RESUMEN
Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, causes self-limited diarrhea in normal hosts but can cause life-threatening diarrhea for immunosuppressed patients. There is an urgent need for new drugs to treat this chronic disease. Cryptosporidium parvum infection is associated with intestinal structural and pathophysiologic changes, including villi blunting and glucose malabsorption. Substance P (SP), a neuropeptide and pain transmitter, is associated with the gastrointestinal tract and is elevated in humans and macaques after experimental C. parvum challenge. To examine the relevance of SP in the pathogenesis of cryptosporidiosis, and to determine if SP receptor antagonism can be employed for treatment of cryptosporidiosis in immunosuppressed hosts, we used an immunosuppressed murine model (dexamethasone-immunosuppressed mice) that is frequently utilized for examining chemotherapeutic potential of drugs. Quantitative ELISA was used to measure intestinal SP levels in immunosuppressed mice with, and without, C. parvum infection. Intestinal physiological alterations, as studied by the Ussing chamber technique, plus weight change, fecal oocyst shedding, and villi measurements, were compared in infected mice with, and without, SP receptor antagonist (aprepitant) treatment. Immunosuppressed mice infected with C. parvum demonstrated increased SP levels as well as physiological alterations (glucose malabsorption), weight loss, fecal oocyst shedding, and structural alterations (increased intestinal villi blunting) compared to uninfected mice. Each of these defects was significantly inhibited by aprepitant treatment. These studies demonstrate the potential of SP receptor antagonism for treatment of pathogenesis of cryptosporidiosis in immunosuppressed hosts.
Asunto(s)
Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/efectos de los fármacos , Morfolinas/uso terapéutico , Antagonistas del Receptor de Neuroquinina-1 , Análisis de Varianza , Animales , Aprepitant , Bovinos , Criptosporidiosis/inmunología , Criptosporidiosis/patología , Cryptosporidium parvum/inmunología , Dexametasona/farmacología , Modelos Animales de Enfermedad , Heces/parasitología , Glucosa/metabolismo , Inmunosupresores/farmacología , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Ratones , Ratones Endogámicos C57BL , Microvellosidades/efectos de los fármacos , Microvellosidades/patología , Morfolinas/farmacología , Recuento de Huevos de Parásitos , Distribución Aleatoria , Sustancia P/metabolismo , Pérdida de Peso/efectos de los fármacosRESUMEN
Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Pérdida del Embrión/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Impresión Genómica/genética , Proteínas Quinasas , Factores de Transcripción , Alelos , Animales , Western Blotting , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Eliminación de Gen , Dosificación de Gen , Orden Génico/genética , Silenciador del Gen , Hibridación Fluorescente in Situ , Factores de Transcripción de Tipo Kruppel , Ratones , Poliploidía , Proteínas/genética , ARN Largo no Codificante , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , Receptor IGF Tipo 2/genética , Células Madre/metabolismoRESUMEN
Platelets are known to contribute to ischemia/reperfusion in several organs, but their role in inflammation and organ injury after hemorrhagic shock (HS) has not been examined. To address this issue, we rendered mice thrombocytopenic (20% of normal platelet count) by treatment with pOp3, a rat monoclonal antibody against platelet glycoprotein Ibalpha, 24 h before subjecting them to either a standard HS or sham protocol. Liver apoptosis increased 3- to 5-fold (P<0.05), and focal liver necrosis increased 11-fold (P<0.01) in placebo-treated shock mice compared with sham; these increased indices of liver injury were completely prevented by pOp3 pretreatment. Neutrophils infiltrating the liver increased nearly 3-fold in placebo-treated shock mice versus sham (P<0.05); this shock-induced increase in neutrophil infiltration was also eliminated by pretreatment with pOp3. Alveolar cross-sectional area, used to histologically assess interstitial lung edema and cellular infiltration, was reduced by 25% in pOp3-treated shock mice versus placebo-treated shock mice (P<0.05). Similar to the results in liver, pOp3 pretreatment decreased neutrophil infiltration in the lung after HS. Thus, platelets contribute to the inflammatory injuries of the liver and lung after HS, in part, perhaps by facilitating neutrophil infiltration into tissues.
