Pharmacological characterization of GLPG1972/S201086, a potent and selective small-molecule inhibitor of ADAMTS5.

OBJECTIVE: A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. METHODS: Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants and on interleukin-1ß-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. RESULTS: GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50 ± SD: 19 ± 2 nM and <23 ± 1 nM, respectively), with 8-fold selectivity over ADAMTS4, and 60->5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20 µM and 10 µM, respectively). In DMM mice, GLPG1972/S201086 (30-120 mg/kg b.i.d) vs vehicle reduced femorotibial cartilage proteoglycan loss (23-37%), cartilage structural damage (23-39%) and subchondral bone sclerosis (21-36%). In MNX rats, GLPG1972/S201086 (10-50 mg/kg b.i.d) vs vehicle reduced cartilage damage (OARSI score reduction, 6-23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77-110%). CONCLUSIONS: GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

The influence of physical activity in water on sleep quality in pregnant women: A randomised trial.

INTRODUCTION: Sleep is a physiological state of self-regulation. The international classification of sleep disorders now includes as a new category those occurring during pregnancy. Regular physical activity is known to improve the quality of life, one aspect of which is sleep quality. During pregnancy, physical activity is decreased but should not be eliminated, as studies have reported a high correlation between sleep disorders and the absence of physical activity. Regular physical exercise during pregnancy, whether performed in water or out of it, provides greater control of gestational weight gain. Furthermore, the reduced weight gain during pregnancy, as a result of physical exercise, is associated with greater physical resistance to the demands of childbirth, combats the fatigue caused by pregnancy and reduces back pain. All of these outcomes tend to enhance sleep quality, among other beneficial effects. OBJECTIVE: To determine whether, in pregnant women, there is an association between moderate-intensity physical activity in an aquatic environment and sleep quality. MATERIAL AND METHODS: A randomised clinical trial was conducted with a sample of 140 pregnant women aged 21-43 years, divided into two groups; Intervention Group and Control Group. The women were recruited in the twelfth week of gestation and took part in the [Study of] Water Exercise in Pregnancy programme from week 20 to week 37. Sleep quality was evaluated in the first and third trimesters of pregnancy, using the Pittsburgh Sleep Quality Index questionnaire. RESULTS: The Mann-Whitney U test showed that the results obtained were statistically significant (p<0.05). In the Intervention Group, 44 of the women (65.67%) were classified as "poor sleepers" versus 62 women (92.54%) in the Control Group. CONCLUSIONS: The [Study of] Water Exercise in Pregnancy method improves the quality of sleep in pregnant women, both subjectively and in terms of latency, duration and efficiency.

[Physical activity by pregnant women and its influence on maternal and foetal parameters; a systematic review]. / Actividad física en embarazadas y su influencia en parámetros materno-fetales; revisión sistemática.

INTRODUCTION: Regular physical activity is known to be very beneficial to health. While it is important at all stages of life, during pregnancy doubts may arise about the suitability of physical exercise, as well as the type of activity, its frequency, intensity and duration. AIMS: To analyse major studies on the influence of physical activity on maternal and foetal parameters. METHOD: Systematic review of physical activity programmes for pregnant women and the results achieved, during pregnancy, childbirth and postpartum. 45 items were identified through an automated database search in PubMed, Scopus and Google Scholar, carried out from October 2013 to March 2014. In selecting the items, the criteria applied included the usefulness and relevance of the subject matter and the credibility or experience of the research study authors. The internal and external validity of each of the articles reviewed was taken into account. CONCLUSIONS: The results of the review highlight the importance of physical activity during pregnancy, and show that the information currently available can serve as an initial benchmark for further investigation into the impact of regular physical exercise, in an aquatic environment, on maternal-foetal health.

Introducción: Las múltiples ventajas que aporta a la salud la práctica de una actividad física de forma continuada son conocidas. Aunque es importante en todas las etapas de la vida, cuando llega la gestación aparecen algunas dudas sobre la conveniencia de realizar ejercicio físico, así como del tipo de actividad, de su frecuencia, intensidad y duración. Objetivo: Analizar los principales estudios sobre la influencia de la actividad física sobre los parámetros materno- fetales. Método: Revisión sistemática de programas de actividad física destinados a las mujeres embarazadas y sus resultados durante el embarazo, el parto y el postparto. Se identificaron 45 artículos a través de la búsqueda automatizada en las bases de datos PUBMED, Scopus y Google Scholar; se llevó a cabo entre los meses de octubre de 2013 y marzo de 2014. Para seleccionar los artículos fue preciso considerar la utilidad y la relevancia del tema estudiado y la credibilidad o experiencia de los autores en la temática. Se tuvo en cuenta la validez interna y externa de cada uno de los artículos revisados. Conclusiones: Los resultados de la revisión ponen de manifiesto la importancia de la actividad física durante el periodo gestacional y se constata que la información disponible en la actualidad puede servir de referente inicial para continuar profundizando en los resultados que sobre la salud materno fetal tiene la práctica de actividad física en el medio acuático.

