Acromegalia y cáncer de tiroides

RESUMEN: Introducción La acromegalia se asocia con un mayor riesgo de morbilidad y mortalidad por cáncer. Sin embargo, los datos respecto de la incidencia de cáncer en acromegalia son controvertidos. Objetivos Describir las características clínicas, bioquímicas e imagenológicas de un grupo de pacientes acromegálicos con carcinoma diferenciado de tiroides (CDT). Analizar las características de riesgo de recurrencia (RR) y respuesta en el seguimiento (RtaSg) y comparar la evolución con la de pacientes con CDT no acromegálicos. Materiales y métodos Se realizó un análisis retrospectivo multicéntrico de pacientes con diagnóstico de acromegalia y CDT. Se realizó un análisis comparativo entre los pacientes de bajo RR inicial acromegálicos con una muestra aleatoria de pacientes no acromegálicos con CDT de bajo RR inicial (1:4). Resultados Se analizaron 16 pacientes con diagnóstico de CDT y acromegalia. En 93,8% se hizo el diagnóstico por ecografía, sólo el 50% tenían un nódulo tiroideo palpable. En el momento del diagnóstico del CDT, los valores de IGF-1 fueron 1,8 ± 1,3 LSN, con 62,5% con acromegalia activa. La histología fue papilar en todos los casos, el 56,3% variedad clásica y el resto papilar variedad folicular. El 75% de los pacientes presentó un Estadio I (12/16), sólo 3 pacientes Estadio II y 1 Estadio IVb. El RR inicial fue bajo en el 87,6% (14/16), intermedio en 1 paciente y alto en 1 paciente. Las respuestas al final del seguimiento fueron: 86,7% (13/15) sin evidencia de enfermedad, 1 paciente bioquímica incompleta y 1 estructural incompleta. La RtaSg no tuvo diferencias con los no acromegálicos. Conclusiones Los pacientes acromegálicos con CDT presentaron predominantemente un bajo RR inicial. Al realizar la comparación con el grupo control, se puede concluir que el CDT en pacientes acromegálicos no presentó una evolución más agresiva.

Primary empty sella (PES): a review of 175 cases.

The term primary empty sella (PES) makes reference to the herniation of the subarachnoid space within the sella turcica in patients with no history of pituitary tumor, surgery or radiotherapy. To retrospectively assess clinical features, radiological findings and the biochemical endocrine function from the records of 175 patients with a diagnosis of PES. One hundred seventy-five patients (150 females) were studied. The mean age at diagnosis was 48.2 ± 14 year. Most diagnoses were made by magnetic resonance imaging (n = 172). In most patients, the pituitary function was assessed by basal pituitary hormones measurements. Pituitary scans were ordered for different reasons: headache (33.1 %), endocrine disorders (30.6 %), neurological symptoms (12.5 %), visual disturbances (8.75 %), abnormalities on sella turcica radiograph (8.75 %) and others (6.25 %). Multiple pregnancies were observed in 58.3 % of women; headaches, obesity, and hypertension were found in 59.4, 49.5, and 27.3 % of the studied population, respectively. Mild hyperprolactinemia (<50 ng/ml) was present in 11.6 % of women and 17.3 % of men. Twenty-eight percent of our patients had some degree of hypopituitarism. In the male population, hypopituitarism represented 64 % of cases, whereas it accounted for 22 % of all females. PES seems to be more commonly found in middle-aged women, with a history of multiple pregnancies. In most patients, PES was discovered as an incidental finding on imaging studies, while in almost a quarter of patients PES was found during the diagnostic evaluation of anterior pituitary deficiency, which was more common in men.

Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses.

Adoptive T-cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, which introduces antigen-specific T-cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T-cell repertoires. Studies have suggested that use of higher-affinity TCRs against class I major histocompatibility complex antigens could drive the activity of both CD4(+) and CD8(+) T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here, we describe a high-throughput platform of 'reverse biochemistry' whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes (TILs) or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8(+) T cells expressing the highest-affinity TCR variants were deleted in both the TIL population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4(+) T cells in the tumor, suggesting they had a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR-binding properties that promote peripheral T-cell survival and tumor elimination.

Single-chain VαVß T-cell receptors function without mispairing with endogenous TCR chains.

