The impact of wound-healing assay, phorbol myristate acetate (PMA) stimulation and siRNA-mediated FURIN gene silencing on endogenous retroviral ERVW-1 expression level in U87-MG astrocytoma cells.

PURPOSE: Human endogenous retroviruses (HERVs) are ubiquitous genomic sequences. Normally dormant HERVs, undergo reactivation by environmental factors. This deregulation of HERVs' transcriptional equilibrium correlates with medical conditions such as multiple sclerosis (MS). Here we sought to explore whether exposing the U-87 MG astrocytoma cells to traumatic injury deregulates the expression of HERV-W family member ERVW-1 encoding syncytin-1. We also examined the expression of FURIN gene that is crucial in syncytin-1 synthesis. MATERIAL AND METHODS: Scratch assay was used as a model of cells injury in U-87 MG cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and migration assay using Boyden chamber were used. Phorbol 12-myristate 13-acetate (PMA) and small interfering RNA (siRNA) were used for cell stimulation and gene expression inhibition, respectively. RESULTS: Results revealed reduced ERVW-1 expression in cells exposed to injury (p â€‹< â€‹0.05) while GFAP gene - a marker of active astrocytes, was upregulated (p â€‹< â€‹0.01). These findings were confirmed by both WB and RT-qPCR. Expression of FURIN gene was not altered after injury, but cell stimulation by PMA strongly increased FURIN expression, simultaneously downregulating ERVW-1 (p â€‹< â€‹0.01). SiRNA-mediated expression inhibition of ERVW-1 and FURIN influenced the mRNA level for SLC1A5 (ASCT2) - primary syncytin-1 receptor, that was significantly lower. FURIN inhibition by siRNA caused strong upregulation of ERVW-1 expression (p â€‹< â€‹0.01). CONCLUSION: Results showed that mechanical impact affects the expression of endogenous retroviruses in U-87 MG astrocytoma cells by scratch assay. Regulation of FURIN, a crucial enzyme in ERVW-1 turnover may support the therapy of some neurological conditions.

Exenatide prevents statin-related LDL receptor increase and improves insulin secretion in pancreatic beta cells (1.1E7) in a protein kinase A-dependent manner.

Statins are primary drugs in the treatment of hyperlipidemias. This group of drugs is known for its beneficial pleiotropic effects (e.g., reduction of inflammatory state). However, a growing body of evidence suggests its diabetogenic properties. The culpable mechanism is not completely understood and might be related to the damage to pancreatic beta cells. Therefore, we conceived an in vitro study to explore the impact of atorvastatin on pancreatic islet beta cells line (1.1.E7). We evaluated the influence on viability, insulin, low-density lipoprotein (LDL) receptor, and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. A significant drop in mRNA for proinsulin and insulin expression was noted. Concurrently, a rise in LDL receptor at the protein level in cells exposed to atorvastatin was noted. Further experiments have shown that exenatide - belonging to glucagon-like peptide 1 (GLP-1) analogs that are used in a treatment of diabetes and known for its weight reducing properties - can alleviate the observed alterations. In this case, the mechanism of action of exenatide was dependent on a protein kinase A pathway. In conclusion, our results support the hypothesis that statin may have diabetogenic properties, which according to our study is related to reduced insulin expression. The concomitant use of GLP-1 receptor agonist seemed to successfully revert insulin expression.

Pleiotropic Effects of PCSK-9 Inhibitors.

