Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients

Introduction

Agonistic autoantibodies (Aabs) against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients with systemic sclerosis (SSc). In our present study, we examined the expression of the AT1R and the ETAR in human immune cells and the pathological effects mediated through these receptors by their corresponding Aabs.

Methods

Protein expression of AT1R and ETAR on peripheral blood mononuclear cells (PBMCs) from healthy individuals and SSc patients was analyzed using flow cytometry, and mRNA expression of both receptors in PBMCs from healthy donors was examined by real-time PCR. In addition, PBMCs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin G (IgG) fractions from SSc patients positive for AT1R and ETAR Aabs, as well as with IgG from healthy donors serving as controls. Alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, enzyme-linked immunosorbent assays and chemotaxis assays, respectively. The results were correlated with the characteristics and clinical findings of the IgG donors.

Results

Both AT1R and ETAR were expressed on PBMCs in humans. Protein expression of both receptors was decreased in SSc patients compared with that of healthy donors and declined during the course of disease. IgG fractions of SSc patients positive for AT1R and ETAR Aabs induced T-cell migration in an Aab level–dependent manner. Moreover, IgG of SSc patients stimulated PBMCs to produce more interleukin 8 (IL-8) and chemokine (C-C motif) ligand 18 (CCL18) than did the IgG of healthy donors. All effects were significantly reduced by selective AT1R and ETAR antagonists. Statistical analysis revealed an association of SSc-IgG induced high IL-8 concentrations with an early disease stage and of high CCL18 concentrations with lung fibrosis onset and vascular complications in the respective IgG donors.

Conclusion

In our present study, we could demonstrate the expression of both AT1R and ETAR on human peripheral T cells, B cells and monocytes. The decreased receptor expression in SSc patients, the inflammatory and profibrotic effects upon Aab stimulation of PBMCs in vitro and the associations with clinical findings suggest a role for Aab-induced activation of immune cells mediated by the AT1R and the ETAR in the pathogenesis or even the onset of the disease.     

Metals and arsenic in marine fish commercialized in the NE Brazil: Risk to human health

Abstract

Arsenic, cadmium, lead, and mercury in fish is the result of long-term biomagnification in the food chain and is of public concern, due to the toxicity they engender. The objective of this research was to determine the concentrations of arsenic, cadmium, lead, and mercury in 13 species of marine fish broadly commercialized in Aracaju, SE, Brazil and to evaluate the risks of fish consumption associated with these trace elements, using the Target Hazard Quotient (THQ). As, Cd, and Pb levels were measured with inductively coupled plasma mass spectrometry (ICP-MS), and mercury was analyzed via cold vapor atomic absorption spectrometry. The results indicate a large variability in concentrations for arsenic (0.07–2.03?mg kg^–1) and mercury (0.01–1.44?mg kg^–1), associated with the animal dietary category. Cadmium (0.04–0.19?mg kg^–1) and lead (<0.01–0.45?mg kg^–1), on the other hand showed a mild variability. None of the evaluated specimens had As, Cd, and Pb THQ values higher than 1. The THQ values for mercury were higher but indicated no consumption risk, except for amberjack, and snook fish. Overall THQ indicates lower risk of consumption in fish that are at the base of the food chain, than in those that are top predators.     

Heat shock protein gp96 regulates Toll-like receptor 9 proteolytic processing and conformational stability

Nucleic acid-sensing Toll-like receptors (TLRs) initiate innate immune responses to foreign RNA and DNA, yet can detect and respond to host DNA. To avoid autoimmune pathologies, nucleic acid sensing TLRs are tightly regulated. TLR9 primarily resides in the endoplasmic reticulum, traffics to endosomes, is proteolytically processed and responds to DNA. The heat shock protein gp96 is one of several accessory proteins that regulate intracellular trafficking of TLR9. In the absence of gp96, TLR9 fails to exit the endoplasmic reticulum, and therefore gp96-deficient macrophages fail to respond to CpG DNA. However, absence of gp96 precludes studies on potential chaperoning functions of gp96 for TLR9. Here we demonstrate that pharmacologic interference with gp96 function inhibits TLR9 signaling. TLR9 remains associated with gp96 during intracellular trafficking, and gp96-specific inhibitors increase TLR9 sensitivity to proteolytic degradation. We propose that gp96 is critical for both TLR9 egress from the ER, and for protein conformational stability in the endosomal compartment. These studies highlight the importance of examining gp96-specific inhibitors for modulating TLR9 activation, and the treatment autoimmune diseases.     

