Visualization of Mutant Aggregates from Clock Neurons by Agarose Gel Electrophoresis (AGERA) in Drosophila melanogaster.

The clock neurons of the fruit fly Drosophila melanogaster have become a useful model for expressing misfolded protein aggregates that accumulate in several human neurodegenerative diseases. One advantage of such an approach is that the behavioral effects can be readily quantified on circadian locomotor rhythms, sleep or activity levels via automated, highly reliable and objective procedures. Therefore, a rapid assay is required to visualize whether these neurons develop aggregates. Here we describe a modified immunoblot method, agarose gel electrophoresis (AGERA) that has been optimized for resolving aggregates from fly clock neurons.

Correlation between inflammatory parameters and pituitary-thyroid axis in patients with COVID-19.

PURPOSE: Inflammation plays a critical role in the progression of COVID-19. Nonthyroidal illness syndrome (NTIS) has been increasingly recognized in affected patients. We aim to evaluate the correlation of thyroid hormones with markers of inflammation and association with disease outcome in hospitalized patients with COVID-19, and in two profiles of NTIS (low T3-normal/low FT4 vs. low T3-high FT4). METHODS: consecutive patients admitted to a nonintensive care unit for COVID-19 were recruited. Infection was mild in 22%, moderate in 27.1% and severe in 50.8%; 7.41% died. T4, T3, FT4, FT3, and their ratios (T3/T4, FT3/FT4) were correlated with albumin, ferritin, fibrinogen, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), lactate dehydrogenase (LDH), and D-dimer. RESULTS: Fifty five patients (50.9% men, median age 56 years) were included. Albumin correlated positively with T3 and hormones ratios, but negatively with FT4. T3, FT3, T3/T4, and FT3/FT4 correlated inversely with ferritin, fibrinogen, ESR, CRP, LDH, and D-dimer. FT4 showed direct correlation with fibrinogen and ESR. T3/T4 was lower in severe compared to mild/moderate disease [7.5 (4.5-15.5) vs. 9.2 (5.8-18.1); p = 0.04], and lower in patients who died than in those discharged [5 (4.53-5.6) vs. 8.1 (4.7-18.1); p = 0.03]. A low T3/high FT4 profile was associated with lower albumin, higher ferritin, and severity. CONCLUSION: In this cohort, thyroid hormones correlated with inflammation and outcome. T3 and T3/T4 correlated inversely with inflammatory markers; a low T3/T4 ratio was associated with severity and poor prognosis. Patients with low T3 but high FT4 had higher ferritin, lower albumin, and more severe disease at presentation.

Rab8 Promotes Mutant HTT Aggregation, Reduces Neurodegeneration, and Ameliorates Behavioural Alterations in a Drosophila Model of Huntington's Disease.

BACKGROUND: Altered cellular vesicle trafficking has been linked to the pathogenesis of Huntington's disease (HD), a fatal, inherited neurodegenerative disorder caused by mutation of the huntingtin (HTT) protein. The Rab GTPase family of proteins plays a key role in regulation of vesicle trafficking, with distinct Rabs helping specify membrane identity and mediating cellular processes including budding, motility and tethering of vesicles to their targets. In recent years several Rab GTPases-notably, Rab5 and Rab11-have been linked to the pathogenesis of neurodegenerative disorders, including HD. OBJECTIVE: We investigated whether Rab8, which regulates post-Golgi vesicle trafficking, is able to improve HD-relevant phenotypes in a well-characterised model. METHODS: We overexpressed Rab8 in a Drosophila model of HD testing cellular, behavioural, and molecular phenotypes. RESULTS: We found that Rab8 overexpression ameliorated several disease-related phenotypes in fruit flies expressing a mutant HTT fragment throughout the nervous system, including neurodegeneration of photoreceptor neurons, reduced eclosion of the adult fly from the pupal case and shortened lifespan. Rab8 overexpression also normalised aberrant circadian locomotor behaviour in flies expressing mutant HTT in a specific population of neurons that regulate the circadian clock. Intriguingly, expression of Rab8 increased the accumulation of SDS-insoluble aggregated species of mutant HTT. CONCLUSION: Collectively, our findings demonstrate that increased Rab8 levels protect against mutant HTT toxicity and potentiate its aggregation, likely reducing the accumulation of downstream toxic soluble species.

