Induction of smooth muscle cell nitric oxide synthase by human leukaemia inhibitory factor: Effects in vitro and in vivo

We have previously shown that human leukaemia inhibitory factor (hLIF) inhibits perivascular cuff-induced neointimal formation in the rabbit carotid artery. Since nitric oxide (NO) is a known inhibitor of smooth muscle growth, NO synthase (NOS) activity in the presence of hLIF was examined in vivo and in vitro. In rabbit aortic smooth muscle cell (SMC) culture, significant NOS activity was observed at 50 pg/ml hLIF, with maximal activity at 5 ng/ml. In the presence of the NOS inhibitor L-NAME, hLIF-induced activation of NOS was greatly decreased, however it was still 63-fold higher than in control (p < 0.05). SMC-DNA synthesis was significantly reduced (-47%) following incubation with hLIF plus L-arginine, the substrate required for NO production (p < 0.05), with no effect observed in the absence of L-arginine. Silastic cuff placement over the right carotid artery of rabbits resulted in a neointima 19.3 +/- 5.4% of total wall cross-sectional area, which was increased in the presence of L-NAME (27.0 +/- 2.0%; p < 0.05) and reduced in the presence of L-arginine (11.3 +/- 2.0%; p < 0.05). The effect of L-arginine was ameliorated by co-administration of L-NAME (16.4 +/- 1.5%). However, administration of L-NAME with hLIF had no effect on the potent inhibition of neointimal formation by hLIF (3.2 +/- 2.5 vs. 2.1 +/- 5.4%, respectively). Similarly, with hLIF administration, NOS activity in the cuffed carotid increased to 269.0 +/- 14.0% of saline-treated controls and remained significantly higher with coadministration of L-NAME (188.5 +/- 14.7%). These results indicate that hLIF causes superinduction of NO by SMC, and that it is, either partially or wholly, through this mechanism that hLIF is a potent inhibitor of neointimal formation in vivo and of smooth muscle proliferation in vitro.

In familial hyperaldosteronism type I, hybrid gene-induced aldosterone production dominates that induced by wild-type genes

We compared the aldosterone-producing potency of the angiotensin II-sensitive wild-type aldosterone synthase genes and the ACTH-sensitive hybrid 11 beta-hydroxylase/aldosterone synthase gene by examining aldosterone, PRA, and cortisol day-curves (2-hourly levels over 24 h) in patients with familial hyperaldosteronism type I, before and during long-term (0.8-13.5 yr) glucocorticoid treatment. In 8 untreated patients, PRA levels were usually suppressed, and aldosterone correlated strongly with cortisol (r = 0.69-0.99). Fourteen studies were performed on 10 patients receiving glucocorticoid treatment that corrected hypertension, hypokalemia, and PRA suppression in all. ACTH was markedly and continuously suppressed in 6 studies, 3 of which demonstrated strong correlations between aldosterone and PRA (r = 0.77-0.92), ACTH was only partially suppressed in the remaining 8 studies; aldosterone correlated strongly: 1) with cortisol alone in 5 (r = 0.71-0.98); 2) with cortisol (r = 0.90) and PRA (r = 0.74) in one; 3) with PRA only in one (r = 0.80); and 4) with neither PRA nor cortisol in one. Unless ACTH is markedly and continuously suppressed, aldosterone is more responsive to ACTH than to renin/angiotensin II, despite the latter being unsuppressed. This is consistent with the hybrid gene being more powerfully expressed than the wild-type aldosterone synthase genes in familial hyperaldosteronism type I.

Vascular endothelial growth factor and beta-human chorionic gonadotropin are associated with trophoblastic invasion into the tubal wall in ectopic pregnancy

Objective: To assess the association between the depth of trophoblastic penetration into the tubal wall with serum concentrations of vascular endothelial growth factor (VEGF) and beta-hCG and to assess its predictive value. Design: Prospective study. Setting: Tertiary care university hospital. Patient(s): Thirty patients with ampullary pregnancy undergoing salpingectomy were analyzed. Intervention(s): Trophoblastic invasion was histologically classified as stage I when limited to the tubal mucosa, stage II when extending to the muscle layer, and stage III in the case of complete tubal wall infiltration. Main Outcome Measure(s): The relation between depth of trophoblastic infiltration into the tubal wall with VEGF and beta-hCG serum concentrations on the day of surgery. Result(s): An association between the depth of trophoblastic invasion and maternal serum concentrations of VEGF and beta-hCG was observed. VEGF levels of 297.2 pg/mL showed 100.0% sensitivity and 90.0% specificity for stage I, and levels of 440.1 pg/mL showed 81.8% sensitivity and 88.8% specificity for stage III. Beta-hCG levels of 2590.0 mIU/mL showed 88.9% sensitivity and 80.0% specificity for stage I, and levels of 10,827.0 mUI/mL showed 72.7% sensitivity and 88.9% specificity for stage III. Conclusion(s): Maternal serum VEGF and beta-hCG concentrations are associated with depth of trophoblastic penetration into the tubal wall. (Fertil Steril (R) 2010;94:1595-600. (C) 2010 by American Society for Reproductive Medicine.)

