RESUMEN
Smoldering multiple myeloma (SMM) is a precursor stage that precedes multiple myeloma (MM). SMM is heterogenous with nearly 40% of patients progressing to MM in the first 5 years. The high rate of progression of SMM patients highlights the need for early intervention, which underscores the importance of identifying SMM patients with the highest risk of progression. Several risk stratification models showed utility in identifying high-risk SMM patients; however, these systems showed limited sensitivity. To date, identifying high-risk SMM patients remains an important clinical need. In this study, we present the 3-dimensional telomere profiling as a structural biomarker capable of stratifying SMM patients as a function of genomic instability. Quantifying telomere dysfunction using the TeloView technology showed utility in risk stratification of cancer patients, particularly hematological malignancies. In this study, we analyzed 168 SMM patients. We report an AUC in ROC analysis of 0.8 using a subset of the patients as a training dataset. We then conducted a blind validation on a different cohort and demonstrated a positive predictive value of 85% and negative predictive value of 73%, with sensitivity and specificity of 83% and 76%, respectively. We examined the correlation between the TeloView prediction and the 20-2-20 scoring system, and cytogenetic abnormalities. We report a correlation of 53% with the 20-2-20 scores and over 60% correlation with cytogenetic abnormalities. The result of this study presents the telomere profiling as an effective biomarker able to stratify SMM patients to their respective risk groups with high sensitivity and specificity.
Asunto(s)
Progresión de la Enfermedad , Mieloma Múltiple Quiescente , Telómero , Humanos , Mieloma Múltiple Quiescente/genética , Mieloma Múltiple Quiescente/diagnóstico , Femenino , Masculino , Persona de Mediana Edad , Anciano , Mieloma Múltiple/genética , Mieloma Múltiple/diagnóstico , Valor Predictivo de las PruebasRESUMEN
Retinopathy of prematurity (ROP) is one of the leading causes of blindness in preterm Infants. Anti-vascular endothelial growth factor (VEGF) is emerging as a promising treatment, but there is insufficient evidence on their safety. We investigate the effect of systemic anti-VEGF in rat pups with equivalent maturity to a 32 week neonate. A single dose of either anti-VEGF antibody (nâ¯=â¯7) or saline (control group; nâ¯=â¯6) was administered to newborn rats intra-peritoneally on the first day of life. 14 days' post treatment, the serum concentration of anti-VEGF was measured and the brain, lung, heart, kidney and liver were harvested and weighed. The heart was processed to measure the Fulton index (a surrogate for pulmonary hypertension). All other organs were processed for mRNA expression of VEGF and VEGF-receptors (R1&R2). No group differences in body and organ weights were noted. The anti-VEGF was still detected in serum 14 days post Injection and resulted in increase in lung (pâ¯<â¯0.002) and kidney (pâ¯<â¯0.01) VEGF mRNA expressions and the lung (pâ¯<â¯0.02) VEGF-R1 and kidney (Pâ¯<â¯0.001) VEGF-R2 mRNA expressions. The treated pups exhibited increased total heart weight (pâ¯<â¯0.01) and Fulton Index (pâ¯<â¯0.05). No changes were seen in the liver and brain. Anti-VEGF antibody did not affect mortality, total body and organ weights, but was associated with pulmonary hypertension. Expression of lung and kidney VEGF and its receptors was increased, whilst the brain and liver did not show changes. Dosing experiments can now be targeted to assess safety threshold and at anti-VEGF dose used in human ROP treatment.
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Inhibidores de la Angiogénesis/administración & dosificación , Animales Recién Nacidos/crecimiento & desarrollo , Encéfalo/crecimiento & desarrollo , Riñón/crecimiento & desarrollo , Hígado/crecimiento & desarrollo , Pulmón/crecimiento & desarrollo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Peso Corporal , Encéfalo/metabolismo , Inyecciones Intraperitoneales , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Tamaño de los Órganos , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
BackgroundHuman milk has a high content of the antimicrobial compound hydrogen peroxide (H2O2). As opposed to healthy full-term infants, preterm neonates are fed previously expressed and stored maternal milk. These practices may favor H2O2 decomposition, thus limiting its potential benefit to preterm infants. The goal of this study was to evaluate the factors responsible for H2O2 generation and degradation in breastmilk.MethodsHuman donors' and rats' milk, along with rat mammary tissue were evaluated. The role of oxytocin and xanthine oxidase on H2O2 generation, its pH-dependent stability, as well as its degradation via lactoperoxidase and catalase was measured in milk.ResultsBreast tissue xanthine oxidase is responsible for the H2O2 generation and its milk content is dependent on oxytocin stimulation. Stability of the human milk H2O2 content is pH-dependent and greatest in the acidic range. Complete H2O2 degradation occurs when human milk is maintained, longer than 10 min, at room temperature and this process is suppressed by lactoperoxidase and catalase inhibition.ConclusionFresh breastmilk H2O2 content is labile and quickly degrades at room temperature. Further investigation on breastmilk handling techniques to preserve its H2O2 content, when gavage-fed to preterm infants is warranted.
