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SummarySystemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10-15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included. Platelet apoptosis, assessed by measurement of loss of mitochondrial membrane potential, active caspase 3 and phosphatidylserine exposure, was found to increase in thrombocytopenic patients. Plasma from 67% SLE patients (thrombocytopenic and non-thrombocytopenic) induced loss of sialic acid (Ricinus communis agglutinin I and/or Peanut agglutinin binding) from normal platelet glycoproteins. Concerning platelet production, SLE plasma increased megakaryopoiesis (evaluated using normal human cord blood CD34+ hematopoietic progenitors), but inhibited thrombopoiesis (proplatelet count). Anti-platelet autoantibody depletion from SLE plasma reverted this inhibition. Overall, abnormalities were more frequently observed in thrombocytopenic than non-thrombocytopenic SLE patients and in those with active disease (SLEDAI≥5). In conclusion, platelet clearance due to apoptosis and desialylation, and impaired platelet production mainly due to inhibition of thrombopoiesis, could be relevant mechanisms leading to thrombocytopenia in SLE. These findings could provide a rational basis for the choice of proper therapies to correct platelet counts in these patients.[Figure: see text].

The extracellular matrix plays a key role in cancer progression. Hyaluronan, the main glycosaminoglycan of the extracellular matrix, has been related to several tumor processes. Hyaluronan acts through the interaction with cell membrane receptors as CD44 and RHAMM and triggers signaling pathways as MEK/ERK. 4-methylumbelliferone (4MU), a well-known hyaluronan synthesis inhibitor, is a promising alternative for cancer therapy. 4MU is a coumarin derivative without adverse effects that has been studied in several tumors. However, little is known about its use in glioblastoma (GBM), the most malignant primary brain tumor in adults. Glioblastoma is characterized by fast growth, migration and tissue invasiveness, and a poor median survival of the patients after treatment. Several reports linked glioblastoma progression with HA levels and even with CD44 and RHAMM expression, as well as MEK/ERK activation. Previously, we showed on a murine GBM cell line that HA enhances GBM migration, while 4MU markedly inhibits it. In this work we showed for the first time, that 4MU decreases cell migration and induces senescence in U251 and LN229 human GBM cell lines. Furthermore, we observed that HA promotes GBM cell migration on both cell lines and that such effects depend on CD44 and RHAMM, as well as MEK/ERK signaling pathway. Interestingly, we observed that the exogenous HA failed to counteract the effects of 4MU, indicating that 4MU effects are independent of HA synthesis inhibition. We found that 4MU decreases total CD44 and RHAMM membrane expression, which could explain the effect of 4MU on cell migration. Furthermore, we observed that 4MU increases the levels of RHAMM inside the cell while decreases the nucleus/cytoplasm relation of p-ERK, associated with 4MU effects on cell proliferation and senescence induction. Overall, 4MU should be considered as a promising therapeutic alternative to improve the outcome of patients with GBM.

Xenopus laevis are able to regenerate the spinal cord during larvae stages through the activation of neural stem progenitor cells (NSPCs). Here we use high-resolution expression profiling to characterize the early transcriptome changes induced after spinal cord injury, aiming to identify the signals that trigger NSPC proliferation. The analysis delineates a pathway that starts with a rapid and transitory activation of immediate early genes, followed by migration processes and immune response genes, the pervasive increase of NSPC-specific ribosome biogenesis factors, and genes involved in stem cell proliferation. Western blot and immunofluorescence analysis showed that mTORC1 is rapidly and transiently activated after SCI, and its pharmacological inhibition impairs spinal cord regeneration and proliferation of NSPC through the downregulation of genes involved in the G1/S transition of cell cycle, with a strong effect on PCNA. We propose that the mTOR signaling pathway is a key player in the activation of NPSCs during the early steps of spinal cord regeneration.

Molecular detection of Eimeria species in fecal samples can be useful for experimental and diagnostic purposes. However, the parasite quantity presence in feces and the oocyst wall are an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the oocysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of six protocols for DNA extraction from Eimeria spp. present in bovine and sheep. Twenty pools of fecal samples from cattle (10 pools) and sheep (10 pools) were distributed to six DNA extraction protocols: commercial kit, commercial kit with modification, DNAzol, cetyl-trimethyl ammonium bromide (CTAB), glass beads and commercial kit for fecal samples. Fecal samples were submitted to DNA extraction and PCR. Among the protocols tested, CTAB was determined to be most suitable for DNA extraction from oocysts (90% of DNA detection by PCR); DNAzol and CTAB resulted in higher DNA detection from bovine samples (80%). CTAB and commercial kit with modification improved PCR detection of Eimeria spp. in sheep samples, with positive amplification of DNA in all tested samples. RESUMO: A detecção molecular de espécies de Eimeria em amostras fecais pode ser útil para fins experimentais e de diagnóstico. No entanto, a quantidade de parasitas nas fezes e a parede do oocisto são um obstáculo nos protocolos de extração de DNA. Portanto, uma amostragem adequada e a ruptura efetiva dos oocistos são essenciais para melhorar a precisão da detecção de DNA por PCR. Os objetivos deste estudo foram avaliar seis protocolos para extração de DNA de Eimeria spp. em amostras de bovinos e ovinos. Foram distribuídos 20 grupos de amostras fecais de bovinos (10 grupos) e ovinos (10 grupos) em seis protocolos de extração de DNA: kit comercial, kit comercial com modificação, DNAzol, brometo de cetil-trimetil amônio (CTAB), pérolas de vidro e kit comercial para amostras fecais. As amostras fecais foram submetidas à extração de DNA e PCR. Entre os protocolos testados, CTAB foi considerado o mais adequado para extração de DNA de oocistos (90% de detecção de DNA por PCR); DNAzol e CTAB resultaram em maior detecção de DNA em amostras de bovinos (80%). CTAB e kit comercial com modificação melhoraram a detecção por PCR de Eimeria spp. em amostras de ovinos, amplificação positiva de DNA em todas as amostras testadas.

AgRP neurons are important players in the control of energy homeostasis and are responsive to several hormones. In addition, STAT5 signalling in the brain, which is activated by metabolic hormones and growth factors, modulates food intake, body fat and glucose homeostasis. Given that, and the absence of studies that describe STAT5 function in AgRP cells, the present study investigated the metabolic effects of Stat5a/b gene ablation in these neurons. We observed that STAT5 signalling in AgRP neurons regulates body fat in female mice. However, male and female STAT5-knockout mice did not exhibit altered food intake, energy expenditure or glucose homeostasis compared to control mice. The counter-regulatory response or glucoprivic hyperphagia induced by 2-deoxy-d-glucose treatment were also not affected by AgRP-specific STAT5 ablation. However, under 60% food restriction, AgRP STAT5-knockout mice had a blunted upregulation of hypothalamic Agrp mRNA expression and corticosterone serum levels compared to control mice, suggesting a possible role for STAT5 in AgRP neurons for neuroendocrine adaptations to food restriction. Interestingly, ad libitum fed knockout male mice had reduced Pomc and Ucp-1 mRNA expression compared to control group. Taken together, these results suggest that STAT5 signalling in AgRP neurons regulates body adiposity in female mice, as well as some neuroendocrine adaptations to food restriction.

