the femoropopliteal segment using covered stent grafts. The blood concentrations of total and kininogen fractions were evaluated using immunoenzymatic methods. Plasma kallikrein was evaluated by the colorimetric method. Tissue kallikrein was evaluated by the spectrophotometric method. The activity of kininase II was measured by fluorometric analysis. Quantification of MMPs was performed by zymography, and tissue inhibitors of metalloproteinases were measured by enzyme-linked immunosorbent assay. RESULTS: Four (15%) of the treated patients developed restenosis at the 6-month follow-up evaluation. These patients had significantly lower levels of high-molecular-weight kininogens (24 hours; P < .05) and low-molecular-weight kininogens (before, P < .05; 24 hours, P < .01; 6 months, P < .05) and lower levels of tissue inhibitor of metalloproteinases-2 (6 months; P < .05) than the patients without restenosis. The activity levels of plasma and tissue kallikrein, kininase II, and MMPs did not differ significantly between the patients with and without restenosis. CONCLUSIONS: This study demonstrates an involvement of the kallikrein-kinin system in in-stent restenosis, although we could not confirm the participation of metalloproteinases in the restenosis process.

Autonomic cardiocirculatory control in mice with reduced expression of the vesicular acetylcholine transporter.

In cardiovascular diseases, sympathetic tone has been comprehensively studied, whereas parasympathetic tone has received minor attention. The vesicular ACh transporter (VAChT) knockdown homozygous (VAChT KD(HOM)) mouse is a useful model for examining the cardiocirculatory sympathovagal balance. Therefore, we investigated whether cholinergic dysfunction caused by reduced VAChT expression could adversely impact hemodynamic parameter [arterial pressure (AP) and heart rate (HR)] daily oscillation, baroreflex sensitivity, hemodynamic variability, sympathovagal balance, and cardiovascular reactivity to restraint stress. Wild-type and VAChT KD(HOM) mice were anesthetized for telemetry transmitter implantation, and APs and HRs were recorded 10 days after surgical recovery. Changes in HR elicited by methylatropine and propranolol provided the indexes of sympathovagal tone. Cardiovascular reactivity in response to a restraint test was examined 24 h after continuous recordings of AP and HR. VAChT KD(HOM) mice exhibited reduced parasympathetic and elevated sympathetic tone. Daily oscillations of AP and HR as well as AP variability were similar between groups. Nevertheless, HR variability, patterns with two dissimilar variations from symbolic analysis, and baroreflex sensitivity were reduced in VAChT KD(HOM) mice. The change in mean AP due to restraint stress was greater in VAChT KD(HOM) mice, whereas the tachycardic response was not. These findings demonstrate that the cholinergic dysfunction present in the VAChT KD(HOM) mouse did not adversely impact basal hemodynamic parameters but promoted autonomic imbalance, an attenuation of baroreflex sensitivity, and a greater pressure response to restraint stress. These results provide a framework for understanding how autonomic imbalance impacts cardiovascular function.

Characterization of the kallikrein-kinin system, metalloproteinases, and their tissue inhibitors in the in-stent restenosis after peripheral percutaneous angioplasty.

