Ammonium accumulation and chemokine decrease in culture media of Gcdh−/− 3D reaggregated brain cell cultures

Glutaric Aciduria type I (GA-I) is caused by mutations in the GCDH gene. Its deficiency results in accumulation of the key metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body tissues and fluids. Present knowledge on the neuropathogenesis of GA-I suggests that GA and 3-OHGA have toxic properties on the developing brain.We analyzed morphological and biochemical features of 3D brain cell aggregates issued from Gcdh^?/? mice at two different developmental stages, day-in-vitro (DIV) 8 and 14, corresponding to the neonatal period and early childhood. We also induced a metabolic stress by exposing the aggregates to 10 mM l-lysine (Lys).Significant amounts of GA and 3-OHGA were detected in Gcdh^?/? aggregates and their culture media. Ammonium was significantly increased in culture media of Gcdh^?/? aggregates at the early developmental stage. Concentrations of GA, 3-OHGA and ammonium increased significantly after exposure to Lys. Gcdh^?/? aggregates manifested morphological alterations of all brain cell types at DIV 8 while at DIV 14 they were only visible after exposure to Lys. Several chemokine levels were significantly decreased in culture media of Gcdh^?/? aggregates at DIV 14 and after exposure to Lys at DIV 8.This new in vitro model for brain damage in GA-I mimics well in vivo conditions. As seen previously in WT aggregates exposed to 3-OHGA, we confirmed a significant ammonium production by immature Gcdh^?/? brain cells. We described for the first time a decrease of chemokines in Gcdh^?/? culture media which might contribute to brain cell injury in GA-I.     

Effects of serine protease inhibitor,tame, on IL-1β in LPS-stimulated human monocytes: Relationship between synthesis and release of a 33-kDa precursor and the 17-kDa biologically active species

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1 (mIL-1) but did not result in release of precursor IL-1 (pIL-1). In contrast, the presence of a serine protease inhibitor, N-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1 as well. These effects were initially discerned from observations that the fraction of the total IL-1 produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1 synthesis, predominantly affecting 31-kDa pIL-1, and a dose-dependent inhibition of TNF- production. Parallel examination of lactate dehydrogenase (LDH) release indicated thatpIL-1 release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1 in situ but are probably not mechanistically related to either maturation of the IL-1 molecule or signaling of IL-1 release. IL-1 release appears to be dependent on the amount of total IL-1 synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1.     

Squamous cell carcinomas arising from adnexal ductal cysts.

Malignant tumors arising from adnexal cysts are rare. We report 2 cases of squamous cell carcinomas that developed within cystic structures arising from adnexal ducts. An in situ hybridization technique for human papillomaviruses (HPV)-6/11, -16, -18, and -31, and immunohistochemical staining for p53 were performed. Both tumors showed focal expression of HPV-16 within areas showing squamoid changes and diffuse expression of p53 within the areas of invasive squamous cell carcinoma. Although nuclear staining for HPV has been identified in tumors of adnexal origin, to our knowledge these are the first cases in which a highly oncogenic HPV subtype, HPV-16, has been identified within squamous cell carcinomas arising from adnexal ductal structures. These cases may help explain primary cutaneous squamous cell carcinomas with no epidermal origin.     

A randomized comparison of manual, mechanical and high-impulse chest compression in a porcine model of prolonged ventricular fibrillation

BACKGROUND: Elevated coronary perfusion pressure (CPP) during CPR is associated with return of spontaneous circulation (ROSC). We compared CPP achieved with three methods of chest compression: manual (MAN), mechanical (MECH) and high-impulse mechanical (HI) in a porcine model of prolonged ventricular fibrillation (VF). We hypothesized that HI (very rapid acceleration of the down-stroke) would produce greater CPPs than MAN or MECH, and that HI would also produce a higher rate of ROSC. METHODS: Twenty-eight domestic swine (mean 27.8 kg) were randomly assigned to three methods of chest compression. Animals were instrumented under anesthesia, and VF was induced and untreated for 8 min. After 2 min of CPR, epinephrine (adrenaline) (0. 1 mg/kg), vasopressin (40 U) and propranolol (1.0 mg) were administered. CPR continued for three more minutes, after which up to three rescue shocks were delivered. CPP was determined in an automated fashion by measuring the difference between aortic and right atrial pressures 0.1s prior to the down-stroke of each compression (i.e. end-relaxation). ROSC was defined as a systolic pressure greater than 80 mmHg sustained for at least 1 min. We analyzed CPP and ROSC using repeated measures ANOVA and Fisher's exact test. RESULTS: Over the 5 min of CPR, CPP increased more with HI compression than with MAN compression (p=0.017). ROSC was attained in 4/9 MAN, 6/9 MECH and 10/10 HI (HI versus MAN p=0.01). CONCLUSIONS: Over the course of CPR, HI compression increased CPP more than MAN compression. HI compression produced a significantly higher rate of ROSC than MAN, but not MECH compression.     