In this study, the ‘yacon’ was dried using pulsed vacuum osmotic dehydration as pretreatment followed by vacuum drying (at different temperatures) or convective drying. The use of osmotic dehydration and vacuum drying had their influence evaluated concerning drying kinetics and quality of the final product, considering fructan retention, color, and water activity. Fick’s second law and Page’s equation were suitable for the fitting of drying evolution. It was observed that higher temperatures (60 °C) resulted in shorter drying time, higher diffusivity, and higher fructan retention when compared to 40 and 50 °C. The osmotic pretreatment and the vacuum drying differed in fructan retention (p ≤ 0.05). Moreover, the dried product, osmotically pretreated, presented a shorter drying time. The best condition was vacuum drying at 60 ºC, preceded by pulsed vacuum osmotic dehydration that resulted in fructan retention of approximately 38% in a quicker, higher diffusivity and lighter color product concerning the other tested conditions. RESUMO Neste estudo, o yacon foi seco pelo uso de desidratação osmótica com pulso de vácuo como pré-tratamento seguida por secagem a vácuo (em diferentes temperaturas) ou secagem convectiva. O uso de desidratação osmótica e de vácuo na secagem teve sua influência avaliada com relação à cinética de secagem e à qualidade do produto final considerando a retenção de frutanos, cor e atividade de água. A segunda lei de Fick e a equação de Page se mostraram adequadas para o ajuste da evolução da secagem. Observou-se que temperaturas mais altas (60 °C) levaram a tempos de secagem menores, maiores difusividades efetivas, e maior retenção de frutanos, quando comparada a 40 e 50 °C. O pré-tratamento osmótico e a secagem a vácuo diferiram (p ≤ 0.05) na retenção de frutanos. Além disto, o produto seco pré-tratado osmoticamente apresentou menores tempos de secagem. A melhor condição foi a secagem a vácuo a 60 ºC precedida por desidratação osmótica com pulso de vácuo, que resultou na retenção de aproximadamente 38% de frutanos em um processo mais rápido, maior difusividade e um produto mais claro que nas demais condições testadas.

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other’s localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies., Author summary Axons are the up-to-meter-long processes of nerve cells that form the cables wiring our nervous system. Once established, they must survive for a century in humans. Improper extension of axons leads to neurodevelopmental defects, and age- or disease-related neurodegeneration usually starts in axons. Axonal architecture and function depend on bundles of filamentous polymers, called microtubules. These bundles run all along the axonal core, and their disruption correlates with axon decay. How these axonal microtubule bundles are formed and dynamically maintained is little understood. We bridge this knowledge gap by studying how different classes of microtubule-binding proteins may regulate these processes. Here we show how three proteins of very different function, Eb1, XMAP215 and Tau, cooperate intricately to promote the polymerisation processes that form new microtubules during axon development and maintenance. If either protein is dysfunctional, polymerisation is slowed down and newly forming microtubules fail to align into proper bundles. These findings provide new explanations for the decay of microtubule bundles, hence axons. To unravel these mechanisms, we used the fruit fly as a powerful organism for biomedical discoveries. We then showed that the same mechanisms act in frog axons, suggesting they might apply also to humans.

The discovery and characterization of novel parasite antigens to improve the diagnosis of Trypanosoma cruzi by serological methods and for accurate and rapid follow-up of treatment efficiency are still needed. TcTASV is a T. cruzi-specific multigene family, whose products are expressed on the parasite stages present in the vertebrate host. In a previous work, a mix of antigens from subfamilies TcTASV-A and TcTASV-C (Mix A + C) was sensitive and specific to identify dogs with active infection of high epidemiological relevance. Here, TcTASV-A and TcTASV-C were assayed separately as well as together (Mix A + C) in an ELISA format on human samples. The Mix A + C presented moderate sensitivity (78%) but high diagnostic accuracy with a 100% of specificity, evaluated on healthy, leishmaniasic, and Strongyloides stercoralis infected patients. Moreover, antibody levels of pediatric patients showed-2 years posttreatment-diminished reactivity against the Mix A + C (P < 0.0001), pointing TcTASV antigens as promising tools for treatment follow-up. Fil: Floridia Yapur, Noelia Aldana del Rosario. Universidad Nacional de Salta. Facultad de Ciencias Naturales; Argentina. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina Fil: Monje Rumi, Maria Mercedes. Universidad Nacional de Salta. Facultad de Ciencias Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: D'Amato, Anahí Alberti. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Cimino, Rubén Oscar. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; Argentina Fil: Gil, José Fernando. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Investigaciones en Energía no Convencional. Universidad Nacional de Salta. Facultad de Ciencias Exactas. Departamento de Física. Instituto de Investigaciones en Energía no Convencional; Argentina Fil: Sanchez, Daniel Oscar. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Nasser, Julio Rubén. Universidad Nacional de Salta. Facultad de Ciencias Naturales; Argentina Fil: Tekiel, Valeria Sonia. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina

BACKGROUND Merkel cell carcinoma (MCC) is a rare but aggressive primary cutaneous carcinoma with high mortality rates. The present study intends to delineate the epidemiological profile of patients with MCC seen at the Clinics Hospital of the Medical School at the University of Sao Paulo, Brazil, and its association with Merkel cell polyomavirus (MCPyV). METHODS This is a retrospective study. A search was performed in the hospital's medical index for all cases of MCC from January 1994 to December 2012. Among patients with MCC, the available tumoral skin specimens were analyzed with two different techniques of polymerase chain reaction (PCR) (conventional and real-time) for detection of MCPyV DNA. Additionally, paraffin-embedded samples of patients with non-MCC skin cancers were also analyzed. Analyses suitable for categorical data (i.e., x² of Fisher) were used to compare the proportion of patients in each group. RESULTS Nineteen patients with MCC and 20 patients with non-MCC skin cancers entered the study. All MCC samples available (13) tested positive for the presence of MCPyV DNA; however, in the non-MCC skin cancer samples, the MCPyV DNA was detected in 4 of 20 samples (20%). MCPyV DNA detection rate was higher in patients with MCC than in the other group, and its analysis was statistically significant (P

Color and polarization provide complementary information about the world and are detected by specialized photoreceptors. However, the downstream neural circuits that process these distinct modalities are incompletely understood in any animal. Using electron microscopy, we have systematically reconstructed the synaptic targets of the photoreceptors specialized to detect color and skylight polarization in Drosophila, and we have used light microscopy to confirm many of our findings. We identified known and novel downstream targets that are selective for different wavelengths or polarized light, and followed their projections to other areas in the optic lobes and the central brain. Our results revealed many synapses along the photoreceptor axons between brain regions, new pathways in the optic lobes, and spatially segregated projections to central brain regions. Strikingly, photoreceptors in the polarization-sensitive dorsal rim area target fewer cell types, and lack strong connections to the lobula, a neuropil involved in color processing. Our reconstruction identifies shared wiring and modality-specific specializations for color and polarization vision, and provides a comprehensive view of the first steps of the pathways processing color and polarized light inputs.

Several works have shown different aspects of the use of the plant Moringa oleifera. However, few review studies bring an approach to its use in food preparation, specifying its role as a functional food and its use as a natural additive, focusing on food biochemistry and including sensory acceptance and safety. Composed by multiple bioactive substances, Moringa oleifera has the potential to be used as a food additive, mainly as a preservative with the potential to prevent lipid oxidation and other unwanted chemical reactions that lead to product deterioration. Furthermore, it can improve the physicochemical characteristics of food, increasing its quality and shelf life. It also promotes nutritional improvement, elevating protein, mineral, and vitamin levels. Despite this, the sensorial characteristics of this plant result in a low consumer acceptance of the fortified products, which is a problem for the food industry. Apart from inconclusive works, some data involving Moringa’s safety are contradictory, resulting in its commercialization prohibition in Brazil in 2019. This review focused on important data about Moringa use to contribute to the literature and to the food industry, describing information about this medicinal plant effects on food products.

