Atorvastatin Inhibited ROS Generation and Increased IL-1ß And IL-6 Release by Mononuclear Cells from Diabetic Patients.

BACKGROUND: Atorvastatin (ATV) inhibits the conversion of 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG-CoA) to mevalonate formation and promotes lowering of the LDL cholesterol fraction. However, ATV exhibits pleiotropic metabolic actions beyond cholesterol-lowering properties. OBJECTIVE: We aimed to evaluate the effect of ATV on oxidizing species generation and cytokine secretion in Peripheral Blood Mononuclear Cells (PBMNC) of Type 2 Diabetes Mellitus (T2DM) patients in comparison to healthy control. METHODS: Both NADPH-oxidase-dependent and mitochondrial ROS generation were assessed by chemoluminescence luminol-dependent assay and fluorometric experiment, using Dichlorofluorescein Assay (DCFH-DA), respectively. IL-1ß and IL-6 were quantified by classical ELISA. RESULTS: ATV inhibited NADPH-oxidase dependent ROS generation, but showed no effect on mitochondrial ROS generation and activated IL-1ß and IL-6 secretions in PBMNC from control and T2DM patients. ROS generation and cytokine secretion in the presence of an inhibitor of Protein Kinase Cß (iPKCß) and ATV led to similar results. The secretion of IL-1ß, PDB-induced in the presence of iPKCß, but not ATV, was increased. ATV and iPKCß exacerbated PDB-induced IL-6 secretion. LPS activated the secretion of IL-1ß and IL-6 which was potentiated by ATV. CONCLUSION: ATV inhibited ROS generation and activated IL-1 ß/IL-6 secretion in PBMNC of diabetes patients. Its effect was not affected by the hyperglycemia.

A non-functional galanin receptor-2 in a multiple sclerosis patient.

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.

Quantification of NGAL in Urine of Endurance Cycling Athletes.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein released during early phases of a postischemic kidney in response to kidney injury, inflammation, and oxidative stress. It can be detected in urine after 2 hours of an ischemic event. The aim was to measure and to correlate the level of urine NGAL (uNGAL) with urea, creatinine, and glomerular filtration rate (GFR) of endurance cycling athletes (n = 19) and physically active individuals (control, n = 17). METHODS: Quantification of urea and creatinine were performed by dry chemical method, and GFR was calculated using the modification of diet in renal disease formula, according to Brazilian Society of Nephrology. uNGAL analyses were performed by enzyme linked immunoabsorbent assay. Analyses were performed 48 hours after exercises. RESULTS: uNGAL (in ng/mL) levels, expressed as median, minimum, and maximum, in cyclist group, 387.7 (109.7-1691.0), was significantly higher than that observed in control (physically active) group, 141.5 (4.8-657.0), (P < .05). No significant correlations were observed between uNGAL and creatinine, urea, or GFR (P > .05). CONCLUSIONS: Results have pointed to increased uNGAL levels in endurance cycling athletes. Increase of uNGAL in absence of clinical signs or alterations in creatinine, urea, or GFR might suggest that there is metabolic adaptation to endurance exercise, or possibly predisposition to acute kidney injury over time.

Inflammasome as a New Therapeutic Target for Diabetic Complications.

BACKGROUND: Inflammation is an innate immune response which is considered a common basis for several diseases such as ageing, diabetes, obesity, gout, neurodegenerative diseases and others. Among other platforms, inflammasomes are part of a superfamily of Pattern Recognition Receptors (PRR) and act as cytoplasmatic sensors for stimulation with Pathogen Associated Molecular Pattern (PAMPs) and/or Danger/Damage-Associated Molecular Patterns (DAMPs) leading to an infectious/ pathogenic or sterile inflammation. Inflammasomes constitute a complex platform with high molecular weight and functionality, divided into two families: NOD-like or NLR and PYHIN (pyrin and HIN200 - hematopoietic interferoninducible nuclear antigens). After activation by PAMPs or DAMPs, NLRP3 inflammasome promotes conversion of procaspase 1 in caspase-1 to form the active complex which is able to cleave pro-IL-1ß and pro-IL-18 in respective active inflammatory cytokines IL-1ß and IL-18 inducing cellular death by pyroptosis. Diabetes has a very intricate pathology with metabolic adaptation and inflammatory components apparently responsible for diabetic complications. OBJECTIVE: The present review evaluates the role of inflammasome, emphasizing NRLP3 on diabetes. An overview on several inflammatory diseases in which inflammasomes appear to play a role is included. Patents on inflammasomes associated with diabetes are evaluated and discussed. CONCLUSION: There are a significant number of patents on inflammation but few of them are specifically on inflammasome and diabetes. The patents WO2015003246; US20130273588; WO2012016145; and CN104258398 are shown and their mechanisms are discussed. In conclusion, deeply studies on inflammasomes mechanisms will help the proposition of new therapeutic targets for controlling inflammatory process in diabetic complications.

