Long-term safety of OnabotulinumtoxinA treatment in chronic migraine patients: a five-year retrospective study.

Background: Real-world studies have shown the sustained therapeutic effect and favourable safety profile of OnabotulinumtoxinA (BoNTA) in the long term and up to 4 years of treatment in chronic migraine (CM). This study aims to assess the safety profile and efficacy of BoNTA in CM after 5 years of treatment in a real-life setting. Methods: We performed a retrospective chart review of patients with CM in relation to BoNTA treatment for more than 5 years in 19 Spanish headache clinics. We excluded patients who discontinued treatment due to lack of efficacy or poor tolerability. Results: 489 patients were included [mean age 49, 82.8% women]. The mean age of onset of migraine was 21.8 years; patients had CM with a mean of 6.4 years (20.8% fulfilled the aura criteria). At baseline, patients reported a mean of 24.7 monthly headache days (MHDs) and 15.7 monthly migraine days (MMDs). In relation to effectiveness, the responder rate was 59.1% and the mean reduction in MMDs was 9.4 days (15.7 to 6.3 days; p < 0.001). The MHDs were also reduced by 14.9 days (24.7 to 9.8 days; p < 0.001). Regarding the side effects, 17.5% experienced neck pain, 17.3% headache, 8.5% eyelid ptosis, 7.5% temporal muscle atrophy and 3.2% trapezius muscle atrophy. Furthermore, after longer-term exposure exceeding 5 years, there were no serious adverse events (AE) or treatment discontinuation because of safety or tolerability issues. Conclusion: Treatment with BoNTA led to sustained reductions in migraine frequency, even after long-term exposure exceeding 5 years, with no evidence of new safety concerns.

Fully automated AI-based cardiac motion parameter extraction - application to mitral and tricuspid valves on long-axis cine MR images.

PURPOSE: In cardiac MRI, valve motion parameters can be useful for the diagnosis of cardiac dysfunction. In this study, a fully automated AI-based valve tracking system was developed and evaluated on 2- or 4-chamber view cine series on a large cardiac MR dataset. Automatically derived motion parameters include atrioventricular plane displacement (AVPD), velocities (AVPV), mitral or tricuspid annular plane systolic excursion (MAPSE, TAPSE), or longitudinal shortening (LS). METHOD: Two sequential neural networks with an intermediate processing step are applied to localize the target and track the landmarks throughout the cardiac cycle. Initially, a localisation network is used to perform heatmap regression of the target landmarks, such as mitral, tricuspid valve annulus as well as apex points. Then, a registration network is applied to track these landmarks using deformation fields. Based on these outputs, motion parameters were derived. RESULTS: The accuracy of the system resulted in deviations of 1.44 ± 1.32 mm, 1.51 ± 1.46 cm/s, 2.21 ± 1.81 mm, 2.40 ± 1.97 mm, 2.50 ± 2.06 mm for AVPD, AVPV, MAPSE, TAPSE and LS, respectively. Application on a large patient database (N = 5289) revealed a mean MAPSE and LS of 9.5 ± 3.0 mm and 15.9 ± 3.9 % on 2-chamber and 4-chamber views, respectively. A mean TAPSE and LS of 13.4 ± 4.7 mm and 21.4 ± 6.9 % was measured. CONCLUSION: The results demonstrate the versatility of the proposed system for automatic extraction of various valve-related motion parameters.

Self-reported periodontitis and migraine: results from a multicenter, cross-sectional survey in Spain.