Background Quorum sensing is an extracellular bacterial communication system used in the density-dependent regulation of gene expression and development of biofilms. Biofilm formation has been implicated in the establishment of catheter-associated urinary tract infections and therefore quorum sensing inhibitors (QSIs) have been suggested as anti-biofilm catheter coating agents. The long-term effects of QSIs in uropathogens is, however, not clearly understood. Objectives We evaluated the effects of repeated exposure to the QSIs cinnamaldehyde, (Z)-4-bromo-5(bromomethylene)-2(5H)-furanone-C30 (furanone-C30) and 4-fluoro-5-hydroxypentane-2,3-dione (F-DPD) on antimicrobial susceptibility, biofilm formation and relative pathogenicity in eight uropathogenic Escherichia coli (UPEC) isolates. Methods MICs, MBCs and minimum biofilm eradication concentrations and antibiotic susceptibility were determined. Biofilm formation was quantified using crystal violet. Relative pathogenicity was assessed in a Galleria mellonella model. To correlate changes in phenotype to gene expression, transcriptomic profiles were created through RNA sequencing and variant analysis of genomes was performed in strain EC958. Results Cinnamaldehyde and furanone-C30 led to increases in susceptibility in planktonic and biofilm-associated UPEC. Relative pathogenicity increased after cinnamaldehyde exposure (4/8 isolates), decreased after furanone-C30 exposure (6/8 isolates) and varied after F-DPD exposure (one increased and one decreased). A total of 9/96 cases of putative antibiotic cross-resistance were generated. Exposure to cinnamaldehyde or F-DPD reduced expression of genes associated with locomotion, whilst cinnamaldehyde caused an increase in genes encoding fimbrial and afimbrial-like adhesins. Furanone-C30 caused a reduction in genes involved in cellular biosynthetic processes, likely though impaired ribonucleoprotein assembly. Conclusions The multiple phenotypic adaptations induced during QSI exposure in UPEC should be considered when selecting an anti-infective catheter coating agent.

Previous studies indicate that leptin receptor (LepR) expression in GABAergic neurons is necessary for the biological effects of leptin. However, it is not clear whether LepR expression only in GABAergic neurons is sufficient to prevent the metabolic and neuroendocrine imbalances caused by LepR deficiency. In the present study, we produced mice that express the LepR exclusively in GABAergic cells (LepRVGAT mice) and compared them with wild-type (LepR+/+) and LepR-deficient (LepRNull/Null) mice. Although LepRVGAT mice showed a pronounced reduction in body weight and fat mass, as compared with LepRNull/Null mice, male and female LepRVGAT mice exhibited an obese phenotype relative to LepR+/+ mice. Food intake was normalized in LepRVGAT mice; however, LepRVGAT mice still exhibited lower energy expenditure in both sexes and reduced ambulatory activity in the females, compared with LepR+/+ mice. The acute anorexigenic effect of leptin and hedonic feeding were normalized in LepRVGAT mice despite the hyperleptinemia they present. Although LepRVGAT mice showed improved glucose homeostasis compared with LepRNull/Null mice, both male and female LepRVGAT mice exhibited insulin resistance. In contrast, LepR expression only in GABAergic cells was sufficient to normalize the density of agouti-related peptide (AgRP) and α-MSH immunoreactive fibers in the paraventricular nucleus of the hypothalamus. However, LepRVGAT mice exhibited reproductive dysfunctions, including subfertility in males and alterations in the estrous cycle of females. Taken together, our findings indicate that LepR expression in GABAergic cells, although critical to the physiology of leptin, is insufficient to normalize several metabolic aspects and the reproductive function in mice.

Bovine alphaherpesvirus 2 (BoHV-2) - the agent of bovine herpetic mamillitis (BHM) – is related to Human alphaherpesviruses 1 and 2 (HHV-1, HHV-2) and, as such, has been proposed as a model for vaccine and drug testing. We herein investigated the anti-viral activity in vitro against BoHV-2 of three anti-herpetic drugs: Cidofovir (CDV), Fanciclovir (FAM), Foscarnet (PFA), and diphenyl disselenide (Ph2Se2), a compound that has showed activity against HHV-2. Plaque reduction assays (PRA) revealed a significant reduction in viral plaques (p

Glioblastoma (GBM), the most frequent primary tumor of the central nervous system, has a median survival of 14.6 months. 4-Methylumbelliferone (4MU) is a coumarin derivative widely used as a hyaluronan synthesis inhibitor with proven antitumor activity and without toxic effects reported. We aim to evaluate the antitumor effect of 4MU alone or combined with temozolomide (TMZ) on a GBM cell line, its absence of toxicity on brain cells and its selectivity for tumor cells. The antitumor effect of 4MU alone or combined with TMZ was evaluated on GL26 cells by assessing the metabolic activity through the XTT assay, cell proliferation by BrdU incorporation assay, migration by the wound healing assay, cell death by fluorescein diacetate/propidium iodide (FDA/PI) staining, apoptosis by membrane asymmetry and DNA fragmentation and metalloproteinase activity by zymography. The levels of hyaluronan and its capacity to counteract the effects of 4MU and the expression of RHAMM and CD44 were also determined. The toxicity and selectivity of 4MU were determined by XTT assay and PI staining on normal brain primary cell culture (NBPC-GFP) and GL26/NBPC-GFP cocultures. The GL26 cells expressed RHAMM but not CD44 while synthetized hyaluronan. 4MU decreased hyaluronan synthesis, diminished proliferation and induced apoptosis while reducing cell migration and the activity of metalloproteinases, which was restored by addition of hyaluronic acid. Furthermore, 4MU sensitized GL26 cells to the TMZ effect and showed selective toxicity on tumor cells without exhibiting neurotoxic effects. We demonstrated for the first time the cytotoxic effect of 4MU on GBM cells, highlighting its potential usefulness to improve GBM treatment. Fil: Pibuel, Matías Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Díaz, Mariángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Molinari, Yamila Azul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Poodts, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Silvestroff, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Lompardía, Silvina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Franco, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Hajos, Silvia Elvira. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina

Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR and MPL, which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting. Fil: Marin Oyarzún, Cecilia Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Goette, Nora Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Lev, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: de Luca, Geraldine. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Baroni Pietto, Maria Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Moiraghi, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; Argentina Fil: Castro Ríos, Miguel A.. Consultorios Hematológicos; Argentina Fil: Vicente, Angeles. Hospital Alemán; Argentina Fil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

Background Genetic exchange in Trypanosoma cruzi is controversial not only in relation to its frequency, but also to its mechanism. Parasexual genetic exchange has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis in T. cruzi. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not fully understood yet. Moreover, hybrid T. cruzi DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents. Methodology/Principal findings In order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the minicircle hypervariable region (mHVR) of the kDNA in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones had almost the same ploidy and the DNA content was similar to the reference strain Sylvio (a nearly diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we detected no polymorphisms in the sequence of three maxicircle genes showing mito-nuclear discordance. Lastly, the hybrid stock shared 66% of its mHVR clusters with one putative parent and 47% with the other one; in contrast, the putative parental stocks shared less than 30% of the mHVR clusters between them. Conclusions/significance The results suggest a reductive division, a natural hybridization, biparental inheritance of the minicircles in the hybrid and maxicircle introgression. The models including such phenomena and explaining the relationships between these three clones are discussed., Author summary Chagas disease, an important public health problem in Latin America, is caused by the parasite Trypanosoma cruzi. Despite being a widely studied parasite, several questions on the biology of genetic exchange remain unanswered. Population genomic studies have inferred meiosis in T. cruzi, but this cellular division mechanism has not been observed in laboratory yet. In addition, previous results suggest that mitochondrial DNA (called kDNA) may be inherited from both parents in hybrids. Here, we analyzed a hybrid strain and its potential parents to address the mechanisms of genetic exchange at nuclear and mitochondrial levels. We observed that the hybrid strain had heterozygous patterns and DNA content compatible with a meiosis event. Also, we observed that the evolutionary histories of nuclear DNA and kDNA maxicircles were discordant and that the three strains shared identical DNA sequences. Mitochondrial introgression of maxicircle DNA from one genotype to another may explain this observation. In addition, we demonstrated that the hybrid strain shared kDNA minicircles with both parental strains. Our results suggest that hybridization implied meiosis and biparental inheritance of the kDNA. Further research is required to address such phenomena in detail.

