Activation of the ET-1/ETA Pathway Contributes to Erectile Dysfunction Associated with Mineralocorticoid Hypertension

The cavernosal tissue is highly responsive to endothelin-1 (ET-1), and penile smooth muscle cells not only respond to but also synthesize ET-1. Considering that ET-1 is directly involved in end-organ damage in salt-sensitive forms of hypertension, we hypothesized that activation of the ET-1/ET(A) receptor pathway contributes to erectile dysfunction (ED) associated with mineralocorticoid hypertension. Wistar rats were uninephrectomized and submitted to deoxycorticosterone acetate (DOCA)-salt treatment for 5 weeks. Control (Uni [uninephrectomized control]) animals were uninephrectomized and given tap water. Uni and DOCA-salt rats were simultaneously treated with vehicle or atrasentan (ET(A) receptor antagonist, 5 mg/Kg/day). Cavernosal reactivity to ET-1, phenylephrine (PE), ET(B) receptor agonist (IRL-1620) and electric field stimulation (EFS) were evaluated in vitro. Expression of ROCK alpha, ROCK beta, myosin phosphatase target subunit 1 (MYPT-1), and extracellular signal-regulated kinase 1/2 (ERK 1/2) were evaluated by western blot analysis. ET-1 and ET(A) receptor mRNA expression was evaluated by real-time reverse-transcriptase polymerase chain reaction. Voltage-dependent increase in intracavernosal pressure/mean arterial pressure (ICP/MAP) was used to evaluate erectile function in vivo. ET(A) receptor blockade prevents DOCA-salt-associated ED. Cavernosal strips from DOCA-salt rats displayed augmented preproET-1 expression, increased contractile responses to ET-1 and decreased relaxation to IRL-1620. Contractile responses induced by EFS and PE were enhanced in cavernosal tissues from DOCA-salt hypertensive rats. These functional changes were associated with increased activation of the RhoA/Rho-kinase and ERK 1/2 pathways. Treatment of rats with atrasentan completely prevented changes in cavernosal reactivity in DOCA-salt rats and restored the decreased ICP/MAP, completely preventing ED in DOCA-salt rats. Activation of the ET-1/ET(A) pathway contributes to mineralocorticoid hypertension-associated ED. ET(A) receptor blockade may represent an alternative therapeutic approach for ED associated with salt-sensitive hypertension and in pathological conditions where increased levels of ET-1 are present. Carneiro FS, Nunes KP, Giachini FRC, Lima VV, Carneiro ZN, Nogueira EF, Leite R, Ergul A, Rainey WE, Webb RC, and Tostes RC. Activation of the ET-1/ETA pathway contributes to erectile dysfunction associated with mineralocorticoid hypertension. J Sex Med **;**:**-**.

Role of cytokines (TNF-alpha, IL-1 beta and KC) in the pathogenesis of CPT-11-induced intestinal mucositis in mice: effect of pentoxifylline and thalidomide

Introduction Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established. Purpose Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11. Materials and methods Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and KC ELISA. Results CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1 beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1 beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea. Conclusion These results suggest an important role of TNF-alpha, IL-1 beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11.

Neutrophil migration induced by IL-1 beta depends upon LTB4 released by macrophages and upon TNF-alpha and IL-1 beta released by mast cells

In the present study, we investigate whether mast cells and macrophages are involved in the control of IL-1 beta-induced neutrophil migration, as well as the participation of chemotactic mediators. IL-1 beta induced a dose-dependent neutrophil migration to the peritoneal cavity of rats which depends on LTB4, PAF and cytokines, since the animal treatment with inhibitors of these mediators (MK 886, PCA 4248 and dexamethasone respectively) inhibited IL-1 beta-induced neutrophil migration. The neutrophil migration induced by IL-1 beta is dependent on mast cells and macrophages, since depletion of mast cells reduced the process whereas the increase of macrophage population enhanced the migration. Moreover, mast cells or macrophages stimulated with IL-1 beta released a neutrophil chemotactic factor, which mimicked the neutrophil migration induced by IL-1 beta. The chemotactic activity of the supernatant of IL-1 beta-stimulated macrophages is due to the presence of LTB4, since MK 886 inhibited its release. Moreover, the chemotactic activity of IL-1 beta-stimulated mast cells supernatant is due to the presence of IL-1 beta and TNF-alpha, since antibodies against these cytokines inhibited its activity. Furthermore, significant amounts of these cytokines were detected in the supernatant. In conclusion, our results suggest that neutrophil migration induced by IL-1 beta depends upon LTB4 released by macrophages and upon IL-1 beta and TNF alpha released by mast cells.

