Evaluación funcional y calidad de vida en megaprótesis implantadas por tumores musculoesqueléticos en miembro inferior / Functional evaluation and quality of life in megaprostheses implanted by musculoskeletal tumors in the lower limb

Resumen: Introducción: La resección amplia es el tratamiento quirúrgico de elección en los tumores malignos musculoesqueléticos, que con frecuencia asientan en la pelvis y en los miembros inferiores. La reconstrucción mediante megaprótesis se ha impuesto en los últimos años como primera opción en la cirugía de preservación de la extremidad. Material y métodos: Estudio retrospectivo descriptivo serie de casos, incluye 30 pacientes intervenidos entre 2011 y 2019 de tumores musculoesqueléticos de pelvis y miembro inferior. En todos ellos valoramos la tasa de complicaciones y los resultados funcionales mediante el índice MSTS (Musculoskeletal Tumor Society). Resultados: Se realizó un seguimiento de 40.8 meses (12-101.7). En nueve pacientes (30%) se realizaron resecciones y reconstrucciones pélvicas, a 11 pacientes (36.7%) se les implantaron megaprótesis de cadera por afectación femoral, en tres de los pacientes (10%) se realizó resección del fémur completo y en siete pacientes (23.3%) reconstrucción protésica de la rodilla. El resultado funcional medio de la escala MSTS fue de 72.5% (rango: 40-95%) y la tasa de complicaciones de 56.7% (17 pacientes), siendo la recurrencia tumoral (29%) la principal complicación. Conclusión: La reconstrucción mediante megaprótesis ofrece buenos resultados funcionales a los pacientes dentro de la cirugía de resección radical, permitiendo realizar una vida relativamente normal.

Abstract: Introduction: Radical resection is the surgical treatment of choice in musculoskeletal malignancies, which often settle in the pelvis and lower limbs. Megaprothesis reconstruction has been imposed in recent years as the gold standard in limb preservation surgery. Material and methods: Descriptive retrospective study series of cases, including 30 patients operated between 2011 and 2019 of musculoskeletal pelvic and lower limb tumors at our institution that underwent limb-sparing reconstruction with the megaprosthesis. Functional results according to the MSTS (Musculoskeletal Tumor Society) index and complication rate were analyzed. Results: The average follow-up was 40.8 months (12-101.7). Nine patients (30%) underwent pelvic resections and reconstructions, 11 patients (36.7%) underwent hip reconstruction with megaprothesis due to femoral involvement, in three patients (10%) complete femur resection was performed, and seven patients (23.3%) underwent prosthetic reconstruction of the knee. The mean MSTS score was 72.5% (range: 40-95%), and the complication rate was 56.7% (17 patients), being de tumoral recurrence (29%) the main complication. Conclusion: Tumor megaprothesis give satisfying functional results, allowing the patients to realize a relatively normal life after a lower limb-sparing surgery.

[Functional evaluation and quality of life in megaprostheses implanted by musculoskeletal tumors in the lower limb]. / Evaluación funcional y calidad de vida en megaprótesis implantadas por tumores musculoesqueléticos en miembro inferior.

INTRODUCTION: radical resection is the surgical treatment of choice in musculoskeletal malignancies, which often settle in the pelvis and lower limbs. Megaprothesis reconstruction has been imposed in recent years as the gold standard in limb preservation surgery. MATERIAL AND METHODS: descriptive retrospective study series of cases, including 30 patients operated between 2011 and 2019 of musculoskeletal pelvic and lower limb tumors at our institution that underwent limb-sparing reconstruction with the megaprosthesis. Functional results according to the MSTS (Musculoskeletal Tumor Society) index and complication rate were analyzed. RESULTS: the average follow-up was 40.8 months (12-101.7). Nine patients (30%) underwent pelvic resections and reconstructions, 11 patients (36.7%) underwent hip reconstruction with megaprothesis due to femoral involvement, in three patients (10%) complete femur resection was performed, and seven patients (23.3%) underwent prosthetic reconstruction of the knee. The mean MSTS score was 72.5% (range: 40-95%), and the complication rate was 56.7% (17 patients), being de tumoral recurrence (29%) the main complication. CONCLUSION: tumor megaprothesis give satisfying functional results, allowing the patients to realize a relatively normal life after a lower limb-sparing surgery.

