Red Blood Cell Malfunction in COVID-19: Molecular Mechanisms and Therapeutic Targets.

The world has been facing a pandemic for the past 2 years. COVID-19 still leads to millions of deaths worldwide, while deteriorating the global economy. The need for therapeutic targets, thus, remains. Interestingly, red blood cells, apart from gas exchange, also serve as modulators of innate and adaptive immunity. This function is accommodated mainly by surface molecules (proteins, lipids, and carbohydrates) and increased antioxidant capacity. However, under the circumstances of a disease state, red blood cells can become proinflammatory cells. Recent evidence has shown that, in the context of COVID-19, erythrocytes present protein oxidation, decreased antioxidant capacity, increased glycolysis, altered membrane lipidome, increased binding of Cytosine-Guanine (CpG) DNA and complement proteins, and low CD47 levels. These changes lead to an erythrocyte-dependent inflammation, which possibly participates in the hyperinflammation status of COVID-19. The current knowledge for the dysfunction of red blood cells during COVID-19 implies that the BAND3 protein and toll-like receptor 9 are potential therapeutic targets for COVID-19.

Lysophospholipid Metabolism and Signalling in Non-Alcoholic Fatty Liver Disease.

Non-alcoholic liver disease (NAFLD) constitutes a global health pandemic. It is estimated that about 25% of the world's population suffers from NAFLD. In the long-term, a subgroup of the patients can develop inflammation and fibrosis. The end result in some cases is cirrhosis and even liver-related death. The epidemiology and natural history of NAFLD lead to extreme financial costs.

Red Blood Cell-Conditioned Media from Non-Alcoholic Fatty Liver Disease Patients Contain Increased MCP1 and Induce TNF-α Release.

Background: Non-alcoholic fatty liver disease (NAFLD) constitutes a global pandemic. An intricate network among cytokines and lipids possesses a central role in NAFLD pathogenesis. Red blood cells comprise an important source of both cytokines and signaling lipids and have an important role in molecular crosstalk during immunometabolic deregulation. However, their role in NAFLD has not been thoroughly investigated. Methods: Conditioned media from erythrocytes derived from 10 NAFLD patients (4 men, 6 women, aged 57.875±15.16) and 10 healthy controls (4 men, 6 women, aged 39.3±15.55) was analyzed for the cytokines IFN-γ, TNF-α, CCL2, CCL5, IL-8, IL-1ß, IL-12p40, IL-17, MIP-1ß, the signaling lipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), and cholesterol. Their effect on the cytokine profile released by RAW 264.7 macrophages was also studied. Results: MCP1 levels were greater in conditioned growth medium from NAFLD patient erythrocytes than in that from healthy controls (37±40 vs 6.51±5.63 pg/ml). No statistically significant differences were found between patients and healthy controls with regard to S1P, LPA, cholesterol, or eight other cytokines. TNF-a release by RAW 264.7 cells was greater after incubation with patient-derived erythrocyte-conditioned medium than in medium without RAW 264.7 cells from either healthy or NAFLD subjects. Conclusion: Erythrocytes may contribute to liver infiltration by monocytes, and macrophage activation, partially due to CCL2 release, in the context of NAFLD..

Molecular Interactions between Erythrocytes and the Endocrine System.

Hormones are secreted by the endocrine glands and reach their targets after circulating in the blood. Many studies have documented that erythrocytes can bind hormones, and possible interactions have been reported. Erythrocytes are responsive to signaling initiated after binding of epinephrine, norepinephrine, estrogen, progesterone, thyroid hormones, parathyroid hormone, and angiotensin. Signaling results in regulation of cellular metabolism and membrane fluidity. In addition, erythrocytes are circulating pools for dopamine, thyroid hormones, cortisol, and aldosterone. Erythrocyte function and structure are regulated by endocrine signals, while erythrocytes are important constituents for the transport of hormones in the body.

Lipotoxicity Disrupts Erythrocyte Function: A Perspective.

BACKGROUND: Lipid accumulation in the liver, skeletal and cardiac muscle, kidneys and pancreas causes cell dysfunction, death and inflammation, a biological phenomenon named lipotoxicity. Erythrocytes participate in the transport of lipids in the circulation, and their lipidome is determined by exchange with blood components. OBJECTIVE: The objective of this study is to summarize the current knowledge regarding the effect of toxic lipid accumulation in erythrocytes. RESULTS: Erythrocyte lipidome is altered in lipotoxic diseases, such as fatty liver disease, heart failure and diabetes. In addition, ceramide, lysophosphatidylcholine, lysophosphatidic acid, palmitic acid and free cholesterol induce erythrocyte malfunction. CONCLUSION: Erythrocytes are an additional cell target of lipotoxicity. Further exploration of the implicated molecular mechanisms could lead to novel therapeutic targets for cardiometabolic and hematological diseases.