Immune cells resident in adipose tissue have important functions in local and systemic metabolic homeostasis. Nevertheless, these immune cell populations remain poorly characterized in bovines. Recently, we described diverse lymphocyte subpopulations in adipose tissue of Holstein-Friesian cows. Here, we aimed at characterising myeloid cell populations present in bovine adipose tissue using multicolour flow cytometry, cell sorting and histochemistry/immunohistochemistry. Macrophages, CD14+CD11b+MHC-II+CD45+ cells, were identified in mesenteric and subcutaneous adipose tissue, though at higher proportions in the latter. Mast cells, identified as SSC-AhighCD11b−/+CD14−MHC-II−CH138A−CD45+ cells, were also observed in adipose tissue and found at higher proportions than macrophages in mesenteric adipose tissue. Neutrophils, presenting a CH138A+CD11b+ phenotype, were also detected in mesenteric and subcutaneous adipose tissue, however, at much lower frequencies than in the blood. Our gating strategy allowed identification of eosinophils in blood but not in adipose tissue although being detected by morphological analysis at low frequencies in some animals. A population not expressing CD45 and with the CH138A+ CD11b−MHC-II− phenotype, was found abundant and present at higher proportions in mesenteric than subcutaneous adipose tissue. The work reported here may be useful for further studies addressing the function of the described cells.

Background. Polycythemia vera (PV) patients are treated with periodic therapeutic phlebotomy (TP) alone or in combination with either hydroxyurea (HU), ruxolitinib (RUX) or interferon (IFN) to maintain hematocrit (HCT) levels below 45% as per NCCN guidelines. Since patients are seen periodically, PV patients may spend significant time with HCT levels above 45%, thereby increasing their risk of thrombosis [Marchioli NEJM 2013]. PV is associated with systemic symptoms with fatigue. These fatigue-related symptoms are found to be the most prevalent and severe as reported in an international survey among PV patients [Scherber Cancer 2016]. Symptomatic iron deficiency represents an unaddressed clinical challenge to PV patients as most PV patients have iron deficiency at diagnosis due to increased iron utilization [Ginzburg Leukemia 2018]. The iron deficiency worsens after repeated TP. We have demonstrated in a phase 2 study that rusfertide (PTG-300) has a good tolerability profile and achieves HCT control in PV patients with improvement in iron deficiency. Methods. This is a Phase 3, multicenter, global, randomized trial that compares the efficacy and safety of rusfertide compared to placebo when added on to current therapy for PV (Figure 1). The study population is PV subjects who require frequent phlebotomies to control their hematocrit with or without concurrent therapy. This is a three-part study in subjects with polycythemia vera: - Part 1a: randomized, double-blind, placebo-controlled, add-on parallel-group period for 32 weeks. Subjects will be stratified by their ongoing PV treatment and randomized 1:1 to rusfertide or placebo added-on to their ongoing PV treatment. - Part 1b: open-label treatment phase during which all subjects who complete Part 1a successfully will receive rusfertide for 20 weeks (Week 32 through Week 52). - Part 2: Long term extension (LTE) phase during which all subjects who complete Part 1b will continue to receive rusfertide for 32 weeks (Week 52 to Week 84). Inclusion Criteria: Approximately 250 subjects will be randomized. Eligibility criteria include PV diagnosis (by 2016 WHO criteria) and frequent phlebotomies with or without concurrent cytoreductive therapy to maintain HCT below 45% in the 6 months prior to enrollment in Part 1. Eligible subjects will continue to receive their therapy at screening for PV (phlebotomy alone (TP) or cytoreductive therapy + TP) and must have a hematocrit The "add on" design allows subjects to receive standard cytoreductive therapy to control WBC and/or platelets and to receive rusfertide/placebo. The dose of cytoreductive therapy in Part 1a and Part 1b may be decreased for safety but may not be increased for efficacy including control of hematocrit, elevated platelets and/or WBC. Primary endpoint: Proportion of subjects achieving a response starting at Week 20 through Week 32 (inclusive) who receive rusfertide compared to placebo. A response is defined as absence of phlebotomy eligibility defined as either: 1. a confirmed hematocrit ≥45% and that is at least 3% higher than the baseline hematocrit (value immediately prior to randomization at Week 0); confirmation required within 1 to 7 days, or 2. a hematocrit ≥48%. Key words: Hepcidin, Hematocrit, Rusfertide, PTG-300, Polycythemia Vera, PV, Therapeutic Phlebotomy Figure 1 Figure 1. Disclosures Verstovsek: Incyte Corporation: Consultancy, Research Funding; Gilead: Research Funding; PharmaEssentia: Research Funding; Protagonist Therapeutics: Research Funding; CTI BioPharma: Research Funding; Ital Pharma: Research Funding; NS Pharma: Research Funding; Roche: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; Celgene: Consultancy, Research Funding; Promedior: Research Funding; AstraZeneca: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Consultancy, Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. Kuykendall: Pharmaessentia: Honoraria; Protagonist: Consultancy, Research Funding; Novartis: Honoraria, Speakers Bureau; Celgene/BMS: Honoraria; Abbvie: Honoraria; Incyte: Consultancy; Blueprint: Honoraria. Hoffman: AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Novartis: Other: Data Safety Monitoring Board, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Kartos Therapeutics, Inc.: Research Funding. Pemmaraju: Incyte: Consultancy; HemOnc Times/Oncology Times: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Consultancy, Other: Research Support, Research Funding; Abbvie Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; MustangBio: Consultancy, Other; Stemline Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Celgene Corporation: Consultancy; DAVA Oncology: Consultancy; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Protagonist Therapeutics, Inc.: Consultancy; Roche Diagnostics: Consultancy; LFB Biotechnologies: Consultancy; ASCO Leukemia Advisory Panel: Membership on an entity's Board of Directors or advisory committees; Plexxicon: Other, Research Funding; Samus: Other, Research Funding; Sager Strong Foundation: Other; Aptitude Health: Consultancy; Affymetrix: Consultancy, Research Funding; CareDx, Inc.: Consultancy; Springer Science + Business Media: Other; ASH Communications Committee: Membership on an entity's Board of Directors or advisory committees; Cellectis S.A. ADR: Other, Research Funding; Daiichi Sankyo, Inc.: Other, Research Funding; Clearview Healthcare Partners: Consultancy; Blueprint Medicines: Consultancy; Bristol-Myers Squibb Co.: Consultancy; ImmunoGen, Inc: Consultancy; Pacylex Pharmaceuticals: Consultancy. Valone: Protagonist Therapeutics: Consultancy, Current equity holder in publicly-traded company. Modi: Protagonist Therapeutics: Current Employment. Khanna: Protagonist: Current Employment, Current equity holder in publicly-traded company. O'Connor: Protagonist Therapeutics: Current Employment. Gupta: Protagonist Therapeutics: Current Employment. Kiladjian: Taiho Oncology, Inc.: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Other: Personal fees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

During neuronal development and regeneration axons extend a cytoskeletal-rich structure known as the growth cone, which detects and integrates signals to reach its final destination. The guidance cues “signals” bind their receptors, activating signaling cascades that result in the regulation of the growth cone cytoskeleton, defining growth cone advance, pausing, turning, or collapse. Even though much is known about guidance cues and their isolated mechanisms during nervous system development, there is still a gap in the understanding of the crosstalk between them, and about what happens after nervous system injuries. After neuronal injuries in mammals, only axons in the peripheral nervous system are able to regenerate, while the ones from the central nervous system fail to do so. Therefore, untangling the guidance cues mechanisms, as well as their behavior and characterization after axotomy and regeneration, are of special interest for understanding and treating neuronal injuries. In this review, we present findings on growth cone guidance and canonical guidance cues mechanisms, followed by a description and comparison of growth cone pathfinding mechanisms after axotomy, in regenerative and non-regenerative animal models.

