RESUMEN

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

Asunto(s)

Pollos , Brotes de Enfermedades , Subtipo H7N7 del Virus de la Influenza A/genética , Gripe Aviar/epidemiología , Gripe Aviar/virología , Mutación , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/virología , Animales , Genoma Viral , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H7N7 del Virus de la Influenza A/patogenicidad , Gripe Aviar/diagnóstico , Gripe Aviar/mortalidad , Filogenia , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/mortalidad , Reino Unido/epidemiología , Virulencia , Secuenciación Completa del Genoma

RESUMEN

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sows and respiratory problems in growing pigs. The disease is present in most countries throughout the world but was not diagnosed in Sweden until the summer of 2007 when it was first detected through the national PRRS surveillance program. The immediate mobilization of veterinary authorities, field veterinarians and the pig industry was a prerequisite for preventing the spread of the disease. Within 10 days seven herds were verified as infected and the measures taken included stamping out, cleaning, disinfection and a vacancy period of 3 weeks before the herds were repopulated. To evaluate the effectiveness of these measures, a national sero-surveillance was carried out during the autumn of 2007. Approximately 90% of the pig production was covered by this screening and all samples tested were negative with regard to antibodies to PRRS virus.

Asunto(s)

Anticuerpos Antivirales/sangre , Brotes de Enfermedades/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/epidemiología , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Aborto Veterinario/virología , Animales , Brotes de Enfermedades/prevención & control , Femenino , Masculino , Síndrome Respiratorio y de la Reproducción Porcina/transmisión , Embarazo , Factores de Riesgo , Estudios Seroepidemiológicos , Suecia/epidemiología , Porcinos

RESUMEN

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Asunto(s)

Unión Europea , Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/diagnóstico , Gripe Aviar/virología , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Aves , Embrión de Pollo , Virus de la Influenza A/genética , Laboratorios , Sensibilidad y Especificidad

RESUMEN

Field canine coronaviruses (CCVs) identified during a series of outbreaks of gastroenteritis in Swedish dogs were subjected to genetic analysis involving the open reading frame 1b (ORF1b) and the membrane (M) and spike (S) protein genes. Four field viruses originating from the Stockholm region presented identical sequences and segregated separately from other CCVs characterized so far and from GOT/05, the variant recovered in Western Sweden. A recombinant origin of the fifth virus identified in the Stockholm region is suggested. In addition, the five viruses originating from the same geographical area displayed atypical 5' S gene sequences.

Asunto(s)

Coronavirus Canino/clasificación , Coronavirus Canino/genética , Brotes de Enfermedades , Enfermedades de los Perros/epidemiología , Gastroenteritis/veterinaria , Variación Genética , Secuencia de Aminoácidos , Animales , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Proteínas M de Coronavirus , Coronavirus Canino/aislamiento & purificación , Enfermedades de los Perros/virología , Perros , Gastroenteritis/epidemiología , Gastroenteritis/virología , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Análisis de Secuencia de ADN , Glicoproteína de la Espiga del Coronavirus , Suecia/epidemiología , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética

RESUMEN

Coronaviruses are etiologic agents of respiratory and enteric diseases in humans and in animals. In this study, a one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on SYBR Green chemistry and degenerate primers was developed for the generic detection of coronaviruses. The primers, designed in the open reading frame 1b, enabled the detection of 32 animal coronaviruses including strains of canine coronavirus, feline coronavirus, transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). A specific amplification was also observed with the human coronaviruses (HCoV) HCoV-NL63, HCoV-OC43, HCoV-229E and severe acute respiratory syndrome coronavirus (SARS-CoV). The real-time RT-PCR detected down to 10 cRNA copies from TGEV, BCoV, SARS-CoV and IBV. In addition, the assay exhibited a high sensitivity and specificity on clinical samples from different animal species. The developed assay represents a potential tool for laboratory diagnostics and for detecting still uncharacterized coronaviruses.