Proprotein convertase subtilisin/kexin type 9 (PCSK-9) inhibitors are a group of drugs whose main mechanism of action is binding to the PCSK-9 molecule, which reduces the degradation of the low-density lipoprotein receptor (LDL-R) and, hence, increases the uptake of low-density lipoprotein cholesterol (LDLc) from the bloodstream as well as reducing its concentration. The effectiveness of three monoclonal antibodies, namely, alirocumab (human IgG1/κ monoclonal antibody, genetically engineered in Chinese hamster ovary cells), evolocumab (the first fully human monoclonal antibody), and bococizumab (humanized mouse antibody), in inhibiting the action of PCSK-9 and reducing LDLc levels has been confirmed. The first two, after clinical trials, were approved by the Food and Drug Administration (FDA) and are used primarily in the treatment of autosomal familial hypercholesterolemia and in cases of statin intolerance. They are currently used both as monotherapy and in combination with statins and ezetimibe to intensify therapy and achieve therapeutic goals following the American Heart Association (AHA) and European Society of Cardiology (ESC) guidelines. However, the lipid-lowering effect is not the only effect of action described by researchers that PCSK-9 inhibitors have. This paper is a review of the literature describing the pleiotropic effects of PCSK-9 inhibitors, which belong to a group of drugs that are being increasingly used, especially when standard lipid-lowering therapy fails. The article focuses on activities other than lipid-lowering, such as the anti-atherosclerotic effect and stabilization of atherosclerotic plaque, the anti-aggregation effect, the anticoagulant effect, the antineoplastic effect, and the ability to influence the course of bacterial infections. In this publication, we try to systematically review the current scientific data, both from our own scientific work and knowledge from international publications.

Epicardial, pericardial fat and glucagon-like peptide-1 and -2 receptors expression in stable patients with multivessel coronary artery disease: an association with the renin-angiotensin-aldosterone system.

INTRODUCTION: The expression of glucagon­like peptide receptors (GLP­Rs) in epicardial fat (EF) and pericardial fat (PF) depots might be involved in the pathogenesis of cardiovascular diseases. OBJECTIVES: We sought to evaluate the messenger RNA (mRNA) expressions of GLP­1R and GLP­2R in EF and PF and their associations with the renin­angiotensin­aldosterone system (RAAS) in patients with multivessel coronary artery disease (CAD). PATIENTS AND METHODS: Consecutive stable patients with multivessel CAD requiring elective coronary artery bypass grafting were enrolled. Clinical data, anthropometric parameters, and the quantity of fat depots (assessed by cardiovascular magnetic resonance and abdominal ultrasound) were obtained. Fat samples (EF, PF, subcutaneous fat) were taken from patients during cardiac surgery. Relative mRNA expression of GLP­1R, GLP­2R, and RAAS components (angiotensin II receptor type 1, angiotensinogen, angiotensin I-converting enzyme 1, and angiotensinI-converting enzyme 2) were assessed in those fat depots. RESULTS: Fifty­three patients (64.7 [7.4] years) were included in the final analysis. We found that only the relative expression of GLP­2R was lower in PF compared with subcutaneous fat (reference). Ultrasound abdominal fat depots were associated with both GLP­1R and GLP­2R in PF. GLP­1R and GLP­2R showed significant correlations with RAAS components in both EF and PF. CONCLUSIONS: In stable patients with MVD, the relative mRNA expression for both GLP receptors revealed significant associations with majority of analysed RAAS components.

Tricyclic Antidepressants Modulate Stressed Mitochondria in Glioblastoma Multiforme Cells.

A common feature of solid tumors, including glioblastoma multiforme (GBM), is mitochondrial dysfunction. However, it is reported that the current standard of anti-GBM therapies may potentiate mitochondrial damage and, in effect, support the aggressive character of cancer. As mitochondria are implicated in the modulation of cellular drug sensitivity and chemoresistance mechanisms, activation-stressed mitochondria in GBM cells may represent a new target for anti-GBM therapy that is nontoxic for normal cells. METHODS: As mitochondria are possible targets for antidepressant drugs used as adjuvant therapy in patients with GBM, we examined their influence on mitochondrial volume and activity, reactive oxygen species level, extracellular lactate concentration, and p65 NF-κB gene expression in GBM cells. RESULTS: Our investigation showed, for the first time, that tricyclic antidepressants, imipramine and amitriptyline, partially reverse GBM abnormalities. CONCLUSION: In the light of reported studies, the mitochondrial disturbance observed in glioma cells is a dynamic process that can be reversed or silenced. Moreover, imipramine and amitriptyline are attractive cellular metabolic modulators and can potentially be used to restoring a proper function of mitochondria in GBM cells.

Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA.

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.

The application of strand invasion phenomenon, directed by peptide nucleic acid (PNA) and single-stranded DNA binding protein (SSB) for the recognition of specific sequences of human endogenous retroviral HERV-W family.

