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OBJECTIVE: The aim of this study was to assess indications for and report outcomes of pancreatic surgery in pediatric patients. BACKGROUND: Indications for pancreatic surgery in children are rare and data on surgical outcomes after pediatric pancreatic surgery are scarce. METHODS: All children who underwent pancreatic surgery at a tertiary hospital specializing in pancreatic surgery between 2003 and 2022 were identified from a prospectively maintained database. Indications, surgical procedures, and perioperative as well as long-term outcomes were analyzed. RESULTS: In total, 73 children with a mean age of 12.8 years (range: 4 mo to 18 y) underwent pancreatic surgery during the observation period. Indications included chronic pancreatitis (n=35), pancreatic tumors (n=27), and pancreatic trauma (n=11). Distal pancreatectomy was the most frequently performed procedure (n=23), followed by pancreatoduodenectomy (n=19), duodenum-preserving pancreatic head resection (n=10), segmental pancreatic resection (n=7), total pancreatectomy (n=3), and others (n=11). Postoperative morbidity occurred in 25 patients (34.2%), including 7 cases (9.6%) with major complications (Clavien-Dindo≥III). There was no postoperative (90-d) mortality. The 5-year overall survival was 90.5%. The 5-year event-free survival of patients with chronic pancreatitis was 85.7%, and 69.0% for patients with pancreatic tumors. CONCLUSION: This is the largest single-center study on pediatric pancreatic surgery in a Western population. Pediatric pancreatic surgery can be performed safely. Centralization in pancreatic centers with high expertise in surgery of adult and pediatric patients is important as it both affords the benefits of pancreatic surgery experience and ensures that surgical management is adapted to the specific needs of children.
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Pancreatectomía , Enfermedades Pancreáticas , Humanos , Niño , Pancreatectomía/métodos , Masculino , Adolescente , Femenino , Preescolar , Lactante , Enfermedades Pancreáticas/cirugía , Resultado del Tratamiento , Complicaciones Posoperatorias/epidemiología , Estudios Retrospectivos , Pancreaticoduodenectomía/métodosRESUMEN
BACKGROUND: Evidence on safety and efficacy of different liver transection techniques in pediatric major hepatectomy is completely lacking, as no study has been conducted so far. The use of stapler hepatectomy has never before been reported in children. METHODS: Three liver transection techniques were compared: (1) ultrasonic dissector (CUSA), (2) tissue sealing device (LigaSure™), and (3) stapler hepatectomy. All pediatric hepatectomies performed at a referral center in a 12-year study period were analyzed, patients were pair-matched in a 1:1:1-fashion. Intraoperative weight-adjusted blood loss, operation time, use of inflow occlusion, liver injury (peak-transaminase levels), postoperative complications (CCI), and long-term outcome were compared. RESULTS: Of 57 pediatric liver resections, 15 patients were matched as triples based on age, weight, tumor stage, and extent of resection. Intraoperative blood loss was not significantly different between the groups (p = 0.765). Stapler hepatectomy was associated with significantly shorter operation time (p = 0.028). Neither postoperative death nor bile leakage occurred, and no reoperation due to hemorrhage was needed in any patient. CONCLUSION: This is the first comparison of transection techniques in pediatric liver resection and the first report on stapler hepatectomy in children. All three techniques can be safely applied and may harbor individual advantages in pediatric hepatectomy each.