Essential Role of CCR2 in Neutrophil Tissue Infiltration and Multiple Organ Dysfunction in Sepsis

Rationale Sepsis is defined as a systemic inflammatory response to infection, which in its severe form is associated with multiple organ dysfunction syndrome (MODS). The precise mechanisms by Which MODS develops remain unclear. Neutrophils have a pivotal role in the defense against infections; however, overwhelming activation of neutrophils is known to elicit tissue damage. Objectives: We investigated the role of the chemokine receptor CCR2 in driving neutrophil infiltration and eliciting tissue damage in remote organs during sepsis. Methods: Sepsis was induced in wild-type mice treated with CCR2 antagonist (RS504393) or CCR2(-/-) mice by cecal ligation and puncture (CLP) model. Neutrophil infiltration into the organs was measured by myeloperoxidase activity and fluorescence-activated cell sorter. CCR2 expression and chemotaxis were determined in neutrophils stimulated with Toll-like receptor agonists or isolated from septic mice and patients. Measurements and Main Results: CCR2 expression and responsiveness to its ligands was induced in circulating neutrophils during CLP-induced sepsis by a mechanism dependent on Toll-like receptor/nuclear factor-kappa B pathway. Genetic or pharmacologic inhibition of CCR2 protected mice from CLP-induced mortality. This protection was associated with lower infiltration of neutrophils into the lungs, heart, and kidneys and reduced serum biochemical indicators of organ injury and dysfunction. Importantly, neutrophils from septic patients express high levels of CCR2, and the severity of patient illness correlated positively with increasing neutrophil chemotaxis to CCR2 ligands. Conclusions: Collectively, these data identify CCR2 as a key receptor that drives the inappropriate infiltration of neutrophils into remote organs during sepsis. Therefore, CCR2 blockade is a novel potential therapeutic target for treatment of sepsis-induced MODS.

Role of regulatory T cells in long-term immune dysfunction associated with severe sepsis

Objective: To investigate the role of regulatory T cells in the modulation of long-term immune dysfunction during experimental sepsis. It is well established that sepsis predisposes to development of a pronounced immunosuppression. Nevertheless, the mechanisms underlying the immune dysfunction after sepsis are still not well understood. Design: Prospective experimental study. Setting: University research laboratory. Interventions: Wild-type mice underwent cecal ligation and puncture and were treated with antibiotic during 3 days after surgery. On days 1, 7, or 15 after cecal ligation and puncture, the frequency of regulatory T cells, proliferation of CD4(+) T cells and bacterial counts were evaluated. Fifteen days after cecal ligation and puncture, surviving mice underwent secondary pulmonary infection by intranasal inoculation of nonlethal dose of Legionella pneumophila. Some mice received agonistic glucocorticoid-induced tumor necrosis factor receptor antibody (DTA-1) before induction of secondary infection. Measurements and Main Results: Mice surviving cecal ligation and puncture showed a markedly increased frequency of regulatory T cells in thymus and spleen, which was associated with reduced proliferation of CD4(+) T cells. Fifteen days after cecal ligation and puncture, all sepsis-surviving mice succumbed to nonlethal injection of L. pneumophila. Treatment of mice with DTA-1 antibody reduced frequency of regulatory T cells, restored CD4(+) T cell proliferation, reduced the levels of bacteria in spleen, and markedly improved survival of L. pneumophila infection. Conclusion: These findings suggest that regulatory T cells play an important role in the progression and establishment of immune dysfunction observed in experimental sepsis. (Crit Care Med 2010; 38: 1718-1725)

Sympathetic-mediated hypertension of awake juvenile rats submitted to chronic intermittent hypoxia is not linked to baroreflex dysfunction

