WLB-87848, a Selective σ1 Receptor Agonist, with an Unusually Positioned NH Group as Positive Ionizable Moiety and Showing Neuroprotective Activity.

The synthesis and pharmacological activity of a new series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as sigma-1 receptor (σ1R) ligands are reported. A hit from a high-throughput screening program was evolved into a highly potent and selective σ1R agonist (14qR) that contains a free NH group as positive ionizable moiety, not fulfilling the usual pharmacophoric features of the σ1R. The compound shows good physicochemical and ADMET characteristics, displays an agonist profile in the binding immunoglobulin protein/σ1R association assay, induces neuron viability in an in vitro model of ß-amyloid peptide intoxication, and presents positive results against recognition memory impairment induced by hippocampal injection of Aß peptide in rats after oral treatment, altogether making 14qR (WLB-87848) an interesting candidate for neuroprotection.

Discovery of WLB-89462, a New Drug-like and Highly Selective σ2 Receptor Ligand with Neuroprotective Properties.

The synthesis and pharmacological activity of a new series of isoxazolylpyrimidines as sigma-2 receptor (σ2R) ligands are reported. Modification of a new hit retrieved in an HTS campaign allowed the identification of the compound WLB-89462 (20c) with good σ2R affinity (Ki = 13 nM) and high selectivity vs both the σ1R (Ki = 1777 nM) and a general panel of 180 targets. It represents one of the first σ2R ligands with drug-like properties, linked to a good physicochemical and ADMET profile (good solubility, no CYP inhibition, good metabolic stability, high permeability, brain penetration, and high oral exposure in rodents). Compound 20c shows neuroprotective activity in vitro and improves short-term memory impairment induced by hippocampal injection of amyloid ß peptide in rats. Together with the promising effects in the chronic models where 20c is currently being evaluated, these results pave the way toward its clinical development as a neuroprotective agent.

Piperazinyl Bicyclic Derivatives as Selective Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels.

The synthesis and pharmacological activities of a new series of piperazinyl quinazolin-4-(3H)-one derivatives acting toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) are reported. Different positions of a micromolar HTS hit were explored, and best activities were obtained for compounds containing a small alkyl group in position 3 of the quinazolin-4-(3H)-one scaffold and a 3-methyl-piperazin-1-yl- or 3,5-dimethyl-piperazin-1-yl-butyl group in position 2. The activity was shown to reside in the R enantiomer of the chain in position 2, and several eutomers reached single digit nanomolar affinities. Final modification of the central scaffold to reduce lipophilicity provided the pyrido[4,3-d]pyrimidin-4(3H)-one 16RR, which showed high selectivity for Cavα2δ-1 versus Cavα2δ-2, probably linked to its improved analgesic efficacy-safety ratio in mice over pregabalin.

Drenaje lumbar externo con bomba volumétrica continua en pacientes con fístula cefalorraquídea. Series de casos / External lumbar drainage with volumetric continuing infusion pump in patients with cerebrospinal fluid leak. A case series

OBJETIVO: Describir las incidencias y complicaciones surgidas en una serie de casos de pacientes con fístula cefalorraquídea tratados mediante drenaje lumbar externo con bomba de infusión (BI) volumétrica continua desde el año 2001 al 2014. Cuantificar las fistulas cefalorraquídeas cerradas mediante drenaje lumbar externo con BI. MÉTODO: Estudio descriptivo retrospectivo de serie de casos. Población: pacientes intervenidos de cirugía hipofisaria transesfenoidal, Chiari y laminectomía, que desarrollaron en el postoperatorio fístula de líquido cefalorraquídeo tratada con drenaje lumbar externo continuo mediante BI. Variables: edad, sexo, tipo de intervención, variables relacionadas con el funcionamiento de la BI y relacionadas con complicaciones. Se calcularon medias y medianas para las variables cuantitativas y frecuencias y porcentajes para las cualitativas. RESULTADOS: La muestra incluyó 11 sujetos. Incidencias en el funcionamiento de la BI: desconexión, oclusión y activación de la alarma acústica de la bomba. La complicación más frecuente fue cefalea; hubo un caso de neumoencéfalo. DISCUSIÓN: El escaso número de sujetos y su heterogeneidad no permiten comparar ni establecer asociaciones entre variables. La resolución de la fístula de líquido cefalorraquídeo con BI continua es menor en este estudio que en otros revisados, pudiendo estar influido por el tamaño de la muestra. Destaca la frecuente activación de la alarma de la BI, sin causa aparente. Implicaciones para la práctica: Desarrollar un protocolo para preparar el equipo de la BI, que disminuya la activación de la alarma acústica. Realizar un estudio prospectivo multicéntrico