INTRODUCCIÓN: la resección amplia es el tratamiento quirúrgico de elección en los tumores malignos musculoesqueléticos, que con frecuencia asientan en la pelvis y en los miembros inferiores. La reconstrucción mediante megaprótesis se ha impuesto en los últimos años como primera opción en la cirugía de preservación de la extremidad. MATERIAL Y MÉTODOS: estudio retrospectivo descriptivo serie de casos, incluye 30 pacientes intervenidos entre 2011 y 2019 de tumores musculoesqueléticos de pelvis y miembro inferior. En todos ellos valoramos la tasa de complicaciones y los resultados funcionales mediante el índice MSTS (Musculoskeletal Tumor Society). RESULTADOS: se realizó un seguimiento de 40.8 meses (12-101.7). En nueve pacientes (30%) se realizaron resecciones y reconstrucciones pélvicas, a 11 pacientes (36.7%) se les implantaron megaprótesis de cadera por afectación femoral, en tres de los pacientes (10%) se realizó resección del fémur completo y en siete pacientes (23.3%) reconstrucción protésica de la rodilla. El resultado funcional medio de la escala MSTS fue de 72.5% (rango: 40-95%) y la tasa de complicaciones de 56.7% (17 pacientes), siendo la recurrencia tumoral (29%) la principal complicación. CONCLUSIÓN: la reconstrucción mediante megaprótesis ofrece buenos resultados funcionales a los pacientes dentro de la cirugía de resección radical, permitiendo realizar una vida relativamente normal.

Quiste óseo aneurismático localizado en el techo acetabular en un varón de 11 años / Aneurysmal bone cyst located on the acetabular roof in an 11-year-old male

Resumen: El quiste óseo aneurismático es una tumoración benigna poco común, de aparición en la infancia generalmente y a nivel de extremidades. El tratamiento más habitual consiste en el curetaje y relleno con injerto. No obstante, localizaciones poco accesibles a la cirugía suponen un reto terapéutico. Se presenta el caso de un paciente de 11 años con cojera y dolor en cadera derecha sin antecedente traumático ni infeccioso. En los estudios de imagen con TAC y RM se evidencia una lesión lítica expansiva que ocupa todo el techo del acetábulo y pala ilíaca derecha, sugestiva de un quiste óseo aneurismático presentando fractura acetabular asociada. Se realizó una biopsia que confirmó el diagnóstico. Se trató mediante embolización guiada por angiografía debido al gran volumen y alto riesgo de fractura, después del curetaje y relleno con aloinjerto evolucionó satisfactoriamente y el paciente se encuentra asintomático al año de la intervención.

Abstract: The aneurysmal bone cyst is a benign rare tumor, which usually develops during childhood and it's more often found in limbs. The most accepted treatment consists in curetagge and filling with graft. However, certain locations may be inaccesible for surgery and represent therapeutical challenges. We present the case of an 11 year-old male patient with limping and right hip pain without any traumatic nor infectious record. In the image studies with CT and MRI a lytic and expansive lession was found in the upper part of the right acetabulum and right iliac wing, all of which suggested an aneurysmal bone cyst with an associated acetabular fracture. A biopsy was performed which confirmed the diagnosis. He was treated with a CT-guided embolization and, due to its size, curetagge and allograft filling afterwards. He was asymptomatic after1 year of follow-up.

[Aneurysmal bone cyst located on the acetabular roof in an 11-year-old male]. / Quiste óseo aneurismático localizado en el techo acetabular en un varón de 11 años.

The aneurysmal bone cyst is a benign rare tumor, which usually develops during childhood and its more often found in limbs. The most accepted treatment consists in curetagge and filling with graft. However, certain locations may be inaccesible for surgery and represent therapeutical challenges. We present the case of an 11 year-old male patient with limping and right hip pain without any traumatic nor infectious record. In the image studies with CT and MRI a lytic and expansive lession was found in the upper part of the right acetabulum and right iliac wing, all of which suggested an aneurysmal bone cyst with an associated acetabular fracture. A biopsy was performed which confirmed the diagnosis. He was treated with a CT-guided embolization and, due to its size, curetagge and allograft filling afterwards. He was asymptomatic after1 year of follow-up.

