Role of the Mevalonate Pathway in Adrenocortical Tumorigenesis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme of the mevalonate pathway, which generates cholesterol and non-sterol compounds such as isoprenoid, which are involved in key steps of tumorigenesis such as cell growth and proliferation. Our aim was to evaluate the role of the mevalonate pathway in adrenocortical tumors (ACTs). Expression pattern of HMGCR, FDFT1, LDLR, SCARB1, StAR, TSPO, CYP11A1, CYP11B1, CYP17A1, CYP21A1, and HSD3B1 genes, involved in the mevalonate pathway and steroidogenesis, was quantified by real-time RT-PCR in 46 ACT [14 adenomas (ACA) and 11 carcinomas (ACC) from adults and 13 ACA and 8 ACC from pediatric patients]. Effects of the mevalonate pathway inhibition on NCI-H295A cell viability was assessed by colorimetric assay. HMGCR was overexpressed in most adult ACT. The expression of TSPO, STAR, CYP11B1, CYP21A1, and HSD3B1 in adult ACC was significantly lower than in ACA (p<0.05). Regarding pediatric ACT, the expression of genes involved in steroidogenesis was not different between ACA and ACC. Inhibition of isoprenoid production significantly decreased the viability of NCI-H295A cells (p<0.05). However, cholesterol synthesis blockage did not show the same effect on cell viability. Low expression of TSPO ,: StAR, CYP11B1, CYP21A1, and HSD3B1 characterized a signature of adult ACCs. Our data suggest that HMGCR overexpression in adult ACC might lead to intracellular isoprenoid accumulation and cell proliferation. Therefore, the mevalonate pathway is a potential target for ACC treatment.

Author Correction: A possible association between fructose consumption and pulmonary emphysema.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Aerobic exercise modulates cardiac NAD(P)H oxidase and the NRF2/KEAP1 pathway in a mouse model of chronic fructose consumption.

The present study investigated the effects of exercise on the cardiac nuclear factor (erythroid-derived 2) factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1) pathway in an experimental model of chronic fructose consumption. Male C57BL/6 mice were assigned to Control, Fructose (20% fructose in drinking water), Exercise (treadmill exercise at moderate intensity), and Fructose + Exercise groups (n = 10). After 12 wk, the energy intake and body weight in the groups were similar. Maximum exercise testing, resting energy expenditure, resting oxygen consumption, and carbon dioxide production increased in the exercise groups (Exercise and Fructose + Exercise vs. Control and Fructose groups, P < 0.05). Chronic fructose intake induced circulating hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia and increased white adipose tissue depots, with no changes in blood pressure. This metabolic environment increased circulating IL-6, IL-1ß, IL-10, cardiac hypertrophy, and cardiac NF-κB-p65 and TNF-α expression, which were reduced by exercise (P < 0.05). Cardiac ANG II type 1 receptor and NAD(P)H oxidase 2 (NOX2) were increased by fructose intake and exercise decreased this response (P < 0.05). Exercise increased the cardiac expression of the NRF2-to-KEAP1 ratio and phase II antioxidants in fructose-fed mice (P < 0.05). NOX4, glutathione reductase, and catalase protein expression were similar between the groups. These findings suggest that exercise confers modulatory cardiac effects, improving antioxidant defenses through the NRF2/KEAP1 pathway and decreasing oxidative stress, representing a potential nonpharmacological approach to protect against fructose-induced cardiometabolic diseases.NEW & NOTEWORTHY This is the first study to evaluate the cardiac modulation of NAD(P)H oxidase (NOX), the NRF2/Kelch-like ECH-associated protein 1 pathway (KEAP), and the thioredoxin (TRX1) system through exercise in the presence of moderate fructose intake. We demonstrated a novel mechanism by which exercise improves cardiac antioxidant defenses in an experimental model of chronic fructose intake, which involves NRF2-to-KEAP1 ratio modulation, enhancing the local phase II antioxidants hemoxygenase-1, thioredoxin reductase (TXNRD1), and peroxiredoxin1B (PDRX1), and inhibiting cardiac NOX2 overexpression.

A possible association between fructose consumption and pulmonary emphysema.