Asunto(s)
Plaquetas/fisiología , Inflamación/fisiopatología , Resucitación , Choque Hemorrágico/fisiopatología , Animales , Hígado/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: Intrauterine exposure to hyperthermia at term is associated with adverse neonatal neurologic outcomes. The objective of this study was to determine whether prophylactic maternal corticosteroid treatment prevents fetal exposure to hyperthermia and inflammatory cytokines after epidural analgesia. STUDY DESIGN: A 2-phase, randomized, institutional review board-approved, double-blind, placebo-controlled trial was performed. Term nulliparous women were enrolled at epidural placement. Patients with a temperature of >99.4 degrees F or with diabetes mellitus were excluded. In phase 1, 25 mg methylprednisolone (low dose) or placebo was administered every 8 hours. In phase 2, the treatment dose was increased to 100 mg every 4 hours (high dose). Our primary outcome was a rate of intrapartum fever of >100.4 degrees F. Secondary outcomes were fetal interleukin-6 levels and the rate of neonatal bacteremia. RESULTS: One hundred one patients were assigned randomly to placebo; 50 patients were assigned to the low-dose group, and 49 patients were assigned to the high-dose group. Treatment with the high dose resulted in a 90% reduction in maternal fever, compared with placebo and low dose therapy (2.0% vs 21.8% vs. 34.0%, respectively; P < .001). Neonatal sepsis evaluations were reduced significantly in the high-dose group (4.1% vs 17.8% vs 24%, respectively; P = .01), but the rates of asymptomatic bacteremia were increased (9.3% vs 0% vs 2.1%, respectively; P = .005). Median cord blood interleukin-6 levels were reduced with the high-dose steroid treatment, but this result was statistically significant only between the high-dose and placebo groups (24.0 +/- 38.5 vs 32.0 +/- 95.0 pg/mL, respectively; P = .02). CONCLUSION: Prophylaxis with high-dose corticosteroids significantly reduces fetal exposure to hyperthermia and inflammation. However, maternal high-dose corticosteroids increase the rate of neonatal asymptomatic bacteremia. Stress-dose corticosteroid use in labor should trigger consideration of a screening neonatal blood culture.
Asunto(s)
Enfermedades Fetales/prevención & control , Fiebre/prevención & control , Inflamación/prevención & control , Metilprednisolona/uso terapéutico , Adulto , Analgesia Epidural/efectos adversos , Bacteriemia/etiología , Método Doble Ciego , Femenino , Fiebre/complicaciones , Humanos , Recién Nacido , Inflamación/complicaciones , Interleucina-6/sangre , EmbarazoRESUMEN
Chronic ethanol use preceding severe trauma and hemorrhagic shock (HS) is associated with an increased incidence of multiorgan failure (MOF) and death; however, the molecular basis for this increased susceptibility is unknown. We previously demonstrated that production of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), mediated by nuclear factor-kappa B (NF-kappa B), each make essential contributions to organ injury and inflammation in a rodent model of controlled HS, and we proposed in this study to examine the hypothesis that the increased susceptibility to MOF after shock/trauma in the setting of chronic ethanol use is due to an exaggerated activation of NF-kappa B and production of these proinflammatory cytokines. We observed increased HS-induced liver injury 4 h after resuscitation in rats fed the ethanol-containing Lieber-DeCarli liquid diet for 8 weeks compared with rats fed the control liquid diet (3-fold increase in serum alanine aminotransferase [ALT], P = 0.008, and 2-fold increase in focal liver necrosis, P = 0.005). The increased liver injury in the ethanol-fed HS rats was accompanied by a 70% increase in liver NF-kappa B activation (P < 0.05), a 3- to 5-fold increase in hepatocyte and Kupffer cell production of IL-6 and G-CSF (P < 0.05 for each), and a 2-fold increase in neutrophil infiltration (P < 0.005) compared with the control diet-fed HS rats. Thus, increased susceptibility to HS-induced liver injury in the setting of chronic ethanol use may be mediated, at least in part, by increased NF-kappa B activation resulting in increased local production of IL-6 and G-CSF and increased infiltration of neutrophils, which can damage liver cells directly and contribute to impaired sinusoidal blood flow.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/fisiología , Interleucina-6/fisiología , Hígado/patología , FN-kappa B/fisiología , Choque Hemorrágico/patología , Animales , Análisis de los Gases de la Sangre , Citocinas/metabolismo , Etanol/administración & dosificación , Etanol/toxicidad , Hepatitis Alcohólica/patología , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Insuficiencia Multiorgánica/etiología , Neumonía/metabolismo , Ratas , Ratas Sprague-Dawley , Resucitación , Choque Hemorrágico/complicaciones , Choque Hemorrágico/metabolismoRESUMEN
