The involvement of anti-inflammatory protein, Annexin A1, in ocular toxoplasmosis

Purpose: The aim of this study was to evaluate the expression of the protein annexin A1 (ANXA1), a potent endogenous regulator of the inflammatory process, in ocular toxoplasmosis. Methods: C57BL/6 female mice were infected using intravitreal injections of either 10 6 tachyzoites of Toxoplasma gondii (RH strain; T. gondii) or PBS only (control groups). After 24, 48, and 72 h, animals were sacrificed and their eyes were harvested for histopathological, immunohistochemical, and ultrastructural immunocytochemical analysis of ANXA1. Human retinal pigment epithelial (RPE) cells (ARPE-19) were infected in vitro with T. gondii and collected after 60, 120, 240 min, and 24 h. Results: Compared with non-infected eyes, an intense inflammatory response was observed in the anterior (24 h after infection) and posterior segments (72 h after infection) of the infected eye, characterized by neutrophil infiltration and by the presence of tachyzoites and their consequent destruction along with disorganization of normal retina architecture and RPE vacuolization. T. gondii infection was associated with a significant increase of ANXA1 expression in the neutrophils at 24, 48, and 72 h, and in the RPE at 48 and 72 h. In vitro studies confirmed an upregulation of ANXA1 levels in RPE cells, after 60 and 120 min of infection with T. gondii. Conclusions: The positive modulation of endogenous ANXA1 in the inflammatory and RPE cells during T. gondii infection suggests that this protein may serve as a therapeutic target in ocular toxoplasmosis. © 2012 Molecular Vision

Effects of myenteric denervation on extracellular matrix fibers and mast cell distribution in normal stomach and gastric lesions

Abstract\ud \ud \ud \ud Background\ud \ud In this study the effect of myenteric denervation induced by benzalconium chloride (BAC) on distribution of fibrillar components of extracellular matrix (ECM) and inflammatory cells was investigated in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Rats were divided in four experimental groups: non-denervated (I) and denervated stomach (II) without MNNG treatment; non-denervated (III) and denervated stomachs (IV) treated with MNNG. For histopathological, histochemical and stereological analysis, sections of gastric fragments were stained with Hematoxylin-Eosin, Picrosirius-Hematoxylin, Gomori reticulin, Weigert's Resorcin-Fuchsin, Toluidine Blue and Alcian-Blue/Safranin (AB-SAF).\ud \ud \ud \ud Results\ud \ud BAC denervation causes an increase in the frequency of reticular and elastic fibers in the denervated (group II) compared to the non-denervated stomachs (group I). The treatment of the animals with MNNG induced the development of adenocarcinomas in non-denervated and denervated stomachs (groups III and IV, respectively) with a notable increase in the relative volume of the stroma, the frequency of reticular fibers and the inflammatory infiltrate that was more intense in group IV. An increase in the frequency of elastic fibers was observed in adenocarcinomas of denervated (group IV) compared to the non-denervated stomachs (group III) that showed degradation of these fibers. The development of lesions (groups III and IV) was also associated with an increase in the mast cell population, especially AB and AB-SAF positives, the latter mainly in the denervated group IV.\ud \ud \ud \ud Conclusions\ud \ud The results show a strong association in the morphological alteration of the ECM fibrillar components, the increased density of mast cells and the development of tumors induced by MNNG in the non-denervated rat stomach or denervated by BAC. This suggests that the study of extracellular and intracellular components of tumor microenvironment contributes to understanding of tumor biology by action of myenteric denervation.We are grateful to Domingos Zanchetta Netto and Luiz Roberto Falleiros-Jr for technical assistance. CFE and CBM were supported by Fundação de Amparo á Pesquisa - FAPESP (grants 08/05722-6 and 03/10634-5, respectively) and SRT by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico - CNPq (grants 301111/05-7 and 300163/2008-8).We are grateful to Domingos Zanchetta Netto and Luiz Roberto FalleirosJr for technical assistance. CFE and CBM were supported by Fundação de Amparo á Pesquisa FAPESP (grants 08/057226 and 03/106345, respectively) and SRT by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico CNPq (grants 301111/057 and 300163/20088)

Deletion of annexin A1 in mice upregulates the expression of its receptor, Fpr2/3, and reactivity to the AnxA1 mimetic peptide in platelets

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 (ANXA1Ac2-26) in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor formyl peptide recep-tor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets as characterised by its ability to increase the levels of fibrinogen binding, and exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out us-ing a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3 -deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflamma-tory responses via leukocytes, ANXA1 modulates platelet function which may influence throm-bosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings

TOLL-LIKE RECEPTORS (TLR) 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS

There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the involvement of Toll-Like Receptors (TLRs) 2 and 4 in localized (LD) and disseminated (DD) dermatophytosis due to T. rubrum. Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4 were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2 expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute to the persistent infection and limited inflammation that are characteristic of dermatophytic infections

A mammalian-specific Alex3/Gαq protein complex regulates mitochondrial trafficking, dendritic complexity, and neuronal survival

Mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neural activity. We investigated how G protein–coupled receptors (GPCRs) and G proteins control mitochondrial dynamics and trafficking. The activation of Gαq inhibited mitochondrial trafficking in neurons through a mechanism that was independent of the canonical downstream PLCβ pathway. Mitoproteome analysis revealed that Gαq interacted with the Eutherian-specific mitochondrial protein armadillo repeat–containing X-linked protein 3 (Alex3) and the Miro1/Trak2 complex, which acts as an adaptor for motor proteins involved in mitochondrial trafficking along dendrites and axons. By generating a CNS-specific Alex3 knockout mouse line, we demonstrated that Alex3 was required for the effects of Gαq on mitochondrial trafficking and dendritic growth in neurons. Alex3-deficient mice had altered amounts of ER stress response proteins, increased neuronal death, motor neuron loss, and severe motor deficits. These data revealed a mammalian-specific Alex3/Gαq mitochondrial complex, which enables control of mitochondrial trafficking and neuronal death by GPCRs.This work was funded by the Ministerio de Ciencia e Innovación (grants BFU2017-83379-R to A.M.A., SAF2016-76340R PID2019-106764RB-C21 and PID2022-138105OB-C21 to E.S., SAF2015-65633-R and RTI2018-099357-B-I00 to J.A.E., RTI2018-096386-B-I00 to X.N., EQC2018-004541-P support to E.R., Severo Ochoa Excellence program to J.A.E., and María de Maeztu Excellence program to E.S.), CSIC13-4E-2065 to the Molecular Imaging Platform, and Instituto de Salud Carlos III (CIBERNED to E.S., C.A., X.N. and A.L.d.M.; CIBERER to G.M.; CIBERFES to J.A.E.; grant PI18/01066 to A.L.d.M.; and a collaborative CIBERNED project to E.S. and A.L.d.M.). J.A.E. is supported by the HFSP (RGP0016/2018) and the Pro CNIC Foundation. A.L.d.M. is supported by EiTB Maratoia, grant number BIO17/ND/023, and by Osasun Saila, Eusko Jaurlaritzako, grant number 2015111122. F.J.G.-B. was supported by Roche Stop Fuga de Cerebros (BIO19/ROCHE/017/BD). I.I.-V. was supported by an FI fellowship from AGAUR.Peer reviewe

DNA and bone structure preservation in medieval human skeletons

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimized a method that is frequently used for isolating DNA from modern samples, Chelex® 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR® Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex® 100 is for isolating ancient DNA from archaeological bones and teeth. This optimized method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250 bp were successfully amplified

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder