Inhibition of ERAP1 represses HLA-B27 free heavy chains expression on polarized macrophages and interrupts NK cells activation and function from ankylosing spondylitis.

BACKGROUND: We aimed to assess if Endoplasmic reticulum aminopeptidase 1 (ERAP1) polymorphisms might impress Human leukocyte antigen (HLA)-B27-free heavy chains (FHCs) expression on macrophages and eventually NK cell activation in Ankylosing spondylitis (AS). METHODS: Blood samples were obtained from 10 HLAB27+ patients with protective and 10 HLAB27+ patients with non-protective genotype. Monocytes were isolated and polarized toward M1 and M2 macrophages. ERAP1 was inhibited in macrophages, which were then co-cultured with autologous NK cells. RESULTS: Expression of HLA-B27-FHCs on M1 and M2 macrophages was reduced in patients with protective ERAP1 genotype. Co-culturing ERAP1-inhibited M1 macrophages and NK cells from patients with protective genotype resulted in downmodulation of CD69 and CD107a markers on NK cells and reduced number of IFN-γ+ NK cells compared to that of patients with non-protective genotypes. CONCLUSION: Inhibition of ERAP1 activity, by diminishing NK activation, may have therapeutic value in treating AS patients.

Photocatalytic nanocomposite membranes for environmental remediation.

We report the design and one-pot synthesis of Ag-doped BiVO4embedded in reduced graphene oxide (BiVO4:Ag/rGO) nanocomposites via a hydrothermal processing route. The binary heterojunction photocatalysts exhibited high efficiency for visible light degradation of model dyes and were correspondingly used for the preparation of photocatalytic membranes using polyvinylidene fluoride (PVDF) or polyethylene glycol (PEG)-modified polyimide (PI), respectively. The surface and cross-section images combined with elemental mapping illustrated the effective distribution of the nanocomposites within the polymeric membranes. Photocatalytic degradation efficiencies of 61% and 70% were achieved after 5 h of visible light irradiation using BiVO4:Ag/rGO@PVDF and BiVO4:Ag/rGO@PI (PEG-modified) systems, respectively. The beneficial photocatalytic performance of the BiVO4:Ag/rGO@PI (PEG-modified) membrane is explained by the higher hydrophilicity due to the PEG modification of the PI membrane. This work may provide a rational and effective strategy to fabricate highly efficient photocatalytic nanocomposite membranes with well-contacted interfaces for environmental purification.

Evaluation of ERAP1 gene single nucleotide polymorphisms in immunomodulation of pro-inflammatory and anti-inflammatory cytokines profile in ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a prototype of chronic inflammatory arthritis termed seronegative spondyloarthropathies that typically affects the joints. Among the non-Human leukocyte antigen (HLA) loci, the strongest association has been observed with Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene single nucleotide polymorphisms (SNPs). Moreover, the effect of ERAP1 gene SNPs on the pro-inflammatory and anti-inflammatory cytokines in AS disease has still been poorly elucidated. In this study, we aimed to determine the association of ERAP1 gene SNPs (rs30187 and rs2287987) with AS risk as well as their effect on the mRNA expression of pro-inflammatory and anti-inflammatory cytokines, with emphasis on the immunoregulation of the IL-17/IL-23 pathway, in an Iranian population. METHODS: We performed Single specific primer (SSP)-PCR for genotyping of 160 AS patients and 160 healthy controls. After isolation of peripheral blood mononuclear cells (PBMCs), total RNA of PBMCs was isolated, complementary DNA (cDNA) was synthesized, and quantitative analyses of mRNA expression of cytokines were performed by Real-time PCR for 40 HLA-B27 positive AS patients and 40 healthy individuals as controls. RESULTS: It was seen that T allele of rs30187 (OR = 1.54, 95% CI = 1.07-2.22, P =  0.017) and C allele of rs2287987 (OR 1.50, 95% CI 1.05-2.14, P = 0.024) were associated with the risk of AS. Both of these alleles were associated more strongly in the HLA-B27 positive AS patients. There was a significant overexpression of mRNAs of pro-inflammatory (IL-17A, IL-17F, IL-23, TNF-α and IFN-γ), while downregulation of anti-inflammatory cytokines (IL-10 and TGF-ß) in PBMCs from 40 HLA-B27 positive AS patients in comparison to controls. AS patients with rs30187 SNP TT genotype expressed mRNA of IL-17A, IL-17F, and IL-23 significantly higher than patents with CT and CC genotypes for this SNP. CONCLUSIONS: This study represented the association of ERAP1 gene rs30187 and rs2287987 polymorphism with the risk of AS. Additionally, it appears that rs30187 polymorphism may be involved in the immunomodulation of the IL-17/IL-23 pathway in the AS disease.