OBJECTIVES: To identify novel approaches to improve innate immunity in the lung following trauma complicated by hemorrhagic shock (T/HS) for prevention of nosocomial pneumonia. METHODS: We developed a rat model of T/HS followed by Pseudomonas aeruginosa (PA) pneumonia to assess the effect of alveolar epithelial cell (AEC) apoptosis, and its prevention by IL-6, on lung surfactant protein (SP)-D protein levels, lung bacterial burden, and survival from PA pneumonia, as well as to determine whether AEC apoptosis is a consequence of the unfolded protein response (UPR). Lung UPR transcriptome analysis was performed on rats subjected to sham, T/HS, and T/HS plus IL-6 protocols. Group comparisons were performed via Kaplan-Meier or ANOVA. RESULTS: T/HS decreased lung SP-D by 1.8-fold (p < 0.05), increased PA bacterial burden 9-fold (p < 0.05), and increased PA pneumonia mortality by 80% (p < 0.001). IL-6, when provided at resuscitation, normalized SP-D levels (p < 0.05), decreased PA bacterial burden by 4.8-fold (p < 0.05), and prevented all mortality from PA pneumonia (p < 0.001). The UPR transcriptome was significantly impacted by T/HS; IL-6 treatment normalized the T/HS-induced UPR transcriptome changes (p < 0.05). CONCLUSIONS: Impaired innate lung defense occurs following T/HS and is mediated, in part, by reduction in SP-D protein levels, which, along with AEC apoptosis, may be mediated by the UPR, and prevented by use of IL-6 as a resuscitation adjuvant.
Asunto(s)
Interleucina-6/farmacología , Neumonía/prevención & control , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Choque Hemorrágico/fisiopatología , Análisis de Varianza , Animales , Apoptosis/fisiología , Modelos Animales de Enfermedad , Interleucina-6/administración & dosificación , Ratas , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Trauma complicated by hemorrhagic shock (T/HS) is the leading cause of morbidity and mortality in the United States for individuals under the age of 44 years. Initial survivors are susceptible to developing multiple organ failure (MOF), which is thought to be caused, at least in part, by excessive or maladaptive activation of inflammatory pathways. We previously demonstrated in rodents that T/HS results in liver injury that can be prevented by IL-6 administration at the start of resuscitation; however, the contribution of the severity of HS to the extent of liver injury, whether or not resuscitation is required, and the mechanism(s) for the IL-6 protective effect have not been reported. In the experiments described here, we demonstrated that the extent of liver inflammation induced by T/HS depends on the duration of hypotension and requires resuscitation. We established that IL-6 administration at the start of resuscitation is capable of completely reversing liver inflammation and is associated with increased Stat3 activation. Global assessment of the livers showed that the main effect of IL-6 was to normalize the T/HS-induced inflammation transcriptome. Pharmacological inhibition of Stat3 activity within the liver blocked the ability of IL-6 to prevent liver inflammation and to normalize the T/HS-induced liver inflammation transcriptome. Genetic deletion of a Stat3ß, a naturally occurring, dominant-negative isoform of the Stat3, attenuated T/HS-induced liver inflammation, confirming a role for Stat3, especially Stat3α, in preventing T/HS-mediated liver inflammation. Thus, T/HS-induced liver inflammation depends on the duration of hypotension and requires resuscitation; IL-6 administration at the start of resuscitation reverses T/HS-induced liver inflammation, through activation of Stat3α, which normalized the T/HS-induced liver inflammation transcriptome.
