Clinical Features of Dominant and Recessive Interferon γ Receptor 1 Deficiencies

Background Interferon ? receptor 1 (IFN? R1) deficiency is a primary immunodeficiency with allelic dominant and recessive mutations characterised clinically by severe infections with mycobacteria. We aimed to compare the clinical features of recessive and dominant IFN?R1 deficiencies. Methods We obtained data from a large cohort of patients worldwide. We assessed these people by medical histories, records, and genetic and immunological studies. Data were abstracted onto a standard form. Findings We identified 22 patients with recessive complete IFN?R1 deficiency and 38 with dominant partial deficiency. BCG and environmental mycobacteria were the most frequent pathogens. In recessive patients, 17 (77%) had environmental mycobacterial disease and all nine BCG-vaccinated patients had BCG disease. In dominant patients, 30 (79%) had environmental mycobacterial disease and 11 (73%) of 15 BCG-vaccinated patients had BCG disease. Compared with dominant patients, those with recessive deficiency were younger at onset of first environmental mycobacterial disease (mean 3·1 years [SD 2·5] vs 13·4 years [14·3], p=0·001), had more mycobacterial disease episodes (19 vs 8 per 100 person-years of observation, p=0·0001), had more severe mycobacterial disease (mean number of organs infected by Mycobacterium avium complex 4·1 [SD 0·8] vs 2·0 [1·1], p=0·004), had shorter mean disease-free intervals (1·6 years [SD 1·4] vs 7·2 years [7·6], p

Kinetic analysis of the cleavage of human protease-activated receptor-1/2/3 and 4 using quenched-fluorescent peptide substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat)/K-m values were determined.

Discovery of novel Agonists and antagonists of the free fatty acid receptor 1 (FFAR1) using virtual screening

The G-protein-coupled receptor free fatty acid receptor 1 (FFAR1), previously named GPR40, is a possible novel target for the treatment of type 2 diabetes. In an attempt to identify new ligands for this receptor, we performed virtual screening (VS) based on two-dimensional (2D) similarity, three-dimensional (3D) pharmacophore searches, and docking studies by using the structure of known agonists and our model of the ligand binding site, which was validated by mutagenesis. VS of a database of 2.6 million compounds followed by extraction of structural neighbors of functionally confirmed hits resulted in identification of 15 compounds active at FFAR1 either as full agonists, partial agonists, or pure antagonists. Site-directed mutagenesis and docking studies revealed different patterns of ligand-receptor interactions and provided important information on the role of specific amino acids in binding and activation of FFAR1.

Up-regulation of GABA transporters and GABAA Receptor α1 subunit in tremor rat hippocampus

The loss of GABAergic neurotransmission has been closely linked with epileptogenesis. The modulation of the synaptic activity occurs both via the removal of GABA from the synaptic cleft and by GABA transporters (GATs) and by modulation of GABA receptors. The tremor rat (TRM; tm/tm) is the parent strain of the spontaneously epileptic rat (SER; zi/zi, tm/tm), which exhibits absence-like seizure after 8 weeks of age. However, there are no reports that can elucidate the effects of GATs and GABAA receptors (GABARs) on TRMs. The present study was conducted to detect GATs and GABAR a1 subunit in TRMs hippocampus at mRNA and protein levels. In this study, total synaptosomal GABA content was significantly decreased in TRMs hippocampus compared with control Wistar rats by high performance liquid chromatography (HPLC); mRNA and protein expressions of GAT-1, GAT-3 and GABAR a1 subunit were all significantly increased in TRMs hippocampus by real time PCR and western blot, respectively; GAT-1 and GABAR a1 subunit proteins were localized widely in TRMs and control rats hippocampus including CA1, CA3 and dentate gyrus (DG) regions whereas only a wide distribution of GAT-3 was observed in CA1 region by immunohistochemistry. These data demonstrate that excessive expressions of GAT-1 as well as GAT-3 and GABAR a1 subunit in TRMs hippocampus may provide the potential therapeutic targets for genetic epilepsy.

Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

Agonist-induced endocytosis of rat somatostatin receptor 1

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand.

Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice

Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

Deletion of Tumor Necrosis Factor-alpha Receptor 1 (TNFR1) Protects against Diet-induced Obesity by Means of Increased Thermogenesis

In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-alpha) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-alpha inflammatory signals are delivered by TNF receptor 1 (R1); however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knock-out (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or a high-fat diet, TNFR1 KO gain significantly less body mass despite increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and blood concentrations of insulin and leptin are lower. Protection from hypothalamic leptin resistance is evidenced by increased leptin-induced suppression of food intake and preserved activation of leptin signal transduction through JAK2, STAT3, and FOXO1. Under the high-fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence of increased thermogenesis includes increased O(2) consumption in respirometry measurements, increased expressions of UCP1 and UCP3 in brown adipose tissue and skeletal muscle, respectively, and increased O(2) consumption by isolated skeletal muscle fiber mitochondria. This demonstrates that TNF-alpha signaling through TNFR1 is an important mechanism involved in obesity-associated defective thermogenesis.

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and alpha-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling. Kidney International (2011) 79, 1217-1227; doi:10.1038/ki.2011.14; published online 16 March 2011

Anxiogenic-like effect induced by TRPV1 receptor activation within the dorsal periaqueductal gray matter in mice

Pharmacological manipulation of TRPV1 (Transient Receptor Potential Vanilloid type-1) receptors has been emerging as a novel target in the investigation of anxiety states. Here, we attempt to show the role played by the TRPV1 receptors within the dorsal periaqueductal gray matter (dPAG), a midbrain structure strongly involved in the modulation of anxiety. Anxiety was assessed by recording spatiotemporal [percent open arm entries (%OE) and percent open arm time (%OT)] and ethological [e.g., head dipping (HD), stretched-attend postures (SAP)] measures in mice exposed to the elevated plus-maze (EPM). Mice received an intra-dPAG injection of the TRPV1 agonist capsaicin (0, 0.01, 0.1 or 1.0. nmol/0.2. μL; Experiment 1) or antagonist capsazepine (0, 10, 30 or 60. nmol/0.2. μL; Experiment 2), or combined injections of capsazepine (30. nmol) and capsaicin (1.0. nmol) (Experiment 3), and were exposed to the EPM to record spatiotemporal and ethological measures. While capsaicin produced an anxiogenic-like effect (it reduced %OE and %OT and frequency of SAP and HD in the open arms), capsazepine did not change any behavior in the EPM. However, when injected before capsaicin (1.0. nmol), intra-dPAG capsazepine (30. nmol-a dose devoid of intrinsic effects) antagonized completely the anxiogenic-like effect of the TRPV1 agonist. These results suggest that the anxiogenic-like effect produced by capsaicin is primarily due to TRPV1 activation within the dPAG in mice, but that dPAG TRPV1 receptors do not exert a tonic control over defensive behavior in mice exposed to the EPM. © 2013 Elsevier B.V.

Expression of Protease Activated Receptor-1 in Chronic Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The orexin receptor 1 in the locus coeruleus modulates the hypercapnic ventilatory response in unanesthetized rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)