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Hepatectomía , Neoplasias Hepáticas , Humanos , Niño , Hepatectomía/métodos , Análisis por Apareamiento , Resultado del Tratamiento , Hígado/cirugía , Pérdida de Sangre Quirúrgica/prevención & control , Neoplasias Hepáticas/cirugíaRESUMEN
BACKGROUND: The impact of hepatic resection for liver metastases (LM) on the survival of pediatric patients with Wilms' tumor (WT) is unclear. So far, there is a lack of studies investigating the best suited treatment for patients with WTLM, and the role of liver resection has rarely been investigated. Thus, the development of evidence-based guidelines concerning indications of liver resection for WTLM remains difficult. AIM: To investigate the role of surgery in the therapy of WTLM. All available data on liver resections and subgroup outcomes of patients with WTLM are analyzed. Main research question is whether liver resection improves survival rates of patients with WTLM compared to non-surgical treatment. METHODS: A systematic literature search of MEDLINE, Web of Science, and Central provided the basis for this PRISMA-compliant systematic review. For the main analysis (I), all studies reporting on surgical treatment of pediatric WTLM were included. To provide a representative overview of the general outcome of WTLM patients, in analysis II all studies with cohorts of at least five WTLM patients, regardless of the kind of treatment, were reviewed and analyzed. A Multiple meta-regression model was applied to investigate the impact liver resection on overall survival. RESULTS: 14 studies with reports of liver resection for WTLM were found (Analysis I). They included a total of 212 patients with WTLM, of which 93 underwent a liver resection. Most studies had a high risk of bias, and the quality was heterogenous. For the analysis II, eight studies with subgroups of at least five WTLM patients were found. The weighted mean overall survival (OS) of WTLM patients across the studies was 55% (SD 29). A higher rate of liver resection was a significant predictor of better OS in a multiple meta-regression model with 4 covariates (I2 29.43, coefficient 0.819, p = 0.038). CONCLUSIONS: This is the first systematic review on WTLM. Given a lack of suited studies that specifically investigated WTLM, ecological bias was high in our analyses. Generating evidence is complicated in rare pediatric conditions and this study must be viewed in this context. Meta-regression analyses suggest that liver resection may improve survival of patients with WTLM compared to non-surgical treatment. Especially patients with persisting disease after neoadjuvant chemotherapy but also patients with metachronous LM seem to benefit from resection. Complete resection of LM is vital to achieve higher OS. Studies that prospectively investigate the impact of surgery on survival compared to non-surgical treatment for WTLM are highly needed to further close the current evidence gap. STUDY REGISTRATION: PROSPERO 2021 CRD42021249763 https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=249763 .
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Hepatectomía/mortalidad , Neoplasias Renales/cirugía , Neoplasias Hepáticas/cirugía , Tumor de Wilms/cirugía , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/secundario , Masculino , Análisis Multivariante , Análisis de Regresión , Tasa de Supervivencia , Resultado del Tratamiento , Tumor de Wilms/mortalidad , Tumor de Wilms/patologíaRESUMEN
AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.
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Mutación Puntual , Receptores AMPA/química , Receptores AMPA/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Sitio Alostérico , Animales , Sitios de Unión , Células HEK293 , Humanos , Cinética , Ligandos , Modelos Teóricos , Técnicas de Placa-Clamp , Unión Proteica , Estructura Terciaria de Proteína , RatasRESUMEN
BACKGROUND: In preterm infants, spinal anesthesia (SpA) is recognized as an alternative to general anesthesia for inguinal hernia repair (IHR); however, some patients require supplemental anesthesia during surgery. The purpose of this study was to investigate the frequency and impact of supplemental anesthesia on perioperative care and adverse respiratory and hemodynamic events. METHODS: A retrospective study of preterm infants undergoing IHR at Heidelberg University Hospital within the first year of life between 2009 and 2018 was carried out. RESULTS: In total, 230 patients (255 surgeries) were investigated. Among 189 procedures completed using SpA 24 patients received supplemental anesthesia. Reasons for supplemental anesthesia included loss of anesthetic effect, returning motor response, and respiratory complications. Compared to SpA alone, no differences were found concerning hemodynamic parameters; however, patients requiring supplemental anesthesia displayed higher rates of postoperative oxygen supplementation and unexpected admission to the intensive care unit. The rate of perioperative apnea was 2.7%. Apneic events exclusively occurred after supplemental anesthesia. Bilateral IHR and duration of surgery were associated with the need for supplemental anesthesia. CONCLUSION: Whereas SpA might be favorable when compared to general anesthesia for IHR, the data indicate that particular caution is required in patients receiving supplemental anesthesia due to the possible risk for adverse respiratory events.