Actividad física en embarazadas y su influencia en parámetros materno-fetales; revisión sistemática / Physical activity by pregnant women and its influence on maternal and foetal parameters; a systematic review

Introducción: Las múltiples ventajas que aporta a la salud la práctica de una actividad física de forma continuada son conocidas. Aunque es importante en todas las etapas de la vida, cuando llega la gestación aparecen algunas dudas sobre la conveniencia de realizar ejercicio físico, así como del tipo de actividad, de su frecuencia, intensidad y duración. Objetivo: Analizar los principales estudios sobre la influencia de la actividad física sobre los parámetros materno-fetales. Método: Revisión sistemática de programas de actividad física destinados a las mujeres embarazadas y sus resultados durante el embarazo, el parto y el postparto. Se identificaron 45 artículos a través de la búsqueda automatizada en las bases de datos PUBMED, Scopus y Google Scholar; se llevó a cabo entre los meses de octubre de 2013 y marzo de 2014. Para seleccionar los artículos fue preciso considerar la utilidad y la relevancia del tema estudiado y la credibilidad o experiencia de los autores en la temática. Se tuvo en cuenta la validez interna y externa de cada uno de los artículos revisados. Conclusiones: Los resultados de la revisión ponen de manifiesto la importancia de la actividad física durante el periodo gestacional y se constata que la información disponible en la actualidad puede servir de referente inicial para continuar profundizando en los resultados que sobre la salud materno fetal tiene la práctica de actividad física en el medio acuático (AU)

Introduction: Regular physical activity is known to be very beneficial to health. While it is important at all stages of life, during pregnancy doubts may arise about the suitability of physical exercise, as well as the type of activity, its frequency, intensity and duration. Aims: To analyse major studies on the influence of physical activity on maternal and foetal parameters. Method: Systematic review of physical activity programmes for pregnant women and the results achieved, during pregnancy, childbirth and postpartum. 45 items were identified through an automated database search in PubMed, Scopus and Google Scholar, carried out from October 2013 to March 2014. In selecting the items, the criteria applied included the usefulness and relevance of the subject matter and the credibility or experience of the research study authors. The internal and external validity of each of the articles reviewed was taken into account. Conclusions: The results of the review highlight the importance of physical activity during pregnancy, and show that the information currently available can serve as an initial benchmark for further investigation into the impact of regular physical exercise, in an aquatic environment, on maternal-foetal health (AU)

[Accelerometer description as a method to assess physical activity in different periods of life; systematic review]. / Descripción del acelerómetro como método para valorar la actividad física en los diferentes periodos de la vida; revisión sistemática.

INTRODUCTION: The accelerometer is shown as one of the most accurate techniques in recording and saving the amount and level of physical activity, by each person in a given period of time. AIMS: This review aims to describe and analyze the main items that use this method to assess physical activity. METHODS: The review articles were identified through the following specialized Internet browser: SCOPUS, PUBMED, GOOGLE SCHOLAR, those were selected for inclusion with a total of 56 items. The validity of the articles was given by the degree of evidence demonstrated by describing the recommendations and the applicability to our context. This review has considered studies evaluating physical activity through accelerometers. RESULTS: The results show that this method can be used in ages 3 to 90 years. It can also be used in subjects with overweight/obesity, articulation injuries, Down syndrome (just children), autism and people with psychological problems. Studies in pregnant women show satisfactory results. CONCLUSION: The literature reviewed provides the accelerometer as a reliable and effective method to assess physical activity.