Platelet GSK3B activity in patients with late-life depression: Marker of depressive episode severity and cognitive impairment?

Objective. Increased GSK3B activity has been reported as a state marker of major affective episodes in patients with depression and bipolar disorder. No study so far has addressed GSK3B activity in late-life depression. The aims of the present study were to determine GSK3B activity in platelets of elderly patients with major depression, and the association between GSK3B activity and the severity of depressive symptoms and cognitive impairment. Methods. Forty drug-free elderly patients with major depressive episode were compared to healthy older adults (n == 13). Severity of the depressive episode and current cognitive state were determined by the Hamilton Depression Scale (HAM-D) and the Cambridge Cognitive Test (CAMCOG), respectively. Total- and ser-9-phosphorylated GSK3B (tGSK3B and pGSK3B) were determined in platelets by enzyme immunometric assays (EIA). GSK3B activity was indirectly inferred by the GSK3B ratio (i.e. pGSK3B/tGSK3B). Results. Elderly depressed patients had significantly lower pGSK3B levels (P == 0.03) and GSK3B ratio (P == 0.03), indicating higher GSK3B activity. Higher GSK3B activity were observed in patients with severe depressive episode (HAM-D scores > 22, P == 0.03) and with cognitive impairment (CAMCOG scores < 86, P == 0.01). Conclusion. The present findings provide additional evidence of the involvement of GSK3B in the pathophysiology of late-life major depression. Higher GSK3B activity may be more relevant in those patients with more severe depressive symptoms and cognitive impairment.

Interleukin-1 beta Serum Levels is Increased in Antidepressant-Free Elderly Depressed Patients

Objective: To assess the serum levels of interleukin 1 beta (IL-1 beta) in elderly depressed patients in comparison with nondepressed healthy elderly subjects. Design: Cross-sectional study. Setting: Tertiary memory clinic. Participants: Twenty-three antidepressant-free elderly depressed patients and 44 nondepressed healthy elderly comparison group were enrolled to this study. Measurement: Serum IL-1 beta levels were determined with highly sensitive colorimetric sandwich enzyme-linked immunosorbent assay. Severity of the depressive episode was determined by scores on the Hamilton Depression Scale-21 item and cognitive performance by the scores on the Cambridge Cognition Examination, Mini Mental State Examination clock drawing test, and verbal fluency. Results: IL-1 beta serum levels were increased in elderly patients versus nondepressed elderly (t = 2.21, df = 65, p = 0.04). After categorizing elderly depressed subjects into late onset (LOD) versus early onset (EOD), patients with EOD had the highest IL-1 beta levels, when compared with nondepressed elderly patients and patients with LOD in analysis of variance (F = 4.9, df = 2, 64, p < 0.01). Conclusions: Late-life depression is associated with higher IL-1 beta levels suggesting that increased proinflammatory state may play a role in the physiopathology of depression in the elderly. The authors further show that this might be more prominent in those patients with EOD geriatric depression. (Am J Geriatr Psychiatry 2010; 18: 172-176)

Collagen Type I may Influence the Expression of E-Cadherin and Beta-catenin in Carcinoma Ex-pleomorphic Adenoma

Carcinoma ex-pleomorphic adenoma (CXPA) is an aggressive salivary gland malignancy, usually derived from a long-standing or a recurrent benign tumor, the pleomorphic adenoma (PA). In the context of dynamic reciprocity, changes in the composition and structure of extracellular matrix proteins and cell surface receptors have been frequently associated with dysfunctional adhesion and invasive behavior of tumor cells. It is not fully understood if these changes are involved in the conversion of PA to CXPA. In this study, different progression stages of CXPA were investigated regarding the expression of the major extracellular matrix proteins, collagen type I, and of E-cadherin and beta-catenin, the components of adherens junctions. By immunohistochemical analysis, we have demonstrated that direct contact of tumor cells with fibrillar type I collagen, particularly near the invasive front and in invasive areas prevailing small nests of CXPA cells, could be associated with reduced expression of the E-cadherin and beta-catenin adhesion molecules and with invasive behavior of epithelial; but not of CXPA with myoepithelial component. Our results also suggested that this association could depend on the organization of collagen molecules, being prevented by high-order polymeric structures. These findings could implicate the local microenvironment in the transition from the premalignant PA to invasive CXPA.

