Markers Of Cell Damage Research Articles

Abstract WMP103: Associations Between Multimorbidity At Stroke/transient Ischaemic Attack Onset With All-cause Mortality Are Not Explained By Blood Biomarkers: A Population-based Study

Introduction: Multimorbidity is highly prevalent in patients with transient ischaemic attack (TIA) or stroke, and is associated with increased short- and long-term mortality thereafter. Underlying mechanisms behind this relationship are unclear, and it is unknown whether increased inflammatory or prothrombotic factors might explain increased mortality. Methods: Using the population-based Oxford Vascular Study (OXVASC), we used a panel of 16 blood biomarkers relating to inflammation (IL-6, CRP, NGAL, sTNF-R1), thrombosis/atherogenesis (TM, fibrinogen, vWF, P-Selectin, PZ, D-Dimer, Anti-PC, ADAMTS-13), and cardiac or neuronal cell damage (NT-proBNP, hFABP, NSE, BDNF). Samples were obtained at stroke/TIA onset, and patients were followed up to 10 years. Biomarker levels were log-transformed to facilitate analysis. Multimorbidity immediately prior to TIA/stroke onset was assessed using the Charlson Comorbidity Index (CCI). Partial correlations (age-adjusted) were obtained between CCI and log-biomarker levels. Crude and adjusted hazard ratios (HRs) for all-cause mortality were obtained using Cox models, censoring at 2, 5, and 10 years. Results: 1297 participants (median age 75.2, interquartile range: 65.3 - 83.2) were recruited from 2002-2012. 596 patients (46.0%) had no CCI comorbidity, 266 (20.5%) had 1, and 435 (33.5%) had 2 or more. After adjustment for age, CCI was correlated with IL-6, NGAL, sTNF-R1, TM, fibrinogen, vWF, D-Dimer, NT-proBNP, and hFABP (all p <0.05). CCI was associated with all-cause death at 10 years follow up (HR 1.51, 95%CI 1.38 - 1.64). This association remained significant after adjustment for age, sex, and stroke severity (HR 1.37, 95%CI 1.26 - 1.49) and after further adjustment for inflammatory markers (HR 1.43, 95%CI 1.26 - 1.62), thrombotic/antiatherogenic markers (HR 1.37, 95%CI 1.20 - 1.56), or markers of cardiac or neuronal cell damage (HR 1.33, 95%CI 1.21 - 1.47). Censoring at 2 or 5 years follow-up provided similar estimates. Conclusion: Multimorbidity was predictive of all-cause mortality after TIA/stroke, but the association was not explained by our panel of biomarkers. Further work is needed to explain the excess risk of mortality associated with multimorbidity in stroke/TIA patients.

Disinhibition-Like Behavior Correlates with Frontal Cortex Damage in an Animal Model of Chronic Alcohol Consumption and Thiamine Deficiency.

Wernicke–Korsakoff syndrome (WKS) is induced by thiamine deficiency (TD) and mainly related to alcohol consumption. Frontal cortex dysfunction has been associated with impulsivity and disinhibition in WKS patients. The pathophysiology involves oxidative stress, excitotoxicity and inflammatory responses leading to neuronal death, but the relative contributions of each factor (alcohol and TD, either isolated or in interaction) to these phenomena are still poorly understood. A rat model was used by forced consumption of 20% (w/v) alcohol for 9 months (CA), TD hit (TD diet + pyrithiamine 0.25 mg/kg, i.p. daily injections the last 12 days of experimentation (TDD)), and both combined treatments (CA+TDD). Motor and cognitive performance and cortical damage were examined. CA caused hyperlocomotion as a possible sensitization of ethanol-induced excitatory effects and recognition memory deficits. In addition, CA+TDD animals showed a disinhibited-like behavior which appeared to be dependent on TDD. Additionally, combined treatment led to more pronounced alterations in nitrosative stress, lipid peroxidation, apoptosis and cell damage markers. Correlations between injury signals and disinhibition suggest that CA+TDD disrupts behaviors dependent on the frontal cortex. Our study sheds light on the potential disease-specific mechanisms, reinforcing the need for neuroprotective therapeutic approaches along with preventive treatments for the nutritional deficiency in WKS.

Polydatin Alleviates Diabetes-Induced Hyposalivation through Anti-Glycation Activity in db/db Mouse.