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Peróxido de Hidrógeno/metabolismo , Fenómenos Fisiológicos Nutricionales del Lactante , Leche Humana/química , Animales , Mama/metabolismo , Catalasa/química , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lactante , Recién Nacido , Recien Nacido Prematuro , Lactoperoxidasa/química , Glándulas Mamarias Animales/metabolismo , Leche/química , Oxitocina/química , Ratas , Ratas Sprague-Dawley , Xantina Oxidasa/químicaRESUMEN
BACKGROUND: When compared with infant formula, human milk enhances gastric emptying in preterm infants. Hydrogen peroxide (H2O2) is present in large quantities in human milk that has an antimicrobial role for the mother and infant. In vitro adult rat studies suggest that H2O2 facilitates gastric motor contraction. Hypothesizing that H2O2 enhances gastric motility, we investigated its effects on the newborn rat stomach tissue. METHODS: Rat newborn and adult gastric fundic segments, or their smooth muscle cells, were used to evaluate the muscle response to H2O2 exposure. Tissue expression of Rho kinase 2 (ROCK-2; Western blot), its catalase activity, and H2O2 content (Amplex Red) were measured. H2O2 gastric mucosal diffusion was evaluated with Ussing chambers. RESULTS: In both newborn and adult rats, H2O2 induced gastric muscle contraction and this response was attenuated by pre-incubation with the antioxidant melatonin. H2O2 passively diffused across the gastric mucosa. Its effect on the muscle was modulated via ROCK-2 activation and inhibited by melatonin. CONCLUSION: H2O2, at a concentration similar to that of human milk, promotes gastric motility in the rat. To the extent that the present findings can be clinically extrapolated, the human milk H2O2 content may enhance gastric emptying in neonates.
Asunto(s)
Vaciamiento Gástrico/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Animales , Animales Recién Nacidos , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Estómago/efectos de los fármacosRESUMEN
Feeding intolerance is a common issue in the care of preterm neonates. The condition manifests as delayed emptying of gastric contents and represents a therapeutic challenge, since the factors accounting for its manifestations are unknown. The main goal of this study was to comparatively investigate the age-related function of rat gastric and pyloric smooth muscle and their putative regulators. We hypothesized that a reduced gastric muscle contraction potential early in life contributes to the delayed gastric emptying of the newborn. Newborn and adult rat gastric (fundus) and pyloric sphincter tissues were comparatively studied in vitro. Shortening of the tissue-specific dissociated smooth muscle cell was evaluated, and expression of the key regulatory proteins Rho-associated kinase 2 and myosin light chain kinase was determined. Gastric and pyloric smooth muscle cell shortening was significantly greater in the adult than the respective newborn counterpart. Expression of myosin light chain kinase and Rho-associated kinase 2 was developmentally regulated and increased with age. Pyloric sphincter muscle expresses a higher neuronal nitric oxide synthase and phosphorylated vasodilator-stimulated phosphoprotein content in newborn than adult tissue. Compared with later in life, the newborn rat gastropyloric muscle has a Ca(2+)-related reduced potential for contraction and the pyloric sphincter relaxation-dependent modulators are overexpressed. To the extent that these rodent data can be extrapolated to humans, the delayed gastric emptying in the newborn reflects reduced stomach muscle contraction potential, as opposed to increased pyloric sphincter tone.
