Estudios del laboratorio de diversos parámetros orientados a tratar de dilucidar la fisiopatología del síndrome de ovarios poliquísticos (SOP). Parte 2 / Laboratory studies of several parameters aimed to elucidate the pathophysiology of polycystic ovary syndrome. Second part

RESUMEN Diversos estudios bioquímicos adicionales a la evaluación de Testosterona total (TT), biodisponible (Tbio) y libre (TL) han sido realizados a los efectos que pudieran resultar de mayor utilidad para el diagnóstico de patologías concomitantes en el SOP, entre otros. En la hormona anti Mülleriana, cuando la concentración supera a los 3,0 ng/ml existen evidencias de que el 79% de las mismas pueden ser identificadas correctamente como SOP. El Antígeno Prostático Específico (PSA), marcador de singular importancia en pacientes con cáncer de Próstata, con técnicas ultrasensibles ha podido ser detectado en más del 50% en mujeres. En un grupo de pacientes con SOP, los niveles circulantes de PSA fueron significativamente mayores que en las mujeres sin SOP. El Kiss-1 aislado de la placenta y demostrado en otros tejidos, presenta niveles aumentados que correlacionan con la LH, TT, TL y resistencia a la insulina (RI) en adolescentes con SOP versus adolescentes sin SOP, sugiriendo que el Kiss-1 podría estar involucrado en el desarrollo del SOP en estas pacientes. Algunas pacientes con SOP están asociadas a patologías relevantes, de las cuales han sido comunicadas el aumento del BMI, mayor grado de dislipemia, adiposidad central, RI y Síndrome Metabólico (SMe). En las pacientes con un fenotipo clásico (hiperandrogenismo, alteración del ciclo menstrual y ovarios poliquísticos), estas patologías son de mayor frecuencia y severidad que en los otros fenotipos, particularmente aquellos sin hiperandrogenismo. Otras determinaciones como TNFα, interleuquinas, test de tolerancia a la glucosa, ApoB, partículas pequeñas de LDL e Inhibidor del Activador del Plasminógeno-1 han sido comunicados que podrían ser de utilidad para tener mayor sensibilidad en la definición de patología concomitantes en el SOP. Actualmente se ha comenzado a evaluar otros marcadores como el Fetuin-A; Quemerina, Nesfatina-1, Neopterina y Endocannabinoides, cuyos resultados preliminares parecerían ser un aporte importante para evaluar SMe y RI en paciente con SOP y tratar de definir su prevalencia en los distintos fenotipos de esta patología.

ABSTRACT Several biochemical studies in addition to the evaluation of total Testosterone (TT), bioavailable (bioT) and free (FT) have been performed to the effects that could be of greater use for the diagnosis of concomitant pathologies in the PCOS, among others. The anti-Müllerian hormone whose concentration when exceeds 3.0 ng/ml, there is evidence that 79% of these patients can be correctly identified as PCOS. The Prostate-Specific Antigen (PSA), a marker of singular importance in patients with prostate cancer, with ultra-sensitive techniques, has been detected in more than 50% of women. In a group of patients with PCOS, circulating levels of PSA are significantly higher than in women without PCOS. The Kiss-1 isolated from the placenta and demonstrated in other tissues, has increased levels that correlate with LH, TT, TL and insulin resistance (IR) in adolescents with PCOS respect to adolescents without PCOS, suggesting that Kiss-1 could be involved in the development of the PCOS in these patients. In some patients with PCOS, they are associated with relevant pathologies, of which the increase in BMI, higher degree of dyslipidemia, central adiposity, IR and Metabolic Syndrome (MeS) have been reported. Those that show a classic phenotype (hyperandrogenism, alteration of the menstrual cycle and polycystic ovaries) these characteristics are of greater frequency and severity than in the other phenotypes, particularly those without hyperandrogenism. Other determinations such as TNFα, interleukins, glucose tolerance test, ApoB, small particles of LDL and Plasminogen Activator Inhibitor-1 have been reported that could be useful to have greater sensitivity in the definition of concomitant pathology in the PCOS. Currently, other markers such as Fetuin-A, Chemerin, Nesfatin-1 Neopterin and Endocannabinoids have been evaluated. The preliminary results suggest to be an important contribution to define MeS and IR in patient with PCOS and to try to determine its prevalence in the different phenotypes of this pathology.

Estudios de laboratorio de diversos parámetros orientados a tratar de dilucidar la fisiopatología del síndrome de ovarios poliquísticos. Parte 1 / Laboratory studies of various parameters aimed at trying to elucidate the pathophysiology of polycystic ovary syndrome. Part 1

Esta revisión fue realizada con el fin de evaluar nuestros resultados de laboratorio así como aquellos de la literatura que constituyen, a nuestro entender, aportes significativos en el síndrome de ovarios poliquísticos (SOP). Nuestro especial énfasis será presentar las limitaciones de las metodologías empleadas por nuestro grupo, comparativamente a las reportadas por otros investigadores. La determinación de andrógenos, en particular de Testosterona (TT), es quizá la de mayor complejidad dado que los resultados con los diferentes inmunoensayos empleados en nuestro medio producen resultados muy variables por los diferentes métodos y aún entre laboratorios que usan la misma metodología. La técnica de referencia es la cromatografía líquida en tándem con espectrometría de masa (LC-MSMS), de difícil aplicación en laboratorios de análisis clínicos debido a su alto costo y la imposibilidad de resolver numerosas muestras. En estudios previos demostramos que de los métodos habitualmente usados para evaluar la TT circulante, solo en 2 inmunoensayos los resultados obtenidos fueron satisfactoriamente validados indirectamente según el criterio del Consenso de los Centros para el Control y Prevención de Enfermedades (CDC, USA) contra LC-MSMS, los cuales fueron comparables a dicha metodología con niveles superiores a 0,5 ng/ml. El SOP puede presentar factores de riesgo aumentados para la enfermedad cardiovascular y la diabetes II. Estos factores no están debidamente categorizados en función de los distintos fenotipos del SOP. Se evaluarán los principales analitos empleados con este objetivo y los nuevos que aporten elementos de mayor especificidad en este sentido