BACKGROUND: The kallikrein-kinin system (KKS) has several direct and indirect effects on cells and cellular mediators involved in the inflammatory process. Studies about inflammation on percutaneous transluminal angioplasty with stent (PTA/stent) to treat peripheral arterial disease (PAD) in humans are scarce. The matrix metalloproteinases (MMPs) are calcium-dependent zinc-containing endopeptidases expressed in various cells and tissues such as fibroblasts, inflammatory cells, and, smooth muscle cells. Changes in the extracellular matrix (ECM) take place in the pathogenesis of many cardiovascular pathologies. MMPs and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]) are crucial in ECM remodeling in both physiologic and pathologic conditions. The aim of this study was to evaluate the role of the KKS and the MMP metabolism, which are important mediators that may contribute to tissue repair, in the process of arterial restenosis due to intimal hyperplasia in the femoropopliteal segment with the aim of developing new interventions. METHODS: Thirty-nine consecutive patients were selected (regardless of ethnic group, age, or sex) for revascularization, who underwent PTA/stent of the femoropopliteal segment. Twenty-five patients with the same clinical characteristics who were scheduled for diagnostic angiography but not subjected to PTA/nitinol stent were also selected. The concentrations in blood of total and kininogen fractions were evaluated using immunoenzymatic methods. Plasma kallikrein was evaluated by the colorimetric method. Tissue kallikrein was evaluated by the spectrophotometric method. The activity of kininase II was measured by fluorometric analysis. Quantification of MMPs was performed by zymography, which is an electrophoresis technique, and TIMPs were measured by enzyme-linked immunosorbent assay. RESULTS: Among the 31 patients who completed the survey, there were 10 cases of angiographically defined restenosis of >50%, and 21 cases without restenosis. There was an increase in the concentrations of the substrates (high-molecular-weight kininogens and lower molecular weight kininogens) and enzymes (plasma and tissue kallikrein) in patients with restenosis, indicating activation of this inflammatory pathway in these patients. The activity of kininase II was not significantly different between the groups of patients studied. There were no statistical differences between restenosis and no restenosis patients for both MMPs and TIMPs dosage, but there is an upward trend of MMPs in time 6 months in patients with restenosis. CONCLUSIONS: With the aim of identifying factors contributing to restenosis after endovascular intervention, this study showed evidence of high activation of the KKS in the pathologic inflammatory process of PTA/stent restenosis. In the other hand, it could not show participation of metalloproteinase metabolism in PTA/stent restenosis.

Intravital Microscopy Evidence That Methylene Blue Should Be a Vasopressor-Sparing Agent in Sepsis Vasoplegia.

Microvasculature failure is expected in sepsis and at higher amine concentrations. Therefore, special attention focused individually on microcirculation is needed. Here, we present that methylene blue can prevent leukocytes from adhering to the endothelium in a rat model of lipopolysaccharide-induced endotoxemia. As hypothesis evidence, an intravital microscopy image is presented.

Incidence of hospital acquired pressure injury in critically ill patients with COVID-19 in prone position admitted to the intensive care unit.

Critical patients have conditions that may favor the occurrence of hospital-acquired pressure injury (HAPI). The objective of this study was to identify the incidence and factors associated with the occurrence of HAPI in patients with coronavirus disease 2019 admitted to the intensive care unit (ICU) who used the prone position. Retrospective cohort study carried out in an ICU of a tertiary university hospital. Two hundred four patients with positive real-time polymerase chain reactions were evaluated, of which 84 were placed in the prone position. All patients were sedated and submitted to invasive mechanical ventilation. Of the prone patients, 52 (62%) developed some type of HAPI during hospitalization. The main place of occurrence of HAPI was the sacral region, followed by the gluteus and thorax. Of the patients who developed HAPI, 26 (50%) had this event in places possibly associated with the prone position. The factors associated with the occurrence of HAPI in patients prone to coronavirus disease 2019 were the Braden Scale and the length of stay in the ICU. The incidence of HAPI in prone patients was extremely high (62%), which denotes the need to implement protocols in order to prevent the occurrence of these events.

Effect of methylene blue on hemodynamic response in the early phase of septic shock: A case series.

RATIONALE: Methylene blue (MB) has been used to increase blood pressure in septic shock, acting on the activity of guanylate cyclase and nitric oxide synthase. PATIENCE CONCERNS: The aim of this study is to demonstrate the benefit of MB in early phase of septic shock.Diagnoses: We report 6 cases of patients with septic shock with up to 72 hours of evolution. INTERVENTIONS: We used MB after fluid replacement, use of norepinephrine and vasopressin. Patients received a loading dose of MB and maintenance for 48 hours. OUTCOMES: All patients presented a reduction in the dose of vasopressors and lactate levels soon after the administration of the loading dose of MB, an effect that was maintained with the maintenance dose for 48 hours. Interleukin 6 and interleukin 8 were elevated at the beginning of the septic condition, with a progressive and marked reduction after the beginning of MB infusion, demonstrating a role of MB in reducing the inflammatory activity. LESSONS: This case series suggests that MB used early in the treatment of septic shock may be useful in reducing vasopressor dose and lactate levels. Further studies are still required to further validate these findings.

Airway epithelial cells and macrophages trigger IL-6-CD95/CD95L axis and mediate initial immunopathology of COVID-19.