Transduction of exogenous T-cell receptor (TCR) genes into patients' activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These 'mixed' heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vß single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T-cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

Silla turca vacía primaria (STVP): estudio de 117 casos / Primary Empty Sella (PES): A review of 117 cases

Introducción: El término Silla Turca Vacía Primaria (STVP) hace referencia a la invaginación del espacio subaracnoideo hacia el interior de la silla turca en pacientes sin antecedentes de tumor, cirugía o radioterapia de la región selar. Aunque usualmente no está asociado con disfunciones endocrinas, diferentes grados de hipopituitarismo e hiperprolactinemia han sido reportados. Objetivo: Analizar retrospectivamente datos clínicos, hallazgos radiológicos y bioquímicos de 117 pacientes con diagnóstico de STVP. Pacientes y Métodos: Se estudiaron 117 pacientes, 98 mujeres (48 ± 14.9 años). Los diagnósticos fueron realizados por Resonancia Magnética Nuclear (n=115) y Tomografía Computada (n=2). La evaluación de la función adenohipofisaria se realizó a través de determinaciones hormonales basales. Resultados: Los motivos que llevaron al pedido de las imágenes fueron: cefaleas (35 %), sospecha clínica y/o bioquímica de deficiencia pituitaria (22 %), trastornos visuales (11 %), anormalidades de la radiografía simple de la silla turca (11 %), hiperprolactinemia (2,6 %), otros (18.4 %). El 48,9 % de las mujeres eran multíparas. Cefaleas, obesidad, hipertensión arterial y autoinmunidad tiroidea fueron halladas en el 60, 67, 24,5 y 22,5 % de la población evaluada respectivamente. Hiperprolactinemia (< 50 ng/ml) estuvo presente en 6,1 % de las mujeres y 15, 8 % de los hombres. El 27 % de los pacientes estudiados presentó algún grado de hipopituitarismo, que fue más frecuente en la población masculina. Conclusiones: STVP fue más frecuente en mujeres multíparas de mediana edad. En la mayoría de los casos fue descubierta incidentalmente por estudios radiológicos, mientras que en un cuarto de los pacientes, fue encontrada durante la evaluación diagnóstica de deficiencia adenohipofisaria, lo cual fue más frecuente en hombres.

Introduction: The term Primary Empty Sella (PES) makes reference to the herniation of the subarachnoid space within the sella turcica in those patients with no history of pituitary tumor, neither surgery, nor radiotherapy. Though it is usually not associated with endocrine abnormalities, different degrees of hypopituitarism and mild hyperprolactinemia have been reported. Objective: To assess clinical features, radiological findings and biochemical endocrine function retrospectively from the records of 117 patients with diagnosis of PES. Patients and Methods: One hundred seventeen patients, 98 females, were studied. The mean age at diagnosis was 48 ± 14.9 yr. Most diagnoses were made with magnetic resonance imaging (n = 115), and only 2 through sellar computed tomography scan. Only pituitary basal hormones determinations were made, except for the TRH and ACTH tests which were performed for the diagnosis of primary hypothyroidism and secondary adrenal failure respectively. Results: Pituitary images were requested because of different reasons: headaches (35 %), clinical and biochemical suspicion of pituitary deficiency (22 %), visual disturbances (11 %), abnormalities on the simple sella turcica radiography (11 %) hyperprolactinemia (2.6 %), others (18.4 %): dizziness, seizures, rhinorrhea, loss of consciousness, skull trauma, galactorrhea. Multiple pregnancies were observed in 48.9 % of women; headaches, obesity, arterial hypertension and thyroid autoimmunity were found in 60 %, 67 %, 24.5 % and 22.5 % of the studied population respectively. Mild hyperprolactinemia (< 50 ng/ml) was present in 6.1 % of women and 15.8 % of men. Twenty seven percent of our patients had some degree of hypopituitarism. For male population hypopituitarism comprised 72 %, whereas it took up 19 % for the whole female group. Conclusions: PES seems to be more commonly found in middle-aged women (sex ratio 5/1) with history of multiple pregnancies. In most patients it was discovered as an incidental finding at image studies, while in almost a quarter of patients PES was found during the diagnosis stage of anterior pituitary deficiency, which was more frequently seen among men.

Acromegalia: comparación de los niveles séricos de IGF-I por dos inmunoensayos y su correlación con la prueba de tolerancia oral a la glucosa / Acromegaly: Comparison of serum IGF-1 levels measured by two immunoassays and their correlation with the Oral Glucose Tolerance Test

Introducción: La determinación de IGF-I en suero o plasma es una herramienta esencial en el diagnóstico y seguimiento de la acromegalia. Sin embargo, se deben tener presentes algunos inconvenientes en su medición por diferentes inmunoensayos. Objetivos: Evaluar dos inmunoensayos para la determinación de IGF-I y su correlación con el nadir de GH en el TTOG en pacientes acromegalicos. Materiales y métodos: Se analizaron 37 pacientes acromegálicos, 20 mujeres y 17 hombres. IGF-I fue determinada por Immulite 1000, (IMM) y por IRMA (DSL). Se realizó el TTOG y se determinó glucosa y GH en todos los tiempos (basal, 30, 60, 90 y 120min). Se consideró respuesta normal un nadir de GH <1ng/ml. Nueve pacientes se encontraban bajo tratamiento y 28 sin tratamiento. Análisis estadístico: se utilizaron el test de Wilcoxon, de Bland y Altman y curvas ROC. Se consideró significativa una p<0,05. Resultados: Las concentraciones basales de glucosa fueron 97,86±10,91 mg/dl, de GH 2,8 (1,59-14,4) ng/ml, de IGF-I por IMM 602±318 ng/ml y por DSL 1006±596 ng/ml. IGF-I por IMM y DSL mostró una diferencia significativa con p <0,01 y un bias de - 403,2 ng/ml con valores menores por IMM. IGF-I elevada por IMM y DSL, se encontró en el 84% y en el 97% respectivamente. IGF-I elevada con nadir de GH >1ng/ml se encontró en el 70%, con nadir de GH normal en el 13,5%. IGF-I normal con nadir >1ng/ml en el 2,7% y con nadir de GH normal en el 13,5%. El área bajo las curvas ROC no mostró diferencias significativas. Conclusiones: Los niveles de IGF-I determinados por IMM y DSL fueron significativamente diferentes mostrando un bias negativo para IMM. La mayoría de los valores del nadir de GH fueron consistentes con los niveles de IGF-I observándose una discrepancia en el 30% de los pacientes, estuvieran o no bajo tratamiento.