OBJECTIVE: To describe the incidence and complications arising in a number of cases of patients with cerebrospinal fluid leak treated by external lumbar drainage with infusion pump (IP) volumetric continuous from 2001 to 2014. Quantify cerebrospinal fluid leak closed by lumbar drainage with IP. METHODS: Retrospective descriptive case series study. Population: patients undergoing transsphenoidal pituitary surgery, Chiari surgery and laminectomy, that developed postoperative cerebrospinal fluid leak treated with continuous external lumbar drainage by IP. Variables: age, sex, type of intervention, variables related to the practice of the pump and complications. Average and medians were calculated for quantitative variables, frequencies and percentages for qualitative. RESULTS: Sample: 11 subjects. Incidence in running IP: disconnection, occlusion and acoustic alarm activation. Most frequently complication is headache; a case of pneumocephalus. DISCUSSION: The small number of subjects and the heterogeneity of these do not allow for comparison or establishing associations between variables. The resolution of the cerebrospinal fluid leak with continuous IP is lower in this study than others, and may be influenced by the small number of subjects. It should be noted the frequent activation of the pump alarm for no apparent cause. Implications for practice: Protocol would be developed for preparing the IP team to reduce the acoustic alarm activation, and would make a prospective multicenter study

[External lumbar drainage with volumetric continuing infusion pump in patients with cerebrospinal fluid leak. A case series]. / Drenaje lumbar externo con bomba volumétrica continua en pacientes con fístula cefalorraquídea. Series de casos.

OBJECTIVE: To describe the incidence and complications arising in a number of cases of patients with cerebrospinal fluid leak treated by external lumbar drainage with infusion pump (IP) volumetric continuous from 2001 to 2014. Quantify cerebrospinal fluid leak closed by lumbar drainage with IP. METHODS: Retrospective descriptive case series study. POPULATION: patients undergoing transsphenoidal pituitary surgery, Chiari surgery and laminectomy, that developed postoperative cerebrospinal fluid leak treated with continuous external lumbar drainage by IP. VARIABLES: age, sex, type of intervention, variables related to the practice of the pump and complications. Average and medians were calculated for quantitative variables, frequencies and percentages for qualitative. RESULTS: Sample: 11 subjects. Incidence in running IP: disconnection, occlusion and acoustic alarm activation. Most frequently complication is headache; a case of pneumocephalus. DISCUSSION: The small number of subjects and the heterogeneity of these do not allow for comparison or establishing associations between variables. The resolution of the cerebrospinal fluid leak with continuous IP is lower in this study than others, and may be influenced by the small number of subjects. It should be noted the frequent activation of the pump alarm for no apparent cause. IMPLICATIONS FOR PRACTICE: Protocol would be developed for preparing the IP team to reduce the acoustic alarm activation, and would make a prospective multicenter study.

Simultaneous inhibition of JAK and SYK kinases ameliorates chronic and destructive arthritis in mice.

