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Beatriz de la Puente, Elizabeth Romero-Alejo, José Miguel Vela, Manuel Merlos, Daniel Zamanillo, Enrique Portillo-Salido Department of Pharmacology, Drug Discovery and Preclinical Development, ESTEVE, Barcelona, SpainAbstract: Reflex-based procedures are important measures in preclinical pain studies that evaluate stimulated behaviors. These procedures, however, are insufficient to capture the complexity of the pain experience, which is often associated with the depression of several innate behaviors. While recent studies have made efforts to evidence the suppression of some positively motivated behaviors in certain pain models, they are still far from being routinely used as readouts for analgesic screening. Here, we characterized and compared the effect of the analgesic ibuprofen (Ibu) and the stimulant, caffeine, in assays of acute pain-stimulated and pain-depressed behavior. Intraperitoneal injection of acetic acid (AA) served as a noxious stimulus to stimulate a writhing response or depress saccharin preference and locomotor activity (LMA) in mice. AA injection caused the maximum number of writhes between 5 and 20 minutes after administration, and writhing almost disappeared 1 hour later. AA-treated mice showed signs of depression-like behaviors after writhing resolution, as evidenced by reduced locomotion and saccharin preference for at least 4 and 6 hours, respectively. Depression-like behaviors resolved within 24 hours after AA administration. A dose of Ibu (40 mg/kg) – inactive to reduce AA-induced abdominal writhing – administered before or after AA injection significantly reverted pain-induced saccharin preference deficit. The same dose of Ibu also significantly reverted the AA-depressed LMA, but only when it was administered after AA injection. Caffeine restored locomotion – but not saccharin preference – in AA-treated mice, thus suggesting that the reduction in saccharin preference – but not in locomotion – was specifically sensitive to analgesics. In conclusion, AA-induced acute pain attenuated saccharin preference and LMA beyond the resolution of writhing behavior, and the changes in the expression of hedonic behavior, such as sweet taste preference, can be used as a more sensitive and translational model to evaluate analgesics. Keywords: saccharin preference, locomotor activity, pain, writhing, analgesia, ibuprofen, caffeine

Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine: glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases.

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73a oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73a, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P) H: quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73 alpha oncosuppressors. We show that p. P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein: protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73 alpha, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p. P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

Introduction: RECIPE study was designed to assess the impact of dietary supplementation with fermented milk enriched with plant sterols (FMPS) on the nutritional behavior, lifestyle and the joint therapeutic responsibility of hypercholesterolemic patients. Methods: Observational, prospective, multicenter, international study, was conducted to assess dietary habits and other lifestyle components in hypercholesterolemic patients in whom the general practitioner indicated FMPS supplementation. Nutritional, lifestyle and clinical data were collected at the initial visit and after a 4 months follow-up. The biochemical and anthropometric data were retrieved from medical records obtained at baseline and 4 months. Results: We report the results of Spain. Two hundred and one physicians who provided valid data of 1044 patients participated. The addition of FMPS was associated with improved overall nutritional index, being adequate initially in 28.2% of patients, and increasing up to 75.2% at the end of the study (P< 001). This nutritional change was associated with an improvement in the lipid profile and anthropometric data. Patients more adherent to therapy generally achieved a better result in all parameters compared to non-compliant ones. Conclusions: The addition of FMPS to a diet designed to reduce the LDL enhances the patient's attitude regarding changes in lifestyle, leading to better overall control of dyslipidemia and anthropometric improvement. © 2012 Elsevier España, S.L. and SEA.

El schwanoma es un tumor poco frecuente, que tiene su origen en las envueltas neurales de los nervios periféricos. Para su diagnóstico es fundamental el hallazgo histológico de zonas Antoni A y B. Presentamos un nuevo caso de localización retroperitoneal, que no es su situación más frecuente, en un varón de 60 años, que presentó dolor en flanco izquierdo. Recibió tratamiento quirúrgico, siguiendo una evolución favorable.