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Anestesia Raquidea , Hernia Inguinal , Lactante , Humanos , Recién Nacido , Recien Nacido Prematuro , Anestesia Raquidea/efectos adversos , Estudios Retrospectivos , Hernia Inguinal/cirugía , Anestesia General/efectos adversos , Apnea/etiologíaRESUMEN
BACKGROUND: The aim of this study was to assess the outcome of total ankle replacement (TAR) regarding revision rates by comparing clinical studies of the last decade to data displayed in arthroplasty registers. The secondary aim was to evaluate whether dependent clinical studies show a superior outcome to independent publications. Additionally, revision rates of mobile bearing implants (MB-TARs) were compared to those of fixed bearing implants (FB-TARs). METHODS: Clinical studies on TARs between 2010 and 2020 were systematically reviewed, with the endpoint being a revision for any reason. The parameter "revision rate per 100 observed component years (CYs)" was calculated for each publication. The pooled revision rate for clinical studies was compared to the data reported in arthroplasty registers. In a second step, revision rates were subdivided and analyzed for independent and dependent publications and for FB-TARs and MB-TARs. RESULTS: A total of 43 publications met the inclusion criteria comprising 5806 TARs. A revision rate of 1.8 per 100 observed CYs was calculated, corresponding to a 7-year revision rate of 12.6%. The 3 arthroplasty registers included showed revision rates ranging from 8.2% to 12.3% after 7 years. No significant difference between dependent and independent publications nor between FB-TARs and MB-TARs was detected. CONCLUSION: Revision rates of clinical studies and arthroplasty registers are comparable. Surgeons can compare their own revision rates with those from this study. Dependent studies do not seem to be biased, and no superiority for one bearing type can be described. LEVEL OF EVIDENCE: Level III, systematic review of level III studies.
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Artroplastia de Reemplazo de Tobillo , Humanos , Falla de Prótesis , Reoperación , Resultado del TratamientoRESUMEN
Background: Techniques to increase the future liver remnant (FLR) have fundamentally changed the indications and criteria of resectability in adult liver surgery. In pediatric patients however, these procedures have rarely been applied and the potential benefit or harm as well as suited indications are unclear. Methods: A systematic literature search of MEDLINE, Web of Science, and CENTRAL was conducted. Based on a PRISMA-compliant, predefined methodology, all studies reporting pediatric patients (< 18y) undergoing liver resection with either associating liver partition and portal vein ligation for stages hepatectomy (ALPPS) or preoperative portal vein embolization or ligation (PVE/PVL) were included. Baseline data, periinterventional morbidity, increase of FLR and outcomes were analyzed. Results: 15 studies reporting on 21 pediatric patients with a mean age of 4 years and 7 months (range 1.8 months - 17 years) were included. 12 ALPPS procedures, 8 PVE and 1 PVL were performed. The applied criteria for performing ALPPS or PVE were heterogenous and thresholds for minimally acceptable FLR varied. Mean FLR [% of total liver volume] before the intervention was 23.6% (range 15.0 - 39.3%) in the ALPPS group and 31.4% (range 21.5 - 56.0%) in the PVE group. Mean increase of FLR before stage 2 resection was 69.4% (range 19.0 - 103.8%) for ALPPS and 62.8% (range 25.0 - 108.0%) after PVE. No postoperative death occurred, one early intrahepatic recurrence after an ALPPS procedure was reported. Overall postoperative morbidity was 23.8%. Conclusion: Validated criteria for minimal FLR in pediatric liver resection are lacking and so are clear indications for ALPPS or PVE. In special cases, ALPPS and PVE can be valuable techniques to achieve complete resection of pediatric liver tumors. However, more data are needed, and future studies should focus on a definition and validation of posthepatectomy liver failure as well as the minimally needed FLR in pediatric patients undergoing extended hepatectomy. Systematic Review Registration: [www.clinicaltrials.gov], identifier [PROSPERO 2021 CRD42021274848].