Introduction: IGF-I determination in serum or plasma is an essential tool in the diagnosis and follow-up of acromegaly. Hepatic production of IGF-I is regulated by GH and circulates bound to several IGF-I binding proteins which extends its half life. IGF-I is not released in a pulsatile pattern and has no significant variability in 24 h. Objective: To evaluate two different methodologies in IGF-I levels determination and their correlation with GH nadir in OGTT in acromegalic patients. Material and methods: We analyzed 37 acromegalic patients, 20 women and 17 men, mean age was 45±12 years. IGF-I levels were assayed by Immulite 1000, DPC (IMM) and DSL-5600 ACTIVE® IGF-I Coated-Tube IRMA (DSL) and OGTTs (at baseline and at 30, 60, 90 and 120 minutes) were performed by measuring plasma glucose and GH assay by immunochemiluminometric assay (Access); we considered a nadir <1ng/ml as normal response. Nine patients were under medical treatment (cabergoline: 4, octeotride: 4, and cabergoline plus octeotrite: 1) and 28 without treatment. Statistical analysis: Wilcoxon and, Bland and Altman tests and ROC curves. Differences were considered significant at p< 0.05. Results: Basal glucose levels were 97.86±10.91 mg/dl and mean GH was 2.8 (1.59-14.4) ng/ml. Mean IGF-I levels performed by IMM were 602±318 ng/ml and 1006±596 ng/ml by DSL. There was a statistically significant difference between both methodologies (p<0.01). Bland and Altman test showed a bias of - 403.2 ng/ml with lower values by IMM. We observed elevated IGF-I levels in 84% by IMM and in 97% by DSL, and only one patient had normal levels with both methodologies. Elevated IGF-I levels and GH nadir >1ng/ml were observed in 70% of the patients, increased IGF-I with normal GH nadir in 13.5%, normal IGF-I with GH nadir >1ng/ml in 2.7% and normal IGF-I with normal GH nadir in 13.5%. Patients under treatment: 3 showed normal GH nadir with elevated IGF-I levels, in 2 of them by both methodologies, and in the other one it was normal by IMM and elevated by DSL; the other 6 showed GH nadir > 1ng/ml, 5 of them presented elevated IGF-I by both methodologies and the other one showed discrepancy in IGF-I levels. The under ROC curve area and confidence interval (CI) of 95% for IGF-I IMM and DSL were 0.96 (0.90-1.00) and 0.91 (0.82-1.00) respectively. Differences between the ROC curves areas were not significant Conclusions: IGF-I levels determined by IMM and DSL were statistically significantly different. IGF-I levels showed a negative bias by IMM. Most of the results of GH nadir were consistent with IGF-I levels but we observed discrepancy in 30% of the patients, regardless of whether they were under treatment or not.

Estudio retrospectivo sobre las posibles complicaciones maternofetales de la exposición a cabergolina / Retrospective Study of any potencial Adverse Effects of Cabergoline on Pregnancy and Embryofetal Development

Objetivos: Estimar la frecuencia de complicaciones maternofetales en mujeres que se embarazaron durante el tratamiento con cabergolina (CAB). Estimar la frecuencia de patología detectada posnacimiento en los niños producto de dichos embarazos. Material y métodos: Estudio retrospectivo y multicéntrico de 86 embarazos en 78 mujeres con hiperprolactinemia idiopática (7) o tumoral (44 micro y 27 macro), en tratamiento con CAB en el momento de la concepción. Edad: 20 a 45 años; PRL inicial: 30 a 1429 ng/ml; duración del tratamiento previo al embarazo 1 a 120 meses; dosis: 0.125 a 4 mg/semana. El rango de exposición embriofetal a la CAB fue de 3 a 27 semanas, el 96.39% de las pacientes la recibió durante el primer trimestre y el 3.61% hasta el segundo. Resultados: No hubo complicaciones mayores durante el embarazo. Se registraron 7 abortos espontáneos (8.1%) y 75 partos, de los cuales 49 fueron vaginales y 26 cesáreas. Se registraron 69 recién nacidos, 63 fueron a término y 6 pretérmino (8.8%), ninguno bajo peso para la edad gestacional. En 3 (5.2%) recién nacidos se observó: 1 malformación mayor (Síndrome de Down) y 2 menores (hernia umbilical e inguinal). Se obtuvo seguimiento de 42 recién nacidos; se diagnosticó epilepsia refractaria en uno y un trastorno generalizado del desarrollo en otro. No se halló una mayor frecuencia de complicaciones en los embarazos ni en los recién nacidos expuestos a CAB que en la población normal. Sería necesario mayor número de pacientes para concluir sobre la seguridad de CAB durante el embarazo.