INTRODUCTION: Despite the broad spectrum of antirheumatic drugs, RA is still not well controlled in up to 30-50 % of patients. Inhibition of JAK kinases by means of the pan-JAK inhibitor tofacitinib has demonstrated to be effective even in difficult-to-treat patients. Here, we discuss whether the efficacy of JAK inhibition can be improved by simultaneously inhibiting SYK kinase, since both kinases mediate complementary and non-redundant pathways in RA. METHODS: Efficacy of dual JAK + SYK inhibition with selective small molecule inhibitors was evaluated in chronic G6PI-induced arthritis, a non-self-remitting and destructive arthritis model in mice. Clinical and histopathological scores, as well as cytokine and anti-G6PI antibody production were assessed in both preventive and curative protocols. Potential immunotoxicity was also evaluated in G6PI-induced arthritis and in a 28-day TDAR model, by analysing the effects of JAK + SYK inhibition on hematological parameters, lymphoid organs, leukocyte subsets and cell function. RESULTS: Simultaneous JAK + SYK inhibition completely prevented mice from developing arthritis. This therapeutic strategy was also very effective in ameliorating already established arthritis. Dual kinase inhibition immediately resulted in greatly decreased clinical and histopathological scores and led to disease remission in over 70 % of the animals. In contrast, single JAK inhibition and anti-TNF therapy (etanercept) were able to stop disease progression but not to revert it. Dual kinase inhibition decreased Treg and NK cell counts to the same extent as single JAK inhibition but overall cytotoxicity remained intact. Interestingly, treatment discontinuation rapidly reversed such immune cell reduction without compromising clinical efficacy, suggesting long-lasting curative effects. Dual kinase inhibition reduced the Th1/Th17 cytokine cascade and the differentiation and function of joint cells, in particular osteoclasts and fibroblast-like synoviocytes. CONCLUSIONS: Concurrent JAK + SYK inhibition resulted in higher efficacy than single kinase inhibition and TNF blockade in a chronic and severe arthritis model. Thus, blockade of multiple immune signals with dual JAK + SYK inhibition represents a reasonable therapeutic strategy for RA, in particular in patients with inadequate responses to current treatments. Our data supports the multiplicity of events underlying this heterogeneous and complex disease.

Análisis multivariable y variabilidad química de los metabolitos volátiles presentes en las partes aéreas y la resina de Bursera graveolens (Kunth) Triana & Planch. de Soledad (Atlántico, Colombia) / Multivariate analysis and chemical variability of the volatile metabolites present in the aerial parts and the resin of Bursera graveolens (Kunth) Triana & Planch from Soledad (Atlántico, Colombia)

The aim of this study was to determine the chemical compositions of the volatile fractions from the immature fruits, leaves, branches, stem bark and resin of Bursera graveolens (Kunth) Triana & Planch. by SDE-GC/MS and their relationship through the multivariate statistical analysis. The most abundant constituents found in the volatile fractions were: (a). Fruits – 3-hydroxy-mint furanone (6.7 percent), mint furanone (6.4 percent), carvone (5.3 percent), limonene (4.2 percent), trans-carveol (4.1), limonene-1,2-diol (3.8 percent), limonene dioxide derivative (3.7 percent), 2-hydroperoxide-2S,4R-p-mentha-6,8-diene (3.4 percent) and caryophyllene oxide (3.0 percent); (b). Leaves – germacrene D (20.7 percent), -caryophyllene (18.0 percent), viridiflorol (8.0 percent), limonene (6.6 percent), linalool (6.5 percent) and dendrolasin (5.3 percent); (c). Branches – mint furanone (43.9 percent), iso-mint furanone (6.8 percent), 3-hydroxy mint furanone (6.2 percent); (d.) Stem bark – mint furanone (44.6 percent), 3-hydroxy-mint furanone (16.2 percent), iso-mint furanone (6.4 percent); and, (e.) Resin – limonene (23.2 percent), mint furanone (15.9 percent), mint furanone derivative (14.8 percent), pulegone (12.1 percent), 3-hydroxy-mint furanone (8.6 percent), and menthofuran (6.2 percent). The principal component analysis (70 percent of related information) and cluster analysis applied to the data allowed to find 3 groups: I. – fruits; II. – leaves; and, III. – resin, stem bark and branches; the latter group showed a close compositional relationship. Thus, the volatile fractions in the branches and stem bark were represented by benzofuranoids compounds (60-69 percent); in the leaves by sesquiterpenoids (60 percent); in the fruits by monoterpenoids (54 percent); and, in the resin by benzofuranoids (48 percent) and monoterpenoids (42 percent).

