Tolerance and autoimmunity: novel therapeutic approaches

La función primaria del sistema inmune es resguardar al individuo de los patógenos potencialmente dañinos que invaden el medio ambiente en el cual nos desarrollamos. Este cuenta con dos grandes ramas, la inmunidad innata y la adaptativa, ambas con la propiedad de diferenciar lo peligroso de aquello inofensivo. Estos procesos se hallan regulados por mecanismos homeostáticos que constituyen la tolerancia inmunológica, a los fines de limitar aquellos procesos prolongados y silenciar los potencialmente autoagresivos. Ante la falla de estos mecanismos de control, surgen las enfermedades autoinmunes. Avances en el conocimiento de la fisiopatología de estas entidades, han abierto un nuevo capítulo en el terreno de la inmunofarmacología. Su prometedor potencial actualmente nos ofrece novedosas herramientas terapéuticas para controlar y atenuar el daño causado por este tipo de respuestas. No obstante, debe continuarse la investigación en el campo de los agentes biológicos, ya que ninguno de ellos se encuentra libre de inconvenientes. Seguramente, futuros hallazgos se concretarán en futuros aciertos. Y los aciertos, en Medicina, equivalen a esperanza.The main function of the immune system is to protect the individual against potentially dangerous pathogens. It comprises innate and adaptive cellular and soluble components, both with the capacity to discriminate between harmful and harmless. These processes are regulated by homeostatic mechanisms that constitute the so-called immunological tolerance, which aims to limit the prolonged action of immune mediators and to silence the generation of potentially autoaggressive components. Failure to silence self-reactive T and B cells results in the generation of autoimmune disease. Recent advances in our knowledge of these pathological entities have opened a new chapter in the pharmacology of the immune system. Its promising potential currently offers new therapeutic agents to control and attenuate pathological tissue damage. Nevertheless, further research regarding these biologic agents is required, since they are not free from inconveniences. It is without question that upcoming findings in this field will instill hope into the quest for the “magic bullet”.Fil: Ciliberti, Esteban. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Carambia, Leandro. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; ArgentinaFil: Cavallin, Sebastian. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; ArgentinaFil: Cerda, Osvaldo L.. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Poderoso, Juan J.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

The Pan-Caspase Inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis

BACKGROUND & AIMS: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. METHODS: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan. RESULTS: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-β, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. CONCLUSION: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.Fil: Barreyro, Fernando Javier. Universidad Nacional de Misiones. Facultad de Ciencias Exactas, Químicas y Naturales. Departamento de Microbiología; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Holod, Silvia. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; ArgentinaFil: Finocchietto, Paola Vanesa. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; ArgentinaFil: Camino, Alejandra M.. DIM Clínica Privada; ArgentinaFil: Aquino, Jorge Benjamin. Universidad Austral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Avagnina, Alejandra. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Carreras, Maria Cecilia. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Poderoso, Juan José. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gores, Gregory J.. Mayo Clinic College of Medicine; Estados Unido

New Algorithm to Determine True Colocalization in Combination with Image Restoration and Time-Lapse Confocal Microscopy to Map Kinases in Mitochondria

The subcellular localization and physiological functions of biomolecules are closely related and thus it is crucial to precisely determine the distribution of different molecules inside the intracellular structures. This is frequently accomplished by fluorescence microscopy with well-characterized markers and posterior evaluation of the signal colocalization. Rigorous study of colocalization requires statistical analysis of the data, albeit yet no single technique has been established as a standard method. Indeed, the few methods currently available are only accurate in images with particular characteristics. Here, we introduce a new algorithm to automatically obtain the true colocalization between images that is suitable for a wide variety of biological situations. To proceed, the algorithm contemplates the individual contribution of each pixel's fluorescence intensity in a pair of images to the overall Pearsońs correlation and Manders' overlap coefficients. The accuracy and reliability of the algorithm was validated on both simulated and real images that reflected the characteristics of a range of biological samples. We used this algorithm in combination with image restoration by deconvolution and time-lapse confocal microscopy to address the localization of MEK1 in the mitochondria of different cell lines. Appraising the previously described behavior of Akt1 corroborated the reliability of the combined use of these techniques. Together, the present work provides a novel statistical approach to accurately and reliably determine the colocalization in a variety of biological images

A Mitochondrial Kinase Complex Is Essential to Mediate an ERK1/2-Dependent Phosphorylation of a Key Regulatory Protein in Steroid Biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein –a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser232. Directed mutagenesis of Ser232 to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined

Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis

Hypoxia induces complex I inhibition and ultrastructural damage by increasing mitochondrial nitric oxide in developing CNS

NO-mediated toxicity contributes to neuronal damage after hypoxia; however, the molecular mechanisms involved are still a matter of controversy. Since mitochondria play a key role in signalling neuronal death, we aimed to determine the role of nitrative stress in hypoxia-induced mitochondrial damage. Therefore, we analysed the biochemical and ultrastructural impairment of these organelles in the optic lobe of chick embryos after in vivo hypoxia- reoxygenation. Also, we studied the NO-dependence of damage and examined modulation of mitochondrial nitric oxide synthase (mtNOS) after the hypoxic event. A transient but substantial increase in mtNOS content and activity was observed at 0-2 h posthypoxia, resulting in accumulation of nitrated mitochondrial proteins measured by immunoblotting. However, no variations in nNOS content were observed in the homogenates, suggesting an increased translocation to mitochondria and not a general de novo synthesis. In parallel with mtNOS kinetics, mitochondria exhibited prolonged inhibition of maximal complex I activity and ultrastructural phenotypes associated with swelling, namely, fading of cristae, intracristal dilations and membrane disruption. Administration of the selective nNOS inhibitor 7-nitroindazole 20 min before hypoxia prevented complex I inhibition and most ultrastructural damage. In conclusion, we show here for the first time that hypoxia induces NO-dependent complex I inhibition and ultrastructural damage by increasing mitochondrial NO in the developing brain.Fil: Giusti, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Converso, Daniela Paola. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Poderoso, Juan J.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Fiszer, Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Nutritional status, systemic inflammatory response and mortality in the elderly hospitalized patient