Chronic Obstructive Pulmonary Disease (COPD) is a syndrome that comprises several distinct and overlapping phenotypes. In addition to persistent airflow limitation and respiratory symptoms, COPD is also characterized by chronic systemic inflammation. Epidemiological studies have shown that dietary fibers, fruits and vegetables intake protects against the COPD development, while fructose-loading is associated with increased risk of asthma and chronic bronchitis. Since dietary factors might affect susceptibility to COPD by modulating oxidative stress and inflammatory responses, we evaluated how fructose feeding might affect the smoking-induced emphysema in mice. We found that chronic fructose intake induced destruction and remodeling of lung parenchyma and impairment of respiratory mechanics, which are associated with distinctive cytokine profiles in bronchoalveolar lavage fluid, blood plasma and skeletal muscle. The combined effects of chronic fructose intake and cigarette smoking on destruction of lung parenchyma are more pronounced than the effects of either alone. Excessive intake of fructose might directly cause pulmonary emphysema in mice rather than just altering its natural history by facilitating the installation of a low-grade systemic inflammatory milieu.

Interesterified Fats Induce Deleterious Effects on Adipose Tissue and Liver in LDLr-KO Mice.

Interesterified fats are being widely used by the food industry in an attempt to replace trans fatty acids. The effect of interesterified fats containing palmitic or stearic acids on lipid metabolism and inflammatory signaling pathways in adipose and hepatic tissues was evaluated. Male LDLr-KO mice were fed a high-fat diet containing polyunsaturated (PUFA), palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR), or stearic interesterified (STEAR INTER) fats for 16 weeks. The expression of genes and protein levels involved in lipid metabolism and inflammatory processes in liver and white adipose tissue was determined by quantitative RT-PCR and by Western blot, respectively. The infiltration of inflammatory cells in hepatic and adipose tissues was determined by eosin and hematoxylin, while liver collagen content was determined by Sirius Red staining. Both interesterified fats increased liver collagen content and JNK phosphorylation. Additionally, the STEAR INTER group developed nonalcoholic steatohepatitis (NASH) associated with higher neutrophil infiltration. PALM INTER induced adipose tissue expansion and enlargement of adipocytes. Furthermore, PALM INTER triggered increased IKK phosphorylation and TNFα protein content, conditions associated with the upstream activation of the NFkB signaling pathway. STEAR INTER induced NASH, while PALM INTER triggered hepatic fibrosis and adipocyte hypertrophy with inflammatory response in LDLr-KO mice.

Th17/Treg imbalance in COPD progression: A temporal analysis using a CS-induced model.

BACKGROUND: The imbalance between pro- and anti-inflammatory immune responses plays a pivotal role in chronic obstructive pulmonary disease (COPD) development and progression. To clarify the pathophysiological mechanisms of this disease, we performed a temporal analysis of immune response-mediated inflammatory progression in a cigarette smoke (CS)-induced mouse model with a focus on the balance between Th17 and Treg responses. METHODS: C57BL/6 mice were exposed to CS for 1, 3 or 6 months to induce COPD, and the control groups were maintained under filtered air conditions for the same time intervals. We then performed functional (respiratory mechanics) and structural (alveolar enlargement) analyses. We also quantified the NF-κB, TNF-α, CD4, CD8, CD20, IL-17, IL-6, FOXP3, IL-10, or TGF-ß positive cells in peribronchovascular areas and assessed FOXP3 and IL-10 expression through double-label immunofluorescence. Additionally, we evaluated the gene expression of NF-κB and TNF in bronchiolar epithelial cells. RESULTS: Our CS-induced COPD model exhibited an increased proinflammatory immune response (increased expression of the NF-κB, TNF-α, CD4, CD8, CD20, IL-17, and IL-6 markers) with a concomitantly decreased anti-inflammatory immune response (FOXP3, IL-10, and TGF-ß markers) compared with the control mice. These changes in the immune responses were associated with increased alveolar enlargement and impaired lung function starting on the first month and third month of CS exposure, respectively, compared with the control mice. CONCLUSION: Our results showed that the microenvironmental stimuli produced by the release of cytokines during COPD progression lead to a Th17/Treg imbalance.

Fructose ingestion impairs expression of genes involved in skeletal muscle's adaptive response to aerobic exercise.