This review was performed in order to evaluate our laboratory results as well as those of the literature that constitute, in our opinion, significant contributions in these pathophysiologies. Our special emphasis will be on presenting the limitations of the methodologies used by our group, compared to those reported by other researchers. The determination of androgens, in particular Testosterone (TT), is perhaps the most complex since the results with the different immunoassays used in our environment produce very variable results by the different methods and even between laboratories that use the same methodology. The reference technique is LC-MSMS, difficult to apply in clinical analysis laboratories because of its high cost and the inability to solve numerous samples. In previous studies, we demonstrated that, in comparison to LC-MSMS with the usual methods for evaluating circulating TT, the results obtained in only 2 immunoassays were satisfactorily validated indirectly according to the criteria of CDC against LC-MSMS, which were comparable to that methodology with levels higher than 0.5 ng/ml. PCOS may have increased risk factors for cardiovascular disease and diabetes II. These factors are not properly categorized according to the different phenotypes of PCOS. The main analytes used for this purpose will be evaluated and new ones that contribute elements of greater specificity in this sense

CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis.

Current therapies to treat inflammatory bowel diseases have limited efficacy, significant side effects, and often wane over time. Little is known about the cellular and molecular mechanisms operative in the process of mucosal healing from colitis. To study such events, we developed a new model of reversible colitis in which adoptive transfer of CD4(+)CD45RB(hi) T cells into Helicobacter typhlonius-colonized lymphopenic mice resulted in a rapid onset of colonic inflammation that was reversible through depletion of colitogenic T cells. Remission was associated with an improved clinical and histopathological score, reduced immune cell infiltration to the intestinal mucosa, altered intestinal gene expression profiles, regeneration of the colonic mucus layer, and the restoration of epithelial barrier integrity. Notably, colitogenic T cells were not only critical for induction of colitis but also for maintenance of disease. Depletion of colitogenic T cells resulted in a rapid drop in tumor necrosis factor α (TNFα) levels associated with reduced infiltration of inflammatory immune cells to sites of inflammation. Although neutralization of TNFα prevented the onset of colitis, anti-TNFα treatment of mice with established disease failed to resolve colonic inflammation. Collectively, this new model of reversible colitis provides an important research tool to study the dynamics of mucosal healing in chronic intestinal remitting-relapsing disorders.

A Comparison of Serum Testosterone (T) Measurements by Various Methods, Employing Current Validated Techniques or Otherwise, and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Multicentric Study / Comparación de los resultados de testosterona (T) por cromatografía líquida en tandem con espectrometría de masa (LC-MS/MS) y por kits comerciales convalidados o no por LC-MS/MS

Objective: To compare normal and hirsute women Testosterone (T) measurements performed at different laboratories by the same or different methods, and the gold standard method LC-MS/MS (Quest Diagnostics, USA). Design: Prospective study. Setting: Hormone Determination Laboratory, Hospital Italiano, La Plata, and each participating laboratory's private practice. Patient(s): Blood samples were obtained from 23 individuals sorted into two groups, namely, normal women, n: 11(NW) and hirsute women, n: 12 (HW). Interventions(s): None. Main Outcome Measure(s): To evaluate whether serum T measurements obtained from each serum by the methods currently employed in our country, some of whose kits exhibit changes in previous presentations, some LC-MS/MS-validated and other non-validated ones are significantly different from those obtained by LC-MS/MS. Result(s): None of the 11 NW showed high T values by LC-MS/MS. Two out of the 12 hirsute patients showed normal T values (LC-MS/MS). Methods and number of participating labs -shown between brackets were: in NW, 1st generation Architect (1), 2nd generation Architect (1); Immulite (1) Cobas (4); Access (1); Centaur (2); Immunotech-RIA (1); and, in HW, 2nd generation Architect (3); Immulite (3); Cobas (4); Access (1); Centaur (2); Immunotech-RIA (1). No false positives resulted from the assays performed. No lab yielded false positive results in the NW group. No false positives were reported from the 10 hirsute women with increased T values by LC-MS/MS. False positives, though, resulted from two female hirsute patients with normal T values studied by four of the methods. Statistically, the serum T measurements obtained were significantly different by Centaur in NW and, in HW, by Immulite and Centaur as compared to LC-MS/MS. In the Bland-Altman plot, Centaur and Cobas showed over 5 % of measurements outside the limits of agreement in the HW group. Assessment by p-Spearman resulted in divergences with LC-MS/MS for all methods in NW, whereas in the HW group there were none. When estimating sampling bias for each laboratory taking LC-MS/MS as the reference method and adopting a ± 6.4 % mean bias acceptability criterion for each method compared to LC-MS/MS, two of the techniques reviewed, 2nd generation Architect and Cobas, met the validation requirement satisfactorily. However, one lab out of three using 2nd generation Architect failed to meet the validation requirement, while two out of four labs using Cobas also failed to meet the requirement. This demonstrates the great variability among methods, even when labs are employing the same technique. Conclusion: From the clinical point of view, the methods currently used in our local environment yielded no false positives or false negatives and therefore did not misdiagnose hyperandrogenism. Still, Immulite, Centaur, RIA and Access did present false positives in two of the T-normal hirsute women. The relation of serum T measurements obtained by each method to measurements obtained by LC-MS/MS reveals that the dispersion of the results was larger with values under 0.3 ng/ml, quite close to the detection limit of the various techniques.