The antioxidant properties of organosulfur compounds (sulforaphane).

Sulforaphane (SFN) is a molecule within the isothiocyanate (ITC) group of organosulfur compounds. SFN is a phytochemical commonly found in cruciferous vegetables such as broccoli, brussels sprouts and cabbages. It has been widely studied in order to evaluate its chemopreventive properties and some of those have already been established by means of animal and human models. The SFN induces Phase I and II enzymes involved in detoxification processes of chemical carcinogens in order to prevent the start of carcinogenesis. It also presents anti-tumor action at post-initiation Phase, suggesting supplementary roles in cancer prevention. In a dose dependent manner, ITC inhibits the viability of human cancer cells, modifies epigenetic events that occur in cancer cells and present antiinflammatory effect acting during the initial of uncontrolled cell proliferation. This protective effect may be due to its antioxidant status, its recognized capacity to induce the expression and/or activity of of different cytoprotective proteins involved in the activating "Nuclear factor erythroid-derived 2-like 2" (Nrf2). Nevertheless, the effects on health and the possible connections among different diet constituents in humans must be carefully studied as there are limitations in the current data in order to better understand the molecular mechanisms responsible for those effects. This survey also includes relevant patents on the use of SFN, like its use in skin cancer treatment (US2015038580); and as an adjuvant in anti-cancer treatment (US2014228419). The use of SFN as an antioxidant dietary supplement, methods for compositions that promote glutathione production (WO2015002279) and methods for extracting and purifying SFN from broccoli seeds (CN104086469) are also included in this review.

Is Innate Immunity and Inflammasomes Involved in Pathogenesis of Amyotrophic Lateral Sclerosis (ALS)?

Amyotrophic Lateral Sclerosis (ALS) or Lou Gehrig's disease is an axonopathy with adultonset, progressive and irreversible degeneration of upper and lower motor neurons. Around 90% of ALS is considered as sporadic ALS (sALS) without apparent genetic cause while in the familial type of ALS (fALS) at least one affected blood relative needs to be identified. Both sALS and fALS show similar progression and pathological profile. Biochemical and immunological roles have been reported for both types of ALS. It has been suggested that mutation in SOD1 gene would be responsible for the oxidative stress and neurotoxicity. Besides, oxidative stress, protein aggregation, altered cholinergic synapse, neuro-inflammation and production of pro-inflammatory cytokines have also been reported. Thus, the focus of the present review was on biochemical and immunological biomarkers and pathogenic mechanism. Regulatory T cells, pro-inflammatory cytokines and activation of pro-inflammatory signaling pathway are discussed. The activation of NRL inflammasomes in ALS and the involvement of IL-18, IL-1ß and caspases-1 are also suggested. The presence and importance of HMGB-1 (DAMP) and activation of Tolllike receptors and/or RAGE also are envisaged. The patents US20140212508, WO2014145776, WO2014145118, US20140255371, US20140194427, US20140243400, WO2014128254, WO2014076702, WO2014071449, WO2014043696, WO2014001742, and WO2013082299 are summarized. This review intends to evaluate the biochemical and immunological responses and the involvement of inflammasomes in the pathogenesis of ALS. In the present review, we suggest hypothetical model for ALS pathogenesis and we discuss some patents that suggest new treatment and/or therapeutic targets. Due to a large number of patents covering therapy and control of neurodegenerative diseases, our focus was restricted only to discuss the latest registered patents in 2014.

Impaired clearance of neutrophils extracellular trap (NET) may induce detrimental tissular effect.

Neutrophils Extracellular Trap (NET) is composed of nuclear chromatin with hyper segmentation of nuclear lobes, citrullination of histone-associated DNA and mixing with cytoplasmic proteins including the enzyme myeloperoxidase. It is believed that neutrophils trap can kill microorganisms and constitutes a new form of innate defense. However, in some conditions, NET formation may be detrimental to the organism due to its association with autoantibody formation. Thus, NETs can be beneficial or detrimental depending of the DNA clearance recent registered patents describing the processes, products, methods and therapeutic indications of the neutrophil extracellular trap (NET) phenomenon have been reported. The patents US8710039; EP2465536; EP2651440; US20130302345; US20140099648; US20130183662; WO2012166611; and RU2463349C2, related to NETosis, suggest an association between NET formation and autoimmunity. However, its function is still not fully understood. Some parasites have learned to escape from NET using nucleases. NET persistence could be due to a possible enzymatic inhibition as suggested in Grabar´s theory for explaining the induction of physiologic or pathologic autoantibodies. In the present mini-review NET persistence due to impairment in the homeostasis clearance of DNA is discussed.

Immunomodulatory properties of green propolis.