El quiste óseo aneurismático es una tumoración benigna poco común, de aparición en la infancia generalmente y a nivel de extremidades. El tratamiento más habitual consiste en el curetaje y relleno con injerto. No obstante, localizaciones poco accesibles a la cirugía suponen un reto terapéutico. Se presenta el caso de un paciente de 11 años con cojera y dolor en cadera derecha sin antecedente traumático ni infeccioso. En los estudios de imagen con TAC y RM se evidencia una lesión lítica expansiva que ocupa todo el techo del acetábulo y pala ilíaca derecha, sugestiva de un quiste óseo aneurismático presentando fractura acetabular asociada. Se realizó una biopsia que confirmó el diagnóstico. Se trató mediante embolización guiada por angiografía debido al gran volumen y alto riesgo de fractura, después del curetaje y relleno con aloinjerto evolucionó satisfactoriamente y el paciente se encuentra asintomático al año de la intervención.

[Surgical treatment of malignant primary tumors of the pelvis on Ennekings zone II]. / Tratamiento quirúrgico de los tumores primarios malignos de pelvis de la zona II de Enneking.

INTRODUCTION: Surgical treatment of malignant bone tumors of the pelvis with periacetabular involvement is constantly evolving. Even though acetabular reconstructions improve quality of life without impairing cancer control, they are not complication free. Our purpose is to describe the functional outcomes and surgical complications of different reconstructive techniques for Enneking zone II tumors. MATERIAL AND METHODS: Fifteen patients underwent surgery for malignant pelvic bone tumors between 2002 and 2012. Seven patients were retrospectively evaluated according to the Enneking and Dunham classification and were found to have periacetabular involvement, as follows: 3 were type II; 1 types I + II; 2 types II + III, and one types I + II + III. Five patients had a standard chondrosarcoma and 2 osteosarcoma. Patients included 4 males and 3 females; mean age was 43 years. The minimum follow-up period was 12 months (mean 6.14 years). All of them underwent cancer resection with periacetabular reconstruction: massive osteochondral allograft in 3 patients, structural allograft and hip arthroplasty in 2 patients, and prosthesis with iliac anchoring in 2. They were clinically, radiologically and functionally evaluated with the MSTS scale (1993). RESULTS: The mean MSTS score 6 months after surgery was 20.71 (69%). Five patients (71.4%) had surgical complications: 2 dislocations of the native femoral head on the allograft; one aseptic prosthetic dislodgement, and 2 deep infections. All patients had free intraoperative borders. CONCLUSIONS: Acetabular reconstructions after oncologic resection for malignant bone tumors seem to provide good functional outcomes. However, only selected cases should undergo surgeries associated with a high complication rate.

El tratamiento quirúrgico de los tumores óseos malignos de pelvis con afectación periacetabular está en continua evolución. Las reconstrucciones acetabulares mejoran la calidad de vida sin perjudicar el control oncológico; sin embargo, no están exentas de complicaciones. Nuestro objetivo es describir los resultados funcionales y las complicaciones quirúrgicas de diferentes técnicas reconstructivas de la zona II de Enneking.

Tratamiento quirúrgico de los tumores primarios malignos de pelvis de la zona II de Enneking / Surgical treatment of malignant primary tumors of the pelvis on Enneking's zone II

Resumen: Introducción: El tratamiento quirúrgico de los tumores óseos malignos de pelvis con afectación periacetabular está en continua evolución. Las reconstrucciones acetabulares mejoran la calidad de vida sin perjudicar el control oncológico; sin embargo, no están exentas de complicaciones. Nuestro objetivo es describir los resultados funcionales y las complicaciones quirúrgicas de diferentes técnicas reconstructivas de la zona II de Enneking. Material y métodos: 15 pacientes fueron intervenidos de tumores óseos malignos de pelvis entre 2002 y 2012. Según la clasificación de Enneking y Dunham, hubo siete individuos con afectación periacetabular: tipo II-3, tipo I + II-1, tipo II + III-2, tipo I + II + III-1, que se evaluaron retrospectivamente. Cinco presentaron un condrosarcoma convencional y dos un osteosarcoma. Cuatro eran varones y tres mujeres, con una edad media de 43 años. El seguimiento mínimo fue 12 meses (6.14 años de media). En todos se realizó una resección oncológica con reconstrucción periacetabular: aloinjerto osteocondral masivo en tres sujetos, aloinjerto estructural más artroplastía de cadera en dos personas y dos con prótesis de anclaje ilíaco. Fueron evaluados clínica, radiológica y funcionalmente mediante la escala MSTS (1993). Resultados: La media MSTS a los seis meses de la cirugía fue 20.71 (69%). Complicaciones quirúrgicas aparecieron en cinco casos (71.4%): dos luxaciones de cabeza femoral nativa sobre aloinjerto, una desimplantación protésica aséptica y dos infecciones profundas. Hubo bordes libres intraoperatorios en todos los casos. Conclusiones: Las reconstrucciones acetabulares después de resecciones oncológicas en tumores óseos malignos parecen ofrecer buenos resultados funcionales. Sin embargo, sólo casos seleccionados deberían ser sometidos a intervenciones con alta tasa de complicaciones.