Asunto(s)

Coronaviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Secuencia de Bases , Benzotiazoles , Coronaviridae/clasificación , Coronaviridae/aislamiento & purificación , Cartilla de ADN/genética , ADN Viral/genética , Diaminas , Colorantes Fluorescentes , Humanos , Datos de Secuencia Molecular , Compuestos Orgánicos , Filogenia , Quinolinas , ARN Complementario/genética , ARN Viral/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y Especificidad

RESUMEN

A real-time PCR assay based on primer-probe energy transfer (PriProET) was developed to detect swine vesicular disease virus (SVDV). Specificity tests of SVDV and heterologous virus showed specific amplification of SVDV strains only. The amplification plot for the closely related Coxsackievirus B5 remained negative. The sensitivity of assay was five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirmed specific hybridisation of the SVDV strains. Eight of twenty SVDV strains tested, revealed shifted melting points that indicated mutations in the probe region. All predicted mutations were confirmed by nucleotide sequencing. With the PriProET system there is a chance to identify phylogenetically divergent strains of SVDV, which may appear negative in other probe-based real-time PCR assays. At the same time, any difference in melting points may provide an indication of divergence in the probe region. The high sensitivity, specificity, and tolerance toward mutations in the probe region of the SVDV PriProET assay may improve the early and rapid detection of a wide range of SVDV strains, allowing reduced turnaround time and the use of high-throughput, automated technology.

Asunto(s)

Enterovirus Humano B/genética , Reacción en Cadena de la Polimerasa/métodos , Enfermedad Vesicular Porcina/virología , Animales , Disparidad de Par Base , Secuencia de Bases , Cartilla de ADN , Transferencia de Energía , Enterovirus Humano B/clasificación , Enterovirus Humano B/aislamiento & purificación , Enterovirus Humano B/patogenicidad , Geografía , Datos de Secuencia Molecular , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Porcinos , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/virología , Enfermedad Vesicular Porcina/diagnóstico

RESUMEN

AIM: We have previously shown that mental and hypoxic stress can trigger the development of myocardial infarction (MI) in atherosclerotic apoE(-/-) x LDLR(-/-) mice. The purpose of the present study was to characterize the interval between stress and MI and determine whether electrophysiological changes precede the precipitation of an infarct by assessing telemetry recordings of the electrocardiogram. METHODS: Isoflurane anaesthetized apoE(-/-) x LDLR(-/-) (n = 16) and C57BL/6J (n = 8) mice were exposed to systemic hypoxia by reducing the inhaled oxygen concentration to 10% for 10 min. Mental stress was induced in eight conscious apoE(-/-) x LDLR(-/-) and eight C57BL/6J mice by blowing air into the cage. Physiological parameters were recorded every 30 min for 2-6 days by implanted transmitters. RESULTS: During stress all mice developed transient ischaemic STU-area changes, which returned to normal at the end of stress. During the recovery phase (6 days) 50% (4/8) of the mentally stressed apoE(-/-) x LDLR(-/-) mice developed increased STU-area variability (P < 0.05) followed by dramatic STU-area elevations and spontaneous death at approximately 12-24 h. In hypoxia-exposed apoE(-/-) x LDLR(-/-) mice 56% (9/16) developed MI as determined by elevated serum levels of the infarction marker troponin T which correlated with increased variability in the STU-area (P < 0.05). CONCLUSION: This is the first mouse model showing that increased STU-area variability is indicative of MI development in atherosclerotic mice following ischaemic stress. Furthermore, our findings suggest a two-phase pathway for the infarction development: an initial phase comprising a transient ischaemic response which triggers a delayed second phase of ischaemia and MI.

