Transcriptional analysis of nasal polyps fibroblasts reveals a new source of pro-inflammatory signaling in CRSwNP.

BACKGROUND: Fibroblasts and others mesenchymal cells have recently been identified as critical cells triggering tissue-specific inflammatory responses. Persistent activation of fibroblasts inflammatory program has been suggested as an underlying cause of chronic inflammation in a wide range of tissues and pathologies. Nevertheless, the role of fibroblasts in the emergence of chronic inflammation in the upper airway has not been previously addressed. We aimed to elucidate whether fibroblasts could have a role in the inflammatory response in chronic rhinosinusitis with nasal polyps (CRSwNP). METHODOLOGY: We performed whole-transcriptome microarray in fibroblast cultured from CRSwNP samples and confirmed our results by qRT-PCR. We selected patients without other associated diseases in upper airway. To investigate shifts in transcriptional profile we used fibroblasts from nasal polyps and uncinate mucosae from patient with CRSwNP, and fibroblasts from uncinate mucosae from healthy subjects as controls. RESULTS: This study exposes activation of a pro-inflammatory and pro-fibrotic transcriptional program in nasal polyps and CRSwNP fibroblasts when compared to controls. Our Gene-set Enrichment Analysis (GSEA) pointed to common up-regulation of several pro-inflammatory pathways in patients-derived fibroblasts, along with higher mRNA expression levels of cytokines, growth factors and extracellular matrix components. CONCLUSIONS: Our work reveals a potential new source of inflammatory signaling in CRSwNP. Furthermore, our results suggest that deregulated inflammatory signaling in tissue-resident fibroblasts could support a Type-2 inflammatory response. Further investigations will be necessary to demonstrate the functionality of these novel results.

Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study.

BACKGROUND: We evaluated a new chemoimmunotherapy combination based on the anti-PD1 monoclonal antibody pembrolizumab and the pyrimidine antimetabolite gemcitabine in HER2- advanced breast cancer (ABC) patients previously treated in the advanced setting, in order to explore a potential synergism that could eventually obtain long term benefit in these patients. METHODS: HER2-negative ABC patients received 21-day cycles of pembrolizumab 200 mg (day 1) and gemcitabine (days 1 and 8). A run-in-phase (6 + 6 design) was planned with two dose levels (DL) of gemcitabine (1,250 mg/m2 [DL0]; 1,000 mg/m2 [DL1]) to determine the recommended phase II dose (RP2D). The primary objective was objective response rate (ORR). Tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumors and myeloid-derived suppressor cells (MDSCs) levels in peripheral blood were analyzed. RESULTS: Fourteen patients were treated with DL0, resulting in RP2D. Thirty-six patients were evaluated during the first stage of Simon's design. Recruitment was stopped as statistical assumptions were not met. The median age was 52; 21 (58%) patients had triple-negative disease, 28 (78%) visceral involvement, and 27 (75%) ≥ 2 metastatic locations. Progression disease was observed in 29 patients. ORR was 15% (95% CI, 5-32). Eight patients were treated ≥ 6 months before progression. Fourteen patients reported grade ≥ 3 treatment-related adverse events. Due to the small sample size, we did not find any clear association between immune tumor biomarkers and treatment efficacy that could identify a subgroup with higher probability of response or better survival. However, patients that experienced a clinical benefit showed decreased MDSCs levels in peripheral blood along the treatment. CONCLUSION: Pembrolizumab 200 mg and gemcitabine 1,250 mg/m2 were considered as RP2D. The objective of ORR was not met; however, 22% patients were on treatment for ≥ 6 months. ABC patients that could benefit of chemoimmunotherapy strategies must be carefully selected by robust and validated biomarkers. In our heavily pretreated population, TILs, PD-L1 expression and MDSCs levels could not identify a subgroup of patients for whom the combination of gemcitabine and pembrolizumab would induce long term benefit. TRIAL REGISTRATION: ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001,779-54, respectively). Registration dates: 20/01/2017 and 18/11/2016, respectively.

Insulin and Leptin Signaling in Placenta from Gestational Diabetic Subjects.