Lagoviruses belong to the Caliciviridae family. They were first recognized as highly pathogenic viruses of the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus) that emerged in the 1970–1980s, namely, rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV), according to the host species from which they had been first detected. However, the diversity of lagoviruses has recently expanded to include new related viruses with varying pathogenicity, geographic distribution and host ranges. Together with the frequent recombination observed amongst circulating viruses, there is a clear need to establish precise guidelines for classifying and naming lagovirus strains. Therefore, here we propose a new nomenclature based on phylogenetic relationships. In this new nomenclature, a single species of lagovirus would be recognized and called Lagovirus europaeus. The species would be divided into two genogroups that correspond to RHDV- and EBHSV-related viruses, respectively. Genogroups could be subdivided into genotypes, which could themselves be subdivided into phylogenetically well-supported variants. Based on available sequences, pairwise distance cutoffs have been defined, but with the accumulation of new sequences these cutoffs may need to be revised. We propose that an international working group could coordinate the nomenclature of lagoviruses and any proposals for revision., This work was supported in part by a grant from the Agence Nationale de la Recherche (France), Calilago and by a grant from the Région des Pays de la Loire (France) Armina to JLP. It was performed within the framework of the ECALEP project selected during the 2nd joint call of the Animal Health and Welfare ERA-Net (Anihwa) initiative, a Coordination Action funded under the European Commission’s ERA-Net scheme within the Seventh Framework Programme (Contract No. 291815). The ECALEP project is funded by the Agence Nationale de la Recherche (France), the Ministry of Health, Depa for Veterinary Public Health, Nutrition and Food Safety (Italy) and the Research council FORMAS (Sweden). FCT-Foundation for Science and Technology, Portugal, supported the FCT Investigator grant of JA (ref. IF/01396/2013) and the Post-doc grant of AML (SFRH/BPD/115211/2016).

Impaired iron homeostasis and the suppressive effects of proinflammatory cytokines on erythropoiesis, together with alterations of the erythrocyte membrane that impair its survival, cause anemia of inflammation. Recent epidemiologic studies have connected inflammatory anemia with critical illness, obesity, aging, kidney failure, cancer, chronic infection, and autoimmune disease. The proinflammatory cytokine, interleukin-6, the iron regulatory hormone, hepcidin, and the iron exporter, ferroportin, interact to cause iron sequestration in the setting of inflammation. Although severe anemia is associated with adverse outcomes in critical illness, experimental models suggest that iron sequestration is part of a natural defense against pathogens.

Multiple neuroendocrine, autonomic and behavioral responses are regulated by the paraventricular nucleus of the hypothalamus (PVH). Previous studies have shown that PVH neurons express the growth hormone (GH) receptor (GHR), although the role of GH signaling on PVH neurons is still unknown. Given the great heterogeneity of cell types located in the PVH, we performed a detailed analysis of the neurochemical identity of GH-responsive cells to understand the possible physiological importance of GH action on PVH neurons. GH-responsive cells were detected via the phosphorylated form of the signal transducer and activator of transcription-5 (pSTAT5) in adult male mice that received an intraperitoneal GH injection. Approximately 51% of GH-responsive cells in the PVH co-localized with the vesicular glutamate transporter 2. Rare co-localizations between pSTAT5 and vesicular GABA transporter or vasopressin were observed, whereas approximately 20% and 38% of oxytocin and tyrosine hydroxylase (TH) cells, respectively, were responsive to GH in the PVH. Approximately 55%, 35% and 63% of somatostatin, thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) neurons expressed GH-induced pSTAT5, respectively. Additionally, 8%, 49% and 75% of neuroendocrine TH, TRH and CRH neurons, and 67%, 32% and 74% of nonneuroendocrine TH, TRH and CRH neurons were responsive to GH in the PVH of Fluoro-Gold-injected mice. Our findings suggest that GH action on PVH neurons is involved in the regulation of the thyroid, somatotropic and adrenal endocrine axes, possibly influencing homeostatic and stress responses.

The relationship among genetic diversity of Trypanosoma cruzi and clinical forms of Chagas disease remain elusive. In order to assess the possible association between different T. cruzi Discrete Typing Units (DTUs) and the clinical pictures of the disease, 205 chronic patients from Salta province, Argentina, were analysed. One hundred and twenty-two of these patients were clinically categorized as: cardiac 38.5% (47/122), digestive 15% (18/122), cardio-digestive 16% (20/122) and asymptomatic 30% (37/122). From each patient, blood samples were taken for both, Polymerase Chain Reaction (PCR) targeting kDNA and blood culture analyses. The presence of T. cruzi kDNA was detected in 43% (88/205) of the patients. T. cruzi DTUs were identified in 74% (65/88) of the kDNA positive patients by PCR-hybridization using specific probes. We detected the presence of DTUs TcI, TcII, TcV and TcVI. Single infections (i.e. presence of only one DTU in the sample) were detected in 38.64% of the samples (34/88), while mixed infections were 35.23% (31/88). TcV was the most prevalent DTU (60.3%- 53/88). The association analyses showed, for the first time to the best of our knowledge, that TcV and TcVI were associated with the digestive form of Chagas Disease (Fisher p = .0001). Fil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Floridia Yapur, Noelia Aldana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Pérez Brandán, Jimena María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Nuñez, Sonia. Gobierno de la Provincia de Salta. Hospital San Bernardo.; Argentina Fil: Barrientos, Natalia. Gobierno de la Provincia de Salta. Hospital San Bernardo.; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina

The importance of immune cells present in the adipose tissue to metabolic homeostasis has been increasingly recognized. Nevertheless, in bovines few studies have so far addressed the immune cell populations resident in this tissue. Here we developed an eight-colour flow cytometry panel to address T cell populations present in bovine adipose tissue. Our results showed that γδ T cells, CD4+ and CD8+ CD3+ non-γδ T cells, as well as NK cells, are present in the mesenteric and subcutaneous adipose tissue of Holstein-Friesian cows. The frequency of both γδ T cells and CD8+ non-γδ T cells was found higher in mesenteric than in subcutaneous adipose tissue. The majority of T cells in adipose tissue presented a CD45RO+CD62L− phenotype, characteristic of effector memory cells, and the frequency of these cellular populations was higher than in the blood. The ratio of CD4+ T cells over CD8+ T cells was similar between subcutaneous and mesenteric adipose tissue but different from the one found in blood. Overall, our results highlight particular phenotypic characteristics of bovine adipose tissue T cell populations.

Gonadotropin-inhibitory hormone, GnIH, is named because of its function in birds and mammals; however, in other vertebrates this function is not yet clearly established. More than half of the vertebrate species are teleosts. This group is characterized by the 3R whole genome duplication, a fact that could have been responsible for the great phenotypic complexity and great variability in reproductive strategies and sexual behavior. In this context, we revise GnIH cell bodies and fibers distribution in adult brains of teleosts, discuss its relationship with GnRH variants and summarize the few reports available about the ontogeny of the GnIH system. Considering all the information presented in this review, we propose that in teleosts, GnIH could have other functions beyond reproduction or act as an integrative signal in the reproductive process. However, further studies are required in order to clarify the role of GnIH in this group including its involvement in development, a key stage that strongly impacts on adult life. Fil: Di Yorio, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina Fil: Muñoz Cueto, José A.. Universidad de Cádiz; España Fil: Paullada Salmerón, José A.. Universidad de Cádiz; España Fil: Somoza, Gustavo Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Tsutsui, Kazuyoshi. Waseda University; Japón Fil: Vissio, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina

AIM To examine frequencies and spectrum of genomic alterations in Ukrainian patients diagnosed with primary myelofibrosis (PMF). MATERIALS AND METHODS We enrolled 30 Ukrainian patients diagnosed with PMF who were previously tested for usual mutations in mye-loproliferative neoplasms driver genes (JAK2, MPL and CALR). Genomic DNA samples were obtained from peripheral blood leukocytes of these patients. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed using a high-density CytoScan HD microarray platform. Statistical significance was evaluated by the Fisher exact test. RESULTS We identified frequent genomic alterations, but no significant difference in the rates of copy-number loss, copy-number gain, cnLOH, or multiple genomic alterations were found in the groups of PMF patients that were positive for one of the usual mutations in driver genes or negative for such mutations (33.3% and 55.6%, p = 0.4181, 19.0% and 11.1%, p = 1.0000, 61.9% and 44.4%, p = 0.4434, 33.3% and 55.6%, p = 0.4181, respectively). The most frequent alterations were cnLOH at 1p36-1p22, 9p24.3-9p13.3 and 11q12.3-11q25; copy number loss at 7q21-7q36.3 and 13q12.3-13q14.3. Copy number alterations and cnLOH commonly affected the EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53 genes, in addition to JAK2, MPL and CALR. CONCLUSION We demonstrated the spectrum of genomic alterations in the groups of the Ukrainian PMF patients with or without the usual mutations in the specific driver genes. We identified several potential genes, which may be involved in the myeloproliferative neoplasms development and their phenotype modification (EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53).