Polydatin (resveratrol-3-O-β-mono-D-glucoside) is a polyphenol that can be easily accessed from peanuts, grapes, and red wine, and is known to have antiglycation, antioxidant, and anti-inflammatory effects. Diabetes mellitus is a very common disease, and diabetic complications are very common complications. The dry mouth symptom is one of the most common oral complaints in patients with diabetes mellitus. Diabetes mellitus is thought to promote hyposalivation. In this study, we aimed to investigate the improvement effect of polydatin on diabetes-induced hyposalivation in db/db mouse model of type 2 diabetes. We examined salivary flow rate, TUNEL assay, PAS staining, and immunohistochemical staining for AGEs, RAGE, HMGB1, 8-OHdG, and AQP5 to evaluate the efficacy of polydatin in the submandibular salivary gland. Diabetic db/db mice had a decreased salivary flow rate and salivary gland weight. The salivary gland of the vehicle-treated db/db mice showed an increased apoptotic cell injury. The AGEs were highly accumulated, and its receptor, RAGE expression was also enhanced. Expressions of HMGB1, an oxidative cell damage marker, and 8-OHdG, an oxidative DNA damage marker, increased greatly. However, polydatin ameliorated this hypofunction of the salivary gland and inhibited diabetes-related salivary cell injury. Furthermore, polydatin improved mucin accumulation, which is used as a damage marker for salivary gland acinar cells, and decreased expression of water channel AQP5 was improved by polydatin. In conclusion, polydatin has a potent protective effect on diabetes-related salivary gland hypofunction through its antioxidant and anti-glycation activities, and its AQP5 upregulation. This result suggests the possibility of the use of polydatin as a therapeutic drug to improve hyposalivation caused by diabetes.

Biofidelic dynamic compression of human cortical spheroids reproduces neurotrauma phenotypes.

ABSTRACTFundamental questions about patient heterogeneity and human-specific pathophysiology currently obstruct progress towards a therapy for traumatic brain injury (TBI). Human in vitro models have the potential to address these questions. Three-dimensional spheroidal cell culture protocols for human-origin neural cells have several important advantages over their two-dimensional monolayer counterparts. Three-dimensional spheroidal cultures may mature more quickly, develop more biofidelic electrophysiological activity and/or reproduce some aspects of brain architecture. Here, we present the first human in vitro model of non-penetrating TBI employing three-dimensional spheroidal cultures. We used a custom-built device to traumatize these spheroids in a quantifiable, repeatable and biofidelic manner, and correlated the heterogeneous mechanical strain field with the injury phenotype. Trauma reduced cell viability, mitochondrial membrane potential and spontaneous synchronous electrophysiological activity in the spheroids. Electrophysiological deficits emerged at lower injury severities than changes in cell viability. Also, traumatized spheroids secreted lactate dehydrogenase, a marker of cell damage, and neurofilament light chain, a promising clinical biomarker of neurotrauma. These results demonstrate that three-dimensional human in vitro models can reproduce important phenotypes of neurotrauma in vitro.

Dose-dependent expression of extracellular microRNAs in HCT116 colorectal cancer cells exposed to high-dose-rate ionising radiation.

Biomarkers of tumour response to radiotherapy may help optimise cancer treatment. The aim of the present study was to identify changes in extracellular microRNAs (miRNAs) as a biomarker of radiation-induced damage to human colorectal cancer cells. HCT116 cells were exposed to increasing doses of X-rays, and extracellular miRNAs were analysed by microarray. The results were correlated with the frequency of micronuclei. A total of 59 miRNAs with a positive correlation and 4 with a negative correlation between dose (up to 6 Gy) and extracellular miRNA expression were identified. In addition, for doses between 0 and 10 Gy, 12 miRNAs among those 59 miRNAs with a positive correlation were identified; for these extracellular miRNAs, a significantly positive correlation was observed between their expression and the frequency of micronuclei for doses up to 10 Gy. These results suggest that specific miRNAs may be considered as cell damage markers and may serve as secreted radiotherapy response biomarkers for colorectal cancer; however, the results must be further validated in serum samples collected from patients undergoing radiotherapy.

Expression of a Crry/p65 is reduced in acute lung injury induced by extracellular histones.