Insulin and leptin receptors are known to share signaling pathways, such as JAK2/STAT-3 (Janus kinase2/signal transduction and activator of transcription3), MAPK (Mitogen activated protein kinase), and PI3K (phosphoinositide 3-kinase). Both positive and negative cross-talk have been previously found in different cellular systems. Gestational diabetes (GDM) is a pathophysiological state with high circulating levels of both insulin and leptin. We have previously found that these 3 signaling pathways are activated in placenta from GDM patients to promote translation, involving the activation of leptin receptor. Now, we have tested the hypothesis that both leptin and insulin receptors might contribute to this activation in a positive way that may become negative when the system is overactivated. We studied the activation of leptin and insulin receptors in placenta from GDM and healthy pregnancies. We have also performed in vitro studies with insulin and leptin stimulation of trophoblast explants from healthy placenta. We have found that both leptin and insulin receptors are activated in placenta from GDM. In vitro stimulation of trophoblast explants with both leptin and insulin at submaximal doses (0.1 nM) potentiated the activation of signaling, whereas preincubation with maximal concentrations of insulin (10 nM) and further stimulation with leptin showed negative effect. Trophoblastic explants from GDM placenta, which presented high signaling levels, had a negative signaling effect when further incubated in vitro with leptin. In conclusion, insulin and leptin receptors have positive effects on signaling, contributing to high signaling levels in GDM placenta, but insulin and leptin have negative effects upon overstimulation.

Increased Expression of Aquaporin 9 in Trophoblast From Gestational Diabetic Patients.

Gestational diabetes mellitus is the most frequent pathophysiological alteration in pregnancy, increasing the incidence of complications in both mother and fetus. The macrosomia that occurs in these fetuses may be related with some changes in nutrient transport mechanism in placenta. The presence of aquaporin 9, an aquaglyceroporin, has previously been demonstrated in placenta. We raised the question whether aquaporin 9 expression may be upregulated in placenta from gestational diabetes, thus providing a faster transport of glycerol and water through placenta. We studied 21 placentas (13 controls and 8 gestational diabetes) from cesarean delivery at term. The expression of aquaporin 9 was analyzed by quantitative PCR, immunoblot, and immunohistochemistry. The median values from quantitative PCR were compared by nonparametric tests for independent samples (Mann-Whitney U-test). We have found that trophoblast from gestational diabetes express higher amount of aquaporin 9, which was found statistically significant (p<0.05). The increase in aquaporin 9 expression was confirmed by immunoblot, and localization in the syncytiotrophoblast was checked by immunohistochemistry. The increase in aquaporin 9 expression in placenta from gestational diabetes may contribute to the higher transport rate in this pathology of pregnancy.

Expression and immunohistochemical localization of leptin receptor in human periapical granuloma.

AIM: To investigate the expression and immunohistochemical localization of leptin receptor (LEPR) in human periapical granulomas. METHODOLOGY: Periapical inflammatory lesions were obtained from extracted human teeth and teeth which underwent periapical surgery. After their histopathological categorization as periapical granulomas (n = 20), they were examined by immunohistochemistry using human LEPR monoclonal antibodies. LEPR mRNA expression was also determined by quantitative real-time PCR (qRT-PCR), and the amount of LEPR protein was analysed by immunoblot. RESULTS: All granuloma samples expressed LEPR. Amongst inflammatory cells, only macrophages showed expression of LEPR. Western blot analysis revealed the presence in the samples of a protein with apparent molecular weight of ~120 kDa, corresponding to the estimated molecular weight of LEPR. The qRT-PCR analysis demonstrated the expression of LEPR mRNA, corresponding the size of the amplified fragment (338 bp), assessed by agarose gel electrophoresis, to that of LEPR mRNA. CONCLUSIONS: Human periapical granulomas express LEPR. In periapical granulomas, only macrophages showed expression of LEPR. This finding suggests that leptin can play a role in inflammatory and immune periapical responses.

Activated translation signaling in placenta from pregnant women with gestational diabetes mellitus: possible role of leptin.

Placentas from gestational diabetes (GDM) suffer from structural and functional changes including overgrowth. That is why we aimed to study [³H]-leucine incorporation into protein in addition to translation signaling in placenta from GDM. Thus, we investigated the expression of leptin and leptin receptor (LEPR), as well as the activation state of signaling proteins regulating protein synthesis, such as mTOR, S6 Kinase, EIF4E-BP1, EIF4E, and eEF2 by measuring protein phosphorylation by immunoblot. [³H]-Leucine incorporation into protein also was determined in trophoblastic placenta explants from GDM and control pregnancy. We found that leptin and LEPR expression are increased in placentas from GDM and the translation machinery activity as well as [³H]-leucine incorporation into protein were higher in placentas from GDM compared with placentas from control pregnancy. In conclusion, protein synthesis rate is increased in placenta from GDM patients, and this may be due, at least in part, by the activation of translation signaling. The increased expression of leptin and LEPR may contribute to these effects. These results may provide a possible mechanism for the previously observed increase in placenta growth in GDM.

