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.

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.

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).

Methylene Blue not Contraindicated in Treating Hemodynamic Instability in Pediatric and Neonate Patients.

The present review was carried out to describe publications on the use of methylene blue (MB) in pediatrics and neonatology, discussing dose, infusion rate, action characteristics, and possible benefits for a pediatric patient group. The research was performed on the data sources PubMed, BioMed Central, and Embase (updated on Aug 31, 2020) by two independent investigators. The selected articles included human studies that evaluated MB use in pediatric or neonatal patients with vasoplegia due to any cause, regardless of the applied methodology. The MB use and 0 to 18-years-old patients with vasodilatory shock were the adopted criteria. Exclusion criteria were the use of MB in patients without vasoplegia and patients ≥ 18-years-old. The primary endpoint was the increase in mean arterial pressure (MAP). Side effects and dose were also evaluated. Eleven studies were found, of which 10 were case reports, and 1 was a randomized clinical study. Only two of these studies were with neonatal patients (less than 28 days-old), reporting a small number of cases (1 and 6). All studies described the positive action of MB on MAP, allowing the decrease of vasoactive amines in several of them. No severe side effects or death related to the use of the medication were reported. The maximum dose used was 2 mg/kg, but there was no consensus on the infusion rate and drug administration timing. Finally, no theoretical or experimental basis sustains the decision to avoid MB in children claiming it can cause pulmonary hypertension. The same goes for the concern of a possible deleterious effect on inflammatory distress syndrome.

Why Methylene Blue Have to Be Always Present in the Stocking of Emergency Antidotes.

Evidence-based review of the existing literature ultimately recommends stocking of Methylene Blue (MB) as an emergency antidote in the United States. The same is reported around the world in Japan, Greece, Italy and Canada. The observation that MB is always present as the main antidote required in emergency and critical care units calls for a revisit on its effects on the NO/cGMP system to reemphasize its multisystem actions. Therefore, the present review aimed to display the role of MB in emergency units, concerning: 1) Polytrauma and circulatory shock; 2) Neuroprotection, 3) Anaphylaxis and, 4) Overdose and poisoning.

Methylene blue reduces progression of burn and increases skin survival in an experimental rat model.

Following burn, increased nitric oxide (NO) combine with superoxide anion forming peroxynitrite. Methylene blue (MB) has NO blocking and antioxidant effects. Male Wistar rats (250g) were burned bilaterally in dorsum with a comb metal plate heated inside boiling water and applied during 30s, creating four rectangular 10×20mm full-thickness burned areas separated by three 5×20mm unburned interspaces (stasis zone). 30 rats were randomized into three groups (n=10): treated groups received one dose of intraperitoneal (IP) MB injections (2mg/kg), one or six hours after injury, and control group received saline. Seven days after injury, wounds were visually analyzed for interspaces necrosis; full-thickness sections were evaluated with Masson staining; tissue fragments were processed for nitrite/nitrate (NOx) and malondialdehyde (MDA) dosages. Photographic analysis: interspaces progression to necrosis were higher in control (64.8%) than in one (44.7%) and six (13.3%) hours MB groups (P=0.0060). Histopathology showed lower necrosis percentage in one (34.85%) and six (41.62%) hours MB groups than control (77.03%) (P=0.0034) and higher normal skin percentage in one (25.33%) and six (26.85%) hours MB groups than control (8.32%) (P=0.0037). Re-epithelialization skin areas were higher in both MB groups (39.94% for one and 31.89% for six hours) than control (14.63%) (P=0.0210). Interspace's NOx increased in both MB groups (P=0.0130) with no difference in burned areas. No MDA difference was observed. IP MB injection one or six hours after injury reduced necrosis progression in stasis area in the rat comb burn model suggesting an antioxidant effect reducing oxidative stress.

Bradykinin Impairs and HOE 140 does not Protect Rat Hindlimb Skeletal Muscle Against Tourniquet-induced Reperfusion Injury.