Objectives: To assess the rate of any potential adverse effects on pregnancy and embryo-fetal development in women who became pregnant under treatment with cabergoline (CAB). To follow up medical data of children who were born from mothers exposed to Cab in early weeks of gestation. Material and methods: Observational, retrospective and multicenter study on 86 pregnancies in 78 women with idiopathic or tumoral hyperprolactinemia. All patients were under Cab at conception. The average age was 29 (range: 20-45). Pituitary images at diagnosis showed 44 microadenomas, 27 macroadenomas and 7 were normal. Serum PRL at baseline was between 30 and 1429 ng/ml. Duration of therapy before pregnancy ranged from 1 to 120 months. Maternal and fetal exposure to cabergoline and doses ranged from 0.125 to 4 mg/week. The mean serum PRL level under which patients achieved pregnancy was 17 ng/ml. Fetal exposure ranged from 3 to 27 weeks; 96.39% of patients received CAB during the first trimester of pregnancy and 3.61% until the second one. Results: No significant complications during pregnancy were found. Seven women (8.1%) had spontaneous abortions. Term deliveries were recorded in 63/69, preterm in six (8.8%), none of them with low weight for gestational age. Neonatal abnormalities were observed in 3 (5.2%): 1 major (Down syndrome) and 2 minor malformations (umbilical and inguinal hernia). Two out of 42, developed abnormalities during the follow- up, one of them was a refractory epilepsy during the second month of life, the other presented a Pervasive Developmental Disorder diagnosed in the third year of life. Conclusion: No significantly higher frequency of complications was found in pregnancies and/or offspring exposed to CAB than in normal population. Larger series of patients are needed to asses the safety.

Experience from the Argentine Pegvisomant Observational Study: preliminary data.

The GH receptor antagonist pegvisomant is an efficient agent to achieve biochemical control of acromegaly in those cases refractory to surgery and medical therapy with somatostatin analogs. We conducted an observational multicenter study consisting of data collection in accordance with the standard management of patients with acromegaly in everyday practice. We reviewed the medical records of 28 patients, 23 females, who were treated with pegvisomant due to the lack of biochemical response or intolerance to the somatostatin analogs. The objective was to monitor long-term safety and efficacy of the antagonist. 82% of the patients had previous pituitary surgery, 53.6% radiotherapy and 96.4% received medical therapy for acromegaly. Only 19.2% of the patients had pituitary residual tumor size larger than 1 cm, the remainder harbored a microadenoma or no visible tumor in the pituitary images. In terms of biochemical efficacy, IGF-I levels decreased to normal ranges in 45% and 58.8% of patients after 3 and 6 months of treatment, respectively, the daily mean dose of pegvisomant being 9.6+/-1.1 mg. Adverse events, potentially related to pegvisomant were reported in 6 patients (21.4%), local injection site reaction and elevated liver enzymes being the most frequent. Tumor size did not show enlargement in the evaluated population (15 patients) during the period of the study. This paper presents preliminary data from a small observational study in Argentina which represents the first database in our country.

Gonadotrofinomas: análisis multicéntrico y retrospectivo de 66 pacientes / Gonadotrophinomas: multicenter and retrospective analysis of 66 patients