The sigma-1 (σ(1) ) receptor is a ligand-regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ(1) receptor ligands used as pharmacological tools are unclear and the demonstration that σ(1) receptor antagonists have efficacy in reversing central sensitization-related pain sensitivity is still missing.The pharmacological properties of a novel σ(1) receptor antagonist (S1RA) were first characterized. S1RA was then used to investigate the effect of pharmacological antagonism of σ(1) receptors on in vivo nociception in sensitizing conditions and on in vitro spinal cord sensitization in mice. Drug levels and autoradiographic, ex vivo binding for σ(1) receptor occupancy were measured to substantiate behavioural data.Formalin-induced nociception (both phases), capsaicin-induced mechanical hypersensitivity and sciatic nerve injury-induced mechanical and thermal hypersensitivity were dose-dependently inhibited by systemic administration of S1RA. Occupancy of σ(1) receptors in the CNS was significantly correlated with the antinociceptive effects. No pharmacodynamic tolerance to the antiallodynic and antihyperalgesic effect developed following repeated administration of S1RA to nerve-injured mice. As a mechanistic correlate, electrophysiological recordings demonstrated that pharmacological antagonism of σ(1) receptors attenuated the wind-up responses in spinal cords sensitized by repetitive nociceptive stimulation.These findings contribute to evidence identifying the σ(1) receptor as a modulator of activity-induced spinal sensitization and pain hypersensitivity, and suggest σ(1) receptor antagonists as potential novel treatments for neuropathic pain.

Several studies have evaluated the prognostic value of the individual expression of certain genes in patients with myelodysplastic syndromes (MDS). However, none of them includes their simultaneous analysis by quantitative polymerase chain reaction (PCR). We evaluated relative expression levels of 14 molecular markers in 193 peripheral blood samples from untreated MDS patients using real-time PCR. Detectable WT1 expression levels, low TET2, and low IER3 gene expression were the only markers showing in univariate analysis a poor prognostic value for all treatment-free (TFS), progression-free (PFS), and overall survival (OS). In multivariate analysis, molecular parameters associated with a shorter TFS were: WT1 detection (p = 0.014), low TET2 (p = 0.002), and low IER3 expression (p = 0.025). WT1 detection (p = 0.006) and low TET2 (p = 0.006) expression were associated with a shorter PFS when multivariate analysis was carried out by including only molecular markers. Molecular values with an independent value in OS were: WT1 detection (p = 0.003), high EVI1 expression (p = 0.001), and undetectatable p15-CDKN2B (p = 0.037). WT1 expressers were associated with adverse clinical-biological features, high IPSS and WPSS scoring, and unfavorable molecular expression profile. In summary, detectable WT1 expression levels, and low TET2 and low IER3 expression in peripheral blood showed a strong association with adverse prognosis in MDS patients at diagnosis. However, WT1 was the only molecular marker displaying an independent prognostic value in both OS and TFS.