Inducible nitric oxide synthase inhibition attenuates lung tissue responsiveness and remodeling in a model of chronic pulmonary inflammation in guinea pigs

We evaluated the influence of iNOS-derived NO on the mechanics, inflammatory, and remodeling process in peripheral lung parenchyma of guinea pigs with chronic pulmonary allergic inflammation. Animals treated or not with 1400W were submitted to seven exposures of ovalbumin in increasing doses. Seventy-two hours after the 7th inhalation, lung strips were suspended in a Krebs organ bath, and tissue resistance and elastance measured at baseline and after ovalbumin challenge. The strips were submitted to histopathological measurements. The ovalbumin-exposed animals showed increased maximal responses of resistance and elastance (p < 0.05), eosinophils counting (p < 0.001), iNOS-positive cells (p < 0.001), collagen and elastic fiber deposition (p < 0.05), actin density (p < 0.05) and 8-iso-PGF2 alpha expression (p < 0.001) in alveolar septa compared to saline-exposed ones. Ovalbumin-exposed animals treated with 1400 W had a significant reduction in lung functional and histopathological findings (p < 0.05). We showed that iNOS-specific inhibition attenuates lung parenchyma constriction, inflammation, and remodeling, suggesting NO-participation in the modulation of the oxidative stress pathway. (C) 2008 Elsevier B.V. All rights reserved.

Macrophage and TGF-beta immunohistochemical expression in Jorge Lobo`s disease

Jorge Lobo`s disease, or lacaziosis, is a chronic deep mycosis that clinically manifests as solid, variable-sized nodular parakeloidal lesions. Few studies have characterized the in situ cellular and humoral immune response, especially the involvement of cytokines with immunosuppressive effects such as TGF-beta. The objective this paper was to analyze the expression of TGF-beta in cutaneous lesions in lacaziosis and investigate its importance in the etiopathogy of the disease. The results indicate that the abundance of collagen bands, together with weak immunolabeling for CD68 seen in macrophages, indicates a concomitant effect of TGF-beta inhibiting macrophages and inducing fibrosis, which is responsible for the keloid aspect frequently acquired by these lesions. Finally, the evolution of the infection supports the hypothesis that TGF-beta plays a fundamental role in the etiopathology of Lacazia loboi infection, either by inhibiting the cellular immune response mainly mediated by macrophages or by inducing fibrosis. Further studies are necessary to better characterize the phenotype of the inflammatory infiltrate as well as the participation of other cytokines and growth factors in the tissue response of the host in Jorge Lobo`s disease. (C) 2008 Published by Elsevier Inc.

Immunohistochemical evaluation of macrophage activity and its relationship with apoptotic cell death in the polar forms of leprosy

The objective of the present study was to investigate the correlation between macrophage activity and apoptosis in the polar forms of leprosy because the immunopathological phenomena involved in these forms are still poorly understood For this purpose, 29 skin biopsy samples obtained from patients with the polar forms of leprosy were analyzed. Macrophage activity and apoptosis were evaluated by immunohistochemistry using lysozyme, CD68, iNOS and caspase 3 as markers The nonparametric Mann-Whitney test and Spearman`s linear correlation test were used for statistical analysis The results suggest that the apoptosis rate is under the direct influence of macrophage activity in lesions of patients with the tuberculoid form In contrast, in lepromatous lesions other factors seem to induce programmed cell death, possibly TGF-beta. Further studies are necessary to identify additional factors involved in the immunopathogenesis of leprosy. (C) 2010 Elsevier Ltd. All rights reserved

Low temperature induced changes in activity and protein levels of the enzymes associated to conversion of starch to sucrose in banana fruit