La desnutrición en el anciano involucra un estado inflamatorio. Con el objetivo de evaluar en el paciente internado la relación con la respuesta inflamatoria sistémica y la mortalidad desarrollamos un estudio de cohortes prospectivo en el que evaluamos un score nutricional (SGA), años de instrucción, capacidad funcional, falla orgánica (Marshall), presencia de sepsis, comorbilidades (Charlson), estado cognitivo (MMSE), albúmina, eritrosedimentación y mortalidad. Se incluyeron 52 pacientes, 19 hombres (36.5%) y 33 mujeres (63.5%) con una mediana de edad de 80 (RI 12.5) años. Los pacientes normonutridos fueron 29 (55.8%) y los desnutridos 23 (44.2%).El 53.8% de los pacientes desarrollaron sepsis al ingreso o en la internación. La mortalidad intrahospitalaria en toda la muestra fue 7.7% (n = 4) y al año fue del 31.8% (n = 14). En el análisis comparativo se evidenció mayor edad (80 vs. 78; p = 0.012), menos años de instrucción (7 vs. 8; p = 0.027), un MMST menor (14 vs. 27; p = 0.017), menor capacidad funcional previa (21 vs. 32; p < 0.0001), menor valor de albumina (3 vs. 3.35; p = 0.014) y mayor score de falla orgánica de ingreso (3 vs. 1; p = 0.01) con mayor número de órganos afectados (2 vs. 1; p = 0.003) en los desnutridos con respecto a los normonutridos. También se observó mayor incidencia de sepsis -al ingreso o durante la internación- (73.9% vs. 37.9%; p = 0.01) y niveles de sepsis más graves en desnutridos. La mortalidad al año fue significativamente mayor en los desnutridos (52.2% vs. 9.5%, log rank test = 0.002). En conclusión, los pacientes desnutridos presentaron mayor respuesta inflamatoria sistémica.In order to evaluate the relationship between systemic inflammatory response and mortality in the older hospitalized patient, we developed a prospective cohort study in which we evaluated a nutritional score (SGA), years of instruction, functional status, organic failure (Marshall), presence of sepsis, comorbidities (Charlson), cognitive state (MMSE), albumin, erythrocyte sedimentation rate and mortality. Fifty two patients were included, 19 men (36.5%) and 33 women (63.5%), mean age was 80 (Interquartile Range 12.5) years. 29 (55.8%) patients were well-nourished and 23 (44.2%) malnourished, 53.8% of patients developed sepsis at admission or during hospitalization. Total nosocomial mortality was 7.7 % (n = 4) and one-year mortality was 31.8% (n = 14). Comparative analyses showed older age (80 vs. 78; p = 0.012), less years of instruction (7 vs. 8; p = 0.027), lower MMST (14 vs. 27; p = 0.017), lower previous functional status (21 vs. 32; p < 0.0001), lower albumin (3 vs. 3.35; p = 0.014) and higher organic failure score at admission (3 vs. 1; p = 0.01) with more number of affected organs (2 vs. 1; p = 0.003) in malnourished patients compared to well nourished ones. Higher incidence of sepsis -at admission or during hospitalization- (73.9% vs. 37.9%; p = 0.01) and more severe stages of sepsis were also observed in malnourished patients. One-year mortality was significantly higher in malnourished (52.2% vs. 9.5%, log rank test = 0.002). In conclusion, malnourished patients presented greater systemic inflammatory response.Fil: Zamora, Rafael J.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Instituto de Neurología Cognitiva; ArgentinaFil: Chavin, Hernán. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Regazzoni, Carlos J.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Instituto de Neurología Cognitiva; ArgentinaFil: Pisarevsky, Ana A.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Petrucci, Enrique. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Poderoso, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Laboratorio de Metabolismo del Oxígeno; Argentin

Redox status modulates ERK cellular traffic through differential phosphorylation.

<p>LP07 cells were incubated in the presence or absence of 1 μM or 50 μM H₂O₂ for the indicated times. Then, mitochondrial, nucleus and cytosolic fractions were obtained and the detection of pTyr (pYERK) and Thr/Tyr phosphorylated ERK1/2 (2pERK) was analyzed by Western blot. Representative images for the cytosolic (Panel A), mitochondrial (Panel B) and nuclear fraction (Panel C) of three independent experiments. Actin, complex III (CIII) and TFIID protein antibodies were used as loading control markers to obtain relative phosphorylated ERK levels. Levels of pYERK and 2pERK relative to actin (Panel A), CIII (Panel B) and TFDII (Panel C) expressed in arbitrary units (A.U.). Panel D shows representative immunoblots with specific antibodies for each subcellular fraction. Data are expressed as the mean ± SD of three independent experiments * p<0.05, **p<0.01 vs. 0 min with H₂O₂.</p