The aim of this study was to evaluate self-reported periodontitis (PD) prevalence in migraineurs as well as to investigate the association between both diseases. A cross-sectional survey was carried out including patients diagnosed with migraine attending 12 Spanish Headache Units. We determined diagnosis of PD administering a validated self-reported questionnaire. Socio-demographic, clinical and medical information, comorbidities, daily habits, migraine characteristics and medication were collected using a questionnaire. Of the 651 consecutive migraineurs included in the study, 393 suffered from chronic migraine (CM). Self-reported PD was detected in 327 patients with migraine (50.2%). Migraineurs with self-reported PD were significantly older and had a previous history of fibromyalgia, stress, anxiety, depression, and allodynia (all P < 0.001). Additionally, this group of patients consumed more topiramate (P = 0.008) and simple analgesics (P < 0.001) than patients with migraine and without self-reported PD. Also, they were less active physically and belonged to a low education level (both P < 0.001). Prevalence of self-reported PD was significantly higher in chronic migraineurs compared to those diagnosed with episodic migraine (EM) (53.9% vs. 44.6%, P = 0.019). Logistic regression analyses showed that self-reported PD was associated with CM (OR 1.456; 95% CI 1.062-1.997, P = 0.020). However, after adjusting for significant confounders, the association was attenuated (OR 1.100; 95% CI 0.784-1.543, P = 0.581). We concluded that self-reported PD was significantly more frequent in CM compared to EM. Self-reported PD was associated with the presence of CM, although some comorbidities shared by both diseases could have an effect on this association.

92R Monoclonal Antibody Inhibits Human CCR9+ Leukemia Cells Growth in NSG Mice Xenografts.

CCR9 is as an interesting target for the treatment of human CCR9+-T cell acute lymphoblastic leukemia, since its expression is limited to immature cells in the thymus, infiltrating leukocytes in the small intestine and a small fraction of mature circulating T lymphocytes. 92R, a new mouse mAb (IgG2a isotype), was raised using the A-isoform of hCCR9 as immunogen. Its initial characterization demonstrates that binds with high affinity to the CCR9 N-terminal domain, competing with the previously described 91R mAb for receptor binding. 92R inhibits human CCR9+ tumor growth in T and B-cell deficient Rag2-/- mice. In vitro assays suggested complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity as possible in vivo mechanisms of action. Unexpectedly, 92R strongly inhibited tumor growth also in a model with compromised NK and complement activities, suggesting that other mechanisms, including phagocytosis or apoptosis, might also be playing a role on 92R-mediated tumor elimination. Taken together, these data contribute to strengthen the hypothesis of the immune system's opportunistic nature.

Estilo de vida y apego al tratamiento con fármacos orales de pacientes con diabetes mellitus tipo 2

Introducción: La diabetes mellitus está al alza en la actualidad debido a múltiples factores en el estilo de vida. Las complicaciones de la enfermedad dicen mucho del adecuado seguimiento al apego del tratamiento y los cambios a su estilo de vida. Objetivo: evaluar el apego al tratamiento farmacológico y estilo de vida en personas con diabetes mellitus tipo 2 (DM2) Métodos: estudio prospectivo realizado en 276 personas con DM2 que acudieron a consulta al Patronato del Diabético zona 1 durante agosto 2016. Para evaluar el apego al tratamiento farmacológico se utilizó la escala de adherencia a medicamentos Morisky-8 y el cuestionario IMEVID para el estilo de vida. La adherencia se clasificó como baja, media y alta y el estilo de vida como desfavorable, poco favorable o favorable dependiendo de la calificación total. Para el análisis de datos se utilizó SPSS y estadísticas descriptivas. Resultados: 56% de los pacientes encuestados tienen un nivel de adherencia bajo en la toma de sus medicamentos. 71% llevan un estilo de vida poco favorable para su enfermedad. Conclusión: el apego al tratamiento en la mayoría de pacientes que acuden al Patronato del Diabético no es óptimo. Palabras clave: Diabetes mellitus, estilo de vida, apego, adherencia

Introduction. Type 2 diabetes is on the rise mainly due to several factors involved but mainly due to changes in life style. Its complications reflex none adequate adherence to recommended treatment. Objective: To evaluate medication adherence and lifestyle in persons with type 2 diabetes mellitus Methods: prospective study carried out in 276 patients of Patronato del Diabético during august 2016. To determine adherence to pharmacologic treatment, the Morisky-8 medication adherence scale was used and to determine lifestyle the IMEVID questionnaire. Depending on the total score medication adherence was classified as low, medium or high; and lifestyle as unfavorable, poor and favorable. Data were analyzed using descriptive statistics and SPSS program. Results: 56% of patients have low adherence in taking their medication for diabetes and 71% have a poor lifestyle for their disease Conclusion: adherence to treatment in the majority of patients that go for medical attention in Patronato del Diabético is not optimal. Keywords: Diabetes mellitus, adherence, life style

Effect of airway acidosis and alkalosis on airway vascular smooth muscle responsiveness to albuterol.

