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Background and AimsIn clinical trials for reducing fibrosis in NASH patients, therapeutics that target macrophages have had variable results. We evaluated intrahepatic macrophages in patients with non-alcoholic steatohepatitis to determine if fibrosis influenced phenotypes and expression of CCR2 and Galectin-3.Approach & ResultsWe used nCounter to analyze liver biopsies from well-matched patients with minimal (n=12) or advanced (n=12) fibrosis to determine which macrophage-related genes would be significantly different. Known therapy targets (e.g., CCR2 and Galectin-3) were significantly increased in patients with cirrhosis.However, several genes (e.g., CD68, CD16, and CD14) did not show significant differences, and CD163, a marker of pro-fibrotic macrophages was significantly decreased with cirrhosis. Next, we analyzed patients with minimal (n=6) or advanced fibrosis (n=5) using approaches that preserved hepatic architecture by multiplex-staining with anti-CD68, Mac387, CD163, CD14, and CD16. Spectral data were analyzed using deep learning/artificial intelligence to determine percentages and spatial relationships. This approach showed patients with advanced fibrosis had increased CD68+, CD16+, Mac387+, CD163+, and CD16+CD163+ populations. Interaction of CD68+ and Mac387+ populations was significantly increased in patients with cirrhosis and enrichment of these same phenotypes in individuals with minimal fibrosis correlated with poor outcomes. Evaluation of a final set of patients (n=4) also showed heterogenous expression of CD163, CCR2, Galectin-3, and Mac387, and significant differences were not dependent on fibrosis stage or NAFLD activity.ConclusionsApproaches that leave hepatic architecture intact, like multispectral imaging, may be paramount to developing effective treatments for NASH. In addition, understanding individual differences in patients may be required for optimal responses to macrophage-targeting therapies.

Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-?) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-? expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-? enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-?-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even disease-promoting. Accordingly, inhibition of STAT3 activation led to a reduced parasite load in infected macrophages. Thus, the STAT3 pathway offers a rational target for adjunctive host-directed therapy to interrupt the pathogenesis of VL.

Visceral leishmaniasis (VL), caused by infection with the intracellular protozoan Leishmania donovani, is a chronic progressive disease with a relentlessly increasing parasite burden in the spleen, liver and bone marrow. The disease is characterized by fever, splenomegaly, cachexia, and pancytopenia, and progresses to death if not treated. Control of Leishmania infection is mediated by Th1 (IFN?-producing) CD4+ T cells, which activate macrophages to produce nitric oxide and kill intracellular parasites. However, despite expansion of CD4+ T cells and increased IFN? expression in the spleen, humans with active VL do not control the infection. We used an experimental model of chronic progressive VL in hamsters, which mimics clinical and pathological features seen in humans, to better understand the mechanisms that lead to progressive disease. Transcriptional profiling of the spleen during chronic infection revealed expression of markers of both T cell activation and inhibition. CD4+ T cells isolated from the spleen during chronic progressive VL showed mixed expression of Th1 and Th2 cytokines and chemokines, and were marginally effective in controlling infection in an ex vivo T cell-macrophage co-culture system. Splenic CD4+ T cells and macrophages from hamsters with VL showed increased expression of inhibitory receptors and their ligands, respectively. Blockade of the inhibitory receptor PD-L2 led to a significant decrease in parasite burden, revealing a pathogenic role for the PD-1 pathway in chronic VL. PD-L2 blockade was associated with a dramatic reduction in expression of host arginase 1, but no change in IFN? and inducible nitric oxide synthase. Thus, the expression of counter-regulatory molecules on splenic CD4+ T cells and macrophages promotes a more permissive macrophage phenotype and attenuates intracellular parasite control in chronic progressive VL. Host-directed adjunctive therapy targeting the PD-1 regulatory pathway may be efficacious for VL.

Introduction.Canines are the principal domestic reservoirs of visceral leishmaniasis in both the Old and New World. The development of highly sensitive and quantitative methods, such as real time reverse transcriptase polymerase chain reaction for measurement of canine cytokines has not been exploited in studies of visceral leishmaniasis. Objective. To standardize the relative quantification of canine IFN-g, IL-4, IL-10, IL-12p40 and IL-12p35 using real time reverse transcriptase polymerase chain reaction. Materials and methods. RNA was isolated from PBMCs from 1 year-old foxhounds and cultured with or without Con A, LPS or Staphylococcus aureus extract. This RNA was used in one-step real time reverse transcriptase polymerase chain reaction to optimize the concentrations of the cytokine primers and probes, generate standard curves for each cytokine, confirm equivalent amplification efficiency of cytokine and normalizer (18S rRNA) RNA, and to quantify the expression of the cytokine RNA. The comparative Ct method was used to determine the relative levels of gene expression in the samples, expressed as the fold-increase relative to the control samples. Results. The regression coefficient for the standard curves and the amplification efficiencies of the cytokine and normalizer RNA indicated that the quantification was reliable over a broad concentration range of input RNA. Relative to control cells, activation of PBMCs led to increased expression of IFN-g (132-fold), IL-4 (8.8-fold), IL-10 (7.2-fold), and IL-12p40 (275-fold). Basal expression of IL-12p35 was also detected. Conclusion. This approach provides several advantages over conventional assays for cytokine measurement and can be exploited in the study of the immunopathogenesis and immunity in canine leishmaniasis.