The HERV-W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV-W-derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin-1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV-W members is highly desirable. A peptide nucleic acid (PNA)-mediated technique for the discrimination between multiple sclerosis-associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis-associated retrovirus (MSRV) template, shows high selective potential. Single-stranded DNA binding protein facilitates the PNA-mediated, sequence-specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single-stranded DNA-specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV-W env sequences have been evaluated. We believe that PNA/single-stranded DNA binding protein-based application has the potential to selectively discriminate particular HERV-W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho-neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto-immunologic background (psoriasis and lupus erythematosus).

Exenatide and metformin express their anti-inflammatory effects on human monocytes/macrophages by the attenuation of MAPKs and NFκB signaling.

Metformin and exenatide are effective antidiabetic drugs, and they seem to have pleiotropic properties improving cardiovascular outcomes. Macrophages' phenotype is essential in the development of atherosclerosis, and it can be modified during antidiabetic therapy, resulting in attenuated atherogenesis. The mechanism orchestrating this phenomenon is not fully clear. We examined the impact of exenatide and metformin on the level of TNF alpha, MCP-1, reactive oxygen species (ROS), and the activation of mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NFκB), and CCAAT/enhancer-binding protein beta (C/EBP beta) in human monocytes/macrophages. We found that both drugs reduced levels of TNF alpha, ROS, and NFκB binding activity to a similar extent. Compared to metformin, exenatide was more effective in reducing MCP-1 levels. We noted that Compound C (AMPK inhibitor) reduced the impact of exenatide on cytokines, ROS, and NFκB in cultures. Both drugs elevated the C/EBP beta phosphorylation level. Experiments on MAPKs showed effective inhibitory potential of exenatide toward p38, JNK, and ERK, whereas metformin inhibited JNK and ERK only. Exenatide was more effective in the inhibition of JNK than metformin. Interestingly, an in vitro setting additive effect of drugs was absent. In conclusion, here, we report that metformin and exenatide inhibit the proinflammatory phenotype of human monocytes/macrophages via influence on MAPK, C/EBP beta, and NFκB. Exenatide was more effective than metformin in reducing MCP-1 expression and JNK activity. We also showed that some effects of exenatide relied on AMPK activation. This shed light on the possible mechanisms responsible for pleiotropic effects of metformin and exenatide.

Exenatide (a GLP-1 agonist) expresses anti-inflammatory properties in cultured human monocytes/macrophages in a protein kinase A and B/Akt manner.

BACKGROUND: Incretin-based therapies in the treatment of type 2 diabetes mellitus are associated with significant improvements in glycemic control, which are accompanied by a beneficial impact on atherosclerosis. Macrophages are essential in the development of atherosclerotic plaques and may develop features that accelerate atherosclerosis (classically activated macrophages) or protect arterial walls against it (alternatively activated macrophages). Therefore, we explored whether beneficial actions of exenatide are connected with the influence on the macrophages' phenotype and synthesis of inflammatory and anti-inflammatory cytokines. METHODS: Monocytes/macrophages were harvested from 10 healthy subjects. Cells were cultured in the presence of exenatide, exendin 9-39 (GLP-1 antagonist), LPS, IL-4, PKI (PKA inhibitor) and triciribine (PKB/Akt inhibitor). We measured the effects of the above-mentioned compounds on markers of macrophages' phenotype (inducible nitrous oxide (iNOS), arginase 1 (arg1) and mannose receptors) and concentration of nitrite, IL-1ß, TNF-α and IL-10. RESULTS: Exenatide significantly increased the level of IL-10 and decreased both TNF-α and IL-1ß in LPS-treated monocytes/macrophages. Furthermore exenatide increased the expression of arg1-a marker of classical activation and reduced the LPS-induced expression of iNOS-a marker of classical activation. According to experiments with protein kinases inhibitors we found that proinflammatory markers were protein kinase A dependent, whereas the activation of alternative activation was similarly reliant on protein kinase A and B/Akt. CONCLUSIONS: We showed that exenatide skewed the macrophages phenotype toward anti-inflammatory phenotype and this effect is predominantly attributable to protein kinase A and to a less extent to B/Akt activation.