El objetivo de este estudio fue determinar las composiciones químicas de las fracciones volátiles de los frutos inmaduros, hojas, ramas, corteza del tallo y resina de Bursera graveolens (Kunth) Triana & Planch. mediante SDE-GC/MS y su relación a través de análisis estadístico multivariable. Los constituyentes más abundantes encontrados en las fracciones volátiles fueron: (a). Frutos – 3-hidroxi-mentofuranona (6.7 por ciento), mentofuranona (6.4 por ciento), carvona (5.3 por ciento), limoneno (4.2 por ciento), trans-carveol (4.1 por ciento), limonen-1,2-diol (3.8 por ciento), derivado de dióxido de limoneno (3.7 por ciento), 2-hidroperóxido-2S,4R-p-menta-6,8-dieno (3.4 por ciento) y óxido de cariofileno (3.0 por ciento); (b). Hojas – germacreno D (20.7 por ciento), trans--cariofileno (18.0 por ciento), viridiflorol (8.0 por ciento), limoneno (6.6 por ciento), linalol (6.5 por ciento) y dendrolasina (5.3 por ciento); (c). Ramas – mentofuranona (43.9 por ciento), iso-mentofuranona (6.8 por ciento), 3-hidroxi-mentofuranona (6.2 por ciento); (d.) Corteza del tallo – mentofuranona (44.6 por ciento), 3-hidroxi-mentofuranona (16.2 por ciento), iso-mentofuranona (6.4 por ciento); y, (e.) Resina – limoneno (23.2 por ciento), mentofuranona (15.9 por ciento), mentofuranona derivado (14.8 por ciento), pulegona (12.1 por ciento), 3-hidroxi-mentofuranona (8.6 por ciento) y mentofurano (6.2 por ciento). El análisis de componentes principales (70 por ciento de información relacionada) y de conglomerado, aplicado a los datos permitió encontrar 3 grupos: I. – frutos; II. – hojas; y, III. – resina, corteza del tallo y ramas; este último grupo mostró estrecha relación composicional. Así, las fracciones volátiles estuvieron representadas por compuestos benzofuranoides (60-69 por ciento) en las ramas y corteza del tallo; por sesquiterpenoides (60 por ciento) en las hojas; por monoterpenoides (54 por ciento) en los frutos; y, por benzofuranoides (48 por ciento) y monoterpenoides...

Estudio químico de los metabolitos secundarios volátiles de flores y hojas de Cyanthillium cinereum (L.) H. Rob recolectada en Juan de Acosta (Atlántico, Colombia) / Chemical study of the volatile secondary metabolites of flowers and leaves of Cyanthillium cinereum (L.) H. Rob from Juan de Acosta (Atlántico, Colombia)

Volatile fractions and essential oils obtained from the flowers and leaves of Cyanthillium cinereum, isolated by simultaneous-distillation extraction (SDE) and conventional hydrodistillation (HD), were analyzed by gas chromatography and mass spectrometry (GC-MS). The most abundant compounds found in the volatile fractions were: (a). flowers – alpha-cadinol (14.4 percent), delta-cadinene (11.0 percent), thymohydroquinone dimethyl ether (7.0 percent), alpha-humulene (6.4 percent), tau-muurolol (5.8 percent), and terpinen-4-ol (5.1 percent); and, (b). leaves – alpha-cadinol (20.3 percent), delta-cadinene (11.7 percent), germacrene D-4-ol (9.1 percent), tau-muurolol (6.5 percent), terpinen-4-ol (5.8 percent), and elemol (5.1 percent). As main compounds in the essential oils (EO) were identified: (c). flowers – delta-cadinene (15.8 percent), alpha-cadinol (15.7 percent), alpha-humulene (9.6 percent), tau-muurolol (6.1 percent), thymohydroquinone dimethyl ether (5.5 percent), and tau-cadinol (4.4 percent); and, (d). leaves – alpha-cadinol (23.2 percent), elemol (10.6 percent), delta-cadinene (9.9 percent), tau-muurolol (8.2 percent), germacrene D-4-ol (6.1 percent), and terpinen-4-ol (4.9 percent). The yield achieved in the isolation of EO was 0.09 percent, for the flowers and leaves. Similarly, volatile fractions and essential oils, classified according to the terpene structures and chemical functionality, were represented by cadinane type sesquiterpene bicyclic hydrocarbons (19.8-30.9 percent) and their alcohols (36.2-46.2 percent).