Abstract: Introduction: Surgical treatment of malignant bone tumors of the pelvis with periacetabular involvement is constantly evolving. Even though acetabular reconstructions improve quality of life without impairing cancer control, they are not complication free. Our purpose is to describe the functional outcomes and surgical complications of different reconstructive techniques for Enneking zone II tumors. Material and methods: Fifteen patients underwent surgery for malignant pelvic bone tumors between 2002 and 2012. Seven patients were retrospectively evaluated according to the Enneking and Dunham classification and were found to have periacetabular involvement, as follows: 3 were type II; 1 types I + II; 2 types II + III, and one types I + II + III. Five patients had a standard chondrosarcoma and 2 osteosarcoma. Patients included 4 males and 3 females; mean age was 43 years. The minimum follow-up period was 12 months (mean 6.14 years). All of them underwent cancer resection with periacetabular reconstruction: massive osteochondral allograft in 3 patients, structural allograft and hip arthroplasty in 2 patients, and prosthesis with iliac anchoring in 2. They were clinically, radiologically and functionally evaluated with the MSTS scale (1993). Results: The mean MSTS score 6 months after surgery was 20.71 (69%). Five patients (71.4%) had surgical complications: 2 dislocations of the native femoral head on the allograft; one aseptic prosthetic dislodgement, and 2 deep infections. All patients had free intraoperative borders. Conclusions: Acetabular reconstructions after oncologic resection for malignant bone tumors seem to provide good functional outcomes. However, only selected cases should undergo surgeries associated with a high complication rate.

Seguridad y eficacia de la cifoplastia en el tratamiento de la enfermedad tumoral de la columna vertebral / Safety and efficacy of kyphoplasty in the treatment of tumoral disease of the spine

Las fracturas vertebrales en pacientes oncológicos generan dolor e incapacidad, con limitación funcional y disminución de la calidad de vida. El objetivo del estudio es valorar la eficacia y seguridad de la cifoplastia en este tipo de fracturas vertebrales en el momento agudo. Material y métodos. Estudio descriptivo retrospectivo de 75 pacientes oncológicos consecutivos con 122 fracturas vertebrales agudas, que fueron tratados mediante cifoplastia percutánea bilateral con balón, con un seguimiento medio de 11 meses. Resultados. Se produjo mejoría del dolor en el 91% de los pacientes. La mejoría media en la Escala Visual Analógica (EVA) fue de 4,28 puntos (valor preoperatorio 7,49 [DE 1,19], postoperatorio 3,21 [DE 0,95]). Antes de la intervención necesitaban opioides mayores un 53% de los pacientes (40 casos) y al mes de la cirugía solo un 12% (9 pacientes). La calidad de vida determinada por el índice de Karnosfky mejoró de 60,2 (DE 10) a 80,7 (DE 12,1). En un 5,7% de las cifoplastias (7 casos) se encontraron fugas de cemento, todas ellas sin repercusión neurológica. Aparecieron nuevas fracturas en un 14% de las cifoplastias (11 casos). Este subgrupo presentó un empeoramiento discreto de la mejoría clínica adquirida inicialmente. No encontramos ninguna complicación neurológica ni pulmonar relacionada con la técnica quirúrgica que no estuviera justificada por la evolución de la enfermedad. Conclusiones. La cifoplastia constituye un procedimiento eficaz y seguro para el tratamiento de las fracturas vertebrales en pacientes con cáncer (AU)