Objetivos: Comparar los resultados de testosterona (T) obtenidos en diferentes laboratorios en mujeres normales (MN) e hirsutas (MH), empleando el mismo o diferentes métodos respecto a la técnica de cromatografía líquida en tándem con espectrometría de masa LC-MS/MS (gold standard) realizada en el laboratorio Quest (USA) Protocolo: Estudio prospectivo Estudio realizado en: Laboratorio de Determinaciones Hormonales; Hospital Italiano de La Plata y en los laboratorios privados de cada participante Pacientes: Se obtuvieron muestras de sangre periférica en 23 mujeres agrupadas en 2 grupos: controles normales (n:11) y en mujeres hirsutas (n: 12) Intervención: Ninguna. Principales resultados a evaluar: Evaluar si con los métodos habituales empleados en nuestro medio con los diferentes kits comerciales, de los cuales algunos han sido convalidados por técnica "gold standard" y otros no, presentan diferencias significativas con los obtenidos por LC-MS/MS Resultados: Los resultados obtenidos por LC-MS/MS mostraron que ninguna de las 11 MN tuvieron niveles aumentado de T y 2 MH tuvieron valores normales de T. Los métodos empleados y el número de laboratorios (entre paréntesis) que emplearon cada método fueron en MN Architect 1st generation (1) Architect 2nd generation (1); Immulite (1); Cobas (4); Access (1); Centauro (2); Immunotech-RIA (1). En las MH Architect 2nd generation (3); Immulite (3); Cobas (4); Access (1); Centauro (2); Immunotech-RIA (1). En el grupo de MN en ningún laboratorio (lab) se obtuvieron resultados falsos positivos. En el grupo de MH no se obtuvieron falsos negativos en las 10 hirsutas con valores aumentados de T por LC-MS/MS. En las 2 pacientes hirsutas con T normal en 4 métodos se obtuvieron falsos positivos Estadísticamente los resultados fueron significativamente diferentes, en las MN por Centauro y en las MH por Immulite y Centauro. En el análisis de Bland-Altman Centauro y Cobas en las MH presentaron más del 5 % de los resultados fuera del límite de acuerdo. Resultados por p-Speerman todos los métodos fueron diferentes a LC-MSMS en las MN y no se obtuvieron diferencias en el grupo de MH. Evaluando el bias de cada muestra en cada laboratorio respecto a LC-MS/MS y adoptando el criterio de aceptabilidad de ± 6,4 % mean bias de cada método respecto al de LC-MS/MS, 2 de las metodologías estudiadas, Architect 2da generación y Cobas pasaron satisfactoriamente el requisito de validación, sin embargo de los 3 laboratorios que emplearon 2da generación, 1 no pasó el criterio de validación y de los 4 que usaron Cobas, 2 tampoco lo pasaron. Esto demuestra la gran variabilidad de los métodos aun entre lab que emplean la misma técnica. Conclusiones: Desde el punto de vista clínico los métodos habitualmente empleados en nuestro medio, no sobrediagnosticaron o subdiagnosticaron hiperandrogenismo, por no presentar falsos positivos o negativos respectivamente. Sin embargo Immulite, Centauro, RIA y Access presentaron falsos positivos en las 2 hirsutas con T normal. En la relación de los resultados de cada muestra en cada método sobre el valor de LC-MS/MS referido a la concentración de T en ese suero por LC-MS/MS, la mayor dispersión de los resultados se observaron con valores menores de 0,3 ng/ml, muy cercano al límite de detección de las diferentes técnicas.

Quantification of Testosterone (T) by 8 Immunoassays and by Liquid Chromatography -Tandem Mass Spectrometry (LC-MSMS) in Normal and Hirsute Women: A Multicenter Study / Cuantificación de testosterona (T) por 8 inmunoensayos y por cromatografía líquida en tandem con espectrometría de masa (LC MS/MS): En mujeres normales e hirsutas. Estudio multicéntrico

Objective: To compare T results in normal and hirsute women, obtained by different laboratories employing the same or different methods, including an in-home RIA, and the gold standard method LC-MS/MS. In addition, T results were referred to a curve obtained by 6 different pools that had been prepared on the basis of LC-MS/MS results. Design: Prospective study Setting: Hormone Determination Laboratory, Hospital Italiano, La Plata, and private practice of each participant laboratory. Patient(s): Blood samples were obtained from 78 individuals sorted into 3 groups, namely, normal men (n:39), normal women (n:24) and hirsute women (n:15) Interventions(s): None Main Outcome Measure(s): To evaluate if the results obtained in each lab for each serum sample by the methods currently employed in our country are significantly different from those obtained by LC-MS/MS (Gold standard) Result(s) One out of the 24 NW showed high T values by LC - MS/MS. In each lab, except in 1 (Architect) T results of this serum sample were normal. Two out of the 15 hirsute patients showed normal T values (LC - MS/MS). Method and number of labs -shown between brackets- and percentages of normal T results (false negatives) are described for each method as follows: Chemiluminescence: Axsym - Abbott (Axn) - (3) 85, Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electrochemiluminescence - Elecsys - Roche- ((EQL); (4) 52; Fluorescent enzymatic - Vidas - Bio-Merieux - (Vidas) (1) 69; Manual coated tube radioimmunoassay (RIA): RIA - Siemmens Coat-a-Count (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; and in-Home RIA (in-H) (1) 12. Statistically significant differences were obtained between different methods and against LC MS/MS. In-H method is the one that comes closest to 1 on the Weighted Deming regression and closest to zero on the SD intercept, (standard deviation of the constant in the straight line equation) indicating that the values match those obtained by LC - MS/MS. The values recorded by the various methods employed showed no significant modifications when plotted against a secondary standard curve. Conclusion(s) This indicates that the techniques in current use in our area underestimate hyperandrogenemia in these patients. Discrepancies are not due to the various calibration curves proposed in the corresponding commercial kits. The fact that the In-H technique affords finer results while employing a larger serum volume suggests that the disparities among the various commercial methods result from their limited sensitivity to the sample volumes they process.(AU)