Objetivo: Analizar la presentación clínica, radiológica, bioquímica y el comportamiento posquirúrgico de una cohorte de pacientes portadores de gonadotrofinomas. Pacientes y Métodos: Se evaluaron pacientes con gonadotrofinomas estudiados en nueve centros endocrinológicos de la ciudad de Bs.As. durante el período 1983 a 2003. El criterio de inclusión fue la inmunohistoquímica (IH) positiva para hormona luteinizante (LH), folículoestimulante (FSH) y/o alfa subunidad (ASU). Los adenomas plurihormonales fueron excluidos. Resultados: Fueron analizados 66 pacientes de 51,8 ± 12,1 (X +/- DS) años (39 varones). Los síntomas mas frecuentemente observados fueron las alteraciones visuales (72,8%), seguidas por el hipogonadismo y las cefaleas. El 10,6% se diagnosticaron en forma incidental. El 98,5% fueron macroadenomas, 56,9% de los cuales correspondieron a un estadio Hardy (EH) 3 y 29,6% a un EH 4. El tiempo de seguimiento fue de 47,8 meses (r: 5-168). El hipogonadismo definido bioquímicamente se presentó en el 82,4% de los pacientes. En su mayoría presentaban niveles bajos o inapropiadamente normales de gonadotrofinas, pero 4 mujeres y 3 varones presentaron niveles séricos elevados y disociados de FSH y LH. La hiperprolactinemia por desconexión fue observada en 45,2% de la población (X: 65.6 ng/ml r: 30-172). El hipopituitarismo se detectó en 25,7% de los casos. La cirugía fue transeptoesfenoidal (TSE) en 80%; una segunda operación fue realizada en el 28% de la población. La IH fue positiva por orden de frecuencia para LH, FSH y ASU o las 3 combinaciones. La evolución posquirúrgica evidenció mejoría en el campo visual (CV) en el 41%. La presencia de restos tumorales y/o recidiva fue del 84%. Se indicó radioterapia en 37% y la sustitución hormonal fue necesaria en el 65% de los pacientes.

The aim of our study was to describe the clinical-biochemical and radiologic presentation and the post surgery outcome in a cohort of patients with gonadotrophinomas. Patients were selected from nine Endocrinology Units of the city of Buenos Aires from 1983 at 2003. The inclusion criteria was defined by nonfunctinoning pituitary adenomas with positive innmunohistochemical (IH) for luteinizing hormone (LH), follicle-stimulating hormone (FSH) and/or alpha subunit (ASU). Innmunohistochemically plurihormonal adenomas were excluded. Sixty six patients were analyzed, aged 51,8 ± 12,1 (X +/- DS) years; (39 men). More prevalent symptoms were visual alterations (72,8%), hypogonadism and headaches. Eleven percent was diagnosed as incidentalomas. Ninety eight percent were macroadenomas, 56,9% was Hardy stage (HS) 3 and 29,6% was HS 4. The patients were followed up for 47,8 months (r: 5-168). Hypogonadism was biochemically found in 82,4%. The majority showed low or inappropriately normal levels of gonadotrophins except for 4 women and 3 men that had high and dissociated levels. Hyperprolactinemia was observed in 45,2% and was interpreted as an interference with normal dopamine inhibition of prolactin secretion (X+/-DS: 65.6+/- ng/ml, r: 30-172). Hypopituitarism was found in 25,7% of the patients. Transsphenoidal surgery was carried out in 80% and in 28% a second surgery was needed. The IH was positive for LH, FSH and ASU in this order of frequency or its combinations. Tumor persistency and/or recurrency were found in 84% of the patients. Forty one percent showed improvement of visual defects. Radiotherapy was indicated in 37% and hormonal replacement was needed in 65% of the patients.

Overcoming the radioresistance of prostate cancer cells with a novel Bcl-2 inhibitor.

Bcl-2 overexpression is an important mechanism underlying the aggressive behavior of prostate cancer cells and their resistance to radio- or chemotherapy. HA14-1, a recently discovered organic Bcl-2 inhibitor, potently induces apoptosis in various human cancer cells. Sequential exposure of radioresistant LNCaP (wild-type (wt) p53), LNCaP/Bcl-2 (wt p53) and PC3 (mutant p53) prostate cancer cells to a minimally cytotoxic concentration of 10 microM HA14-1 for 1 h followed by 1-6 Gy gamma radiation, resulted in a highly synergistic (combination index <1.0) induction of cell death as determined by an apoptosis assay at 72 h, and a clonogenicity assay at 12 days, after the initial treatment. The reverse treatment sequence did not cause a synergistic induction of cell death. When compared to individual treatments, cell death induced by the combined treatment was associated with dramatically increased reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, Bcl-2 phosphorylation, cytochrome c release, caspase-3 activation and DNA fragmentation. Exposure to either 200 microg/ml of the antioxidant alpha-tocopherol or 10 microM JNK inhibitor SP600125 before the combined treatment resulted in decreased activation of JNK and caspase-3 as well as decreased DNA fragmentation. However, treatment with the pancaspase inhibitor carbobenzoxyl-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone before the combined treatment inhibited apoptosis without affecting JNK activation, and this inhibitory effect was enhanced in the presence of alpha-tocopherol or SP600125. Taken together, our results indicate that HA14-1 potently sensitizes radioresistant LNCaP and PC3 cells to gamma radiation, regardless of the status of p53. ROS and JNK are important early signals that trigger both caspase-dependent and -independent cell death pathways and contribute to the apoptotic synergy induced by the combined treatments.

Estradiol stimulates vascular endothelial growth factor and interleukin-6 in human lactotroph and lactosomatotroph pituitary adenomas.