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Background: Rhabdoid liver tumors in children are rare and have a devastating prognosis. Reliable diagnosis and targeted treatment approaches are urgently needed. Immunohistochemical and genetic studies suggest that tumors formerly classified as small cell undifferentiated hepatoblastoma (SCUD) belong to the entity of malignant rhabdoid tumors of the liver (MRTL), in contrast to hepatoblastomas with focal small cell histology (F-SCHB). This may have relevant implications on therapeutic approaches. However, studies with larger cohorts investigating the clinical relevance of the histological and genetic similarities for patients are lacking. Purpose: To analyze possible similarities and differences in patient characteristics, tumor biology, response to treatment, and clinical course of patients with MRTL, SCUD and F-SCHB. Applied therapeutic regimens and prognostic factors are investigated. Methods: A systematic literature search of MEDLINE, Web of Science, and CENTRAL was performed for this PRISMA-compliant systematic review. All studies of patients with MRTL, SCUD and F-SCHB that provided individual patient data were included. Demographic, histological, and clinical characteristics of the three subgroups were compared. Overall survival (OS) was estimated with the Kaplan-Meier method and prognostic factors investigated in a multivariable Cox regression model. Protocol registered: PROSPERO 2021 CRD42021258760. Results: Fifty-six studies with a total of 118 patients were included. The two subgroups MRTL and SCUD did not differ significantly in baseline patient characteristics. However, heterogenous diagnostic and therapeutic algorithms were applied. Large histological and clinical overlap between SCUD and MRTL could be shown. Two-year OS was 22% for MRTL and 13% for SCUD, while it was significantly better in F-SCHD (86%). Chemotherapeutic regimens for hepatoblastoma proved to be ineffective for both SCUD and MRTL, but successful in F-SCHB. Soft tissue sarcoma chemotherapy was associated with significantly better survival for MRTL and SCUD, but was rarely applied in SCUD. Patients who did not undergo surgical tumor resection had a significantly higher risk of death. Conclusions: While F-SCHB is a subtype of HB, SCUD should be classified and treated as a type of MRTL. Surgical tumor resection in combination with intensive, multi-agent chemotherapy is the only chance for cure of these tumors. Targeted therapies are highly needed to improve prognosis. Currently, aggressive regimens including soft tissue sarcoma chemotherapy, extensive resection, radiotherapy or even liver transplantation are the only option for affected children.
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Because of anatomical and physiological similarities to humans, porcine small bowel transplantation (SBTx) can be used as an appropriate experimental model in the field of surgical research. Various approaches to SBTx have been described in literature. The aim of this work is to present a review of different surgical techniques of SBTx which have been developed using the porcine model. Our analysis of Medline-cited studies dealing with different techniques of SBTx in porcine models was particularly focused on surgical aspects. With regard to graft procurement and enterectomy, the reported techniques vary widely. Arterial reconstruction is mainly conducted by performing the anastomosis between the superior mesenteric artery (SMA) of the donor and SMA or infrarenal aorta of the recipient. Alternatively, an aortic segment of the donor can be anastomosed to the infrarenal aorta of the recipient. Venous anastomosis is frequently performed between the superior mesenteric vein (SMV) of the donor and SMV or the inferior vena cava (IVC) of the recipient. Some studies also report venous anastomosis between the portal vein of the donor and the recipient. Bowel continuity is then restored by end-to-end or end-to-side anastomosis. Remarkable results were generated thanks to improved techniques which include proximal side-to-side ileo-ileal anastomosis with double-barrel ileostomy, or so-called "Paul-Mikulicz-Ileostomy". Most frequently used were jejunostomy and the "Bishop-Koop-Ileostomy"--where the proximal part of the bowel is anastomosed end-to-side to the distal part, which is then exteriorized as an ostomy. Based on the techniques presented in this review, one must select the most suitable surgical technique of porcine SBTx among those various models.
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Intestino Delgado/trasplante , Modelos Animales , Trasplante de Órganos/métodos , Porcinos , Anastomosis Quirúrgica/métodos , AnimalesRESUMEN
Purpose: To establish comparable reporting of surgical results in pediatric liver surgery, the recently introduced composite outcome measures Textbook Outcome (TO) and Comprehensive Complication Index (CCI) are applied and validated in a pediatric surgery context for the first time. In a representative cohort of pediatric patients undergoing liver resection, predictive factors for TO and CCI are investigated, and outcomes are compared to available literature on surgical outcomes of pediatric liver resection. Methods: All liver resections for patients under 21 years of age performed at the Department of General, Visceral, Transplantation and Pediatric Surgery of the University of Heidelberg between 2009 and 2020 were included in the analysis. Criteria for TO were defined prior to the analysis. Univariate and Multivariate regression was applied to identify factors associated with TO and CCI. Results: Fifty-three pediatric patients underwent liver resections during the observation period. No 30- or 90-day mortality occurred. Twenty-three patients (43.4%) had a TO. CCI and TO showed highly significant correlation (b = -30.33, 95% CI [-37.44; -23.22], p < 0.001). Multivariate analyses revealed significant association between intraoperative blood loss (adjusted for circulating blood volume) and CCI (b = 0.70, 95%CI [0.22; 1.32], p = 0.008) and failure to achieve TO (OR = 0.85, 95%CI [0.69; 0.97], p = 0.048). Conclusion: TO and CCI are suited outcome measures in pediatric surgical studies and offer objective comparability of results. Their application in clinical studies will be a major step forward to establish evidence-based therapies in pediatric surgery. Systematic utilization of TO and CCI can aid in generating comparable studies on surgical techniques and outcomes in pediatric liver resection.