BACKGROUND: The inverse relationship between exercise capacity and its variation over time and both cardiovascular and all-cause mortality suggests the existence of an etiological nexus between cardiometabolic diseases and the molecular regulators of exercise capacity. Coordinated adaptive responses elicited by physical training enhance exercise performance and metabolic efficiency and possibly mediate the health benefits of physical exercise. In contrast, impaired expression of genes involved in mitochondrial biogenesis or protein turnover in skeletal muscle-key biological processes involved in adaptation to physical training-leads to insulin resistance and obesity. Ingestion of fructose has been shown to suppress the exercise-induced GLUT4 response in rat skeletal muscle. To evaluate in greater detail how fructose ingestion might blunt the benefits of physical training, we investigated the effects of fructose ingestion on exercise induction of genes that participate in regulation of mitochondrial biogenesis and protein turnover in rat's skeletal muscle. METHODS: Eight-week-old Wistar rats were randomly assigned to sedentary (C), exercise (treadmill running)-only (E), fructose-only (F), and fructose + exercise (FE) groups and treated accordingly for 8 weeks. Blood and quadriceps femoris were collected for biochemistry, serum insulin, and gene expression analysis. Expression of genes involved in regulation of mitochondrial biogenesis and autophagy, GLUT4, and ubiquitin E3 ligases MuRF-1, and MAFbx/Atrogin-1 were assayed with quantitative real-time polymerase chain reaction. RESULTS: Aerobic training improved exercise capacity in both E and FE groups. A main effect of fructose ingestion on body weight and fasting serum triglyceride concentration was detected. Fructose ingestion impaired the expression of PGC-1α, FNDC5, NR4A3, GLUT4, Atg9, Lamp2, Ctsl, Murf-1, and MAFBx/Atrogin-1 in skeletal muscle of both sedentary and exercised animals while expression of Errα and Pparδ was impaired only in exercised rats. CONCLUSIONS: Our results show that fructose ingestion impairs the expression of genes involved in biological processes relevant to exercise-induced remodeling of skeletal muscle. This might provide novel insight on how a dietary factor contributes to the genesis of disorders of glucose metabolism.

Time-course effects of aerobic physical training in the prevention of cigarette smoke-induced COPD.

A previous study by our group showed that regular exercise training (ET) attenuated pulmonary injury in an experimental model of chronic exposure to cigarette smoke (CS) in mice, but the time-course effects of the mechanisms involved in this protection remain poorly understood. We evaluated the temporal effects of regular ET in an experimental model of chronic CS exposure. Male C57BL/6 mice were divided into four groups: Control (sedentary + air), Exercise (aerobic training + air), Smoke (sedentary + smoke), and Smoke + Exercise (aerobic training + smoke). Mice were exposed to CS and ET for 4, 8, or 12 wk. Exercise protected mice exposed to CS from emphysema and reductions in tissue damping and tissue elastance after 12 wk (P < 0.01). The total number of inflammatory cells in the bronchoalveolar lavage increased in the Smoke group, mainly due to the recruitment of macrophages after 4 wk, neutrophils and lymphocytes after 8 wk, and lymphocytes and macrophages after 12 wk (P < 0.01). Exercise attenuated this increase in mice exposed to CS. The protection conferred by exercise was mainly observed after exercise adaptation. Exercise increased IL-6 and IL-10 in the quadriceps and lungs (P < 0.05) after 12 wk. Total antioxidant capacity and SOD was increased and TNF-α and oxidants decreased in lungs of mice exposed to CS after 12 wk (P < 0.05). The protective effects of exercise against lung injury induced by cigarette smoke exposure suggests that anti-inflammatory mediators and antioxidant enzymes play important roles in chronic obstructive pulmonary disease development mainly after the exercise adaptation.NEW & NOTEWORTHY These experiments investigated for the first time the temporal effects of regular moderate exercise training in cigarette smoke-induced chronic obstructive pulmonary disease. We demonstrate that aerobic conditioning had a protective effect in emphysema development induced by cigarette smoke exposure. This effect was most likely secondary to an effect of exercise on oxidant-antioxidant balance and anti-inflammatory mediators.

Optimization of total RNA isolation from human urinary sediment.

Extracting RNA from human urinary sediment is notoriously challenging because of cell paucity and hostile environment and column-based commercial kits using silica technology are commonly used. Nonetheless, in our experience, this methodology yields low amounts of total RNA and has low rates of success. We replaced the column-based commercial kit by a protocol using guanidine isothiocyanate-phenol-chloroform buffer (Trizol reagent) followed by addition of glycogen as a carrier and precipitation with isopropanol plus sodium acetate. This methodology was more affordable and efficient for urinary sediment total RNA isolation than silica technology, resulting in higher concentrations of total RNA of better quality.