Acute lung injury (ALI) occurs in patients with severe sepsis and has a mortality rate of 40%–60%. Severe sepsis promotes the release of histones from dying cells, which can induce platelet aggregation, activate coagulation and cause endothelial cell (EC) death. We previously reported that the expression of membrane complement receptor type 1‐related gene Y (Crry)/p65, which plays a principal role in defence against abnormal activation of complement in the blood, is reduced in response to peritoneal mesothelial cell injury, and we hence hypothesized that a similar mechanism occurs in pulmonary ECs. In this study, we examined the role of Crry/p65 in histone‐mediated ALI using an experimental animal model. In ALI model mice, exposure to extracellular histones induces lung injury and results in a decrease in Crry/p65 expression. The levels of lactic acid dehydrogenase (LDH), a marker of cell damage, were significantly increased in the serum of ALI model compared with vehicle mice. The significant inverse correlation between the expression of Crry/p65 and LDH levels in plasma revealed an association between Crry/p65 expression and cell damage. The levels of complement component 3a (C3a) were also significantly increased in the serum of the ALI model compared with vehicle mice. Notably, a C3a receptor antagonist ameliorated lung injury induced by histones. We hypothesize that extracellular histones induce complement activation via down‐regulation of Crry/p65 and that C3a might serve as a therapeutic target for the treatment of ALI.

Quercetin Modulates IGF-I and IGF-II Levels After Eccentric Exercise-Induced Muscle-Damage: A Placebo-Controlled Study.

BackgroundProlonged or unaccustomed eccentric exercise may cause muscle damage and depending from its extent, this event negatively affects physical performance.ObjectivesThe aim of the present investigation was to evaluate, in humans, the effect of the flavonoid quercetin on circulating levels of the anabolic insulin-like growth factor 1 (IGF-I) and insulin-like growth factor 2 (IGF-II), produced during the recovery period after an eccentric-induced muscle damage (EIMD).MethodsA randomized, double-blind, crossover study has been performed; twelve young men ingested quercetin (1 g/day) or placebo for 14 days and then underwent an eccentric-induced muscle damaging protocol. Blood samples were collected, and cell damage markers [creatine kinase (CK), lactate dehydrogenase (LDH) and myoglobin (Mb)], the inflammatory responsive interleukin 6 (IL-6), IGF-I and IGF-II levels were evaluated before the exercise and at different recovery times from 24 hours to 7 days after EIMD.ResultsWe found that, in placebo treatment the increase in IGF-I (72 h) preceded IGF-II increase (7 d). After Q supplementation there was a more marked increase in IGF-I levels and notably, the IGF-II peak was found earlier, compared to placebo, at the same time of IGF-I (72 h). Quercetin significantly reduced plasma markers of cell damage [CK (p<0.005), LDH (p<0.001) and Mb (p<0.05)] and the interleukin 6 level [IL-6 (p<0.05)] during recovery period following EIMD compared to placebo.ConclusionsOur data are encouraging about the use of quercetin as dietary supplementation strategy to adopt in order to mitigate and promote a faster recovery after eccentric exercise as suggested by the increase in plasma levels of the anabolic factors IGF-I and IGF-II.

Custodiol® Supplemented with Synthetic Human Relaxin Decreases Ischemia-Reperfusion Injury after Porcine Kidney Transplantation.

Objective. Ischemia-reperfusion injury (IRI) is inevitable after kidney transplantation (KT), impairing outcomes. Relaxin-2 (RLX) is a promising insulin-related peptide hormone that protects against renal IRI in rodents, although large animal models are needed before RLX can be tested in a human setting. Methods. In this blinded, randomized, and placebo-controlled experimental study kidneys from 19 donor pigs were retrieved after perfusion with Custodiol® ± RLX (5 or 20 nmol/L) and underwent static cold storage (SCS) for 24 and 48 h, respectively. Subsequently, KT was performed after unilateral right nephrectomy. Study outcomes included markers for kidney function, oxidative stress, lipid peroxidation, and endothelial cell damage. PCR analysis for oxidative stress and apoptosis-related gene panels as well as immunohistochemistry were performed. Results. RLX upregulated SOD2 and NFKB expression to 135% (p = 0.042) and 125% (p = 0.019), respectively, while RIPK1 expression was downregulated to 82% (p = 0.016) of corresponding controls. Further RLX significantly downregulated RIPK1 and MLKL expression and decreased the number of Caspase 3- and MPO-positive cells in grafts after SCS. Conclusions. RLX supplemented Custodiol® significantly decreased IRI via both antioxidant and anti-apoptotic mechanisms. Clinical trials are warranted to implement synthetic human RLX as a novel additive to preservation solutions against IRI.

