Molecular characterization of lipoamide dehydrogenase gene in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Lipoamide dehydrogenase (LipDH) is a flavin-containing disulfide oxidoreductase from the same group of thioredoxin reductase, glutathione reductase and trypanothione reductase. This enzyme is found in the mitochondria of all aerobic organisms where it takes part in at least three important multienzyme complexes from the citric acid cycle. In this study, we performed a phylogenetic analysis comparing the amino acid sequence of the LipDH from Trypanosoma cruzi (TcLipDH) with the LipDH from other organisms. Subsequently, the copy number of the TcLipDH gene, the mRNA and protein levels, and the enzymatic activity of the LipDH were determined in populations and strains of T. cruzi that were either resistant or susceptible to benznidazole (BZ). In silico analysis showed the presence of two TcLipDH alleles in the T. cruzi genome. It also showed that TcLipDH protein has less than 55% of identity in comparison to the human LipDH, but the active site is conserved in both of them. Southern blot results suggest that the TcLipDH is a single copy gene in the genome of the T. cruzi samples analyzed. Northern blot assays showed one transcript of 2.4 kb in all T. cruzi populations. Northern blot and Real Time RT-PCR data revealed that the TcLipDH mRNA levels were 2-fold more expressed in the BZ-resistant T. cruzi population (17LER) than in its susceptible pair (17WTS). Western blot results revealed that the TcLipDH protein level is 2-fold higher in 17LER sample in comparison to 17WTS sample. In addition, LipDH activity was higher in the 17LER population than in the 17WTS. Sequencing analysis revealed that the amino acid sequences of the TcLipDH from 17WTS and 17LER populations are identical. Our findings show that one of the mechanisms associated with in vitro-induced BZ resistance to T. cruzi correlates with upregulation of LipDH enzyme.

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.

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.

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.

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.

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.