Hiperaldosteronismo primario: puntos de corte del cociente aldosterona/renina / Primary aldosteronism: Aldosterone/renin ratio cut-off points

Introducción: El hiperaldosteronismo primario (HAP) es la causa más frecuente de hipertensión endocrina, con una prevalencia del 6-12% en pacientes hipertensos. El cociente aldosterona/renina es el método de cribado de elección. Dada la variabilidad de sus puntos de corte, se sugiere contar con valores de referencia propios. Objetivos: 1) Optimizar los puntos de corte del cociente aldosterona/renina para el cribado de hiperaldosteronismo con la metodología actual; 2)evaluar la correlación y la sensibilidad diagnóstica de los cocientes aldosterona/actividad de renina plasmática (RAA) y aldosterona/concentración de renina (RAC) para el cribado de hiperaldosteronismo, y 3)determinar la prevalencia de hiperaldosteronismo en nuestra población. Materiales y métodos: Se determinaron los niveles de aldosterona (RIA competitivo en fase sólida RIAZENco Zentech), actividad de renina plasmática (RIA en fase sólida DiaSorin) y concentración de renina (quimioluminiscencia Liaison DiaSorin) en 345 sujetos (136 controles y 209 hipertensos). Se calcularon los cocientes RAA y RAC. Resultados: La prevalencia de HAP, tras confirmación diagnóstica, fue del 5,9% de los hipertensos. El valor de corte para sospecha de HAP determinado por curvas ROC fue 48,9(ng/dl)/(ng/ml/h) para RAA (sensibilidad 100% y especificidad 93,6%) y 2,3(ng/dl)/(μUI/ml) para RAC (sensibilidad 100% y especificidad 90,9%). Se observó buena correlación entre RAA y RAC (ρ=0,83; p<0,0001), con una concordancia diagnóstica presuntiva del 96,6%. Conclusiones: Hemos determinado en nuestra población nuevos valores de corte de RAA y RAC para el cribado de HAP, con buena sensibilidad y concordancia como métodos de cribado. Es importante contar con rangos de normalidad propios para evitar errores diagnósticos

Introduction: Primary aldosteronism (PA) is the most common cause of endocrine hypertension, with a prevalence rate of 6-12% in hypertensive patients. Aldosterone/renin ratio (ARR) is the screening test of choice for PA. Because of the variable cut-off points of ARR, reference values related to the populations and methods considered are recommended. Objectives: (i)To optimize the ARR cut-off points for PA screening with current methods; (ii)to assess the correlation and diagnostic sensitivity of the plasma aldosterone concentration/plasma renin activity (ARR) ratio and the aldosterone concentration/renin concentration (ARC) ratios for PA screening, and (iii)to determine the prevalence of PA in our population. Materials and methods: Plasma aldosterone concentration and plasma renin activity levels were measured using radioimmunoassays (RIAZENco Zentech and RIA DiaSorin respectively), while a chemiluminescence assay (Liaison Diasorin) was used to test renin concentration. ARR and ARC ratios were calculated in 345 subjects (136 healthy subjects and 209 hypertensive patients). Results: Prevalence of PA was 5.9% after diagnostic confirmation. ROC curve analysis suggested an ARR threshold of 48.9(ng/dL)/(ng/mL/h) (100% sensitivity, 93.6% specificity) and an ARC threshold of 2.3(ng/dL)/(μIU/mL) (100% sensitivity, 90.9% specificity). Good correlation was seen between ARR and ARC (ρ=.83, P<.0001), with a presumptive diagnostic concordance of 96.6%. Conclusions: New cut-off values of ARR and ARC for screening of PA, with high sensitivity and good diagnostic concordance, were determined in the study population. It is important to have valid normal ranges to avoid diagnostic errors

Primary aldosteronism: Aldosterone/renin ratio cut-off points. / Hiperaldosteronismo primario: puntos de corte del cociente aldosterona/renina.