Immune and Metabolic Interactions of Human Erythrocytes: A Molecular Perspective.

Apart from their main function as oxygen carriers in vertebrates, erythrocytes are also involved in immune regulation. By circulating throughout the body, the erythrocytes are exposed and interact with tissues that are damaged as a result of a disease. In this study, we summarize the literature regarding the contribution of erythrocytes to immune regulation and metabolism. Under the circumstances of a disease state, the erythrocytes may lose their antioxidant capacity and release Damage Associated Molecular Patterns, resulting in the regulation of innate and adaptive immunity. In addition, the erythrocytes scavenge and affect the levels of chemokines, circulating cell-free mtDNA, and C3b attached immune complexes. Furthermore, through surface molecules, erythrocytes control the function of T lymphocytes, macrophages, and dendritic cells. Through an array of enzymes, red blood cells contribute to the pool of blood's bioactive lipids. Finally, the erythrocytes contribute to reverse cholesterol transport through various mechanisms. Our study is highlighting overlooked molecular interactions between erythrocytes and immunity and metabolism, which could lead to the discovery of potent therapeutic targets for immunometabolic diseases.

Cholesterol and Phospholipid Distribution Pattern in the Erythrocyte Membrane of Patients with Hepatitis C and Severe Fibrosis, before and after Treatment with Direct Antiviral Agents: A pilot Study.

Objectives: Hepatitis C virus requires and induces changes in liver lipidome for its life cycle. In addition, alterations in plasma and erythrocyte lipidome are observed during a range of chronic liver diseases. Methods: A total of six subjects (three males and three females) were included in our study. All subjects were HCV positive according to virus RNA detection. Erythrocyte ghosts were prepared from blood and collected upon diagnosis and also at the end of the treatment with Direct Antiviral Agents (DAA). Lipids were extracted from the erythrocyte ghosts, and cholesterol and phospholipids were analyzed by thin layer chromatography. A semi-quantitative estimation of cholesterol (CHOL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and sphingomyelin (SM) was performed by densitometric analysis of the chromatographs. Results: After the antiviral treatment, PE percentage decreased, whereas the PC/PE and CHOL/PE ratio increased significantly. There were also other weaker differences for CHOL, PI, PS, PC and SM. Before DAA there was a very weak correlation between ALT and PC/PE ratio. In contrast, there was a steep negative correlation between these two parameters after DAA. Conclusion: Red blood cell lipid composition and especially the PC/PE ratio could be a candidate real time biological marker for inflammation resolution during hepatitis C treatment.

Red Blood Cell Dysfunction in Non-Alcoholic Fatty Liver Disease: Marker and Mediator of Molecular Mechanisms.

Despite efforts to unravel the pathogenetic mechanisms of non-alcoholic fatty liver disease (NAFLD), there is still a need for approved treatments and biomarkers. Interestingly, red blood cells present alterations in their characteristics during NAFLD. The phosphatidylcholine to phosphatidylethanolamine ratio, fatty acid profile, red blood cell count and red cell distribution width reflect molecular changes that are taking place in the liver. In addition, glycosylated hemoglobin, chemokine binding and release, and phosphatidylserine exposure actively participate in NAFLD pathogenesis. In this review, we describe the neglected red blood cell dysfunction in NAFLD, with the aim to unveil potent biomarkers and therapeutic targets.

Correlation of Baseline Plasma and Inguinal Connective Tissue Metalloproteinases and Their Inhibitors With Late High-Pressure Endoleak After Endovascular Aneurysm Repair: Long-term Results.