Asunto(s)

Arteriosclerosis/fisiopatología , Infarto del Miocardio/fisiopatología , Estrés Psicológico/fisiopatología , Animales , Arteriosclerosis/complicaciones , Arteriosclerosis/patología , Presión Sanguínea/fisiología , Vasos Coronarios/patología , Electrocardiografía/métodos , Frecuencia Cardíaca/fisiología , Hipoxia/complicaciones , Hipoxia/patología , Hipoxia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Estrés Psicológico/complicaciones , Estrés Psicológico/patología

RESUMEN

Nineteen pregnant cows were experimentally infected with bovine viral diarrhoea virus (BVDV) between day 74 and 81 of pregnancy. All cows became infected and developed serum antibodies. Sixteen of the cows delivered persistently infected (PI) offspring, whereas the remaining three gave birth to calves with detectable serum antibodies and free from BVDV. The 16 cows with PI foetuses developed higher levels of antibodies in serum during pregnancy than did their three peers carrying non-PI calves. Multivariate analysis showed that the antibody levels in these two groups of cows were significantly different from day 135 of pregnancy. Foetal fluid was successfully collected from 18 of the 19 infected cows and from five uninfected control cows between 10 and 24 days before delivery by use of a percutaneous, blind puncture technique. No negative effects were observed in the cows or their offspring. BVDV was isolated and detected with an immunoperoxidase test in foetal fluid from 13 of the 16 cows carrying PI foetuses, and from 15 of the cows when a quantitative fluorescent polymerase chain reaction (PCR) technique was used. The negative sample in the PCR assay was positive for BVDV antibodies. The number of viral copies per microlitre in foetal fluids varied between 103 and 1080 in the positive samples. All samples taken from the cows carrying non-PI foetuses were negative for BVDV in both assays. In this experiment, examination of either serum or foetal fluids could identify the cows carrying a PI foetus. Examination of serum for BVDV antibodies was a reliable indicator of a PI foetus if the serum was collected during the last 2 months of pregnancy. For examination of foetal fluids, both viral and serological analyses should be performed. For viral analysis, PCR should be the test of choice. High levels of BVDV antibodies in conjunction with a negative result in the PCR may be indicative of a false-negative virus result. Further experience with the method of collection of foetal fluids is necessary for evaluation of its safety. Investigation of pregnant cows in order to discover a PI offspring before it is born could be a useful tool in control and eradication of BVDV.

Asunto(s)

Diarrea Mucosa Bovina Viral/diagnóstico , Diarrea Mucosa Bovina Viral/transmisión , ADN Viral/sangre , Virus de la Diarrea Viral Bovina/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Transmisión Vertical de Enfermedad Infecciosa/veterinaria , Complicaciones Infecciosas del Embarazo/veterinaria , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Bovinos , ADN Viral/análisis , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina/aislamiento & purificación , Virus de la Diarrea Viral Bovina/patogenicidad , Femenino , Feto/virología , Reacción en Cadena de la Polimerasa/veterinaria , Valor Predictivo de las Pruebas , Embarazo , Complicaciones Infecciosas del Embarazo/diagnóstico

RESUMEN

During the development of a Langendorff preparation of isolated mouse hearts, hitherto undescribed cyclic fluctuations in left ventricular pressure and coronary flow were independently observed in three laboratories. Isolated mouse hearts were perfused with crystalloid glucose-containing Krebs-Hensleit buffer in a constant pressure model, and left ventricular pressures were measured via an intraventricular balloon catheter. After acquiring technical skill in preparing the mouse hearts, the perfusionists observed that fluctuations in cardiac performance with a cycle period lasting 5-10 min occurred shortly after initiation of perfusion. Each fluctuation cycle consisted of a phase of increase and a phase of decrease. Synchronized with the fluctuations in left ventricular pressure, increases and decreases in dP/dt max took place. Analogous fluctuations in coronary flow occurred, with onset 1-2 min later than changes in left ventricular systolic pressure. In some preparations a gradual ST-segment elevation was seen on the electrocardiogram during the systolic pressure increase phase. The amplitude of the fluctuations could be augmented by increasing the perfusion pressure, and reduced, but not abolished, by lowering the pressure. Changes in buffer calcium, magnesium, or sodium concentration did not alter the fluctuations, nor did any change of anaesthetics, mouse strain, or left ventricular drainage. Altering the perfusion mode from constant pressure to constant flow did not prevent the occurrence of the cyclic fluctuations. The hearts became stable and the fluctuations disappeared when the buffer was supplemented with 2 mm pyruvate. In the present study, pyruvate given throughout stabilization and reperfusion also markedly attenuated the ischaemic insult, as evidenced by the delayed ischaemic contracture and a reduced magnitude of ischaemic contracture. A cardioprotective effect was only visible at early reperfusion, did not affect the final functional recovery. In conclusion, a phenomenon of cyclic fluctuations in left ventricular pressure followed by fluctuations in coronary flow was observed in isolated mouse hearts. These could be abolished by adding 2 mm pyruvate to the perfusion buffer. Pyruvate in the buffer also markedly attenuated the post-ischaemic deterioration of cardiac performance seen in this mouse model.