Airway epithelial cells (AEC) infected with SARS-CoV-2 may drive the dysfunction of macrophages during COVID-19. We hypothesized that the direct interaction of AEC with macrophages mediated by CD95/CD95L or indirect interaction mediated by IL-6 signaling are key steps for the COVID-19 severe acute inflammation. The interaction of macrophages with apoptotic and infected AEC increased CD95 and CD163 expression, and induced macrophage death. Macrophages exposed to tracheal aspirate with high IL-6 levels from intubated patients with COVID-19 or to recombinant human IL-6 exhibited decreased HLA-DR expression, increased CD95 and CD163 expression and IL-1ß production. IL-6 effects on macrophages were prevented by both CD95/CD95L antagonist and by IL-6 receptor antagonist and IL-6 or CD95 deficient mice showed significant reduction of acute pulmonary inflammation post-infection. Our findings show a non-canonical CD95L-CD95 pathway that simultaneously drives both macrophage activation and dysfunction and point to CD95/CD95L axis as therapeutic target.

Indigo Carmine Hemodynamic Studies to Treat Vasoplegia Induced by Compound 48/80 in a Swine Model of Anaphylaxis.

INTRODUCTION: There are many reasons to believe that the nitric oxide/guanosine 3'5' - cyclic monophosphate (or NO/cGMP) pathway on vasoplegic states is underestimated. To study indigo carmine (IC) as an alternative to methylene blue was the investigation rationale. METHODS: The IC (3mg/kg intravenous infusion) study protocol included five experimental groups; 1) Control group - saline was injected at 0 and 10 minutes; 2) IC group - IC was injected at 0 and saline at 10 minutes; 3) compound 48/80 (C48/80) group - C48/80 was injected at 0 minute and saline at 10 minutes; 4) C48/80 + IC group - C48/80 was injected at 0 minute and IC at 10 minutes; and 5) IC + C48/80 group - IC was injected at 0 minute and C48/80 at 10 minutes. The studies were carried out by registering and measuring hemodynamic and blood gasometric parameters, including continuous cardiac output. RESULTS: 1) The effects of the drugs (IC and C48/80) were more evident in the first 20 minutes of recording; 2) hypotensive responses were more pronounced in the C48/80 groups; 3) IC isolated or applied before C48/80 caused transient pulmonary hypertension; and 4) after the first 20 minutes, the pressure responses showed stability with apparent hypotension more pronounced in the C48/80 groups. Clinical observations showed significant hemodynamic instability and catastrophic anaphylactic reactions (agitation, pulmonary hypertension, severe bronchospasm, urticaria, high-intensity cyanosis, violent gastric hypersecretion, and ascites). CONCLUSION: A global results analysis showed differences between groups only in the first 20 minutes of the experiments.

Assessment of physicians' knowledge about brain death and organ donation and associated factors.

Precocity and assertiveness when diagnosing brain death are essential for identifying potential donors. To assess the knowledge of physicians about brain death and organ donation, cross-sectional web-based survey was carried out with physicians from different specialties. The knowledge about brain death and organ donation was assessed by a questionnaire with 12 multiple-choice or multiple-answer questions (possible range from 0 to 12). The nonparametric Mann-Whitney and Kruskal-Wallis tests were performed to verify the association between the physicians' knowledge and others variables. The project was approved by the Research Ethics Committee of the Hospital das Clínicas, Faculty of Medicine of Ribeirão Preto, University of São Paulo, under number 4.022.657, and all patients agreed to participate and provided free prior-informed consent. Three hundred sixty physicians were included in this study, most of them have postgraduate (55%) and 59.2% were intensive care physicians. The median of responses was 5 (obtained range from 0 to 10). The participants were classified in 2 groups: with satisfactory knowledge (scores above 5) or without satisfactory knowledge (scores equal/below 5). There was better performance among participants who: completed graduation between 6 and 10 years (P < .012); were intensive care physicians (P < .002); had participated in training courses (P < .001); and those who had worked in intensive care unit (ICU) from 6 to 10 years (P < .023); had performed over 10 brain death protocols (P < .001), and felt safe to talk to family members about brain death (P < .001). The results showed that the participants had low knowledge about diagnosis of brain death and organ donation protocols despite the majority working in ICUs. Be an intensive care physician, had large time experience in ICU, and had performed brain death protocols were associated with unsatisfactory knowledge concerning the subject.

Effect of methylene blue on hemodynamic and metabolic response in septic shock patients.