BACKGROUND: In vitro and animal experiments have shown that the transport and signaling of ß2-adrenergic agonists are pH-sensitive. Inhaled albuterol, a hydrophilic ß2-adrenergic agonist, is widely used for the treatment of obstructive airway diseases. Acute exacerbations of obstructive airway diseases can be associated with changes in ventilation leading to either respiratory acidosis or alkalosis thereby affecting albuterol responsiveness in the airway. The purpose of this study was to determine if airway pH has an effect on albuterol-induced vasodilation in the airway. METHODS: Ten healthy volunteers performed the following respiratory maneuvers: quiet breathing, hypocapnic hyperventilation, hypercapnic hyperventilation, and eucapnic hyperventilation (to dissociate the effect of pH from the effect of ventilation). During these breathing maneuvers, exhaled breath condensate (EBC) pH and airway blood flow response to inhaled albuterol (ΔQ̇aw) were assessed. RESULTS: Mean ± SE EBC pH (units) and ΔQ̇aw (µl.min(-1).mL(-1)) were 6.4 ± 0.1 and 16.8 ± 1.9 during quiet breathing, 6.3 ± 0.1 and 14.5 ± 2.4 during eucapnic hyperventilation, 6.6 ± 0.2 and -0.2 ± 1.8 during hypocapnic hyperventilation (p = 0.02 and <0.01 vs. quiet breathing), and 5.9 ± 0.1 and 2.0 ± 1.5 during hypercapnic hyperventilation (p = 0.02 and <0.02 vs quiet breathing). CONCLUSIONS: Albuterol responsiveness in the airway as assessed by ΔQ̇aw is pH sensitive. The breathing maneuver associated with decreased and increased EBC pH both resulted in a decreased responsiveness independent of the level of ventilation. These findings suggest an attenuated response to hydrophilic ß2-adrenergic agonists during airway disease exacerbations associated with changes in pH. TRIAL REGISTRATION: Registered at clinicaltrials.gov: NCT01216748 .

Hyaluronan and layilin mediate loss of airway epithelial barrier function induced by cigarette smoke by decreasing E-cadherin.

Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS.

MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium.

Cigarette smoke represents a major risk factor for the development of chronic obstructive pulmonary disease (COPD), a respiratory condition associated with airflow obstruction, mucus hypersecretion, chronic inflammation, and upregulation of inflammatory mediators such as the monocyte chemotactic protein-1 (MCP-1). MCP-1 through its receptor CCR2 induces chemotaxis and activates (44/42)MAPK, a kinase known to play a key role in mucin regulation in bronchial epithelium. In the present study we used differentiated primary cultures of normal human bronchial epithelial (NHBE) cells to test whether MCP-1 through its receptor CCR2 induces mucin upregulation. We have provided evidence that NHBE cells release MCP-1 to the epithelial surface and express the CCR2B isoform of the receptor mainly at the apical pole. In addition, we found that MCP-1 has a novel function in airway epithelium, increasing the two major airway mucins MUC5AC and MUC5B, an effect mediated, at least in part, by a cascade of events initiated by interaction of its receptor CCR2B with G(q) subunits in caveolae, followed by PLCß, PKC, and (44/42)MAPK activation. We also have shown that MCP-1 is able to induce its own expression using the same receptor but through a different pathway that involves RhoA GTPase. Furthermore, we found that a single exposure to MCP-1 is enough to induce MCP-1 secretion and sustained mucin upregulation up to 7 days after initial exposure, an effect mediated by CCR2B as confirmed using short hairpin RNA. These results agree with our data in smoker's airway epithelium, where CCR2B is present in MUC5AC- and MUC5B-expressing cells and augmented MCP-1 expression is associated with increased MUC5AC and MUC5B immunolabeling, suggesting that the mechanisms described in primary cell cultures in the present study are operative in vivo. Therefore, therapeutic approaches targeting MCP-1/CCR2B may be useful in preventing not only influx of inflammatory cells to the airways but also mucus hypersecretion and goblet cell hyperplasia.