El diagnóstico de hiperandrogenemia requiere la demostración de niveles aumentados de Testosterona Total (TT) en suero. Los inmuno ensayos comerciales dan resultados divergentes a niveles bajos de TT como los obtenidos en mujeres. Valoramos los niveles de TT en 24 mujeres normales (MN) y 15 hirsutas (MH) en 18 laboratorios por métodos comúnmente empleados en nuestro medio. Los métodos y número de laboratorio que emplearon cada método se muestra entre paréntesis así como los porcentajes de T normal (falsos negativos) obtenidos en cada método fueron: Quimioluminiscencia: Axsym - Abbott (Axn)- (3) (85), Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electroquimioluminiscencia - Elecsys - Roche- ((EQL); (4) 52; Enzimático acoplado a fluorescencia Vidas - Bio-Merieux - (Vidas) (1) 69, Radioinmunoiensayo en tubo recubierto (RIA): RIA - Siemmens (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; y un método desarrollado en uno de los laboratorios (in-H) (1) 12. Comparativamente a LC MS/MS los niveles fueron en todas las muestras significativamente más bajos por Axn y en 18 de las 24 MN por DiaS. En 7 casos; 3 por RIA S, 2 por IMM y 1 por EQL y Arch los valores de TT fueron superiores al límite superior de sus respectivos métodos. En todos los casos se obtuvo una gran variación entre los mismos y con diferentes métodos. Trece de las 15 MH tuvieron niveles altos de TT por LC MS/MS. De las MH con TT aumentada de acuerdo a la determinación por LC MS/MS entre el 12 y el 85 % de las mismas por los distintos métodos fueron normales, indicando que en la mayoría de los métodos habitualmente utilizados en nuestro medio subvaloran la hipernadrogenemia en estas pacientes. Estas diferencias se hacen más notorias a niveles más bajos de TT (Se obtuvieron valores normales en el 71 % de los casos con valores de TT entre 0,47 y 0,74 ng/ml y en el 38 % de los casos, con niveles de TT mayor a 0,98 ng/ml). ...(AU)

Quantification of Testosterone (T) by 8 Immunoassays and by Liquid Chromatography -Tandem Mass Spectrometry (LC-MSMS) in Normal and Hirsute Women: A Multicenter Study / Cuantificación de testosterona (T) por 8 inmunoensayos y por cromatografía líquida en tandem con espectrometría de masa (LC MS/MS): En mujeres normales e hirsutas. Estudio multicéntrico

Objective: To compare T results in normal and hirsute women, obtained by different laboratories employing the same or different methods, including an in-home RIA, and the gold standard method LC-MS/MS. In addition, T results were referred to a curve obtained by 6 different pools that had been prepared on the basis of LC-MS/MS results. Design: Prospective study Setting: Hormone Determination Laboratory, Hospital Italiano, La Plata, and private practice of each participant laboratory. Patient(s): Blood samples were obtained from 78 individuals sorted into 3 groups, namely, normal men (n:39), normal women (n:24) and hirsute women (n:15) Interventions(s): None Main Outcome Measure(s): To evaluate if the results obtained in each lab for each serum sample by the methods currently employed in our country are significantly different from those obtained by LC-MS/MS (Gold standard) Result(s) One out of the 24 NW showed high T values by LC - MS/MS. In each lab, except in 1 (Architect) T results of this serum sample were normal. Two out of the 15 hirsute patients showed normal T values (LC - MS/MS). Method and number of labs -shown between brackets- and percentages of normal T results (false negatives) are described for each method as follows: Chemiluminescence: Axsym - Abbott (Axn) - (3) 85, Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electrochemiluminescence - Elecsys - Roche- ((EQL); (4) 52; Fluorescent enzymatic - Vidas - Bio-Merieux - (Vidas) (1) 69; Manual coated tube radioimmunoassay (RIA): RIA - Siemmens Coat-a-Count (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; and in-Home RIA (in-H) (1) 12. Statistically significant differences were obtained between different methods and against LC MS/MS. In-H method is the one that comes closest to 1 on the Weighted Deming regression and closest to zero on the SD intercept, (standard deviation of the constant in the straight line equation) indicating that the values match those obtained by LC - MS/MS. The values recorded by the various methods employed showed no significant modifications when plotted against a secondary standard curve. Conclusion(s) This indicates that the techniques in current use in our area underestimate hyperandrogenemia in these patients. Discrepancies are not due to the various calibration curves proposed in the corresponding commercial kits. The fact that the In-H technique affords finer results while employing a larger serum volume suggests that the disparities among the various commercial methods result from their limited sensitivity to the sample volumes they process.