Purpose: To investigate whether plasma and connective tissue matrix metalloproteinases (MMP) and their inhibitors (TIMP) may predict late high-pressure endoleak after endovascular aneurysm repair (EVAR). Materials and Methods: Samples of inguinal fascia and blood were collected in 72 consecutive patients (mean age 73.1 years; 68 men) undergoing primary EVAR with the Endurant stent-graft. Baseline plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 and baseline MMP-2 and MMP-9 activity estimated using gelatin zymography (GZ) were compared between patients who developed late endoleak in follow-up and those who did not. Subgroup analyses were performed between patients with (n=18) and without inguinal hernias and between patients with moderate-diameter (50-59 mm; n=45) or large-diameter (≥60 mm; n=27) abdominal aortic aneurysms (AAA) at primary EVAR. Results: The mean follow-up period was 63.1 months (range 7.5-91.5), during which time 13 (18.1%) patients developed type I (6 Ia and 5 Ib) or 2 type III endoleaks. Only GZ-analyzed proMMP-9 concentrations were higher in the endoleak group than in patients without endoleak (mean difference 8.44, 95% CI -19.653 to -1.087, p=0.03). The patients with primary inguinal hernia at presentation had significantly higher tissue TIMP-2 values (0.8±0.7 vs 0.5±0.4, p=0.018) but lower plasma total (pro- + active) MMP-9 values (11.9±7.8 vs 16.2±7.4, p=0.042) than patients without hernias at the time of EVAR. Patients with AAAs ≥60 mm had significantly higher mean tissue homogenate levels of total (pro- + active) MMP-9 (p=0.025) and total (pro- + active) MMP-2 (p=0.049) as well as higher proMMP-9 (p=0.018) and total (pro- + active) MMP-9 (p=0.021) levels based on GZ compared to patients with moderate-diameter AAAs. Regression analysis revealed a significant association between total (pro- + active) MMP-9 plasma samples and the presence of hernia (OR 0.899, 95% CI 0.817 to 0.989, p=0.029) and between GZ-analyzed proMMP-9 and late endoleak (OR 1.055, 95% CI 1.007 to 1.106, p=0.025). GZ-analyzed proMMP-9 and active MMP-9 were strong predictors of late endoleak in patients with hernia (p=0.012 and p=0.044, respectively) and in patients with AAAs ≥60 mm (p=0.018 and p=0.041 respectively). Conclusion: Inguinal fascial tissue proMMP-9 significantly predicted late endoleak. ProMMP-9 and active MMP-9 biomarkers are significantly associated with late endoleak in hernia patients and in patients with AAAs ≥60 mm. Considering the clinical association between hernia and AAA and the fact that the AAA wall connective tissue environment remains exposed to systemic circulation after EVAR, inguinal fascia extracellular matrix dysregulation and altered MMP activity may reflect similar changes in AAA biology, leading to complications such as endoleak.

Gut Microbial Signatures Underline Complicated Crohn's Disease but Vary Between Cohorts; An In Silico Approach.

Microflora dysbiosis is implicated in the pathophysiology of Crohn's disease. This work analyzes differences in microbial communities and relevant metabolic pathways among the nonstricturing nonpenetrating (B1), stricturing (B2), and penetrating (B3) subphenotypes of Crohn's disease vs healthy controls. We conducted a bioinformatics analysis using the QIIME pipeline and the Calypso, linear discriminant analysis effect size, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, and STAMP tools on publicly available 16S bacterial rRNA sequencing data from terminal ileum mucosal biopsies of healthy controls and the 3 subphenotypes of Crohn's disease. We analyzed differences in microbial diversity and taxonomy, inferred active metabolic pathways via relevant genes' abundance, and detected bacterial families that could serve as biomarkers. Microbiota α-diversity was decreased within all 3 Crohn's disease subphenotypes vs control samples, with more significant reductions in B2 and B3 compared with B1. ß-diversity analysis identified similar microbial patterns in B2 and B3 samples, different from those of B1 and from those of healthy controls. Abundance analysis of microbial families in cohorts, beyond altered abundances compared with healthy controls, highlighted significant differences between the B2 and B3 subphenotypes and the B1 subphenotype. A similar pattern was observed in the inference of microbial metabolomics: the B2 and B3 cohorts had different predicted metabolotypes from the B1 cohort, in addition to differences observed in Crohn's disease vs healthy controls. Our findings indicate distinct microbiome signatures in complicated Crohn's disease subphenotypes and provide the basis for further investigation into the role of gut microflora in the natural course of Crohn's disease.

Retrobulbar hemodynamics and aqueous humor levels of endothelin-1 in exfoliation syndrome and exfoliation glaucoma.