Introducción: La acelerometría, se muestra como una de las técnicas más fiables, en el registro y almacenamiento de la cantidad y el nivel de actividad física, realizada por cada persona y en un periodo de tiempo determinado. Objetivo: Esta revisión tiene como objetivo describir y analizar los principales artículos que utilizan este método para valorar la actividad física. Método: Los artículos seleccionados para ser incluidos en esta revisión se identificaron a través de los siguientes motores de búsqueda especializados: SCOPUS, PUBMED, GOOGLE SCHOLAR; con un total de 56 artículos. La validez de los artículos estuvo dada por el grado de evidencias demostrado, por las recomendaciones que describen y por la aplicabilidad a nuestro contexto. Esta revisión ha considerado los estudios que evalúan la actividad física a través de acelerómetros. Resultados: Los resultados demuestran que este método se puede utilizar en edades comprendidas entre 3 y 90 años. También se puede utilizar en personas con sobrepeso/ obesidad, con lesiones articulares, niños con síndrome de Down, autistas, así como personas con problemas psicológicos. Los estudios en mujeres embarazadas muestran datos fiables. Conclusión: La literatura analizada establece la acelerometría como un método eficaz para valorar la actividad física.

Descripción del acelerómetro como método para valorar la actividad física en los diferentes periodos de la vida; revisión sistemática / Accelerometer description as a method to assess physical activity in different periods of life; systematic review

Introducción: La acelerometría, se muestra como una de las técnicas más fiables, en el registro y almacenamiento de la cantidad y el nivel de actividad física, realizada por cada persona y en un periodo de tiempo determinado. Objetivo: Esta revisión tiene como objetivo describir y analizar los principales artículos que utilizan este método para valorar la actividad física. Método: Los artículos seleccionados para ser incluidos en esta revisión se identificaron a través de los siguientes motores de búsqueda especializados: SCOPUS, PUBMED, GOOGLE SCHOLAR; con un total de 56 artículos. La validez de los artículos estuvo dada por el grado de evidencias demostrado, por las recomendaciones que describen y por la aplicabilidad a nuestro contexto. Esta revisión ha considerado los estudios que evalúan la actividad física a través de acelerómetros. Resultados: Los resultados demuestran que este método se puede utilizar en edades comprendidas entre 3 y 90 años. También se puede utilizar en personas con sobrepeso/obesidad, con lesiones articulares, niños con síndrome de Down, autistas, así como personas con problemas psicológicos. Los estudios en mujeres embarazadas muestran datos fiables. Conclusión: La literatura analizada establece la acelerometría como un método eficaz para valorar la actividad física (AU)

Introduction: The accelerometer is shown as one of the most accurate techniques in recording and saving the amount and level of physical activity, by each person in a given period of time. Aims: This review aims to describe and analyze the main items that use this method to assess physical activity. Methods: The review articles were identified through the following specialized Internet browser: SCOPUS, PUBMED, GOOGLE SCHOLAR, those were selected for inclusion with a total of 56 items. The validity of the articles was given by the degree of evidence demonstrated by describing the recommendations and the applicability to our context. This review has considered studies evaluating physical activity through accelerometers. Results: The results show that this method can be used in ages 3 to 90 years. It can also be used in subjects with overweight/obesity, articulation injuries, Down syndrome (just children), autism and people with psychological problems. Studies in pregnant women show satisfactory results. Conclusion: The literature reviewed provides the accelerometer as a reliable and effective method to assess physical activity (AU)

[Identification of the epidural space: usual practice among Spanish anesthesiologists]. / Identificación del espacio epidural en la práctica habitual de los anestesiólogos españoles.

OBJECTIVE: To determine what methods are habitually used by Spanish anesthesiologists to identify the epidural space. MATERIAL AND METHODS: Spanish anesthesiologists were asked to fill in an Internet questionnaire about the methods they had learned during residency training for identifying the epidural space, the method they currently use most often, and the one they consider best for residents to learn during training. RESULTS: Responses were received from 617 anesthesiologists. The techniques the respondents had learned during training were loss of resistance to air (LOR-A), 58.5%; LOR to saline (LOR-S), 31.9%; LOR and air bubble (LOR-B), 6.1%; and the hanging drop method, 2.4%. Those who had learned the LOR-A technique had changed in 14.2% of the cases, as had 28.4% of those who learned the LOR-S procedure. LOR-A is currently used by 59.2% of the respondents, LOR-S is used by 32.4%, and LOR-B by 6%. The respondents recommended that new residents physicians learn the LOR-A (48.5%) and LOR-S (37.8%) techniques. The LOR-B is recommended by 12.6% a much larger percentage of anesthesiologists than it is habitually used by. Of those who use the LORA technique habitually, 26.4% recommend that new residents use a different procedure. CONCLUSIONS: The procedure used most often by Spanish anesthesiologists to identify the epidural space is the LOR-A technique; however, over a quarter of Spanish users of LOR-A do not recommend it.