INTRODUCTION: Septic shock is a lethal disease responsible for a large proportion of deaths in the Intensive Care Unit (ICU), even with therapy centered on fluid resuscitation, use of vasopressors and empirical antibiotic therapy applied within the first hour of diagnosis. Considering the multifactorial pathophysiology of septic shock and the mechanism of action of vasopressors, some patients may not respond adequately, which can lead to the maintenance of vasodilatation, hypotension and increased morbidity, and mortality. This protocol aims to verify whether the use of methylene blue in septic patients with an early diagnosis can contribute to an earlier resolution of a shock compared to standard treatment. METHODS AND ANALYSIS: This is a study protocol for a single-center randomized clinical trial design in an ICU of a tertiary university hospital. In this study, we intend to include 64 patients aged between 18 and 80 years with a diagnosis of septic shock, of any etiology, with up to 72 hours of evolution after volume restoration, using norepinephrine at a dose ≥0.2 µg/kg/min and vasopressin at a dose of 0.04 IU/min. After the initial approach, we will randomize patients into two groups, standard care, and standard care plus methylene blue. The sample size was calculated in order to show 30% differences in septic shock resolution between groups. The Research Ethics Committee approved the study, and all patients included will sign an informed consent form (Clinical registration: RBR-96584w4).

Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage.

BACKGROUND AND PURPOSE: Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography. KEY RESULTS: SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities. CONCLUSION AND APPLICATIONS: SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

Timeline Kinetics of Systemic and Airway Immune Mediator Storm for Comprehensive Analysis of Disease Outcome in Critically Ill COVID-19 Patients.

In the present study, the levels of serum and airway soluble chemokines, pro-inflammatory/regulatory cytokines, and growth factors were quantified in critically ill COVID-19 patients (total n=286) at distinct time points (D0, D2-6, D7, D8-13 and D>14-36) upon Intensive Care Unit (ICU) admission. Augmented levels of soluble mediators were observed in serum from COVID-19 patients who progress to death. An opposite profile was observed in tracheal aspirate samples, indicating that systemic and airway microenvironment diverge in their inflammatory milieu. While a bimodal distribution was observed in the serum samples, a unimodal peak around D7 was found for most soluble mediators in tracheal aspirate samples. Systems biology tools further demonstrated that COVID-19 display distinct eccentric soluble mediator networks as compared to controls, with opposite profiles in serum and tracheal aspirates. Regardless the systemic-compartmentalized microenvironment, networks from patients progressing to death were linked to a pro-inflammatory/growth factor-rich, highly integrated center. Conversely, patients evolving to discharge exhibited networks of weak central architecture, with lower number of neighborhood connections and clusters of pro-inflammatory and regulatory cytokines. All in all, this investigation with robust sample size landed a comprehensive snapshot of the systemic and local divergencies composed of distinct immune responses driven by SARS-CoV-2 early on severe COVID-19.

Angiotensin-converting enzyme inhibition augments the expression of rat elastase-2, an angiotensin II-forming enzyme.

Mounting evidence suggest that tissue levels of angiotensin (ANG) II are maintained in animals submitted to chronic angiotensin-converting enzyme (ACE) inhibitor treatment. We examined the expression levels of transcripts for elastase-2, a chymostatin-sensitive serine protease identified as the alternative pathway for ANG II generation from ANG I in the rat vascular tissue and the relative role of ACE-dependent and -independent pathways in generating ANG II in the rat isolated carotid artery rings of spontaneously hypertensive rats (SHR) and Wistar normotensive rats (WNR) treated with enalapril for 7 days. Enalapril treatment decreased blood pressure of SHR only and resulted in significantly more elastase-2 mRNA expression in carotid artery of both enalapril-treated WNR and SHR. Captopril induced a comparable rightward shift of concentration-response curves to ANG I in vehicle and enalapril-treated rats, although this effect was of lesser magnitude in SHR group. Chymostatin induced a rightward shift of the dose response to ANG I in vehicle-treated and a decrease in maximal effect of 22% in enalapril-treated WNR group. Maximal response induced by ANG I was remarkably reduced by chymostatin in enalapril-treated SHR carotid artery (by 80%) compared with controls (by 23%). Our data show that chronic ACE inhibition was associated with augmented functional role of non-ACE pathway in generating ANG II and increased elastase-2 gene expression, suggesting that this protease may contribute as an alternative pathway for ANG II generation when ACE is inhibited in the rat vascular tissue.