The Role Vascular Endothelial Growth Factor, Control Glycemic, Lipid Profile, and Hypoxia-inducible Factor 1-Alpha at Type 2 Diabetic Patients in North Sumatera, Indonesia

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder whose prevalence continues to increase worldwide. Chronic hyperglycemia increases the area of hypoxia that can be measured by markers of hypoxia-inducible factor 1-alpha (HIF-1α) and endothelial cell damage by vascular endothelial growth factor (VEGF) secretion and the association of the course of diabetes mellitus, dyslipidemia is also a risk factor that can aggravate the condition. diabetes mellitus. AIM: The aim of this study was to correlate VEGF with HIF-1α and other metabolic markers in T2DM. METHODS: Examination such as blood pressure, height, and body mass index, and duration of diabetes were recorded. Laboratory examination like blood sugar levels and glycated hemoglobin (Hba1C) levels, lipid profile such as cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides were evaluated by Paramitha Laboratory Clinic and VEGF and HIF-1α we examined by ELISA methods in the Integrated laboratory of Medical Faculty, Universitas Sumatera Utara. The study was done by cross-sectional analytic methods, among 135 patients with T2DM who were admitted from the various primary health-care centers in Medan city and surrounding areas in North Sumatera. The inclusion criteria of the samples were all the patients diagnosed with T2DM, both the sexes, and the exclusion criteria of the samples with T1DM and severe disease. The data of the samples were processed using a computer with the SPSS program. RESULTS: There was a positive significant correlation between VEGF with HIF-1α, with a strong correlation, and found a negative correlation between VEGF with fasting blood sugar and HDL (p &lt; 0.005). CONCLUSION: By finding strongly and positive correlation between VEGF and HIF-1α, the samples of menunjukkan bahwa peningkatan konsentrasi VEGF meningkat selaras dengan peningkatan konsentrasi dari HIF-1α _dan ini menunjukkan bahwa proses angiogenesis pada sample sedang berlangsung sebagai mekanisme kompensasi pertahanan vascular.

Biomarkers of Early Liver Graft Damage in Circulatory Death and Brain Death Donors: A Propensity Score Matching Analysis

Donation after circulatory death (DCD) is related to a warm ischemia time and more complications compared with traditional donors (donation after brain death [DBD]). This study included biopsy samples retrospectively collected from November 2014 to December 2018 to compare histologic and biological markers of DCD and DBD liver grafts. The analysis includes marker of early apoptosis (p21), senescence (telomerase reverse transcriptase [TERT]), cell damage (caspase-3 active), endothelial damage (vascular endothelial growth factor), stem cell (CD90), hypoxia (HIF1A), inflammatory activation (COX-2), and cross-organ allograft rejection (CD44). A propensity score matching (PSM) was used to match patients receiving DCD livers to those receiving DBD livers. We analyzed the immunohistochemical initial liver damage-related warm ischemia time. Positive staining expression of liver damage biomarkers (COX-2, CD44, TERT, HIF1A, and CD90) was found, but no significant differences were found between DCD and DBD and with ischemic cholangiopathy. After PSM, there was a significant relationship between CD90 and male donors (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.07-0.91), TERT with donor sodium (OR, 1.11; 95% CI, 1.02-1.2), HIF1A with steatosis (OR, 0.33; 95% CI, 0.13-0.83), and CD44 with donor vasoactive drugs (OR, 0.36; 95% CI, 0.13-1) and glutamic oxaloacetic transaminase 1 week increase (OR, 1.01; 95% CI, 1-1.03). DCD immunohistochemical initial liver damage was found to behave similarly to DBD. The increase in complications and cholangiopathy associated with warm ischemia could be related to a different later phenomenon.

Role of Oxidative Stress in the Senescence Pattern of Auditory Cells in Age-Related Hearing Loss.

Age-related hearing loss (ARHL) is an increasing and gradual sensorineural hearing dysfunction. Oxidative stress is an essential factor in developing ARHL; additionally, premature senescence of auditory cells induced by oxidative stress can produce hearing loss. Hydrogen peroxide (H2O2) represents a method commonly used to generate cellular senescence in vitro. The objective of the present paper is to study H2O2-induced senescence patterns in three auditory cell lines (House Ear Institute-Organ of Corti 1, HEI-OC1; organ of Corti, OC-k3, and stria vascularis, SV-k1 cells) to elucidate the intrinsic mechanisms responsible for ARHL. The auditory cells were exposed to H2O2 at different concentrations and times. The results obtained show different responses of the hearing cells concerning cell growth, β-galactosidase activity, morphological changes, mitochondrial activation, levels of oxidative stress, and other markers of cell damage (Forkhead box O3a, FoxO3a, and 8-oxoguanine, 8-oxoG). Comparison between the responses of these auditory cells to H2O2 is a helpful method to evaluate the molecular mechanisms responsible for these auditory cells’ senescence. Furthermore, this in vitro model could help develop anti-senescent therapeutic strategies for the treatment of AHRL.

