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Like human language, song in songbirds is learned during an early sensitive period and is facilitated by motivation to seek out social interactions with vocalizing adults. Songbirds are therefore powerful models with which to understand the neural underpinnings of vocal learning. Social motivation and early social orienting are thought to be mediated by the oxytocin system; however, the developmental trajectory of oxytocin receptors in songbirds, particularly as it relates to song learning, is currently unknown. This gap in knowledge has hindered the development of songbirds as a model of the role of social orienting in vocal learning. In this study, we used quantitative PCR to measure oxytocin receptor expression during the sensitive period of song learning in zebra finches (Taeniopygia guttata). We focused on brain regions important for social motivation, attachment, song recognition, and song learning. We detected expression in these regions in both sexes from post-hatch day 5 to adulthood, encompassing the entire period of song learning. In this species, only males sing; we found that in regions implicated in song learning specifically, oxytocin receptor mRNA expression was higher in males than females. These sex differences were largest during the developmental phase when males attend to and memorize tutor song, suggesting a functional role of expression in learning. Our results show that oxytocin receptors are expressed in relevant brain regions during song learning, and thus provide a foundation for developing the zebra finch as a model for understanding the mechanisms underlying the role of social motivation in vocal development. This article is protected by copyright. All rights reserved.

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR., Integrin-linked kinase (ILK) is an important mediator of integrin signaling. Here Park et al. show that mice with endothelial-specific deletion of Ilk develop vascular defects that resemble familial exudative vitreoretinopathy, and identify mutations in ILK in patients with exudative vitreoretinopathy suggesting a potential role in human pathogenesis.

Background Braak’s hypothesis states that sporadic Parkinson’s disease (PD) follows a specific progression of pathology from the peripheral to the central nervous system, and this progression can be monitored by detecting the accumulation of alpha-Synuclein (α-Syn) protein. Consequently, there is growing interest in understanding how the gut (commensal) microbiome can regulate α-Syn accumulation, as this could potentially lead to PD. Methods We used 16S rRNA and shotgun sequencing to characterise microbial diversity. 1H-NMR was employed to understand the metabolite production and intestinal inflammation estimated using ELISA and RNA-sequencing from feces and the intestinal epithelial layer respectively. The Na+ channel current and gut permeability were measured using an Ussing chamber. Immunohistochemistry and immunofluorescence imaging were applied to detect the α-Syn protein. LC-MS/MS was used for characterization of proteins from metabolite treated neuronal cells. Finally, Metascape and Ingenuity Pathway Analysis (IPA) bioinformatics tools were used for identification of dysregulated pathways. Results We studied a transgenic (TG) rat model overexpressing the human SNCA gene and found that a progressive gut microbial composition alteration characterized by the reduction of Firmicutes to Bacteroidetes ratio could be detected in the young TG rats. Interestingly, this ratio then increased with ageing. The dynamics of Lactobacillus and Alistipes were monitored and reduced Lactobacillus and increased Alistipes abundance was discerned in ageing TG rats. Additionally, the SNCA gene overexpression resulted in gut α-Syn protein expression and increased with advanced age. Further, older TG animals had increased intestinal inflammation, decreased Na+ current and a robust alteration in metabolite production characterized by the increase of succinate levels in feces and serum. Manipulation of the gut bacteria by short-term antibiotic cocktail treatment revealed a complete loss of short-chain fatty acids and a reduction in succinate levels. Although antibiotic cocktail treatment did not change α-Syn expression in the enteric nervous system of the colon, however, reduced α-Syn expression was detected in the olfactory bulbs (forebrain) of the TG rats. Conclusion Our data emphasize that the gut microbiome dysbiosis synchronous with ageing leads to a specific alteration of gut metabolites and can be modulated by antibiotics which may affect PD pathology.

Antibiotics are widely used in zoo technical and veterinary practices as feed supplementation to ensure wellness of farmed animals and livestock. Several evidences have been suggesting both the toxic role for tetracyclines, particularly for oxytetracycline (OTC). This potential toxicity appears of great relevance for human nutrition and for domestic animals. This study aimed to extend the evaluation of such toxicity. The biologic impact of the drug was assessed by evaluating the proinflammatory effect of OTC and their bone residues on cytokine secretion by in vitro human peripheral blood lymphocytes. Our results showed that both OTC and OTC‐bone residues significantly induced the T lymphocyte and non‐T cell secretion of interferon (IFN)‐γ, as cytokine involved in inflammatory responses in humans as well as in animals. These results may suggest a possible implication for new potential human and animal health risks depending on the entry of tetracyclines in the food‐processing chain.