Leptin expression in healthy and inflamed human dental pulp.

AIM: To investigate the expression of leptin in healthy and inflamed human dental pulp. METHODOLOGY: Twenty-one pulp samples were obtained from freshly caries- and restoration-free extracted human third molars. In seven-third molars (inflamed pulp group), inflammation was induced prior to extraction. Pulp samples were processed, and leptin expression was determined by quantitative real-time PCR (qRT-PCR) and the amount of leptin by immunoblot. RESULTS: All healthy and inflamed dental pulp samples expressed leptin. Western blot analysis revealed the presence of a protein with an apparent molecular weight of ~16 kDa in human dental pulp, which corresponds to the estimated molecular weight of leptin. The expression of leptin mRNA in dental pulp was confirmed by qRT-PCR analysis, and the size of the amplified fragments (296 bp for leptin and 194 bp for cyclophilin) was confirmed by agarose gel electrophoresis. The expression of leptin in the inflamed pulp group was significantly greater (P < 0.05) than in healthy teeth. The relative amount of leptin in inflamed pulps was almost twice than in healthy pulps. CONCLUSIONS: For the first time, the presence of leptin in human dental pulp tissues has been demonstrated. The upregulation of leptin expression in inflamed pulp samples suggests that leptin can play a role in pulpal inflammatory and immune responses.

Sam68 mediates leptin-stimulated growth by modulating leptin receptor signaling in human trophoblastic JEG-3 cells.

BACKGROUND: Sam68, a member of the signal transduction and activation of RNA metabolism (STAR) family of RNA-binding proteins, has been previously implicated as an adaptor molecule in different signaling systems, including leptin receptor (LEPR) signaling. LEPR activation is known to stimulate JAK-STAT, MAPK and PI3K signaling pathways, thus mediating the biological effects of leptin in different cell types, including trophoblastic cells. We have recently found that leptin stimulation also promotes the overexpression and tyrosine phosphorylation of Sam68 in human trophoblastic JEG-3 cells, suggesting a role for Sam68 in leptin signaling and action in these cells. In the present work, we have studied the participation of Sam68 in the main signaling pathways activated by LEPR to increase growth and proliferation in trophoblastic JEG-3 cells. METHODS: We used an antisense strategy to down-regulate Sam68 expression in these cells, and we studied LEPR signaling by immunoprecipitation and poly-U affinity precipitation and by analyzing phosphorylation levels of signaling proteins by immunoblot. The effect of leptin on protein synthesis and proliferation was studied by ³[H]-leucine and ³[H]-thymidine incorporation. RESULTS: Sam68 knockdown impaired leptin activation of JAK-STAT, PI3K and MAPK signaling pathways in JEG-3 cells. We have also found that leptin-stimulated Sam68 tyrosine phosphorylation is dependent on JAK-2 activity, since the pharmacological inhibitor AG490 prevents the phosphorylation of Sam68 in JEG-3 cells. Finally, the trophic and proliferative effect of leptin in trophoblastic cells is dependent on Sam68 expression, since its down-regulation impaired the leptin-stimulated DNA and protein synthesis. CONCLUSIONS: These data demonstrate that Sam68 participates in the main signaling pathways of LEPR to mediate the trophic and proliferative effect of leptin in human trophoblastic cells.

New horizons in breast cancer: the promise of immunotherapy.

Immunology and immunotherapy of cancer is an expanding field in oncology, with recent great achievements obtained through the new successful approaches implemented to circumvent immune evasion, which is undoubtedly considered a novel hallmark of cancer. Translational research in this topic has revealed targets that can be modulated in the clinical setting with new compounds and strategies. Like most of the tumors, breast cancer is considered a complex and heterogeneous disease in which host immune responses have been also recently demonstrated of critical relevance. T infiltrating lymphocyte measurement is suggested as a powerful new tool necessary to predict early breast cancer evolution, especially for the her2-positive and triple-negative subtypes. Other biomarkers in tissue and peripheral blood are under intense scrutiny to ascertain their eventual role as prognostic and/or predictive factors. This background has fueled the interest in developing clinical research strategies to test activity of modern immunotherapy in breast cancer, which constitutes the main focus of this review.