Introducción: El oncocitoma renal es un tumor benigno cuya frecuencia de presentación oscila entre el 3-7% de las masas renales sólidas. Muestra especiales características celulares y evolutivas, con posibilidad de metacronicidad, bilateralidad y multifocalidad, además de un difícil diagnóstico diferencial con neoplasias malignas. Material y Método: Revisamos retrospectivamente en 428 tumores renales intervenidos desde Enero de 1986 hasta Abril de 05 la proporción de oncocitomas diagnosticados, analizando sus características anatomopatológicas y comportamiento clínico, comparándolas con las presentadas por los carcinomas de células renales. Observamos su posible bilateralidad, multifocalidad y asociación con otros tumores. Determinamos en cuántos casos los métodos diagnósticos de imagen permitieron sospechar la naturaleza tumoral benigna previamente a la intervención. Seguimiento medio y mediano 13 y 36,86 meses respectivamente (1-193). Resultados: Hemos encontrado 24 oncocitomas (5,67%), en 10 hombres y 12 mujeres con una edad media de 59 años (34-84). 12 de ellos en riñón izquierdo y 12 en derecho, además de una oncocitomatosis renal. Tamaño medio de 4,64 cm (1-12,5). En 17 pacientes el diagnóstico ha sido incidental. En los 5 restantes el síntoma de debut fue hematuria. Supervivencia específica 100%. Se sospechó la naturaleza tumoral benigna o específicamente oncocitomatosa previamente a la cirugía en 22,73% (5/22) y 9,9% (2/22) respectivamente. Conclusiones: En nuestra serie de tumores renales intervenidos el porcentaje de oncocitomas coincide con lo publicado en la literatura. Hemos encontrado dos casos de tumor sincrónico, pero ninguno metacrónico, bilateral ni metastásico. Todos han presentado un comportamiento benigno. Summary: Renal oncocytoma (OR) is a benign tumor. It may represent up to 3-7% of solid kidney masses, and shows specifics cellular and evolutive characteristics. Metacronicity, multifocality and bilateralism has been reported. Materials and methods: Between 1986 and 2005, 478 kidney tumors have been surgically treated at our institution. We report the frequency and characteristics of OR in our patients, compared with renal cell carcinomas (RCC). We try to find out the rate of multifocality, bilateralism and other tumor association, and the number of neoplasms originally diagnosed as OR before surgery. Mean and median follow up: 36.86 and 13 months (1-193). Specific survival rate 100%. Results: We found 24 OR in 10 men and 12 women with a mean age of 59 years (34-84). 12 in the left kidney and 12 in the right one, one patient presenting oncocytomatosis. Tumor mean size was 4.64cm (1-12.5 cm). Tumors were discovered incidentally in 17 cases. Presentation symptoms in the rest of patients were gross hematuria. Conclusions: The rate of OR found in our sample population of renal tumors undergoing surgery matches other series already published. Two synchronic OR, but not metacronous, bilateral or metastatic tumors were found. All cases presented a benign evolution.

Objetivos: Analizar la proporción de reflujos secundarios o complejos en el total de reflujos vesicoureterales tratados endoscópicamente en nuestro hospital. Determinar el éxito conseguido y su relación con el grado de reflujo presentado. Material y método: Presentamos nuestra experiencia entre 1992 y 2003 con tres tipos de sustancias (politetrafluoroetileno, polidimetilsiloxane y dextranomero-copolímero de ácido hialurónico). Tratamos endoscópicamente con la inyección de estas sustancias a 74 pacientes con reflujo vesicoureteral complejo. Todos ellos se siguieron sistemáticamente mediante cistografía a los 3 y 9 meses tras la inyección. Definimos éxito del tratamiento como aquellos reflujos grado 0 ó I. Resultados: El reflujo fue solucionado endoscópicamente en el 86,25% de las ocasiones en el primer intento, 93,75% tras el segundo y 96,25% tras el tercero. El éxito según el grado de reflujo fue 88,9% para los de grado II, 83,3% para los de grado III y 100% para los de IV. Conclusiones: Concluimos que la inyección subureteral de distintas sustancias (Teflon®, Macroplastique® o Deflux®) constituye una alternativa útil de tratamiento en la mayoría de reflujos vesicoureterales complejos. Opinamos que debe considerarse como el primer escalón del tratamiento. Objective: To analize the proportion of complex reflux in the whole amount of patients treated endoscopically of vesicoureteral reflux in our hospital. To determine the endoscopic treatment success in complex reflux, and the influence of reflux grade in it. Material and methods: We present our experience between 1992 and 2003 with three kinds of substances (polytetrafluoroethylene, polydimethylsiloxane and dextranomer-hyaluronic acid copolymer). We treated complex reflux in 74 patients with endoscopic injection. All patients were scheduled to have voiding cystourethrogram 3 and 9 moths after injection. A positive response was defined as grade 0 or I reflux. Results: Reflux was solved using the endoscopic procedure in 86,25% after first injection, 93,75% after second and 96,25% after third. The corresponding results for reflux grade II, III and IV were 88.9%, 83.3% and 100%. Conclusions: We conclude that subureteral injection of different sustances (Teflon®, Macroplastique® or Deflux®) is a useful treatment for most cases of vesicoureteral reflux. We propose it as first step of treatment.