Reactive oxygen species and hyaluronidase 2 regulate airway epithelial hyaluronan fragmentation.

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction was confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. In summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress.

Hyaluronan fragments/CD44 mediate oxidative stress-induced MUC5B up-regulation in airway epithelium.

Mucus hypersecretion with elevated MUC5B mucin production is a pathologic feature in many airway diseases associated with oxidative stress. In the present work, we evaluated MUC5B expression in airways and in primary cultures of normal human bronchial epithelial (NHBE) cells, as well as the mechanisms involved in its regulation. We found that oxidative stress generated by cigarette smoke or reactive oxygen species (ROS) induces MUC5B up-regulation in airway epithelium from smokers and in NHBE cells, respectively. We have previously shown that ROS-induced MUC5AC expression in NHBE cells is dependent on hyaluronan depolymerization and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) activation. Since hyaluronan fragments can activate MAPK through the hyaluronan receptor CD44, and CD44 heterodimerizes with EGFR, we tested whether ROS and/or hyaluronan fragments induce MUC5B mRNA and protein expression through CD44/EGFR. We found that ROS promotes CD44/EGFR interaction, EGFR/MAPK activation, and MUC5B up-regulation that are prevented by blocking CD44 and/or EGFR. These results were mimicked by hyaluronan fragments. In summary, our results show that oxidative stress in vivo (cigarette smoke) or in vitro (ROS) induces MUC5B up-regulation. This ROS-induced MUC5B expression requires CD44 as well as EGFR and MAPK activation. In addition, we also provide evidence that hyaluronan fragments are sufficient to induce CD44/EGFR interaction and downstream signaling that results in MUC5B up-regulation, suggesting that hyaluronan depolymerization during inflammatory responses could be directly involved in the induction of mucus hypersecretion.

Effect of an inhaled glucocorticoid on endothelial function in healthy smokers.

Cigarette smoking is associated with attenuated endothelium-dependent vasodilation (endothelial dysfunction) in the systemic circulation, including the airway circulation. We wished to determine whether an inhaled corticosteroid could restore endothelial function in the airway of lung-healthy current smokers, ex-smokers, and nonsmokers. We measured baseline airway blood flow (Qaw) and Qaw reactivity to inhaled albuterol as an index of endothelium-dependent vasodilation and to sublingual nitroglycerin as an index of endothelium-independent vasodilation in lung-healthy current smokers, ex-smokers, and nonsmokers. Current smokers were then treated with inhaled fluticasone for 3 wk, and all measurements were repeated after fluticasone treatment and after a subsequent 3-wk fluticasone washout period. Baseline mean Qaw and endothelium-independent Qaw reactivity were similar in the three groups. Mean endothelium-dependent Qaw reactivity was 49.5% in nonsmokers, 42.7% in ex-smokers, and 10.8% in current smokers (P < 0.05 vs. nonsmokers). In current smokers, mean baseline Qaw was unchanged after fluticasone treatment, but endothelium-dependent Qaw reactivity significantly increased to 34.9%. Qaw reactivity was again blunted after fluticasone washout. Endothelial dysfunction, as assessed by vascular reactivity, can be corrected with an inhaled corticosteroid in the airway of lung-healthy current smokers. This proof of concept can serve as the basis for future clinical investigations on the effect of glucocorticoids on endothelial function in smokers.

Hyaluronidase expression and activity is regulated by pro-inflammatory cytokines in human airway epithelial cells.

Hyaluronan (HA) is present at the apical surface of airway epithelium as a high-molecular-weight polymer. Since HA depolymerization initiates a cascade of events that results in kinin generation and growth factor processing, in the present work we used primary cultures of human bronchial epithelial (HBE) cells grown at the air-liquid interface (ALI) to assess hyaluronidase (Hyal) activity by HA zymography, gene expression by quantitative real-time PCR, and localization by confocal microscopy. Because TNF-alpha and IL-1beta induce Hyals in other cells, we tested their effects on Hyals expression and activity. We found that Hyal-like activity is present in the apical and basolateral secretions from HBE cells where Hyals 1, 2, and 3 are expressed, and that IL-1beta acts synergistically with TNF-alpha to increase gene expression and activity. Confocal microscopy showed that Hyals 1, 2, and 3 were localized intracellularly, while Hyal2 was also expressed at the apical pole associated with the plasma membrane, and in a soluble form on the apical secretions. Tissue sections from normal individuals and from individuals with asthma showed a Hyal distribution pattern similar to that observed on nontreated HBE cells or exposed to cytokines, respectively. In addition, increased expression and activity were observed in tracheal sections and in bronchoalveolar lavage (BAL) obtained from subjects with asthma when compared with normal lung donors and healthy volunteers. Our observations indicate that Hyal 1, 2, and 3 are expressed in airway epithelium and may operate in a coordinated fashion to depolymerize HA during inflammation associated with up-regulation of TNF-alpha and IL-1beta, such as allergen-induced asthmatic responses.