清开灵与利巴韦林治疗小儿呼吸道合胞病毒肺炎的比较：单盲、随机、平行对照试验

目的:比较清开灵与利巴韦林对呼吸道合胞病毒肺炎患儿治疗效果的差异。方法:选择2005-02/2006-04在北京儿童医院分中心治疗的小儿呼吸道合胞病毒肺炎97例,患儿法定监护人知情同意。采用单盲、随机、平行对照试验的原则,按区组随机化方法分为2组,清开灵注射液组49例,利巴韦林组48例。①清开灵注射液组:清开灵注射液静脉滴注加口服中成药。②利巴韦林组:利巴韦林注射液静脉滴注加口服复方愈创木酚磺酸钾口服液。两组疗程均为10d,比较两组患儿的疗效。结果:清开灵注射液组脱落3例,利巴韦林组脱落1例,进入结果分析清开灵注射液组46例,利巴韦林组47例。①清开灵注射液组发热患儿体温恢复正常时间比利巴韦林组短[(2.72±1.86)d,(6.29±2.41)d(P<0.01)]。②清开灵注射液组患儿咳嗽、痰壅、气促症状积分改善方面优于利巴韦林组(P<0.05～0.01)。③清开灵注射液组的呼吸道合胞病毒转阴时间明显优于利巴韦林组。④咳嗽、痰壅、病毒转阴时间、气促均进入Logistic模型,其中前两个症状的回归系数绝对值较大。结论:清开灵注射液治疗小儿呼吸道合胞病毒肺炎在退热、止咳平喘、呼吸道合胞病毒转阴时间等方面均具有明显优势,咳嗽、痰壅这两个症状更能反映清开灵注射液的疗效优于利巴韦林。     

人羊膜间充质细胞具有分化成软骨及成骨细胞的潜能

目的:人羊膜间充质细胞具有比骨髓间充质干细胞更强的扩增能力和免疫原性低等优势。建立体外适宜的诱导培养条件,观察人羊膜间充质细胞定向分化为软骨细胞和成骨细胞的能力。方法:实验于2005-09/2006-12在贵州省细胞工程重点实验室完成。①材料来源:经产妇知情同意,无菌采集健康足月分娩新生儿胎盘6份,实验经医院医学伦理委员会批准。②实验方法:采用机械法剥离羊膜组织,二步酶消化法分离收获人羊膜间充质细胞,按2.2×10~8L~(-1)密度接种,传至第1～2代用于诱导分化实验。向软骨细胞诱导分化时,人羊膜间充质细胞按3×10~8L~(-1)密度接种,诱导培养液为含体积分数0.01的胎牛血清、10 mg/L转化生长因β1、100 nmol/L地塞米松、50 mg/L抗坏血酸、1%培养基添加物。向成骨细胞诱导分化时,人羊膜间充质细胞按6×10~7L~(-1)密度接种,诱导培养液为含体积分数0.1的胎牛血清、100 nmol/L地塞米松、50 mg/L抗坏血酸、5 mmol/Lβ-甘油磷酸。③实验评估:原代细胞用流式细胞仪分析表型,免疫细胞化学染色进行波形蛋白表达鉴定。分别于体外诱导第7,14,21,28天采用免疫细胞化学法检测软骨特异性Ⅱ型胶原的表达,细胞化学法检测蛋白聚糖的表达,钙-钴法检测成骨细胞特异性碱性磷酸酶的表达,茜素红S检测钙盐沉积情况。结果:①免疫组化与表型特征:人羊膜间充质细胞高表达间充质干细胞表面标志CD29、CD44和间充质细胞标志波形蛋白。②向软骨细胞诱导分化:诱导14 d后,人羊膜间充质细胞由长梭型逐渐变为多角形,可检测到Ⅱ型胶原蛋白表达及软骨细胞特异性细胞外基质蛋白聚糖。③向成骨细胞诱导分化:诱导21 d后,可观察到人羊膜间充质细胞的胞浆内有碱性磷酸酶表达,且可见钙盐沉积。结论:人羊膜间充质细胞具有分化成软骨细胞和成骨细胞的特性,可作为骨及软骨组织工程种子细胞的新来源。     