It has long been established that neuronal growth cone navigation depends on changes in microtubule (MT) and F-actin architecture downstream of guidance cues. However, the mechanisms by which MTs and F-actin are dually coordinated remain a fundamentally unresolved question. Here, we report that the well-characterized MT polymerase, XMAP215 (also known as CKAP5), plays an important role in mediating MT–F-actin interaction within the growth cone. We demonstrate that XMAP215 regulates MT–F-actin alignment through its N-terminal TOG 1–5 domains. Additionally, we show that XMAP215 directly binds to F-actin in vitro and co-localizes with F-actin in the growth cone periphery. We also find that XMAP215 is required for regulation of growth cone morphology and response to the guidance cue, Ephrin A5. Our findings provide the first strong evidence that XMAP215 coordinates MT and F-actin interaction in vivo. We suggest a model in which XMAP215 regulates MT extension along F-actin bundles into the growth cone periphery and that these interactions may be important to control cytoskeletal dynamics downstream of guidance cues. This article has an associated First Person interview with the first author of the paper.

Several hypothalamic neuronal populations are directly responsive to growth hormone (GH) and central GH action regulates glucose and energy homeostasis. However, the potential role of GH signaling in proopiomelanocortin (POMC) neurons has not been studied yet. Thus, we investigated whether POMC neurons are responsive to GH and if ablation of GH receptor (GHR) or STAT5 in POMC cells leads to metabolic imbalances. Approximately 60% of POMC neurons of the arcuate nucleus exhibited STAT5 phosphorylation after intracerebroventricular GH injection. Ablation of GHR or STAT5 in POMC cells did not affect energy or glucose homeostasis. However, glucoprivic hyperphagia was blunted in male and female GHR knockout mice, and in male POMC-specific STAT5 knockout mice. Additionally, the absence of GHR in POMC neurons decreased glycemia during prolonged food restriction in male mice. Thus, GH action in POMC neurons regulates glucoprivic hyperphagia as well as blood glucose levels during prolonged food restriction.

The SDF-1-CXCR4 axis plays an essential role in the regulation of platelet production, by directing megakaryocyte (MK) migration toward the vascular niche, thus allowing terminal maturation and proplatelet formation, and also regulates platelet function in an autocrine manner. Inherited thrombocytopenias (IT) comprise a spectrum of diverse clinical conditions caused by mutations in genes involved in platelet production and function. We assessed CXCR4 expression and SDF-1 levels in a panel of well-characterized forms of IT. Decreased surface CXCR4 levels were found in 8 of 27 (29.6%) IT patients by flow cytometry, including 4 of 6 patients with ANKRD26-RT, 3 of 3 patients with GPS and 1 of 6 patients with FPD/AML. Low CXCR4 levels were associated with impaired SDF-1-triggered platelet aggregation, indicating that this decrease is functionally relevant, whereas a normal platelet response was shown in patients harbouring preserved membrane CXCR4. Reduced CXCR4 was not due to decreased gene expression, as platelet RNA levels were normal or increased, suggesting a post-transcriptional defect. Increased ligand-induced receptor internalization was ruled out, as circulating SDF-1 levels were similar to controls. MK CXCR4 expression was normal, indicating that the defect in CXCR4 arises after the step of platelet biogenesis. In conclusion, the finding of defective CXCR4 expression specifically associated with certain IT disorders highlights the fact that abnormalities in several megakaryocytic regulators underlie IT pathogenesis and further reveal the heterogeneous nature of these conditions. Fil: Salim, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Lev, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Marin Oyarzún, Cecilia Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Goette, Nora Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Molinas, Felisa Concepción. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina

Acute graft-versus-host disease (aGVHD) is the main complication of allogeneic hematopoietic stem cell transplantation, and many efforts have been made to overcome this important limitation. We showed previously that G-CSF treatment generates low-density splenic granulocytes that inhibit experimental aGVHD. In this article, we show that aGVHD protection relies on incoming IL-10+ neutrophils from G-CSF–treated donor spleen (G-Neutrophils). These G-Neutrophils have high phagocytic capacity, high peroxide production, low myeloperoxidase activity, and low cytoplasmic granule content, which accounts for their low density. Furthermore, they have low expression of MHC class II, costimulatory molecules, and low arginase1 expression. Also, they have low IFN-γ, IL-17F, IL-2, and IL-12 levels, with increased IL-10 production and NO synthase 2 expression. These features are in accordance with the modulatory capacity of G-Neutrophils on regulatory T cell (Treg) generation. In vivo, CD25+ Treg depletion shortly after transplantation with splenic cells from G-CSF–treated donors blocks suppression of aGVHD, suggesting Treg involvement in the protection induced by the G-Neutrophils. The immunocompetence and specificity of the semiallogeneic T cells, long-term after the bone marrow transplant using G-Neutrophils, were confirmed by third-party skin graft rejection; importantly, a graft-versus-leukemia assay showed that T cell activity was maintained, and all of the leukemic cells were eliminated. We conclude that G-CSF treatment generates a population of activated and suppressive G-Neutrophils that reduces aGVHD in an IL-10– and Treg-dependent manner, while maintaining immunocompetence and the graft versus leukemia effect.

In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans. Fil: Floridia Yapur, Noelia Aldana del Rosario. Universidad Nacional de Salta. Facultad de Cs.naturales. Escuela de Biología. Cátedra de Química Biologica; Argentina. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Alberti D'amato, Anahí Maitén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Cimino, R.. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Marco, Jorge Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Barroso, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Sanchez, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Nasser, Julio Rubén. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Tekiel, Valeria Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Summary Background Gut bacteria might predispose to or protect from necrotising enterocolitis, a severe illness linked to prematurity. In this observational prospective study we aimed to assess whether one or more bacterial taxa in the gut differ between infants who subsequently develop necrotising enterocolitis (cases) and those who do not (controls). Methods We enrolled very low birthweight (1500 g and lower) infants in the primary cohort (St Louis Children's Hospital) between July 7, 2009, and Sept 16, 2013, and in the secondary cohorts (Kosair Children's Hospital and Children's Hospital at Oklahoma University) between Sept 12, 2011 and May 25, 2013. We prospectively collected and then froze stool samples for all infants. Cases were defined as infants whose clinical courses were consistent with necrotising enterocolitis and whose radiographs fulfilled criteria for Bell's stage 2 or 3 necrotising enterocolitis. Control infants (one to four per case; not fixed ratios) with similar gestational ages, birthweight, and birth dates were selected from the population after cases were identified. Using primers specific for bacterial 16S rRNA genes, we amplified and then pyrosequenced faecal DNA from stool samples. With use of Dirichlet multinomial analysis and mixed models to account for repeated measures, we identified host factors, including development of necrotising enterocolitis, associated with gut bacterial populations. Findings We studied 2492 stool samples from 122 infants in the primary cohort, of whom 28 developed necrotising enterocolitis; 94 infants were used as controls. The microbial community structure in case stools differed significantly from those in control stools. These differences emerged only after the first month of age. In mixed models, the time-by-necrotising-enterocolitis interaction was positively associated with Gammaproteobacteria (p=0·0010) and negatively associated with strictly anaerobic bacteria, especially Negativicutes (p=0·0019). We studied 1094 stool samples from 44 infants in the secondary cohorts. 18 infants developed necrotising enterocolitis (cases) and 26 were controls. After combining data from all cohorts (166 infants, 3586 stools, 46 cases of necrotising enterocolitis), there were increased proportions of Gammaproteobacteria (p=0·0011) and lower proportions of both Negativicutes (p=0·0013) and the combined Clostridia–Negativicutes class (p=0·0051) in infants who went on to develop necrotising enterocolitis compared with controls. These associations were strongest in both the primary cohort and the overall cohort for infants born at less than 27 weeks' gestation. Interpretation A relative abundance of Gammaproteobacteria (ie, Gram-negative facultative bacilli) and relative paucity of strict anaerobic bacteria (especially Negativicutes) precede necrotising enterocolitis in very low birthweight infants. These data offer candidate targets for interventions to prevent necrotising enterocolitis, at least among infants born at less than 27 weeks' gestation. Funding National Institutes of Health (NIH), Foundation for the NIH, the Children's Discovery Institute.