El diagnóstico de hiperandrogenemia requiere la demostración de niveles aumentados de Testosterona Total (TT) en suero. Los inmuno ensayos comerciales dan resultados divergentes a niveles bajos de TT como los obtenidos en mujeres. Valoramos los niveles de TT en 24 mujeres normales (MN) y 15 hirsutas (MH) en 18 laboratorios por métodos comúnmente empleados en nuestro medio. Los métodos y número de laboratorio que emplearon cada método se muestra entre paréntesis así como los porcentajes de T normal (falsos negativos) obtenidos en cada método fueron: Quimioluminiscencia: Axsym - Abbott (Axn)- (3) (85), Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electroquimioluminiscencia - Elecsys - Roche- ((EQL); (4) 52; Enzimático acoplado a fluorescencia Vidas - Bio-Merieux - (Vidas) (1) 69, Radioinmunoiensayo en tubo recubierto (RIA): RIA - Siemmens (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; y un método desarrollado en uno de los laboratorios (in-H) (1) 12. Comparativamente a LC MS/MS los niveles fueron en todas las muestras significativamente más bajos por Axn y en 18 de las 24 MN por DiaS. En 7 casos; 3 por RIA S, 2 por IMM y 1 por EQL y Arch los valores de TT fueron superiores al límite superior de sus respectivos métodos. En todos los casos se obtuvo una gran variación entre los mismos y con diferentes métodos. Trece de las 15 MH tuvieron niveles altos de TT por LC MS/MS. De las MH con TT aumentada de acuerdo a la determinación por LC MS/MS entre el 12 y el 85 % de las mismas por los distintos métodos fueron normales, indicando que en la mayoría de los métodos habitualmente utilizados en nuestro medio subvaloran la hipernadrogenemia en estas pacientes. Estas diferencias se hacen más notorias a niveles más bajos de TT (Se obtuvieron valores normales en el 71 % de los casos con valores de TT entre 0,47 y 0,74 ng/ml y en el 38 % de los casos, con niveles de TT mayor a 0,98 ng/ml). ...

TRAIL enhances paracetamol-induced liver sinusoidal endothelial cell death in a Bim- and Bid-dependent manner.

Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage.

Quantification of Testosterone (t) by 8 Immunoassays and by Liquid Chromatography -Tandem mass Spectrometry (lc -msms) in Normal and Hirsute Women: A Multicenter Study / Cuantificación de testosterona (t) por 8 inmunoensayos y por cromatografía líquida en tandem con espectrometría de masa (Lc ms/ms): En mujeres normales e hirsutas. Estudio multicéntrico

Objective: To compare T results in normal and hirsute women, obtained by different laboratories employing the same or different methods, including an in-home RIA, and the gold standard method LC-MS/MS. In addition, T results were referred to a curve obtained by 6 different pools that had been prepared on the basis of LC-MS/MS results. Design: Prospective study Setting: Hormone Determination Laboratory, Hospital Italiano, La Plata, and private practice of each participant laboratory. Patient(s): Blood samples were obtained from 78 individuals sorted into 3 groups, namely, normal men (n:39), normal women (n:24) and hirsute women (n:15) Interventions(s): None Main Outcome Measure(s): To evaluate if the results obtained in each lab for each serum sample by the methods currently employed in our country are significantly different from those obtained by LC-MS/MS (Gold standard) Result(s) One out of the 24 NW showed high T values by LC - MS/MS. In each lab, except in 1 (Architect) T results of this serum sample were normal. Two out of the 15 hirsute patients showed normal T values (LC - MS/MS). Method and number of labs -shown between brackets- and percentages of normal T results (false negatives) are described for each method as follows: Chemiluminescence: Axsym - Abbott (Axn) - (3) 85, Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electrochemiluminescence - Elecsys - Roche- ((EQL); (4) 52; Fluorescent enzymatic - Vidas - Bio-Merieux - (Vidas) (1) 69; Manual coated tube radioimmunoassay (RIA): RIA - Siemmens Coat-a-Count (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; and in-Home RIA (in-H) (1) 12. Statistically significant differences were obtained between different methods and against LC MS/MS. In-H method is the one that comes closest to 1 on the Weighted Deming regression and closest to zero on the SD intercept, (standard deviation of the constant in the straight line equation) indicating that the values match those obtained by LC - MS/MS. The values recorded by the various methods employed showed no significant modifications when plotted against a secondary standard curve. Conclusion(s) This indicates that the techniques in current use in our area underestimate hyperandrogenemia in these patients. Discrepancies are not due to the various calibration curves proposed in the corresponding commercial kits. The fact that the In-H technique affords finer results while employing a larger serum volume suggests that the disparities among the various commercial methods result from their limited sensitivity to the sample volumes they process. No financial conflicts of interest exist.(AU)

El diagnóstico de hiperandrogenemia requiere la demostración de niveles aumentados de Testosterona Total (TT) en suero. Los inmuno ensayos comerciales dan resultados divergentes a niveles bajos de TT como los obtenidos en mujeres. Valoramos los niveles de TT en 24 mujeres normales (MN) y 15 hirsutas (MH) en 18 laboratorios por métodos comúnmente empleados en nuestro medio, Quimioluminiscencia: Axsym - Abbott (Axn)- (3 ), Architect - Abbott - (Arch); (2); Immulite - Siemmens - (IMM); (2); Electroquimioluminiscencia - Elecsys - Roche- ((EQL); (4); Enzimático acoplado a fluorescencia Vidas - Bio-Merieux - (Vidas) (1), Radioinmunoiensayo en tubo recubierto (RIA): RIA - Siemmens (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; y un metodo desarrollado en uno de los laboratorios (in-H) (1).El número entreparéntesis indica elnúmero de laboratorios que emplearon la misma técnica,y comparamos los resultados por LC MS/MS. Comparativamente a LC MS/MS los niveles fueron en todas las muestras significativamente más bajos por AXS y en 18 de las 24 MN por DiaS. En 7 casos; 3 por RIA S, 2 por IMM y 1 por EQL y Arch los valores de TT fueron superiores al límite superior de sus respectivos métodos. En todos los casos se obtuvo una gran variación entre los mismos y con diferentes métodos. Trece de las 15 MH tuvieron niveles altos de TT por LC MS/MS. De las MH con TT aumentada de acuerdo a la determinación por LC MS/MS entre el 12 y el 85 % de las mismas por los distintos métodos fueron normales, indicando que en la mayoría de los métodos habitualmente utilizados en nuestro medio subvaloran la hipernadrogenemia en estas pacientes. Estas diferencias se hacen más notorias a niveles más bajos de TT (Se obtuvieron valores normales en el 71 % de los casos con valores de TT entre 0.47 y 0.74 ng/ml y en el 38 % de los casos, con niveles de TT mayor a 0.98 ng/ml). En 9 muestras se determinó la TT empleando una curva en el rango de 0.21 a 6.44 ng/ml preparada con de una mezcla de 78 sueros cuyos valores fueron obtenidos por LC MS/MS. No se obtuvo una modificación significativa de los valores indicando que la diferencia entre los distintos métodos no es debida a las diferentes curvas de calibración de los kit comerciales. En conclusión ninguno de los métodos mayormente empleados en nuestro medio son aceptables para la evaluación de niveles menores a 1.5 ng/ml.(AU)