Leptin promotes cell survival and activates Jurkat T lymphocytes by stimulation of mitogen-activated protein kinase.

Leptin (Ob) is a non-glycosylated peptide hormone that regulates energy homeostasis centrally, but also has systemic effects including the regulation of the immune function. We have reported previously that leptin activates human peripheral blood lymphocytes co-stimulated with phytohaemagglutinin (PHA) (4 microg/ml), which prevented the employment of pharmacological inhibitors of signalling pathways. In the present study, we used Jurkat T cells that responded to leptin with minimal PHA co-stimulation (0.25 microg/ml). The long isoform of leptin receptor is expressed on Jurkat T cells and upon leptin stimulation, the expression of early activation marker CD69 increases in a dose-dependent manner (0.1-10 nM). We have also found that leptin activates receptor-associated kinases of the Janus family-signal transucers and activators of transcription (JAK-STAT), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K) signalling pathways. Moreover, we sought to study the possible effect of leptin on cell survival and apoptosis of Jurkat T cells by culture in serum-free conditions. We have assayed the early phases of apoptosis by flow cytometric detection of fluorescein isothiocyanate (FITC)-labelled annexin V simultaneously with dye exclusion of propidium iodide (PI). As well, we have assayed the activation level of caspase-3 by inmunoblot with a specific antibody that recognizes active caspase-3. We have found that leptin inhibits the apoptotic process dose-dependently. By using pharmacological inhibitors, we have found that the stimulatory and anti-apoptotic effects of leptin in Jurkat T cells are dependent on MAPK activation, rather than the PI3K pathway, providing new data regarding the mechanism of action of leptin in T cells, which may be useful to understand more clearly the association between nutritional status and the immune function.

Breast Cancer Immunology and Immunotherapy: Current Status and Future Perspectives.

Cancer immunology has gained renewed interest in the past few years due to emerging findings on mechanisms involved in tumoral immune evasion. Indisputably, immune edition is currently considered a critical hallmark of cancer. Basic research has revealed new targets which can be modulated in the clinical setting with new compounds and strategies. As recent evidence confirms, breast cancer (BC) is a complex and heterogeneous disease in which host immune responses play a substantial role. T-infiltrating lymphocytes measurement is suggested as a powerful new tool necessary to predict early BC evolution, especially in HER2-positive and triple negative subtypes. However, T-infiltrating lymphocytes, genomic platforms, and many other biomarkers in tissue and peripheral blood (e.g., regulatory T cells and myeloid-derived suppressor cells) are not the only factors being evaluated regarding their potential role as prognostic and/or predictive factors. Many ongoing clinical trials are exploring the activity of immune checkpoint modulators in BC treatment, both in the advanced and neoadjuvant setting. Although this field is expanding with exciting new discoveries and promising clinical results-and creating great expectations-there remain many uncertainties yet to be addressed satisfactorily before this long awaited therapeutic promise can come to fruition.

Characterization of pancreastatin receptors and signaling in adipocyte membranes.

Pancreastatin (PST), a chromogranin A derived peptide with an array of effects in different tissues, has a role as a counterregulatory hormone of insulin action in hepatocytes and adipocytes, regulating glucose, lipid and protein metabolism. We have previously characterized PST receptors and signaling in rat hepatocytes, in which PST functions as a calcium-mobilizing hormone. In the present work we have studied PST receptors as well as the signal transduction pathways generated upon PST binding in adipocyte membranes. First, we have characterized PST receptors using radiolabeled PST as a ligand. Analysis of binding data indicated the existence of one class of binding sites, with a B(max) of 5 fmol/mg of protein and a K(d) of 1 nM. In addition, we have studied the G protein system that couples the PST receptor by gamma-(35)S-GTP binding studies. We have found that two G protein systems are involved, pertussis toxin-sensitive and -insensitive respectively. Specific anti-G protein alpha subtype sera were used to block the effect of pancreastatin receptor activation. Galpha(q/11) and to a lesser extent Galpha(i1,2) are activated by PST in rat adipocyte membranes. On the other hand, adenylate cyclase activity was not affected by PST. Finally, we have studied the specific phospholipase C isoform that is activated in response to PST. We have found that PST receptor is coupled to PLC-beta(3) via Galpha(q/11) activation in adipocyte membranes.