BACKGROUND: Bradykinin (BK) is used in different tissues. Dose-dependent studies have demonstrated that low doses protect against ischemia/reperfusion (I/R) injury while higher doses lead to adverse effects. Although the beneficial effects of BK infusion were observed in myocardium, its role on the I/R impact in skeletal muscle (SM) has not been fully clarified. OBJECTIVE: This study was carried out to evaluate the effects of BK, administered in the hindlimbs of rats subjected to I/R. METHODS: The study design included three experimental groups: Group 1 control (saline), Group 2 (bradykinin), and Group 3 (HOE 140, a BK2 receptor blocker). In all three groups, rats were subjected to hindlimb ischemia for a total of 2 h followed by continuous 4 h of reperfusion with pharmacological interventions. The methods include analysis of enzymes (lactate dehydrogenase-LDH and creatinine phosphokinase-CPK), cell membrane marker of injury (malondialdeyde-MDA), recruitment of neutrophils (myeloperoxidase-MPO), and apoptosis index (immunohistochemistry TUNEL in situ peroxidase dead end). RESULTS: Except for the apoptotic index, all parameters studied were shown to be elevated in the reperfusion group intervened with BK. The blocking of BK2 receptors by HOE 140 did not affect the I/R injury. CONCLUSION: After 2 h of total ischemia, infusion of bradykinin during 4 h of reperfusion, worsened the I/R injury in the hindlimb skeletal muscle.

Chronic fluoxetine treatment increases NO bioavailability and calcium-sensitive potassium channels activation in rat mesenteric resistance arteries.

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has effects beyond its antidepressant properties, altering, e.g., mechanisms involved in blood pressure and vasomotor tone control. Although many studies have addressed the acute impact of fluoxetine on the cardiovascular system, there is a paucity of information on the chronic vascular effects of this SSRI. We tested the hypothesis that chronic fluoxetine treatment enhances the vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling and activation of potassium (K+) channels. Wistar rats were divided into two groups: (I) vehicle (water for 21 days) or (II) chronic fluoxetine (10 mg/kg/day in the drinking water for 21 days). Fluoxetine treatment increased endothelium-dependent and independent vasorelaxation (analyzed by mesenteric resistance arteries reactivity) as well as constitutive NO synthase (NOS) activity, phosphorylation of eNOS at Serine1177 and NO production, determined by western blot and fluorescence. On the other hand, fluoxetine treatment did not alter vascular expression of neuronal and inducible NOS or guanylyl cyclase (GC). Arteries from fluoxetine-treated rats exhibited increased relaxation to pinacidil. Increased acetylcholine vasorelaxation was abolished by a calcium-activated K+ channel (KCa) blocker, but not by an inhibitor of KATP channels. On the other hand, vascular responses to Bay 41-2272 and 8-bromo-cGMP were similar between the groups. In conclusion, chronic fluoxetine treatment increases endothelium-dependent and independent relaxation of mesenteric resistance arteries by mechanisms that involve increased eNOS activity, NO generation, and KCa channels activation. These effects may contribute to the cardiovascular effects associated with chronic fluoxetine treatment.

The effect of extracellular pH changes on intracellular pH and nitric oxide concentration in endothelial and smooth muscle cells from rat aorta.

AIMS: It has been known for more than a century that pH changes can alter vascular tone. However, there is no consensus about the effects of pH changes on vascular response. In this study, we investigated the effects of extracellular pH (pHo) changes on intracellular pH (pHi) and intracellular nitric oxide concentration ([NO]i) in freshly isolated endothelial cells and cross sections from rat aorta. MAIN METHODS: The HCl was used to reduce the pHo from 7.4 to 7.0 and from 7.4 to 6.5; the NaOH was used to increase the pHo from 7.4 to 8.0 and from 7.4 to 8.5. The fluorescent dyes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were employed to measure the pHi and [NO]i, respectively. The fluorescence intensity was measured in freshly isolated endothelial cells by flow cytometry and in freshly obtained aorta cross sections by confocal microscopy. KEY FINDINGS: The endothelial and vascular smooth muscle pHi was increased at pHo 8.5. The extracellular acidification did not change the endothelial pHi, but the smooth muscle pHi was reduced at pHo 7.0. At pHo 8.5 and pHo 6.5, the endothelial [NO]i was increased. Both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]i. SIGNIFICANCE: Not all changes in pHo did result in pHi changes, but disruption of acid-base balance in both directions induced NO synthesis in the endothelium and/or vascular smooth muscle.