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BACKGROUND: The biliary tree is a rare location of pediatric rhabdomyosarcoma. Due to the low incidence, there is a lack of evidence concerning therapeutic guidelines for this tumor location. In particular, the impact of surgery is discussed controversially. PURPOSE: Objective is to generate evidence-based treatment guidelines for pediatric biliary rhabdomyosarcoma (BRMS). All available published data on therapeutic regimens and important prognostic factors are investigated with a focus on the role of surgery. METHODS: A systematic literature search of MEDLINE, Web of Science, and CENTRAL was performed. Patient data were entered individually. Data was pooled and qualitative and quantitative analyses of demographic data, therapy, postoperative/interventional outcomes, relapse, and survival were conducted. In an individual patient data analysis, cox regression was applied to identify key factors predicting the outcome of patients with BRMS. RESULTS: 65 studies met the inclusion criteria, providing data on 176 patients with BRMS. Individual patient data analysis showed a 5-year overall survival and progression-free survival of 51% and 50% for the total study population. For patients treated after 2000, 5-year OS and PFS was 65% and 59%, respectively. Absence of surgical tumor resection was an independent risk factor for death (Hazard ratio 8.9, 95%-CI 1.8-43.6, p = 0.007) and significantly associated with recurrent disease and disease-related death. CONCLUSION: This analysis provides comprehensive information on the largest number of patients hitherto reported in the literature. BRMS is still associated with high morbidity and mortality. Surgical tumor resection is essential for appropriate oncological treatment of BRMS. International cooperation studies are needed to enhance evidence and improve the outcome of this orphan disease. PROTOCOL REGISTRATION: PROSPERO (CRD42021228911) https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021228911.
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Ampakines are cognitive enhancers that potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents and synaptic responses by slowing receptor deactivation. Their efficacy varies greatly between classes of neurons and brain regions, but the factor responsible for this effect remains unclear. Ampakines also increase agonist affinity in binding tests in ways that are related to their physiological action. We therefore examined 1) whether ampakine effects on agonist binding vary across brain regions and 2) whether they differ across receptor subunits expressed alone and together with transmembrane AMPA receptor regulatory proteins (TARPs), which associate with AMPA receptors in the brain. We found that the maximal increase in agonist binding (E(max)) caused by the prototypical ampakine 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546) differs significantly between brain regions, with effects in hippocampus and cerebellum being nearly three times larger than that in thalamus, brainstem, and striatum, and cortex being intermediate. These differences can be explained at least in part by regional variations in receptor subunit and TARP expression because combinations prevalent in hippocampus (GluA2 with TARPs gamma3 and gamma8) exhibited E(max) values nearly twice those of combinations abundant in thalamus (GluA4 with gamma2 or gamma4). TARPs seem to be critical because GluA2 and GluA4 alone had comparable E(max) and also because hippocampal and thalamic receptors had similar E(max) after solubilization with Triton X-100, which probably removes associated proteins. Taken together, our data suggest that variations in physiological drug efficacy, such as the 3-fold difference previously seen in recordings from hippocampus versus thalamus, may be explained by region-specific expression of GluA1-4 as well as TARPs.
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Encéfalo/efectos de los fármacos , Dioxoles/farmacología , Nootrópicos/farmacología , Piperidinas/farmacología , Subunidades de Proteína/agonistas , Receptores AMPA/agonistas , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Ligandos , Plásmidos , Unión Proteica , Subunidades de Proteína/biosíntesis , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores AMPA/biosíntesis , Tálamo/efectos de los fármacos , Tálamo/metabolismo , TransfecciónRESUMEN
Transmembrane AMPA receptor-associated regulatory proteins (TARPs) modulate the kinetics of AMPA receptors and increase their surface expression. This study compared the effects of the TARPs gamma2, gamma3, gamma4 and gamma8 on the AMPA receptor subunits GluR1-3. We found that the deactivation and desensitization time constants of GluR3 receptors were greatly increased by gamma4 and gamma8 (approximately 3 fold) whereas gamma2 and gamma3 caused minimal changes. This stands in contrast to the effects on GluR1 and GluR2 receptors which were similar across all four TARPs. Other response parameters like the current density, the EC(50) for glutamate-induced peak currents, and kainate-induced currents were in general more effectively modulated by gamma2 and gamma3 in all subunits, including GluR3. All TARPs were effective in increasing the unitary conductance. The differential TARP effects on deactivation time constants of the GluR3 subunit are likely to have a significant impact on synaptic responses across different neurons in the brain.