Pancreastatin modulates insulin signaling in rat adipocytes: mechanisms of cross-talk.

Pancreastatin (PST), a chromogranin A-derived peptide, has counterregulatory effects on insulin in the hepatocyte and the adipocyte, suggesting a possible role in insulin resistance. The mechanism of PST action on glucose and lipid metabolism is typical of a calcium-mobilizing hormone and involves a receptor Gq/11 protein-phospholipase C (PLC)-beta pathway. In the rat adipocyte, PST inhibits insulin-mediated glucose transport, glucose utilization, and lipid synthesis, and it has a lipolytic effect but stimulates basal and insulin-stimulated protein synthesis. We have also recently studied the PST receptor-effector system in adipocyte membranes. To further investigate the mechanisms of PST effect on insulin action, we studied the cross-talk of PST with insulin signaling in the rat adipocyte. We found that PST inhibits insulin-stimulated GLUT4 translocation to the membrane, which may explain the reported inhibition of glucose transport. Tyrosine phosphorylation of the activated insulin receptor, insulin receptor substrate (IRS)-1, and p60-70 was also blunted, preventing their association with p85 phosphatidylinositol 3-kinase (PI3K) and their activity. The mechanism of this inhibition involves the activation of the "classical" protein kinase C isoforms and the serine phosphorylation of insulin receptor and IRS-1. On the other hand, PST activates the mitogen-activated protein kinase (MAPK) signaling module and enhances the effect of insulin. This pathway may account for the described effect of PST on protein synthesis. In conclusion, PST seems to inhibit the insulin-stimulated PI3K pathway in the adipocyte, whereas it activates the MAPK pathway. These data provide some clues to the PST cross-talk with insulin signaling that may explain the PST effects on glucose metabolism and protein synthesis.

Homocysteine thiolactone inhibits insulin-stimulated DNA and protein synthesis: possible role of mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation.

Hyperhomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of homocysteine are related to the formation of homocysteine thiolactone and the consequent increase in oxidative stress. We have recently found that homocysteine thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase (PI3K) activity and inhibition of glycogen synthesis. Oxidative stress seemed to be the mechanism underlying these effects, since glutathione was able to restore the insulin signaling as well as the insulin-mediated glycogen synthesis. In the present work we have further investigated insulin receptor signaling studying mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation. Again, homocysteine thiolactone (50 microM) prevented insulin-mediated MAPK, GSK-3 and p70 S6K phosphorylation and these effects were blocked by glutathione (250 microM). Since MAPK and PI3K pathways, including GSK3 and S6K, seem to mediate insulin-mediated growth and proliferation, we measured DNA and protein synthesis. We have found that homocysteine thiolactone (50 microM) inhibits insulin-mediated growth and proliferation, as previously shown for glycogen synthesis. Again, these effects seem to be mediated by oxidative stress, since 250 microM glutathione completely abolished the effects of homocysteine thiolactone on insulin-stimulated DNA and protein synthesis. In conclusion, these data suggest that homocysteine thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in the action of insulin on growth and proliferation.

Pancreastatin action in the liver: dual coupling to different G proteins.

Pancreastatin is a 49 amino acid peptide first isolated, purified and characterized from porcine pancreas. Its biological activity in different tissues can be assigned to the C-terminal part of the molecule. Pancreastatin has a prohormonal precursor, chromogranin A, which is a glycoprotein present in neuroendocrine cells, including the endocrine pancreas. We have been interested in pancreastatin action in the liver. We found that pancreastatin has a glycogenolytic effect in the hepatocyte both in vivo and in vitro. We then studied and characterized the specific pancreastatin receptor in the rat liver plasma membrane, as well as the specific signal transduction. This receptor appears to be coupled to two different G proteins. A pertussis toxin-insensitive G proteins leads to the activation of phospholipase C, and therefore mediates the glycogenolytic effect in the liver by increasing cytoplasmic free calcium and stimulating protein kinase C. The role of cyclic GMP in the action of pancreastatin is not known yet, although it seems to regulate negatively the activation of phospholipase C. The precise mechanism by which pancreastatin stimulates guanylate cyclase activity remains to be studied.