Superoxide dismutase 1 and glutathione peroxidase 1 are involved in the protective effect of sulodexide on vascular endothelial cells exposed to oxygen-glucose deprivation.

Sulodexide (SDX) is widely used in the treatment of both arterial and venous thrombotic disorders. In addition to its recognized antithrombotic action, SDX has endothelial protective potential, which is independent of the coagulation/fibrinolysis system. However, the detailed molecular mechanisms of the endothelioprotective action of the drug are still unresolved. The aim of the present study was to determine whether treatment with SDX at concentrations of 0.125-0.5 lipase releasing unit (LRU)/ml have on the expression and activity of antioxidant enzymes in ischemic endothelial cells and how these effects might be related to the antiapoptotic properties of SDX. In the present study, human umbilical vein endothelial cells (HUVECs) were subjected to ischemia-simulating conditions (combined oxygen and glucose deprivation, OGD) for 6h to determine the protective effects of SDX. SDX (0.25 and 0.5LRU/ml) in OGD significantly increased the cell viability and prevented mitochondrial depolarization in the HUVECs. Moreover, SDX protected the HUVECs against OGD-induced apoptosis. At concentrations of 0.25 and 0.5LRU/ml, the drug increased both superoxide dismutase 1 (SOD1) and glutathione peroxidase 1 (GPx1) mRNA/protein expression together with a significant attenuation of oxidative stress in ischemic HUVECs. Our findings also demonstrate that an increase in both SOD and GPx activity is involved in the protective effect of SDX on ischemic endothelial cells. Altogether, these results suggest that SDX has a positive effect on ischemia-induced endothelial damage because of its antioxidant and antiapoptotic properties.

Exenatide (a GLP-1 agonist) improves the antioxidative potential of in vitro cultured human monocytes/macrophages.

Macrophages are dominant cells in the pathogenesis of atherosclerosis. They are also a major source of reactive oxygen species (ROS). Oxidative stress, which is particularly high in subjects with diabetes, is responsible for accelerated atherosclerosis. Novel antidiabetic drugs (e.g., glucagon-like peptide-1 (GLP-1) agonists) were shown to reduce ROS level. Therefore, we conceived a study to evaluate the influence of exenatide, a GLP-1 agonist, on redox status in human monocytes/macrophages cultured in vitro, which may explain the beneficial effects of incretin-based antidiabetic treatment. Human macrophages obtained from 10 healthy volunteers were in vitro subjected to the treatment with GLP-1 agonist (exenatide) in the presence of lipopolysaccharide (LPS), antagonist of GLP-1 receptors (exendin 9-39), or protein kinase A inhibitor (H89). Afterwards, reactive oxygen species, malondialdehyde level, NADPH oxidase, and antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase] expression was evaluated. Finally, we estimated the activity of the abovementioned enzymes in the presence of H89. According to our findings, exenatide reduced ROS and malondialdyhyde (MDA) level by decreasing the expression of ROS-generating NADPH oxidase and by increasing the expression and activities of SOD and GSH-Px. We also showed that this effect was significantly inhibited by exendin 9-39 (a GLP-1 antagonist) and blocked by H89. Exenatide improved the antioxidative potential and reduced oxidative stress in cultured human monocytes/macrophages, and this finding may be responsible for the pleiotropic effects of incretin-based therapies. This effect relied on the stimulation of GLP-1 receptor.

A Novel, Highly Selective RT-QPCR Method for Quantification of MSRV Using PNA Clamping Syncytin-1 (ERVWE1).