Asunto(s)
Inflamación/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Choque Hemorrágico/complicaciones , Choque Hemorrágico/metabolismo , Animales , Immunoblotting , Inflamación/genética , Masculino , Ratones , Análisis por Micromatrices , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/genética , Choque Hemorrágico/genéticaRESUMEN
Acute lung injury (ALI) occurs in up to 37% of patients following trauma/hemorrhagic shock (T/HS) and, in other settings, is due to alveolar epithelial cell (AEC) apoptosis. To determine if AEC apoptosis is a key contributor to ALI following T/HS and whether or not signal transducer and activator of translation (Stat)3 activation can prevent it, rats were pretreated with a Stat3 inhibitor or placebo and subjected to T/HS or sham protocol and resuscitated without or with interleukin (IL)-6. T/HS induced apoptosis in up to 15% of lung cells, 82% of which were AEC. Apoptosis increased with increasing duration of shock and required resuscitation. IL-6 treatment stimulated lung Stat3 activation and prevented AEC apoptosis. Pretreatment of rats with a Stat3 inhibitor blocked the antiapoptotic effect of IL-6. Mice deficient in Stat3 beta, a naturally occurring dominant negative isoform of Stat3, were resistant to T/HS-induced lung apoptosis. T/HS altered the expression of 87% of apoptosis-related genes. IL-6 treatment normalized expression of 75% of the genes altered by T/HS; Stat3 inhibition prevented normalization of 65% of the gene whose expression was normalized by IL-6. Thus, T/HS-induced AEC apoptosis, which depended on the duration of hypotension, required resuscitation and was prevented by IL-6-mediated activation of Stat3, which acted to normalize the apoptosis transcriptome.
Asunto(s)
Apoptosis/efectos de los fármacos , Interleucina-6/farmacología , Pulmón/efectos de los fármacos , Pulmón/patología , Factor de Transcripción STAT3/genética , Choque Hemorrágico/complicaciones , Heridas y Lesiones/complicaciones , Animales , Apoptosis/genética , G-Cuádruplex , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/administración & dosificación , Ratones , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/patología , Ratas , Ratas Sprague-Dawley , Resucitación , Factor de Transcripción STAT3/metabolismoRESUMEN
Trauma is a major cause of mortality in the United States. Death among those surviving the initial insult is caused by multiple organ failure (MOF) with the liver among the organs most frequently affected. We previously demonstrated in rodents that trauma complicated by hemorrhagic shock (trauma/HS) results in liver injury that can be prevented by IL-6 administration at the start of resuscitation; however, the contribution of the severity of HS to the extent of liver injury, whether or not resuscitation is required and the mechanism for the IL-6 protective effect have not been reported. In the experiments reported here, we demonstrated that the extent of liver apoptosis induced by trauma/HS depends on the duration of hypotension and requires resuscitation. We established that IL-6 administration at the start of resuscitation is capable of completely reversing liver apoptosis and is associated with increased Stat3 activation. Microarray analysis of the livers showed that the main effect of IL-6 was to normalize the trauma/HS-induced apoptosis transcriptome. Pharmacological inhibition of Stat3 activity within the liver blocked the ability of IL-6 to prevent liver apoptosis and to normalize the trauma/HS- induced liver apoptosis transcriptome. Genetic deletion of a Stat3beta, a naturally occurring, dominant-negative isoform of the Stat3, attenuated trauma/HS-induced liver apoptosis, confirming a role for Stat3, especially Stat3alpha, in preventing trauma/HS-mediated liver apoptosis. Thus, trauma/HS-induced liver apoptosis depends on the duration of hypotension and requires resuscitation. IL-6 administration at the start of resuscitation reverses HS-induced liver apoptosis, through activation of Stat3alpha, which normalizes the trauma/HS-induced liver apoptosis transcriptome.
RESUMEN
Half of trauma deaths are attributable to hypovolemic circulatory collapse (HCC). We established a model of HCC in rats involving minor trauma plus severe hemorrhagic shock (HS). HCC in this model was accompanied by a 50% reduction in peak acceleration of aortic blood flow and cardiomyocyte apoptosis. HCC and apoptosis increased with increasing duration of hypotension. Apoptosis required resuscitation, which provided an opportunity to intervene therapeutically. Administration of IL-6 completely reversed HCC, prevented cardiac dysfunction and cardiomyocyte apoptosis, reduced mortality 5-fold and activated intracardiac signal transducer and activator of transcription (STAT) 3. Pre-treatment of rats with a selective inhibitor of Stat3, T40214, reduced the IL-6-mediated increase in cardiac Stat3 activity, blocked successful resuscitation by IL-6 and reversed IL-6-mediated protection from cardiac apoptosis. The hearts of mice deficient in the naturally occurring dominant negative isoform of Stat3, Stat3beta, were completely resistant to HS-induced apoptosis. Microarray analysis of hearts focusing on apoptosis related genes revealed that expression of 29% of apoptosis related genes was altered in HS vs. sham rats. IL-6 treatment normalized the expression of these genes, while T40214 pretreatment prevented IL-6-mediated normalization. Thus, cardiac dysfunction, cardiomyocyte apoptosis and induction of apoptosis pathway genes are important components of HCC; IL-6 administration prevented HCC by blocking cardiomyocyte apoptosis and induction of apoptosis pathway genes via Stat3 and warrants further study as a resuscitation adjuvant for prevention of HCC and death in trauma patients.