Aerobic Exercise Reduces Asthma Phenotype by Modulation of the Leukotriene Pathway.

INTRODUCTION: Leukotrienes (LTs) play a central role in asthma. Low- to moderate-intensity aerobic exercise (AE) reduces asthmatic inflammation in clinical studies and in experimental models. This study investigated whether AE attenuates LT pathway activation in an ovalbumin (OVA) model of asthma. METHODS: Sixty-four male, BALB/c mice were distributed into Control, Exercise (Exe), OVA, and OVA + Exe groups. Treadmill training was performed at moderate intensity, 5×/week, 1 h/session for 4 weeks. Quantification of bronchoalveolar lavage (BAL) cellularity, leukocytes, airway remodeling, interleukin (IL)-5, IL-13, cysteinyl leukotriene (CysLT), and leukotriene B4 (LTB4) in BAL was performed. In addition, quantitative analyses on peribronchial leukocytes and airway epithelium for LT pathway agents: 5-lypoxygenase (5-LO), LTA4 hydrolase (LTA4H), CysLT1 receptor, CysLT2 receptor, LTC4 synthase, and LTB4 receptor 2 (BLT2) were performed. Airway hyperresponsiveness (AHR) to methacholine (MCh) was assessed via whole body plethysmography. RESULTS: AE decreased eosinophils (p < 0.001), neutrophils (p > 0.001), lymphocytes (p < 0.001), and macrophages (p < 0.01) in BAL, as well as eosinophils (p < 0.01), lymphocytes (p < 0.001), and macrophages (p > 0.001) in airway walls. Collagen (p < 0.01), elastic fibers (p < 0.01), mucus production (p < 0.01), and smooth muscle thickness (p < 0.01), as well as IL-5 (p < 0.01), IL-13 (p < 0.01), CysLT (p < 0.01), and LTB4 (p < 0.01) in BAL were reduced. 5-LO (p < 0.05), LTA4H (p < 0.05), CysLT1 receptor (p < 0.001), CysLT2 receptor (p < 0.001), LTC4 synthase (p < 0.001), and BLT2 (p < 0.01) expression by peribronchial leukocytes and airway epithelium were reduced. Lastly, AHR to MCh 25 mg/mL (p < 0.05) and 50 mg/mL (p < 0.01) was reduced. CONCLUSION: Moderate-intensity AE attenuated asthma phenotype and LT production in both pulmonary leukocytes and airway epithelium of OVA-treated mice.

Prevalence of thyroiditis and immunohistochemistry study searching for a morphologic consensus in morphology of autoimmune thyroiditis in a 4613 autopsies series.

We sought to verify the prevalence of lymphocytic thyroiditis (LT) and Hashimoto's thyroiditis (HT) in autopsy materials. Cases examined between 2003 and 2007 at the Department of Pathology of Faculty of Medicine of São Paulo University were studied. Immunohistochemical analyses were conducted in selected cases to characterize the type of infiltrating mononuclear cells; in addition, we evaluated the frequency of apoptosis by TUNEL assay technique and caspase-3 immunostaining. Significant increase in overall thyroiditis frequency was observed in the present series when compared with the previous report (2.2978% vs. 0.0392%). Thyroiditis was more prevalent among older people. Selected cases of LT and HT (40 cases each) had their infiltrating lymphocytes characterized by immunohistochemical analyses. Both LT and HT showed similar immunostaining patterns for CD4, CD8, CD68, thus supporting a common pathophysiology mechanism and indicating that LT and HT should be considered different presentations of a same condition, that is, autoimmune thyroiditis. Moreover, apoptosis markers strongly evidenced that apoptosis was present in all studied cases. Our results demonstrated an impressive increase in the prevalence of thyroiditis during recent years and our data support that the terminology of autoimmune thyroiditis should be used to designate both LT and HT. This classification would facilitate comparison of prevalence data from different series and studies.

Dietary phytosterol does not accumulate in the arterial wall and prevents atherosclerosis of LDLr-KO mice.