Vertebral fractures in oncology patients cause significant pain and disability, with decreased quality of life. The aim of the study is to assess the efficacy and safety of kyphoplasty in this type of vertebral fracture in the acute phase. Materials and methods. A retrospective study was conducted on 75 consecutive oncology patients with 122 acute vertebral fractures, who underwent bilateral balloon kyphoplasty, with a mean follow up of 11 months. Results. Almost all (91%) of the patients improved their pain level. The mean improvement in the Visual Analogue Scale (VAS) was 4.28 points (preoperative value 7.49 [SD 1.19], postoperative 3.21 [SD 0.95]). Before surgery, 53% of patients needed major opioids (40 cases), and one month after surgery only 12% (9 patients) required them. Quality of life determined by the Karnofsky index improved from 60.2 (SD 10) to 80.7 (SD 12.1). Cement leaks were found in 5.7% (7 cases), all without neurological repercussions. New fractures appeared in 11 patients. This subgroup showed a slight worsening of the initially acquired clinical improvement. No neurological or pulmonary complications related to surgical technique were found. Conclusions. Kyphoplasty is an effective and safe for treating vertebral fractures in patients with cancer (AU)

[Safety and efficacy of kyphoplasty in the treatment of tumoral disease of the spine]. / Seguridad y eficacia de la cifoplastia en el tratamiento de la enfermedad tumoral de la columna vertebral.

UNLABELLED: Vertebral fractures in oncology patients cause significant pain and disability, with decreased quality of life. The aim of the study is to assess the efficacy and safety of kyphoplasty in this type of vertebral fracture in the acute phase. MATERIALS AND METHODS: A retrospective study was conducted on 75 consecutive oncology patients with 122 acute vertebral fractures, who underwent bilateral balloon kyphoplasty, with a mean follow up of 11 months. RESULTS: Almost all (91%) of the patients improved their pain level. The mean improvement in the Visual Analogue Scale (VAS) was 4.28 points (preoperative value 7.49 [SD 1.19], postoperative 3.21 [SD 0.95]). Before surgery, 53% of patients needed major opioids (40 cases), and one month after surgery only 12% (9 patients) required them. Quality of life determined by the Karnofsky index improved from 60.2 (SD 10) to 80.7 (SD 12.1). Cement leaks were found in 5.7% (7 cases), all without neurological repercussions. New fractures appeared in 11 patients. This subgroup showed a slight worsening of the initially acquired clinical improvement. No neurological or pulmonary complications related to surgical technique were found. CONCLUSIONS: Kyphoplasty is an effective and safe for treating vertebral fractures in patients with cancer. LEVEL OF EVIDENCE: Level IV.

First report of anthelmintic resistance in gastrointestinal nematodes of sheep from costa rica.

As the prevalence and severity of anthelmintic resistance continue to rise, nematode infections in sheep correspondingly reduce the profitability of the sheep industry. In Costa Rica, sheep production systems are increasing in both number and importance. A field trial study was carried out to detect the level of anthelmintic resistance to albendazole and ivermectin in gastrointestinal nematodes (GIN) of sheep from seven farms in Costa Rica. Resistance was determined using the fecal egg count reduction test (FECRT). Three treatment groups were assessed on each farm: control, albendazole, and ivermectin. Haemonchus spp. (71%), Strongyloides sp. (57%), and Trichostrongylus spp. (43%) presented resistance levels to albendazole, whereas Strongyloides sp. (43%), Haemonchus spp. (29%), and Trichostrongylus spp. (29%) were resistant to ivermectin. Haemonchus spp., Strongyloides sp., and Trichostrongylus spp. were the most resistant GIN to both products. This study suggests that frequency of treatment, exclusive chemical control, and visual estimation of animal weight to calculate dosage may contribute to the high levels of anthelmintic resistance that were observed on the farms analyzed herein.

Analysis of the presence of cardiovascular and analgesic/anti-inflammatory/antipyretic pharmaceuticals in river- and drinking-water of the Madrid Region in Spain.