Asunto(s)

Contracción Miocárdica/fisiología , Ácido Pirúvico/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Calcio/análisis , Electrocardiografía/métodos , Femenino , Magnesio/análisis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Contracción Miocárdica/efectos de los fármacos , Daño por Reperfusión Miocárdica/fisiopatología , Pentobarbital/farmacología , Perfusión , Sodio/análisis , Función Ventricular

RESUMEN

Thyroid hormone governs a diverse repertoire of physiological functions through receptors encoded in the receptor genes alpha and beta, which each generate variant proteins. In mammals, the alpha gene generates, in addition to the normal receptor TRalpha1, a non-hormone-binding variant TRalpha2 whose exact function is unclear. Here, we present the phenotype associated with the targeted ablation of TRalpha2 expression. Selective ablation of TRalpha2 resulted in an inevitable, concomitant overexpression of TRalpha1. Both TRalpha2 +/- and -/- mice show a complex phenotype with low levels of free T3 and free T4, and have inappropriately normal levels of TSH. The thyroid glands exhibit mild morphological signs of dysfunction and respond poorly to TSH, suggesting that the genetic changes affect the ability of the gland to release thyroid hormones. However, the phenotype of the mutant mice also has features of hyperthyroidism, including decreased body weight, elevated heart rate, and a raised body temperature. Furthermore, TRalpha2-/- and TRalpha2+/- mice are obese and exhibit skeletal alterations, associated with a late-onset growth retardation. The results thus suggest that the overexpression of TRalpha1 and the concomitant decrease in TRalpha2 expression lead to a mixed hyper- and hypothyroid phenotype, dependent on the tissue studied. The phenotypes suggest that the balance of TRalpha1:TRalpha2 expressed from the TRalpha gene provides an additional level of tuning the control of growth and homeostasis in mammalian species.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Hipertiroidismo/genética , Hipotiroidismo/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Hormona Tiroidea , Animales , Northern Blotting , Composición Corporal , Densidad Ósea , Cruzamientos Genéticos , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Femenino , Histocitoquímica , Hipertiroidismo/metabolismo , Hipotiroidismo/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fenotipo , ARN/química , ARN/aislamiento & purificación , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telemetría , Tiroxina/sangre , Triyodotironina/sangre

RESUMEN

Evolutionary conflicts among social hymenopteran nestmates are theoretically likely to arise over the production of males and the sex ratio. Analysis of these conflicts has become an important focus of research into the role of kin selection in shaping social traits of hymenopteran colonies. We employ microsatellite analysis of nestmates of one social hymenopteran, the primitively eusocial and monogynous bumblebee Bombus hypnorum, to evaluate these conflicts. In our 14 study colonies, B. hypnorum queens mated between one and six times (arithmetic mean 2.5). One male generally predominated, fathering most of the offspring, thus the effective number of matings was substantially lower (1-3.13; harmonic mean 1.26). In addition, microsatellite analysis allowed the detection of alien workers, those who could not have been the offspring of the queen, in approximately half the colonies. Alien workers within the same colony were probably sisters. Polyandry and alien workers resulted in high variation among colonies in their sociogenetic organization. Genetic data were consistent with the view that all males (n = 233 examined) were produced by a colony's queen. Male parentage was therefore independent of the sociogenetic organization of the colony, suggesting that the queen, and not the workers, was in control of the laying of male-destined eggs. The population-wide sex ratio (fresh weight investment ratio) was weakly female biased. No evidence for colony-level adaptive sex ratio biasing could be detected.