Estrogens are considered to be critically involved in lactotroph and lactosomatotroph pituitary tumor development. In addition to direct effects, estradiol-induced tumor formation may involve alterations in growth factor and cytokine production. We have studied whether estradiol stimulates the production of the angiogenic vascular endothelial growth factor and the potential tumor progression factor interleukin-6 in 5 lactotroph (LA) and 5 lactosomatotroph (LSA) human pituitary adenoma cell cultures. All tumors secreted heterogenous basal amounts of VEGF (18.0 +/- 1.4 to 425 +/- 26 pg/ml per 24 h) and IL-6 (18.1 +/- 1.5 to 604 +/- 17 pg/ml per 24 h). Estradiol (100 nM) significantly enhanced VEGF release in all LA and LSA cell cultures (47 to 168 % above basal). IL-6 secretion was stimulated in 3 out of 5 LA and in all LSA cell cultures (31 to 287 % above basal). In cell cultures obtained from tumors from which sufficient cells could be isolated, a dose-dependent effect of estradiol (1 to 100 nM) on VEGF and IL-6 production was observed. Stimulation of IL-6 and/or VEGF secretion by estradiol in the majority of human lactotroph and lactosomatotroph adenoma cell cultures studied, suggests that estrogens may contribute to adenoma expansion through the stimulation of these auto-/paracrine-acting adenoma progression factors.

Effects of the gp130 cytokines ciliary neurotropic factor (CNTF) and interleukin-11 on pituitary cells: CNTF receptors on human pituitary adenomas and stimulation of prolactin and GH secretion in normal rat anterior pituitary aggregate cultures.

Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor (LIF) and interleukin (IL)-6, which belong to the cytokine family using the common gp130 signal transducer. Recently, the expression and action of two other members of this family, IL-11 and ciliary neurotrophic factor (CNTF), on different cell lines has also been demonstrated. We studied the expression of the specific receptor subunits for CNTF in mammotropic, non-functioning and somatotropic tumors and the action of CNTF and IL-11 in the regulation of hormone secretion in these and normal pituitary cells. The mRNA for the alpha chain specific for the CNTF receptor was detected by Northern blot in tumors secreting prolactin (PRL) and GH and in non-functioning tumors. We found that both IL-11 and CNTF exerted a similar stimulatory effect on GH mRNA expression in somatotropic monolayer cell cultures from acromegalic tumors, but these cytokines had no significant influence on GH secretion. CNTF stimulates prolactin secretion in lactotropic monolayer cell cultures from patients with prolactinoma. In monolayer cell cultures from normal rat anterior pituitary, IL-11 and CNTF had no significant effect on the release of either GH or PRL, or on GH mRNA. However, when the cells were cultured in aggregate cultures, in which the three-dimensional structure of the cells is reconstituted, both cytokines, in doses at which they had no effect on monolayer cultures, significantly stimulated both PRL and GH secretion. These data show that IL-11 and CNTF may act as regulatory factors in anterior pituitary cells, in which the three-dimensional structure of the gland is of critical importance.

Interleukin-6 is inhibited by glucocorticoids and stimulates ACTH secretion and POMC expression in human corticotroph pituitary adenomas.

Interleukins and their receptors are expressed intrinsically in the anterior pituitary and regulate hormone production and cell proliferation. It has previously been shown that interleukin-6 (IL-6) regulates hormone secretion in normal pituitary cells and cell lines. Here we examined the effects of IL-6 on propiomelanocortin (POMC) expression and ACTH production in corticotroph adenoma cells in vitro. We found that IL-6 stimulates both ACTH secretion and POMC gene expression in corticotroph adenoma cell cultures. This first demonstration of the stimulatory action of IL-6 on human corticotroph adenoma cell function provides further evidence for a direct action of IL-6 on corticotroph pituitary cells. We have confirmed previous reports of IL-6 production by corticotroph adenoma cells and in addition, demonstrated for the first time that the synthetic glucocorticoid dexamethasone is a potent suppressor of intratumoral IL-6 production. This intratumoral produced IL-6 may be in part responsible, in an autocrine manner, for the stimulation of ACTH synthesis and secretion. Our results suggest that IL-6 might play a role in the pathogenesis of Cushing's disease. However, elevated glucocorticoid levels in patients with Cushing's disease may prevent excessive action of IL-6 on ACTH production and tumor progression of corticotroph adenomas in vivo.

Postsynaptic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: maintenance of core components independent of actin filaments and microtubules.