Quantification of Testosterone (t) by 8 Immunoassays and by Liquid Chromatography -Tandem mass Spectrometry (lc -msms) in Normal and Hirsute Women: A Multicenter Study / Cuantificación de testosterona (t) por 8 inmunoensayos y por cromatografía líquida en tandem con espectrometría de masa (Lc ms/ms): En mujeres normales e hirsutas. Estudio multicéntrico

Objective: To compare T results in normal and hirsute women, obtained by different laboratories employing the same or different methods, including an in-home RIA, and the gold standard method LC-MS/MS. In addition, T results were referred to a curve obtained by 6 different pools that had been prepared on the basis of LC-MS/MS results. Design: Prospective study Setting: Hormone Determination Laboratory, Hospital Italiano, La Plata, and private practice of each participant laboratory. Patient(s): Blood samples were obtained from 78 individuals sorted into 3 groups, namely, normal men (n:39), normal women (n:24) and hirsute women (n:15) Interventions(s): None Main Outcome Measure(s): To evaluate if the results obtained in each lab for each serum sample by the methods currently employed in our country are significantly different from those obtained by LC-MS/MS (Gold standard) Result(s) One out of the 24 NW showed high T values by LC - MS/MS. In each lab, except in 1 (Architect) T results of this serum sample were normal. Two out of the 15 hirsute patients showed normal T values (LC - MS/MS). Method and number of labs -shown between brackets- and percentages of normal T results (false negatives) are described for each method as follows: Chemiluminescence: Axsym - Abbott (Axn) - (3) 85, Architect - Abbott - (Arch); (2) 70; Immulite - Siemmens - (IMM); (2) 42; Electrochemiluminescence - Elecsys - Roche- ((EQL); (4) 52; Fluorescent enzymatic - Vidas - Bio-Merieux - (Vidas) (1) 69; Manual coated tube radioimmunoassay (RIA): RIA - Siemmens Coat-a-Count (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; and in-Home RIA (in-H) (1) 12. Statistically significant differences were obtained between different methods and against LC MS/MS. In-H method is the one that comes closest to 1 on the Weighted Deming regression and closest to zero on the SD intercept, (standard deviation of the constant in the straight line equation) indicating that the values match those obtained by LC - MS/MS. The values recorded by the various methods employed showed no significant modifications when plotted against a secondary standard curve. Conclusion(s) This indicates that the techniques in current use in our area underestimate hyperandrogenemia in these patients. Discrepancies are not due to the various calibration curves proposed in the corresponding commercial kits. The fact that the In-H technique affords finer results while employing a larger serum volume suggests that the disparities among the various commercial methods result from their limited sensitivity to the sample volumes they process. No financial conflicts of interest exist.

El diagnóstico de hiperandrogenemia requiere la demostración de niveles aumentados de Testosterona Total (TT) en suero. Los inmuno ensayos comerciales dan resultados divergentes a niveles bajos de TT como los obtenidos en mujeres. Valoramos los niveles de TT en 24 mujeres normales (MN) y 15 hirsutas (MH) en 18 laboratorios por métodos comúnmente empleados en nuestro medio, Quimioluminiscencia: Axsym - Abbott (Axn)- (3 ), Architect - Abbott - (Arch); (2); Immulite - Siemmens - (IMM); (2); Electroquimioluminiscencia - Elecsys - Roche- ((EQL); (4); Enzimático acoplado a fluorescencia Vidas - Bio-Merieux - (Vidas) (1), Radioinmunoiensayo en tubo recubierto (RIA): RIA - Siemmens (RIA S); (3) 64; RIA - DSL Inc (RIA DSL); (1) 31; RIA - DIASource - (DiaS); (1) 31; y un metodo desarrollado en uno de los laboratorios (in-H) (1).El número entreparéntesis indica elnúmero de laboratorios que emplearon la misma técnica,y comparamos los resultados por LC MS/MS. Comparativamente a LC MS/MS los niveles fueron en todas las muestras significativamente más bajos por AXS y en 18 de las 24 MN por DiaS. En 7 casos; 3 por RIA S, 2 por IMM y 1 por EQL y Arch los valores de TT fueron superiores al límite superior de sus respectivos métodos. En todos los casos se obtuvo una gran variación entre los mismos y con diferentes métodos. Trece de las 15 MH tuvieron niveles altos de TT por LC MS/MS. De las MH con TT aumentada de acuerdo a la determinación por LC MS/MS entre el 12 y el 85 % de las mismas por los distintos métodos fueron normales, indicando que en la mayoría de los métodos habitualmente utilizados en nuestro medio subvaloran la hipernadrogenemia en estas pacientes. Estas diferencias se hacen más notorias a niveles más bajos de TT (Se obtuvieron valores normales en el 71 % de los casos con valores de TT entre 0.47 y 0.74 ng/ml y en el 38 % de los casos, con niveles de TT mayor a 0.98 ng/ml). En 9 muestras se determinó la TT empleando una curva en el rango de 0.21 a 6.44 ng/ml preparada con de una mezcla de 78 sueros cuyos valores fueron obtenidos por LC MS/MS. No se obtuvo una modificación significativa de los valores indicando que la diferencia entre los distintos métodos no es debida a las diferentes curvas de calibración de los kit comerciales. En conclusión ninguno de los métodos mayormente empleados en nuestro medio son aceptables para la evaluación de niveles menores a 1.5 ng/ml.