Dehydrozingerone ameliorates Lipopolysaccharide induced acute respiratory distress syndrome by inhibiting cytokine storm, oxidative stress via modulating the MAPK/NF-κB pathway

BackgroundInflammation-mediated lung injury is a major cause of health problems in many countries and has been the leading cause of morbidity/mortality in intensive care units. In the current COVID-19 pandemic, the majority of the patients experienced serious pneumonia resulting from inflammation (Acute respiratory distress syndrome/ARDS). Pathogenic infections cause cytokine release syndrome (CRS) by hyperactivation of immune cells, which in turn release excessive cytokines causing ARDS. Currently, there are no standard therapies for viral, bacterial or pathogen-mediated CRS. PurposeThis study aimed to investigate and validate the protective effects of Dehydrozingerone (DHZ) against LPS induced lung cell injury by in-vitro and in-vivo models and to gain insights into the molecular mechanisms that mediate these therapeutic effects. MethodsThe therapeutic activity of DHZ was determined in in-vitro models by pre-treating the cells with DHZ and exposed to LPS to stimulate the inflammatory cascade of events. We analysed the effect of DHZ on LPS induced inflammatory cytokines, chemokines and cell damage markers expression/levels using various cell lines. We performed gene expression, ELISA, and western blot analysis to elucidate the effect of DHZ on inflammation and its modulation of MAPK and NF-κB pathways. Further, the prophylactic and therapeutic effect of DHZ was evaluated against the LPS induced ARDS model in rats. ResultsDHZ significantly (p < 0.01) attenuated the LPS induced ROS, inflammatory cytokine, chemokine gene expression and protein release in macrophages. Similarly, DHZ treatment protected the lung epithelial and endothelial cells by mitigating the LPS induced inflammatory events in a dose-dependent manner. In vivo analysis showed that DHZ treatment significantly (p < 0.001) mitigated the LPS induced ARDS pathophysiology of increase in the inflammatory cells in BALF, inflammatory cytokine and chemokines in lung tissues. LPS stimulated neutrophil-mediated events, apoptosis, alveolar wall thickening and alveolar inflammation were profoundly reduced by DHZ treatment in a rat model. ConclusionThis study demonstrates for the first time that DHZ has the potential to ameliorate LPS induced ARDS by inhibiting cytokine storm and oxidative through modulating the MAPK and NF-κB pathways. This data provides pre-clinical support to develop DHZ as a potential therapeutic agent against ARDS.

Targeting of fibroblast activation protein in rheumatoid arthritis patients: imaging and ex vivo photodynamic therapy.

ObjectiveActivated synovial fibroblasts are key effector cells in RA. Selectively depleting these based upon their expression of fibroblast activation protein (FAP) is an attractive therapeutic approach. Here we introduce FAP imaging of inflamed joints using 68Ga-FAPI-04 in a RA patient, and aim to assess feasibility of anti-FAP targeted photodynamic therapy (FAP-tPDT) ex vivo using 28H1-IRDye700DX on RA synovial explants.MethodsRemnant synovial tissue from RA patients was processed into 6 mm biopsies and, from several patients, into primary fibroblast cell cultures. Both were treated using FAP-tPDT. Cell viability was measured in fibroblast cultures and biopsies were evaluated for histological markers of cell damage. Selectivity of the effect of FAP-tPDT was assessed using flow cytometry on primary fibroblasts and co-cultured macrophages. Additionally, one RA patient intravenously received 68Ga-FAPI-04 and was scanned using PET/CT imaging.ResultsIn the RA patient, FAPI-04 PET imaging showed high accumulation of the tracer in arthritic joints with very low background signal. In vitro, FAP-tPDT induced cell death in primary RA synovial fibroblasts in a light dose-dependent manner. An upregulation of cell damage markers was observed in the synovial biopsies after FAP-tPDT. No significant effects of FAP-tPDT were noted on macrophages after FAP-tPDT of neighbouring fibroblasts.ConclusionIn this study the feasibility of selective FAP-tPDT in synovium of rheumatoid arthritis patients ex vivo is demonstrated. Furthermore, this study provides the first indication that FAP-targeted PET/CT can be used to image arthritic joints, an important step towards application of FAP-tPDT as a targeted locoregional therapy for RA.