Interest in the presence of pharmaceuticals in wastewater, in the water of our rivers and, to a lesser extent, in our drinking water, has been growing in recent decades. Many of these substances, currently classified as "emerging pollutants", are biologically active compounds and continuously released in effluents. As sewage treatment plants (STPs) are not adequately equipped to eliminate all of these substances completely, some are discharged directly into rivers. In Spain, as in most of its neighbouring countries, there is an elevated use of pharmaceuticals for the treatment of cardiovascular diseases (which are extremely prevalent among the older adult population) and anti-inflammatory medications, which are obtainable over the counter without a medical prescription. This study therefore sought to determine to what degree pharmaceuticals with the highest regional prescription and/or use rates, such as cardiovascular and analgesic/anti-inflammatory/antipyretic medications, were present in the principal rivers (Jarama, Manzanares, Guadarrama, Henares and Tagus) and tap-water samples of the Madrid Region (MR). Samples were taken downstream the discharge of 10 of the most important region's STPs and the most frequently used drugs in the region were analysed for. Of the 24 drugs analysed, 21 were detected at concentrations ranging from 2 ng L⁻¹ to 18 µg L⁻¹. The highest drug concentrations corresponded to ibuprofen, diclofenac, naproxen, atenolol, frusemide (furosemide), gemfibrozil and hydrochlorthiazide, and in most cases exceeded the amounts reported in the scientific literature. No traces of these groups of pharmaceuticals were detected in the drinking water analysed. On the basis of the high concentrations detected, we believe that an environmental surveillance system should be implemented to assess the continuous discharge of these pharmaceuticals and their possible ecotoxicological effects. At the same time, efforts to raise the awareness of the public about responsible use and the proper disposal of such substances at purpose-designated collection points should be increased. Furthermore sewage treatment processes should be suitably adapted to increase the rates of removal of these drugs.

Tomografía axial computarizada facial

Varón de 35 años de edad con antecedentes personales de rinitis sin predominio estacional, poliposis nasal, fumador de 2 paquetes al día, trabajador en tienda de calzado. Consulta hace 2 meses por rinorrea purulenta por fosa nasal derecha, tumefacción maxilar y epífora. Se realizó radiografía simple evidenciándose ocupación del seno maxilar derecho, sin nivel hidroaéreo. Se trató como una sinusitis aguda con antibioterapia durante 10 días; así la rinorrea mejoró pero persistió la epífora, la hipoestesia y la algia periorbitaria. Se remitió a otorrinolaringología donde se realizó endoscopia nasal observándose pólipos nasales. Ante la imposibilidad de completar el estudio por vía endoscópica, se solicitó una tomografía axial computarizada (TAC) de cráneo, que mostró una masa de partes blandas heterodensa que ocupaba la totalidad de la cavidad nasal, el seno maxilar derecho, y las celdillas etomoidales, con destrucción de paredes óseas (orbitaria inferior, superior y media de seno maxilar derecho).Se evidenció una imagen polipoidea y un nivel hidroaéreo en el seno maxilar izquierdo. El estudio anatomopatológico fue compatible con un papiloma invertido (AU)

Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction.

Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate approximately 0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-induced- maturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca(2+) does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.

Embarazo abdominal primario de presentación atípica / Atypical presentation of primary abdominal pregnancy

No disponible

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death.

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.

BCL-2-related protein expression in apoptosis: oxidative stress versus serum deprivation in PC12 cells.

Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H2O2 treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H2O2-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H2O2 but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H2O2 exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.

Analogies and differences between omega-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells.

The characteristics of the binding sites for the Conus magus toxins omega-conotoxin MVIIC and omega-conotoxin MVIID, as well as their effects on K+-evoked 45Ca2+ entry and whole-cell Ba2+ currents (IBa), and K+-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [125I] omega-conotoxin GVIA to bovine adrenal medullary membranes was displaced by omega-conotoxins GVIA, MVIIC and MVIID with IC50 values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [125I] omega-conotoxin MVIIC, which was displaced by omega-conotoxins MVIIC, MVIID and GVIA with IC50 values of around 30, 80 and 1.200 nM, respectively. The sites recognized by omega-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC50 values of around 1 nM). Both omega-conotoxin MVIIC and MVIID blocked IBa by 70-80%; the higher the [Ba2+]o of the extracellular solution the lower the blockade induced by omega-conotoxin MVIIC. This was not the case for omega-conotoxin MVIID; high Ba2+ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba2+. A further difference between the two toxins concerns their reversibility; washout of omega-conotoxin MVIIC did not reverse the blockade of IBa while in the case of omega-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by omega-conotoxin GVIA, suggesting that it is associated with N-type Ca2+ channels. Blockade of K+-evoked 45Ca2+ entry produced results which paralleled those obtained by measuring IBa. Thus, 1 microM of each of omega-conotoxin GVIA and MVIIA inhibited Ca2+ uptake by 25%, while 1 microM of each of omega-conotoxin MVIIC and MVIID caused a 70% blockade. K+-evoked catecholamine secretory responses were not reduced by omega-conotoxin GVIA (1 microM). In contrast, at 1 microM both omega-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca2+ channels that contribute to the regulation of secretion and are sensitive to omega-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for omega-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca2+ channels or an entirely new subtype of voltage-dependent high-threshold Ca2+ channel.