Storage at low temperature is the most frequently used method to extend the shelf life of banana fruit, and is fundamental for extended storage and transport over long distances. However, storage and transport conditions must be carefully controlled because of the high susceptibility of many commercial cultivars to chilling injury. The physiological behavior of bananas at low temperatures has been studied to identify possible mechanisms of resistance to chilling injury. The aim of this work was to evaluate differences in the starch-to-sucrose metabolism of a less tolerant and susceptible (Musa acuminata, AAA cv. Nanicao) and a more tolerant (M. acuminata x Musa balbusiana, AAB, cv. Prata) banana cultivar to chilling injury. Fruits of these cultivars were stored in chambers at 13 degrees C for 15 d, at which point they were transferred to 19 degrees C, where they were left until complete ripening. The low temperature induced significant changes in the metabolism of starch and sucrose in comparison to fruit ripened only at 19 degrees C. The sucrose accumulation was slightly higher in cv. Prata, and different patterns of starch degradation, sucrose synthesis, activity and protein levels of the alpha-and beta-amylases, starch phosphorylase, sucrose synthase and sucrose phosphate synthase were detected between the cultivars. Our results suggest that starch-to-sucrose metabolism is likely part of the mechanism for cold acclimation in banana fruit, and the cultivar-dependent differences contribute to their ability to tolerate cold temperatures. (C) 2011 Elsevier B.V. All rights reserved.

Increased platelet GSK3B activity in patients with mild cognitive impairment and Alzheimer`s disease

The disruption of glycogen synthase kinase 3-beta (GSK3B) homeostasis has implications in the pathophysiology of neuropsychiatric disorders, namely Alzheimer`s disease (AD). GSK3B activity is increased within the AD brain, favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. Such abnormality has also been detected in leukocytes of patients with cognitive disorders. The aim of the present study was to determine the expression of total and phosphorylated GSK3B at protein level in platelets of older adults with varying degrees of cognitive impairment, and to compare GSK3B activity in patients with AD, mild cognitive impairment (MCI) and healthy controls. Sixty-nine older adults were included (24 patients with mild to moderate AD, 22 patients with amnestic MCI and 23 elderly controls). The expression of platelet GSK3B (total- and Ser-9 phosphorylated GSK3B) was determined by Western blot. GSK3B activity was indirectly assessed by means of the proportion between phospho-GSK3B to total GSK3B (GSK3B ratio), the former representing the inactive form of the enzyme. Ser-9 phosphorylated GSK3B was significantly reduced in patients with MCI and AD as compared to controls (p = 0.04). Platelet GSK3B ratio was significantly decreased in patients with MCI and AD (p = 0.04), and positively correlated with scores on memory tests (r = 0.298, p = 0.01). In conclusion, we corroborate previous evidence of increased GSK activity in peripheral tissues of patients with MCI and AD, and further propose that platelet GSK may be an alternative peripheral biomarker of this abnormality, provided samples are adequately handled in order to preclude platelet activation. (C) 2010 Elsevier Ltd. All rights reserved.

Similar Intracellular Peptide Profile of TAP1/beta 2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Cells produce and use peptides in distinctive ways. In the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/beta 2m(-/-) (transporter associated with antigen-processing 1/beta 2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some peptides showed a modest increase or decrease (similar to 2-fold), whereas a glycine-rich peptide derived from the C-terminal of neurogranin (KGPGPGGPGGAGGARGGAGGGPSGD) showed a substantial increase (6-fold) in TAP1/beta 2m(-/-) mice. Thus, TAP1 and beta 2microglobulin have a small influence on the peptide profile of neuronal tissue, suggesting that the presence of peptides derived from intracellular proteins in neuronal tissue is not associated with antigens of the class I major histocompatibility complex. Therefore, it is possible that these intracellular peptides play a physiological role.

Congenital Hyperinsulinism in Brazilian Neonates: A Study of Histology, KATP Channel Genes, and Proliferation of beta Cells

Congenital hyperinsulinism (CHI) is a rare pancreatic beta-cell disease of neonates, characterized by inappropriate insulin secretion with severe persistent hypoglycemia, with regard to which many questions remain to be answered, despite the important acquisition of its molecular mechanisms in the last decade. The aim of this study was to examine pancreatic histology, beta-cell proliferation (immunohistochemistry with double staining for Ki-67/insulin), and beta-cell adenosine triphosphate-sensitive potassium channels genes from 11 Brazilian patients with severe medically unresponsive CHI who underwent pancreatectomy. Pancreatic histology and beta-cell proliferation in CHI patients were compared to pancreatic samples from 19 age-matched controls. Ten cases were classified as diffuse form (D-CHI) and 1 as focal form (F-CHI). beta-cell nucleomegaly and abundant cytoplasm were absent in controls and were observed only in D-CHI patients. The Ki-67 labeling index (Ki-67-LI) was used to differentiate the adenomatous areas of the F-CHI case (10.15%) from the ""loose cluster of islets`` found in 2 D-CHI samples (2.29% and 2.43%) and 1 control (1.54%) sample. The Ki-67-LI was higher in the F-CHI adenomatous areas, but D-CHI patients also had significantly greater Ki-67-LI (mean value = 2.41%) than age-matched controls (mean value = 1.87%) (P = 0.009). In this 1st genetic study of CHI patients in Brazil, no mutations or new polymorphisms were found in the 33-37 exons of the ABCC8 gene (SUR1) or in the entire exon of the KCNJ11 gene (Kir 6.2) in 4 of 4 patients evaluated. On the other hand, enhanced beta-cell proliferation seems to be a constant feature in CHI patients, both in diffuse and focal forms.

