Development of an antigen-driven colitis model to study presentation of antigens by antigen presenting cells to T cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/-recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts. This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

Laminar and cellular distribution of AMPA, kainate, and NMDA receptor subunits in monkey sensory-motor cortex

In situ hybridization histochemistry and immunocytochemistry were used to examine lamina- and cell-specific expression of glutamate receptor (GluR) mRNAs and polypeptide subunits in motor and somatosensory cortex of macaque monkeys. Radioactive complementary RNA (cRNA) probes were prepared from cDNAs specific for α-amino-3-hydroxy-5-methylisoxozolepropionate (AMPA)/kainate (GluR1-GluR4), kainate (GluR5-GluR7), and N-methylD-aspartate (NMDA; NR1, NR2A-NR2D) receptor subunits. AMPA/kainate and NR1, NR2A, and NR2B receptor transcripts show higher expression than other transcripts. All transcripts show lamina-specific patterns of distribution. GluR2 and GluR4 mRNAs show higher expression than do GluR1 and GluR3 mRNAs. GluR6 transcript expression is higher than that of GluR5 and GluR7. NR1 mRNA expression is much higher than that of NR2 mRNAs. NR2C subunit expression is very low except for a very distinct band of high expression in layer IV of area 3b. Immunocytochemistry, using subunit-specific antisera and double labeling for calbindin, parvalbumin, or α type II Ca2+/calmodulin-dependent protein kinase (CaMKII-α), allowed identification of cell types expressing different subunit genes. GluR1 and GluR5/6/7 immunoreactivity is found in both pyramidal cells and gamma-amino butyric acid (GABA) cells; GluR2/3 immunoreactivity is preferentially found in pyramidal cells, whereas GluR4 immunoreactivity is largely restricted to GABA cells; NMDA receptor subunit immunoreactivity is far greater in excitatory cells than in GABA cells. The density of expression of AMPA/kainate, kainate, and NMDA receptor subunit mRNAs differed within and across the architectonic fields of sensory-motor cortex. This finding and the lamina- and cell-specific patterns of expression suggest assembly of functional receptors from different arrangements of available subunits in specific neuronal populations.

Factores asociados a embarazo no deseado en mujeres en edad reproductiva en Colombia 2010

Objetivo: Identificar factores sociodemográficos y de fecundidad, asociados a ocurrencia de embarazo no deseado en mujeres colombianas en edad reproductiva en el año 2010. Métodos: Se realizó estudio retrospectivo de corte transversal, basado en los datos de la ENDS Colombia-2010, del total de mujeres en edad fértil (13-49 años) que al momento de la encuesta se encontraban en embarazo. La variable de interés fue embarazo no deseado, se describió la población a estudio y se evaluó la posible asociación con variables sociodemográficas y de fecundidad, a través de análisis bivariado y multivariado. Se realizaron los mismos análisis por grupo de edad (adolescentes vs adultas). Resultados: La prevalencia de embarazo no deseado en las mujeres colombianas en el 2010 fue de 61,4 %. De acuerdo al modelo de regresión logística, no estar en unión a una pareja (OR: 4,01 IC95%: 3,066-5,269), tener hijos (OR: 2,040 IC95%: 1,581 – 2,631), estar en el quintil de menor riqueza (OR: 2,137 IC95%: 1,328-3,440), y ser adolescente (OR: 1,599 IC95%: 1,183-2,162), son factores que aumentan la probabilidad de tener un embarazo no deseado. Se encontraron diferencias en los factores asociados al realizar segmentación por edad. Conclusiones: La prevalencia de embarazo no deseado permanece alta en Colombia respecto a años anteriores y a otros países. Los resultados pueden ser de utilidad para el desarrollo de políticas en salud sexual y reproductiva teniendo en cuenta los factores asociados identificados priorizando a la población adolescente y de menor estatus socioeconómico, para la prevención de embarazo no deseado en Colombia.

Efeito de diferentes pesticidas sobre os ácaros predadores Phytoseiulus macropilis e Amblyseius idaeus (Acari: Phytoseiidae).