Novel insights into mechanisms of food allergy and allergic airway inflammation using experimental mouse models.

Over the last decades, considerable efforts have been undertaken in the development of animal models mimicking the pathogenesis of allergic diseases occurring in humans. The mouse has rapidly emerged as the animal model of choice, due to considerations of handling and costs and, importantly, due to the availability of a large and increasing arsenal of genetically modified mouse strains and molecular tools facilitating the analysis of complex disease models. Here, we review latest developments in allergy research that have arisen from in vivo experimentation in the mouse, with a focus on models of food allergy and allergic asthma, which constitute major health problems with increasing incidence in industrialized countries. We highlight recent novel findings and controversies in the field, most of which were obtained through the use of gene-deficient or germ-free mice, and discuss new potential therapeutic approaches that have emerged from animal studies and that aim at attenuating allergic reactions in human patients.

Local glucocorticoid production in the mouse lung is induced by immune cell stimulation.

BACKGROUND: Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive steroid hormones, mainly produced by the adrenal glands. However, increasing evidence supports the idea of additional extra-adrenal sources of bioactive GC. The lung epithelium is constantly exposed to a plethora of antigenic stimuli, and local GC synthesis could contribute to limit uncontrolled immune reactions and tissue damage. METHODS: Expression of steroidogenic enzymes and GC synthesis in ex vivo organ cultures was studied in mouse lung tissue after in vivo stimulation of immune cells. RESULTS: Mouse lung tissue was found to express steroidogenic enzymes required for the synthesis of corticosterone from cholesterol and to synthesize corticosterone in large quantities after immune cell activation by anti-CD3 antibody, lipopolysaccharide, or TNFα. In marked contrast, ovalbumin-induced allergic airway inflammation failed to promote lung GC synthesis. Although the lung expresses all steroidogenic enzymes necessary for de novo synthesis of corticosterone from cholesterol, functional data indicated that inactive serum-derived dehydrocorticosterone is converted to active corticosterone by 11ß-hydroxysteroid dehydrogenase 1. CONCLUSION: Our results support the notion that local GC synthesis represents a novel immunoregulatory mechanism to limit uncontrolled immune responses in the lung and indicate that defective local steroidogenesis may contribute to the pathogenesis of allergic airway inflammation.

Thiazolide-induced apoptosis in colorectal cancer cells is mediated via the Jun kinase-Bim axis and reveals glutathione-S-transferase P1 as Achilles' heel.

Glutathione-S-transferase of the Pi class (GSTP1) is frequently overexpressed in a variety of solid tumors and has been identified as a potential therapeutic target for cancer therapy. GSTP1 is a phase II detoxification enzyme and conjugates the tripeptide glutathione to endogenous metabolites and xenobiotics, thereby limiting the efficacy of antitumor chemotherapeutic treatments. In addition, GSTP1 regulates cellular stress responses and apoptosis by sequestering and inactivating c-Jun N-terminal kinase (JNK). Thiazolides are a novel class of antibiotics for the treatment of intestinal pathogens with no apparent side effects on the host cells and tissue. Here we show that thiazolides induce a GSTP1-dependent and glutathione-enhanced cell death in colorectal tumor cell lines. Downregulation of GSTP1 reduced the apoptotic activity of thiazolides, whereas overexpression enhanced it. Thiazolide treatment caused strong Jun kinase activation and Jun kinase-dependent apoptosis. As a critical downstream target of Jun kinase we identified the pro-apoptotic Bcl-2 homolog Bim. Thiazolides induced Bim expression and activation in a JNK-dependent manner. Downregulation of Bim in turn significantly blocked thiazolide-induced apoptosis. Whereas low concentrations of thiazolides failed to induce apoptosis directly, they potently sensitized colon cancer cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic drug-induced cell death. Although GSTP1 overexpression generally limits chemotherapy and thus antitumor treatment, our study identifies GSTP1 as Achilles' heel and thiazolides as novel interesting apoptosis sensitizer for the treatment of colorectal tumors.

Role of TRAIL and the pro-apoptotic Bcl-2 homolog Bim in acetaminophen-induced liver damage.

Acetaminophen (N-acetyl-para-aminophenol (APAP), paracetamol) is a commonly used analgesic and antipyretic agent. Although considered safe at therapeutic doses, accidental or intentional overdose causes acute liver failure characterized by centrilobular hepatic necrosis with high morbidity and mortality. Although many molecular aspects of APAP-induced cell death have been described, no conclusive mechanism has been proposed. We recently identified TNF-related apoptosis-inducing ligand (TRAIL) and c-Jun kinase (JNK)-dependent activation of the pro-apoptotic Bcl-2 homolog Bim as an important apoptosis amplification pathway in hepatocytes. In this study, we, thus, investigated the role of TRAIL, c-JNK and Bim in APAP-induced liver damage. Our results demonstrate that TRAIL strongly synergizes with APAP in inducing cell death in hepatocyte-like cells lines and primary hepatocyte. Furthermore, we found that APAP strongly induces the expression of Bim in a c-JNK-dependent manner. Consequently, TRAIL- or Bim-deficient mice were substantially protected from APAP-induced liver damage. This study identifies the TRAIL-JNK-Bim axis as a novel target in the treatment of APAP-induced liver damage and substantiates its general role in hepatocyte death.

Colon cancer cells produce immunoregulatory glucocorticoids.