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Ácido Glutámico/fisiología , Proteínas de la Membrana/fisiología , Subunidades de Proteína/fisiología , Receptores AMPA/metabolismo , Canales de Calcio , Línea Celular , Regulación de la Expresión Génica/fisiología , Ácido Glutámico/metabolismo , Humanos , Ácido Kaínico/metabolismo , Cinética , Proteínas de la Membrana/agonistas , Subunidades de Proteína/agonistas , Receptores AMPA/agonistas , Receptores AMPA/biosíntesis , Receptores AMPA/genética , Receptores AMPA/fisiologíaRESUMEN
Radioligand binding studies have shown that AMPA receptors exist in two variants that differ about twenty-fold in their binding affinities, with brain receptors being mainly of the low-affinity type and recombinantly expressed receptors having almost exclusively high affinity. However, the physiological correlate of high- and low-affinity binding is not yet known. In this study we examined if physiological experiments similarly reveal evidence for two distinct receptor variants. We therefore measured equilibrium desensitization by glutamate and determined IC(50) values for neuronal receptors and for the homomeric receptors GluR1-4 expressed in HEK293 cells. Contrary to the prediction that these IC(50) values exhibit large differences commensurate with those of high- and low-affinity binding, values for homomeric receptors (1-18 microM) were on an average not different from those of neuronal receptors (3-10 microM). Moreover, simulations with kinetic receptor models suggest that the IC(50) values for neuronal and recombinant receptors correspond to the binding affinity of the low-affinity receptor variant. These findings indicate that the high-affinity binding measured in heterologous expression systems represents an immature receptor variant that does not contribute to the currents recorded from these cells, and that the functional low-affinity receptors are present in such small number that they are effectively masked in binding assays by the high-affinity receptors. Thus, in order to compare experimentally determined saturation binding profiles with those predicted by kinetic receptor models and with dose-response curves from physiological studies, it will be imperative to develop methods for isolating first the low-affinity receptors.
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Unión Competitiva/fisiología , Encéfalo/metabolismo , Membrana Celular/metabolismo , Neuronas/metabolismo , Receptores AMPA/agonistas , Receptores AMPA/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Agonistas de Aminoácidos Excitadores/farmacología , Humanos , Cinética , Modelos Moleculares , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores AMPA/efectos de los fármacos , Receptores AMPA/genética , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fracciones SubcelularesRESUMEN
Organotypic cerebellar cultures were maintained on multi-electrode dishes (MED) with an 8x8 array of electrodes and examined for physiological activity. The cultures remained viable for up to seven months and exhibited spontaneous discharges most likely originating from Purkinje cells. Spike frequencies varied but were mostly around 10-30 Hz and were often stable over weeks with average drifts of <20% per week. Spontaneous firing was significantly reduced by blockers of sodium channels (riluzole) and several potassium channels (iberiotoxin, TEA, 4-amino-pyridine), but blockers of calcium channels, GIRK channels, and SK-type potassium channels were ineffective. Inhibitors of excitatory and inhibitory synaptic transmission made spike discharges more regular. Particularly robust changes in spike frequency were produced by agents that increase cGMP. Bromo-cGMP, the NO donor SNAP, the guanylate cyclase activator YC-1, and the phosphodiesterase inhibitor zaprinast greatly reduced spike frequency. Activation of the metabotropic receptor mGluR1 and inhibition of I(h) channels caused a majority of cells to switch from tonic firing to a cyclic activity mode in which intense firing alternated with silence. Agonists for cholinergic, serotonergic, histamine, opiate, and CRF receptors had no effect, but those for adrenergic and adenosine A1 receptors reduced firing. Moreover, brief application of bromocriptine caused a delayed decrease in firing that reached a minimum after 24 to 48 h and recovered after 1-2 weeks. Taken together, our results demonstrate that long-term cultures maintained on multi-electrode arrays retain many essential features of cerebellar physiology and that they provide a test system that is well suited for broad screening of pharmacological agents as well as for studying long-term effects of drugs, tissue factors, and pathogens.