Entre julho de 1991 e junho de 1992 foram realizados no laboratorio de Entomologia do CNPDA, testes de seletividade dos produtos Malation, Triclorfon, Mancozeb, Cardaril, Deltametrina, Enxofre, Permetrina, Oxicloreto de cobre, Bacillus thuringiensis, Pirimicarbe, Carbamoil e Benomil aos ácaros predadores fitoseídeos Phytoseiulus macropilis (Banks) e Amblyseius idaeus (Demark & Muma). Estes testes foram realizados utilizando-se o método de imersão de laminas. Em cada lamina foram fixados 20 ácaros predadores sobre fita adesiva com dupla face. Com cada produto foram preparadas soluções com diferentes concentrações: x (dose recomendada); 0,0 x (testemunha, H2O destilada); 0,01x; 0,1x; 10x e 100x. As lâminas foram mergulhadas durante 5 segundos nas soluções e as leituras realizadas 5 e 24 horas após. O produto que apresentou maior toxicidade a P. macropilis foi o piretroide Permetrina, que causou mortalidade de 100% dos predadores, em todas as doses, 24 horas após o tratamento. O produto que apresentou menor toxicidade foi o enxofre. O produto de maior toxicidade a A. idaeus foi o piretróide Deltametrina que provocou mortalidade de 100% em todas as doses, 5 horas após o tratamento. O produto que causou menor toxicidade a esse predador foi também o enxofre. Os resultados obtidos neste ensaio indicam que aplicações de piretróides não são recomendáveis no manejo integrado do ácaro rajado com uso daqueles fitoseídeos.

Unbalanced R-loops and micronuclei induced by DNA topoisomerase I poisons in cancer cells

Topoisomerase I (Top1) poisons are among the most clinically-effective drugs used for colon, ovary and lung cancers. Unpublished data from our lab have recently revealed that the structurally-unrelated Top1 poisons, Camptothecin (CPT) and Indimitecan (LMP776), induce the formation of micronuclei (MNi) in human cancer cells. In addition, MNi trigger an innate immune gene response by stimulating the cGAS/STING pathway. As the mechanisms of MNi formation are not fully determined, our aim is here to establish how MNi form after Top1 poisoning. Using immunofluorescence assays and EdU labelling of nascent DNAs, our results show that, after 24 hours of recovery, a short treatment with sub-cytotoxic doses of Top1 poisons induces the formation of MNi that do not contain newly synthetized (EdU+) DNA. We also saw that Top1 poisons delay replication machinery reducing EdU incorporation and produce significant levels of the damage markers γH2AX and p53BP1 in S-phase cells but not in G1 and G2/M cells. The results also show that MNi formation is dependent on R-loops, as RNaseH1 overexpression markedly reduces Top1 induced MNi. Genome-wide mapping of R-loops by DRIP-seq technique revealed that R-loop levels are both decreased and increased by CPT. In particular, increased R-loops are mainly found at active genes and always overlapped with Top1cc sites. We also found that increased R-loops overlap with lamina-associated chromatin domains while decreased R-loops correlate with replication origin sites. Overall, our data are consistent with the formation of MNi due to R-loop increase and under-replication at specific regions caused by Top1 poisons. These results will eventually help in developing new strategies for effective personalized interventions by using Top1-targeted compounds as immuno-modulators in cancer patients.

Nuclear shape instability caused by lamin A deregulation promotes invasiveness in pediatric bone sarcomas: from nucleo-cytoskeleton dynamics to novel therapeutic opportunities

Osteosarcoma (OS) and Ewing sarcoma (EWS) are the two most frequent primary bone tumors, in which metastases remain the most relevant adverse prognostic factor. Lamin A is the main constituent of the nuclear lamina, with a fundamental role in maintaining the connection between nucleus and cytoskeleton (through LINC complex proteins interactions), and its alterations can be implicated in tumor progression. We investigated how nucleo-cytoskeleton dynamics is influenced by lamin A modulation in OS and EWS, demonstrating that both these cancer models had low levels of lamin A, which are linked to a significantly more marked nuclear misshaping. In our in vitro studies, reduced levels of lamin A promoted migratory abilities in these tumors. Moreover, these findings were corroborated by gene expression analyses on EWS patient samples, showing that LMNA levels were significantly lower in metastatic lesions compared to primary tumors and that patients with low LMNA had a significant worse overall survival. We also found that LMNA expression significantly impaired EWS metastases formation in vivo. We demonstrated that low lamin A expression was linked to a severe mislocalization of LINC complex proteins, thus disrupting nucleo-cytoskeleton interactions, with a corresponding gain in malignant properties, which resulted in increased invasiveness. Lamin A overexpression or its accumulation by a statin-based pharmacological treatment allowed us to reconstitute a functional nucleo-cytoskeleton interplay, which resulted in significant downmodulation of ROCK2 and YAP, two crucial drivers of EWS aggressiveness. Our study demonstrated that lamin A is a favorable mediator of nuclear shape stability in bone sarcomas, and its modulation rescues LINC complex protein localization and regulates mechano-signaling pathways, thus promoting a less aggressive cancer phenotype. We also identified statins, already employed in clinical practice, as a tool capable to increase lamin A levels, and to reconstitute functional nucleo-cytoskeletal dynamics, resulting in reduced cellular migration.