Trypanosoma cruzi is the etiological agent of Chagas disease, a major health problem in Latin America. Polyamines are polycationic compounds that play a critical role as regulators of cell growth and differentiation. In contrast with other protozoa, T. cruzi is auxotrophic for polyamines because of its inability to synthesize putrescine due to the lack of both, arginine and ornithine decarboxylase; therefore, the intracellular availability of polyamines depends exclusively on transport processes. In this work, the polyamine transporter TcPAT12 was overexpressed in T. cruzi epimastigotes demonstrating that growth rates at different concentrations of polyamines strongly depend on the regulation of the polyamine transport. In addition, parasites overexpressing TcPAT12 showed a highly increased resistance to hydrogen peroxide and the trypanocidal drugs nifurtimox and benznidazole, which act by oxidative stress and interfering the synthesis of polyamine derivatives, respectively. Finally, the presence of putative polyamine transporters was analyzed in T. cruzi, Trypanosoma brucei, and Leishmania major genomes identifying 3-6 genes in these trypanosomatids.

Chagas disease is a neglected tropical disease endemic to Latin America, though migratory movements have recently spread it to other regions. Here, we have applied a cascade virtual screening campaign combining ligand- and structure-based methods. In order to find novel inhibitors of putrescine uptake in Trypanosoma cruzi, an ensemble of linear ligand-based classifiers obtained by has been applied as initial screening filter, followed by docking into a homology model of the putrescine permease TcPAT12. 1,000 individual linear classifiers were inferred from a balanced dataset. Subsequently, different schemes were tested to combine the individual classifiers: MIN operator, average ranking, average score, average voting, with MIN operator leading to the best performance. The homology model was based on the arginine/agmatine antiporter (AdiC) from Escherichia coli as template. It showed 64% coverage of the entire query sequence and it was selected based on the normalized Discrete Optimized Protein Energy parameter and the GA341 score. The modeled structure had 96% in the allowed area of Ramachandran's plot, and none of the residues located in non-allowed regions were involved in the active site of the transporter. Positivity Predictive Value surfaces were applied to optimize the score thresholds to be used in the ligand-based virtual screening step: for that purpose Positivity Predictive Value was charted as a function of putative yields of active in the range 0.001-0.010 and the Se/Sp ratio. With a focus on drug repositioning opportunities, DrugBank and Sweetlead databases were subjected to screening. Among 8 hits, cinnarizine, a drug frequently prescribed for motion sickness and balance disorder, was tested against T. cruzi epimastigotes and amastigotes, confirming its trypanocidal effects and its inhibitory effects on putrescine uptake. Furthermore, clofazimine, an antibiotic with already proven trypanocidal effects, also displayed inhibitory effects on putrescine uptake. Two other hits, meclizine and butoconazole, also displayed trypanocidal effects (in the case of meclizine, against both epimastigotes and amastigotes), without inhibiting putrescine uptake., Facultad de Ciencias Exactas, Laboratorio de Investigación y Desarrollo de Bioactivos

We agree with and expand on the points made by Costanza and Finkelstein (2015) regarding the definition of “generation” and its measurement, the lack of theory in this area; the harmful effects of propagating generationally based differences to organizations, society, and individuals; and the future directions to make this line of research more applicable to organizations and the workplace. Examining age differences at work through the lens of well-established within-person changes in physical ability (e.g., Maertens, Putter, Chen, Diehl, & Huang, 2012), cognition (e.g., Schaie, 1994), motivation (e.g., Kooij, De Lange, Jansen, Kanfer, & Dikkers, 2011), personality (e.g., Roberts, Walton, & Viechtbauer, 2006), and lifespan development theories (e.g., Baltes & Baltes, 1990; Carstensen, Isaacowitz, & Charles, 1999) would likely be more productive than using loose, relatively atheoretical generational stereotypes to understand age differences at work.

Inducible regulatory T cells (Tregs) exert a timely and efficient immunosuppressive action at the critical peri-implantation stage essential for maternal tolerance to the conceptus. Vasoactive intestinal peptide (VIP) promotes anti-inflammatory and tolerogenic profiles through binding to VIP receptors on immune cells. We evaluated whether VIP produced by trophoblast cells induces Tregs during the early interaction of maternal leukocytes with trophoblast cells, thus contributing to maternal tolerance. We used an in vitro model of maternal leukocyte–trophoblast cell interaction represented by cocultures of fertile women’s PBMCs with a human trophoblast cell line (Swan-71) and evaluated the effect of VIP added exogenously and of the endogenous polypeptide. VIP increased the frequency of CD4+CD25+FoxP3+ cells after coculture, and these cells were able to suppress the maternal alloresponse. VIP also increased the frequency of CD4+IL10+ and CD4+TGFβ+ cells, but it did not modulate IFN-γ or IL-17 production. Swan-71 secreted VIP, and their coculture with maternal PBMCs significantly increased the frequency of Tregs. This effect was even more pronounced if the trophoblast cells had been pretreated with VIP. In both situations, the VIP antagonist prevented the increase in the frequency of CD4+Foxp3+ cells, reflecting a specific effect of the polypeptide after the interaction with Swan-71 cells. Finally, the increase in CD4+CD25+FoxP3+ frequency was prevented by an anti–TGF-β Ab and a VIP antagonist. These results suggest that VIP could have an active role in the immunoregulatory processes operating in the maternal–placental interface by contributing to the induction of Tregs through a mechanism involving TGF-β1. Fil: Fraccaroli, Laura Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Grasso, Esteban Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hauk, Vanesa Cintia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paparini, Daniel Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Soczewski, Elizabeth Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Mor, Gil. University of Yale. School of Medicine; Estados Unidos Fil: Perez Leiros, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Ramhorst, Rosanna Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Supplemental Digital Content is available in the text., Rationale: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. Objective: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. Methods and Results: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv–deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell–extracellular matrix adhesion structures and an altered actin cytoskeleton. Conclusions: Endothelial α-pv is essential for vessel sprouting and for vessel stability.

Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients) to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression., Author summary In spite of its old discovery, more than one hundred years ago, Trypanosoma cruzi, the causative agent of Chagas’ disease, is still prevalent in the world, infecting more than 6 million people mostly in Latin America, where this illness is endemic. Only two approved drugs, benznidazole and nifurtimox, are currently used for the treatment of Chagas’ disease. Although efficient for the acute phase, they are poorly effective in the chronic period of the disease and they cause many undesirable side effects. There is an urgent need for therapeutic alternatives. To this end, identifying and validating novel molecular targets is critically relevant. This study describes the effect of different inhibitors on the T. cruzi autophagic pathway, a process required for parasite differentiation. Herein, we demonstrate that the regulation of parasite autophagy exhibits similarities and differences with host cell autophagy. Our study provides new insights that could be used to avoid T. cruzi cycle progression in both insect and mammalian hosts.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by salivary and lacrimal gland dysfunction. Clinical observations and results from animal models of SS support the role of aberrant epithelial cell apoptosis and immune homeostasis loss in the glands as triggering factors for the autoimmune response. Vasoactive intestinal peptide (VIP) promotes potent anti-inflammatory effects in several inflammatory and autoimmune disease models, including the non-obese diabetic (NOD) mouse model of SS. With the knowledge that VIP modulates monocyte function through vasoactive intestinal peptide receptors (VPAC) and that immune homeostasis maintenance depends strongly upon a rapid and immunosuppressant apoptotic cell clearance by monocytes/macrophages, in this study we explored VPAC expression on monocytes from primary SS (pSS) patients and the ability of VIP to modulate apoptotic cell phagocytic function and cytokine profile. Monocytes isolated from individual pSS patients showed an increased expression of VPAC2 subtype of VIP receptors, absent in monocytes from control subjects, with no changes in VPAC1 expression. VPAC2 receptor expression could be induced further with lipopolysaccharide (LPS) in pSS monocytes and VIP inhibited the effect. Moreover, monocytes from pSS patients showed an impaired phagocytosis of apoptotic epithelial cells, as evidenced by reduced engulfment ability and the failure to promote an immunosuppressant cytokine profile. However, VIP neither modulated monocyte/macrophage phagocytic function nor did it reverse their inflammatory profile. We conclude that monocytes from pSS patients express high levels of VPAC2 and display a deficient clearance of apoptotic cells that is not modulated by VIP. Fil: Hauk, Vanesa Cintia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fraccaroli, Laura Virginia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Grasso, Esteban Nicolas. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Eimon, Alicia. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: Ramhorst, Rosanna Elizabeth. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hubscher, Osvaldo. Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”; Argentina Fil: Perez Leiros, Claudia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Several extrinsic factors, like drugs and chemicals, can foster autoimmunity. Tetracyclines, in particular oxytetracycline (OTC), appear to correlate with the emergence of immune-mediated diseases. Accumulation of OTC, the elective drug for gastrointestinal and respiratory infectious disease treatment in broiler chickens, was reported in chicken edible tissues and could represent a potential risk for pets and humans that could assume this antibiotic as residue in meat or in meat-derived byproducts. We investigated thein vitroanti-inflammatory properties of a pool of thirteen botanicals as a part of a nutraceutical diet, with proven immunomodulatory activity.In addition, we evaluated the effect of such botanicals in contrasting thein vitroproinflammatory toxicity of OTC. Our results showed a significant reduction in interferon- (INF-)γproduction by human and canine lymphocytes in presence of botanicals (p⁎<0.05). Increased INF-γproduction, dependent on 24-hour OTC-incubation of T lymphocytes, was significantly reduced by the coincubation withHaematococcus pluvialis, withGlycine max, and with the mix of all botanicals (p⁎<0.05). In conclusion, the use of these botanicals was shown to be able to contrast OTC-toxicity and could represent a new approach for the development of functional foods useful to enhance the standard pharmacological treatment in infections as well as in preventing or reducing the emergence of inflammatory diseases.