Asunto(s)

Abejas/genética , Abejas/fisiología , Conducta Sexual Animal/fisiología , Animales , Femenino , Masculino , Repeticiones de Microsatélite/genética , Reproducción , Razón de Masculinidad

RESUMEN

Lactose is widely used as a carrier of drugs in inhalation devices for asthmatic patients, but some clinicans have suspected that it may cause bronchoconstriction. Only a few studies have been done to examine this and the results are not uniform. This study was conducted to determine the effects of inhalation grade lactose delivered by Diskhaler on lung function and airway conductance in asthmatic subjects. The effect of five doses of lactose ranging from 6.25 mg to 100 mg and placebo were investigated using spirometry and constant volume plethysmography. Nineteen subjects (nine females) with stable asthma and a proven reversibility of at least 12% in forced expiratory volume in 1 sec (FEV) (compared to baseline) in the last 6 months, were included in this single-centre, randomized, placebo-controlled, double-blind, cross-over study. The subjects received placebo plus five doses of lactose on one study day and six doses of placebo on another study day. Both doses and study days were assigned in a random order, and intervals of 1 h were allowed between each dose and at least 36 h between study days. Specific airways conductance (sGaw) and FEV were measured periodically over the course of 1 h after each dose of lactose or placebo. Administration of lactose at four or eight times the concentration in the Diskus and Diskhaler dry powder inhalers did not result in any statistically significant changes in FEV1. sGaw also showed no statistical difference between lactose and placebo at 1 or 3 min post-dosing. Both placebo and lactose produced both dilatation and constriction of the airways in the same patients, with no consistency in direction and no dose-response relationship. No adverse effect of lactose on a rways conductance or FEV1 of stable asthmatic patients was found in this study when given at higher than normal clinical doses.

Asunto(s)

Asma/tratamiento farmacológico , Portadores de Fármacos/farmacología , Lactosa/farmacología , Sistema Respiratorio/efectos de los fármacos , Adolescente , Adulto , Estudios Cruzados , Método Doble Ciego , Portadores de Fármacos/administración & dosificación , Femenino , Volumen Espiratorio Forzado/efectos de los fármacos , Humanos , Lactosa/administración & dosificación , Masculino , Persona de Mediana Edad , Nebulizadores y Vaporizadores , Pletismografía Total

RESUMEN

Nitric oxide (NO) may play an essential role for maintenance of cardiac function and perfusion, while endothelial dysfunction of atherosclerotic vessels may aggravate ischaemia/reperfusion injury. This paper investigates the role of nitric oxide in ischaemia/reperfusion injury in hearts with coronary atherosclerosis. Hearts of apolipoprotein E/LDL receptor double knockout (ApoE/LDLr KO) mice fed an atherogenic diet for 7-9 months were isolated and Langendorff-perfused with 40 minutes of global ischaemia and 60 minutes reperfusion, and funtion and infarction compared with hearts of C57BL/6 controls in the prescence or abscence of the NO-donor SNAP or the NOS inhibitor L-NAME. Hearts of animals with atherosclerosis were more susceptible to ischaemia/reperfusion injury than hearts of animals with healthy vessels, evident as more impaired left ventricular performance. SNAP protected function and reduced infarct size in atherosclerotic hearts, but the same concentration of SNAP was detrimental in normal hearts, perhaps due to NO-overproduction and peroxynitrite formation demonstrated immunohistochemically as increased formation of nitrosylated tyrosine. A low concentration of SNAP protected against ischaemia/reperfusion dysfunction in normal hearts. L-NAME decreased left ventricular performance in atherosclerotic hearts. These findings suggest that impaired endothelium dependent function contributes to reperfusion injury in coronary atherosclerosis.