Asunto(s)
Interleucina-6/uso terapéutico , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Choque/prevención & control , Animales , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Hipovolemia , Miocitos Cardíacos/patología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, causes self-limited diarrhea in immunocompetent hosts and severe life-threatening diarrhea in AIDS patients. Highly active antiretroviral therapy has been used to effectively treat cryptosporiosis in some but not all AIDS patients. Therefore, there is an urgent need for innovative drugs to treat this disease. Cryptosporidium infection results in intestinal pathophysiological changes such as glucose malabsorption, increased chloride ion (Cl(-)) secretion, and epithelial barrier disruption, leading to disease pathogenesis. In order to develop tools to combat this opportunistic pathogen, it is vital to understand mediators involved in disease pathogenesis. Substance P (SP), a neuropeptide and pain transmitter, is located in the gastrointestinal tract. SP can cause Cl(-) secretion in human gastrointestinal explants. However, its role in cryptosporidiosis has not been fully studied. Jejunal samples from macaques before and after Cryptosporidium parvum infection were assayed for SP and SP receptor mRNA and protein levels by reverse transcription-PCR and by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The role of SP in pathophysiological alterations, such as Cl(-) secretion and glucose malabsorption, was studied using tissues derived from macaques infected with C. parvum by the Ussing chamber technique. SP and SP receptor mRNA and protein expression levels were increased in jejunal samples following C. parvum infection and were accompanied by increased basal ion secretion and glucose malabsorption. In vitro treatment of samples obtained from infected macaques with the SP receptor antagonist aprepitant (Emend; Merck, Whitehouse Station, NJ) completely reversed the increase in basal ion secretion and corrected the glucose malabsorption. Our findings raise the possibility of using SP receptor antagonists for the treatment of symptoms associated with cryptosporidiosis.
Asunto(s)
Cloruros/metabolismo , Criptosporidiosis/metabolismo , Glucosa/metabolismo , Síndromes de Malabsorción/metabolismo , Sustancia P/fisiología , Animales , Enfermedades del Yeyuno/metabolismo , Enfermedades del Yeyuno/parasitología , Macaca , Enfermedades de los Monos/metabolismo , Enfermedades de los Monos/parasitología , Receptores de Neuroquinina-1/fisiologíaRESUMEN
BACKGROUND: Resuscitation from hemorrhagic shock (HS) predisposes to subsequent infections. Susceptibility to infection following sepsis has been attributed to apoptosis. Interleukin (IL)-6 has been shown to have antiapoptotic properties and to decrease postresuscitation inflammation in rodent and porcine models of HS. OBJECTIVE: The objective was to determine if HS increases host susceptibility to infection, if IL-6 administration at resuscitation reduces this susceptibility, and if changes in susceptibility to infection are accompanied by parallel changes in apoptosis. SUBJECTS AND INTERVENTIONS: Mice were randomized into three groups-HS, sham, and no-surgery control-and each group was further randomized to receive either IL-6 (3 microg/kg; HS/IL-6) or placebo (HS/P) at the start of resuscitation. In the HS-infection protocol, each mouse was challenged intraperitoneally the next day with a sublethal dose of Staphylococcus aureus (4x107 colony-forming units); 24 hrs later, the peritoneal cavity was lavaged and the major organs were harvested for culture. In the HS-apoptosis protocol, the livers were harvested the next day and analyzed by means of the terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling (TUNEL) assay. RESULTS: HS/P mice had a six- to eight-fold increase in total bacterial counts in comparison with sham and control mice that was attributable to a seven- to nine-fold increase in liver burden. IL-6 treatment reduced total and liver bacterial counts in HS/IL-6 mice by 62% and 69%, respectively, to levels statistically indistinguishable from IL-6-treated sham and control mice. The number of TUNEL-positive liver cells in the HS/P group was increased eight-fold vs. that in the sham group (p=.002); IL-6 resuscitation completely reversed the HS-induced increase in TUNEL-positive cells in the HS/IL-6 group (p=.002). CONCLUSIONS: IL-6 treatment at resuscitation eliminated the HS-mediated increase in total and liver bacterial burden and protected the liver from HS-induced apoptosis. Reduced liver apoptosis may explain the ability of IL-6 to blunt the HS-induced increase in susceptibility to bacterial challenge.