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Potenciales de Acción/fisiología , Cerebelo/citología , Células de Purkinje/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Animales Recién Nacidos , GMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Electrodos , Moduladores del Transporte de Membrana/farmacología , Técnicas de Cultivo de Órganos , Parvalbúminas/metabolismo , Células de Purkinje/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Factores de TiempoRESUMEN
AIM: We describe the clinical features of a 6-year-old boy with sexual precocity caused by a somatic activating mutation of the luteinizing hormone (LH) receptor gene preceding gonadotropin-releasing hormone (GnRH)-dependent sexual precocity. STUDY DESIGN: Genomic DNA was extracted from the right testis and from the peripheral leukocytes followed by DNA amplification and sequencing of the LH receptor gene. We described the clinical characteristics including anthropometric parameters, bone age, and endocrine evaluation when the boy presented with sexual precocity. These data were compared with the clinical and hormonal evaluation after orchiectomy preceding GnRH-dependent sexual precocity and after subsequent treatment with GnRH agonist. RESULTS: No mutation was found in the sequence of the LH receptor gene extracted from peripheral leukocytes. Interestingly, sequencing of the tumor LH receptor gene revealed a heterozygous mutation in exon 11 encoding a replacement of Asp(578)His. Despite normalization of plasma testosterone, true precocious puberty was triggered within a year. CONCLUSIONS: Inmales with GnRH-independent sexual precocity the presence of small testicular Leydig cell tumorous lesions harboring a somatic mutation of the LH receptor gene should be considered. A close follow-up of affected patients should be instigated in order to monitor recurrence or subsequent true precocity.
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Tumor de Células de Leydig/genética , Mutación Missense , Proteínas de Neoplasias/genética , Pubertad Precoz/genética , Receptores de HL/genética , Neoplasias Testiculares/genética , Sustitución de Aminoácidos , Niño , Exones/genética , Hormona Liberadora de Gonadotropina/sangre , Heterocigoto , Humanos , Tumor de Células de Leydig/sangre , Tumor de Células de Leydig/fisiopatología , Masculino , Proteínas de Neoplasias/metabolismo , Pubertad Precoz/sangre , Pubertad Precoz/fisiopatología , Receptores de HL/metabolismo , Neoplasias Testiculares/sangre , Neoplasias Testiculares/fisiopatologíaRESUMEN
Glutamate receptors are competitively inhibited by guanine nucleotides. Insight into the physiological function of this inhibition would be greatly advanced if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. In the present study we mutated the equivalent lysine in the rat AMPA receptor subunit GluR1 flip to alanine (K445A) and assessed changes in nucleotide affinity from the displacement of [(3)H]fluorowillardiine. As in the cKBP, the affinity for nucleotides was greatly reduced while the binding affinity for agonists remained unchanged. The reduction in affinity was largest for GTP (factor of 5.8) and GDP (4.4) and minor for GMP and guanosine. This suggests that K445 is involved in stabilizing the second phosphate of the nucleotide. Given that bulkier analogs like GDP-fucose are also accommodated at this site, it seems likely that nucleotides bind in such a way that their phosphates project out of the cleft. In excised-patch recordings using short pulses of glutamate, the K445A mutation increased the EC(50) for the peak response 1.8-fold and accelerated desensitization and deactivation. This indicates that the effects of this mutation are not as specific as previously suggested. Efforts to selectively eliminate inhibition by nucleotides may therefore depend on mapping out further the docking site. In a first attempt using point mutations we ruled out several amino acids around the cleft as being involved in nucleotide binding. Also, the AMPA receptor modulator PPNDS which competitively inhibits nucleotide binding to purinergic receptors did not affect nucleotide inhibition, suggesting that there are major differences in the topography between purinergic and glutamate receptors. Thus new approaches, including crystallography, may be called for to identify residues uniquely involved in nucleotide binding.