Asunto(s)

Arteriosclerosis/fisiopatología , Óxido Nítrico/fisiología , Daño por Reperfusión/fisiopatología , Animales , Apolipoproteínas E/genética , Arritmias Cardíacas/fisiopatología , Arteriosclerosis/enzimología , Arteriosclerosis/metabolismo , Western Blotting , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Penicilamina/análogos & derivados , Penicilamina/farmacología , Receptores de LDL/genética , Daño por Reperfusión/enzimología , Daño por Reperfusión/metabolismo , Troponina T/metabolismo

RESUMEN

The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from alpha-helix to antiparallel beta-pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed.

Asunto(s)

Proteínas Portadoras/química , Fosfolípidos/química , Membrana Celular/metabolismo , Péptidos de Penetración Celular , Dicroismo Circular , Cinética , Modelos Químicos , Péptidos/química , Conformación Proteica , Estructura Secundaria de Proteína , Transporte de Proteínas , Espectrofotometría , Factores de Tiempo

RESUMEN

BACKGROUND: Vascular endothelial growth factors (VEGFs) and their receptors are essential regulators of vasculogenesis and angiogenesis in both embryos and adults. One of the factors with a still unknown physiological function is VEGF-B, which is expressed in many tissues, including the heart. METHODS AND RESULTS: Mice carrying a targeted deletion in the VEGF-B gene were developed. In VEGF-B(-/-) animals, no gross abnormalities were observed in organs that normally show high expression of VEGF-B, such as the heart, muscle, and kidney. Analysis of heart function by ECG showed that adult VEGF-B(-/-) mice have an atrial conduction abnormality characterized by a prolonged PQ interval. VEGF- or basic fibroblast growth factor-induced corneal angiogenesis was similar in normal and VEGF-B(-/-) mice. CONCLUSIONS: VEGF-B seems to be required for normal heart function in adult animals but is not required for proper development of the cardiovascular system either during development or for angiogenesis in adults.

Asunto(s)

Factores de Crecimiento Endotelial/deficiencia , Atrios Cardíacos/fisiopatología , Sistema de Conducción Cardíaco/fisiopatología , Animales , Recuento de Células Sanguíneas , Electrocardiografía , Técnicas Electrofisiológicas Cardíacas , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Factores de Crecimiento Endotelial/farmacología , Ojo/irrigación sanguínea , Ojo/efectos de los fármacos , Femenino , Fertilidad/genética , Viabilidad Fetal/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/fisiología , Marcación de Gen , Atrios Cardíacos/crecimiento & desarrollo , Homocigoto , Linfocinas/farmacología , Masculino , Ratones , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Tamaño de los Órganos , Fenotipo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factor B de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

In the multistep process of leukocyte extravasation, the mechanisms by which leukocytes establish the initial contact with the endothelium are unclear. In parallel, there is a controversy regarding the role for L-selectin in leukocyte recruitment. Here, using intravital microscopy in the mouse, we investigated leukocyte capture from the free flow directly to the endothelium (primary capture), and capture mediated through interactions with rolling leukocytes (secondary capture) in venules, in cytokine-stimulated arterial vessels, and on atherosclerotic lesions in the aorta. Capture was more prominent in arterial vessels compared with venules. In venules, the incidence of capture increased with increasing vessel diameter and wall shear rate. Secondary capture required a minimum rolling leukocyte flux and contributed by approximately 20-50% of total capture in all studied vessel types. In arteries, secondary capture induced formation of clusters and strings of rolling leukocytes. Function inhibition of L-selectin blocked secondary capture and thereby decreased the flux of rolling leukocytes in arterial vessels and in large (>45 microm in diameter), but not small (<45 microm), venules. These findings demonstrate the importance of leukocyte capture from the free flow in vivo. The different impact of blockage of secondary capture in venules of distinct diameter range, rolling flux, and wall shear rate provides explanations for the controversy regarding the role of L-selectin in various situations of leukocyte recruitment. What is more, secondary capture occurs on atherosclerotic lesions, a fact that provides the first evidence for roles of L-selectin in leukocyte accumulation in atherogenesis.