Asunto(s)
Vaciamiento Gástrico , Fundus Gástrico/fisiología , Píloro/fisiología , Animales , Fundus Gástrico/crecimiento & desarrollo , Fundus Gástrico/metabolismo , Contracción Muscular , Músculo Liso/crecimiento & desarrollo , Músculo Liso/metabolismo , Músculo Liso/fisiología , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Píloro/crecimiento & desarrollo , Píloro/metabolismo , Ratas , Ratas Sprague-Dawley , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
The factors accounting for the pathological maintenance of a high pulmonary vascular (PV) resistance postnatally remain elusive, but neonatal stressors may play a role in this process. Cross-fostering in the immediate neonatal period is associated with adult-onset vascular and behavioral changes, likely triggered by early-in-life stressors. In hypothesizing that fostering newborn rats induces long-lasting PV changes, we evaluated them at 14 days of age during adulthood and compared the findings with animals raised by their biological mothers. Fostering resulted in reduced maternal-pup contact time when compared with control newborns. At 2 wk of age, fostered rats exhibited reduced pulmonary arterial endothelium-dependent relaxation secondary to downregulation of tissue endothelial nitric oxide synthase expression and tetrahydrobiopterin deficiency-induced uncoupling. These changes were associated with neonatal onset-increased ANG II receptor type 1 expression, PV remodeling, and right ventricular hypertrophy that persisted into adulthood. The pulmonary arteries of adult-fostered rats exhibited a higher contraction dose response to ANG II and thromboxane A2, the latter of which was abrogated by the oxidant scavenger Tempol. In conclusion, fostering-induced neonatal stress induces long-standing PV changes modulated via the renin-angiotensin system.
Asunto(s)
Pulmón/irrigación sanguínea , Arteria Pulmonar/fisiopatología , Animales , Animales Recién Nacidos , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Femenino , Pulmón/enzimología , Conducta Materna , Contracción Muscular , Relajación Muscular , Músculo Liso Vascular/fisiopatología , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Sistema Renina-Angiotensina , Estrés Psicológico/fisiopatología , Resistencia VascularRESUMEN
Inhaled nitric oxide (NO) and other cGMP- or cAMP-dependent pulmonary vasodilators are often used in combination for the treatment of the persistent pulmonary hypertension of the newborn syndrome. There is in vitro evidence to indicate that NO downregulate the pulmonary vascular response to cGMP-dependent agonists raising concern as to whether a synergistic effect is observed when employing a combined strategy in newborns. Hypothesizing that a synergistic effect is absent, we evaluated newborn and juvenile rat pulmonary arteries to determine the individual and combined vasodilatory effect of cGMP- and cAMP-dependent agonists. In precontracted near-resistance pulmonary arteries, the addition of sildenafil reduced vasorelaxation response to NO donor S-nitroso-N-acetyl penicillamine (SNAP). A similar decrease in SNAP-induced vasodilation was observed in arteries pretreated with BAY 41-2272 (10(-9) M), a soluble guanylate cyclase stimulator cGMP, and its downstream protein kinase activator. cGMP also reduced the vasorelaxant response to the cAMP-dependent forskolin. Inhibition of endogenous vascular NO generation enhanced SNAP-induced relaxation. The present data suggest that the mechanism involved in the cGMP desensitization to other relaxant agonists involves downregulation of the small heat shock protein HSP20 and is evident in rat pulmonary and systemic vascular smooth muscle cells. In newborn rats with chronic hypoxia-induced pulmonary hypertension, the combination of sildenafil and inhaled NO resulted in a lesser reduction in pulmonary vascular resistance compared with their individual effect. These data suggest that clinical exposure to one cGMP-dependent pulmonary vasodilator may affect the response to other cGMP- or cAMP-mediated agonists.