Hypoxia alters the expression of ACE2 and TMPRSS2 SARS-CoV-2 cell entry mediators in hCMEC/D3 brain endothelial cells.

The mechanisms by which the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induces neurological complications remain to be elucidated. We aimed to identify possible effects of hypoxia on the expression of SARS-CoV-2 cell entry mediators, angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease serine 2 (TMPRSS2) protein, in human brain endothelial cells, in vitro. hCMEC/D3 cells were exposed to different oxygen tensions: 20% (Control group), 8% or 2% O2 (Hypoxia groups). Cells were harvested 6-, 24- and 48 h following hypoxic challenge for assessment of mRNA and protein, using qPCR and Western Blot. The response of the brain endothelial cells to hypoxia was replicated using modular incubator chambers. We observed an acute increase (6 h, p < 0.05), followed by a longer-term decrease (48 h, p < 0.05) in ACE2 mRNA and protein expression, accompanied by reduced expression of TMPRSS2 protein levels (48 h, p < 0.05) under the more severe hypoxic condition (2% O2). No changes in levels of von Willebrand Factor (vWF – an endothelial cell damage marker) or interleukin 6 (IL-6 – a pro-inflammatory cytokine) mRNA were observed. We conclude that hypoxia regulates brain endothelial cell ACE2 and TMPRSS2 expression in vitro, which may indicate human brain endothelial susceptibility to SARS-CoV-2 infection and subsequent brain sequelae.

The effects of cold, dry and heated, humidified amniotic insufflation on sheep fetal membranes

IntroductionUterine distension with pressurised carbon dioxide (CO2) (amniotic insufflation) is used clinically to improve visibility during keyhole fetal surgery. However, there are concerns that amniotic insufflation with unconditioned (cold, dry) CO2 damages the fetal membranes which leads to post-operative preterm prelabour rupture of membranes (iatrogenic PPROM). We assessed whether heating and humidifying the insufflated CO2 could reduce fetal membrane damage in sheep. MethodsThirteen pregnant ewes at 103–106 days gestation underwent amniotic insufflation with cold, dry (22 °C, 0–5% humidity, n = 6) or heated, humidified (40 °C, 95–100% humidity, n = 7) CO2 at 15 mmHg for 180 min. Twelve non-insufflated amniotic sacs acted as controls. Fetal membrane sections were collected after insufflation and analysed for molecular and histological markers of cell damage (caspase 3 and high mobility group box 1 [HMGB1]), inflammation (interleukin 1-alpha [IL1-alpha], IL8 and vascular cell adhesion molecule [VCAM]) and collagen weakening (matrix metalloprotease 9 [MMP9]). ResultsExposure to cold, dry CO2 increased mRNA levels of caspase 3, HMGB1, IL1-alpha, IL8, VCAM and MMP9 and increased amniotic epithelial caspase 3 and HMGB1 cell counts relative to controls. Exposure to heated, humidified CO2 also increased IL8 levels relative to controls however, HMGB1, IL1-alpha and VCAM mRNA levels and amniotic epithelial HMGB1 cell counts were significantly lower than the cold, dry group. DiscussionAmniotic insufflation with cold, dry CO2 damaged the amniotic epithelium and induced fetal membrane inflammation. Heated, humidified insufflation partially mitigated this damage and inflammation in sheep and may prove an important step in reducing the risk of iatrogenic PPROM following keyhole fetal surgery.

Markers of oxidative stress in patients with coronary heart disease and dyslipidaemia

Background and Aims: Changes in the balance between the pro-and antioxidant systems lead to the formation of the earliest markers of cell damage – oxidized modified proteins (OMP), which can lead to a violation of the integrity of the endothelium. Since dyslipidemia is a risk factor for CHD, it is therefore of interest to study the indicators of OMP in patients with CHD and dyslipidemia.