Identificación del espacio epidural en la práctica habitual de los anestesiólogos españoles / Identification of the epidural space: usual practice among Spanish anesthesiologists

OBJETIVO: Indagar los métodos de identificación del espacio epidural que utilizan los anestesiólogos españoles en su práctica habitual. MATERIAL Y MÉTODOS: Mediante una encuesta por Internet se interrogó a los anestesiólogos españoles acerca de los métodos con los que habían aprendido la técnica durante su período de formación, el método que utilizan con mayor frecuencia en la actualidad y el método que consideraban más conveniente para el aprendizaje de los anestesiólogos en período de formación. RESULTADOS: Respondieron la encuesta 617 anestesiólogos. Las técnicas más frecuentes con las que aprendieron a realizar la anestesia epidural fueron: pérdida de resistencia con aire (LOR-A = 58,5%), LOR con solución salina (LOR-S = 31,9%); LOR con salino + burbuja de aire (LOR-B = 6,1%), método de la gota pendiente = 2,4%. El 14,2% de quienes aprendieron la técnica con LOR-A ha cambiado de método, al igual que el 28,4% de quienes aprendieron con LOR-S. Actualmente utilizan LOR-A = 59,2%, LOR-S = 32,4% y LOR-B = 6%. Para el aprendizaje de los nuevos médicos internos residentes (MIR), los encuestados recomiendan: LOR-A (48,5%) y LOR-S (37,8%). La técnica de LOR-B es aconsejada por una proporción de anestesiólogos mucho mayor de la que lo emplean en su práctica habitual (12,6%). El 26,4% de quienes utilizan la técnica de LOR-A recomiendan a los MIR que utilicen otro método. CONCLUSIONES: El método de identificación del espacio epidural más utilizado en España es el de LOR-A; sin embargo, más de una cuarta parte de sus usuarios no lo aconsejan (AU)

OBJECTIVE: To determine what methods are habitually used by Spanish anesthesiologists to identify the epidural space. MATERIAL AND METHODS: Spanish anesthesiologists were asked to fill in an Internet questionnaire about the methods they had learned during residency training for identifying the epidural space, the method they currently use most often, and the one they consider best for residents to learn during training. RESULTS: Responses were received from 617 anesthesiologists. The techniques the respondents had learned during training were loss of resistance to air (LOR-A), 58.5%; LOR to saline (LOR-S), 31.9%; LOR and air bubble (LOR-B), 6.1%; and the hanging drop method, 2.4%. Those who had learned the LOR-A technique had changed in 14.2% of the cases, as had 28.4% of those who learned the LOR-S procedure. LOR-A is currently used by 59.2% of the respondents, LOR-S is used by 32.4%, and LOR-B by 6%. The respondents recommended that new residents physicians learn the LOR-A (48.5%) and LOR-S (37.8%) techniques. The LOR-B is recommended by 12.6% a much larger percentage of anesthesiologists than it is habitually used by. Of those who use the LORA technique habitually, 26.4% recommend that new residents use a different procedure. CONCLUSIONS: The procedure used most often by Spanish anesthesiologists to identify the epidural space is the LOR-A technique; however, over a quarter of Spanish users of LOR-A do not recommend it (AU)

[Analysis of bibliographic citations received by Spanish anesthesiologists for work indexed by Science Citation Index, 1988-2002]. / Análisis de las citas bibliográficas recibidas por las publicaciones de los anestesiólogos españoles (incluidas en el Science Citation Index) en el período 1988-2002.

OBJECTIVE: To analyze the international impact of articles published by authors in Spanish anesthesiology departments. METHOD: Citable articles indexed by Science Citation Index between 1988 and 2002 and authored by members of Spanish departments of anesthesiology were considered. Citations were counted 2 years and 5 years after publication. Authors and institutions were ranked according to number of citations received. We also determined the journals Spanish anesthesiologists most often chose for publishing their work. RESULTS: Of the 322 citable articles identified, 61.8% were cited in the 2 years following publication (total 587 citations), and 79.5% were cited within 5 years (total 1472 citations). The most frequently cited articles received 17 citations in 2 years and 45 in 5 years after publication. Articles from the Department of Anesthesiology of Hospital Clinic i Provincial of Barcelona received the largest number of citations (333 citations in 5 years). The author with the highest rate of citations received 11.57 per article. The author with the largest number of citations received 86. Anesthesia & Analgesia was the journal publishing the largest number of articles by Spanish anesthesiologists (35 articles). CONCLUSION: This citation analysis shows the international impact of publications by Spanish anesthesiologists.