Asunto(s)
GMP Cíclico/metabolismo , Hipoxia/metabolismo , Músculo Liso Vascular/fisiología , Circulación Pulmonar/efectos de los fármacos , Circulación Pulmonar/fisiología , Vasodilatadores/farmacología , Animales , Animales Recién Nacidos , Enfermedad Crónica , Femenino , Proteínas del Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Hipoxia/fisiopatología , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Fosforilación/fisiología , Piperazinas/farmacología , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Ratas , Ratas Sprague-Dawley , Citrato de Sildenafil , Sulfonas/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
Infantile hypertrophic pyloric stenosis (IHPS) is a common disease of unknown etiology. The tetrahydrobiopterin (BH4)-deficient hyperphenylalaninemia-1 (hph-1) newborn mouse has a similar phenotype to the human condition. For hph-1 and wild-type control animals, pyloric tissue agonist-induced contractile properties, reactive oxygen species (ROS) generation, cGMP, neuronal nitric oxide synthase (nNOS) content, and Rho-associated protein kinase 2 (ROCK-2) expression and activity were evaluated. Primary pyloric smooth muscle cells from wild-type newborn animals were utilized to evaluate the effect of BH4 deficiency. One-week-old hph-1 mice exhibited a fourfold increase (P < 0.01) in the pyloric sphincter muscle contraction magnitude but similar relaxation values when compared with wild-type animals. The pyloric tissue nNOS expression and cGMP content were decreased, whereas the rate of nNOS uncoupling increased (P < 0.01) in 1-wk-old hph-1 mice when compared with wild-type animals. These changes were associated with increased pyloric tissue ROS generation and elevated ROCK-2 expression/activity (P < 0.05). At 1-3 days of age and during adulthood, the gastric emptying rate of the hph-1 mice was not altered, and there were no genotype differences in pyloric tissue ROS generation, nNOS expression, or ROCK-2 activity. BH4 inhibition in pyloric smooth muscle cells resulted in increased ROS generation (P < 0.01) and ROCK-2 activity (P < 0.05). Oxidative stress upregulated ROCK-2 activity in pyloric tissue, but no changes were observed in newborn fundal tissue in vitro. We conclude that ROS-induced upregulation of ROCK-2 expression accounts for the increased pyloric sphincter tone and nNOS downregulation in the newborn hph-1 mice. The role of ROCK-2 activation in the pathogenesis of IHPS warrants further study.
Asunto(s)
Contracción Muscular/fisiología , Estenosis Hipertrófica del Piloro/etiología , Píloro/fisiopatología , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Vaciamiento Gástrico/fisiología , Humanos , Peróxido de Hidrógeno/metabolismo , Lactante , Ratones , Óxido Nítrico Sintasa de Tipo I/metabolismo , Estenosis Hipertrófica del Piloro/metabolismo , Estenosis Hipertrófica del Piloro/fisiopatología , Píloro/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
Proton pump inhibitors reduce gastric acid secretion and are commonly utilized in the management of gastroesophageal reflux disease across all ages. Yet a decrease in lower esophageal sphincter tone has been reported in vitro in rats through an unknown mechanism; however, their effect on the gastroesophageal muscle tone early in life was never studied. Hypothesizing that proton pump inhibitors also reduce gastroesophageal muscle contraction in newborn and juvenile rats, we evaluated the in vitro effect of pantoprazole on gastric and lower esophageal sphincter muscle tissue. Electrical field stimulation and carbachol-induced force were significantly (P < 0.01) reduced in the presence of pantoprazole, whereas the drug had no effect on the neuromuscular-dependent relaxation. When administered in vivo, pantoprazole (9 mg/kg) significantly (P < 0.01) reduced gastric emptying time at both ages. To ascertain the signal transduction pathway responsible for the reduction in muscle contraction, we evaluated the tissue ROCK-2 and CPI-17 activity. Pantoprazole reduced myosin light chain phosphatase MYPT-1, but not CPI-17 phosphorylation of gastric and lower esophageal sphincter tissue, strongly suggesting that it is a ROCK-2 inhibitor. To the extent that these findings can be extrapolated to human neonates, the use of pantoprazole may impair gastric and lower sphincter muscle tone and thus paradoxically exacerbate esophageal reflux. Further studies addressing the effect of proton pump inhibitors on gastroesophageal muscle contraction are warranted to justify its therapeutic use in gastroesophageal reflux disease.