Targeting Filamin A Reduces Macrophage Activity and Atherosclerosis.

BACKGROUND: The actin-binding protein FLNA (filamin A) regulates signal transduction important for cell locomotion, but the role of macrophage-specific FLNA during atherogenesis has not been explored. METHODS: We analyzed FLNA expression in human carotid atherosclerotic plaques by immunofluorescence. We also produced mice with Flna-deficient macrophages by breeding conditional Flna-knockout mice ( Flna o/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter ( LC). Atherosclerosis in vivo was studied by transplanting bone marrow from male Flna o/fl/ LC mice to atherogenic low-density lipoprotein receptor-deficient ( Ldlr-/-) mice; and by infecting Flna o/fl and Flna o/fl/ LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Furthermore, C57BL/6 mice were infected with AdPCSK9 and then treated with the calpain inhibitor calpeptin to inhibit FLNA cleavage. RESULTS: We found that macrophage FLNA expression was higher in advanced than in intermediate human atherosclerotic plaques. Flna o/fl/ LC macrophages proliferated and migrated less than controls; expressed lower levels of phosphorylated AKT and ERK1/2; exhibited reduced foam cell formation and lipid uptake; and excreted more lipids. The deficiency of Flna in macrophages markedly reduced the size of aortic atherosclerotic plaques in both Ldlr-/-BMT: Flnao/fl/LC and AdPCSK9-infected Flna o/fl/ LC mice. Intima/media ratios and numbers of CD68-positive macrophages in atherosclerotic plaques were lower in Flna-deficient mice than in control mice. Moreover, we found that STAT3 interacts with a calpain-cleaved carboxyl-terminal fragment of FLNA. Inhibiting calpain-mediated FLNA cleavage with calpeptin in macrophages reduced nuclear levels of phosphorylated STAT3, interleukin 6 secretion, foam cell formation, and lipid uptake. Finally, calpeptin treatment reduced the size of atherosclerotic plaques in C57BL/6 mice infected with AdPCSK9. CONCLUSIONS: Genetic inactivation of Flna and chemical inhibition of calpain-dependent cleavage of FLNA impaired macrophage signaling and function, and reduced atherosclerosis in mice, suggesting that drugs targeting FLNA may be useful in the treatment of atherosclerosis.

Blocking the Cleavage of Filamin A by Calpain Inhibitor Decreases Tumor Cell Growth.

BACKGROUND/AIM: Filamin A (FLNA) is the most abundant and widely expressed isoform of filamin in human tissues. It is cleaved by calpain at the hinge 1 and 2 domains, producing a 90-kDa carboxyl-terminal fragment (FLNACT). Recently, it has been shown that FLNACT mediates cell signaling and transports transcription factors into the cell nucleus. However, the significance of cleavage of FLNA by calpain has not been studied in cancer cell growth. Calpeptin is a chemical inhibitor of both calpain 1 and 2 that cleaves FLNA. In this study, we questioned if inhibiting calpain using calpeptin would decrease tumor cell proliferation, migration, invasion, and colony formation. MATERIALS AND METHODS: Human melanoma (A7), prostate cancer (PC3), mouse fibrosarcoma (T241) and endothelial (MS1) cells were assayed for proliferation, migration, invasion and colony formation after treatment with calpeptin. Cell lysates were immunoblotted for FLNA and FLNACT Results: Calpeptin treatment of these cells resulted in a decreased production of FLNACT Calpeptin-treated human and mouse tumor cells displayed impaired proliferation, migration, and colony formation. CONCLUSION: These data suggest that the cleavage of FLNA by calpain is an important cellular event in the regulation of tumor cell growth.