INTRODUCTION: Primary aldosteronism (PA) is the most common cause of endocrine hypertension, with a prevalence rate of 6-12% in hypertensive patients. Aldosterone/renin ratio (ARR) is the screening test of choice for PA. Because of the variable cut-off points of ARR, reference values related to the populations and methods considered are recommended. OBJECTIVES: (i)To optimize the ARR cut-off points for PA screening with current methods; (ii)to assess the correlation and diagnostic sensitivity of the plasma aldosterone concentration/plasma renin activity (ARR) ratio and the aldosterone concentration/renin concentration (ARC) ratios for PA screening, and (iii)to determine the prevalence of PA in our population. MATERIALS AND METHODS: Plasma aldosterone concentration and plasma renin activity levels were measured using radioimmunoassays (RIAZENco Zentech and RIA DiaSorin respectively), while a chemiluminescence assay (Liaison Diasorin) was used to test renin concentration. ARR and ARC ratios were calculated in 345 subjects (136 healthy subjects and 209 hypertensive patients). RESULTS: Prevalence of PA was 5.9% after diagnostic confirmation. ROC curve analysis suggested an ARR threshold of 48.9(ng/dL)/(ng/mL/h) (100% sensitivity, 93.6% specificity) and an ARC threshold of 2.3(ng/dL)/(µIU/mL) (100% sensitivity, 90.9% specificity). Good correlation was seen between ARR and ARC (ρ=.83, P<.0001), with a presumptive diagnostic concordance of 96.6%. CONCLUSIONS: New cut-off values of ARR and ARC for screening of PA, with high sensitivity and good diagnostic concordance, were determined in the study population. It is important to have valid normal ranges to avoid diagnostic errors.

Related quality of life questionnaire specific to dysthyroid ophthalmopathy evaluated in a population of patients with Graves' disease

ABSTRACT Objective The aim of this study was to measure quality of life (QOL) impairment in individuals currently suffering from Graves' ophthalmopathy (GO) and to determine the correlation of GO-specific QOL scores with disease severity and activity. Subjects and methods Seventy three GO-specific QOL surveys were prospectively analysed and compared with GO status. The GO-specific QOL survey was translated into Spanish and applied to Argentine patients with Graves' disease (GD). Results were compared with presence or absence of GO, Clinical Activity Score (CAS), severity score, age, gender and thyroid function. Results Fifty-six patients answered the survey and underwent complete ophthalmic evaluation, 15 did not have GO and were considered to be a control group. Appearance QOL score for patients with GO (53 ± 31.4) was lower than the control group (88.3 ± 17) (p < 0,000), no difference was observed in functional QOL score. There was a negative correlation between GO severity and both functional (r = -0.575; p < 0.000) and appearance QOL (r = -0.577; p < 0.000). Functional QOL differed between patients with active GO vs control group (p = 0.043). Patients with active and inactive GO had lower appearance QOL scores than control group (p < 0.000, p < 0.001 respectively). Conclusions GO has significant impact on the life of these Argentine patients. QOL was worse in GO patients than in control group, functional QOL was mostly affected by the activity and appearance QOL was mainly altered by the effects of the disease. Patients with more severe GO had lower scores on both QOL scales.

Related quality of life questionnaire specific to dysthyroid ophthalmopathy evaluated in a population of patients with Graves' disease.

OBJECTIVE: The aim of this study was to measure quality of life (QOL) impairment in individuals currently suffering from Graves' ophthalmopathy (GO) and to determine the correlation of GO-specific QOL scores with disease severity and activity. SUBJECTS AND METHODS: Seventy three GO-specific QOL surveys were prospectively analysed and compared with GO status. The GO-specific QOL survey was translated into Spanish and applied to Argentine patients with Graves' disease (GD). Results were compared with presence or absence of GO, Clinical Activity Score (CAS), severity score, age, gender and thyroid function. RESULTS: Fifty-six patients answered the survey and underwent complete ophthalmic evaluation, 15 did not have GO and were considered to be a control group. Appearance QOL score for patients with GO (53 ± 31.4) was lower than the control group (88.3 ± 17) (p < 0,000), no difference was observed in functional QOL score. There was a negative correlation between GO severity and both functional (r = -0.575; p < 0.000) and appearance QOL (r = -0.577; p < 0.000). Functional QOL differed between patients with active GO vs control group (p = 0.043). Patients with active and inactive GO had lower appearance QOL scores than control group (p < 0.000, p < 0.001 respectively). CONCLUSIONS: GO has significant impact on the life of these Argentine patients. QOL was worse in GO patients than in control group, functional QOL was mostly affected by the activity and appearance QOL was mainly altered by the effects of the disease. Patients with more severe GO had lower scores on both QOL scales.