Pancreastatin, a chromogranin A-derived peptide, activates Galpha(16) and phospholipase C-beta(2) by interacting with specific receptors in rat heart membranes.

Pancreastatin (PST) is one of the chromogranin A (CGA)-derived peptides with known biological activity. It has a general inhibitory effect on secretion in many exocrine and endocrine systems including the heart atrium. Besides, a role of PST as a counter-regulatory peptide of insulin action has been proposed in the light of its effects on glucose and lipid metabolism in the liver and adipose tissue, where receptors and signaling have been described. Galpha(q/11) pathway seems to mediate PST action. Since PST has been shown to function as a typical calcium-dependent hormone, and increased plasma levels have been found in essential hypertension correlating with catecholamines, we sought to study its possible interaction and signaling in heart membranes. Here, we are characterizing specific PST binding sites and signaling in rat heart membranes. We have found that PST receptor has a K(d) of 0.5 nM and a B(max) of 34 fmol/mg of protein. The PST binding is inhibited by guanine nucleotides, suggesting the functional coupling of the receptor with GTP binding proteins (G proteins). Moreover, PST dose-dependently increases GTP binding to rat heart membranes. Finally, we have studied PST signaling-effector system by measuring phospholipase C (PLC) activity using blocking antibodies against different G proteins and PLC isoforms. We have found that PST stimulates PLCbeta(2)>PLCbeta(1)>PLCbeta(3) by activating Galpha(16) in rat heart membranes. These data suggest that PST may modulate the cardiac function.

Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor to insulin receptor substrate 1.

After insulin stimulation of cells, signaling complexes are formed, containing the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and phosphatidylinositol-3-kinase. To study the nature of these complexes, we employed purified IR, recombinant IRS-1, antibodies to IR and IRS-1, and fusion proteins containing the two SH2 domains of p85. In intact cells, insulin increased tyrosine phosphorylation of both the IR and IRS-1. Both of these proteins were immunoprecipitated with antibodies to p85. Also, fusion proteins containing the two SH2 domains of p85 directly precipitated both the IR and IRS-1. Next, these signaling complexes were reconstituted in vitro with purified IR, recombinant IRS-1, and the two SH2 domains of p85. In the presence of both SH2 domains of p85, the IR associated with IRS-1. Other data, both in intact cells and in vitro, demonstrated that N- and C-terminal SH2 domains of p85 had preferential binding affinities for the IR and IRS-1, respectively. Studies with an IR mutant truncated in the C terminus indicated that the C-terminal phosphotyrosines of the IR play a major role in interacting with the SH2 domains of p85. In conclusion, both in vivo and in vitro data support a role for p85 in directly linking the IR to IRS-1 via its SH2 domains. The formation of these complexes, therefore, may provide a mechanism for the translocation to the plasma membrane of phosphatidylinositol-3-kinase and other molecules that are involved in IR signaling.

[Vascular damage in chronic renal failure. The increase of vascular nitrotyrosine and cytochines accumulation is accompanied by an increase of endothelial nitric oxide synthase (eNOS) expression]. / Daño vascular en la insuficiencia renal crónica. El aumento de depósitos vasculares de nitrotirosina y citocinas se acompaña de una elevación de la expresión vascular de la óxido nítrico sintasa endotelial (eNOS).