The actions of ouabain and lithium chloride on cytosolic Ca2+ in single chromaffin cells.

The effects of ouabain, Li+ and veratridine on the concentration of cytosolic free Ca2+ ([Ca2+]i) were studied in single fura-2-loaded bovine adrenal chromaffin cells. Superfusion of cells with ouabain (10 microM for 60 min) caused only a delayed mild increase of the Ca2+]i, from around 0.1 microM to 0.2-0.3 microM; this increase was Nao(+)-dependent. Replacement of all NaCl of the Krebs-Hepes solution by LiCl (144 mM) produced a gradual increase of [Ca2+]i, which remained elevated at a stable plateau of 0.4-0.5 microM for 40-50 min. When ouabain (in the presence of normal Nao+) or Li+ (in the absence of Nao+) was given in Krebs-Hepes solution containing no Ca2+, the reintroduction of 2.5 mM Ca2+ produced a fast elevation of the [Ca2+]i. In the case of ouabain-treated cells, the [Ca2+]i curve exhibited an initial phasic component which inactivated to a tonic component. omega-Conotoxin MVIIC (3 microM) and R56865 (10 microM) inhibited the phasic but not the tonic component. Veratridine (30 microM) induced large [Ca2+]i oscillations. Both ouabain or Li+ abolished such oscillations. These results are compatible with ouabain causing elevation of [Ca2+]i in bovine chromaffin cells through a dual mechanism, i.e. cell depolarisation and slowing down of the Na(+)-Ca2+ exchanger of their plasmalemma. Through its binding to the Na+ site on the Na(+)-Ca2+ exchanger, Li+ ions generate powerful Cai2+ signals that might be relevant to its known effects on neurosecretory mechanisms.

Effects of omega-conotoxin MVIIC on veratridine-induced cytotoxicity and cytosolic Ca(2+) oscillations.

External Ca(2+) entry through various Ca(2+)-channel subtypes is responsible for the large oscillations of the cytosolic Ca(2+) concentrations, [Ca(2+)](i), and cell death induced by veratridine in primary cultures of bovine chromaffin cells. Blockade by omega-conotoxin GVIA (GVIA) of N-type Ca(2+) channels, by omega-agatoxin IVA (IVA) of P-type Ca(2+) channels, or by furnidipine of L-type Ca(2+) channels did not afford cytoprotection. However, (omega-conotoxin MVIIC (MVIIC), a wide-spectrum blocker of N-, P- and Q-type Ca(2+) channels greatly protected the cells against the cytotoxic effects of veratridine. Furnidipine further enhanced the cytoprotecting effects of MVIIC. MVIIC but not furuidipine, markedly reduced the oscillations of [Ca(2+)](i) induced by veratridine in single fura-2-loaded chromaffin cells. The results suggest that Ca(2+) entry through any of the different Ca(2+) channel subtypes present in bovine chromaffin cells might be cytotoxic. They also support two ideas: (i) that wide-spectrum neuronal Ca(2+) channel blockers (i.e. MVIIC) might be better cytoprotecting agents than more specific neuronal Ca(2+) channel blockers (i.e., GVIA, IVA, furnidipine); and (ii) that combined Ca(2+) channel blockers may provide greater cytoprotection than single compounds.

Contribution of SK and BK channels in the control of catecholamine release by electrical stimulation of the cat adrenal gland.