Aims Cholinergic neurons are distributed in brain areas containing growth hormone (GH)-responsive cells. We determined if cholinergic neurons are directly responsive to GH and the metabolic consequences of deleting the GH receptor (GHR) specifically in choline acetyltransferase (ChAT)-expressing cells. Main methods Mice received an acute injection of GH to detect neurons co-expressing ChAT and phosphorylated STAT5 (pSTAT5), a well-established marker of GH-responsive cells. For the physiological studies, mice carrying ablation of GHR exclusively in ChAT-expressing cells were produced and possible changes in energy and glucose homeostasis were determined when consuming regular chow or high-fat diet (HFD). Key findings The majority of cholinergic neurons in the arcuate nucleus (60%) and dorsomedial nucleus (84%) of the hypothalamus are directly responsive to GH. Approximately 34% of pre-ganglionic parasympathetic neurons in the dorsal motor nucleus of the vagus also exhibited GH-induced pSTAT5. GH-induced pSTAT5 in these ChAT neurons was absent in GHR ChAT knockout mice. Mice carrying ChAT-specific GHR deletion, either in chow or HFD, did not exhibit significant changes in body weight, body adiposity, lean body mass, food intake, energy expenditure, respiratory quotient, ambulatory activity, serum leptin levels, glucose tolerance, insulin sensitivity and metabolic responses to 2-deoxy- d -glucose. However, GHR deletion in ChAT neurons caused decreased hypothalamic Pomc mRNA levels in HFD mice. Significance Cholinergic neurons that regulate the metabolism are directly responsive to GH, although GHR signaling in these cells is not required for energy and glucose homeostasis. Thus, the physiological importance of GH action on cholinergic neurons still needs to be identified.

Trypanosomes are a group of parasitic flagellates with medical and veterinary importance. Despite many species having been described in this genus, little is known about many of them. Here, we report a genetic and morphological characterization of trypanosomatids isolated from wild mammals from the Argentine Chaco region. Parasites were morphologically and ultrastructurally characterized by light microscopy and transmission electron microscopy. Additionally, 18s rRNA and gGAPDH genes were sequenced and analyzed using maximum likelihood and Bayesian inference. Morphological characterization showed clear characteristics associated with the Trypanosoma genus. The genetic characterization demonstrates that the studied isolates have identical sequences and a pairwise identity of 99% with Trypanosoma lainsoni, which belongs to the clade of lizards and snakes/rodents and marsupials. To date, this species had only been found in the Amazon region. Our finding represents the second report of T. lainsoni and the first record for the Chaco region. Furthermore, we ultrastructurally described for the first time the species. Finally, the host range of T. lainsoni was expanded (Leopardus geoffroyi, Carenivora, Felidae, and Calomys sp., Rodentia, Cricetidae), showing a wide host range for this species.

The use of CD3/target bispecific molecules has great therapeutic potential in oncology. This potential, however, is diminished due to serious on-target drug-related toxicities, restricting the development of bispecific T cell engagers to targets that have limited expression in normal tissues. Conditionally Active Biologic (CAB) technology is a proprietary platform that generates antibodies which have no or very little binding to the target antigen in healthy tissue (normal physiological conditions), but have strong binding in the context of diseased tissues (tumor microenvironment) based on the glycolytic tumor metabolism (including Warburg effect). Using our CAB technology, we have developed CD3/target bispecific antibodies that bind to recombinant CD3 and to CD3 expressing cells under in vitro tumor microenvironment conditions, but not in physiologic conditions (CAB CD3). CAB CD3 bispecific antibodies were generated against various well-established tumor associated antigens (TAA), including EpCAM, B7-H3, HER-2 and EGFR. All of these molecules play important roles in cancer biology and are attractive targets for the development of therapeutic bispecific antibodies. In addition, we have also developed CAB TAA / CAB CD3 bispecific antibodies. In vitro and in vivo efficacy data as well as toxicology studies in non-human primates for CAB-T cell engagers will be presented. Our data demonstrates that CAB bispecific antibodies are efficacious in vivo and have lower toxicity comparing to non-CAB bispecific antibodies. The CAB technology will allow the generation of a new class of T-cell engagers with increased safety margin and therapeutic index in the clinic. Citation Format: Ana Paula G. Cugnetti, Haizhen Liu, Jing Wang, Charles Xing, Christina Wheeler, Matthew Lucas, Cathy Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Novel conditionally active bispecific T cell engagers targeting solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5698.

Neospora caninum is an apicomplexan parasite that in cattle assumes particular importance, as it is responsible for abortions reported worldwide. Leptin is an adipokine mainly secreted by adipocytes, which beside its role in maintaining metabolic homeostasis also has important effects in both innate and adaptive immunity. In previous work, we showed that mice chronically infected with N. caninum had elevated serum leptin levels. Here, we sought to assess whether acute infection with N. caninum infection influenced the production of this adipokine as well as leptin receptor mRNA levels. Our results show that acute infection with N. caninum led to decreased leptin serum levels and mRNA expression in adipose tissue. A decrease in leptin receptor transcript variant 1 mRNA (long isoform) and leptin receptor transcript variant 3 mRNA (one of the short isoforms) expression was also observed. An increase in the number of cells staining positive for leptin in the liver of infected mice was observed, although this increase was less marked in Interleukin (IL)-12/IL-23 p40-deficient mice. Overall, our results show that N. caninum infection also influences leptin production during acute infection.

BACKGROUND The mammalian guanine deaminase (GDA), called cypin, is important for proper neural development, by regulating dendritic arborization through modulation of microtubule (MT) dynamics. Additionally, cypin can promote MT assembly in vitro. However, it has never been tested whether cypin (or other GDA orthologs) binds to MTs or modulates MT dynamics. Here, we address these questions and characterize Xenopus laevis GDA (Gda) for the first time during embryonic development. RESULTS We find that exogenously expressed human cypin and Gda both display a cytosolic distribution in primary embryonic cells. Furthermore, while expression of human cypin can promote MT polymerization, Xenopus Gda has no effect. Additionally, we find that the tubulin-binding collapsin response mediator protein (CRMP) homology domain is only partially conserved between cypin and Gda. This likely explains the divergence in function, as we discovered that the cypin region containing the CRMP homology and PDZ-binding domain is necessary for regulating MT dynamics. Finally, we observed that gda is strongly expressed in the kidneys during late embryonic development, although it does not appear to be critical for kidney development. CONCLUSIONS Together, these results suggest that GDA has diverged in function between mammals and amphibians, and that mammalian GDA plays an indirect role in regulating MT dynamics. Developmental Dynamics 248:296-305, 2019. © 2019 Wiley Periodicals, Inc.

The anemia of chronic disease is an old disease concept, but contemporary research in the role of proinflammatory cytokines and iron biology has shed new light on the pathophysiology of the condition. Recent epidemiologic studies have connected the anemia of chronic disease with critical illness, obesity, aging, and kidney failure, as well as with the well-established associations of cancer, chronic infection, and autoimmune disease. Functional iron deficiency, mediated principally by the interaction of interleukin-6, the iron regulatory hormone hepcidin, and the iron exporter ferroportin, is a major contributor to the anemia of chronic disease. Although anemia is associated with adverse outcomes, experimental models suggest that iron sequestration is desirable in the setting of severe infection. Experimental therapeutic approaches targeting interleukin-6 or the ferroportin–hepcidin axis have shown efficacy in reversing anemia in either animal models or human patients, although these agents have not yet been approved for the treatment of the anemia of chronic disease.