PSNAV2.0-TNFα重组质粒的构建及其在体外的表达

目的:在前期微囊化基因工程细胞制备平台的基础上,构建分泌型人肿瘤坏死因子α的真核表达载体PSNAV2.0-TNFα重组质粒,并鉴定其蛋白的体外瞬时表达,为进一步利用该基因进行微囊化细胞移植治疗和改善疾病奠定基础。方法:实验于2006-06/2007-05在解放军总医院老年医学研究所细胞生物学实验室完成。①以含有人肿瘤坏死因子αcDNA序列的质粒为模板,通过PCR扩增获得人肿瘤坏死因子α基因片段;将其定向插入真核表达载体PSNAV2.0中,获得重组质粒PSNAV2.0-TNFα。采用SalⅠ和EcoRⅠ双酶切法、PCR法及插入片段序列测定法鉴定该质粒。②利用阳离子脂质体介导法,将其转染到人胚胎肾细胞HEK-293细胞中,构建可持续分泌人肿瘤坏死因子α的基因工程细胞,采用RT-PCR法和Western blot法检测转染细胞培养上清液中人肿瘤坏死因子蛋白的体外瞬时表达。结果:①通过SalⅠ和EcoRⅠ双酶切、PCR及测序鉴定证明:在HEK-293中插入片段正确。②采用RT-PCR和Western blot法检测表明HEK-293细胞培养上清中有人肿瘤坏死因子α蛋白,Mr17000。结论:成功构建了重组质粒PSNAV2.0-TNFα真核表达载体,转染HEK-293细胞后可有效分泌人肿瘤坏死因子α蛋白,并能分泌到细胞外。     

β-blockers reduce bone resorption marker in early postmenopausal women

Background: There is evidence to suggest that β-blockers used in the management of cardiovascular disease may also modulate bone metabolism and reduce bone fragility.

Aim: The study aimed to determine the association between β-blocker use, serum markers of bone turnover and bone loss in early postmenopausal women.

Subjects and methods: In this observational study, we evaluated β-blocker exposure in association with serum levels of C-telopeptide and bone-specific alkaline phosphatase, and rates of bone loss. β-blocker use, concomitant therapy and lifestyle were documented for 197 women (50–59 years), 175 of whom had changes in whole body bone mineral density monitored over a 2–year period.

Results: Twenty-four β-blocker users were identified at baseline. After controlling for concomitant use of hormone therapy, C-telopeptide levels were 6.7% lower among β-blocker users (p?=?0.02). No association was detected between bone-specific alkaline phosphatase and β-blocker use. Analysis of 15 β-blocker users and 152 non-users identified 2 years post-baseline showed that levels of C-telopeptide but not bone-specific alkaline phosphatase were predictors of adjusted rates of bone loss (p?=?0.008 and p>0.05, respectively). Adjusted rates of bone loss were??0.001?±?0.026?g?cm^?2 over 2 years for the users and??0.004?±?0.025?g?cm^?2 over 2 years for non-users, but this difference was not significant.

Conclusion: β-blockers might suppress bone resorption with relative preservation of bone formation. A study with greater power is required to determine whether β-blocker use is associated with lower rates of bone loss.

Résumé. Arrière plan: Il semble que les β-bloquants employés pour le traitement des maladies cardiaques, puissant aussi moduler le métabolisme osseux et réduire la fragilité de l'os.

But: Cette étude a pour objet de déterminer l'association de l'usage des β-bloquants avec les marqueurs sériques du remplacement osseux et la perte osseuse juste après la ménopause.

Sujets et méthodes:.On a évalué l'exposition aux β-bloquants en association avec les niveaux sériques de C-telopeptide et l'alcaline phosphatase osseuse spécifique ainsi qu'avec les taux de perte osseuse. L'utilisation de β-bloquants, la thérapie concomitante et le mode de vie ont été enregistrés pour 197 femmes âgée de 50 à 59 ans, parmi lesquelles 175 ont subi un contrôle suivi des changements de leur densité minérale osseuse totale sur une période de deux ans.

Résultats: 34 utilisateurs de β-bloquants ont été identifiés comme base de référence. Après contrôle de l'emploi concomitant d'une thérapie hormonale, les niveaux de C-telopeptides sont de 6,7% plus bas chez les utlilisateurs de β-bloquants (p?=?0,02). On ne trouve pas d'association entre l'alcaline-phosphatase et l'utilisation de β-bloquants. L'analyse de 15 utilisateurs de β-bloquants et de 152 non utilisateurs identifiés deux ans après la base de référence, montre que les niveaux de C-telopeptides mais non pas l'alcaline phosphatase, sont prédicateurs des taux ajustés de perte osseuse (respectivement p?=?0,008 et p?=?0,05)). Les taux ajustés de perte osseuse sur 2 ans sont de??0,001?±?0,026 g/cm^?2 pour les utilisateurs et??0,004?±?0,025?g/cm^?2 pour les non utilisateurs, mais cette différence n'est pas significative.

Conclusion: Les β-bloquants pourraient supprimer la résorption osseuse par une préservation relative de la formation de l'os. Une étude de plus vaste envergure est nécessaire afin de déterminer si les β-bloquants sont associés à des taux plus faibles de perte osseuse.