1. Transmural electrical stimulation (10 Hz, 1 ms, 40 V for 10 s) of cat adrenal glands perfused at room temperature with Krebs-Hepes solution produced catecholamine secretory responses which were reproducible when stimulations were applied at 5 min intervals. Such responses were inhibited about 20% by atropine (1 microM) and 80% by hexamethonium (30 microM). Apamin (100 nM) increased the secretory response 2.5-fold in the presence of atropine and 8-fold in the presence of hexamethonium. 2. Potentiation by apamin of secretory responses evoked by 100-pulse trains was similar at 5, 10 and 20 Hz (about 2-fold). When glands were continuously stimulated at 3 Hz, apamin increased 4-fold the initial secretion plateau. Continuous stimulation at a higher frequency (20 Hz) produced a sharp secretory peak followed by a small, sustained plateau; apamin did not alter this plateau. Apamin also enhanced the secretory responses obtained with sustained stimulation with acetylcholine (10 or 200 microM). 3. Secretion peaks induced by brief acetylcholine pulses (10 microM for 10 s) applied to isolated and superfused cat adrenal chromaffin cells were enhanced more than 3-fold by 100 nM apamin. Charybdotoxin (10 nM) did not enhance these secretory peaks. 4. In perfused cat adrenal glands, charybdotoxin (10 nM) affected neither the secretion evoked by trains of electrical stimulation applied at different frequencies nor the secretion evoked by acetylcholine pulses. 5. In 0.5 mM [Ca2+]o, apamin enhanced 3-fold the secretion evoked by electrical stimulation trains of 100 pulses (10 Hz, 10 s) and almost 6-fold the acetylcholine (10 microM for 10 s)-induced secretion. In 5 mM Ca2+, apamin enhanced the secretory responses to electrical stimulation and acetylcholine 2- and 10-fold, respectively. Charybdotoxin enhanced 2.5-fold the secretory response to electrical stimulation in 0.5 mM Ca2+, although this effect was not statistically significant. A synergistic interaction between the two toxins on catecholamine release induced by electrical stimulation was observed at low but not at high [Ca2+]o. 6. Simultaneous release of acetylcholine and catecholamines upon electrical stimulation was achieved in glands in which the endogenous acetylcholine stores in the splanchnic nerve terminals had been prelabelled by perfusion with [3H]choline. While apamin enhanced more than 2-fold the postsynaptic release of catecholamines, the presynaptic release of acetylcholine remained unaffected. 7. The results are compatible with the hypothesis that, under physiological conditions, Ca(2+)-activated SK channels present in chromaffin cells control the firing patterns of action potentials induced by the acetylcholine released from splanchnic nerves during stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological protection against the cytotoxicity induced by 6-hydroxydopamine and H2O2 in chromaffin cells.

We present in this report the characteristics of the damage induced by 6-hydroxydopamine and H2O2 on bovine chromaffin cells in primary culture. Cytotoxicity was quantified using catecholamine cell contents, lactate dehydrogenase (LDH) release, trypan blue exclusion and morphological appearance. An excellent correlation between these four parameters was found. The cytotoxic effects of 6-hydroxydopamine were Ca(2+)-independent. In spite of this, the Ca2+ channel antagonists R56865 (N-[1-(4-(fluorophenoxy)butyl)]-4-piperidinyl-N-methyl-2-benzo-thiazo lamine) lidoflazine exhibited marked cytoprotective effects against both 6-hydroxydopamine and H2O2. The selective dopamine uptake blocker, bupropion, increased the viability of 6-hydroxydopamine and H2O2-treated cells from 20% to around 80%. Catalase drastically protected against the cytotoxic effects of 6-hydroxydopamine and H2O2. In contrast, desferrioxamine gave better protection against H2O2 cytotoxicity; glutathione and N-acetylcysteine only afforded substantial protection against 6-hydroxydopamine. Three main conclusions emerge from this study. (1st) 6-Hydroxydopamine causes chromaffin cell damage via a mechanism probably related to the production of free radicals, but unrelated to Ca2+ ions. Cytoprotection afforded by R56865 and lidoflazine must be unrelated to their Ca2+ antagonist properties. This suggests a novel component in the cytoprotective mechanism of action of these drugs. (2nd) The strong cytoprotective effects of bupropion seem to be unrelated to its ability to block the plasmalemmal dopamine carrier. (3rd) Bovine adrenal chromaffin cells in primary cultures are a suitable model for adult neurons to study the basic mechanism of cell damage, and to screen new drugs with putative neuroprotective properties.