SCOPE: There have been conflicting reports on the usefulness of phytosterols (PS) in preventing atherosclerosis. We evaluated the effects of dietary PS supplementation in LDLr-KO male mice on the plasma and aorta sterol concentrations and on atherosclerotic lesion development. METHODS AND RESULTS: Mice were fed a high fat diet (40% of energy) supplemented with or without PS (2% w/w, n = 10). Plasma and arterial wall cholesterol and PS concentrations, lesion area, macrophage infiltration, and mRNA expression from LOX-1, CD36, ABCA1 and ABCG1 in peritoneal macrophages were measured. After 16 weeks, the plasma cholesterol concentration in PS mice was lower than that in the controls (p = 0.02) and in the arterial wall (p = 0.03). Plasma PS concentrations were higher in PS-fed animals than in controls (p < 0.0001); however, the arterial wall PS concentration did not differ between groups. The atherosclerotic lesion area in the PS group (n = 5) was smaller than that in controls (p = 0.0062) and the macrophage area (p = 0.0007). PS correlates negatively with arterial lipid content and macrophage (r = -0.76; p < 0.05). PS supplementation induced lower ABCG1 mRNA expression (p < 0.05). CONCLUSIONS: Despite inducing an increase in PS plasma concentration, PS supplementation is not associated with its accumulation in the arterial wall and prevents atherosclerotic lesion development.

Iodine increases and predicts incidence of thyroiditis in NOD mice: Histopathological and ultrastructural study.

Prolonged intake of large amounts of iodine has been reported to increase the incidence of hypothyroidism in humans, as well as in animals which are prone to spontaneously developing autoimmune thyroiditis. We sought to investigate the histopathological consequences of large amounts of dietary iodine on the thyroid gland and observe the occurrence of lymphocytic infiltration associated with the time of exposure to iodine. An experimental model using non-obese diabetic (NOD) mice was analyzed. A potassium iodide intake of 0.2 mg/animal/day was administered via drinking water, in experimental groups of 60 and 90 days (EG60 and EG90). Distended rough endoplasmic reticulum, degenerated mitochondria, debris and amorphous spaces or 'ill-defined' spaces were observed with electron microscopy (EM). Lymphocyte infiltration was observed in the two groups and the time of exposure to iodine did not increase the appearance of lymphocyte infiltration but significantly associated with the development of necrosis. The results of the present study demonstrated that the NOD mouse is a feasible experimental model for thyroiditis induced by iodine administration and may represent an opportunity to analyze the steps and factors associated with genetic autoimmune thyroiditis. High doses of ingested iodine were observed to precdict and increase the incidence of the thyroiditis process.

Possible role of a radiation-induced p53 mutation in a Nelson's syndrome patient with a fatal outcome.

Nelson's syndrome (NS) is characterized by the appearance and/or progression of ACTH-secreting pituitary macroadenomas in patients who had previously undergone bilateral adrenalectomy for the treatment of Cushing's disease. Such corticotroph macroadenomas respond poorly to currently available therapeutic options which include surgery, radiotherapy and chemotherapy. P53 protein accumulation may be detected by immunohistochemistry in pituitary corticotroph adenomas and it has been suggested that it might be causally related to tumor development. Wild type P53 protein plays an important role in the cellular response to ionizing radiation and other DNA damaging agents and is mutated in many human tumors. In this study we report an adult male patient with NS who underwent both transsphenoidal and transcranial pituitary surgeries, conventional and stereotaxic radiotherapy and brachytherapy. Despite of the efforts to control tumor mass and growth, this macroadenoma displayed relentless growth and aggressive behavior. DNA extracted from the first two surgical samples, as well as DNA from peripheral blood leukocytes disclosed normal p53 sequence. DNA extracted from tumor samples obtained at surgeries performed after pituitary irradiation carried a somatic heterozygous mutation, consisting of a deletion of four cytosines between nucleotides 12,144-12,149 in exon 4 of the p53 gene. This frameshift mutation creates a stop codon in exon 4 excluding the expression of a functional protein from the defective allele. These data demonstrate a possible association between the P53 protein loss of function induced by radiotherapy and the aggressive course of the disease in this patient.

A method to immunolabel rodent spinal cord neurons and glia for molecular study in specific laser microdissected cells involved in neurodegenerative disorders.

Neuron-glia interaction is involved in physiological function of neurons, however, recent evidences have suggested glial cells as participants in neurotoxic and neurotrophic mechanisms of neurodegenerative/neuroregenerative processes. Laser microdissection offers a unique opportunity to study molecular regulation in specific immunolabeled cell types. However, an adequate protocol to allow morphological and molecular analysis of rodent spinal cord astrocyte, microglia and motoneurons remains a big challenge. In this paper we present a quick method to immunolabel those cells in flash frozen sections to be used in molecular biology analyses after laser microdissection and pressure catapulting.