BACKGROUND: Endothelin-1 (ET-1) is the most potent vasoconstrictor in the body. There are reports in the literature correlating plasma levels of ET-1 and its impact on retrobulbar hemodynamics. This study evaluates aqueous humor levels of ET-1 and retrobulbar hemodynamics in patients with exfoliation syndrome (XFS) and exfoliation glaucoma (XFG). PATIENTS AND METHODS: Patients scheduled for cataract surgery were included. Patients were allocated to non-exfoliation non-glaucoma group (controls), XFG and XFS groups. Peak systolic velocity (PSV), end diastolic velocity, and resistivity index of the ophthalmic artery (OA), short posterior ciliary arteries, and central retinal artery (CRA) were determined preoperatively using color Doppler imaging. Aqueous humor samples obtained at the beginning of cataract surgery were analyzed with enzyme-linked immunosorbent assay. RESULTS: Sixty-nine participants of similar age were included (controls: n=24, XFG: n=22, XFS: n=23). Multiple regression analysis showed that ET-1, OA-PSV, OA-resistivity index, CRA-PSV, and CRA-end diastolic velocity were influenced by group but not by sex or age. ET-1 for the XFG group (15.93±2.8 pg/mL) was significantly higher than for the XFS (8.92±2.7 pg/mL) and control (8.44±2.6 pg/mL) groups. The difference in ET-1 titers between the control and XFS groups was not statistically significant. All four hemodynamic parameters affected by group status significantly correlated with ET-1 levels in eyes with XFS. In eyes with XFG, two of the four hemodynamic parameters significantly correlated with ET-1 levels. In control eyes, no correlation between ET-1 and hemodynamic parameters affected by group status was detected. CONCLUSION: ET-1 aqueous levels in eyes with XFG were significantly higher than those of age-matched controls. Reduced blood flow and increased vascular resistivity were detected in the OA and the CRA in eyes with XFG/XFS. ET-1 levels in eyes with XFG/XFS exhibit a significant correlation with hemodynamic parameters that indicate reduced perfusion.

Intravasation of SW620 colon cancer cell spheroids through the blood endothelial barrier is inhibited by clinical drugs and flavonoids in vitro.

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.

Prepartum Psychosis and Neonaticide: Rare Case Study and Forensic-Psychiatric Synthesis of Literature.

Peripartum psychosis is a rare but serious psychiatric disorder characterized by the presence of a mood episode with psychotic features. Although controversy surrounds the nosological status of peripartum mental disorders, these conditions continue to be of exceptional interest to the medical and forensic mental health communities. The aim of this study was to report a rare case of prepartum psychosis which escalated to the endpoint of neonaticide and summarize literature on peripartum mental disorders and infanticide. A 30-year-old mother murdered her newborn with the spike of her serum delivery system and planned to commit suicide while in hospital after hallucinating due to an acute puerperal psychotic disorder with a prepartum onset and postpartum deterioration. Her disorder was not managed until neonaticide. Throughout this paper, the significance of a multidisciplinary approach for the optimal management of these incidents is highlighted and diagnostic as well as therapeutic issues are addressed.

Room air versus carbon dioxide pneumoperitoneum: effects on oxidative state, apoptosis and histology of splanchnic organs.

BACKGROUND: Although CO2 is the insufflation gas of choice in laparoscopic procedures, room air is usually used in natural orifice transluminal endoscopic surgery. The aim of the present study was to compare the safety of room air versus CO2 pneumoperitoneum in terms of their effect on the oxidative state, apoptosis and tissue injury of splanchnic organs. METHODS: Eighteen Wistar rats were assigned to three groups (n = 6 per group) and were subjected to 8 mm Hg room air (group Pne-Air) or CO2 pneumoperitoneum (group Pne-CO2) or sham operation for 60 min. Forty-five minutes postdeflation, tissue samples were excised from the liver, stomach, ileum and kidneys for reduced glutathione-to-glutathione disulfide (GSH/GSSG) ratio, caspase-8 and caspase-3 and hypoxia-inducible factor-1α (HIF-1α) immunohistochemical assessment and histopathologic examination. RESULTS: GSH/GSSG ratio substantially declined in both pneumoperitoneum groups. No change was noted in HIF-1α expression. Mild upregulation of caspase-8 and caspase-3 was noted in both pneumoperitoneum groups being less pronounced in group Pne-Air. Histopathologic score was increased in all organs studied, but the stomach, in both pneumoperitoneum groups. CONCLUSION: Pneumoperitoneum established by either room air or CO2 induced substantial oxidative stress, mild apoptosis and mild tissue injury in splanchnic organs. While air pneumoperitoneum conferred a less pronounced apoptotic effect, the oxidative state and histopathologic profile of splanchnic organs did not differ between insufflation gases.

Regional disparities in cancer mortality across the rural-urban axis: a case study from north-eastern Greece.