Análisis de las citas bibliográficas recibidas por las publicaciones de los anestesiólogos españoles (incluidas en el Science Citation Index) en el período 1988-2002 / Analysis of bibliographic citations received by Spanish anesthesiologists for work indexed by Science Citation Index, 1988-2002

OBJETIVO: Analizar la repercusión que han tenido los artículos producidos por los Servicios de Anestesiología españoles en la comunidad científica internacional. MÉTODO: Se consideraron los artículos citables procedentes de Servicios de Anestesiología españoles incluidos en la base de datos del Science Citation Index entre los años 1988 y 2002. Se contabilizaron las citas recibidas en los 2 y 5 años posteriores a su publicación. Se clasificaron los artículos, autores e instituciones que han recibido mayor cantidad de citas, así como las revistas más frecuentemente utilizadas por los anestesiólogos españoles para publicar sus trabajos de investigación. RESULTADOS: De los 322 artículos citables identificados, el 61,8 por ciento recibió alguna cita en los 2 años siguientes al de su publicación (587 citas en total), mientras que el 79,5 por ciento recibió alguna cita en los 5 años siguientes (1.472 citas en total). Los artículos más citados han recibido 17 y 45 citas en los 2 y 5 años siguientes al de su publicación, respectivamente. Los artículos procedentes del Servicio de Anestesiología del Hospital Clinic i Provincial de Barcelona fueron los que más citas recibieron (333 citas en 5 años). El autor con mayor índice de citas ha recibido 11,57 citas por artículo, en 5 años. El anestesiólogo más citado ha recibido 86 citas en 5 años. Anesthesia & Analgesia fue la revista que publicó mayor cantidad de artículos de anestesiólogos españoles (35 artículos). CONCLUSIÓN: Se ha aplicado el análisis de citas para determinar el impacto que han tenido las publicaciones de los anestesiólogos españoles en el contexto científico internacional (AU)

Bone-resorbing cytokines from peripheral blood mononuclear cells after hormone replacement therapy: a longitudinal study.

Conflicting results have been reported in several cross-sectional studies measuring cytokine production from adherent monocytes in pre- and postmenopausal women. Furthermore, the target cells for the action of estrogen are still debated. We therefore assessed in a longitudinal manner the cytokine production from different fractions of peripheral blood mononuclear cells (PBMC) cultured for 48 h. PBMC were obtained from 30 postmenopausal women before and after 6 months of hormone replacement therapy (HRT). Women were randomly allocated to two groups: an adherent PBMC group (n = 20) and a total PBMC group (n = 9). After 6 months of treatment, urinary pyridinoline levels were markedly decreased in both groups (353+/-24 vs 114+/-13 microg/mmol creatinine and 325+/-35 vs 164+/-31 microg/mmol creatinine respectively, p<0.01). Culture supernatants were assayed for interleukin 1beta (IL-1beta), interleukin 6 (IL-6), soluble IL-6 receptor (IL-6rs) and tumor necrosis factor alpha (TNF-alpha). In the adherent PBMC group, HRT induced a nonsignificant trend toward decreased levels of IL-1beta (35+/-10 vs 13+/-5 pg/ml), TNF-alpha (333+/-58 vs 222+/-30 pg/ml) and IL-6 (115+/-70 vs 17+/-10 pg/ml). In contrast, in the total PBMC group, HRT induced a consistent and dramatic decrease in levels of IL-1beta (104+/-22 vs 25+/-8 pg/ml), IL-6 (5950+/-1041 vs 1011+/-361 pg/ml), IL-6rs (148+/-33 vs 35+/-12 pg/ml) (p<0.01) and TNF-alpha (1468+/-315 vs 585+/-207 pg/ml, p = 0.05). We then evaluated whether HRT had the same effect in vitro. Adherent or total PBMC of 8 postmenopausal women were cultured with or without 10(-8) M 17beta-estradiol or tibolone for 48 h. Production of IL-1beta, TNF-alpha, IL-6 and IL-6rs was not affected by the presence of 17beta-estradiol or tibolone in cultures of these cell fractions. In conclusion, our data indicate that non-adherent PBMC could mediate the response to HRT. HRT may exert its action indirectly via noncirculating cells, as suggested by the absence of an in vitro effect.