Imunofenotipagem e rearranjo gênico em doenças pulmonares linfocíticas e linfoproliferativas / Immunophenotyping and gene rearrangement analysis in lymphoid/lymphoproliferative disorders of the lungs

OBJETIVO: Determinar a utilidade, na prática rotineira, da análise da clonalidade dos linfócitos T e B nos tecidos pulmonares por reação em cadeia da polimerase no diagnóstico das doenças linfoproliferativas pulmonares. MÉTODOS: Avaliaram-se, mediante análise imunohistoquímica e rearranjo molecular dos genes, 8 casos de pneumonia intersticial linfocítica (PIL) e 7 casos de doenças linfoproliferativas pulmonares. RESULTADOS: Todos os 8 casos de PIL expressaram imunocoloração moderada a forte para CD3, em contraste com apenas 2 casos de linfoma e 1 caso de pseudolinfoma. Rearranjo gênico foi detectado em 4 de 8 casos de PIL, o que mudou o diagnóstico de PIL para linfoma, indicando, assim, a importância da detecção de rearranjo gênico em casos de PIL. Nesta situação, rearranjo gênico usando-se os pares de primers VH/JH e Vgama11/Jgama12 foi detectado em 3 e 1 casos de PIL, respectivamente, e não foram detectadas anormalidades gênicas usando-se as pares Dbeta1/Jbeta2 e Vgama101/Jgama12. Uma associação positiva foi detectada entre a intensidade de imunoexpressão CD20 e CD68 e rearranjo gênico usando-se o par de primers VH/JH. Antes do rearranjo gênico, 4 pacientes com PIL morreram rapidamente, enquanto que, após o rearranjo gênico, apenas 1 paciente com PIL morreu. CONCLUSÕES: A detecção de células B e T monoclonais por imunofenotipagem e reação em cadeia da polimerase mostrou impacto no diagnóstico de linfomas pulmonares em pacientes previamente diagnosticados com PIL. Portanto, imunofenotipagem e reação em cadeia da polimerase devem ser incluídas como métodos de 'padrão ouro' na rotina diagnóstica.

OBJECTIVE: To determine the usefulness, in routine practice, of using polymerase chain reaction to analyze B and T lymphocyte clonality in pulmonary tissue as a tool for the diagnosis of pulmonary lymphoproliferative disorders. METHODS: Immunohistochemistry and molecular gene rearrangement analysis were performed in order to assess 8 cases of lymphoid interstitial pneumonia (LIP) and 7 cases of pulmonary lymphoproliferative disorders. RESULTS: All 8 cases of LIP presented moderate to strong immunostaining for CD3, compared with only 2 cases of lymphoma and 1 case of pseudolymphoma (p = 0.02). Gene rearrangement was detected in 4 of the 8 cases, which changed the diagnosis from LIP to lymphoma, showing the importance of gene rearrangement detection in cases of LIP. In this situation, gene rearrangement using the VH/JH and Vgamma11/Jgamma12 primer pairs was detected in 3 cases and 1 case, respectively, and no gene abnormalities were found using the Dbeta1/Jbeta2 and Vgamma101/Jgamma12 primer pairs in any of the cases. A significant positive association was found between the intensity of CD20 and CD68 expression and gene rearrangement using the VH/JH primer pair. Prior to the gene rearrangement, 4 patients with LIP died quickly, whereas only one patient with LIP died after the gene rearrangement. CONCLUSIONS: Detection of monoclonal B and T cells by immunophenotyping and polymerase chain reaction had an impact on the diagnosis of pulmonary lymphomas in patients previously diagnosed with LIP. Therefore, immunophenotyping and polymerase chain reaction should be used as 'gold standard' techniques in routine practice.

[Immunophenotyping and gene rearrangement analysis in lymphoid/lymphoproliferative disorders of the lungs]. / Imunofenotipagem e rearranjo gênico em doenças pulmonares linfocíticas e linfoproliferativas.