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2-Piridinilmetilsulfinilbencimidazoles/toxicidad , Esfínter Esofágico Inferior/efectos de los fármacos , Vaciamiento Gástrico/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/toxicidad , Inhibidores de la Bomba de Protones/toxicidad , Estómago/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Carbacol/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Esfínter Esofágico Inferior/enzimología , Esfínter Esofágico Inferior/inervación , Proteínas Musculares/metabolismo , Pantoprazol , Fosfoproteínas/metabolismo , Fosforilación , Proteína Fosfatasa 1/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Estómago/enzimología , Estómago/inervación , Factores de Tiempo , Quinasas Asociadas a rho/metabolismoRESUMEN
Feeding intolerance resulting from delayed gastric emptying is common in premature neonates. Metoclopramide (MCP), the most frequently used prokinetic drug in neonates, enhances gastric muscle contractility through inhibition of dopamine receptors. Although its therapeutic benefit is established in adults, limited data are available to support its clinical use in infants. Hypothesizing that developmentally dependent differences are present, we comparatively evaluated the effect of MCP on fundus muscle contractility in newborn, juvenile, and adult rats. The muscle strips were either contracted with electrical field stimulation (EFS) to induce cholinergic nerve-mediated acetylcholine release or carbachol, a cholinergic agonist acting directly on the muscarinic receptor. Although in adult rats MCP increased EFS-induced contraction by 294 ± 122% of control (P < 0.01), no significant effect was observed in newborn fundic muscle. MCP had no effect on the magnitude of the carbachol-induced and/or bethanechol-induced gastric muscle contraction at any age. In response to dopamine, an 80.7 ± 5.3% relaxation of adult fundic muscle was observed, compared with only a 8.4 ± 8.7% response in newborn tissue (P < 0.01). Dopamine D2 receptor expression was scant in neonates and significantly increased in adult gastric tissue (P < 0.01). In conclusion, the lack of MCP effect on the newborn fundic muscle contraction potential relates to developmental differences in dopamine D2 receptor expression. To the extent that these novel data can be extrapolated to neonates, the therapeutic value of MCP as a prokinetic agent early in life requires further evaluation.
Asunto(s)
Antagonistas de Dopamina/farmacología , Metoclopramida/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Animales , Animales Recién Nacidos , Fenómenos Electrofisiológicos/efectos de los fármacos , Tránsito Gastrointestinal , Probucol/análogos & derivados , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , EstómagoRESUMEN
BACKGROUND: Vasopressin is a systemic vasoconstrictor. Its pulmonary vasodilatory effect is controversial, and limited data are available on its use in neonates with pulmonary hypertension. Hypothesizing that the vasopressin-induced pulmonary vasodilation is developmentally regulated, we evaluated its pulmonary and systemic arterial response in newborn and adult rats. METHODS: Vessels were mounted on a wire myograph, and the vasopressin-induced changes in vasomotor tone measured. The vessel- and age-dependent differences in vasopressin V1a and V2 receptors' expression were evaluated by western blotting. RESULTS: Vasopressin induced a dose-dependent increase in mesenteric arterial tone at both ages, but of greater magnitude in adult vessels (P < 0.01). At lower concentrations, vasopressin induced pulmonary vasodilation in adult vessels and vasoconstriction in newborn arteries. The adult vasopressin-induced pulmonary vasodilation was inhibited by ibuprofen, suggesting that the response is prostaglandin mediated. Pulmonary tissue V1a receptor protein expression was higher in adult, when compared with newborn arteries (P < 0.01). The adult vessels V1a expression predominated in the pulmonary arteries, and V2 was only detected in mesenteric arteries. CONCLUSION: The vasopressin-induced pulmonary vasodilation is absent in newborn rats likely due to the lower tissue V1a expression early in life. These animal data challenge the therapeutic use of vasopressin in neonatal pulmonary hypertension.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Vasodilatadores/química , Vasopresinas/química , Factores de Edad , Animales , Animales Recién Nacidos , Arterias/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hipertensión Pulmonar/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Classic Hodgkin's lymphoma (cHL) is a curable cancer with a disease-free survival rate of over 10 years. Over 80% of diagnosed patients respond favorably to first-line chemotherapy, but few biomarkers exist that can predict the 15-20% of patients who experience refractory or early relapsed disease. To date, the identification of patients who will not respond to first-line therapy based on disease staging and traditional clinical risk factor analysis is still not possible. Three-dimensional (3D) telomere analysis using the TeloView® software platform has been shown to be a reliable tool to quantify genomic instability and to inform on disease progression and patients' response to therapy in several cancers. It also demonstrated telomere dysfunction in cHL elucidating biological mechanisms related to disease progression. Here, we report 3D telomere analysis on a multicenter cohort of 156 cHL patients. We used the cohort data as a training data set and identified significant 3D telomere parameters suitable to predict individual patient outcomes at the point of diagnosis. Multivariate analysis using logistic regression procedures allowed for developing a predictive scoring model using four 3D telomere parameters as predictors, including the proportion of t-stumps (very short telomeres), which has been a prominent predictor for cHL patient outcome in a previously published study using TeloView® analysis. The percentage of t-stumps was by far the most prominent predictor to identify refractory/relapsing (RR) cHL prior to initiation of adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD) therapy. The model characteristics include an AUC of 0.83 in ROC analysis and a sensitivity and specificity of 0.82 and 0.78 respectively.