Las fracciones volátiles y aceites esenciales obtenidos de flores y hojas de Cyanthillium cinereum, aislados por destilación-extracción simultánea con solvente (SDE) e hidrodestilación convencional (HD), fueron analizados por cromatografía de gases y espectrometría de masas (GC-MS). Los componentes más abundantes encontrados en las fracciones volátiles fueron: (a). flores – alfa-cadinol (14.4 por ciento), delta-cadineno (11.0 por ciento), timohidroquinona dimetil éter (7.0 por ciento), alfa-humuleno (6.4 por ciento), tau-muurolol (5.8 por ciento) y terpinen-4-ol (5.1 por ciento); y (b). hojas – alfa-cadinol (20.3 por ciento), delta-cadineno (11.7 por ciento), germacreno D-4-ol (9.1 por ciento), tau-muurolol (6.5 por ciento), terpinen-4-ol (5.8 por ciento) y elemol (5.1 por ciento). En los aceites esenciales (AE) se identificaron como compuestos mayoritarios: (c). flores – delta-cadineno (15.8 por ciento), alfa-cadinol (15.7 por ciento), alfa-humuleno (9.6 por ciento), tau-muurolol (6.1 por ciento), timohidroquinona dimetil éter (5.5 por ciento) y tau-cadinol (4.4 por ciento); y (d). hojas – alfa-cadinol (23.2 por ciento), elemol (10.6 por ciento), delta-cadineno (9.9 por ciento), tau-muurolol (8.2 por ciento), germacreno D-4-ol (6.1 por ciento) y terpinen-4-ol (4.9 por ciento). El rendimiento de los AE fue 0.09 por ciento, para las flores y hojas. Asimismo, las fracciones volátiles y aceites esenciales, clasificadas de acuerdo con las estructuras terpénicas y funcionalidad química, estuvieron representados por hidrocarburos (19.8-30.9 por ciento) sesquiterpénicos bicíclicos tipo cadinano y sus alcoholes (36.2-46.2 por ciento).

Caracterización química de las fracciones volátiles y aceites esenciales de hojas y flores de Chromolaena barranquillensis encontrada en Sabanalarga (Atlántico, Colombia) / Chemical characterization of the volatile fractions and essential oils of leaves and flowers of Chromolaena barranquillensis from Sabanalarga (Atlántico, Colombia)