INTRODUCTION: The aim of this study was to identify differences in cancer mortality in north-eastern Greece, to describe potential drivers operating at the population level and to propose practical interventions and mitigation strategies. METHODS: Cancer mortality data were collected from local registries using the WHO 10th edition of International Classification of Disease (ICD-10). The direct standardization method was used to address demographic differences in the two regions, with the Standard European Population as reference. Rate ratios (RR) were employed for comparisons and 95% confidence intervals (95%CI) were calculated according to the Poisson approximation method. RESULTS: An increased risk of digestive system cancers (excluding liver neoplasms) was observed in rural versus urban areas (RR=1.25, 95%CI=1.02-1.54). Stomach cancer, in particular, was more prevalent in the older cohorts (>65 years), suggesting a historical epidemiological perspective. A more pronounced discrepancy was observed for prostate cancer mortality (RR=1.86, 95%CI=1.10-3.14), indicating a strong positive correlation with rurality. CONCLUSIONS: Cancer mortality disparities have been observed between rural and urban regions of north-eastern Greece. Health promotion and education, including improved access to medical facilities and early cancer screening, can help mitigate the burden and extend survival rates. Decreasing cancer staging at the time of diagnosis and reversing social and economic inequalities is key for combating these types of malignancy.

Erythrocyte membrane cholesterol and lipid core growth in a rabbit model of atherosclerosis: modulatory effects of rosuvastatin.

BACKGROUND: Lipid core expansion is partly responsible for the conversion of a stable atherosclerotic lesion to a rupture-prone plaque. Intraplaque hemorrhage contributes to the accumulation of cholesterol within unstable plaques. In the present study, we investigated, using a rabbit model of atherosclerosis, the extent to which diet-induced increases in cholesterol content of erythrocyte membranes (CEM) contribute to lipid core expansion and the modulatory effect of rosuvastatin use. METHODS AND RESULTS: Rabbits fed with atherogenic diet (0.75% cholesterol) for 5 months exhibited advanced atherosclerotic lesions (mean plaque area, 0.39 ± 0.03 mm(2)), and lipid core size was associated with the concentration-time integral (CTI) of CEM levels (r=0.567, P=0.004) independent of other established predictors of lipid core size. Further experiments were performed by feeding rabbits atherogenic diet (1% cholesterol) for 3 months, followed by either normal diet or normal diet plus rosuvastatin for the next 3 months. Although no differences were observed in total plaque area between both groups, administration of rosuvastatin was associated with significantly smaller lipid cores, fewer macrophages within the lipid core, less microvessels as well as with lower CTI of CEM levels compared to normal diet alone. Moreover, intraplaque erythrocyte membranes covered a smaller lipid core area in rabbits under rosuvastatin plus normal diet as opposed to rabbits under diet alone. CONCLUSIONS: Increased CEM levels, induced by high-cholesterol diet, are associated with lipid core growth. Ingestion of a potent HMG-CoA reductase inhibitor (rosuvastatin) may decrease CEM levels, and this effect may contribute to regression of the lipid core.

Lipopolysaccharide-binding protein: a potential marker of febrile urinary tract infection in childhood.

BACKGROUND: Urinary tract infections (UTIs) are encountered frequently in children, and their early diagnosis and treatment are important. This study evaluates the diagnostic value of serum concentrations of lipopolysaccharide-binding protein (LBP), an acute-phase protein, in children with febrile UTI and compares it to those of the total white blood cell count (WBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6). METHODS: The study population comprised 77 consecutive patients with a first-episode febrile UTI (33 boys) with a median age of 11 months [interquartile range (IQR), 5.5-33 months], 21 healthy controls (11 boys) with a median age of 10 months (IQR, 5-20.5 months) and 58 febrile controls with a fever due to other causes (28 boys) with a median age of 12.5 months (IQR, 7-30 months). LBP, IL-6, PCT, and CRP were measured for both patients and control groups. RESULTS: The serum levels of LBP (p < 0.001), CRP (p < 0.001), PCT (p = 0.001), IL-6 (p = 0.002), ESR (p = 0.020), and WBC (p < 0.001) were higher in patients with febrile UTI than in the healthy and febrile control groups. The LPB cut-off value for best sensitivity and specificity in patients with febrile UTI was >43.23 mg/l. Furthermore, the area under the receiver operating characteristic curve was significantly greater for LBP than for CRP (p = 0.014), PCT (p < 0.001), ESR (p < 0.001), WBC (p = 0.002) and IL-6 (p = 0.006). CONCLUSIONS: The results of this study suggest that the serum LBP concentration constitutes a reliable biologic marker for the diagnosis of a febrile UTI in children.