BON cattle derived from the domestic animals brought by Christopher Columbus on his second trip have gone through 500 years of adaptation to the tropical coffee producing areas of Colombia. This breed posseses several important characteristics such as: tameness, maternal ability, longevity, high fertility, increased productivity in F1 crosses, and ability to take advantage of rough relatively poor pastures. These qualities together with recent molecular findings suggesting high genetic variability and resistance to viral and bacterial infections make of this breed, now classified as “at risk” of extinction, an invaluable genetic resource from which a national cattle industry adapted to our tropical conditions could be established. The aim of this review it is to compile information about this breed and indicate some research perspectives to the light of advances made at University of Antioquia demonstrating the genetic potential of this breed, that maybe has not been expressed before due to the husbandry conditions to which it has been subjected throughout the five centuries. RESUMEN: El ganado Blanco Orejinegro descendiente del ganado traído por Colón durante su segundo viaje, ha sobrevivido durante casi 500 años en las áreas tropicales colombianas productoras de café. Además de su capacidad adaptativa este ganado ha mostrado otras características como: docilidad, habilidad para aprovechar forrajes de mala calidad, gran habilidad materna, mayor precocidad sexual, alta fertilidad, mayor productividad en cruces F1 (carne y leche) y marcada resistencia a ectoparásitos. Estas características, en conjunto con hallazgos moleculares recientes que sugieren alta variabilidad genética y resistencia a patógenos bacterianos y vírales demuestran que esta raza, que en la actualidad se encuentra en vía de extinción, es portadora de información genética importante que la convierte en una alternativa para la producción en las condiciones tropicales. El objetivo de esta revisión es hacer una recopilación de trabajos realizados con ganado BON; además, mostrar las perspectivas de investigación, basados en los trabajos actualmente llevados a cabo en la Universidad de Antioquia, con el fin de demostrar el potencial genético de esta raza, que quizás no ha podido ser expresado debido a las condiciones de manejo a que esta raza ha sido sometida.

The Salmonella species are intracellular facultative pathogens that affect several animals, including birds, pigs, cattle and humans. After ingestion of contaminated water or food, the bacteria reach the last portion of the small gut. These microorganisms need to express coordinately many of their genes in order to establish a productive infection. The expression begins when the bacteria get in contact with the hostile environment that represent the host gut tract, where they found a great variety of conditions such as: osmolality, oxygen tension and pH. These conditions induce the transcription of genes that codify virulence factors required during the pathogenesis. Additionally, the Salmonella uses a type III secretion system as a basic mechanism of virulence. Through this system a variety of unrelated proteins are secreted and translocated into the citosol of eukaryotic cells, interfer18 ing with the transduction signals and other cellular processes, favoring the bacteria pathogenesis. Some of these genes have been described and characterized by in vitro isolation of mutants, which have shown defects in several characteristics that might be important to carry out some basic functions and also for virulence; for instance: the capability to colonize epithelial cells in culture, the survival inside fagocitic cells, the regulation of inflammation processes and the secretion of fluids. Most of these genes that encode virulence factors are regulated by systems that can be found in both, pathogens and no pathogens species. Several strains of Salmonella have plasmids that encode virulence genes, which are highly associated with bacteremia and systemic infections. A better understanding of these genes, of their proteins products and their functions will improve our knowledge of the pathogenecity pathways used by these microorganisms when they infect a susceptible host. Additionally the development of strategies to successfully prevent such infections will be more easily accomplished. RESUMEN: Las serovariedades de Salmonella, patógenos intracelulares gram negativos, afectan varias especies animales como las aves, los cerdos, los bovinos y el hombre. Después de la ingestión de alimentos o aguas contaminadas, la bacteria se localiza en la porción distal del intestino delgado donde hace contacto con la mucosa intestinal. Estos microorganismos requieren la expresión coordinada de muchos de sus genes para causar una infección productiva en su hospedero. La expresión se inicia cuando la Salmonella entra en contacto con el medio ambiente hostil que representa el tracto gastrointestinal del hospedero, donde encuentra una gran variedad de condiciones como: la osmolaridad, la tensión de oxígeno y el pH; que actúan como señales para que inicie la transcripción de genes que codifican factores de virulencia, los cuales favorecen la interacción con la célula blanco durante la patogénesis. La Salmonella utiliza, además, un sistema de secreción tipo III como un mecanismo básico de virulencia, este sistema es el encargado de translocar proteínas hacia el citosol, las cuales interfieren con las señales de transducción y otros procesos celulares, facilitando la patogénesis de la bacteria, algunos de estos genes han sido descritos y caracterizados mediante la obtención de mutantes in vitro, las cuales han mostrado defectos en ciertas características que parecen ser importantes para cumplir algunas funciones básicas y para la virulencia in vivo, por ejemplo: la capacidad para invadir células epiteliales en cultivo, la sobrevivencia dentro de células fagocíticas, la citotoxicidad de macrófagos, la regulación de la inflamación y la secreción de fluidos. Muchos de los genes que codifican estos factores de virulencia son regulados por sistemas presentes en especies patógenas y no patógenas. Algunas cepas de Salmonella contienen plásmidos que codifican genes de virulencia que están altamente asociados con bacteremia y con la diseminación de la infección. El conocimiento de los genes que conforman el genoma bacteriano, de las proteínas que codifican y de sus funciones, permitirá comprender mejor los mecanismos de patogenecidad de estos microorganismos para generar conocimiento que permita prevenir exitosamente estas infecciones. COL0012444