Asunto(s)
Apoptosis/efectos de los fármacos , Infección Hospitalaria/prevención & control , Interleucina-6/uso terapéutico , Sepsis/prevención & control , Choque Hemorrágico/tratamiento farmacológico , Animales , Recuento de Colonia Microbiana , Infección Hospitalaria/fisiopatología , Susceptibilidad a Enfermedades , Inflamación/prevención & control , Interleucina-6/farmacología , Hígado/citología , Hígado/metabolismo , Hígado/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Proteínas Recombinantes , Resucitación , Sepsis/fisiopatologíaRESUMEN
In mammalian heterochromatin, cytosine bases of CpG dinucleotides are symmetrically modified by methylation. Patterns of CpG methylation are maintained by the action of Dnmt1, the mammalian maintenance cytosine methyltransferase enzyme. We genetically manipulated the levels of CpG methylation and found that extensive chromatin alterations occur in pericentric heterochromatin. Homozygous mutations in Dnmt1 cause severe hypomethylation of pericentric heterochromatin and concomitant chromatin reorganization involving the histone variant macroH2A. Demethylation-induced alterations in macroH2A localization occur in both interphase and mitotic embryonic stem (ES) cells. Heterochromatin protein 1 (HP1) marks interphase pericentric heterochromatin (chromocenters). MacroH2A immunostaining in Dnmt1(-/-) cells becomes coincident with chromocenters detected by HP1 content. MacroH2A, but not HP1, is enriched in nuclease-resistant chromatin fractions extracted from Dnmt1(-/-) cells. Normal localization of macroH2A was restored upon reintroduction of a Dnmt1 transgene into Dnmt1(-/-) cells. MacroH2A localization was also affected in T-antigen-transformed fibroblasts subjected to the conditional mutation of Dnmt1. Together, these results suggest that pericentric heterochromatin can be maintained in the absence of CpG methylation, but in a significantly altered configuration.
Asunto(s)
Islas de CpG/fisiología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Heterocromatina/metabolismo , Histonas/metabolismo , Animales , Línea Celular , Centrómero/genética , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Eliminación de Gen , Silenciador del Gen/fisiología , Heterocromatina/genética , Interfase/fisiología , Masculino , Ratones , Mutación/fisiología , Células Madre Pluripotentes/metabolismo , Transfección , Transgenes/genéticaRESUMEN
The patterns of transcription and translation of the ribosomal protein L32 (Rpl32) mRNA differ greatly in adult testis and somatic tissues. Northern blots reveal that the levels of Rpl32 mRNA are four- to five-fold higher in prepubertal and adult testes, and purified pachytene spermatocytes and round spermatids than in a variety of nongrowing adult somatic tissues. 5' RACE demonstrates that transcription in 8-day prepubertal testis, which lacks meiotic and haploid cells, strongly prefers the same start site in the 5' terminal oligopyrimidine tract (5' TOP) that is used is somatic cells. The 5' TOP is a cis element that inhibits translation of many mRNAs in nongrowing somatic cells. Although the sizes of deadenylated Rpl32 mRNAs are indistinguishable in somatic and spermatogenic cells, transcription initiates at 11 sites over a 31-nt segment in adult testis and approximately 62% of Rpl32 mRNAs lack a 5' TOP. In agreement with previous studies, low levels of cycloheximide increase the proportions and sizes of polysomes in absorbance profiles, and increase the proportions and sizes of polysomes translating four 5' TOP mRNA species including the Rpl32 mRNA in 8-day seminiferous tubules. In contrast, cycloheximide has little or no effect on the absorbance profiles and distribution of Rpl32 mRNA and 5' TOP mRNAs in adult seminiferous tubules. The failure of cycloheximide to increase the size of polysomes in adult seminiferous tubules implies a block in the pathway by which ribosomes are recruited onto translationally active mRNAs.