Summary Successful embryo implantation occurs followed by a local inflammatory/T helper type 1 (Th1) response, subsequently redirected towards a tolerogenic predominant profile. The lack of control of this initial local inflammatory response may be an underlying cause of early pregnancy complications as recurrent spontaneous abortions (RSA). Considering that vasoactive intestinal peptide (VIP) mediates anti-inflammatory and tolerogenic effects in several conditions we hypothesized that VIP might contribute to tolerance towards trophoblast antigens during the early interaction of maternal leucocytes and trophoblast cells. In this study we investigated VIP/VPAC system activity and expression on maternal peripheral blood mononuclear cells (PBMCs) after interaction with immortalized trophoblast cells (Swan-71 cell line) as an in-vitro model of feto–maternal interaction, and we analysed whether it modulates maternal regulatory T cell (Treg)/Th1 responses. We also investigated the contribution of the endogenous VIP/VPAC system to RSA pathogenesis. VIP decreased T-bet expression significantly, reduced monocyte chemotactic protein-1 (MCP-1) and nitrite production in co-cultures of PBMCs from fertile women with trophoblast cells; while it increased the frequency of CD4+CD25+ forkhead box protein 3 (Foxp3)+ cells, transforming growth factor (TGF)-β expression and interleukin (IL)-10 secretion. These effects were prevented by VIP-specific antagonist. Interestingly, PBMCs from RSA patients displayed significantly higher T-bet expression, lower Treg frequency and lower frequency of VIP-producer CD4 lymphocytes after the interaction with trophoblast cells. Moreover, the patients displayed a significantly lower frequency of endometrial CD4+VIP+ cells in comparison with fertile women. VIP showed a Th1-limiting and Treg-promoting response in vitro that would favour early pregnancy outcome. Because RSA patients displayed defects in the VIP/VPAC system, this neuropeptide could be a promising candidate for diagnostic biomarker or surrogate biomarker for recurrent spontaneous abortions.

BACKGROUND Dendritic cells (DCs), which are biased toward a tolerogenic profile, play a pivotal role in tissue-remodeling processes and angiogenesis at the maternal-fetal interface. Here, we analyzed the effect of trophoblast cells on the functional profile of DCs to gain insight on the tolerogenic mechanisms underlying the human placental-maternal dialog at early stages of gestation. METHODS DCs were differentiated from peripheral blood monocytes obtained from fertile women (n = 21), in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor during 5 days in culture. Then, DCs were cultured with trophoblast cells (Swan-71 cell line obtained from normal cytotrophoblast, at 7 weeks) for 24 h and for an additional 24 h in the absence or presence of lipopolysaccharide (LPS) from Escherichia coli. DCs were recovered and used for flow cytometry, enzyme-linked immunosorbent assay, RT-PCR and suppression and migration assays. RESULTS Trophoblast cells significantly prevented the increase in CD83 expression induced by LPS without affecting the expression of CD86, CD40 and human leukocyte antigen-DR (P < 0.05). Trophoblast cells significantly decreased the production of IL-12p70 and tumor necrosis factor-α, while it increased the production of IL-10 (P < 0.05). No changes were observed in the production of IL-6 and monocyte chemotactic protein-1. The culture of DCs with trophoblast cells, also suppressed the stimulation of the allogeneic response triggered by LPS (P < 0.05). Conditioned DCs were able to increase the frequency of CD4 + CD25 + Foxp3 cells and this effect was accompanied by an increase in indoleamine 2, 3-dioxygenase expression in DCs (P < 0.05). CONCLUSIONS The interaction of DCs with trophoblast cells promotes the differentiation of DCs into cells with a predominantly tolerogenic profile that could contribute to a tolerogenic microenvironment at the maternal-fetal interface.

Citation Ramhorst R, Fraccaroli L, Aldo P, Alvero AB, Cardenas I, Leiros CP, Mor G. Modulation and recruitment of inducible regulatory T cells by first trimester trophoblast cells. Am J Reprod Immunol 2012; 67: 17–27 Problem The specialized regulatory T-cells (Treg) population, essential for maternal tolerance of the fetus, performs its suppressive actions in the critical peri-implantation phase of pregnancy. In the present work, we investigated whether trophoblast cells are able to induce Treg recruitment, differentiation, and whether these mechanisms are modified by a bacterial or viral infection. Method of Study Human T-regulatory cells were differentiated from naive CD45RA+ CCR7+ cells obtained from peripheral blood mononuclear cells cultured with IL-2 and TGFβ over 5 days. Induction of iTregs (CD4+ Foxp3+ cells) was evaluated using low serum conditioned media (LSCM), obtained from two first trimester trophoblast cell lines, Swan-71 and HTR8. Coculture experiments were carried out using transwell assays where trophoblast cells were in the absence or presence of PGN, LPS, or Poly [I:C]. Cytokine production was measured by multiplex analysis. Results Trophoblast cells constitutively secrete high levels of TGFβ and induced a significant increase of Foxp3 expression accompanied by a specific T-reg cytokine profile. Moreover, trophoblast cells were able to recruit iTregs in a specific manner. Conclusion We demonstrate that trophoblast cells have an active role on the recruitment and differentiation of iTregs, therefore, contributing to the process of immune regulation at the placental–maternal interface.