Disease activity accounts for long-term efficacy of IL-1 blockers in pyogenic sterile arthritis pyoderma gangrenosum and severe acne syndrome.

OBJECTIVE: To provide a rationale for anti-IL-1 treatment in pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA) by defining whether IL-1ß secretion is enhanced; requires NLRP3; and correlates with proline-serine-threonine phosphatase-interacting protein 1 mutations, disease activity and/or the clinical picture in PAPA. METHODS: Monocytes were isolated from 13 patients and 35 healthy donors and studied at baseline and following activation. Secretion pattern of IL-1ß, IL-1α, IL-1Ra, IL-6, IL-18 and TNF-α was assessed in supernatants by ELISA. The NLRP3 requirement for IL-1ß secretion was investigated by silencing technique in PAPA and healthy donor monocytes. Long-term follow-up (mean 26 months, range 4-38) was performed in five patients enrolled in an anti-IL-1 regimen. RESULTS: IL-1ß secretion in PAPA is increased, requires NLRP3 and correlates with disease activity. Patients with a history of osteoarticular flares release more IL-1ß, IL-6 and TNF-α compared with those with predominant cutaneous recurrences. Monocytes from patients in anti-IL-1 treatment dramatically reduced IL-1ß secretion after ex vivo activation, and long-term follow-up demonstrated decreased frequency of flares and normalization of acute phase reactants in all the patients. A straightforward correlation between genotype and IL-1ß signalling was not observed suggesting that factors other than mutation itself may play a role in regulating IL-1ß secretion and response to treatment in PAPA. CONCLUSION: PAPA patients with active lesions display increased NLRP3-mediated IL-1ß secretion, and long-term efficacy of IL-1 blockade was demonstrated. Even if other mechanisms related to the complex proline-serine-threonine phosphatase-interacting protein 1 protein networking might play additional roles, this study further supports the potential of IL-1 blockade as an effective therapeutic strategy in PAPA syndrome.

Correlación clínico patológica y evolución de la nefropatía lúpica en niños y adolescentes / Clinicopathologic correlation and evolution of lupus nephritis in children and adolescents

El lupus eritematoso sistémico (LES) tiene una prevalencia baja en la población general y es menor en la edad pediátrica. La nefropatía lúpica (NL) es más frecuente y de mayor severidad que en adultos, condicionando la morbimortalidad de la enfermedad. Se realizó un estudio descriptivo prospectivo de 20 niños y adolescentes con NL controlados en la Policlínica de Colagenopatías del Centro Hospitalario Pereira Rossell en el período desde octubre de 2003 hasta setiembre de 2013 con el objetivo de describir las características clínico-serológicas y evolutivas de pacientes con NL y correlacionarlas con los hallazgos anátomopatológicos. La NL se observó en el 52,6% de los casos con LES. El 70% fueron de sexo femenino, relación femenino/masculino de 2,3/1, 85% de raza blanca, la mediana del diagnóstico fue de 12 años. Las formas de presentación fueron: alteraciones urinarias menores (AUM) en 14 pacientes (0,7), en cuatro casos síndrome nefrótico (SN), con o sin insuficiencia renal (IR) y/o hipertensión arterial. Un paciente se manifestó con síndrome nefrítico. Un paciente tenía un examen de orina normal. Las formas histopatológicas proliferativas graves se presentaron en 18 (0,9); los casos con AUM presentaban NL grado III-IV en 13 (0,93); todos los casos con SN con o sin IR tenían NL III-IV. No hubo casos de NL aislada como forma de comienzo. En el momento del diagnóstico, los anticuerpos antinucleares fueron positivos en 19 (0,95) y los anti DNA doble cadena en 16 (0,8); C3 y C4 estuvieron descendidos en 19 (0,95) y en 15 (0,75) respectivamente. El seguimiento promedio fue 4,2 años. Al final del seguimiento estaban en remisión 16 pacientes (0,8), cuatro en remisión parcial, todos con función renal normal, excepto un caso que presentó IR extrema, fue trasplantado y tuvo una excelente evolución. Un paciente falleció con hemorragia pulmonar. La sobrevida de la función renal y la de los pacientes fue 0,95 respectivamente. El tratamiento se realizó en base a corticoides, hidroxicloroquina asociados a azatioprina o micofenolato mofetilo. En ocho pacientes con cuadros graves se usó la ciclofosfamida I/V. Esta serie constituye la primera serie nacional de nefropatía lúpica en niños y adolescentes. Conclusión: predominó la presentación clínica con AUM y formas histopatológicas severas, clases III y IV, evidenciando una disociación clínico anatomopatológica. A pesar del elevado porcentaje de NL severas, el manejo adecuado y oportuno y la adherencia al tratamiento y a los controles médicos fueron fundamentales para la evolución favorable de la NL.