In the present study, we evaluated the mechanisms underpinning the hypertension observed in freely moving juvenile rats submitted to chronic intermittent hypoxia (CIH). Male juvenile Wistar rats (20-21 days old) were submitted to CIH (6% O(2) for 40 s every 9 min, 8 h day(-1)) for 10 days while control rats were maintained in normoxia. Prior to CIH, baseline systolic arterial pressure (SAP), measured indirectly, was similar between groups (86 +/- 1 versus 87 +/- 1 mmHg). After exposure to CIH, SAP recorded directly was higher in the CIH (n = 28) than in the control group (n = 29; 131 +/- 3 versus 115 +/- 2 mmHg, P < 0.05). This higher SAP of CIH rats presented an augmented power of oscillatory components at low (10.05 +/- 0.91 versus 5.02 +/- 0.63 mmHg(2), P < 0.05) and high (respiratory-related) frequencies (12.42 +/- 2.46 versus 3.28 +/- 0.61 mmHg(2), P < 0.05) in comparison with control animals. In addition, rats exposed to CIH also exhibited an increased cardiac baroreflex gain (-3.11 +/- 0.08 versus -2.1 +/- 0.10 beats min(-1) mmHg(-1), P < 0.0001), associated with a shift to the right of the operating point, in comparison with control rats. Administration of hexamethonium (ganglionic blocker, i.v.), injected after losartan (angiotensin II type 1 receptor antagonist) and [beta-mercapto-beta,beta-cyclopenta-methylenepropionyl(1), O-Me-Tyr(2), Arg(8)]-vasopressin (vasopressin type 1a receptor antagonist), produced a larger depressor response in the CIH (n = 8) than in the control group (n = 9; -49 +/- 2 versus -39 +/- 2 mmHg, P < 0.05). Fifteen days after the cessation of exposure to CIH, the mean arterial pressure of CIH rats returned to normal levels. The data indicate that the sympathetic-mediated hypertension observed in conscious juvenile rats exposed to CIH is not secondary to a reduction in cardiac baroreflex gain and exhibits a higher respiratory modulation, indicating that an enhanced respiratory-sympathetic coupling seems to be the major factor contributing to hypertension in rats exposed to CIH.

Mast cells, TGF-beta 1 and alpha-SMA expression in IgA nephropathy

IgA nephropathy (IgAN) is a kidney disease with a varying renal prognosis. Recently, many studies have demonstrated that renal alpha-smooth muscle actin (alpha-SMA) and transforming growth factor (TGF-beta 1) expression, as well interstitial mast cell infiltrates could represent a prognostic marker in several renal diseases. The aim of our study was to analyze the prognostic value of mast cell, TGF-beta 1 and alpha-SMA expression in IgAN. A survey of the medical records and renal biopsy reports of 62 patients with a diagnosis of IgAN followed-up from 1987 to 2003 was performed. The mean follow-up time was 74.7 +/- 50.0 months. The immunohistochemical studies were performed using a monoclonal antibody anti-human mast cell tryptase, a polyclonal antibody anti-human TGF-beta 1, and a monoclonal antibody anti-human alpha-SMA. An unfavorable clinical course of IgAN was related to interstitial mast cell infiltrates and alpha-SMA expression in the tubulointerstitial area. Expression of glomerular TGF-beta 1 and alpha-SMA, and interstitial TGF-beta 1 is not correlated with clinical course in IgAN. In conclusion, the increased number of mast cells and higher alpha-SMA expression in the tubulointerstitial area may be predictive factors for the poor prognosis of patients with IgAN.

Ethinylestradiol and estradiol have different effects on oxidative stress and nitric oxide synthesis in human endothelial cell cultures

Study objective: To compare the effects of ethinylestradiol (EE) and 17 beta-estradiol (E(2)) on nitric oxide (NO) production and protection against oxidative stress in human endothelial cell cultures. Design: Experimental study. Settings: Research laboratory. Material: Human ECV304 endothelial cell cultures. Intervention(s): The NO synthesis was determined by flow cytometry, and oxidative stress was determined by a cell viability assay, after exposure to hydrogen peroxide (H(2)O(2)) and stimulation of endothelial cells with EE at concentrations similar to those of a contraceptive containing 30 mu g EE. Main Outcome Measure(s): The effects of EE were compared with those of E(2) at concentrations similar to those occurring during the follicular phase. Result(s): Ethinylestradiol did not increase NO synthesis and did not protect cells against oxidative stress. The viability of the cells incubated with E(2) in combination with H(2)O(2) was greater than the viability obtained with H(2)O(2) only or with H(2)O(2) in combination with EE. The cells stimulated with E(2) presented a significant increase in NO production compared with control. Conclusion(s): In contrast to the effects of E(2), EE did not protect human ECV304 endothelial cells against oxidative stress and did not increase their production of NO. (Fertil Steril (R) 2010; 94: 1578-82. (C) 2010 by American Society for Reproductive Medicine.)