Dispositivi cardiaci impiantabili senza batteria: le nuove frontiere per l'autoalimentazione

Il testo esamina tre studi svolti nello sviluppo di sistemi di autoalimentazione per pacemaker e defibrillatori impiantabili, volti a risolvere il problema della longevità della batteria. Sono state inizialmente approfondite le modalità di generazione del battito cardiaco, regolato dal passaggio del potenziale d’azione e presentati i tipi di scompenso cardiaco esponendo le modifiche che questi causano alla forma d’onda dell’ECG. Successivamente sono state descritte le tecnologie adottate per trattare i disturbi del ritmo cardiaco, pacemaker e defibrillatori impiantabili. Vengono dunque introdotti gli studi che presentano le innovazioni più recenti che riguardano la possibilità di autoalimentare i dispositivi cardiaci impiantabili. Il primo mostra la realizzazione di un trasduttore di energia meccanica in elettrica che utilizza una lamina di materiale piezoelettrico. Tale dispositivo è stato testato con successo in vitro ed in vivo. Nel secondo studio viene invece sfruttato un dispositivo automatico di un orologio per convertire l’accelerazione generata dal cuore in energia elettrica. Sono stati realizzati tre prototipi ed eseguiti test in vitro ed in vivo. Il terzo studio si focalizza su una microturbina che sfrutta il flusso sanguigno per generare energia elettrica. È stato eseguito un test in vivo su un suino nel quale il sistema è stato connesso ad un pacemaker senza batteria. Questo è riuscito ad aumentare i battiti al minuto producendo più energia di quella necessaria, con il difetto della formazione di trombi. La tesi si conclude con una valutazione critica dei metodi illustrati e la proposta di potenziali sviluppi miglioramenti.

Effetto dell'emilaminectomia sul rachide lombare: un'analisi biomeccanica in vitro

La stenosi spinale lombare (LSS) è un processo degenerativo frequente nell’attuale popolazione anziana e può essere considerata come la causa principale di chirurgia spinale nei pazienti di età superiore ai sessantacinque anni. Comporta un restringimento del forame vertebrale, che, nei casi più gravi, causa una compressione del midollo spinale e degli elementi neurali e vascolari situati nel tratto lombo-sacrale. Uno dei trattamenti chirurgici di decompressione utilizzato è l’emilaminectomia, che, prevedendo la resezione della lamina di un solo lato della vertebra e di parte del legamento giallo, può portare a una riduzione della stabilità spinale. L’obiettivo di questo studio in vitro è quello di analizzare l’impatto dell’emilaminectomia sulla biomeccanica del rachide. Sei provini, estratti da rachide umano e costituiti da quattro vertebre lombari e una sacrale (L2-S1), sono stati testati meccanicamente in flessione, estensione e flessione laterale sinistra e destra in due condizioni di prova: intatti e post emilaminectomia. La stabilità spinale è stata valutata calcolando il Range of Motion tra le vertebre L2 e S1 agli estremi dei provini. Mediante l’algoritmo di Digital Image Correlation (DIC), sono state estratte e valutate le distribuzioni delle deformazioni dell’intero provino, valutando, in particolare, le deformazioni principali massime e minime sulla superficie del disco intervertebrale L4-L5. I risultati hanno mostrato che l’emilaminectomia ha causato una diminuzione significativa della stabilità spinale solo in flessione, con un aumento del Range of Motion del 54%. L’emilaminectomia non ha causato variazioni nelle distribuzioni delle deformazioni in ogni configurazione di carico. Le deformazioni principali minime sul disco intervertebrale L4-L5, tra le due vertebre in cui è stata eseguita l’emilaminectomia, sono aumentate, in modo statisticamente significativo, del 38% nella flessione nel lato in cui è stato svolto l’intervento di emilaminectomia.