Exhaled nitrite/nitrate levels as a marker of respiratory complications after heart valve surgery.

PURPOSE: The purposes of this study are to measure the nitric oxide metabolites nitrite and nitrate (NOx) in the exhaled breath condensates (EBCs) of patients submitted to heart valve surgery and to assess the correlation between NOx levels and postoperative respiratory complications. MATERIALS AND METHODS: Exhaled breath condensate and blood samples were collected from each patient during spontaneous breathing preoperatively, during invasive mechanical ventilation in the fourth hour after surgery and 12, 24, 48, and 72 hours after the operation. Nitrite and nitrate levels in the EBC and serum were measured by chemiluminescence. RESULTS: Thirty-two patients were included in the study. In patients who presented with postoperative respiratory complications, the postoperative levels of NOx were significantly higher in the EBC from the fourth postoperative hour compared with those who experienced uneventful postoperative periods (P = .027). However, the preoperative and postoperative serum levels of NOx were not significantly different in between-group analyses (P = .995). CONCLUSION: Our results suggest that the postoperative NOx level in the EBC is an early marker of respiratory complications after heart valve surgery. Additional studies using large cohorts are necessary to corroborate our results and to better define the clinical usefulness of assessing NOx in the EBC after cardiac surgery.

Effects of elevated perfusion pressure and pulsatile flow on human saphenous veins isolated from diabetic and non-diabetic patients.

OBJECTIVE: This study was carried out to determine high pressure and pulsatile flow perfusion effects on human saphenous vein (HSV) segments obtained from diabetic and non-diabetic patients. METHODS: The veins were perfused with oxygenated Krebs solution for 3 h, with a pulsatile flow rate of 100 mL/min and pressures of 250 × 200 or 300 × 250 mmHg. After perfusion, veins were studied by light microscopy; nitric oxide synthase (NOS) isoforms, CD34 and nitrotyrosine immunohistochemistry and tissue nitrite/nitrate (NO(x)) and malondialdehyde (MDA) quantification. RESULTS: Light microscopy revealed endothelial denuding areas in all HSV segments subjected to 300 × 250 mmHg perfusion pressure, but the luminal area was similar. The percentage of luminal perimeter covered by endothelium decreased as perfusion pressures increased, and significant differences were observed between groups. The endothelial nitric oxide synthase (eNOS) isoform immunostaining decreased significantly in diabetic patients' veins independent of the perfusion pressure levels. The inducible NOS (iNOS), neuronal NOS (nNOS) and nitrotyrosine immunostaining were similar. Significant CD34 differences were observed between the diabetic 300 × 250 mmHg perfusion pressure group and the non-diabetic control group. Tissue nitrite/nitrate and MDA were not different among groups. CONCLUSIONS: Pulsatile flow and elevated pressures for 3 h caused morphological changes and decreased the eNOS expression in the diabetic patients' veins.

Imbalanced matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 activities in patients with thromboangiitis obliterans.