Apical oxidative hyaluronan degradation stimulates airway ciliary beating via RHAMM and RON.

Hyaluronan (HA) is synthesized in high-molecular-weight form at the apical pole of airway epithelial cells, covering the luminal surface. When human airway epithelial cells grown and redifferentiated at the air-liquid interface (ALI) were exposed to xanthine/xanthine oxidase (X/XO), ciliary beat frequency (CBF) increased. This effect was blocked by superoxide dismutase (SOD) and catalase. Inhibition of hyaluronan synthesis inhibited the CBF response to X/XO, while addition of exogenous HA amplified it. A functionally blocking antibody to the receptor for hyaluronic acid-mediated motility (RHAMM) reduced the CBF response to X/XO. Since RHAMM has no transmembrane domain and thus cannot signal on its own, the association of RHAMM with recepteur d'origine nantais (RON), a member of the hepatocyte growth factor receptor family, was explored. Immunohistochemistry of human airway epithelium showed co-localization of RHAMM and RON at the apex of ciliated cells. Physical association of RHAMM and RON was confirmed with co-immunoprecipitations. Macrophage-stimulating protein (MSP), an agonist of RON, stimulated CBF. Genistein, a nonspecific tyrosine kinase inhibitor, and MSP beta chain (beta-MSP), a specific RON inhibitor, blocked the X/XO-induced CBF increase. HA present in the apical secretions of human airway epithelial cells was shown to degrade upon exposure to X/XO, a process inhibited by SOD. Low-molecular-weight HA fragments stimulated CBF, an effect blocked by anti-RHAMM antibody and genistein. These data suggest that high molecular form HA is broken down by reactive oxygen species to form low-molecular-weight fragments that signal via RHAMM and RON to stimulate CBF.

TSG-6 potentiates the antitissue kallikrein activity of inter-alpha-inhibitor through bikunin release.

TSG-6 (the protein product of TNF-stimulated gene-6), an inflammation-associated protein, forms covalent complexes with heavy chains (HCs) from inter-alpha-inhibitor and pre-alpha-inhibitor and associates noncovalently with their common bikunin chain, potentiating the antiplasmin activity of this serine protease inhibitor. We show that TSG-6 and TSG-6.HC complexes are present in bronchoalveolar lavage fluid from patients with asthma and increase after allergen challenge. Immunodetection demonstrated elevated TSG-6 in the airway tissue and secretions of smokers. Experiments conducted in vitro with purified components revealed that bikunin.HC complexes (byproducts of TSG-6.HC formation) release bikunin. Immunoprecipitation revealed that bikunin accounts for a significant proportion of tissue kallikrein inhibition in bronchoalveolar lavage after allergen challenge but not in baseline conditions, confirming that bikunin in its free state, but not when associated with HCs, is a relevant protease inhibitor in airway secretions. In primary cultures of differentiated human airway epithelial and submucosal gland cells, TSG-6 is induced by TNF-alpha and IL-1beta, which suggests that these cells are responsible for TSG-6 release in vivo. Bikunin and HC3 (i.e., pre-alpha-inhibitor) were also induced by TNF-alpha in primary cultures. Our results suggest that TSG-6 may play an important protective role in bronchial epithelium by increasing the antiprotease screen on the airway lumen.

Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium.