Volatile secondary metabolites obtained from the leaves and flowers (fresh and dried) of Chromolaena barranquillensis (native specie of the Departamento del Atlántico), and isolated by simultaneous-distillation extraction (SDE) and conventional hydrodistillation (HD), were characterized chemically by gas chromatography coupled with mass spectrometry (GC-MS). The main compounds identified in the volatile fractions were: (a). fresh flowers – beta - elemene (24.4 percent), a-pinene (19.6 percent), and limonene (15.7 percent); (b). dried flowers – beta-caryophyllene (21.4 percent), germacrene D (16.6 percent), and caryophyllene oxide (13.6 percent); (c). Fresh leaves – myrcene (39.0 percent), y-curcumene (17.8 percent), and limonene (10.2 percent); and, (d). Dried leaves – beta-caryophyllene (13.8 percent), -curcumene (9.8 percent), beta-elemene (7.7 percent), and caryophyllene oxide (6.4 percent). In the essential oils (EO) were recognized as major components: (e). Fresh flowers – beta-caryophyllene (22.9 percent), beta-elemene (14.3 percent), and germacrene D (14.0 percent); (f). Dried flowers – beta-caryophyllene (23.6 percent), -elemene (20.6 percent), and germacrene D (15.8 percent); (g). Fresh leaves – beta-caryophyllene (22.0 percent), limonene (11.8 percent), -cadinene (6.8 percent), and germacrene D (6.1 percent); and, (h). Dried leaves – beta-caryophyllene (29.1 percent), germacrene D (13.1 percent), and caryophyllene oxide (12.0 percent). The yields achieved in the isolation of EO were 0.2 percent/0.4 percent and 0.06 percent/0.1 percent for fresh/dried flowers and fresh/dried leaves, respectively.

Los metabolitos secundarios volátiles obtenidos de hojas y flores (frescas y secas) de Chromolaena barranquillensis (especie nativa del departamento del Atlántico), aislados por destilación-extracción simultánea con solvente (SDE) e hidrodestilación convencional (HD), se caracterizaron químicamente por cromatografía de gases acoplada a espectrometría de masas (GC-MS). Los compuestos mayoritarios que se identificaron en las fracciones volátiles fueron: (a). flores frescas – beta-elemeno (24.4 por ciento), a-pineno (19.6 por ciento) y limoneno (15.7 por ciento); (b). Flores secas – trans-beta-cariofileno (21.4 por ciento), germacreno D (16.6 por ciento) y óxido de cariofileno (13.6 por ciento); (c). Hojas frescas – beta-mirceno (39.0 por ciento), y-curcumeno (17.8 por ciento) y limoneno (10.2 por ciento); y, (d). Hojas secas – trans-beta-cariofileno (13.8 por ciento), y-curcumeno (9.8 por ciento), beta-elemeno (7.7 por ciento) y óxido de cariofileno (6.4 por ciento). En los aceites esenciales (AE) se reconocieron como componentes principales: (e). Flores frescas – trans-beta-cariofileno (22.9 por ciento), beta-elemeno (14.3 por ciento) y germacreno D (14.0 por ciento); (f). Flores secas – trans-beta-cariofileno (23.6 por ciento), beta-elemeno (20.6 por ciento) y germacreno D (15.8 por ciento); (g). Hojas frescas – trans-beta-cariofileno (22.0 por ciento), limoneno (11.8 por ciento), -cadineno (6.8 por ciento) y germacreno D (6.1 por ciento); y, (h). Hojas secas – trans-beta-cariofileno (29.1 por ciento), germacreno D (13.1 por ciento) y óxido de cariofileno (12.0 por ciento). Los rendimientos alcanzados en el aislamiento de los AE fueron 0.2 por ciento/0.4 por ciento y 0.06 por ciento/0.1 por ciento para las flores frescas/secas y hojas frescas/secas, respectivamente.

Intestinal epithelial barrier dysfunction and dendritic cell redistribution during early stages of inflammation in the rat: role for TLR-2 and -4 blockage.

