IL-1ß induces changes in expression of core circadian clock components PER2 and BMAL1 in primary human chondrocytes through the NMDA receptor/CREB and NF-κB signalling pathways.

The circadian clock is a specialised cell signalling circuit present in almost all cells. It controls the timing of key cell activities such as proliferation and differentiation. In osteoarthritis, expression of two components of the circadian clock, BMAL1 and PER2 is altered in chondrocytes and this change has been causally linked with the increase in proliferation and altered chondrocyte differentiation in disease. IL-1ß, an inflammatory cytokine abundant in OA joints, has previously been shown to induce changes in BMAL1 and PER2 expression in chondrocytes. The purpose of this study is to identify the mechanism involved. We found IL-1ß treatment of primary human chondrocytes led to activation of NMDA receptors as evidenced by an increase in phosphorylation of GluN1 and an increase in intracellular calcium which was blocked by the NMDAR antagonist MK801. Levels of phosphorylated CREB were also elevated in IL-1ß treated cells and this effect was blocked by co-treatment of cells with IL-1ß and the NMDAR antagonist MK-801. Knockdown of CREB or inhibition of CREB activity prevented the IL-1ß induced increase in PER2 expression in chondrocytes but had no effect on BMAL1. Phosphorylated p65 levels were elevated in IL-1ß treated chondrocytes indicating increased NF-κB activation. Inhibition of NF-κB activity prevented the IL-1ß induced reduction in BMAL1 expression and partially mitigated the IL-1ß induced increase in PER2 expression in chondrocytes. These data indicate that the NMDAR/CREB and NF-κB signalling pathways regulate the core circadian clock components PER2 and BMAL1 in chondrocytes. Given that changes in expression of these clock components have been observed in a wide range of diseases, these findings may be broadly relevant for understanding the mechanism leading to circadian clock changes in pathology.

Altered N-methyl D-aspartate receptor subunit expression causes changes to the circadian clock and cell phenotype in osteoarthritic chondrocytes.

The chondrocyte circadian clock is altered in osteoarthritis. This change is implicated in the disease-associated changes in chondrocyte phenotype and cartilage loss. Why the clock is changed is unknown. N-methyl-D-aspartate receptors (NMDAR) are critical for regulating the hypothalamic clock. Chondrocytes also express NMDAR and the type of NMDAR subunits expressed changes in osteoarthritis. OBJECTIVE: To determine if NMDAR regulate the chondrocyte clock and phenotype. DESIGN: Chondrocytes isolated from macroscopically-normal (MN) and osteoarthritic human cartilage were treated with NMDAR antagonists or transfected with GRIN2A or GRIN2B-targetting siRNA. H5 chondrocytes were transfected with GluN2B-expression plasmids. Clock genes and chondrocyte phenotypic markers were measured by RT-qPCR. RESULTS: PER2 amplitude was higher and BMAL1 amplitude lower in osteoarthritic compared to MN chondrocytes. In osteoarthritic chondrocytes, NMDAR inhibition restored PER2 and BMAL1 expression to levels similar to MN chondrocytes, and resulted in reduced MMP13 and COL10A1. Paradoxically, NMDAR inhibition in MN chondrocytes resulted in increased PER2, decreased BMAL1 and increased MMP13 and COL10A1. Osteoarthritic, but not MN chondrocytes expressed GluN2B NMDAR subunits. GluN2B knockdown in osteoarthritic chondrocytes restored expression of circadian clock components and phenotypic markers to levels similar to MN chondrocytes. Ectopic expression of GluN2B resulted in reduced BMAL1, increased PER2 and altered SOX9, RUNX2 and MMP13 expression. Knockdown of PER2 mitigated the effects of GluN2B on SOX9 and MMP13. CONCLUSIONS: NMDAR regulate the chondrocyte clock and phenotype suggesting NMDAR may also regulate clocks in other peripheral tissues. GluN2B expression in osteoarthritis may contribute to pathology by altering the chondrocyte clock.

Cell differentiation versus cell death: extracellular glucose is a key determinant of cell fate following oxidative stress exposure.