Silibinin protects H9c2 cardiac cells from oxidative stress and inhibits phenylephrine-induced hypertrophy: potential mechanisms.

Cardiac hypertrophy is the main response of the heart to various extrinsic and intrinsic stimuli, and it is characterized by specific molecular and phenotypic changes. Recent in vitro and in vivo studies indicate the involvement of reactive oxygen species in the hypertrophic response. In this study, silibinin, a plant flavonolignan extracted from milk thistle with potent antioxidant activity, was evaluated for its effects in (a) preventing hydrogen peroxide (H2O2)-induced cellular damage and (b) blocking the phenylephrine-induced hypertrophic response. Using the in vitro model of embryonic rat heart-derived H9c2 cells, we showed that silibinin has a rather safe profile as concentrations up to 200µM did not affect cell viability. Pretreatment of H9c2 cells with silibinin resulted in better protection of H9c2 cells under conditions of H2O2-induced cellular stress compared to untreated cells as indicated by cell viability and DNA fragmentation assays. Furthermore, silibinin attenuated the phenylephrine-induced hypertrophic response as evidenced by the measurement of cell surface, up-regulation of atrial natriuretic peptide and increase of cellular protein levels. Moreover, silibinin repressed the phenylephrine-induced phosphorylation of ERK1/2 kinases, while it appeared to inhibit the weakly activated by phenylephrine phosphorylation of Akt. Based on our results, silibinin may attenuate the phenylephrine-induced hypertrophic response of H9c2 cells via antioxidant mechanisms involving mainly the inhibition of the intracellular signaling pathways mediated by ERK1/2 MAPKs and Akt.

Tissue inhibitor of metalloproteinase 4 in aqueous humor of patients with primary open angle glaucoma, pseudoexfoliation syndrome and pseudoexfoliative glaucoma and its role in proteolysis imbalance.

BACKGROUND: To quantify the levels of tissue inhibitor of metalloproteinase 4 (TIMP4) and its ratios with free metalloproteinases (MMP) in the aqueous humor of patients with primary open angle glaucoma (POAG), pseudoexfoliation syndrome (PXS) and pseudoexfoliative glaucoma (PXG) and to evaluate a possible imbalance between MMPs and TIMPs in these samples. METHODS: Free MMP2, MMP3, MMP9, TIMP1, TIMP2, TIMP4 concentrations and active levels of MMP2 and MMP3 were determined with immunoassay ELISA and activity assay kits in 168 aqueous samples. RESULTS: TIMP4 was elevated in glaucoma patients(POAG: 0.95 ± 0.49 PXG: 1.28 ± 1.38 pg/ml. p < 0.001). POAG, PXS and PXG samples demonstrated higher MMP2, TIMP1 and TIMP2 concentrations (p < 0.001). Samples from the PXS and PXG groups had a lower total/active MMP2 ratio (p < 0.004 and p < 0.008 respectively). Stoichiometric analysis showed an overbalance of TIMPsover MMPs in both POAG & PXG groups,especially of TIMP4. CONCLUSION: TIMP4 elevation is a novel finding in glaucomatous eyes. A disregulation of extracellular matrix homeostasis is suggested in POAG, PXS and PXG.

Overall survival and clinicopathological characteristics of patients with breast cancer in relation to the expression pattern of HER-2, IL-6, TNF-α and TGF-ß1.

The present study was conducted to investigate the prognostic significance of co-expression patterna of HER-2, IL-6, TNF-a and TGF-ß1 in breast cancer, by correlating the number of markers with positive expression with clinicopathological characteristics indicative of tumor progression and overall survival. One hundred thirty consecutive patients with primary breast cancer were prospectively included and evaluated. Serum concentrations of the above markers were measured by ELISA. Median split was used to subdivide patients with marker positive or negative expression. The presence of ≥ 3 positive markers was independently associated with extended lymph node (>3) involvement (aOR, 11.94, p=0.001) and lymphovascular invasion (aOR, 12.04, p=0.018), increasing the prognostic significance of each marker considered separately. Additional prognostic information regarding survival was also provided; as the number of positive markers increased, a gradually reduction of survival time was observed. In addition, patients with 4 positive markers had significantly shorter survival (25 vs 39 months, p=0.006) and a more than 4 fold increased risk of death (aHR, 4.35, p=0.003) compared to patients with 3 positive markers. Our findings suggest that the coexpression pattern of these four markers could be used clinically as a useful marker for tumor extension and outcome of breast cancer.