OBJECTIVE: To determine the usefulness, in routine practice, of using polymerase chain reaction to analyze B and T lymphocyte clonality in pulmonary tissue as a tool for the diagnosis of pulmonary lymphoproliferative disorders. METHODS: Immunohistochemistry and molecular gene rearrangement analysis were performed in order to assess 8 cases of lymphoid interstitial pneumonia (LIP) and 7 cases of pulmonary lymphoproliferative disorders. RESULTS: All 8 cases of LIP presented moderate to strong immunostaining for CD3, compared with only 2 cases of lymphoma and 1 case of pseudolymphoma (p = 0.02). Gene rearrangement was detected in 4 of the 8 cases, which changed the diagnosis from LIP to lymphoma, showing the importance of gene rearrangement detection in cases of LIP. In this situation, gene rearrangement using the VH/JH and Vgamma11/Jgamma12 primer pairs was detected in 3 cases and 1 case, respectively, and no gene abnormalities were found using the Dbeta1/Jbeta2 and Vgamma101/Jgamma12 primer pairs in any of the cases. A significant positive association was found between the intensity of CD20 and CD68 expression and gene rearrangement using the VH/JH primer pair. Prior to the gene rearrangement, 4 patients with LIP died quickly, whereas only one patient with LIP died after the gene rearrangement. CONCLUSIONS: Detection of monoclonal B and T cells by immunophenotyping and polymerase chain reaction had an impact on the diagnosis of pulmonary lymphomas in patients previously diagnosed with LIP. Therefore, immunophenotyping and polymerase chain reaction should be used as 'gold standard' techniques in routine practice.

Avanços recentes no conhecimento dos mecanismos moleculares envolvidos na tumorigênese adrenocortical / Molecular mechanisms envolved in adrenocortical tumorigenesis

A tumorigênese adrenal é um fenômeno complexo, que envolve múltiplas alterações genéticas. Uma melhor compreensão dos mecanismos que levam ao desenvolvimento dos tumores adrenocorticais possibilitaria não só a identificação precoce dos casos de má evolução, mas também o desenvolvimento de novas estratégias terapêuticas. Embora nos últimos anos tenham surgido vários estudos sobre a tumorigênese adrenocortical, o processo permanece em grande parte desconhecido. A maior parte dos trabalhos disponíveis estudou apenas um ou poucos genes. Por se tratar de um fenômeno complexo, técnicas que avaliam múltiplos, como os microarrays, possivelmente possibilitarão o entendimento de aspectos que até o momento são desconhecidos. Nesta revisão, tentamos resumir de forma abrangente os principais trabalhos científicos produzidos nos últimos anos a respeito do processo de tumorigênese adrenocortical.

[Molecular mechanisms involved in adrenocortical tumorigenesis]. / Avanços recentes no conhecimento dos mecanismos moleculares envolvidos na tumorigênese adrenocortical.

The adrenocortical tumorigenesis is a complex process, which involves multiple genetic changes. A better knowledge on the mechanisms involved in tumor development would enable an early identification of malignant disease and also lead to the development of new treatment strategies. Although in the recent years a large amount of data was produced, the exact mechanisms that lead to adrenocortical tumor development remains poorly understood. Most of the studies produced were based on the candidate-gene strategy, which has its own limitations. A genome-wide approach, such as microarrays, will surely shed some light into the mechanisms responsible for adrenocortical tumorigenesis. In this review, we summarize the most recent data available on this complex process.

Silenciamento de genes com RNA interferência: um novo instrumento para investigação da fisiologia e fisiopatologia do córtex adrenal / Gene silencing with RNA interference: a novel tool for the study of physiology and pathophysiology of adrenal cortex

A inativação de genes por knock-out ou por bloqueio da tradução de seus transcritos (silenciamento) constitui uma estratégia extremamente poderosa tanto para atribuir função aos genes como para mapear a inter-relação dos diferentes componentes das vias regulatórias intracelulares. Um dos meios para se obter o silenciamento pós-transcricional consiste na ativação de um mecanismo mediado por RNAs fita-dupla (dsRNA) conhecido como RNA interferência (RNAi). O RNAi se mostrou um instrumento extremamente versátil em pesquisa biomédica, podendo ser utilizado em experimentos de silenciamento pontual de genes ou ser adaptado para estudos em larga escala de genômica funcional, podendo, inclusive, ser utilizado como meio de terapia gênica. Neste trabalho, os autores discutem as vias intracelulares envolvidas no RNAi, bem como as principais estratégias e limitações técnicas para se obter o silenciamento em células de mamíferos. Fazem, também, uma revisão das principais aplicações do RNAi na terapêutica de doenças humanas e na investigação de fenômenos fisiológicos e fisiopatológicos do córtex adrenal.