Asunto(s)
ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Espermatocitos/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Codón de Terminación/genética , Cicloheximida/farmacología , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Polirribosomas/efectos de los fármacos , Polirribosomas/genética , Polirribosomas/metabolismo , Biosíntesis de Proteínas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Secuencia de Oligopirimidina en la Región 5' Terminal del ARN/genética , ARN Mensajero/genética , Espermátides/citología , Espermátides/metabolismo , Espermatocitos/citología , Testículo/citología , Transcripción Genética/genéticaRESUMEN
INTRODUCTION: Hypotension causes diffuse liver injury accompanied by increased local production of interleukin-6 (IL-6) in swine models of uncontrolled hemorrhagic shock (HS). IL-6 is transcriptionally up-regulated by nuclear factor (NF)-kappaB and results in activation of signal transducer and activator of transcription-3 (Stat3) in a murine model of controlled HS. Our objectives were: 1). to determine if increased IL-6 production and NF-kappaB and Stat3 activation occurs in a swine model of uncontrolled HS, and 2). to assess whether or not levels of IL-6 mRNA and activity of NF-kappaB and Stat3 correlate with shock severity. MATERIALS AND METHODS: Swine were assigned to four groups: 1). control animals (n = 6): no intervention, 2). sham operation (n = 6): celiotomy and splenectomy, 3). uncontrolled hemorrhagic shock (UHS) (n = 6): sham plus grade V vascular liver injury and resuscitation, 4). profound uncontrolled hemorrhagic shock (PUHS) (n = 8): UHS after dilutional hypothermia. Following euthanasia at 2 h, livers were harvested, total RNA isolated, and IL-6 mRNA levels quantified by Q-RT-PCR (ABI Prism 7700, Applied Biosystems International, Foster City, CA). Protein was extracted for measurement of NF-kappaB and Stat3 activity by electrophoretic mobility shift assay (EMSA). RESULTS: Compared to shams, IL-6 mRNA levels increased 4.5-fold in UHS and 90-fold in PUHS (P < 0.001). Compared with shams; NF-kappaB activity increased 2-fold in both UHS and PUHS (P < 0.05). Stat3 activity was equivalent (not significant) in UHS when compared with shams but increased 5.3-fold in PUHS. (P < 0.05). CONCLUSION: These findings suggest that regional proinflammatory cytokine production results from and perpetuates a proinflammatory transcription factor cascade in a swine model of uncontrolled hemorrhagic shock and indicate that this process is proportional to the severity of shock.
Asunto(s)
Choque Hemorrágico/inmunología , Factores de Transcripción/inmunología , Animales , Proteínas de Unión al ADN/metabolismo , Interleucina-6/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT3 , Índice de Severidad de la Enfermedad , Porcinos , Transactivadores/metabolismoRESUMEN
Stat3 is an Src homology (SH)2-containing protein constitutively activated in a wide variety of human cancers following its recruitment to YXXQ-containing motifs, which results in resistance to apoptosis. Despite resolution of the crystal structure of Stat3 homodimer bound to DNA, the structural basis for the unique specificity of Stat3 SH2 for YXXQ-containing phosphopeptides remains unresolved. We tested three models of this interaction based on computational analysis of available structures and sequence alignments, two of which assumed an extended peptide configuration and one in which the peptide had a beta-turn. By using peptide immunoblot affinity assays and mirror resonance affinity analysis, we demonstrated that only phosphotyrosine (Tyr(P)) peptides containing +3 Gln (not Leu, Met, Glu, or Arg) bound to wild type Stat3. Examination of a series of wild type and mutant Stat3 proteins demonstrated loss of binding to pYXXQ-containing peptides only in Stat3 mutated at Lys-591 or Arg-609, whose side chains interact with the Tyr(P) residue, and Stat3 mutated at Glu-638, whose amide hydrogen bonds with oxygen within the +3 Gln side chain when the peptide ligand assumes a beta-turn. These findings support a model for Stat3 SH2 interactions that could form the basis for anticancer drugs that specifically target Stat3.