Exercise training inhibits inflammatory cytokines and more than prevents myocardial dysfunction in rats with sustained beta-adrenergic hyperactivity

Myocardial hypertrophy and dysfunction occur in response to excessive catecholaminergic drive. Adverse cardiac remodelling is associated with activation of proinflammatory cytokines in the myocardium. To test the hypothesis that exercise training can prevent myocardial dysfunction and production of proinflammatory cytokines induced by beta-adrenergic hyperactivity, male Wistar rats were assigned to one of the following four groups: sedentary non-treated (Con); sedentary isoprenaline treated (Iso); exercised non-treated (Ex); and exercised plus isoprenaline (Iso+Ex). Echocardiography, haemodynamic measurements and isolated papillary muscle were used for functional evaluations. Real-time RT-PCR and Western blot were used to quantify tumour necrosis factor alpha, interleukin-6, interleukin-10 and transforming growth factor beta(1) (TGF-beta(1)) in the tissue. NF-kappa B expression in the nucleus was evaluated by immunohistochemical staining. The Iso rats showed a concentric hypertrophy of the left ventricle (LV). These animals exhibited marked increases in LV end-diastolic pressure and impaired myocardial performance in vitro, with a reduction in the developed tension and maximal rate of tension increase and decrease, as well as worsened recruitment of the Frank-Starling mechanism. Both gene and protein levels of tumour necrosis factor alpha and interleukin-6, as well as TGF-beta(1) mRNA, were increased. In addition, the NF-kappa B expression in the Iso group was significantly raised. In the Iso+Ex group, the exercise training had the following effects: (1) it prevented LV hypertrophy; (ii) it improved myocardial contractility; (3) it avoided the increase of proinflammatory cytokines and improved interleukin-10 levels; and (4) it attenuated the increase of TGF-beta(1) mRNA. Thus, exercise training in a model of beta-adrenergic hyperactivity can avoid the adverse remodelling of the LV and inhibit inflammatory cytokines. Moreover, the cardioprotection is related to beneficial effects on myocardial performance.

E-cadherin and beta-catenin Loss of Expression Related to Bone Metastasis in Prostate Cancer

Objectives: E-cadherin and beta-catenin are adhesion molecules responsible for the maintenance of normal epithelial cell phenotype. A disturbance in epithelial cell adhesion, which leads to a more invasive and metastatic phenotype, is a hallmark of tumor progression. Several immunohistochemical studies have reported a strong correlation between loss of their expression to higher stage and grade in prostate carcinoma, but their influence in metastatic process is not yet known. The aim of this study is to verify the role of adhesion molecules in the progression of prostate cancer (PC), assessing the expression of E-cadherin and beta-catenin in bone metastasis. Materials and Methods: Twenty-eight bone metastases of prostate carcinoma were submitted to immunohistochemistry analysis for E-cadherin and beta-catenin expression. In 6 patients, we were able to assess the expression of the adhesion molecules in the primary tumors and their respective metastases. The definition of normal expression for both antibodies was strong and diffuse expression in more than 70% of tumor cells. Results: In bone metastases, there was loss of expression of E-cadherin and beta-catenin in 86% and 82%, respectively. Among the primary tumors, E-cadherin and beta-catenin expression was normal in 83% and 50% cases, respectively. Considering the 6 patients with paired primary and bone metastasis, we found loss of expression for both E-cadherin and beta-catenin in most of the cases. Conclusions: Comparing primary PC and its metastasis, we showed persistent loss of E-cadherin and beta-catenin expression. This phenomenon may be related to metastatic potential in PC, because we have shown underexpression for E-cadherin and beta-catenin in 86% and 82% of bone metastases.