The impact of fluid status and decremental PEEP strategy on cardiac function and lung and kidney damage in mild-moderate experimental acute respiratory distress syndrome

BackgroundWe evaluated the effects of abrupt versus gradual PEEP decrease, combined with standard versus high-volume fluid administration, on cardiac function, as well as lung and kidney damage in an established model of mild-moderate acute respiratory distress syndrome (ARDS).MethodsWistar rats received endotoxin intratracheally. After 24 h, they were treated with Ringer’s lactate at standard (10 mL/kg/h) or high (30 mL/kg/h) dose. For 30 min, all animals were mechanically ventilated with tidal volume = 6 mL/kg and PEEP = 9 cmH2O (to keep alveoli open), then randomized to undergo abrupt or gradual (0.2 cmH2O/min for 30 min) PEEP decrease from 9 to 3 cmH2O. Animals were then further ventilated for 10 min at PEEP = 3 cmH2O, euthanized, and their lungs and kidneys removed for molecular biology analysis.ResultsAt the end of the experiment, left and right ventricular end-diastolic areas were greater in animals treated with high compared to standard fluid administration, regardless of PEEP decrease rate. However, pulmonary arterial pressure, indicated by the pulmonary acceleration time (PAT)/pulmonary ejection time (PET) ratio, was higher in abrupt compared to gradual PEEP decrease, independent of fluid status. Animals treated with high fluids and abrupt PEEP decrease exhibited greater diffuse alveolar damage and higher expression of interleukin-6 (a pro-inflammatory marker) and vascular endothelial growth factor (a marker of endothelial cell damage) compared to the other groups. The combination of standard fluid administration and gradual PEEP decrease increased zonula occludens-1 expression, suggesting epithelial cell preservation. Expression of club cell-16 protein, an alveolar epithelial cell damage marker, was higher in abrupt compared to gradual PEEP decrease groups, regardless of fluid status. Acute kidney injury score and gene expression of kidney injury molecule-1 were higher in the high versus standard fluid administration groups, regardless of PEEP decrease rate.ConclusionIn the ARDS model used herein, decreasing PEEP abruptly increased pulmonary arterial hypertension, independent of fluid status. The combination of abrupt PEEP decrease and high fluid administration led to greater lung and kidney damage. This information adds to the growing body of evidence that supports gradual transitioning of ventilatory patterns and warrants directing additional investigative effort into vascular and deflation issues that impact lung protection.

It’s About Time: Time-Dependent Tissue Damage in the Adult Porcine Retina After Enucleation

The ex vivo large animal retina is extensively used in research ranging from discovery of disease mechanisms to future treatment paradigms. Due to limited standardization when harvesting the tissue, the time after enucleation is often extended for several hours, a factor that so far has not yet been fully characterized. The purpose of this study was to investigate the relationship between time after enucleation and retinal tissue damage. Adult, porcine retinal explants were dissected and fixed 90 or 240 min after enucleation. In a separate experiment, explants were cultured for 48 h, following dissection either 90 or 240 min after enucleation. Retinas were analyzed morphologically using hematoxylin and eosin for overall tissue damage, TUNEL staining for detection of apoptosis, and RBPMS immunohistochemistry for evaluation of ganglion cell survival. In addition, medium from the cultured explants was sampled after 2, 24, and 48 h of culture and assessed for the cell damage marker lactate dehydrogenase (LDH). Retinas examined 240 min after enucleation displayed a significant increase in overall tissue damage, increased apoptosis, and decreased ganglion cell survival compared with 90-min counterparts. In the culture experiment, no significant difference in overall tissue damage was found between the 2 groups, however, apoptosis was significantly increased, and ganglion cell survival decreased in the cultured 240-min group. In addition, a significantly increased LDH medium activity was found in the 240-min group compared with the 90-min counterpart at all time points. The adult porcine retina is relatively resistant to tissue damage 90 min after enucleation but displays distinct signs of injury after 240 min. The importance of these time points is further highlighted when retinal explants are cultured. Our results strongly suggest that time after enucleation is a crucial factor that should be considered in experiments involving the ex vivo adult porcine retina.

Beta cell protective effect of Curcuma longa and Piper nigrum in cytokine cocktail induced apoptosis in Min6 pancreatic beta cells