Citation Fraccaroli L, Grasso E, Zeitler E, Lombardi E, Gogorza S, Etchepareborda JJ, Nagle C, Cortelezzi M, Leiros CP, Ramhorst R. Modulation of maternal LIF producers T cells by trophoblast and paternal antigens. Am J Reprod Immunol 2011; 65: 133–145 Problem Fetal implantation enhances the production of essential growth factors such as LIF (leukaemia inhibitory factor), hence we investigated the contribution of maternal CD4 cells, activated by paternal or trophoblast antigens and its modulation by VIP (vasoactive intestinal peptide) and progesterone. Method of study We performed co cultures of trophoblast cells (Swan-71 cell line) or paternal antigens and PBMCs from patients with recurrent spontaneous abortions (RSA) and fertile women. Result Fertile-CD4+LIF+ cells were increased by VIP and progesterone in response to paternal and trophoblast antigens. Also MMP-9 activity was decreased and pSTAT3/STAT3 ratio was increased. RSA patients have decreased levels of LIF expression which could not be modulated by VIP and progesterone and displayed a reduced number of endometrial infiltrated CD4+LIF+ cells compared with fertile women. Conclusion The decrease of CD4+LIF+ cells in RSA patients could be related with the exacerbated inflammatory response observed in the maternal-fetal dialogue model.

Human herpesvirus 6 (HHV-6) is prevalent in healthy persons, causes disease in immunosuppressed carriers, and may be involved in autoimmune disease. Cytotoxic CD8 T cells are probably important for effective control of infection. However, the HHV-6-specific CD8 T cell repertoire is largely uncharacterized. Therefore, we undertook a virus-wide analysis of CD8 T cell responses to HHV-6. We used a simple anchor motif-based algorithm (SAMBA) to identify 299 epitope candidates potentially presented by the HLA class I molecule B*08:01. Candidates were found in 77 of 98 unique HHV-6B proteins. From peptide-expanded T cell lines, we obtained CD8 T cell clones against 20 candidates. We tested whether T cell clones recognized HHV-6-infected cells. This was the case for 16 epitopes derived from 12 proteins from all phases of the viral replication cycle. Epitopes were enriched in certain amino acids flanking the peptide. Ex vivo analysis of eight healthy donors with HLA-peptide multimers showed that the strongest responses were directed against an epitope from IE-2, with a median frequency of 0.09% of CD8 T cells. Reconstitution of T cells specific for this and other HHV-6 epitopes was also observed after allogeneic hematopoietic stem cell transplantation. We conclude that HHV-6 induces CD8 T cell responses against multiple antigens of diverse functional classes. Most antigens against which CD8 T cells can be raised are presented by infected cells. Ex vivo multimer staining can directly identify HHV-6-specific T cells. These results will advance development of immune monitoring, adoptive T cell therapy, and vaccines., Author summary This paper deals with the immune response to a very common virus, called human herpesvirus 6 (HHV-6). Most people catch HHV-6 in early childhood, which often leads to a disease known as three-day fever. Later in life, the virus stays in the body, and an active immune response is needed to prevent the virus from multiplying and causing damage. It is suspected that HHV-6 contributes to autoimmune diseases and chronic fatigue. Moreover, patients with severely weakened immune responses, for example after some forms of transplantation, clearly have difficulties controlling HHV-6, which puts them at risk of severe disease and shortens their survival. This can potentially be prevented by giving them HHV-6-specific "killer" CD8 T cells, which are cells of the immune system that destroy body cells harboring the virus. However, little is known so far about such T cells. Here, we describe 16 new structures that CD8 T cells can use to recognize and kill HHV-6-infected cells. We show that very different viral proteins can furnish such structures. We also observe that such T cells are regularly present in healthy people and in transplant patients who control the virus. Our results will help develop therapies of disease due to HHV-6.

Despite current efforts worldwide to develop new medications against Chagas disease, only two drugs are available, nifurtimox and benznidazole. Both drugs require prolonged treatment and have multiple side effects and limited efficacy on adult patients chronically infected with Trypanosoma cruzi. Recently, computer-guided drug repositioning led to the discovery of the trypanocidal effects of clofazimine and benidipine. These compounds showed inhibitory effects on cruzipain, the major cysteine protease of T. cruzi, of different parasite stages and in a murine model of acute Chagas disease. The aim of this work was to determine the efficacy of these novel cruzipain inhibitors when administered in a murine model of chronic Chagas disease. Benidipine and clofazimine were able to reduce the parasite burden in cardiac and skeletal muscles of chronically infected mice compared with untreated mice as well as diminish the inflammatory process in these tissues. Further studies should be performed to study the synergism with benznidazole and nifurtimox in view of combined therapies.

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.

The ability to identify the largest amount of prey available is fundamental for optimizing foraging behaviour in several species. To date, this cognitive skill has been observed in all vertebrate groups except reptiles. In this study we investigated the spontaneous ability of ruin lizards to select the larger amount of food items. In Experiment 1, lizards proved able to select the larger food item when presented with two alternatives differing in size (0.25, 0.50, 0.67 and 0.75 ratio). In Experiment 2 lizards presented with two groups of food items (1 versus 4, 2 versus 4, 2 versus 3 and 3 versus 4 items) were unable to select the larger group in any contrast. The lack of discrimination in the presence of multiple items represents an exception in numerical cognition studies, raising the question as to whether reptiles' quantitative abilities are different from those of other vertebrate groups.