RESUMEN
Pyloric stenosis, the most common infant gastrointestinal disease, has no known etiology and clinically presents as abnormal gastric emptying with evidence of pyloric muscle hypertrophy. Whether abnormalities in gastric muscle contraction and/or relaxation have a role in this condition is poorly known, but gastroparesis is commonly observed in association with delayed gastric emptying in adults. Therefore, we evaluated the tetrahydrobiopterin (BH4)-deficient newborn mouse model of this disease (hph-1) and hypothesized that their gastric muscle properties are impaired, when compared with wild-type control animals. In vitro studies evaluating the age-dependent gastric fundus muscle contraction and relaxation potential were conducted. Compared with wild-type mice, the hph-1 stomach content/body weight ratio was significantly increased in newborn but not juvenile or adult animals, confirming abnormal gastric emptying. Gastric tissue neuronal nitric oxide synthase (nNOS) protein expression was upregulated in both newborn and adult hph-1 mice, but in the former there was evidence of enzyme uncoupling and higher tissue superoxide generation when compared with same age-matched animals. As opposed to the lack of strain differences in the U46619-induced force, the newborn hph-1 gastric muscle carbachol-induced contraction and nNOS-dependent relaxation were significantly reduced (P < 0.01). These group differences were not present in juvenile or adult mice. Preincubation with BH4 significantly enhanced the newborn hph-1, but not wild-type, gastric muscle contraction. In conclusion, changes compatible with gastroparesis are present in the newborn mouse model of pyloric stenosis. The role of BH4 deficiency and possibly associated gastroparesis in the pathogenesis of infantile pyloric stenosis warrants further investigation.
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Biopterinas/análogos & derivados , Gastroparesia/metabolismo , Animales , Animales Recién Nacidos , Biopterinas/deficiencia , Biopterinas/metabolismo , Óxidos N-Cíclicos/farmacología , Femenino , Mucosa Gástrica/metabolismo , Gastroparesia/etiología , Masculino , Ratones , Contracción Muscular/fisiología , Músculo Liso/fisiología , NG-Nitroarginina Metil Éster/farmacología , Marcadores de Spin , Estómago/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Cells in mechanically challenged environments cope with high-amplitude exogenous forces that can lead to cell death, but the mechanisms that mediate force-induced apoptosis and the identity of mechanoprotective cellular factors are not defined. We assessed apoptosis in NIH 3T3 and HEK (human embryonic kidney)-293 cells exposed to tensile forces applied through ß1-integrins. Apoptosis was mediated by Rac-dependent activation of p38α. Depletion of Pak1 (p21-activated kinase 1), a downstream effector of Rac, prevented force-induced p38 activation and apoptosis. Rac was recruited to sites of force transfer by filamin A, which inhibited force-induced apoptosis mediated by Rac and p38α. We conclude that, in response to tensile force, filamin A regulates Rac-dependent signals, which induce apoptosis through Pak1 and p38.
Asunto(s)
Apoptosis , Proteínas Contráctiles/metabolismo , Mecanotransducción Celular , Proteínas de Microfilamentos/metabolismo , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Caspasa 3/metabolismo , Células Cultivadas , Filaminas , Humanos , Immunoblotting , Riñón/citología , Riñón/metabolismo , Ratones , Células 3T3 NIH , ARN Interferente Pequeño/genética , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/genéticaRESUMEN
Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface beta1 integrin expression, and beta1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and beta1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-epsilon. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-epsilon, which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-epsilon, thereby enabling vimentin phosphorylation, which is important for beta1 integrin activation and cell spreading on collagen.