Cells, particularly mechano-sensitive musculoskeletal cells such as tenocytes, routinely encounter oxidative stress. Oxidative stress can not only stimulate tissue repair, but also cause damage leading to tissue degeneration. As diabetes is associated with increased oxidative damage as well as increased risk of tendon degeneration, the aim of this study was to determine if extracellular glucose levels alter the response of tendon cells to oxidative stress. Primary human tenocytes were cultured in either high (17.5 mM) or low (5 mM) glucose and treated with 100 µM hydrogen peroxide. In low glucose, peroxide-treated cells remained fully viable and collagen synthesis was increased, suggesting an anabolic response. In high glucose, however, peroxide treatment led to increased bim-mediated apoptosis. The activities of both forkhead box O (FOXO1) and p53 were required for upregulation of bim RNA expression in high glucose. We found that both p53-mediated inhibition of the bim repressor micro RNA (miR17-92) and FOXO1-mediated upregulation of bim transcription were required to permit accumulation of bim RNA. High glucose coupled with oxidative stress resulted in upregulation of miR28-5p, which directly inhibited expression of the p53 deacetylase sirtuin 3, resulting in increased levels of acetylated p53. In peroxide-treated cells in both high and low glucose, protein levels of acetylated FOXO1 as well as HIF1α (hypoxia-inducible factor 1α) were increased. However, under low-glucose conditions, peroxide treatment resulted in activation of p38, which inhibited FOXO1-mediated but promoted HIF1α-mediated transcriptional activity. In low glucose, HIF1α upregulated expression of sox9 and scleraxis, two critical transcription factors involved in establishing the tenocyte phenotype, and increased collagen synthesis. The switch from FOXO1-mediated (proapoptosis) to HIF1α-mediated (prodifferentiation) transcription occurred at an extracellular glucose concentration of 7 mM, a concentration equivalent to the maximum normal blood glucose concentration. Extracellular glucose has a profound effect on the cellular response to oxidative stress. A level of oxidative stress normally anabolic may be pathological in high glucose.

An ink surgical marker pen is damaging to tendon cells.

OBJECTIVES: Surgical marking during tendon surgery is often used for technical and teaching purposes. This study investigates the effect of a gentian violet ink marker pen, a common surgical marker, on the viability of the tissue and cells of tendon. METHODS: In vitro cell and tissue methods were used to test the viability of human hamstring explants and the migrating tenocytes in the presence of the gentian violet ink. RESULTS: The outcome of this study was that a constituent of the surgical marker pen causes cell and tissue death in culture, implying the same would occur in vivo. CONCLUSIONS: This is a cause for concern when marking tendon during surgical procedures, as it may compromise healing and repair and potentially contribute to a poor outcome. The authors suggest that an alternative surgical marking procedure should be found, or that all marker pens should undergo testing on human tendon tissue in vitro prior to use.

Post-conditioning reduces infarct size in an open-chest porcine acute ischemia-reperfusion model.

BACKGROUND: Timely reperfusion is a prerequisite for myocardial salvage; however, re-oxygenation of the ischemic myocardium initiates reperfusion injury. Post-conditioning diminishes the detrimental aftermath of an acute myocardial infarction through alleviation of reperfusion injury. Ischemic post-conditioning consists of a series of brief interruptions in the coronary blood supply that has to be applied within the first minutes after re-establishing the coronary flow. METHODS: Sixteen female mixed Danish Landrace and Yorkshire pigs weighing 20 kg were included. The heart was exposed through a midline sternotomy. A snare was positioned around the left anterior descending coronary artery downstream of the second diagonal branch. After randomization to either no treatment (control group) or treatment by ischemic post-conditioning (post-conditioning group), the pigs underwent 45 min of ischemia and 180 min of reperfusion. The post-conditioning group had a post-conditioning algorithm applied consisting of 15 s of reperfusion alternating with 15 s of re-occlusion repeated 10 times. RESULTS: The groups were comparable with regard to body weight, hemodynamics and the size of the area at risk. The post-conditioning group had an absolute reduction in infarct size of 18.1% [confidence interval (CI): 6.2: 30.0%] compared with the control group (P=0.0056). In the post-conditioning group, infarction developed in 39.6+/-12.0% (1 SD) of the area at risk compared with 57.8+/-10.2% (1 SD) in the control group. CONCLUSION: When ischemic post-conditioning was applied at reperfusion, we found an absolute reduction in infarct size of 18.1% presumably attributable to a diminished reperfusion injury. The model we have developed is suitable for further studies of this promising intervention.