Problem: Women with celiac disease (CD) are often affected by atypical presentations of the disease associated with reproductive disorders as a main extra-digestive complaint. Here, we analyzed if autoantibodies against tissue transglutaminase (tTG) in sera from CD patients with reproductive disorders could display direct effects through their interaction with tTG expressed on trophoblast cells and phagocytes inducing tissue damage and interfering in the clearance of trophoblast apoptotic bodies. Method of study: Sera from CD women with reproductive disorders were obtained, and their ability to induce apoptosis of Swan-71 (cytotrophoblast cell line) and to modulate the wound-healing and phagocytes process was tested. Results: Swan-71 cells expressed tTG and CD sera displayed a significant decrease in trophoblast cell migration and a delay in injury healing on trophoblast cells, compared with those observed with control sera. Moreover, CD sera significantly reduced trophoblast cell proliferation and increased apoptosis levels in comparison with those observed in the control sera. Finally, autoantibodies against tTG interfere in the clearance of trophoblast apoptotic bodies through a mechanism involving MFG-E8 (milk fat globulin-EGF factor 8)-tTG binding. Conclusion: The anti-tTG antibodies might contribute to trophoblast damage and disrupt the phagocytosis process of apoptotic bodies that could promote a pro-inflammatory microenvironment. Fil: Sóñora, Cecilia. Universidad de la República. Facultad de Ciencias; Uruguay Fil: Calo, Guillermina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fraccaroli, Laura Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Perez Leiros, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hernández, Ana. Universidad de la República. Facultad de Ciencias; Uruguay Fil: Ramhorst, Rosanna Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Sjogren's syndrome (SS) is a chronic inflammatory disease characterized by salivary and lacrimal gland dysfunction although extraglandular manifestations are also found. Suitable study models and in vitro cell culture designs are used to approach SS pathogenic mechanisms. Cellular and molecular pathways involved in gland homeostasis loss and the autoimmune response are focused in the search of novel drug targets and biomarkers. Vasoactive intestinal peptide (VIP) has trophic, pro-secretory and immunomodulatory effects in several chronic and autoimmune disease models. Here we review evidence pointing to its role as an endogenous modulator of gland homeostasis at early stages of the disease. Particularly, mechanisms involving VIP/VPAC system in the course of salivary function impairment in the non obese diabetic (NOD) mouse model of Sjogren's syndrome are described.

Background/Aims: The maternal-fetal interface is a unique immunological site that generates an adequate microenvironment during pregnancy, recognizing and eliminating infections and tolerating the trophoblast/placenta unit. For that purpose, trophoblast cells display several tolerogenic mechanisms to allow fetal survival, such as production of the neuropeptide vasoactive intestinal peptide (VIP). Here we investigated the contribution of VIP to maintain homeostasis at the maternal-placental interface under lipopolysaccharide (LPS) stimulation. Methods: We performed cocultures between trophoblast cells (Swan-71 cell line) and maternal leukocytes obtained from fertile women as an in vitro model of maternal-placental interaction, and we focused on the effects of LPS on the modulation of VIP and their receptors (VPAC1 and VPAC2). Results: VIP could prevent the upregulation of IL-6, MCP-1, and nitrite production and maintain the production of IL-10 and TGF-β under LPS (10 µg/ml) stimulation after 48 h of coculture. To gain deeper insight into the mechanisms of how VIP could contribute to a tolerogenic microenvironment even in the presence of LPS, we investigated VIP production by maternal leukocytes and observed a significant increase in the frequency of CD4+VIP+ cells after interaction with Swan-71 cells in the presence of LPS. LPS increased VIP and inducible receptor VPAC2 expression directly on trophoblast cells in a dose- and time-dependent manner. Conclusions: The present results suggest that VIP might act as an additional homeostatic mechanism during early stages at the maternal-placental interface to control exacerbated inflammatory responses such as the ones observed in intrauterine infections. Fil: Fraccaroli, Laura Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Grasso, Esteban Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hauk, Vanesa Cintia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Cortelezzi, M.. Sociedad Argentina de Endocrinología Ginecológica y Reproductiva; Argentina Fil: Perez Leiros, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Ramhorst, Rosanna Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.

Mechanisms accounting for protection of the fetal semiallograft from maternal immune cells remain incompletely understood. In previous studies we showed that galectin-1 (Gal1), an immunoregulatory glycan-binding protein, plays key roles in modulating immune responses during murine pregnancies. Here we show that Gal1 fosters immune privilege at the human fetomaternal interface through modulation of multiple tolerance mechanisms. Gal1 was mainly expressed in invasive extravillous trophoblast cells of human first trimester and term placenta in direct contact with maternal tissue. Trophoblast-derived Gal1 was primarily controlled by progesterone and pro-inflammatory cytokines and fostered T cell tolerance by favoring the expansion of CD4+CD25+FoxP3+ regulatory T (Treg) cells, suppressing the secretion of Th1-type cytokines and limiting T cell viability. Targeted inhibition of Gal1 expression through different strategies including Ab-mediated blockade, addition of the specific disaccharide lactose and silencing through retroviral-mediated siRNA strategies prevented these immunoregulatory effects. Consistent with a tolerogenic function for endogenous Gal1, patients with recurrent pregnancy loss showed considerably lower levels of circulating Gal1 and had greatly enhanced frequency of anti-Gal1 autoantibodies in their sera compared with fertile women. Thus, endogenous Gal1 confers immune privilege to human trophoblasts by triggering a broad tolerogenic program, with potential implications in threatened pregnancies. Fil: Ramhorst, Rosanna Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina Fil: Giribaldi, María Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Fraccaroli, Laura Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina Fil: Toscano, Marta Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Stupirski, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Romero, Marta D.. Laboratorio Inmunopatologia Investigación y Docencia; Argentina Fil: Durand, Edith Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Rubinstein, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Blaschitz, Astrid. Medizinische Universität Graz; Austria Fil: Sedlmayr, Peter. Medizinische Universität Graz; Austria Fil: Genti de Raimondi, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Fainboim, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires; Argentina