The pathogenic mechanisms of thromboangiitis obliterans (TAO) are not entirely known and the imbalance of matrix metalloproteinases (MMPs) plays a role in vascular diseases. We evaluated the MMP-2 and MMP-9 circulating levels and their endogenous tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in TAO patients with clinical manifestations. The study included 20 TAO patients (n = 10 female, n = 10 male) aged 38-59 years under clinical follow-up. The patients were classified into two groups: (1) TAO former smokers (n = 11) and (2) TAO active smokers (n = 9); the control group included normal volunteer non-smokers (n = 10) and active smokers without peripheral artery disease (n = 10). Patient plasma samples were used to analyze MMP-2 and MMP-9 levels using zymography, and TIMP-1 and TIMP-2 concentrations were determined by enzyme-linked immunosorbent assays. The analysis of MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios (which were used as indices of net MMP-2 and MMP-9 activity, respectively) showed significantly higher MMP-9/TIMP-1 ratios in TAO patients (p < 0.05). We found no significant differences in MMP-2/TIMP-2 ratios (p > 0.05). We found higher MMP-9 levels and decreased levels of TIMP-1 in the TAO groups (active smokers and former smokers), especially in active smokers compared with the other groups (all p < 0.05). MMP-2 and TIMP-2 were not significantly different in patients with TAO as compared to the control group (p > 0.05). In conclusion, our results showed increased MMP-9 and reduced TIMP-1 activity in TAO patients, especially in active smokers compared with non-TAO patients. These data suggest that smoke compounds could activate MMP-9 production or inhibit TIMP-1 activity.

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway.

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/ reperfusion conditions following cilostazol administration. METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/ Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence. RESULTS: Acetylcholine-and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/ Cilostazol/Reperfusion groups. CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/ reperfusion conditions following cilostazol administration. METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/ Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence. RESULTS: Acetylcholine-and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/ Cilostazol/Reperfusion groups. CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endotheliumdependent vascular reactivity under ischemia/reperfusion conditions.

Comparação do uso da pressão positiva com a fisioterapia convencional e incentivadores respiratórios após cirurgia cardíaca: revisão de literatura / Comparison among positive pressure, standard physioterapy and incentive spirometry after cardiac surgery: literature review

Introdução: No pós-operatório de cirurgia cardíaca frequentemente ocorrem complicações pulmonares, as quais podem ser prevenidas e tratadas com técnicas específicas de fisioterapia respiratória. Porém não se sabe qual a técnica mais efetiva. Objetivo: Revisão de literatura com o objetivo de verificara efetividade da pressão positiva (CPAP, VNI-2P, RPPI) comparada às técnicas de fisioterapia convencional e incentivador respiratório (IR) na recuperação da função pulmonar em pacientes no pós-operatório de cirurgia cardíaca. Métodos: Seleção de referências em inglês e português com descritores específicos ao tema nas seguintes fontes de dados: BIREME, SciELO Brazil, LILACS, PUBMED, de 1985 até 2010. Foram incluídos apenas ensaios clínicos randomizados. Resultados: Dez ensaios clínicos randomizadosforam incluídos para revisão. Em relação à superioridade de uma técnica sobre a outra, doisestudos verificaram que a modalidade CPAP e VNI-2P mostrou-se mais efetiva do que a fisioterapia convencional e o IR, enquanto que em dois outros estudos, demonstrou-se a superioridade da VNI-2P, em relação ao uso de cateter de oxigênio e à fisioterapia convencional. Apenas um estudo demonstrou diferença significativa ao comparar duas modalidades de pressão positiva, sendo a RPPI mais efetiva que a CPAP...

Introduction: Postoperative pulmonary complications in patients undergoing cardiac surgeries are usually a clinical challenge, which can be prevented and treated with specific physical therapy techniques. However, it is not known which technique is the most effective. Objective: Literature review with the objective of assessing the effectiveness of positive pressure (CPAP, IPPB, NIV-2P) compared to standard physioterapy therapy and incentive spirometry on improving pulmonary function in postoperative cardiac surgery patients. Methods: English and Portuguese studies were used as references, searching for specific descriptors on the following data sources: BIREME, SciELO Brazil, LILACS, PUBMED, from 1985 to 2010. Only randomized clinical trials were included. Results: Ten randomized control trials were included in this review. About the most effective technique, two studies showed that CPAP and NIV-2P were more effective than standard physioterapy and incentive spirometry. In other two studies, NIV-2P were more effective than nasal oxygen catheter and standard physioterapy...