The mechanisms responsible for anchoring molecular components of postsynaptic specializations in the mammalian brain are not well understood but are presumed to involve associations with cytoskeletal elements. Here we build on previous studies of neurotransmitter receptors (Allison et al., 1998) to analyze the modes of attachment of scaffolding and signal transducing proteins of both glutamate and GABA postsynaptic sites to either the microtubule or microfilament cytoskeleton. Hippocampal pyramidal neurons in culture were treated with latrunculin A to depolymerize actin, with vincristine to depolymerize microtubules, or with Triton X-100 to extract soluble proteins. The synaptic clustering of PSD-95, a putative NMDA receptor anchoring protein and a core component of the postsynaptic density (PSD), was unaffected by actin depolymerization, microtubule depolymerization, or detergent extraction. The same was largely true for GKAP, a PSD-95-interacting protein. In contrast, the synaptic clustering of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)alpha, another core component of the PSD, was completely dependent on an intact actin cytoskeleton and was partially disrupted by detergent. Drebrin and alpha-actinin-2, actin-binding proteins concentrated in spines, were also dependent on F-actin for synaptic localization but were unaffected by detergent extraction. Surprisingly, the subcellular distributions of the inhibitory synaptic proteins GABA(A)R and gephyrin, which has a tubulin-binding motif, were unaffected by depolymerization of microtubules or actin or by detergent extraction. These studies reveal an unsuspected heterogeneity in the modes of attachment of postsynaptic proteins to the cytoskeleton and support the idea that PSD-95 and gephyrin may be core scaffolding components independent of the actin or tubulin cytoskeleton.

The gp130 cytokines interleukin-11 and ciliary neurotropic factor regulate through specific receptors the function and growth of lactosomatotropic and folliculostellate pituitary cell lines.

Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor and interleukin-6 (IL-6), which belong to the cytokine receptor family using the common gp130 signal transducer. We studied the actions of two other members of this family, IL-11 and ciliary neurotropic factor (CNTF), on folliculostellate (FS) cells (TtT/GF cell line) and lactosomatotropic cells (GH3 cell line). The messenger RNA (mRNA) for the alpha-chain specific for the IL-11 receptor (1.7 kb) and CNTF receptor (2 kb) are expressed on both cell types. In addition, we detected CNTF receptor mRNA in normal rat anterior pituitary cells. IL-11 (1.25-5 nM) dose dependently stimulated the proliferation of FS cells. CNTF, at doses from 0.4-2 nM, also significantly stimulated the growth of these cells. In addition, both cytokines significantly stimulated proliferation of lactosomatotropic GH3 cells, and CNTF stimulated hormone production (GH and PRL) at 24 h by these cells. At 16-72 h, IL-11 stimulates the secretion of the angiogenic factor vascular endothelial growth factor by FS cells. In addition, both GH3 and FS cells express CNTF mRNA. These data suggest that IL-11 and CNTF may act as growth and regulatory factors in anterior pituitary cells.

High levels of matrix metalloproteinases regulate proliferation and hormone secretion in pituitary cells.

Beside the digestion of the extracellular matrix during tumor invasion and metastasis, more recently, new functions for matrix metalloproteinases (MMPs) have been proposed. We studied the expression and function of these enzymes in pituitary cells. We observed the activities of MMP-2 and MMP-9 together with expression of membrane-type MMP and tissue inhibitor of metalloproteinase-1 in all types of human pituitary adenomas. We found surprisingly high levels of MMP activity and low levels of tissue inhibitor of metalloproteinases, indicating a high level of extracellular matrix-degrading activity in pituitary adenomas. To examine the function of metalloproteinase activity in pituitary cells we used the synthetic MMP inhibitor batimastat. These studies demonstrate that MMPs secreted by pituitary cells can release growth factors anchored to the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. These results define a new additional mechanism for the control of pituitary hormone secretion and indicate new potential therapeutic targets for pituitary adenomas.

Evaluation of disease activity by IGF-I and IGF binding protein-3 (IGFBP3) in acromegaly patients distributed according to a clinical score.

UNLABELLED: To facilitate the estimation of acromegalic activity a prospective study was done comparing, against a clinical score, the effectiveness of serum IGF-I, IGFBP3 and the IGF-I/IGFBP3 molar ratio. Sixty nine observations were distributed in three groups: Group I=patients before surgery; group II=patients improved but still clinically active; group III=patients clinically inactive. Suppression of serum GH levels one hour after an oral glucose load was in agreement with the clinical score in 21/22 observations. Increases in serum IGF-I and IGFBP3 levels were similarly frequent: both 100% in group I, 80% and 95% in group II, 9% and 36% in group III, respectively. The frequency of abnormal molar ratios was 95%, 40% and 0% in the same groups. Log IGF-I, log IGFBP3, and log molar ratio correlated significantly with the clinical scores (r=0.873, r=0.692, and r=0.829, respectively). CONCLUSIONS: The IGF-I/IGFBP3 molar ratio was not better than either IGF-I or IGFBP3 in detecting activity in the three groups of patients studied. Both IGF-I and IGFBP3 appear comparably useful for the diagnosis and follow-up of acromegalic patients. Since IGF-I is a more biologically meaningful parameter it might be preferable.

Elevated proenkephalin-derived peptide levels in ACTH-producing adenomas: nucleus and cytoplasm localization.