Asunto(s)

Arteriosclerosis/etiología , Inflamación/etiología , Selectina L/fisiología , Leucocitos/patología , Animales , Arterias/patología , Arterias/fisiopatología , Arteriosclerosis/patología , Arteriosclerosis/fisiopatología , Adhesión Celular , Movimiento Celular , Inflamación/patología , Inflamación/fisiopatología , Selectina L/genética , Leucocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía por Video , Vénulas/patología , Vénulas/fisiopatología

RESUMEN

A decreased exercise tolerance is a common symptom in patients with congestive heart failure (CHF). This decrease has been suggested to be partly due to altered skeletal muscle function. Therefore, we have studied contractile function and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i), measured with the fluorescent dye indo 1) in isolated muscles from rats in which CHF was induced by ligation of the left coronary artery. The results show no major changes of the contractile function and [Ca(2+)](i) handling in unfatigued intact fast-twitch fibers isolated from flexor digitorum brevis muscles of CHF rats, but these fibers were markedly more susceptible to damage during microdissection. Furthermore, CHF fibers displayed a marked increase of baseline [Ca(2+)](i) during fatigue. Isolated slow-twitch soleus muscles of CHF rats displayed slower twitch contraction and tetanic relaxation than did muscles from sham-operated rats; the slowing of relaxation became more pronounced during fatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteins and sarcolemma Na(+),K(+)-ATPase showed no difference in flexor digitorum brevis muscles of sham-operated versus CHF rats. In conclusion, functional impairments can be observed in limb muscle isolated from rats with CHF. These impairments seem to mainly involve structures surrounding the muscle cells and sarcoplasmic reticulum Ca(2+) pumps, the dysfunction of which becomes obvious during fatigue.

Asunto(s)

Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Contracción Muscular , Músculo Esquelético/metabolismo , Animales , ATPasas Transportadoras de Calcio/metabolismo , Modelos Animales de Enfermedad , Estimulación Eléctrica , Electrocardiografía , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/farmacocinética , Pruebas de Función Cardíaca , Immunoblotting , Técnicas In Vitro , Isoenzimas/metabolismo , Masculino , Microinyecciones , Fatiga Muscular , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Ratas , Ratas Wistar , Sarcolema/enzimología , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Estrés Mecánico

RESUMEN

Leukocyte infiltration in atherosclerosis has been extensively investigated by using histological techniques on fixed tissues. In this study, intravital microscopic observations of leukocyte recruitment in the aorta of atherosclerotic mice were performed. Interactions between leukocytes and atherosclerotic endothelium were highly transient, thereby limiting the ability for rolling leukocytes to firmly adhere. Leukocyte rolling was abolished by function inhibition of P-selectin (P<0.001, n=8), whereas antibody blockage of E-selectin (n=10) decreased rolling leukocyte flux to 51 +/- 9.9% (mean+/-SE, P<0.01) and increased leukocyte rolling velocity to 162 +/- 18% (P<0.01) of pretreatment values. Notably, function inhibition of the integrin alpha(4) subunit (n=5) had no effect on rolling flux (107+/-25%, P=0.782) or rolling velocity (89+/-6.1%, P=0.147), despite endothelial expression of vascular cell adhesion molecule 1 (VCAM-1). Leukocytes interacting with atherosclerotic endothelium were predominantly neutrophils, because treatment with antineutrophil serum decreased rolling and neutrophil counts in peripheral blood to the same extent. In conclusion, we present the first direct observations of atherosclerosis in vivo. We show that transient dynamics of leukocyte-endothelium interactions are important regulators of arterial leukocyte recruitment and that leukocyte rolling in atherosclerosis is critically dependent on the endothelial selectins. This experimental technique and the data presented introduce a novel perspective for the study of pathophysiological events involved in large-vessel disease.