The role of gut microbiota and IL-23/IL-17 pathway in ankylosing spondylitis immunopathogenesis: New insights and updates.

Ankylosing spondylitis (AS) is a type of arthritis that is referred to a group of chronic immune-mediated inflammatory diseases termed as seronegative spondyloarthropathies or spondyloarthritides. It typically affects the joints of the spinal and axial skeleton and exhibits common clinical features and genetic factors such as human leukocyte antigen class I allele HLA-B27, the Endoplasmic Reticulum Aminopeptidase 1 (ERAP1), and environmental factors such as microbial triggers. Although the precise etiopathogenic mechanisms that implicate the pathogenesis of AS have still remained to be clarified, the IL-23/IL-17 immune axis has been detected as an important factor in the immunopathogenesis of AS. Moreover, therapeutic options targeting this signaling pathway have been demonstrated to be effective in various other inflammatory diseases that share similar genetic etiology and pathogenetic pathways. In mammalian intestinal, there are trillions of commensal microbes that create the intricate symbiotic relationship with host well-known as the microbiota and play the major role in human health and disease. Several publications have appeared in recent years documenting the pivotal role of the gut microbiota and the IL-23/IL-17 pathway in the pathogenesis of spondyloarthritides. In this review, several points are discussed and summarized including recent advances on the role of the IL-17/IL-23 immune pathway in the pathogenesis of AS, HLA-B27, and ERAP 1 and 2 mediated pathogenesis, AS-related microbiota compositions, and new potential therapies for AS.

Mutation/SNP analysis in EF-hand calcium binding domain of mitochondrial Ca[Formula: see text] uptake 1 gene in bipolar disorder patients.

Calcium signaling is important for synaptic plasticity, generation of brain rhythms, regulating neuronal excitability, data processing and cognition. Impairment in calcium homeostasis contributed to the development of psychiatric disorders such as bipolar disorder (BP). MCU is the most important calcium transporter in mitochondria inner membrane responsible for influx of Ca[Formula: see text]. MICU1 is linked with MCU and has two canonical EF hands that are vital for its activity and regulates MCU-mediated Ca[Formula: see text] influx. In the current study, we aimed to investigate the role of genetic alteration of EF hand calcium binding motifs of MICU1 on the development of BP. We examined patients with BP, first degree relatives of these patients and healthy volunteers for mutations and polymorphisms in EF hand calcium binding motifs of MICU1. The result showed no SNP/mutation in BP patients, in healthy subjects and in first degree relatives. Additionally, alignment of the EF hand calcium binding regions among species (Gallus-gallus, Canis-lupus-familiaris, Bos-taurus, Mus-musculus, Rattus-norvegicus, Pan-troglodytes, Homosapiens and Danio-rerio) showed exactly the same amino acids (DLNGDGEVDMEE and DCDGNGELSNKE) except in one of the calcium binding domain of Danio-rerio that there was only one difference; leucine instead of Methionine. Our results showed that the SNP on EF-hand Ca[Formula: see text] binding domains of MICU1 gene had no effect in phenotypic characters of BP patients.

Use and Evaluation of Newly Synthesized Fluorescence Probes to Detect Generated OH• Radicals in Fibroblast Cells.