HERV-W is a multi-locus family of human endogenous retroviruses (HERVs) that has been found to play an important role in human physiology and pathology. Two particular members of HERV-W family are of special interests: ERVWE1 (coding syncytin-1, which is a glycoprotein essential in the formation of the placenta) and MSRV (multiple sclerosis-associated retrovirus that is thought to play a significant role in human pathology as a result of its increased expression in the brain tissue and blood cells derived from patients with multiple sclerosis (MS)). Both ERVWE1 and MSRV mRNA share high level of similarity and hence a method that allows to exclusively quantify the MSRV expression in clinical samples would be desirable. We developed a quantitative polymerase chain reaction (QPCR) technique for the detection and quantification of the multiple sclerosis-associated retrovirus. The assay utilises fluorescently labelled oligonucleotide probe, which is complementary to the conservative fragment of MSRV env gene and a peptide nucleic acid (PNA) probe, fully complementary to the ERVWE1 sequence fragment that efficiently blocks the polymerase action on ERVWE1 templates. The PNA molecule, if used parallel with hydrolysis probe in QPCR analysis, greatly facilitates the detection efficiency of MSRV even if ERVWE1 is present abundantly in respect to MSRV in the analysed sample. We achieved a wide and measurable range from 1 × 10 e(2) to 1 × 10 e(8) copies/reaction; the linearity of the technique was maintained even at the low MSRV level of 1% in respect to ERVWE1. Using our newly developed method we confirmed that the expression of MSRV takes place in normal human astrocytes and in human umbilical vein endothelial cells in vitro. We also found that the stimulation of human monocytes did not influence the specific expression of MSRV but it caused changes in mRNA level of distinct HERV-W templates.

TREATMENT WITH CINACALCET INCREASES PLASMA ADIPONECTIN CONCENTRATION IN HEMODIALYZED PATIENTS WITH CHRONIC KIDNEY DISEASE AND SECONDARY HYPERPARATHYROIDISM.

OBJECTIVE: Cinacalcet increases calcium-sensing receptor (CaSR) sensitivity to serum calcium. CaSR is expressed by adipocytes, and adiponectin is an adipokine with antiatherogenic and insulin-sensitizing properties. The aim of this study was to assess the influence of a 3-month cinacalcet regimen on plasma adiponectin concentration in hemodialyzed patients (HDP) with chronic kidney disease (CKD) and secondary hyperparathyroidism (sHPT). METHODS: Plasma adiponectin, advanced oxidation protein products (AOPP), serum interleukin-6 (IL-6) and C-reactive protein (CRP) concentrations were assessed in 65 HDP with sHPT treated with cinacalcet (30-120 mg/day) before the first dose and after 3 months of treatment. RESULTS: There was a significant decrease in serum parathyroid hormone (PTH) concentration: from 1,089 (891-1,286) pg/mL to 775 (574-976) pg/mL after 3 months of treatment (P<.0001). The treatment was associated with a significant (P = .048) increase in plasma adiponectin concentration from 16.9 (14.4-19.5) µg/mL to 17.8 (15.0-20.6) µg/mL. Significant (P = .03) reduction of plasma AOPP concentration was observed from 186.7 (156.7-216.7) pg/mL to 162.6 (141.2-183.9) pg/mL. CONCLUSIONS: A 3-month cinacalcet regimen increased plasma adiponectin concentrations in HDP with sHPT. Increased adiponectinemia in these patients may be related to reduced oxidative stress.

Metformin affects macrophages' phenotype and improves the activity of glutathione peroxidase, superoxide dismutase, catalase and decreases malondialdehyde concentration in a partially AMPK-independent manner in LPS-stimulated human monocytes/macrophages.

BACKGROUND: Diabetic patients experience accelerated atherosclerosis. Metformin is a cornerstone of the current therapy of type 2 diabetes. Macrophages are the key cells associated with the development of atherosclerotic plaques. Therefore, our aim was to assess the in vitro effects of metformin on macrophages and its influence on the mechanisms involved in the development of atherosclerosis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained from the group including 16 age-matched healthy non-smoking volunteers aged 18-40 years. Monocytes were further incubated with metformin, LPS and compound C--a pharmacological inhibitor of AMPK. The impact of metformin on oxidative stress markers, antioxidative properties, inflammatory cytokines and phenotypical markers of macrophages was studied. RESULTS: We showed that macrophages treated with metformin expressed less reactive oxygen species (ROS), which resulted from increased antioxidative potential. Furthermore, a reduction in inflammatory cytokines was observed. We also observed a phenotypic shift toward the alternative activation of macrophages that was induced by metformin. All the aforementioned results resulted from AMPK activation, but a residual activity of metformin after AMPK blockade was still noticeable even after inhibition of AMPK by compound C. CONCLUSIONS: Authors believe that metformin-based therapy, a cornerstone in diabetes therapy, not only improves the prognosis of diabetics by reducing blood glucose but also by reducing oxidative stress, inflammatory cytokine production and the shift toward alternative activation of macrophages.