Asunto(s)

Aorta/patología , Arteriosclerosis/fisiopatología , Movimiento Celular/fisiología , Selectina E/metabolismo , Endotelio Vascular/patología , Leucocitos/fisiología , Microscopía/métodos , Selectina-P/metabolismo , Animales , Anticuerpos Anticitoplasma de Neutrófilos/farmacología , Antígenos CD/metabolismo , Aorta/metabolismo , Aorta/ultraestructura , Arteriosclerosis/patología , Selectina E/inmunología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Integrina alfa4 , Leucocitos/efectos de los fármacos , Leucocitos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Molécula 1 de Adhesión Celular Vascular/metabolismo

RESUMEN

In annual hymenopteran societies headed by a single outbred queen, paternity (determined by queen mating frequency and sperm use) is the sole variable affecting colony kin structure and is therefore a key predictor of colony reproductive characteristics. Here we investigate paternity and male production in five species of Dolichovespula wasps. Twenty workers from each of 10 colonies of each of five species, 1000 workers in total, were analysed at three DNA microsatellite loci to estimate paternity. To examine the relationship between kin structure and reproductive behaviour, worker ovary activation was assessed by dissection and the maternal origin of adult males was assessed by DNA microsatellites. Effective paternity was low in all species (D. media 1.08, D. maculata 1.0, D. sylvestris 1.15, D. norwegica 1.08 and D. saxonica 1.35), leading to the prediction of queen-worker conflict over male production. In support of this, workers with full-size eggs in their ovaries (four out of five species) and adult males that were workers' sons (all five species) were found in queenright colonies. However, workers were only responsible for a minority of male production (D. media 7.4%, D. maculata 20.9%, D. sylvestris 9.8%, D. norwegica 2.6% and D. saxonica 34.6%) suggesting that the queen maintains considerable reproductive power over the workers. Kin structure and reproductive conflict in Dolichovespula contrast with their sister group Vespula. Dolichovespula is characterized by low paternity, worker reproduction, and queen-worker conflict and Vespula by high paternity, effective worker policing and absence of worker reproduction. The trend revealed by this comparison is as predicted by kin selection theory suggesting that colony kin structure has been pivotal in the evolution of the yellowjacket wasps.

Asunto(s)

Repeticiones de Microsatélite/genética , Avispas/genética , Avispas/fisiología , Animales , Femenino , Variación Genética/genética , Masculino , Ovario/fisiología , Reproducción , Conducta Sexual Animal

RESUMEN

Septic shock, a major cause of death, is characterized by a pathophysiologic increased production of nitric oxide (NO), which leads to vasodilation and myocardial toxicity. Septic Escherichia coli frequently express proteinaceous curli fibers. In this study, curliated E. coli induced high levels of NO by directly inducing type 2 nitric oxide synthase (NOS2) both in vitro and in vivo. More severe hypotension and higher plasma nitrite/nitrate levels were seen in wild type mice systemically infected with curliated E. coli than in animals infected with E. coli mutants that lacked curli proteins. Blood pressure remained stable in NOS2-deficient mice with curliated bacteria. Increased heart rates, transient hypothermia, and loss of gross activity were seen in all mice, regardless of curli expression. Study results suggest that expression of curli fibers by E. coli activates the NO/NOS2 arm of the innate immune system, which leads to a significant fall in blood pressure.

Asunto(s)

Proteínas Bacterianas/fisiología , Infecciones por Escherichia coli/fisiopatología , Proteínas de Escherichia coli , Escherichia coli/patogenicidad , Hipotensión/fisiopatología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Aorta Torácica/citología , Proteínas Bacterianas/genética , Presión Sanguínea , Temperatura Corporal , Células Cultivadas , Escherichia coli/genética , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Frecuencia Cardíaca , Ratones , Ratones Endogámicos C57BL , Nitratos/sangre , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Nitritos/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Choque Séptico/microbiología , Choque Séptico/fisiopatología