Specific effects of gamma-linolenic, eicosapentaenoic, and docosahexaenoic ethyl esters on bone post-ovariectomy in rats.

Long chain polyunsaturated fatty acids (LCPUFAs) are involved in the regulation of bone metabolism. Increased dietary consumption of n-3, and possibly some n-6, LCPUFAs may limit postmenopausal bone loss. The aim of this study was to determine the effects on bone of specific fatty acids within the n-3 and n-6 LCPUFA families in ovariectomized (OVX) rats. Rats were OVX or sham-operated and fed either a control diet (OVX and sham) or a diet supplemented with 0.5 g/kg body weight/day of gamma-linolenic (GLA), eicosapentaenoic (EPA), docosahexaenoic (DHA) ethyl esters or a mixture of all three (MIX) for 16 weeks. Bone mineral content (BMC), area, and density and plasma concentrations of insulin-like growth factor-I, vitamin D, selected biochemical markers of bone metabolism, and parathyroid hormone (PTH) were determined. The OVX-induced decrease in lumbar spine BMC was significantly attenuated by DHA but not by EPA or GLA supplementation or supplementation with a mixture of all three LCPUFAs. Endosteal circumferences of tibiae were significantly greater in DHA and EPA compared to OVX. Plasma C-terminal telopeptide of type I collagen and osteocalcin concentrations were not significantly different in the DHA group compared to OVX. Femur BMC decreased by a significantly greater amount in GLA than OVX, and final plasma PTH concentrations were significantly higher in GLA compared to all other groups. In conclusion, DHA ameliorated OVX-induced bone mineral loss. GLA exacerbated post-OVX bone mineral loss, possibly as a result of PTH-induced bone catabolism.

Synchrotron charge-density studies in materials chemistry: 16 K X-ray charge density of a new magnetic metal-organic framework material, [Mn2(C8H4O4)2(C3H7NO)2].

A new magnetic metal-organic framework material, [Mn(2)(C(8)OH(4)(4))(2)(C(3)H(7)NO)(2)], has been synthesized. The structure consists of chains of carboxylate-bridged Mn atoms interconnected with acid linkers, giving much larger interchain than intrachain Mn...Mn distances. Magnetic susceptibility data fitted to a Curie-Weiss law give Theta = -5.7 K and a total magnetic moment of 5.96 micro(B). The heat capacity provides no evidence of magnetic ordering down to 2 K. The X-ray charge density was determined from multipole modeling of 16 (1) K single-crystal synchrotron-radiation data. The structural surroundings of the two unique Mn centers are different, but orbital population analysis reveals close to single electron occupation in all 3d orbitals of both Mn sites, in agreement with the magnetic susceptibility measurements. Bader topological analysis shows the presence of direct chemical Mn...Mn interactions only in two out of three intrachain contacts, which suggests a 'broken' chain. The topological measures and approximate energy densities at the metal-ligand bond critical points (rho, nabla(2)rho, G, V and H) indicate ionic interactions. Formal electron counting suggests mixed-valence Mn sites, but this hypothesis is not supported by the Bader atomic charges [q(Mn) = +2.035 and +2.031].

Síndrome de Goldenhar asociado a embarazo / Goldenhar syndrome and pregnancy

El síndrome de Goldenhar es una rara condición, de aparición esporádica, y con componente genético débil. Se caracteriza por un espectro de malformaciones faciales, especialmente deformaciones oculares (ausencia o hipotrofia ocular) y malformaciones auriculares, que característicamente comprometen una hemicara, con presencia o ausencia de anomalías vertebrales. Se presenta un caso clínico de la asociación de Síndrome de Goldenhar y embarazo.

Talasemia asociada a embarazo / Thalassemia associated to pregnancy

La Talasemia es un desorden congénito hemolítico causado por una deficiencia parcial o completa de la síntesis de las cadenas alfa o beta de las globinas de la hemoglobina. Se manifiesta en una amplia gama de cuadros clínicos que van desde la muerte intrauterina hasta la microcitosis asintomática sin anemia. El depósito de hierro constituye la complicación más importante de la talasemia y su mayor preocupación en el manejo. Existen escasos reportes de esta condición asociada a embarazo, se sabe poco acerca de su manejo y de las complicaciones que puede tener en el embarazo. Presentamos una paciente portadora de Talasemia, en la cual se manejó su embarazo en nuestro servicio.