Asunto(s)
Adhesión Celular , Forma de la Célula , Proteínas Contráctiles/metabolismo , Fibroblastos/metabolismo , Proteínas de Microfilamentos/metabolismo , Vimentina/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Colágeno/metabolismo , Proteínas Contráctiles/genética , Fibroblastos/efectos de los fármacos , Filaminas , Humanos , Indoles/farmacología , Integrina beta1/metabolismo , Maleimidas/farmacología , Ratones , Proteínas de Microfilamentos/genética , Células 3T3 NIH , Fosforilación , Unión Proteica , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteína Quinasa C-epsilon/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Vimentina/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Cápsulas Bacterianas/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Factores de Virulencia/metabolismo , Animales , Adhesión Bacteriana , Cápsulas Bacterianas/ultraestructura , Línea Celular , Escherichia coli Enteropatógena/metabolismo , Escherichia coli Enteropatógena/ultraestructura , Células Epiteliales/microbiología , Eritrocitos/microbiología , Infecciones por Escherichia coli , Escherichia coli O157/metabolismo , Escherichia coli O157/ultraestructura , Proteínas de Escherichia coli/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Humanos , Intestino Grueso/microbiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Mutagénesis Insercional , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Conejos , Transactivadores/metabolismoRESUMEN
Tetrahydrobiopterin (BH4) is a cofactor of a number of regulatory enzymes. Although there are no known BH4 exogenous sources, the tissue content of this biopterin increases with age in GTP cyclohydrolase 1-deficient hyperphenylalaninemia-1 (hph-1) mice. Since certain bacteria are known to generate BH4, we hypothesize that generation of this biopterin by the intestinal microbiota contributes to its tissue increase in hph-1 adult mice. The goal of this study was to comparatively evaluate hph-1 mice and wild-type C57Bl/6 controls for the presence of intestinal BH4-producing bacteria. Newborn and adult mice fecal material was screened for 6-pyruvoyltetrahydropterin synthase (PTPS-2) an enzyme only present in BH4-generating bacteria. Adult, but not newborn, wild-type control and hph-1 mouse fecal material contained PTPS-2 mRNA indicative of the presence of BH4-generating bacteria. Utilizing chemostat-cultured human fecal bacteria, we identified the PTPS-2-producing bacteria as belonging to the Actinobacteria phylum. We further confirmed that at least two PTPS-2-producing species, Aldercreutzia equolifaciens and Microbacterium schleiferi, generate BH4 and are present in hph-1 fecal material. In conclusion, intestinal Actinobacteria generate BH4. This finding has important translational significance, since manipulation of the intestinal flora in individuals with congenital biopterin deficiency may allow for an increase in total body BH4 content.
Asunto(s)
Actinobacteria/fisiología , Biopterinas/análogos & derivados , Distonía/tratamiento farmacológico , Heces/microbiología , Microbioma Gastrointestinal , Errores Innatos del Metabolismo/tratamiento farmacológico , Complejo Represivo Polycomb 1/metabolismo , Trastornos Psicomotores/tratamiento farmacológico , Animales , Animales Recién Nacidos , Biopterinas/genética , Biopterinas/metabolismo , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Complejo Represivo Polycomb 1/genéticaRESUMEN
Cells in mechanically active environments are subjected to high-amplitude exogenous forces that can lead to cell death. Filamin A (FLNa) may protect cells from mechanically induced death by mechanisms that are not yet defined. We found that mechanical forces applied through integrins enhanced Rac-mediated lamellae formation in FLNa-null but not FLNa-expressing cells. Suppression of force-induced lamella formation was mediated by repeat 23 of FLNa, which also binds FilGAP, a recently discovered Rac GTPase-activating protein (GAP). We found that FilGAP is targeted to sites of force transfer by FLNa. This force-induced redistribution of FilGAP was essential for the suppression of Rac activity and lamellae formation in cells treated with tensile forces. Depletion of FilGAP by small interfering RNA, inhibition of FilGAP activity by dominant-negative mutation or deletion of its FLNa-binding domain, all resulted in a dramatic force-induced increase of the percentage of annexin-V-positive cells. FilGAP therefore plays a role in protecting cells against force-induced apoptosis, and this function is mediated by FLNa.
Asunto(s)
Proteínas Contráctiles/fisiología , Proteínas Activadoras de GTPasa/fisiología , Mecanotransducción Celular/fisiología , Proteínas de Microfilamentos/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Anexina A5/metabolismo , Apoptosis , Sitios de Unión , Línea Celular , Forma de la Célula , Proteínas Contráctiles/genética , Proteínas Contráctiles/metabolismo , Filaminas , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Transporte de Proteínas , Estrés Fisiológico , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Upon infection of host cells, enteropathogenic Escherichia coli (EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn10kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including cesD, escC, escJ, escV, espD, sepL, espB, and escF. Other mutants contained insertions in micC or upstream of bfpP, yehL, or ydeP. The insertion upstream of ydeP was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes, ydeP and ydeO expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both ydeP and ydeO, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both ydeP and ydeO expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.