BACKGROUND: Dendritic cell (DC) redistribution during early stages of enteritis may be related to ileal barrier dysfunction. We used a rat model of ileitis to examine this hypothesis. METHODS: Sprague-Dawley rats were injected with indomethacin or saline and euthanized 2, 6, 12, or 24 hours later. Ileal segments and mesenteric lymph nodes were obtained for morphological, bacterial, or functional studies. To determine the role of Toll-like receptor (TLR)-2 and -4 blockages, rats were pretreated with normal IgG, anti-TLR-2, or anti-TLR-4 antibodies prior to indomethacin or saline, and ileal segments were collected 24 hours later. RESULTS: In control rats, CD103+DC were mainly located in the lamina propria (LP) and some expressed TLR-2. TLR-4+ cells with different morphology and distribution from CD103+DC were also detected. In indomethacin-treated rats at 6-24 hours, inflammation was evident as was redistribution of CD103+DC from LP to Peyer's patches. We also observed TLR-2+ monocyte depletion and changes in TLR-4 distribution. At 2-6 hours we detected opened tight junctions as well as abnormal trans- and para-epithelial enteric bacterial infiltration, while macromolecular permeability was not significantly enhanced until 24 hours. In the absence of indomethacin, anti-TLR-2 blockage induced a significant increase of LP CD103+DC, while in the presence of indomethacin, anti-TLR-2 or -4 blockages significantly inhibited (P < 0.05) the reduction of LP CD103+DC. CONCLUSIONS: During the early stages of indomethacin-induced ileitis, epithelial barrier damage and abnormal bacterial infiltration into the mucosa occurred in conjunction with initial redistribution of CD103+DC. Furthermore, we showed that TLR-2 and -4 blockade regulates CD103+DC distribution during early phases in this experimental model.

The nitric oxide donor LA-419 [S-(6-Nitro-oxi-hexahydro-furo[3,2-b]furan-3-1-il)thioacetate] prevents intestinal dysmotility, bacterial translocation, and inflammation in a rat model of enteritis.

Indomethacin induces a chronic model of inflammatory bowel disease (IBD) characterized by spontaneous relapses of inflammation, bacterial translocation, and long-lasting motor disturbances derived from cyclical up-regulated inducible nitric-oxide synthase (iNOS) and sustained down-regulated neuronal NOS (nNOS). The aims of this study were to evaluate whether LA-419 [S-(6-nitro-oxi-hexahydro-furo[3,2-b]furan-3-1-il)thioacetate], a NO-donor drug, could re-establish the normal expression of NOS and, hence, prevent the development of intestinal dysmotility, bacterial translocation, and relapses of inflammation associated to this model. Enteritis was induced in rats by administration of indomethacin with and without treatment with a novel NO-donor: LA-419 (0.5 mg/ml in the drinking water). Inflammatory reaction was evaluated by measuring blood leukocytes, serum tumor necrosis factor, and tissue myeloperoxidase. Intestinal motor activity was evaluated using strain-gauges. Ileal expression of iNOS and nNOS mRNA was determined by reverse transcription-polymerase chain reaction. Bacterial translocation was evaluated in cultures from mesenteric lymph nodes. The indomethacin-induced acute inflammatory reaction was associated with a rise in blood leukocytes and tumor necrosis factor. In the chronic stage, blood leukocyte monitoring allowed the selection of animals in active and inactive phases. Active phase was associated with iNOS up-regulation, high myeloperoxidase levels, hypomotility, and bacterial translocation. In contrast, inactive phase was associated with hypermotility and absence of bacterial translocation. LA-419 treatment restored nitric-oxide synthase isoenzyme expression and prevented the oscillation of both inflammatory and motor parameters that could be cyclically observed in inflamed rats. LA-419 also prevented intestinal dysmotility, bacterial translocation, and relapses of intestinal inflammation. LA-419 might be a novel therapeutic approach to prevent acute inflammatory relapses in patients with IBD.

Correlation between cyclical epithelial barrier dysfunction and bacterial translocation in the relapses of intestinal inflammation.

BACKGROUND: Although several factors have been implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanisms underlying the recurrent relapses have not yet been clarified. We hypothesized that epithelial barrier dysfunction, associated with intestinal motor disturbances, could play a key role in exacerbation of inflammatory illness due to an increased uptake of luminal antigen and bacterial translocation. METHODS: Indomethacin administration to rats induced a long-lasting oscillation of active and quiescent phases of inflammation associated with phases of hypo and hypermotility. Rats selected at either active or quiescent phase and from 2 to 60 days after indomethacin treatment were used. Short-circuit current; conductance and HRP flux were evaluated in small intestinal segments mounted in Ussing Chambers. Enterocyte endosomes containing HRP and ultrastructural damage were assessed by electron microscopy. Bacterial translocation was determined by cultures from mesenteric lymph nodes. RESULTS: Rats with induced enteritis in both phases demonstrated a long-lasting increase of epithelial paracellular permeability. In contrast, transcellular permeability was only disturbed during the active phases, coinciding with bacterial translocation and the increase in inflammatory parameters. Furthermore, although mithochondrial damage was observed throughout the inflammatory state, alterations were worse during the active phase. CONCLUSIONS: The sustained enhancement of paracellular permeability could facilitate the constant passage of luminal antigens through the mucosa, and hence, be the basis for chronicity. By contrast, transcellular permeability only increases during the active phases, when hypomotility and bacterial translocation are also present, suggesting this factor may play a critical role in the course of acute relapses in IBD.