Alteraciones de las extremidades inferiores en el síndrome de Prune-Belly / Alterations of the lower extremities in the Prune-Belly syndrome

El síndrome de Prune-Belly es una rara condición congénita de causa desconocida, que fue descrita por primera vez en 1839. Tiene una prevalencia de 20:1 en hombres versus mujeres, situación probablemente debida a un defecto superficial en el cromosoma X. Se caracteriza por: 1) ausencia de la pared abdominal en forma completa o parcial. 2) criptorquidia y 3) anomalías del tracto urinario. A veces, también está asociado a algunas anomalías ortopédicas debidas a la presencia de oligohidroamnios, pero raramente ocurren anormalidades de las extremidades inferiores. El pronóstico es malo sólo un 20 por ciento sobreviven al parto y un 50 por ciento muere dentro de los primeros 5 años de vida principalmente debido a las consecuencias de falla renal.Reportamos el caso de un niño con síndrome de Prune-Belly con una amputación congénita de una de sus extremidades inferiores que fue manejada con punción vesical en el período antenatal.

Angiomixoma de la vulva

El angiomixoma agresivo es una neoplasia rara, de etiología indeterminada que ocurre principalmente en el vulva y el periné femeninos (1, 2). Dado que sus límites son siempre impreciosos y por lo tanto la resección no siempre es completa, tiene una gran tendencia a la recurrencia local. Su presentación habitual es como un tumor sólido, gelatinoso, de bordes poco claros. Como tratamiento se preconiza la resección amplia con bordes quirúrgicos libres. La quimioterapia y radioterapia son de escasa utilidad por el mínimo índice mitótico. A continuación presentamos el caso de una mujer con Angiomixoma que fue manejado en nuestro servicio. Se comenta su manejo, evolución y resultado.

Colitis ulcerosa asociada al embarazo

En los últimos 15 años la enfermedad inflamatoria intestinal no ha sido reproducible a nivel de laboratorio, no existen nuevas luces sobre su patogénesis, y tampoco se ha facilitado el diagnóstico. Estudios en gemelos idénticos esclarecen que el desarrollo de la enfermedad depende de factores adicionales. La activación del sistema inmune central está eventualmente acompañado por la producción de una amplia variedad de mediadores no específicos de la inflamación. Estos mediadores mantienen el proceso inflamatorio y desencadenan la destrucción tisular, lo cual eventualmente se manifiesta clínicamente como enfermedad.

Rabdomiosarcoma alveolar de utero

Los rabdomiosarcomas constituyen un tipo raro de sarcoma que se origina en las células mesenquimáticas que van a derivar en músculo estriado. De ocurrencia mayoritaria en la infancia (50-55% de los sarcomas de los niños, de ellos el 70% ocurre en la primera década). Se clasifican en forma histológica en embriones, botriodes, alveolar y pleomórficas. Estos tumores se diagnostican en raras ocasiones. Se etapifican según la IRSG de Estados Unidos. Debido a los pobres resultados se evalúa la sobrevida a los 2 años (90% de mortalidad a los dos años). A continuación presentamos el poco afortunado caso de una paciente manejada en nuestro servicio, en cuyo estudio patológico se demostró un rabdomiosarcoma alveolar de útero.

Dermatomiositis juvenil y embarazo / Juvenile dermatomyositis and pregnancy

La dermatomiositis juvenil es un desorden inflamatorio crónico multisistémico del tejido conectivo. Tiene una incidencia de 2-3/100.000/año. Con la disminución en la mortalidad experimentada en los últimos decenios, la atención está cifrada en la morbilidad a largo plazo y en las alteraciones funcionales. Con un tratamiento agresivo los niños con dermatomiositis juvenil generalmente tienen un futuro promisorio, sin incapacidad o con incapacidad mínima. La mortalidad actualmente se estima cercana al 2% y está frecuentemente relacionada con vasculitis descontrolada, infección pulmonar, o septicemia producida en áreas de calcificación patológica. Los reportes de dermatomiosis que complican el embarazo son raros, y según algunos reportes clínicos el resultado fetal puede ser adverso. A continuación presentamos el caso del primer embarazo en paciente con dermatomiositis juvenil en tratamiento en el Servicio de Obstetricia y Ginecología del Hospital Clínico de Antofagasta.