Background: Type 1 diabetes mellitus is characterized by loss of beta cell mass and insulin insufficiency. Beta cell apoptosis and destruction by cytotoxic T lymphocytes and pro-inflammatory cytokines like IL-1β, TNF-α and IFN-γ appears to represent a key event in pathogenesis of type 1 diabetes mellitus. Purpose: The aim of present study was to evaluate the prophylactic effect of aqueous extract of Curcuma longa and Piper nigrum (CL-PN) in ratio 2:1 on pancreatic beta cell apoptosis and dysfunction induced by cytokine cocktail of IL-1β (50 ng/ml), TNF-α (25 ng/ml) and IFN-γ (25 ng/ml). Methods: We used Min6 beta cell line as an in vitro model and the cells were pre-treated with herbal extract of CL-PN and then exposed to cytokine cocktail for 6 hrs to mimic beta cell destruction as in type 1 diabetes. Beta cell apoptosis and protective activity were assessed by flow cytometer based assay of annexin-V FITC and propidium iodide staining. Results: Aqueous extract of the combination of CL-PN at 1 μg/ml concentration can effectively inhibit apoptosis in Min6 beta cell line by 1.6 fold as compared to cytokine cocktail. It also demonstrated 2.6 fold lesser production of nitric oxide, 1.4 fold reduced ROS and increasing mitochondrial membrane potential which are major markers of beta cell damage. The caspase 3 mRNA expression is also reduced by 4.7 fold despite cytokine challenge. Conclusion: Therefore, the present study paves the way for establishing herbal oral treatment for protection of beta cells in type 1 diabetes mellitus.

ACE2 and Proinflammatory Signaling by S1 Protein of SARS‐Cov‐2 in Human Endothelial Cells

IntroductionCOVID‐19 is primarily a respiratory disease associated with cardiovascular risk. SARS‐CoV‐2, the virus causing COVID‐19, uses ACE2, an important enzyme in the cardiovascular system that regulates the conversion of Ang II (deleterious/pro‐hypertensive) to Ang 1‐7 (protective/anti‐hypertensive), as a receptor for host cell entry and infection. Considering the relationship between the viral S1‐protein and the host's ACE2, it is unclear whether this interaction is merely a mechanism of infection or whether it also contributes to cardiovascular damage associated with COVID‐19. We hypothesisedthat SARS‐Cov‐2‐ACE2 interaction induces activation of vascular cell inflammatory responses that are influenced by ACE2 dependent and/or independent enzymatic Ang‐(1‐7) production.MethodsHuman microvascular endothelial cells (MEC) were used and stimulated with SARS‐CoV‐2 recombinant S1 protein (rS1p) (0.66 μg/mL) at 10/30 min (acute) and 5/24h (chronic). Activation of pro‐inflammatory signaling pathways (immunoblotting, real‐time PCR), microparticle (MP) generation (NanoSight), and cytokine production (ELISA) were assessed. In some experiments, cells were pre‐incubated with an ACE2 activator (DIZE – 190 nM) and inhibitor (MLN‐4760 – 440 pM).ResultsrS1P increased NFκB activation (Control ©=0.99±0.06 vs. 1.38±0.19 AU; p<0.05) and MP formation (C=1.01±0.17 vs. 2.06±0.21, x109/mL; p<0.05), a marker of endothelial cell damage. mRA expression of IL‐1β (C=1.07±0.13 vs. 50.04±4.63 2^‐ddCT), IL6 (C=1.15±0.0.19 vs. 13.52±2 2^‐ddCT), TNFα (C=1.12±0.16 vs. 20.29±2.15 2^‐ddCT), VCAM‐1 (C=1.21±0.24 vs. 13.39±1.57 2^‐ddCT) and MCP‐1 (C=1.02±0.06 vs. 7.01±2.16 2^‐ddCT) was increased by rS1p (p<0.05). This was associated with an increased production of IL6 (C=22.77±3.27 vs. 1221±18.33 pg/mL) and MCP‐1 (C=876.9±33.47 vs. 1110±13.33 pg/mL, (p<0.05). rS1p did not change gene levels of TGFβ, galectin‐3 or ACE2. While DIZE did not influence rS1p pro‐inflammatory markers; MLN‐4760 potentiated rS1p‐induced increase in IL‐1β gene levels (rS1p=27.59±5.05 vs. MLN=62.11±11.17 2^‐ddCT) and blocked rS1p effects on MCP‐1 (rS1p=5.08±0.47 vs. MLN=2.43±0.6 2^‐ddCT) and VCAM‐1 (rS1p=72.82±13.76 vs. MLN=20.74±4.53 2^‐ddCT). ACE2 inhibition did not interfere with rS1p‐induced increase in IL6 or TNFα mRNA expression. Analysis of MP content revealed the expression of ACE2 short form (60 kDa) and flotillin‐1, suggesting that ACE2 localizes in membrane microdomains.ConclusionsOur data demonstrate that in human endotheial cells rS1P induces a pro‐inflammatory response and stimulates generation of ACE2‐containing microparticles. These findings suggest that the spike protein 1 of SARS‐CoV‐2 has a direct injurious effect on the endothelium that may contribute to endotheliitis in COVID‐19. Whether these processes are associated with viral infection await clarification.