Fil: Antony Debre, I.. Albert Einstein College of Medicine; Estados Unidos. Universite de Paris XI; Francia

The adipose tissue can make important contributions to immune function. Nevertheless, only a limited number of reports have investigated in lean hosts the immune response elicited in this tissue upon infection. Previous studies suggested that the intracellular protozoan Neospora caninum might affect adipose tissue physiology. Therefore, we investigated in mice challenged with this protozoan if immune cell populations within adipose tissue of different anatomical locations could be differently affected. Early in infection, parasites were detected in the adipose tissue and by 7 days of infection increased numbers of macrophages, regulatory T (Treg) cells and T-bet(+) cells were observed in gonadal, mesenteric, omental and subcutaneous adipose tissue. Increased expression of interferon-γ was also detected in gonadal adipose tissue of infected mice. Two months after infection, parasite DNA was no longer detected in these tissues, but T helper type 1 (Th1) cell numbers remained above control levels in the infected mice. Moreover, the Th1/Treg cell ratio was higher than that of controls in the mesenteric and subcutaneous adipose tissue. Interestingly, chronically infected mice presented a marked increase of serum leptin, a molecule that plays a role in energy balance regulation as well as in promoting Th1-type immune responses. Altogether, we show that an apicomplexa parasitic infection influences immune cellular composition of adipose tissue throughout the body as well as adipokine production, still noticed at a chronic phase of infection when parasites were already cleared from that particular tissue. This strengthens the emerging view that infections can have long-term consequences for the physiology of adipose tissue.

Predicting drug-induced liver injury with in vitro cell culture models more accurately would be of significant value to the pharmaceutical industry. To this end we have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop and bidirectional fluid flow (average flow rate of 650 μL/min, and a maximum shear stress of 0.64 dyne/cm2). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures derived from human tissues increase their metabolic activity in response to bidirectional fluidic flow. Since bidirectional flow drastically changes the behavior of other cells types that are shear sensitive, the finding that bidirectional flow increases the metabolic activity of primary liver cells also supports the theory that this increase in metabolic activity is likely caused by increased levels of gas and metabolite exchange or by the accumulation of soluble growth factors rather than by shear sensing. Our results indicate that device operation with bi-directional gravity-driven medium flow supports the 14-day culture of a mix of primary human liver cells with the benefits of enhanced metabolic activity. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% – 36/98) and a previously not detected high proportion of mixed infections (47.9% – 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% – 30/98) in human samples; while TcVI (42.8% – 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% – 32/98); and TcI/TcVI (14.3% – 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco. Fil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Perez Brandan, Cecilia Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Alberti D'Amato, Anahi M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina Fil: Cimino, Ruben O.. Universidad Nacional de Salta. Facultad de Cs.naturales. Escuela de Biologia. Catedra de Quimica Biologica; Argentina. Universidad Nacional de Salta. Sede Regional Oran. Instituto de Investigación de Enfermedades Tropicales; Argentina Fil: Orellana, Viviana. Universidad Nacional de Salta. Facultad de Ciencias de la Salud; Argentina Fil: Basombrio, Miguel Angel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentina

Triatoma virus occurs infecting Triatominae in the wild (Argentina) and in insectaries (Brazil). Pathogenicity of Triatoma virus has been demonstrated in laboratory; accidental infections in insectaries produce high insect mortality. When more than one microorganism enters the same host, the biological interaction among them differs greatly depending on the nature and the infection order of the co-existing species of microorganisms. We studied the possible interactions between Triatoma virus (TrV) and Trypanosoma cruzi (the etiological agent of Chagas disease) in three different situations: (i) when Triatoma virus is inoculated into an insect host (Triatoma infestans) previously infected with T. cruzi, (ii) when T. cruzi is inoculated into T. infestans previously infected with TrV, and (iii) when TrV and T. cruzi are inoculated simultaneously into the same T. infestans individual. Trypanosoma cruzi infection was found in 57% of insects in the control group for T. cruzi, whereas 85% of insects with previous TrV infection were infected with T. cruzi. TrV infection was found in 78.7% of insects in the control group for TrV, whereas insects previously infected with T. cruzi showed 90% infection with TrV. A total of 67.9% of insects presented simultaneous infection with both types of microorganism. Our results suggest that TrV infection could increase adhesion of T. cruzi to the intestinal cells of triatomines, but presence of T. cruzi in intestinal cells would not increase the possibility of entry of TrV into cells. Although this study cannot explain the mechanism through which TrV facilitates the infection of triatomines with T. cruzi, we conclude that after TrV replication, changes at cellular level should occur that increase the adhesion of T. cruzi. Fil: Marti, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Balsalobre, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Ceccarelli, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Susevich, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Echeverria, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina Fil: Rabinovich, Jorge Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina

Integrated multi-organ microphysiological systems are an evolving tool for preclinical evaluation of the potential toxicity and efficacy of drug candidates. Such systems, also known as Body-on-a-Chip devices, have a great potential to increase the successful conversion of drug candidates entering clinical trials into approved drugs. Systems, to be attractive for commercial adoption, need to be inexpensive, easy to operate, and give reproducible results. Further, the ability to measure functional responses, such as electrical activity, force generation, and barrier integrity of organ surrogates, enhances the ability to monitor response to drugs. The ability to operate a system for significant periods of time (up to 28 d) will provide potential to estimate chronic as well as acute responses of the human body. Here we review progress towards a self-contained low-cost microphysiological system with functional measurements of physiological responses. Impact statement Multi-organ microphysiological systems are promising devices to improve the drug development process. The development of a pumpless system represents the ability to build multi-organ systems that are of low cost, high reliability, and self-contained. These features, coupled with the ability to measure electrical and mechanical response in addition to chemical or metabolic changes, provides an attractive system for incorporation into the drug development process. This will be the most complete review of the pumpless platform with recirculation yet written.

People living in areas with active vector-borne transmission of Chagas disease have multiple contacts with its causative agent, Trypanosoma cruzi. Reinfections by T. cruzi are possible at least in animal models leading to lower or even hardly detectable parasitaemia. In humans, although reinfections are thought to have major public health implications by increasing the risk of chronic manifestations of the disease, there is little quantitative knowledge about their frequency and the timing of parasite re-inoculation in the course of the disease. Here, we implemented stochastic agent-based models i) to estimate the rate of re-inoculation in humans and ii) to assess how frequent are reinfections during the acute and chronic stages of the disease according to alternative hypotheses on the adaptive immune response following a primary infection. By using a hybrid genetic algorithm, the models were fitted to epidemiological data of Argentinean rural villages where mixed infections by different genotypes of T. cruzi reach 56% in humans. To explain this percentage, the best model predicted 0.032 (0.008–0.042) annual reinfections per individual with 98.4% of them occurring in the chronic phase. In addition, the parasite escapes to the adaptive immune response mounted after the primary infection in at least 20% of the events of re-inoculation. With these low annual rates, the risks of reinfection during the typically long chronic stage of the disease stand around 14% (4%-18%) and 60% (21%-70%) after 5 and 30 years, with most individuals being re-infected 1–3 times overall. These low rates are better explained by the weak efficiency of the stercorarian mode of transmission than a highly efficient adaptive immune response. Those estimates are of particular interest for vaccine development and for our understanding of the higher risk of chronic disease manifestations suffered by infected people living in endemic areas., Author summary Chagas disease is caused by a unicellular parasite mainly transmitted by blood-feeding bugs. The disease starts with an acute phase when the parasite multiplies in tissues. The immune system is then activated and almost clears the parasites before the host enters the chronic phase of the disease. As hosts remain exposed to the vectors, secondary contacts may result in re-inoculation with the parasite. The re-inoculated parasites then have to face an adaptive immune response that may prevent reinfection. There is little quantitative knowledge about the frequency of such re-inoculation and the efficiency of the immune system to prevent them. Since reinfections can lead to people carrying two or more strains of the parasite, we can potentially infer on the frequency of reinfection from the detection of such mixed infections. We fitted agent-based models to field data to estimate the frequency of reinfection in humans and its timing after primary infection. Our models predicted that most reinfections occur during the chronic phase as the adaptive immune response fails to prevent them in 20% of re-inoculations. Such a rate of failure is particularly important as reinfections are thought to increase the risk of heart damage in the chronic phase of the disease.