Reactive oxygen species (ROS) are pro-oxidant molecules synthesized in body with various functions and are essential for life. Increasing in reactive oxygen species or decreasing in antioxidants level cause oxidative stress which is very harmful. OH• radical is one of ROS's, with tendency to bind to lipids, DNA and proteins which cause irreversible damage in cells. The most devastating consequences related to excess OH• radicals occur via direct binding to nucleic acids and proteins. Quantification of this high reactive radical with short life time is difficult. Electron Spin Resonance, Fluorescence, and Luminescence Spectroscopy are commonly used to determine the level of ROS. Fluorescence Probes have higher specificity and sensitivity with their excellent sensors to detect ROS's compare to the other methods. Also, there are different probes specifically designed for each radical. The purpose of this study was to identify the probe better suiting for detection of OH• radical levels. The two most recommended fluorescence probes, 2-[6-(4 V-Hydroxy) phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and coumarin-3-carboxylic acid (3-CCA) to determine OH• radical levels were compared. Following the formation of OH• radical with Fenton reaction, HPF and 3-CCA probes were added to cells and spectrofluorometric measurements were performed in their respective wavelengths. The mean amplitude of fluorescence for HPF was 32.72 ± 2.37 F.I (n = 40) and for 3-CCA was 52.11 ± 0.5 F.I (n = 40). This difference was statistically significant. 3-CCA also demonstrated more stable measurements at different days compered to HPF.

Deficiency of filamin A in endothelial cells impairs left ventricular remodelling after myocardial infarction.

AIMS: Actin-binding protein filamin A (FLNA) regulates signal transduction important for cell locomotion, but the role of FLNA after myocardial infarction (MI) has not been explored. The main purpose of this study was to determine the impact of endothelial deletion of FLNA on post-MI remodelling of the left ventricle (LV). METHODS AND RESULTS: We found that FLNA is expressed in human and mouse endothelial cells (ECs) during MI. To determine the biological significance of endothelial expression of FLNA, we used mice that are deficient for endothelial FLNA by cross-breeding adult mice expressing floxed Flna (Flna(o/fl)) with mice expressing Cre under the vascular endothelial-specific cadherin promoter (VECadCre+). Male Flna(o/fl) and Flna(o/fl)/VECadCre+ mice were subjected to permanent coronary artery ligation to induce MI. Flna(o/fl)/VECadCre+ mice that were deficient for endothelial FLNA exhibited larger and thinner LV with impaired cardiac function as well as elevated plasma levels of NT-proBNP and decreased secretion of VEGF-A. The number of capillary structures within the infarcted areas was reduced in Flna(o/fl)/VECadCre+ hearts. ECs silenced for Flna mRNA expression exhibited impaired tubular formation and migration, secreted less VEGF-A, and produced lower levels of phosphorylated AKT and ERK1/2 as well as active RAC1. CONCLUSION: Deletion of FLNA in ECs aggravated MI-induced LV dysfunction and cardiac failure as a result of defective endothelial response and increased scar formation by impaired endothelial function and signalling.

Micro-emulsion synthesis, surface modification, and photophysical properties of Zn(1-x) Mn(x)S nanocrystals for biomolecular recognition.

In this research, we mainly focused on the micro-emulsion synthesis of biotinylated ZnS (zinc sulfide) nanocrystals for avidin recognition. Various samples of Zn(1-x)Mn(x) S, with x = 0.0001, 0.007, 0.02, 0.03, 0.055, 0.09 and 0.13, prepared by quaternary W/O (water-in-oil) microemulsion system. Cyclohexane was used as oil, Triton X-100 as surfactant, n-hexanol as a co-surfactant and mercaptoethanol and thioglycolic acid as linking agents. The obtained products were evaluated by commonly techniques such as: scanning electron microscopy (SEM), transmission electron microscopy (TEM), zeta meter for measurement ZP (zeta potential) and fluorescence spectroscopy analyses. The above-experimental results indicated that the optimum doping concentration of Mn was ~ 5.5% . The fluorescence spectra of the doped crystals consist of orange-red emissions. Eventually, this research showed with increasing more than 18 µl biotin to nanocrystals, no changes were observed in the emission intensity spectra.