Phasic production of urinary pyridinium crosslinks in mice: the effect of ovariectomy.

Considerable sample to sample variability in deoxypyridinoline crosslink/creatinine (Dpd/CREAT) ratio values was confirmed when twice-weekly sampling for 77 days was performed in C57 mice. Analysis of samples from individual mice indicated that, in the majority of mice in a given group (54-67%), phasic changes occurred with periodic peaks as much as 4-5 times basal values. Alignment of peaks in the individual time courses of mice revealed a clear cyclic crosslink production (periodicity 12-14 days) for the population, although not all mice gave a peak in every case. Ovariectomy (OVX) (compared to sham-operated mice) increased mean values of crosslink production by either C57 or C57 x 129 F1 mice from about 10 days after operation with highest values between 21 and 35 days, and then a decrease in the difference between sham and OVX, particularly in C57 mice. Analysis of both time courses for individual mice and distribution curves of the data from groups of mice indicated that OVX consistently increased basal crosslink values (6-9 ratio units) with phasic peak values superimposed. The peak alignment analysis reinforced this observation. The influence of the background variability was avoided by pooling data over one cycle time and the use of nonparametric statistics, and the effect of OVX was analyzed in several strains of mice. Crosslink levels and phasic production declined with age but were also influenced by manipulation of the mice, suggesting that experimental conditions should be rigorously controlled when this urinary crosslink measure is used as a parameter in mouse models of OVX-induced osteoporosis.

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria.

Prostaglandins (PG) E1, E2 and F2alpha induce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1 and misoprostol, but blocked the effects of PGF2alpha and the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50-500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5-5 microg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.

Factors influencing the effect of the soluble IL-6 receptor on IL-6 responses in HepG2 hepatocytes.

The soluble IL-6 receptor (sIL-6R) can increase IL-6-induced signalling by forming a complex with IL-6 and membrane-bound gp130 (the receptor beta chain which transduces signals). The conditions affecting this response to sIL-6R were studied using fibrinogen release from HepG2 hepatocytes. Exogenous sIL-6R had no effect alone or in the presence of a submaximal concentration of IL-6, but increased responses to supramaximal IL-6 concentrations in a concentration-related manner. Dexamethasone increased the expression of the membrane IL-6R and endogenous sIL6R release, and increased responses to supramaximal but not submaximal IL-6 concentrations. The amount of endogenous sIL-6R released is relatively small and is unlikely to influence the effects of the exogenous sIL-6R. The observed concentration-related decrease in sIL-6R production in the presence of IL-6 may indicate internalization of ligand/receptor complexes. This would significantly decrease the amount of IL-6R (soluble or membrane) available for signalling and limit continued functional response later in the cultures. These data indicate that the major factor influencing responses to exogenous sIL-6R is an excess of IL-6 which is necessary to form complexes with the sIL-6R, which can then interact with gp130 to increase signalling.

The use of TNF family ligands and receptors and agents which modify their interaction as therapeutic agents.

The earlier known TNF family cytokines have fairly wide physiological actions, mainly in inflammation and immune responses. It is now considered feasible to develop these large proteins themselves as therapeutic agents, but in addition, modular organisation of structures of biological proteins, and the identification of localised ligand-receptor interaction sites, allow rational design of smaller, preferably nonpeptide molecules which interfere with these protein:protein interactions. Neutralising anti-TNF antibodies and soluble TNF receptor preparations were shown to have striking anti-inflammatory activities in clinical studies, particularly in rheumatoid arthritis. As the TNF beta:TNFR1 co-crystal structure was the first in the family to be solved, rational drug design based on the ligand:receptor interaction sites is more advanced. Ligand mutations and a peptide sequence from TNF-alpha have given useful information regarding ligand-receptor interactions. Small peptide sequences from TNFR I which interact with the ligand have shown some activity in blocking the biological actions of TNF. The physiological activities of several recently-discovered ligands are more limited, giving possibilities for selective treatment of several diseases. For example, TRAIL can induce apoptosis in a wide range of tumour cells with little effects on normal tissues, both in vitro and in vivo. The co-crystal structure of TRAIL with one of its signalling receptors TRAILR 2 has been solved, opening the way to rational small molecule drug design. TRANCE (RANKligand) has modulatory effects on the dendritic cell:T cell interaction in immune responses. However, it plays a more major controlling role in the development of osteoclasts and their bone resorbing activity. In this way, it is a very interesting drug development target for the treatment of bone disorders such as osteoporosis. A recombinant secreted inhibitory receptor, osteoprotegerin (OPG), is in Phase 1 clinical trial for the treatment of hyper-resorptive bone diseases. However, OPG also blocks TRAIL and may not be sufficiently specific in long term therapy, but it is hoped that inhibitors of the interaction of TRANCE and its specific signalling receptor, RANK, can be rationally designed.