Knockdown of AKT3 (PKBγ) and PI3KCA suppresses cell viability and proliferation and induces the apoptosis of glioblastoma multiforme T98G cells.

Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor that is difficult to treat and has a very poor prognosis. Thus, new therapeutic strategies that target GBM are urgently needed. The PI3K/AKT/PTEN signaling pathway is frequently deregulated in a wide range of cancers. The present study was designed to examine the inhibitory effect of AKT3 or PI3KCA siRNAs on GBM cell growth, viability, and proliferation.T98G cells were transfected with AKT3 and/or PI3KCA siRNAs. AKT3 and PI3KCA protein-positive cells were identified using FC and Western blotting. The influence of specific siRNAs on T98G cell viability, proliferation, cell cycle, and apoptosis was evaluated as well using FC. Alterations in the mRNA expression of AKT3, PI3KCA, and apoptosis-related genes were analyzed using QRT-PCR. Knockdown of AKT3 and/or PI3KCA genes in T98G cells led to a significant reduction in cell viability, the accumulation of subG1-phase cells and, a reduced fraction of cells in the S and G2/M phases. Additionally, statistically significant differences in the BAX/BCL-2 ratio and an increased percentage of apoptotic cells were found. The siRNA-induced AKT3 and PI3KCA mRNA knockdown may offer a novel therapeutic strategy to control the growth of human GBM cells.

The influence of ezetimibe on classical and alternative activation pathways of monocytes/macrophages isolated from patients with hypercholesterolemia.

Macrophages are crucial for the development of atherosclerotic plaques. Classically activated macrophages contribute to plaque growth and destabilization, while alternatively activated macrophages increase plaque stability. Here, we assessed the influence of ezetimibe on the activation of monocyte-derived macrophages isolated from patients with hypercholesterolemia (total cholesterol 263.4 ± 12.5 mg/dl, low-density lipoprotein cholesterol 179.7 ± 11.3 mg/dl, triglycerides 123.9 ± 11.4 mg/dl). Cells were stimulated with 1 µg/ml lipopolysaccharide (LPS) or 1 µg/ml LPS plus 22 ng/ml ezetimibe. Control cells were left unstimulated. The expression of classical activation markers (interleukin-1ß (IL-1ß), nitric oxide (NO), and inducible nitric oxide synthase (iNOS)) and alternative activation markers (mannose receptor (MR) and arginase-1 (Arg1)) was determined after 48 h. The employed analytical methods included enzyme-linked immunosorbent assay, Griess reaction, real-time polymerase chain reaction, and Western blotting. LPS increased the secretion of IL-1ß and NO and the expression of iNOS mRNA, iNOS protein, and Arg1 protein. It did not affect the expression of MR or Arg1 mRNA. In comparison to LPS stimulation, co-stimulation with ezetimibe decreased the secretion of IL-1ß and the expression of iNOS mRNA and protein, while it increased MR mRNA and protein expression. Co-stimulation with ezetimibe did not change the secretion of NO or the expression of Arg1. The results suggest that ezetimibe in inflammatory in vitro conditions contributes to the suppression of classical and promotion of the alternative macrophage activation.

A peptide nucleic acid (PNA)-mediated polymerase chain reaction clamping allows the selective inhibition of the ERVWE1 gene amplification.

A new peptide nucleic acid (PNA) mediated QPCR technique for the detection and quantification of the Multiple Sclerosis-Associated Retrovirus (MSRV) belonging to the human endogenous retrovirus-W (HERV-W) family has been developed. The assay utilizes a PNA probe which is fully complementary to the ERVWE1 sequence, another member of the HERV-W family which is closely related to MSRV. Due to its excellent affinity to a completely matched template, PNA probe selectively blocks the amplification of ERVWE1 thus allowing the specific and exclusive detection and quantification of the MSRV as PNA does not interfere with the amplification of MSRV. Using this newly developed method we found that MSRV is predominantly expressed over ERWVE1 in astrocyte-derived U-87 MG cell line but not in human umbilical vein endothelial cells or human placental cells.