The biosynthesis of met-enkephalin in human pituitary and human pituitary adenomas is still not well known. In this work, we studied the processing of proenkephalin-derived peptides in postmortem human pituitary (PMHP), ACTH-producing adenomas (ACTH-PA), nonfunctioning adenomas (NFA), and GH-producing adenomas (GH-PA). ACTH-PA contained at least 10 times more proenkephalin-derived peptides than PMHP, NFA,and GH-PA. Proenkephalin processing was different in the four tested tissues. In ACTH-PA, proenkephalin was processed to high-, intermediate-, and low-mol-wt products. The highest met-enkephalin-containing peptides levels corresponded to intermediate and low-mol-wt materials, although met-enkephalinArg-Phe and synenkephalin immunoreactivity appeared only in high-mol-wt peptides. In PMHP and NFA, met-enkephalin-Arg-Phe immunoreactivity was detected in intermediate- and low-mol-wt materials, and it was absent in GH-PA. Immunoblotting of ACTH-PA showed that met-enkephalin-Arg-Phe immunoreactivity corresponded to peptides of 44, 32-30, 27, and 17 kDa. The 32-30 and 17-kDa molecules were localized in the nuclear fraction where they were extracted after enzymatic digestion with DNase I. Plasmatic met-enkephalin levels did not increase in patients with Cushing's disease, suggesting that the pentapeptide stored in ACTH-PA was not released to the general circulation. In conclusion, we demonstrated that only ACTH-PA contained high levels of proenkephalin peptides, which were stored in cytoplasm organelles and in the nucleus, probably bound to chromatin. These results suggest an adenoma-specific physiological role of proenkephalin products.

[Treatment of hyperprolactinemic amenorrhea with cabergoline]. / Tratamiento de amenorreas hiperprolactinémicas con cabergolina.

Cabergoline (CAB) is a long-acting dopamine agonist. In the first national study with CAB--as part of an international multicentric study--39 adult and adolescent females (16 to 44 years old) with hyperprolactinemic amenorrhea (18 microadenomas and 21 idiopathic hyperprolactinemias) were evaluated. CAB or bromocriptine (BEC) was administered for 24 weeks: over 8 weeks, treatment was given under double-blind conditions, and over the remaining 16 weeks (open period) 18 patients received CAB and 21 received BEC as a result of a random distribution. Maximum dosage: CAB = 1.5 mg in 2 or 3 weekly doses; BEC = up to 10 mg in 2 daily doses. Prolactin was measured at base line and 2, 4, 6, 8, 12, 14, 16, 20 and 24 weeks after the initiation of treatment. When vaginal bleeding was restored, progesterone was measured as an ovulation sign. The 4 adolescents continued with CAB treatment for 1 more year. Prolactin was statistically evaluated according to Man Whitney Test (general population) or Wilcoxon Test (adolescents). There were no significant differences between basal levels of prolactin (ng/ml) in patients treated with BEC or CAB: (173.86 +/- 28.23 and 152.11 +/- 14.06 respectively); at the fourth week of treatment the decrease was smaller (p = 0.005) in patients treated with BEC (36.36 +/- 5.71) than in those treated with CAB (14.06 +/- 3.60) and at 24 weeks differences disappeared: BEC = 19.88 +/- 4.48 and CAB = 9.63 +/- 2.62 (p = NS). The adolescents showed a marked decrease in prolactin with no significant differences between BEC and CAB: basal levels = 168.17 +/- 75.47 and 213 +/- 96.99 (p = NS); 4 weeks = 48.00 +/- 8.72 and 35.00 +/- 12.58 (p = NS); 24 weeks = 34.33 +/- 10.17 and 21.75 +/- 7.23 respectively. At 48 weeks (23.25 +/- 11.23) levels remained the same as those of week 24 (p = NS). Some patients treated with BEC had nausea, vomits and epigastralgia; these symptoms were not observed with CAB. All patients resumed menstrual cycles, except one treated with BEC; 6 patients treated with CAB became pregnant, and the 5 patients who continued under our control gave birth to healthy infants. It is concluded that CAB is a useful therapy. This is specially true for adolescents (an age group difficult to manage) because of its easy administration and the almost complete absence of side effects.

Interleukin-2 (IL-2) and IL-6 regulate c-fos protooncogene expression in human pituitary adenoma explants.

We have previously shown that interleukin-2 (IL-2) and IL-6, which are expressed in the anterior pituitary, affect anterior pituitary cell proliferation in normal rats and cell lines. Here we examined their effects on the c-fos expression by human anterior pituitary adenomas. Adenoma cells in culture do not express c-fos mRNA. In adenoma explants, however, c-fos expression was detected and was regulated by IL-2 or IL-6. In different tumors (ACTH-, PRL-, GH-secreting and non functioning adenomas), these interleukins had inhibitory or stimulatory effects but the kind of response does not seem to be associated to tumor type or size. Using blocking antibodies, we observed that intrinsic IL-2 and IL-6 regulate c-fos expression in the same way. Our data suggest that IL-2 and IL-6 are not only involved in the regulation of pituitary adenoma function but may also, given the role of c-fos in cell proliferation, be implicated in the development of human pituitary adenomas.