Systemic lupus erythematosus (SLE) has a low prevalence in the overall general population and this is lower in children. Child lupus nephropathy (LN) is more frequent and severe than in SLE adult patients, with greater disease morbidity and mortality. A prospective descriptive study of 20 children and adolescents with LN monitored in the Collagen Diseases Office of the Pereira Rossell Hospital between October, 2003 and September, 2013 was performed. The objective of this study was to describe clinical-serological features and the evolution of these patients and to correlate them with its anatomopathological findings. LN was diagnosed in 52,6% of the SLE patients, 70% were female with a female/male correlation of 2,3/1; 85% were Caucasian; median age at diagnosis was 12 years old. The clinical presentations were minor urinary findings (MUF) in 14 patients (0,7) and nephrotic syndrome (NS) in 4 (0,2), and another one nephritic syndrome. One patient presented no symptoms and had normal urinalysis. Severe proliferative classes predominated in18 patients (0.9); 13 (0.93) patients with MUF and all the patients with NS had LN classes III or IV. Isolated LN was not seen in the initial presentation. At the time of diagnosis antinuclear antibodies were positive in 19 patients (0.95); and anti DNA double stranded in 16 (0.8). Low C3 was found in 19 (0.95) and C4 in 15 (0.75), respectively. Average follow-up time was 4.2 years. At the end of follow-up 16 (0.8) were in remission, 4 of them in partial remission; all patients presented normal renal function except for one who evidenced severe renal failure and required hemodialysis and transplantation and had an excellent evolution. One patient died with pulmonary hemorrhage. The renal and patient survivals were 0.95 respectively. Treatment consisted in corticosteroids and hydroxychloroquine associated with azathioprine or mycophenolate mofetil. Cyclophosphamide was administered to 8 patients with severe illness. This is the first national report of LN in children and adolescents. Conclusions: the predominant clinical presentation of LN was MUF with severe anatomopathological findings, classes III and IV, showing a clinical-pathological dissociation. Despite the high percentage of severe LN, early and adequate treatment, as well as a good compliance to it with periodic medical follow-up, were essential to achieve a favorable outcome of LN.

Chloroplasts play a central role in plant defence and are targeted by pathogen effectors.

Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. Here we show that the chloroplast is a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the inhibitor of photosynthetic electron transport, DCMU, abolishes the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

TLR costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production.

IL-1ß acts in concert with anti-inflammatory cytokines, in particular, IL-1R antagonist (IL-1Ra), to ensure the correct development and outcome of the inflammation: imbalance in the IL-1ß/IL-1Ra ratio is implicated in many human diseases and may lead to dramatic consequences. In this article, we show that single TLR engagement induces IL-1ß and, with a little delay, IL-1Ra. Differently, costimulation of TLR2, TLR4, and TLR7/8 enhances IL-1ß secretion but severely inhibits IL-1Ra production. The IL-1ß/IL-1Ra unbalance after activation of multiple TLRs depends on the insurgence of oxidative stress, because of enhanced production of reactive oxygen species and failure of the antioxidant systems. Increased reactive oxygen species levels increase ATP externalization by monocytes, resulting in enhanced inflammasome activation and IL-1ß secretion. Oxidative stress then induces cell responses to stress, including inhibition of protein synthesis, which, in turn, is responsible for the impaired production of IL-1Ra. IL-1Ra secretion is restored by exogenous antioxidants that oppose oxidative stress. Similar effects are evident also on other cytokines: TNF-α is induced, whereas IL-6 is inhibited by costimulation. Our findings provide a molecular basis to the imbalance between proinflammatory and regulatory cytokine circuits that occur in various pathologic conditions, and suggest new strategies for controlling inflammation.