Effect of N-acetylcysteine administration on the expression and activities of antioxidant enzymes and the malondialdehyde level in the blood of lead-exposed workers.

We investigated whether treatment with N-acetylcysteine (NAC) reduces oxidative stress intensity and restores the expression and activities of superoxide dismutase (Sod1, SOD), catalase (Cat, CAT) and glutathione peroxidase (Gpx1, GPx) in lead-exposed workers. The exposed population was divided randomly into two groups. Workers in the first group (reference group, n=49) were not administered any drugs, while workers in the second group (n=122) were treated with NAC at three doses for 12 weeks (200 mg, 400 mg, 800 mg/day). NAC administered orally to lead-exposed workers normalized antioxidant enzyme activities in blood cells. Oxidative stress intensity measured as malondialdehyde (MDA) levels in serum, leukocytes and erythrocytes significantly decreased after NAC administration. NAC may be an alternative therapy for chronic lead intoxication.

Eplerenone mimics features of the alternative activation in macrophages obtained from patients with heart failure and healthy volunteers.

Alternative activation of macrophages plays protective role in cardiac remodelling in heart failure and the activity of mineralocorticoid receptor may determine the phenotype of these cells. We examined the influence of eplerenone, aldosterone, and IL-4 on descriptors of alternative activation in blood monocytes collected from 19 patients with heart-failure and 20 healthy volunteers. "Heart failure" macrophages in comparison with "healthy" macrophages had increased mineralocorticoid activity, NO and reactive oxygen species production, expression of iNOS mRNA and protein, but decreased expression of arginase I and mannose receptor proteins, and activity of MnSOD and CuZnSOD. Aldosterone increased mineralocorticoid activity, NO and reactive oxygen species production, iNOS mRNA and protein expression, MnSOD and CuZnSOD activity. Eplerenone attenuated the effects of aldosterone on all but MnSOD and CuZnSOD variables. Eplerenone alone increased the production of NO, MnSOD and CuZnSOD activity, arginase I gene and protein expression, and mannose receptor gene and protein expression, but decreased mineralocorticoid activity only in "heart failure" macrophages. The latter suggests altered function of mineralocorticoid receptor in heart failure. Increased mineralocorticoid activity accounts for increased NO production, iNOS gene and protein expression but does not explain the increased basal reactive oxygen species production and decreased markers of alternative activation in "heart failure" macrophages. In the lack of change in basal mineralocorticoid activity, eplerenone increases markers of alternative activation in a mineralocorticoid receptor-independent manner. Because of changes in iNOS and NO variable, eplerenone induced qualitatively different activation of macrophages from that obtained with IL-4.

[Hypoparathyroidism: the present state of art]. / Niedoczynnosc przytarczyc: wspólczesny stan wiedzy.

Hypoparathyroidism is a result of reduced secretion or impaired action of parathyroid hormone (PTH). Although considered a rare condition, hypoparathyroidism seems to occur much more frequently than reported. In most cases, hypoparathyroidism remains a complication of neck surgery. However, there is a growing incidence of the autoimmune form of hypoparathyroidism, which may occur in combination with other autoimmune diseases. As parathyroid glands are necessary to sustain life and maintain homeostasis, undetected or misdiagnosed hypoparathyroidism may pose a significant threat to health outcomes, as its presence may increase morbidity and mortality in affected individuals. The clinical consequences of PTH deficiency or impaired receptor action are multidirectional and include nervous hyperexcitability, paresthesias, cramps, tetany, hyperreflexia, convulsions, cataract, weakened tooth enamel, brittle nails and basal ganglia calcifications. In some patients, however, its manifestation may be non-specific, and in these cases the correct diagnosis may be easily missed. Laboratory measurements show hypocalcemia, hyperphosphatemia, and, with the exception of pseudohypoparathyroidism, inappropriately low or undetectable PTH levels. Treatment consists of oral calcium supplementation and vitamin D derivatives. In this review article, we discuss the causes, clinical picture, diagnosis and treatment of hypoparathyroidism and provide the reader with some practical guidance concerning dealing with a patient suffering from this disorder.