Probability of mast cells to mediate anaphylaxis in skeletal muscle

Are there enough mast cells in denervated skeletal muscle to account for autopharmacological mediation of the antigen potentials (APs) elicited by microtaps? Through rough qualitative estimations, some authors have suggested a positive answer to this question. However, in view of measurements performed in this investigation of both the density of mast cells and the diffusion coefficient of antigens, the probability of such mediated effects was found to be relatively low:P=0.016 for egg albumin andP=0.004 for ferritin. Therefore, most APs induced by microtaps should be attributed to the direct effect of antigen over the sensitized muscle fibers. Yet, both the density of mast cells found in this work and the known amount of histamine they are capable of releasing when challenged with antigen, support the hypothesis regarding the involvement of these cells when antigen is massively superfused so as to induce Schultz-Dale reactions in muscle strips. Under this circumstance, the direct and mediated mechanisms may coexist.     

Noninvasive Ultrasound Molecular Imaging of the Effect of Statins on Endothelial Inflammatory Phenotype in Early Atherosclerosis

Background/Objectives

Inflammatory changes on the endothelium are responsible for leukocyte recruitment to plaques in atherosclerosis. Noninvasive assessment of treatment-effects on endothelial inflammation may be of use for managing medical therapy and developing novel therapies. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) with contrast enhanced ultrasound (CEU) could assess treatment effects on endothelial phenotype in early atherosclerosis.

Methods

Mice with atherosclerosis produced by gene deletion of the LDL-receptor and Apobec-1-editing protein were studied. At 12 weeks of age, mice received 8 weeks of regular chow or atorvastatin-enriched chow (10 mg/kg/day). At 20 weeks, CEU molecular imaging for aortic endothelial VCAM-1 expression was performed with VCAM-1-targeted (MB_VCAM) and control microbubbles (MB_Ctr). Aortic wall thickness was assessed with high frequency ultrasound. Histology, immunohistology and Western blot were used to assess plaque burden and VCAM-1 expression.

Results

Plaque burden was reduced on histology, and VCAM-1 was reduced on Western blot by atorvastatin, which corresponded to less endothelial expression of VCAM-1 on immunohistology. High frequency ultrasound did not detect differences in aortic wall thickness between groups. In contrast, CEU molecular imaging demonstrated selective signal enhancement for MB_VCAM in non-treated animals (MB_VCAM 2±0.3 vs MB_Ctr 0.7±0.2, p<0.01), but not in statin-treated animals (MB_VCAM 0.8±0.2 vs MB_Ctr 1.0±0.2, p = ns; p<0.01 for the effect of statin on MB_VCAM signal).

Conclusions

Non-invasive CEU molecular imaging detects the effects of anti-inflammatory treatment on endothelial inflammation in early atherosclerosis. This easily accessible, low-cost technique may be useful in assessing treatment effects in preclinical research and in patients.     

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.     

Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF-/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-gamma by CD4(+) T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.     

A Bayesian Hierarchical Model for Estimation of Abundance and Spatial Density of Aedes aegypti

Strategies to minimize dengue transmission commonly rely on vector control, which aims to maintain Ae. aegypti density below a theoretical threshold. Mosquito abundance is traditionally estimated from mark-release-recapture (MRR) experiments, which lack proper analysis regarding accurate vector spatial distribution and population density. Recently proposed strategies to control vector-borne diseases involve replacing the susceptible wild population by genetically modified individuals’ refractory to the infection by the pathogen. Accurate measurements of mosquito abundance in time and space are required to optimize the success of such interventions. In this paper, we present a hierarchical probabilistic model for the estimation of population abundance and spatial distribution from typical mosquito MRR experiments, with direct application to the planning of these new control strategies. We perform a Bayesian analysis using the model and data from two MRR experiments performed in a neighborhood of Rio de Janeiro, Brazil, during both low- and high-dengue transmission seasons. The hierarchical model indicates that mosquito spatial distribution is clustered during the winter (0.99 mosquitoes/premise 95% CI: 0.80–1.23) and more homogeneous during the high abundance period (5.2 mosquitoes/premise 95% CI: 4.3–5.9). The hierarchical model also performed better than the commonly used Fisher-Ford’s method, when using simulated data. The proposed model provides a formal treatment of the sources of uncertainty associated with the estimation of mosquito abundance imposed by the sampling design. Our approach is useful in strategies such as population suppression or the displacement of wild vector populations by refractory Wolbachia-infected mosquitoes, since the invasion dynamics have been shown to follow threshold conditions dictated by mosquito abundance. The presence of spatially distributed abundance hotspots is also formally addressed under this modeling framework and its knowledge deemed crucial to predict the fate of transmission control strategies based on the replacement of vector populations.