The juvenile dermatomyositis is a chronic inflammatory multisystemic disorder of the connective tissue. It has an incidence of 2-3/100.000/year. With the decrease in the mortality that has experienced in the last decade, the attention is been paid in the long term morbidity and functional alterations. With an aggressive treatment the children with juvenile dermatomyositis generally have a promising future, with no or minimal disability. The mortality now is considered near to 2% and this frequently happens with out of control vasculitis, lung infection, or septicemia taking place in areas of pathological calcification. The dermatomyositis reports that complicat pregnancy are uncommon and according to some clinical reports the fetal outcome can be adverse. We present the case of the first pregnancy in patient with juvenile dermatomyositis being treated in the Department Obstetrics and Gynecology of the Clinical Hospital of Antofagasta.

Enfermedad de Von Recklinghausen y embarazo / Von Recklinghausen disease and pregnancy

La enfermedad de Von Recklinghausen (neurofibromatosis) es una condición autosómica dominantela cualha tenido variables expresiones clínicas, con manifestaciones que van de lesiones cutáneas moderadas a complicaciones ortopédicas severas y lateraciones funcionales. El 50 por ciento de los casos de neurofibromatosis son esporádicos, ya que no se encuentran lesiones en ninguno de los progenitores. Estos casos esporádicos se deben a mutaciones de novo, la mayoría de las cuales se producen en los gametos paternos. La incidencia de neurofibromatosis tipo 1 asociada al embarazo es de 1 cada 4000 nacimientos (1). Las pacientes con neurifibromatosis tiene un incremento en el riesgo de complicaciones perinatales (2,3,4). Se presenta el caso de una paciente con neurofibromatosis atendida en nuestro servicio, se comenta la fisiopatología, resultados perinatales y manejo

Síndrome de Wolf Parkinson White asociado a embarazo / Wolf Parkinson White syndrome associated to pregnancy

El embarazo se ha asociado con un incremento en la incidencia de arritmias. Aunque las palpitaciones, la ansiedad y eventualmente el síncope son síntomas relativamente común en el embarazo normal, estos serán raramente asociados con las arritmias cardíacas. Una reducción en la presión sanguínea asociadas con tales arritmias pueden resultar en bradicardia fetal y necesitan un tratamiento inmediato con drogas antiarrítmicas, cardioversión eléctrica o cesárea de urgencia. En el puerperio existe una asociación con un aumento significativo del riesgo de eventos cardíaco, incluyendo muerte, infartos cardíacos y síncopes. Algunos pacientes tienen conexiones anatómicas anómalas, que pueden evitar total o parcialmente el paso a través del nódulo aurículo ventricular. El Haz de Kent (comunicación anómala entre la aurícula y el ventrículo) constituyen la base anatómica del síndrome de Wolf Parkinson White (WPW). Este es una enfermedad congénita con incidencia familiar y en ocasiones se asocia a otras anomalías congénitas. La prevalencia de este síndrome oscila entre 0,1 y 3 por cada 1.000 personas. Puede constituir un hallazgo cardiográfico fortuito y cursar de forma asintomática durante toda la vida del paciente. No obstante, en estos enfermos existe una incidencia elevada de taquiarritmias

Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork.

BACKGROUND: Antibiotic-resistant enterococci are often present in retail meats, but it is unclear whether the ingestion of these contaminants leads to sustained intestinal carriage. METHODS: We conducted a randomized, double-blind study in 18 healthy volunteers. Six ingested a mixture of 10(7) colony-forming units (CFU) of two glycopeptide-resistant strains of Enterococcus faecium obtained from chicken purchased at a grocery store, six ingested 10(7) CFU of a streptogramin-resistant strain of E. faecium obtained from a pig at slaughter, and six ingested 10(7) CFU of a glycopeptide-susceptible and streptogramin-susceptible strain of E. faecium from chicken purchased at a grocery store. Suspensions of enterococci were prepared in 250 ml of whole milk and were well within the amounts deemed acceptable by Danish food regulations. Stool samples were collected before exposure, daily for 1 week after ingestion, and at 14 and 35 days. Resistant enterococci in stools were identified by selective culture techniques; further molecular characterization of the organisms was also conducted. RESULTS: At the outset, none of the subjects were colonized with glycopeptide-resistant or streptogramin-resistant E. faecium. After ingestion of the study strains, these same strains were isolated from the stools of all subjects, in various concentrations. The test strain was isolated in stool from 8 of 12 subjects on day 6, and from 1 of 12 on day 14. All stool samples were negative at 35 days. CONCLUSIONS: The ingestion of resistant E. faecium of animal origin leads to detectable concentrations of the resistant strain in stools for up to 14 days after ingestion. The organisms survive gastric passage and multiply.