Increased NLRP3-dependent interleukin 1ß secretion in patients with familial Mediterranean fever: correlation with MEFV genotype.

OBJECTIVES: To define in patients affected by familial Mediterranean fever (FMF) whether or not interleukin (IL)-1ß secretion (1) is enhanced, (2) correlates with the type of MEFV mutation and (3) is mediated by NLRP3. METHODS: Freshly isolated monocytes from 21 patients with FMF (12 homozygous and 9 heterozygous), 14 MEFV healthy carriers and 30 healthy donors (HDs), unstimulated or after lipopolysaccharide (LPS)-induced activation, were analysed for redox state (production of reactive oxygen species (ROS) and antioxidant responses) and IL-1ß and IL-1 receptor antagonist (IL-1Ra) secretion. NLRP3 down-modulation was induced by in vitro silencing of the NLRP3 gene. RESULTS: LPS-stimulated monocytes from patients with FMF displayed enhanced IL-1ß secretion, which correlated with number and penetrance of MEFV mutations. Silencing of NLRP3 consistently inhibited IL-1ß secretion. As in other autoinflammatory diseases, FMF monocytes produced more ROS than genetically negative cells from HDs. Unlike in cryopyrin-associated periodic fever syndromes (CAPS), however, they were characterised by a conserved and sustained antioxidant response. Consistent with this finding, activated MEFV-mutated monocytes did not exhibit the functional indicators of oxidative stress observed in CAPS, including accelerated IL-1ß secretion and deficient production of IL-1Ra. CONCLUSIONS: MEFV-mutated monocytes display enhanced IL-1ß secretion, which correlates with number of high-penetrance mutations and level of endogenous ROS. Unlike NLRP3-mutated cells, monocytes carrying MEFV mutations withstand oxidative stress and preserve IL-1Ra production, thereby limiting inflammation. Finally, in contrast with that found in the animal model, the increased secretion of IL-1ß by LPS-stimulated FMF monocytes is NLRP3-dependent.

The pharmacologic inhibition of the xc- antioxidant system improves the antitumor efficacy of COX inhibitors in the in vivo model of 3-MCA tumorigenesis.

The chemopreventive and therapeutic efficacy of the cyclooxygenase (COX) inhibitor ibuprofen (IB) and of sulfasalazine (SASP), a drug that targets the antioxidant xc- system, were exploited in the experimental model of 3-methylcholantrene (3-MCA)-induced mouse sarcoma. The chemopreventive treatments gave unsatisfactory results because administration of IB one day after the 3-MCA injection only slightly delayed the tumor development, whereas SASP dispensed under the same conditions resulted in accelerated tumorigenesis. Similarly, the therapeutic treatment with either drug, administrated daily from the tumor detection, decreased the proliferation rate of tumor cells and increased the survival of treated mice only at a low extent. Remarkably, the combined chemopreventive treatment with IB and therapeutic treatment with SASP displayed a better efficacy, with strong delay of sarcoma growth, reduced tumor size and increased survival of treated mice. The two drugs target not only tumor cells but also tumor-associated macrophages that were dramatically decreased in the tumor infiltrate of mice subjected to the combined treatment. The synergistic effects of the association between a broad anti-inflammatory compound, such as IB, and a redox-directed drug, such as SASP, shed new light in the role of inflammation and of the redox response in chemical tumorigenesis and point to the combined chemopreventive plus therapeutic treatment with IB and SASP as a promising novel approach for antitumor therapy.

Deficient production of IL-1 receptor antagonist and IL-6 coupled to oxidative stress in cryopyrin-associated periodic syndrome monocytes.