Dual mechanisms of action of interferon-gamma in potentiating responses to LPS in mice: IL1, TNFalpha and IL6 production in serum and hypothermia.

IFNgamma potentiates the production of serum cytokines and mortality induced by LPS, but these responses do not change in parallel, and the underlying mechanisms are not clear. Pretreatment of mice with 15 microg rrIFNgamma intraperitoneally (IP) resulted in potentiation of LPS-induced serum cytokine production and hypothermia, but these changes depended on the pretreatment time and did not occur in parallel. TNFalpha and IL1beta levels showed peak potentiation after 8-h-IFNgamma pretreatment which may result from a process of sensitization of mechanisms involved in LPS responses. IL6 levels were most markedly potentiated after 3- and 6-h-IFNgamma-pretreatment and hypothermia was markedly potentiated after 0-8 h pretreatments. These effects may result from an additional synergistic action of IFNgamma with other mediators when it is present at significant levels earlier after its injection, given that IFNgamma had little (hypothermia) or no effect (cytokines) alone. The degree of potentiation induced by 18-h-IFNgamma pretreatment was related to the dose of LPS, the maximum response having been increased. Two injections of IFNgamma at 42 and 18 h prior to LPS induced greater increases in TNFalpha and IL1beta production than 18-h pretreatment alone, but not in IL6 production or hypothermia. There may be a maximum level of IL6 production which was surpassed under these conditions. These findings suggest that a balance of sensitizing and synergistic actions of IFNgamma with other mediators such as IL1 and TNFalpha, are the major mechanisms underlying its potentiation of LPS responses in mice.

Selective enhancement of LPS-induced serum TNF-alpha production by carrageenan pretreatment in mice.

1. Bacterial lipopolysaccharide (LPS)-induced increases in serum cytokines (TNF-alpha, IL-1 and IL-6) and hypothermia were studied in mice sensitized by carrageenan pretreatment and compared with mice sensitized with heat-killed P. acnes or IFN-gamma, all given IP at appropriate intervals (24 hr, 7 days and 12-18 hr, respectively) before LPS. 2. In mice with localized peritoneal inflammation induced by carrageenan, peak TNF-alpha levels (1.5-2 h after LPS) were markedly enhanced after both doses of LPS tested (50 and 200 microg/mouse IP). However, IL-1beta levels were not changed and IL-6 levels were decreased only after the higher dose of LPS. Hypothermia showed weak and inconsistent changes in carrageenan-sensitized mice. 3. IL-1beta levels in spleen lysates were higher but paralleled those in the serum, being increased in IFN-gamma-sensitized but not in carrageenan-sensitized mice. The levels of both TNF-alpha and IL-1beta were high in the peritoneum of carrageenan-sensitized mice, suggesting that the increased serum TNF-alpha did not emanate from the peritoneum. 4. In mice sensitized with the other two agents, as expected, the levels of all three cytokines increased, but peak levels were attained at the same times post-LPS (TNF-alpha: 1-1.5 hr; IL-1: 3-4 hr; IL-6: 3-4 hr). In addition, hypothermia was increased with both of these methods of sensitization. 5. The lack of consistent correlation of the levels of cytokines studied, particularly TNF-alpha, with the degree of hypothermia, raises questions as to their causative role in its induction in these models. 6. The mechanisms underlying these models of sensitization are clearly different, and further understanding of these mechanisms would aid in the interpretation of the effects of drugs in the models.

Increases in osteocalcin after ovariectomy are amplified by LPS injection: strain differences in bone remodelling.