Echinacea angustifolia cell suspension cultures are usually grown and maintained in the dark, but we also exposed cells to light for one culture cycle (14 days) and then compared the metabolomes of dark-grown and illuminated cells by liquid chromatography–mass spectrometry. Among 256 signals, we putatively identified 159 molecules corresponding to 56 different metabolites plus their fragments, adducts and isotopologs. The E. angustifolia metabolome consisted mainly of caffeic acid derivatives, comprising (a) caffeic acid conjugated with tartaric, quinic and hexaric acids; and (b) caffeic acid conjugated with hydroxytyrosol glycosides (e.g., echinacoside, verbascoside and related molecules). Many of these metabolites have not been previously described in E. angustifolia, which currently lacks detailed metabolic profiles. Exposure to light significantly increased the levels of certain caffeic acid derivatives (particularly caffeoylquinic acids and hydroxytyrosol derivatives lacking rhamnose residues) and reduced the level of hydroxytyrosol derivatives with rhamnose residues, revealing that light specifically inhibits the rhamnosylation of caffeoyl phenylethanoid glycosides. These results are significant because they suggest that the metabolic profile of cell cultures can be manipulated by controlling simple environmental variables such as illumination to modulate the levels of potentially therapeutic compounds.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by a progressive oral and ocular dryness that correlates poorly with the autoimmune damage of the glands. It has been proposed that a loss of homeostatic equilibrium in the glands is partly responsible for salivary dysfunction with acinar cells involved actively in the pathogenesis of SS. The non-obese diabetic (NOD) mouse model of Sjögren's syndrome develops secretory dysfunction and early loss of glandular homeostatic mechanisms, with mild infiltration of the glands. Based on the vasodilator, prosecretory and trophic effects of the vasoactive intestinal peptide (VIP) on acini as well as its anti-inflammatory properties we hypothesized that the local expression of VIP/vasoactive intestinal peptide receptor (VPAC) system in salivary glands could have a role in acinar cell apoptosis and macrophage function thus influencing gland homeostasis. Here we show a progressive decline of VIP expression in submandibular glands of NOD mice with no changes in VPAC receptor expression compared with normal mice. The deep loss of endogenous VIP was associated with a loss of acinar cells through apoptotic mechanisms that could be induced further by tumour necrosis factor (TNF)-α and reversed by VIP through a cyclic adenosine-5'-monophosphate (cAMP)/protein kinase A (PKA)-mediated pathway. The clearance of apoptotic acinar cells by macrophages was impaired for NOD macrophages but a shift from inflammatory to regulatory phenotype was induced in macrophages during phagocytosis of apoptotic acinar cells. These results support that the decline in endogenous VIP/VPAC local levels might influence the survival/apoptosis intracellular set point in NOD acinar cells and their clearance, thus contributing to gland homeostasis loss. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology. Fil:Hauk, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Calafat, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Larocca, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Fraccaroli, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Grasso, E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ramhorst, R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Leirós, C.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

During normal early pregnancy circulating monocytes are recruited to the maternal-placental interface where they differentiate to macrophages expressing different functional phenotypes for the maintenance of tissue homeostasis. Pregnancy in the nonobese diabetic (NOD) mouse model presents some pathological features in the pre-diabetic stage. The aim of this work was to analyze the functional profile of peritoneal macrophages faced with inflammatory and phagocytic stimuli in early pregnant pre-diabetic NOD mice and their modulation by vasoactive intestinal peptide (VIP). Pregnant NOD mouse macrophages showed no basal NFκB activation, lower IL-12 and nitrites production compared with the macrophages from non-pregnant NOD mice. Their pro-inflammatory aberrant response to LPS and apoptotic cell challenge was reduced and VIP inhibited macrophage residual deleterious responses to apoptotic cells. A functional phenotype switch in macrophages during pregnancy in NOD mice and a promoting effect of VIP towards this regulatory phenotype would be in line with the central role of macrophages in the maternal-placental dialogue. Fil: Larocca, Luciana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hauk, Vanesa Cintia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Calafat, Mario Jose. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Roca, Valeria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fraccaroli, Laura Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Ramhorst, Rosanna Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Perez Leiros, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