Asunto(s)
Nucleótidos de Guanina/metabolismo , Receptores AMPA/metabolismo , Alanina/análogos & derivados , Alanina/fisiología , Sustitución de Aminoácidos , Animales , Línea Celular , ADN Complementario/genética , Nucleótidos de Guanina/genética , Guanosina Difosfato/metabolismo , Humanos , Lisina/fisiología , Mutagénesis , Plásmidos/genética , Antagonistas Purinérgicos , Pirimidinas , Ratas , Receptores AMPA/agonistas , Receptores AMPA/genética , Receptores Purinérgicos/genética , Receptores Purinérgicos/fisiología , TransfecciónAsunto(s)
Ejercicio Físico , Actividad Motora , Aptitud Física , Programas Informáticos , Juegos de Video , Adolescente , Niño , Alemania , Encuestas Epidemiológicas , Humanos , Estados UnidosRESUMEN
Compounds which modulate AMPA receptor function through allosteric mechanisms were examined for their effect on the binding of the agonist [3H]fluorowillardiine (FW). Benzamide-type positive modulators (ampakinestrade mark) under all experimental circumstances increased [3H]FW binding to native receptors in rat brain membranes. Benzothiadiazide drugs had more variable effects ranging from large reductions with cyclothiazide and JM-13 to increases produced by more recent compounds like PEPA, D1 and LY392098. These effects on binding were moderately influenced by the assay conditions, including temperature and the presence or absence of thiocyanate. Significant changes in agonist binding were also produced by other modulatory agents such as noncompetitive blockers (GYKI 53655, SYM 2206), polycationic compounds (spermine, Naspm, philanthotoxin) and polyanionic compounds (Evans Blue, suramin, PPNDS). EC50 values usually were similar to those from physiological studies, which validates using binding tests to assess drug potencies. Moreover, direction and magnitude of the binding change (Emax) provide information about which kinetic aspects are affected by a drug. For example, the magnitude of the binding increase produced by positive modulators was strongly correlated with their ability to slow response deactivation in excised patch recordings. Binding also provides a reliable method to examine whether interactions between agents are competitive. Thus, thiocyanate did not significantly influence the EC50 of cyclothiazide, suggesting distinct sites of action. Taken together, [3H]FW binding can yield important information about drug-receptor and drug-drug interactions for a wide range of modulatory agents. One potential limitation of [3H]FW is a large preference for subunits GluR1 and GluR2 (KD 4-10 nM) over GluR3 and GluR4 (160-600 nM) which implies that tests with brain membranes preferentially reveal drug effects produced at the former two subunits. Lastly, data are shown which highlight the importance of optimizing experimental conditions in filtration assays, for instance by always including thiocyanate in wash buffers.
Asunto(s)
Alanina/análogos & derivados , Sitio Alostérico/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Pirimidinas/farmacología , Receptores AMPA/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Alanina/farmacología , Sitio Alostérico/fisiología , Animales , Benzodiazepinas/farmacología , Unión Competitiva , Encéfalo/citología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Agonistas de Aminoácidos Excitadores/química , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Membranas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Tritio/farmacocinética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
BACKGROUND: Articular cartilage contributes to transferring enormous loads as uniformly as possible from one skeletal segment to the next. Whether it manages this task when subjected to the high repetitive loading cycles occurring during long-distance running and can remain intact is still the topic of controversy. PURPOSE: To investigate the changes in cartilaginous volumes of the tibia, patella, and medial and lateral menisci after extreme dynamic loading as occurs in long-distance runners. STUDY DESIGN: Controlled laboratory study. METHODS: Forty-eight knees of male athletes were studied (38 +/- 14 years). The subjects ran around a predetermined and precisely measured course (5, 10, 20 km), the beginning and end of the run being in the magnetic resonance imaging investigation room. The scan protocol was 60-minute rest period, first measurement, run, 3-minute delay, and second measurement. RESULTS: Overall, there were significant reductions in volume (P < .05) for the patella, tibia, and menisci. There was evidence of significant change after a running distance of 5 km. A further statistical reduction of the volume could only be demonstrated for the medial meniscus after 10 and 20 km. CONCLUSION: Tibial, patellar, and meniscal cartilaginous volumes show not only load-dependent volume changes but also an asymptotic curve. This is the first time that meniscal volume changes due to loading have been used as an indicator of the important mechanical contribution that the menisci make to sustaining the knee during repetitive loading. CLINICAL RELEVANCE: On the basis of the results of this study, the authors assume that the cartilage is able to adapt well to the loads caused by running.