Characterization of ileal dendritic cell distribution in a rat model of acute and chronic inflammation.

We examined ileal dendritic cell (DC) subpopulations in a rat model of indomethacin-induced enteritis to determine changes in phenotype and distribution associated with increased mucosal permeability during acute and chronic stages of inflammation. Sprague-Dawley rats were treated with indomethacin (7.5 mg/kg subcutaneously, 2 injections 48 h apart). Animals were killed at day 4 (acute stage) or at day 15 or 30 (chronic stages); control rats were injected with saline. DC distribution was evaluated by immunohistochemistry for CD103, CD11b, CD83, and CD163; inflammation was assessed by light microscopy; and permeability was determined by flux of horseradish peroxidase in Ussing chambers. In controls, both immature DC subpopulations, CD103+CD11b+CD163-CD83- and CD103+CD11b-CD163-CD83-, were observed in the lamina propria, and the CD11b- population also was present in Peyer's patches. In acute inflammation, permeability was increased (P<0.01), and inflamed areas with or without ulcers were observed. CD103+ and CD11b+ (CD83-) DCs were absent from inflamed areas, reduced in noninflamed tissues, but present in Peyer's patches. In the chronic stage at day 15, CD103+ and CD11b+ cells were located in inflamed and noninflamed areas and in Peyer's patches. In addition, CD83+ DCs were detected in inflamed areas. At day 30, when we observed a complete microscopic resolution of inflammation, numbers of CD103+ and CD11b+ DCs were increased, and there were CD83+ DCs beneath the epithelial cell layer. We conclude that antigen uptake in acute inflammation may activate resident immature DCs, inducing their migration to lymphoid tissue where they mature and then return to the intestine to play a role in the local inflammatory response.

Intestinal motor disorders associated with cyclical bacterial overgrowth in a rat model of enteritis.

The aims of this study were: 1) to obtain an experimental model reproducing the characteristics of chronicity and spontaneous relapses found in inflammatory bowel disease (IBD) and 2) to correlate these changes with intestinal motility and bacteria translocation. For this purpose, two groups of Sprague-Dawley rats were used: a treated group that received two subcutaneous injections of indomethacin (7.5 mg/kg) 48 h apart and a control group that received saline. Blood leukocytes, TNF, and fecal parameters were monitored for 90 days after treatment. In treated rats, a cyclic oscillation of blood leukocytes and TNF concomitant with an inverse correlation of fecal output was observed. Treated rats were then selected either during their highest or lowest blood leukocyte values for motor activity and microbiological evaluation. Controls were obtained in age-matched rats. Rats with high leukocyte levels showed a decrease of motor activity. In contrast, animals with low leukocyte levels presented hypermotility. Bacterial overgrowth accompanied by bacterial translocation was found in the group with high leukocytes, whereas no differences were observed between the control and indomethacin groups during the lowest leukocyte phase. We obtained a model of IBD characterized by a chronic cyclic oscillation of intestinal motility, flora, and inflammatory blood parameters. During the high-leukocyte stage, motor activity decrease is related to bacterial translocation. This phase is followed by a reactive one characterized by hypermotility associated with a decrease in both bacterial growth and leukocytes. However, as in IBD, this reaction seems unable to prevent a return to relapse.