Propolis is a resinous material collected by honeybees from numerous plants and serves as a defense against intruders. Because of its relevant curative properties, it is now gaining popularity in health foods and in cosmetic products. Understanding the underlying molecular mechanisms of phytochemicals has become a good strategy in bioprospection for new anti-inflammatory compounds. The biological activity of propolis derives from its high levels of phenolic acids, while flavonoids are thought to account for the activity of propolis extracts. The comprehension of the relationship between propolis and the immune system has progressed in the last years, recent articles have provided important contributions to this investigation field. Studies have shown that propolis suppressed the "IL-6-induced phosphorylation of signal transducer and STAT3", an essential cytokine-activated transcription factor in Th17 development. Therefore, action mechanisms of "propolis on Th17 differentiation could be instrumental in controlling disturbed cytokine networks in inflammation, autoimmune diseases, and infections." The use of propolis has been proposed in some patents as: WO201363714; CN102885854, WO2013142936, US20130266521, and US20130129808, which are related to the treatment of dental diseases; adjuvant in anti-cancer treatment; in cosmetic products; as an anti-inflammatory agent and natural antibiotic. Although there are many publications regarding the propolis efficacy, its applicability to human health and mechanisms of action are not completely understood, creating opportunities for new studies.

Effects of nitric oxide synthase inhibition on glutamine action in a bacterial translocation model.

Glutamine may be a precursor for NO synthesis, which may play a crucial role in bacterial translocation (BT). The goal of the present study was to investigate the potential effects of glutamine on BT and the immunological response in an experimental model of NO synthase inhibition by NG-nitro-L-arginine methyl ester (l-NAME). Mice were randomly assigned to four groups: sham; intestinal obstruction (IO); IO+500 mg/kg per d glutamine (GLN); IO+GLN plus 10 mg/kg per d l-NAME (GLN/LN). The groups were pretreated for 7 d. BT was induced by ileal ligation and was assessed 18 h later by measuring the radioactivity of 99mTc-Escherichia coli in the blood and organs. Mucosal damage was determined using a histological analysis. Intestinal permeability (IP) was assessed by measuring the levels of 99mTc-diethylenetriaminepentaacetic acid in the blood at 4, 8 and 18 h after surgery. IgA and cytokine concentrations were determined by ELISA in the intestinal fluid and plasma, respectively. BT was increased in the GLN/LN and IO groups than in the GLN and sham groups. IP and intestinal mucosa structure of the sham, GLN and GLN/LN groups were similar. The GLN group had the highest levels of interferon-γ, while IL-10 and secretory IgA levels were higher than those of the IO group but similar to those of the GLN/LN group. The present results suggest that effects of the glutamine pathway on BT were mediated by NO. The latter also interferes with the pro-inflammatory systemic immunological response. On the other hand, IP integrity preserved by the use of glutamine is independent of NO.

The dual role of free fatty acid signaling in inflammation and therapeutics.

Obesity, type 2 diabetes, insulin resistance, dyslipidemia, cardiovascular diseases and atherosclerosis have all been associated with high levels of free fatty acid (FFA). In the present review, we suggest that FFA may act as either pro- or anti-inflammatory agents depending on the chemical structure. Saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) significantly differ in their contributions to inflammation. While SFAs have been shown to induce inflammation, PUFAs have anti-inflammatory effects by downregulating NF-kappaB, IL-1ß, TNF-α and IL-6 despite upregulating of IL-10. It is suggested that FFA may activate Toll Like Receptor-4 (TLR4) and G protein-coupled receptors (GPCR) activating signaling pathways that promote production and release of inflammatory cytokines (IL-6 and TFN-α). Fatty acid action on TLR4, peroxisome proliferator-activated receptors (PPARs) and GPCRs are potential therapeutic targets for controlling FFA-induced inflammation. Approaches that downregulate the inflammatory properties of free fatty acid are discussed in this manuscript. In this review, some patents associated with controlling FFA effects are also reported.

The anti-oxidant properties of isothiocyanates: a review.

Cruciferous vegetables, such as broccoli and watercress, have been studied extensively aiming to evaluate their chemopreventive properties. Some of them have already been established using animal models. The ITCs induce Phase II enzymes related to detoxification processes of chemical carcinogens to prevent the start of carcinogenesis. They also exhibit antitumor activity at post-initiation phase, suggesting their additional role(s) in cancer prevention. Sulforaphane is the most extensively studied isothiocyanate, focused in its anti-tumoral activity and it is mainly found in great amounts in broccoli and other cruciferous. In a dose dependent manner, ITCs inhibit the cell viability of human cervical cancer cells, human pancreatic cancer cells, human hepatocellular carcinoma cells, human ovarian cancer cells, and have antiinflammatory properties in the treatment of human T-cell leukemia cells. This protective effect may be due to improved antioxidant status. Although the health effects of diet in humans are generally considered promising, there are definite challenges and limitations of the current data in better understanding of the molecular mechanisms responsible for this effect, together with the possible interactions between different dietary constituents. The survey of relevant patents on the use of isothiocyanates such as sulforaphane for cancer and cardiovascular diseases treatments is also included in this review.