Masa anexiales durante el embarazo / Adnexal masses during pregnancy

Las masas anexiales son un hallazgo raro durante el embarazo (0,5 por ciento y 2,2 por ciento). El ultrasonido es el medio de elección en la evaluación de una masa pélvica sospechosa, es segura tanto para la madre como para el feto, y permite sospechar entre tumores benignos y malignos. La manera tradicional de enfocar las masas pélvicas persistentes mayores de 5 cm. durante el embarazo ha sido a través de la intervención quirúrgica, esto debido a la tasa de complicaciones de 10 a 30 por ciento. En general se prefiere realizar en el segundo trimestre, puesto que el riesgo de pérdida fetal y de parto prematuro son menores, y existe la posibilidad de una intervención menos dificultosa (5). Se realizó un estudio descriptivo retrospectorio entre el 1 de julio de 1999 y el 30 de junio del 2001 de las pacientes atendidas con esta patología en nuestra maternidad

Impact on human intestinal microflora of an Enterococcus faecium probiotic and vancomycin.

The aims of this study were to evaluate the impact of a fermented milk product containing viable Enterococcus faecium on human intestinal microflora and to evaluate any risk of development of vancomycin-resistant enterococci (VRE). Twenty Danish and 20 Swedish healthy volunteers were given 150 ml of the fermented milk product once daily, equivalent to a daily dose of 4.5 x 10(9) to 7.5 x 10(9) CFU E. faecium, for 10 d. Half of the volunteers also received 125 mg vancomycin orally q.i.d. for 10 d. Faecal samples were collected on day 0 before intake, on day 10 directly after end of intake and on day 31, 3 weeks after the end of the experiment. There was a significant increase in the total number of enterococci on day 10 (p < 0.01) in the group receiving only the E. faecium supplement, but 3 weeks later the level was as before intake. In the vancomycin group, the total number of enterococci was reduced on day 10 (p < 0.01) but had increased on day 31 (p < 0.01) in relation to day 0. In none of the Swedish and 4 of the Danish volunteers, VRE were sporadically detected, but without relation to intake of the probiotic or vancomycin. In healthy young Danish individuals the VRE carrier rate tended to be higher than previously found.

Evaluation of a new 3-h hybridization method for detecting the mecA gene in Staphylococcus aureus and comparison with existing genotypic and phenotypic susceptibility testing methods.

A new 3-h hybridization assay for detection of the staphylococcal mecA gene and the Staphylococcus aureus nuclease gene was evaluated by comparing the assay with existing genotypic and phenotypic methods. A total of 275 S. aureus strains were tested, including 257 epidemiologically unrelated strains (135 mecA-positive and 122 mecA-negative; collection I), and 18 strains with known borderline resistance to methicillin (collection II). Complete agreement was obtained for both collections when comparing the new assay with genotypic methods. We further evaluated a range of phenotypic susceptibility methods recommended in Europe and/or USA using the presence of the mecA gene as the defining standard. For collection I a high degree of agreement was found for both Etests (256 strains) and the oxacillin screen plate test (255 strains); the degree of agreement was lower for agar dilution methicillin (250 strains) and oxacillin 1 microg discs (239 strains). For the borderline strains a high degree of agreement was only obtained by the oxacillin screen plate test (17 of 18 strains). The other tests were less accurate, in the following order: agar dilution methicillin, Etest methicillin, Etest oxacillin and oxacillin discs with disagreement for four, five, nine and 13 strains, respectively. In conclusion, the new hybridization assay is a rapid and exact method for detecting the mecA gene and the S. aureus nuclease gene. This study confirms that phenotypic tests for methicillin resistance in S. aureus strains creates both false-susceptible and false-resistant results, especially for borderline resistant strains.