OBJECTIVE: To determine whether dysregulated production of cytokines downstream of interleukin (IL)-1 participates in the pathophysiology of cryopyrin-associated periodic syndromes (CAPS). METHODS: Primary monocytes from patients with CAPS, unstimulated or after stimulation with lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists, were examined for signs of stress and production of IL-1ß, IL-1 receptor antagonist (IL-1Ra) and IL-6 in comparison with monocytes from patients with autoimmune diseases and from healthy donors. RESULTS: Unstimulated CAPS monocytes showed mild signs of stress including elevated levels of reactive oxygen species and fragmented mitochondria. Stress signs were worsened by TLR stimulation and eventually led to protein synthesis inhibition with strong impairment of production of cytokines downstream of IL-1, such as IL-1Ra and IL-6. These defects were not detected in monocytes from autoimmune patients and healthy donors. CONCLUSIONS: The stress state of LPS-stimulated CAPS monocytes and the consequent inhibition of translation are likely to be responsible for the impaired production of IL-1Ra and IL-6. The deficient secretion of these cytokines coupled with increased IL-1ß release explains the severity of the IL-1-related clinical manifestations and the predominant implication of innate immunity in CAPS.

The rate of interleukin-1beta secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering.

Human myeloid cells activate the NLRP3 inflammasome and secrete interleukin (IL)-1ß in response to various Toll-like receptor (TLR) ligands, but the rate of secretion is much higher in primary human monocytes than in cultured macrophages or THP-1 cells. The different myeloid cells also display different redox status under resting conditions and redox response to TLR activation. Resting monocytes display a balanced redox state, with low production of reactive oxygen species (ROS) and antioxidants. TLR engagement induces an effective redox response with increased ROS generation followed by a sustained antioxidant response, parallelled by efficient IL-1ß secretion. Drugs blocking ROS production or the antioxidant response prevent the secretion of mature IL-1ß but not the biosynthesis of pro-IL-1ß, indicating that redox remodeling is responsible for IL-1ß processing and release. Unlike monocytes, THP-1 cells and cultured macrophages have up-regulated antioxidant systems that buffer the oxidative hit provided by TLR triggering and suppress the consequent redox response. This aborted redox remodeling is paralleled by low efficiency IL-1ß processing and secretion. High doses (5 mM) of H(2)O(2) overcome the high antioxidant capacity of THP-1 cells, restore an efficient redox response, and increase the rate of IL-1ß secretion. Together these data indicate that a tightly controlled redox homeostasis in resting cells is a prerequisite for a robust redox response to TLR ligands, in turn necessary for the efficient inflammasome activation. Inflammasome activation by bacterial DNA is not modulated by redox responses, suggesting that redox-dependent regulation of IL-1ß secretion is restricted to some inflammasomes including NLRP3 but excluding AIM-2.

The cystine/cysteine cycle and GSH are independent and crucial antioxidant systems in malignant melanoma cells and represent druggable targets.

AIMS: Cancer chemoresistance is often due to upregulation of antioxidant systems. Therapeutic targeting of these systems is however hampered by their redundancy. Here, we have performed a functional dissection of the antioxidant systems in different melanoma cases aimed at the identification of the most effective redox active drug. RESULTS: We have identified two crucial antioxidant mechanisms: glutathione (GSH), the major intracellular redox buffer, and the cystine/cysteine cycle, which switches the extracellular redox state from an oxidized to a reduced state. The two mechanisms are independent in melanoma cells and may be substitutes for each other, but targeting both of them is lethal. Exposure to the pro-oxidant compound As(2)O(3) induces an antioxidant response. However, while in these cells the intracellular redox balance remains almost unaffected, a reduced environment is generated extracellularly. GSH depletion by buthioninesulfoximine (BSO), or cystine/cysteine cycle inhibition by (S)-4-carboxyphenylglycine (sCPG), enhanced the sensitivity to As(2)O(3). Remarkably, sCPG also prevented the remodeling of the microenvironment redox state. INNOVATION: We propose that the definition of the prevalent antioxidant system(s) in tumors is crucial for the design of tailored therapies involving redox-directed drugs in association with pro-oxidant drugs. CONCLUSION: In melanoma cells, BSO is the best enhancer of As(2)O(3) sensitivity. However, since the strong remodeling of the microenvironmental redox state caused by As(2)O(3) may promote tumor progression, the concomitant use of cystine/cysteine cycle blockers is recommended.

Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1beta secretion.