Patients with chronic renal failure (CRF) are at a greatly increased risk of cardiovascular mortality. This fact could be due to the presence of conventional risk factor and specific uremic as increase of oxidative stress, hyperhomocystaenemia, deranged calcium-phosphate metabolism and chronic inflammatory state. In order to analyze the vascular effects of CRF, we studied the histomorphometric characteristics (intima-media thickness and monocyte chemoattractant protein (MCP-1) accumulation (inmunohistochemical) on radial artery from 13 patients with CRF. We determined by Western blot analysis, the vascular nitrotyrosin abundance (footprint of nitric oxide (NO) inactivation by reactive oxygen species (ROS), and the endothelial nitric oxide synthase (eNOS) expression. The NOS activity was, also, determined. The results were compared with those obtained in pudenda artery from a healthy control group (n: 16). The CRF group showed a significant increase in intima and media thickness 108 +/- 16 vs 14 +/- 2.5 microm, p < 0.001 and 291 +/- 19 vs 153 +/- 15 microm, p < 0.001, respectively). The CRF group exhibited a marked elevation of MCP-1 vascular expression (2 +/- 0.15 vs 0.6 +/- 0.12 u, p < 0.001). A significant positive correlation was found between MCP-1 vascular expression and its inmunohistochemical deposits (r: 0.98, p < 0.0001). Nitrotyrosin abundance (western blot) was significantly increased in artery of CRF patients (2.1 +/- 0.1 vs 0.42 +/- 0.1 u, p < 0.0001). No significant differences was found in NOS activity between CRF and control groups. However, eNOS expression was greatly increased in the CRF patients (1.73 +/- 0.1 vs 0.67 +/- 0.1 u, p < 0.001). A significant positive correlation was found between nitrotyrosin and eNOS expression and systolic arterial pressure. However, the differences between CRF and control groups persisted after statistically fitting to arterial pressure. The present study demonstrate that in CRF there are arterial preatherosclerotic changes and an increase of vascular nitrotyrosin accumulation, which is the footprint of NO inactivation by ROS. The secondary NO inactivation can, in turn, contribute to eNOS vascular upregulation.

Increased plasma pancreastatin-like levels in gestational diabetes: correlation with catecholamine levels.

OBJECTIVE: To investigate plasma pancreastatin (a chromogranin A-derived peptide) and catecholamine levels (counterregulatory hormones) in subjects with gestational diabetes compared with normal pregnant subjects. RESEARCH DESIGN AND METHODS: Fasting blood samples were obtained from 11 normal pregnant and 12 nonobese gestational diabetic subjects at late pregnancy (30+/-1 weeks). Selection criteria were those recommended by the National Diabetes Data Group (modified from O'Sullivan original criteria). Plasma glucose, insulin, glucagon, pancreastatin-like, epinephrine, and norepinephrine were measured. RESULTS: Gestational diabetic subjects had significantly higher insulin levels than control pregnant subjects (18+/-1 vs. 15+/-1 microU/ml), whereas glucose and glucagon levels where comparable in the two groups. However, increased catecholamine levels (epinephrine and norepinephrine) were found in the gestational diabetic group. We also found increased pancreastatin-like levels in these patients compared with the pregnant control group (46+/-2 vs. 30+/-2 pmol/l). Actually, pancreastatin levels positively correlated with both epinephrine (r = 0.34) and norepinephrine (r = 0.80) levels. CONCLUSIONS: Catecholamine and pancreastatin-like levels were found elevated in gestational diabetic subjects. These counterregulatory hormones may play a role in the insulin resistance syndrome of gestational diabetes.

Leptin promotes HLA-G expression on placental trophoblasts via the MEK/Erk and PI3K signaling pathways.

INTRODUCTION: The development of the human haemochorial placenta requires complex regulatory mechanisms to protect invasive trophoblast cells from cytotoxic responses elicited by maternal immune cells. Leptin, the adipocyte derived hormone encoded by the Lep gene, is synthesized by placental trophoblasts and exerts pleiotropic effects on the immune system, including the promotion of inflammation and the activation of T cell responses. METHODS: To address its possible involvement in the modulation of maternal immune responses during pregnancy, we investigated the effect of leptin on the expression of the class Ib histocompatibility antigen HLA-G as one of the chief immunosuppressive strategies used by trophoblast cells. RESULTS: In vitro incubation of the trophoblast derived Swan 71 and JEG-3 cell lines with 25-50 ng/ml recombinant leptin significantly boosted HLA-G mRNA and protein expression, and this effect was abrogated upon pharmacological inhibition of the PI3K-Akt and MEK-Erk signaling pathways. A similar stimulatory effect of leptin was observed in term placental tissue explants, though 10-fold higher doses were required for stimulation. Further, JEG-3 cells treated with a leptin antisense oligodeoxynucleotide displayed decreased HLA-G expression levels, which were partially recovered by addition of stimulating doses of exogenous hormone. Immunofluorescence and qPCR analysis confirmed leptin biosynthesis in placental tissue, further showing that invasive extravillous trophoblast cells were a main source of this hormone during the first trimester of normal pregnancies. DISCUSSION: Taken together, our results show that leptin acts as an autocrine/paracrine signal promoting HLA-G expression in placental trophoblasts suggesting an important role in the regulation of immune evasion mechanisms at the fetal maternal interface.