Glucocorticoids (GC) have important anti-inflammatory and pro-apoptotic activities. Initially thought to be exclusively produced by the adrenal glands, there is now increasing evidence for extra-adrenal sources of GCs. We have previously shown that the intestinal epithelium produces immunoregulatory GCs and that intestinal steroidogenesis is regulated by the nuclear receptor liver receptor homolog-1 (LRH-1). As LRH-1 has been implicated in the development of colon cancer, we here investigated whether LRH-1 regulates GC synthesis in colorectal tumors and whether tumor-produced GCs suppress T-cell activation. Colorectal cancer cell lines and primary tumors were found to express steroidogenic enzymes and regulatory factors required for the de novo synthesis of cortisol. Both cell lines and primary tumors constitutively produced readily detectable levels of cortisol, as measured by radioimmunoassay, thin-layer chromatography and bioassay. Whereas overexpression of LRH-1 significantly increased the expression of steroidogenic enzymes and the synthesis of cortisol, downregulation or inhibition of LRH-1 effectively suppressed these processes, indicating an important role of LRH-1 in colorectal tumor GC synthesis. An immunoregulatory role of tumor-derived GCs could be further confirmed by demonstrating a suppression of T-cell activation. This study describes for the first time cortisol synthesis in a non-endocrine tumor in humans, and suggests that the synthesis of bioactive GCs in colon cancer cells may account as a novel mechanism of tumor immune escape.

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid.

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL-Jun kinase-Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.

The multiple roles of major histocompatibility complex class-I-like molecules in mucosal immune function.

The human major histocompatibility complex (MHC) on chromosome 6 encodes three classical class-I genes: human leukocyte antigens (HLA) A, B, and C. These polymorphic genes encode a 43- to 45-kDa cell surface glycoprotein that, in association with the 12-kDa beta2-microglobulin molecule, functions in the presentation of nine amino acid peptides to the T-cell receptor of CD8-bearing T lymphocytes and killer inhibitory receptors on natural killer cells. In addition to these ubiquitously expressed, polymorphic proteins, the human genome also encodes several nonclassical MHC class-I-like, or class Ib, genes that, in general, encode nonpolymorphic molecules involved in various specific immunological functions. Many of these genes, including CD1, the neonatal Fc receptor for IgG, HLA-G, HLA-E, the MHC class-I chain-related gene A, and Hfe, are prominently displayed on epithelial cells, suggesting an important role in epithelial cell biology.

Differential contribution of Fas- and perforin-mediated mechanisms to the cell-mediated cytotoxic activity of naive and in vivo-primed intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) are known to exert strong constitutive cytotoxic activity. In the present study we compared the Ag-specific cytotoxic activity and the effector mechanisms involved in non-Ag-primed, naive and in in vivo-primed IELs and splenic CD8 T cells. Ex vivo isolated naive CD8alphaalpha TCRalphabeta IELs, CD8alphabeta IELs, and splenocytes from lymphocytic choriomeningitis virus (LCMV)-specific TCR transgenic mice exert Ag-specific cytotoxic activity in a long-term, but not in a short-term, cytotoxicity assay. This cytotoxic activity is mainly Fas-Fas ligand mediated and is significantly reduced in the presence of 20 microg/ml Fas-Fcgamma1 fusion protein. Both CD8alphabeta IELs and CD8alphabeta splenocytes isolated from LCMV-infected C57BL/6 mice exert potent perforin-dependent cell-mediated cytotoxicity. CD8alphaalpha TCRalphabeta IELs from LCMV-infected animals, however, show only minimal Ag-specific cytotoxicity. The potent cytotoxic activity of in vivo activated CD8alphabeta IELs is not affected by the addition of Fas-Fcgamma1. Nevertheless CD8alphabeta IELs from LCMV-infected perforin-deficient mice exert Ag-specific cytotoxicity in a short-term cytotoxicity assay, and this cytotoxicity is almost completely blocked by the addition of Fas-Fcgamma1. These results demonstrate that naive CD8alphabeta IELs exert Ag-specific, Fas-Fas ligand-mediated, constitutive cytotoxic activity in a long-term cytotoxicity assay, whereas primed CD8alphabeta IELs primarily use the perforin-dependent exocytosis pathway to exert their potent cytotoxic activity. Furthermore, these results clearly illustrate the requirement for Ag-specific determination of IEL-mediated cytotoxicity, because the elevated, but variable, frequencies of memory-type T cells in this compartment may lead to ambiguous results when polyclonal activation or redirected assays are used.

Noncleavable transmembrane mouse tumor necrosis factor-alpha (TNFalpha) mediates effects distinct from those of wild-type TNFalpha in vitro and in vivo.

Tumor necrosis factor-alpha (TNFalpha) exists in two biologically active forms, a 26-kDa transmembrane form and a proteolytically cleaved and secreted form. We sequentially inactivated all three known cleavage sites of mouse TNFalpha by mutating the corresponding DNA sequences. A murine T cell hybridoma transfected with the nonsecretable mutant TNFalpha efficiently lysed L929 target cells in a cell contact-dependent manner and induced expression of vascular cell adhesion molecule-1 on mouse endothelioma cells. A genomic mouse TNFalpha clone encoding this mutant was subsequently introduced as a transgene into TNFalpha(-/-) lymphotoxin-alpha(-/-) mice. The 3' AU-rich regulatory elements of the TNF locus were maintained in the transgene to assure adequate gene regulation. Transmembrane TNFalpha transgenic mice were fully protected from endotoxic shock, and no TNFalpha bioactivity was detectable in the serum after stimulation with lipopolysaccharide. Activated CD4 T cells from these animals, however, lysed L929 cells in a cell contact-dependent way. After administration of lipopolysaccharide, transmembrane TNFalpha transgenic mice produced significantly higher levels of interleukin-12 than wild-type mice or TNF-deficient mice. This indicates that transmembrane TNFalpha may greatly affect the course of a cellular immune responses in vivo and exerts quantitatively and qualitatively distinct functions from secreted TNFalpha in vitro and in vivo.