1. LPS (Escherichia coli serotype 0111:B4, 300 micrograms/mouse IP) increases serum osteocalcin in normal female C57B16 mice from 2 to 6 hr after its injection, with peak levels at 2-4 hr after LPS. 2. Both basal and LPS-stimulated serum osteocalcin were markedly inhibited by dexamethasone (10 mg/kg IP). 3. When observed 3 hr after LPS injection, serum osteocalcin was increased by ovariectomy (OVX) (with respect to sham-operated mice) and this increase was amplified in LPS-treated mice. This increase in osteocalcin was maximal 14 days after OVX, whereas urinary deoxypyridinoline cross-link levels were increased at all observation times (11-28 days). 4. All these changes were also observed in Balb/c mice but their magnitudes were consistently lower than those in C57B16 mice. 5. We propose that, (1) osteocalcin is a useful marker of bone remodelling in mice and the precision of measurement of changes in its levels after OVX is increased by LPS treatment and (2) C57B16 mice give greater magnitude and more consistent changes in both serum osteocalcin and urinary deoxypyridinoline cross-links after OVX, and may be a better strain for development of an in vivo model of post-menopausal osteoporosis.

Effects of SK&F 86002 on cytokine-stimulated IL6 production in cultured neonatal mouse calvaria and SaOS2 osteoblastic cells: the role of prostaglandins and other mechanisms of action.

The cytokine-suppressant anti-inflammatory drug (CSAID) SK&F 86002 inhibits bone resorption in vitro and this effect cannot be totally explained by its inhibition of IL 1 beta and TNF alpha release. IL6 is another cytokine important in the mechanisms of bone resorption and could be a target for the actions of SK&F 86002. IL6 release and resorption (45Ca release) were induced by IL 1 beta in neonatal mouse calvaria bones in culture. Both indomethacin (5 x 10(-8)-5 x 10(-6) M) and SK&F 86002 (5 x 10(-7)-10(-5) M) markedly inhibited the IL6 release and totally inhibited resorption at all concentrations tested. This may result from inhibition of prostaglandin production by both compounds. In human osteoblastic cells (SaOS2) both basal and TNF alpha-stimulated IL6 production were inhibited in a concentration-related manner by SK&F 86002 but not by indomethacin. The effect of SK&F 86002 was greatest in 6 h cultures where relatively low levels of IL6 are produced and progressively less in 24 and 48 h cultures which produce higher levels of IL6. This is unlikely to be an indirect effect via inhibition of IL 1 beta production as no IL 1 beta (< 0.3 pg/ml) was detected in non-stimulated or stimulated culture supernatants. Therefore, SK&F 86002 may inhibit IL6 production in osteoblastic cells via a more direct mechanism, possibly involving inhibition of the p38 MAP kinase, the mechanism proposed for its inhibition of IL 1 beta and TNF alpha release.

Hypothermia as an indicator of the acute effects of lipopolysaccharides: comparison with serum levels of IL1 beta, IL6 and TNF alpha.

1. Hypothermia was investigated as a parameter indicating the severity of the acute effects of lipopolysaccharides (LPS) in BALB/c mice, and was compared with the induction of serum levels of IL1 beta, TNF alpha and IL6. 2. Hypothermia induced by low doses of LPS (10-50 micrograms/mouse IP LPS E. coli 0111:B4) peaked at 2 hr after LPS and then either plateaued (50 micrograms) or declined. LPS, 100 and 300 mu, induced greater degrees of hypothermia that plateaued or continued to increase with time for 8 hr. Higher doses of LPS induced similar levels of hypothermia until 4 hr but then continued to increase markedly until 8 hr. 3. TNF alpha levels peaked early (1-2 hr) and declined rapidly, IL6 levels peaked at 3 hr and then declined slowly, and IL1 beta levels peaked at 4 hr, declined at lower doses of LPS, plateaued at higher doses and continued to slowly increase at highest doses. 4. The peak levels of the cytokines (IL1 beta up to 4 hr) and hypothermia (4 hr) increased in relation to the dose of LPS and maximum responses were apparently achieved in all cases at 300-1000 micrograms LPS. 5. A similar parallel between hypothermia and induction of cytokines was observed in C57BL6 and OF1 mice, which were good and poor responders to LPS, respectively, and with the more potent Shigella dysenteria LPS in BALB/c mice. 6. In conclusion, hypothermia is a useful parameter for indicating the strength of the acute effects of LPS. Further studies are necessary to determine whether or not the cytokines studied here play a causative role in hypothermia.