BACKGROUND AND PURPOSE: Successful embryo implantation is followed by a local pro-inflammatory and Th1 response, subsequently controlled by a Th2 response. Vasoactive intestinal peptide (VIP) has anti-inflammatory effects and promotes tolerogenic/Th2 responses while favouring embryonic development. We investigated the potential regulatory role of VIP on human trophoblast cells, maternal pro-inflammatory responses and trophoblast-maternal leukocyte interactions. EXPERIMENTAL APPROACH: We tested VIP effects directly on a trophoblast cell line (Swan 71 cells) and after co-culture with maternal peripheral blood mononuclear cells (PBMCs) as models of the feto-maternal dialogue. We also co-cultured maternal and paternal PBMCs to test effects of endogenous VIP on maternal alloresponses. KEY RESULTS: Swan 71 cells express VPAC(1) receptors and VIP induced their proliferation and the expression of leukaemia inhibitor factor, a pro-implantatory marker. After interaction with trophoblast cells, VIP increased Foxp3, the proportion of CD4+CD25+Foxp3+ cells within maternal PBMCs and transforming growth factor beta expression. Also, during the trophoblast-maternal PBMCs interaction, VIP reduced pro-inflammatory mediators [interleukin (IL)-6, monocyte chemoattractant protein 1, nitric oxide], while increasing IL-10. Trophoblast cells produced VIP which dose-dependently suppressed allomaternal responses, accompanied by reduced expression of the T cell transcription factor, T-bet. CONCLUSIONS AND IMPLICATIONS: Vasoactive intestinal peptide induced pro-implantatory markers and trophoblast cell proliferation, while controlling the initial pro-inflammatory response, by increasing maternal regulatory T cells and anti-inflammatory cytokines. As an autocrine regulatory peptide VIP might contribute to fetal survival through two mechanisms; a direct trophic effect on trophoblast cells and an immunomodulatory effect that favours tolerance to fetal antigens Fil: Fraccaroli, Laura Virginia. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alfieri, Julio Armando. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Larocca, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina Fil: Calafat, Mario Jose. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Roca, Valeria Ines. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Lombardi, Eduardo. Instituto de Fertilidad; Argentina Fil: Ramhorst, Rosanna Elizabeth. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Perez Leiros, Claudia. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Química Biológica. Laboratorio de Inmunofarmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

BACKGROUND Successful implantation is followed by a local pro-inflammatory and Th1 response, subsequently controlled by Th2. Regulated upon activation, normal T cell expressed and secreted (RANTES) promotes a Th1 response and is implicated as a physiologic tolerogenic factor; therefore, we studied its potential role in the trophoblast-maternal leukocyte dialog. METHODS We performed co-cultures of immortalized trophoblast cell line (Swan 71) and peripheral blood mononuclear cells (PBMCs) from fertile women (n = 23) or with recurrent spontaneous abortions (n = 18, RSA). After 24 and 48 h, supernatant and cells were analyzed by enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, Western blot and apoptosis assay. To investigate the physiological effects at peripheral level, we co-cultured maternal and paternal PBMCs with conditioned media from Swan cells and progesterone. RESULTS Following interaction of maternal PBMCs and trophoblast cells, RANTES production increased (P < 0.05) and was accompanied by low levels of interferon gamma, interleukin-12 p70 and high levels of tumor necrosis factor-alpha, nitrites and leukemia-inhibitory factor. RANTES production resulted in elevated apoptosis of potentially deleterious maternal CD3+ lymphocytes, accompanied by a decrease in the proliferative maternal response. During fetal-maternal dialog, the anti-RANTES antibody significantly reduced the frequency of CD4+CD25+Foxp3+ cells (P < 0.05) and was associated with trophoblast cell survival. However, co-cultures of Swan cells and RSA-PBMCs displayed a differential RANTES kinetics, lower levels of regulatory T cells (Tregs) and CD3+annexin-V+cells, accompanied by higher levels of apoptotic trophoblast cells. CONCLUSIONS RANTES promotes an adequate pro-implantatory microenvironment that influences trophoblast cell survival and modulates the balance of maternal Treg/T effector lymphocytes in favor of maternal tolerance.

Successful implantation requires a functionally normal embryo at the blastocyst stage and a receptive endometrium as well as adequate communication between them throughout the implantation process. This cross-talk is highly regulated by a number of different kinds of molecules. Particularly, chemokines, small polypeptides that attract specific leukocyte subsets by binding to cell-surface receptors, are also required to maintain immune-privileged sites as the feto-maternal interface. Chemokines expression involves an interdependent network with the absence of a single chemokine affecting the expression of multiple other chemokines, we have chosen to focus on just two representative examples: RANTES (regulated on normal T cell expressed and secreted) and MCP-1 (Monocyte chemo-attractant protein). Here, we present updated information on their expression levels and regulation on three different levels: 1) systemic effects on maternal allogeneic response; 2) local effects on endometrial cells; and 3) during an early stage of the feto-maternal dialogue. For each of the three levels, we analyzed data from both fertile women and patients having experienced recurrent spontaneous abortions as representative of physiological and pathological situations respectively.