Zusammenfassung. Hintergrund: Es gibt deutliche Hinweise darauf, dass die Einnahme von β-Blockern bei der Behandlung kardiovaskulärer Erkrankungen auch den Knochenstoffwechsel beeinflussen und zu erhöhter Knochenbrüchigkeit führen können.

Ziel: Die Studie zielte auf die Bestimmung der Beziehung zwischen Einnahme von β-Blockern, Serummarkern des Knochenumsatzes und Knochenverlusts bei Frauen kurz nach der Menopause.

Probanden und Methoden: In dieser Beobachtungsstudie beurteilten wir den Zustand unter Einnahme β-Blockern in Verbindung mit Serumspiegeln von C-Telopeptiden und knochenspezifischer alkalischer Phosphatase, und Knochenabbauraten. Die Einnahme von β-Blockern, Begleittherapie und Lebensumstände wurden bei 197 Frauen (50–59 Jahre) dokumentiert, von denen 175 Veränderungen der Ganzkörperknochendichte über einen Beobachtungszeitraum von 2 Jahren aufwiesen.

Ergebnisse: Zu Beginn wurden 24 Patienten identifiziert, die β-Blocker einnahmen. Nach rechnerischem Ausschluss von Effekten, die sich durch begleitende Hormonbehandlung ergeben könnten, zeigten Patienten, die β-Blocker einnahmen, um 6,7% niedrigere C-Telopeptidspiegel (p?=?0,02). Es fand sich keine Beziehung zwischen knochenspezifischer alkalischer Phosphatase und der Einnahme von β-Blockern. Die Analyse von 15 Patienten, die β-Blocker einnahmen, und 152 Personen, die dies über einen Zeitraum von 2 Jahren nach Studienbeginn nicht taten, zeigte, dass C-Telopeptidspiegel, nicht aber die knochenspezifische alkalische Phosphatase ein Kriterium war, um korrigierte Knochenabbauraten vorherzusagen (p?=?0,008, bzw. p?>?0,05). Korrigierte Knochen-abbauraten waren –0,001?±?0,026?g?cm^?2 über 2 Jahre für Patienten, die β-Blocker einnahmen, und –0,004?±?0,025?g?cm^?2 über 2 Jahre für solche, die keine β-Blocker einnahmen, aber diese Differenz war nicht signifikant.

Zusammenfassung: β-Blocker können die Knochenresorption supprimieren, wobei die Knochenformation im Prinzip beibehalten wird. Eine Untersuchung mit größerer power ist notwendig, um zu klären, ob die Einnahme von β-Blockern mit erniedrigten Knochenabbauraten vergesellschaftet ist.

Resumen. Antecedentes: Existen evidencias que sugieren que los ß-bloqueantes usados en el tratamiento de la enfermedad cardiovascular también pueden modular el metabolismo óseo y reducir la fragilidad de los huesos.

Objetivos: El estudio trata de determinar la asociación entre el uso de ß-bloqueantes, los marcadores séricos de remodelado y la pérdida ósea, en mujeres postmenopaúsicas tempranas.

Sujetos y métodos: En este estudio de observación, evaluamos la exposición a ß-bloqueantes en asociación con los niveles séricos del telopéptido C y de la fosfatasa alcalina óseo-específica, y las tasas de pérdida ósea. La utilización de ß-bloqueantes, la terapia concomitante y el estilo de vida se documentaron en 197 mujeres (de 50 a 59 años), 175 de las cuales habían tenido cambios en la densidad mineral ósea de todo el cuerpo monitorizados durante un periodo de 2 años.

Resultados: Inicialmente se identificaron veinticuatro usuarias de ß-bloqueantes. Tras controlar el uso conjunto de la terapia hormonal, los niveles del telopéptido C fueron un 6,7% menores entre las usuarias de ß-bloqueantes (p?=?0,2). No se detectó ninguna asociación entre la fosfatasa alcalina óseo-específica y el uso de ß-bloqueantes. El análisis de 15 usuarias de ß-bloqueantes y de 152 no usuarias, identificadas durante 2 años después del inicio, mostró que los niveles del telopéptido C, pero no los de la fosfatasa alcalina óseo-específica, eran predictores de las tasas ajustadas de pérdida ósea (p?=?0,008 y p?>?0,05, respectivamente). Las tasas ajustadas de pérdida ósea fueron de –0,001?±?0,026?g?cm^?2 tras 2 años en las usuarias y de –0,004?±?0,025?g?cm^?2 tras 2 años en las no usuarias, aunque esta diferencia no fue significativa.

Conclusión: Los β-bloqueantes podrían suprimir la resorción del hueso con una relativa preservación de la formación ósea. Se requiere un estudio con mayor potencia para determinar si el uso de ß-bloqueantes está asociado con tasas más bajas de pérdida ósea.