In healthy monocytes, Toll-like receptor (TLR) engagement induces production of reactive oxygen species (ROS), followed by an antioxidant response involved in IL-1beta processing and secretion. Markers of the antioxidant response include intracellular thioredoxin and extracellular release of reduced cysteine. Cryopyrin-associated periodic syndromes (CAPS) are autoinflammatory diseases in which Nod-like receptor family pyrin domain-containing 3 (NLRP3) gene mutations lead to increased IL-1beta secretion. We show in a large cohort of patients that IL-1beta secretion by CAPS monocytes is much faster than that by healthy monocytes. This accelerated kinetics is caused by alterations in the basal redox state, as well as in the redox response to TLR triggering displayed by CAPS monocytes. Indeed, unstimulated CAPS monocytes are under a mild oxidative stress, with elevated levels of both ROS and antioxidants. The redox response to LPS is quickened, with early generation of the reducing conditions favoring IL-1beta processing and secretion, and then rapidly exhausted. Therefore, secretion of IL-1beta is accelerated, but reaches a plateau much earlier than in healthy controls. Pharmacologic inhibition of the redox response hinders IL-1beta release, confirming the functional link between redox impairment and altered kinetics of secretion. Monocytes from patients with juvenile idiopathic arthritis display normal kinetics of redox response and IL-1beta secretion, excluding a role of chronic inflammation in the alterations observed in CAPS. We conclude that preexisting redox alterations distinct from CAPS monocytes anticipate the pathogen-associated molecular pattern molecule-induced generation of the reducing environment favorable to inflammasome activation and IL-1beta secretion.

Redox remodeling allows and controls B-cell activation and differentiation.

During their differentiation to antibody-secreting plasma cells, B lymphocytes undergo dramatic changes in metabolism, structure, and function. Here we show that this transition entails extensive intra- and extracellular redox changes. Lipopolysaccharide (LPS)-driven activation and differentiation of naïve murine B splenocytes is paralleled by increased production of reactive oxygen species (ROS) from different sources, followed by a strong antioxidant response. This response includes upregulation of thioredoxin and of the cystine transporter xCT, and increased production and extracellular release of nonprotein thiols, mainly glutathione (GSH) and cysteine. Although ROS levels are higher in late-differentiating B cells, an early oxidative step is likely required to start the differentiation program, because inhibition of NADPH oxidase-dependent early ROS production impairs B-cell activation and differentiation. Addition of reducing agents such as 2-ME results in increased IgM secretion per cell, suggesting that the antioxidant response not only is aimed at restoring the redox homeostasis but also plays a functional role. A highly reduced environment coincident with the presence of large ROS-producing cells is observed in histologic sections of spleens from immunized mice, indicating that the redox modifications observed in LPS-induced B-cell differentiation in vitro occur also in vivo during physiologic immune responses.

DAMPs and inflammatory processes: the role of redox in the different outcomes.

Inflammation is deeply entangled with redox modulation. Triggering of PRRs on inflammatory cells induces ROS generation. As a consequence, activated cells mount antioxidant responses to counteract the possible harmful effects of oxidation. Therefore, when repair is completed, homeostasis is restored. Here, we describe some recent results showing that an exuberant antioxidant response to pro-oxidant inflammatory stimuli modifies not only the intra- but also the extracellular redox and contributes to the outcome of the inflammatory process. In particular, the role of redox modulation in IL-1beta secretion, in B lymphocyte differentiation to plasma cells, and in tumor progression will be discussed, and the potential consequences of extracellular redox alterations on DAMP activity will be considered.

Pathogen-induced interleukin-1beta processing and secretion is regulated by a biphasic redox response.

In this study, we show that IL-1beta processing and secretion induced by pathogen-associated molecular pattern (PAMP) molecules in human monocytes is regulated by a biphasic redox event including a prompt oxidative stress and a delayed antioxidant response. Namely, PAMPs induce an early generation of reactive oxygen species (ROS) followed by increase of intracellular thioredoxin and release of reduced cysteine: this antioxidant phase is paralleled by secretion of mature IL-1beta. ROS production and antioxidant response are both required, because either inhibitors of NADPH oxidase and of thioredoxin reductase impair IL-1beta secretion. These inhibitors also hinder cysteine release and consequently prevent reduction of the extracellular medium: addition of exogenous reducing agents restores IL-1beta secretion. Not only silencing of thioredoxin, but also of the ROS scavenger superoxide dismutase 1 results in inhibition of IL-1beta secretion. Thus, PAMP-induced ROS trigger an antioxidant response involving intracellular redox enzymes and release of cysteine, ultimately required for IL-1beta processing and secretion.