HMGB-1 as a target for inflammation controlling.

In the present study, we evaluated recent patents that describe products or methods able to down-regulate the pro-inflammatory action of HMGB-1, also called as amphoterin. High Mobility Group Box-1 (HMGB-1) has been implicated in the pathogenesis of inflammatory diseases. HMGB-1 has been proposed to be a crucial mediator in the pathogenesis of many diseases including sepsis, arthritis, cancer, autoimmunity diseases and diabetes. It has been suggested that HMGB-1 itself can signal through RAGEs (receptor for advanced glycation end products) and through the Toll-Like Receptors TLR2 and TLR4. Activation of these receptors results ultimately in the activation of Nuclear Factor-kappaB (NFkappaB), inducing the up-regulation of leukocyte adhesion molecules, production of pro-inflammatory cytokines and angiogenic factors in both hematopoietic and endothelial cells, thereby promoting inflammation. There are several patents proposed for controlling the production, secretion and neutralization of HMGB-1 and consequently the inflammatory process. We have divided the patents in six groups based on mechanism of action. The group 1 is associated with inhibition of HMGB-1 using anti-HMGB-1 antibodies; group 2: inhibition of HMGB-1 releases from the nucleus into the extracellular space; group 3: HMGB-A box as a competitive antagonist of HMGB-1; group 4: blockage of RAGE-HMGB-1 signaling using RAGE antagonists; group 5: blockage of TLR-HMGB-1 signaling using anti-TLR2 antibodies and group 6: other molecules that modulate HMGB-1 activity using e.g. human soluble thrombomodulin. The mechanism of HMGB-1 action, its role and efficiency of each group of patents proposed for controlling inflammation are discussed.

Soluble RAGE and malondialdehyde in type 1 diabetes patients without chronic complications during the course of the disease.

Malondialdehyde (MDA), an end product of lipid peroxidation and biomarker for oxidative stress, and its soluble receptor (sRAGE) were evaluated in 42 patients with type 1 diabetes mellitus, but without chronic complications, during the early years after diagnosis (0-10 years) and through the further progression of the disease (10-20 and > 20 years after diagnosis). Clinical and biochemical parameters of the cohort of diabetic patients were compared with those determined in 24 healthy individuals. The median levels of MDA in plasma were similar in type 1 diabetes patients and in healthy subjects. In contrast, statistically significant increases were detected in the median values of sRAGE in patients with type 1 diabetes compared with healthy subjects (2423.75 versus 1472.75 pg/ml; p=0.001, Mann-Whitney test). However, no significant between-group differences (p>0.05) were observed in levels of sRAGE when diabetic patients were grouped according to time elapsed after diagnosis. It is concluded that increased plasma levels of sRAGE in type 1 diabetes may provide protection against cell damage and may be sufficient to eliminate excessive circulating MDA during early years after disease onset.

Determination of the antioxidant status of plasma from type 2 diabetic patients.

An increase in oxidizing response above a certain threshold produces, in the absence of a concomitant rise in antioxidant/reducing response, oxidative stress that is associated with complications in diabetes. A simple technique involving reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye has been developed in order to determine quantitatively the antioxidant status of plasma. MTT (50microL; 5.0mg/mL in PBS) was incubated with plasma (100microL) in PBS for 30, 60 or 120min at 37 degrees C, the reaction terminated by addition of 1.0mL of 0.04M hydrochloric acid in isopropanol and the absorbance measured at 570nm. The modulation by plasma of the generation of reactive oxygen species (ROS) in 12,13-phorbol dibutyrate (PDB)-stimulated granulocytes was evaluated using a chemiluminescence luminol-dependent assay. Plasma from healthy subjects (n=15) showed significantly higher antioxidant status (p<0.05) over all time periods studied compared with plasma from diabetic patients (n=27). MTT was directly reduced by plasma although platelets were not involved. Moreover, the reduction of MTT by bovine serum albumin at levels equivalent to the concentration of human serum albumin in plasma was much lower. The antioxidant status of plasma, as evaluated by MTT dye reduction, may reflect an antioxidant response since ROS generation in PDB-stimulated granulocytes was rapidly down-regulated by the presence of plasma (3.3-fold in diabetic patients and 5.8-fold in healthy subjects) confirming the lower antioxidant activity of plasma from diabetic patients. The results demonstrate that extracellular reduction of MTT by plasma may occur via enzymatic and non-enzymatic processes.