Mucus overproduction in inflammatory and obstructive airway diseases is associated with goblet cell (GC) metaplasia in airways. Although the mechanisms involved in GC metaplasia and mucus hypersecretion are not completely understood, association with oxidative stress and epidermal growth factor receptor (EGFR) signaling has been reported. To explore the mechanisms involved in oxidative stress-induced GC metaplasia, cultures of differentiated normal human bronchial epithelial cells grown at the air-liquid interface were exposed to reactive oxygen species (ROS) generated by xanthine/xanthine oxidase. EGFR activation and signaling was assessed by measuring EGF and transforming growth factor-alpha release and EGFR and (44/42)MAPK phosphorylation. The GC population was evaluated by confocal microscopy. ROS-induced EGFR activation resulted in GC proliferation and increased MUC5AC gene and protein expression. Signaling was due to pro-EGF processing by tissue kallikrein (TK), which was activated by ROS-induced hyaluronan breakdown. It was inhibited by catalase, a TK inhibitor, and EGF-blocking antibodies. Exposure to recombinant TK mimicked the ROS effects, increasing the expression of MUC5AC and lactoperoxidase. In addition, ROS induced the antiapoptotic factor Bcl-2 in a TK-dependent fashion. In conclusion, ROS-induced GC metaplasia in normal human bronchial epithelial cells is associated with HA depolymerization and EGF processing by TK followed by EGFR signaling, suggesting that increases in TK activity could contribute to GC metaplasia and mucus hypersecretion in diseases such as asthma and chronic bronchitis. The data also suggest that increases in GC population could be sustained by the associated upregulation of Bcl-2 in airway epithelial cells.

Identification of glycosaminoglycans in human airway secretions.

Glycosaminoglycans (GAGs), known to be present in airway mucus, are macromolecules with a variety of structural and biological functions. In the present work, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to identify and relatively quantify GAGs in human tracheal aspirates (HTA) obtained from healthy volunteers. Primary cultures of normal human bronchial epithelial (NHBE) and submucosal gland (SMG) cells were used to assess their differential contribution to GAGs in mucus. Distribution was further assessed by immunofluorescence in human trachea tissue sections and in cell cultures. HTA samples contained keratan sulfate (KS), chondroitin/dermatan sulfate (CS/DS), and hyaluronan (HA), whereas heparan sulfate (HS) was not detected. SMG cultures secreted CS/DS and HA, CS/DS being the most abundant GAGs in these cultures. NHBE cells synthesized KS, HA, and CS/DS. Confocal microscopy showed that KS was exclusively found at the apical border of NHBE cells and on the apical surface of ciliated epithelial cells in tracheal tissues. CS/DS and HA were present in both NHBE and SMG cells. HS was only found in the extracellular matrix in trachea tissue sections. In summary, HTA samples contain KS, CS/DS, and HA, mirroring a mixture of secretions originated in surface epithelial cells and SMGs. We conclude that surface epithelium is responsible for most HA and all KS present in secretions, whereas glands secrete most of CS/DS. These data suggest that, in diseases where the contribution to secretions of glands versus epithelial cells is altered, the relative concentration of individual GAGs, and therefore their biological activities, will also be affected.

Role of hyaluronan and reactive oxygen species in tissue kallikrein-mediated epidermal growth factor receptor activation in human airways.

In human airways, oxidative stress-induced submucosal gland cell hypertrophy and hyperplasia, histological features of chronic bronchitis, have been linked to epidermal growth factor receptor (EGFR) activation. To explore mechanisms of oxidative stress-induced EGFR activation and signaling, primary cultures of human tracheal submucosal gland (SMG) cells were used to assess EGFR ligand release, EGFR phosphorylation, p44/42 MAPK phosphorylation, and mucin 5AC synthesis in response to reactive oxygen species generated by xanthine/xanthine oxidase (X/XO). Exposure to X/XO increased release of epidermal growth factor (EGF) from these cells, thereby activating EGFR, phosphorylating MAPK, and increasing mucin 5AC production. The importance of EGF was confirmed by transfection of small interfering RNA inhibiting pro-EGF production, which resulted in inhibition of EGFR and MAPK phosphorylation despite X/XO exposure. Blocking signaling by using specific protease inhibitors showed that tissue kallikrein (TK) processed pro-EGF in response to X/XO. Airway TK is bound and inactivated by luminal hyaluronan (HA), and treatment of submucosal gland cells with X/XO induced HA depolymerization and TK activation. These events were blocked by reactive oxygen species scavengers and addition of exogenous excess HA and TK inhibitors. Thus, HA plays a crucial role in regulating airway TK activity and thereby TK-mediated release of active EGF from human SMG cells. Sustained HA depolymerization is expected to cause TK activation, EGF release, and EGFR signaling and to lead to SMG cell hypertrophy and hyperplasia as well as mucus hypersecretion with subsequent airflow obstruction.