La pielonefritis xantogranulomatosa es una rara inflamación crónica del parénquima renal. Predomina en mujeres de edad media y es extremadamente rara en niños. Presentamos el caso de una niña de 4 años de edad, con infecciones del tracto urinario de repetición y mala respuesta a tratamiento médico. La paciente tenía retraso pondo-estatural y una masa en hemiabdomen izquierdo a la exploración y en las pruebas de imagen. Se realizó heminefrectomía superior izquierda, con posterior confirmación histopatológica de pielonefritis xantogranulomatosa focal. El tratamiento más frecuente de la forma focal es la nefrectomía parcial, aunque a veces responde a tratamiento antibiótico. Como conclusión la pielonefritis xantogranulomatosa debe ser considerada dentro de un diagnóstico diferencial de un niño con un riñón aumentado de tamaño e infecciones recurrentes. Xanthogranulomatous pyelonephritis is an uncommon chronic inflammatory renal disorder. Most cases have been described in middle aged women and it is extremely rare in children. We report a case of a 4 years old girl who suffered from recurrent urinary tract infection and antibiotic therapy resistance. She had low growth-rate and palpable left renal mass on examination and was confirmed by radiological findings. We performed left kidney partial resection and then histological examination showed focal xanthogranulomatous pyelonephritis. The focal form of the disease may respond to antibiotic treatment although usually an enucleation or partial resection must be performed. In conclusion xanthogranulomatous pyelonephritis should be considered in the differential diagnosis of a renal mass and recurrent urinary tract infection in childhood.

La ureteritis quística es una patología muy infrecuente, sin que hasta el momento se haya establecido su patogénesis exacta. Se presenta con sintomatología inespecífica, pero sin embargo los hallazgos radiológicos son bastante orientativos. No hay un tratamiento específico para estos pacientes, siendo el trasplante renal el paso final para los que desarrollen insuficiencia renal crónica terminal (IRCT). Presentamos un caso de ureteritis crónica diagnosticado durante el estudio pretrasplante de una paciente con IRCT. Cystic ureteritis is a very uncommon pathology, whose pathogenesis is not well established. It is usually asociated with chronic infectious factors. It presents unspecific symptoms but characteristic radiologic findings. There is not an especific treatment for this disease. Kidney trasplant is the final pathway for patients with chronic renal failure. We report a case of cystic ureteritis diagnosed during pre-trasplant study.

Presentamos un nuevo caso de neoplasia retroperitoneal: un histiocitoma fibroso maligno, en su variedad mixoide, la menos agresiva de ellas. Este es un tumor individualizado de forma relativamente reciente del conjunto de sarcomas retroperitoneales, de escasa frecuencia, clínica inespecífica y mal pronóstico por su alta tendencia a la recidiva y a la producción de metástasis. We report a new case of retroperitoneal tumor: a malignant fibrous histiocytoma, in its less agressive histologyc type, the mixoyd variety. This is a recently isolated histologyc presentation from other sarcomas, with a low incidence, non-specific clinic findings and poor prognosis due to its tendency to reappear and to produce metastasis.

El schwanoma es un tumor poco frecuente, que tiene su origen en las envueltas neurales de los nervios periféricos. Para su diagnóstico es fundamental el hallazgo histológico de zonas Antoni A y B. Presentamos un nuevo caso de localización retroperitoneal, que no es su situación más frecuente, en un varón de 60 años, que presentó dolor en flanco izquierdo. Recibió tratamiento quirúrgico, siguiendo una evolución favorable. Schwannoma is an uncommon tumor arising from neural sheath of peripheral nerves. Histological findings showing Antoni A and B zones are the main diagnostic features. We report a new case of a retroperitoneal located one, which is not its most usual place to appear, in a 60 years old male, presenting left flank pain. He recieved surgical treatment, having a satisfactory evolution.