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P<0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO (P=0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women (P<0.01). We found significant positive correlations between pHb and pNOc (r=0.61; P<0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations (P=0.02; r=-0.32 and P=0.01: r=-0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations (P=0.03; r=-0.36 and P=0.01: r=-0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women. (C) 2010 Elsevier Inc. All rights reserved.

Mast cells and transforming growth factor-beta expression: a possible relationship in the development of porphyria cutanea tarda skin lesions

Background Porphyria cutanea tarda (PCT) is a metabolic disease characterized by vesicles and blisters in sun-exposed areas and scleroderma-like lesions in sun-exposed and non-sun-exposed areas. Mast cells participate in the pathogenesis of bullous diseases and diseases that show sclerosis, including PCT. Moreover, transforming growth factor-beta (TGF-beta) is the main cytokine in the development of tissue sclerosis. The correlation of mast cells and TGF-beta with the lesions of PCT has not been examined, however. The possible role of mast cells and TGF-beta (and the relationship between them) in the development of PCT lesions is discussed. Methods To quantify mast cells and cells expressing TGF-beta in skin samples from patients with PCT and controls, immunohistochemical studies were performed in tissue sections allied to morphometric analyses. Results The numbers of mast cells and cells expressing TGF-beta per square millimiter were increased in the PCT group relative to controls, and there was a direct and significant correlation between the mast cell number and cells expressing TGF-beta in PCT. Conclusions The results suggest that the increased number of mast cells and of cells expressing TGF-beta, as well as their direct correlation, may contribute to the pathogenesis of the skin lesions in PCT.

beta 1 Integrin and VEGF expression in an experimental model of brain tissue heterotopia in the lung

Integrins and vascular endothelial growth factor (VEGF) are crucially involved in interaction, proliferation, migration, and survival of the cells. However, there is no report in the literature about beta 1 integrin and VEGF expression in heterotopic brain tissue. The aim of this study was to assess beta 1 integrin and VEGF expression in experimental brain tissue heterotopia in the lung during both fetal and neonatal periods. Twenty-four pregnant female Swiss mice were used to induce brain tissue heterotopia on the 15th gestational day. Briefly, the brain of one fetus of each dam was extracted, disaggregated, and injected into the right hemithorax of siblings. Six of these fetuses with pulmonary brain tissue implantation were collected on the 18th gestational day (group E18) and six other on the eighth postnatal day (group P8). Immunohistochemistry of the fetal trunks showed implantation of glial fibrillary acidic protein- and neuronal nuclei-positive heterotopic brain tissue, which were also positive for beta 1 integrin and VEGF in both groups E18 and P8. These results indicate that brain tissue heterotopia during fetal and postnatal period is able to complete integration with the lung tissue as well as to induce vascular proliferation which are the necessary steps for a successful implantation.

Occurrence of TRGV-BJ hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.

Effect of beta-lactoglobulin polymorphism and seasonality on bovine milk composition

The objective was to evaluate the effect of beta-lactoglobulin (beta-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine beta-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of beta-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and beta-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein :true protein ratio were found.

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17 beta on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17 beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 mu g ml(-1)) of estradiol-17 beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17 beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17 beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.

Developing human minor salivary glands: morphological parallel relation between the expression of TGF-beta isoforms and cytoskeletal markers of glandular maturation

Morphogenesis of salivary glands involves complex coordinated events. Synchronisation between cell proliferation, polarisation and differentiation, which are dependent on epithelial-mesenchymal interactions and on the microenvironment, is a requirement. Growth factors mediate many of these orchestrated biological processes and transforming growth factor-beta (TGF-beta) appear to be relevant. Using immunohistochemistry and immunofluorescence, we have mapped the distribution of TGF-beta 1, 2 and 3 and compared it with the expression of maturation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. TGF-beta 1 first appeared during canalisation stage in the surrounding mesenchyme and, in the more differentiated stages, was expressed in the cytoplasm of acinar cells throughout the adult gland. TGF-beta 2 was detected since the bud stage of the salivary gland. Its expression was observed in ductal cells and increased along gland differentiation, TGF-beta 3 was detected from the canalisation stage of the salivary gland, being weakly expressed on ductal cells, and it was the only factor detected on myoepithelial cells. The data suggest that TGF-beta have a role to play in salivary gland development and differentiation.