Ghrelin increases anxiety-like behavior and memory retention in rats.

Ghrelin is a peptide found in the hypothalamus and stomach that stimulates food intake and whose circulating concentrations are affected by nutritional state. Very little is known about other central behavioral effects of ghrelin, and thus, we investigated the effects of ghrelin on anxiety and memory retention. The peptide was injected intracerebroventricularly in rats and we performed open-field, plus-maze, and step-down tests (inhibitory avoidance). The administration of ghrelin increased freezing in the open field and decreased the number of entries into the open spaces and the time spent on the open arms in the plus-maze, indicating an anxiogenic effect. Moreover, the peptide increased in a dose-dependent manner the latency time in the step-down test. A rapid and prolonged increase in food intake was also observed. Our results indicate that ghrelin induces anxiogenesis in rats. Moreover, we show for the first time that ghrelin increases memory retention, suggesting that the peptide may influence processes in the hippocampus.

Melanin-concentrating hormone, hippocampal nitric oxide levels and memory retention.

The present study attempts to determine, if the effect of melanin-concentrating hormone (MCH) upon memory retention is correlated with changes in nitric oxide synthase (NOS) activity and tissue levels of nitric oxide (NO) and cGMP. We used a behavioral experiment using a step-down inhibitory avoidance test, the biochemical determinations of NO and cGMP, and electrophysiological model. Results of behavioral studies (step-down test) showed that MCH administration reverts the amnesic effects induced by N(G)-nitro-L-arginine (L-NOArg). Moreover, electrophysiological studies demonstrated that L-NOArg did not block the potentiation induced by the peptide. Hippocampal NO and cGMP levels increased after MCH injection.

Evaluación de la técnica de hemocultivo y epidemiología de la enfermedad de Chagas en pacientes agudos y crónicos / Annual reports 1998 / Evaluation of the hemoculture technique and epidemiological characteristic of acute and chronic Chagas disease patients

El diagnóstico de la enfermedad de Chagas por métodos parasitológicos se realiza mediante la visualización directa o indirecta del parásito. La detección del mismo es relativamente fácil en la fase aguda, cuando la parasistemia es alta; sin embargo, durante las fases intermedias y la cronica se debe recurrir a métodos que requieren una observación más prologada como el xenodiagnóstico y el hemocultivo, siendo la sensibilidad de estos últimos menor. En este trabajo se determino la sensibilidad del hemocultivo en paciente chagasicos y se provee informaciones complementaria sobre las carácteristicas epidemiólogicas de los mismos. Se realizó un levantamiento retrospectivos de datos obtenidos de fichas de 100 pacientes chagasicos que consultaron en el Departamento de Medicina Tropical del IICS durante el periodo de enero 1984 a enero 1992. A todos estos pacientes se le realizo la prueba de hemocultivo en forma de rutina. El 21 por ciento de estos pacientes presentaron resultados positivos para T.cruzi por la tecnica de hemocultivo. Teniendo en cuenta el cuadro clinico de los pacientes, se observo que el 100 por ciento (4/4) de los casos agudos, el 73,2 por ciento (6/25) de los pacientes con lesiones cardiacas o digestivas y el 16,4 por ciento (11/67) de los individuos en la etapa indeterminada fueron positivos a la T.cruzi y el resto de los pacientes sin diagnostico clinico fue negativo por esta técnica. Teniendo en cuenta la edad de estos pacientes se encontro que los menores de 30 años dieron hemocultivo positivo más frecuentemente que aquellos que pertenecieron a grupos de mayor edad. En este estudio, la psibilidad del hemocultivo mostro cierta asociación con la edad y la procedencia del paciente chagasico