Risk of ultrasound-detected neonatal brain abnormalities in intrauterine growth-restricted fetuses born between 28 and 34 weeks' gestation: relationship with gestational age at birth and fetal Doppler parameters.

OBJECTIVE: To estimate the value of gestational age at birth and fetal Doppler parameters in predicting the risk of neonatal cranial abnormalities in intrauterine growth-restricted (IUGR) fetuses born between 28 and 34 weeks' gestation. METHODS: Fetal Doppler parameters including umbilical artery (UA), middle cerebral artery (MCA), aortic isthmus, ductus venosus and myocardial performance index were evaluated in a cohort of 90 IUGR fetuses with abnormal UA Doppler delivered between 28 and 34 weeks' gestation and in 90 control fetuses matched for gestational age. The value of gestational age at birth and fetal Doppler parameters in predicting the risk of ultrasound-detected cranial abnormalities (CUA), including intraventricular hemorrhage, periventricular leukomalacia and basal ganglia lesions, was analyzed. RESULTS: Overall, IUGR fetuses showed a significantly higher incidence of CUA than did control fetuses (40.0% vs 12.2%, respectively; P < 0.001). Within the IUGR group, all predictive variables were associated individually with the risk of CUA, but fetal Doppler parameters rather than gestational age at birth were identified as the best predictor. MCA Doppler distinguished two groups with different degrees of risk of CUA (48.5% vs 13.6%, respectively; P < 0.01). In the subgroup with MCA vasodilation, presence of aortic isthmus retrograde net blood flow, compared to antegrade flow, allowed identification of a subgroup of cases with the highest risk of CUA (66.7% vs 38.6%, respectively; P < 0.05). CONCLUSION: Evaluation of fetal Doppler parameters, rather than gestational age at birth, allows identification of IUGR preterm fetuses at risk of neonatal brain abnormalities.

Fetal brain MRI texture analysis identifies different microstructural patterns in adequate and small for gestational age fetuses at term.

OBJECTIVES: We tested the hypothesis whether a texture analysis (TA) algorithm applied to MRI brain images identified different patterns in small for gestational age (SGA) fetuses as compared with adequate for gestational age (AGA). STUDY DESIGN: MRI was performed on 83 SGA and 70 AGA at 37 weeks' GA. Texture features were quantified in the frontal lobe, basal ganglia, mesencephalon, cerebellum and cingulum. A classification algorithm based on discriminative models was used to correlate texture features with clinical diagnosis. RESULTS: Region of interest delineation in all areas was achieved in 61 SGA (12 vasodilated) and 52 AGA; this was the sample for TA feature extraction which allowed classifying SGA from AGA with accuracies ranging from 90.9 to 98.9% in SGA versus AGA comparison and from 93.6 to 100% in vasodilated SGA versus AGA comparison. CONCLUSIONS: This study demonstrates that TA can detect brain differences in SGA fetuses. This supports the existence of brain microstructural changes in SGA fetuses.

Hipotermia cerebral moderada en la encefalopatía hipóxico-isquémica. Experiencia en el primer año de su puesta en marcha / Moderate cerebral hypothermia in hypoxic-ischaemic encephalopathy: Experience after one year

Introducción: La hipotermia cerebral moderada ha probado ser una intervención eficaz para reducir la mortalidad y la discapacidad mayor en los neonatos con encefalopatía hipóxico-isquémica (EHI) moderada-grave. Objetivos: Describir la experiencia en el primer año de su utilización y valorar la factibilidad y seguridad de esta intervención. Métodos: Revisión de los 20 pacientes con EHI moderada-grave tratados con hipotermia corporal total en la Agrupación Sanitaria Hospital Sant Joan de Déu-Hospital Clínic, entre enero de 2009 y junio de 2010. Resultados: Durante este periodo ingresaron 50 neonatos con EHI perinatal, en 26 de ellos moderada-grave. Un total de 20 neonatos recibieron hipotermia (13 con EHI grave y 7 moderada). En todos ellos se encontró un antecedente de riesgo de hipoxia-isquemia perinatal y algún signo clínico de EHI. Quince neonatos presentaron convulsiones clínicas y/o en el registro electroencefalográfico. La temperatura rectal se mantuvo en 33,5±0,5°C en el 76,5% de las determinaciones para los neonatos con control manual de la temperatura y en el 93,6% para los manejados con servocontrol (p<0,0001). El recalentamiento se realizó en una mediana de 10,5 horas. No se produjo ninguna complicación potencialmente grave relacionada con la hipotermia. Fallecieron 7 neonatos (35%), todos ellos con EHI grave. Conclusiones: No se han apreciado dificultades en ninguna de las fases de esta intervención terapéutica ni se ha registrado ninguna complicación potencialmente grave relacionada con ella. Tanto el control manual de la temperatura como su servocontrol son eficaces para mantener la temperatura diana, pero esta muestra una menor variabilidad con el equipo servocontrolado(AU)

Introduction: Moderate cerebral hypothermia has been shown to be an effective intervention in decreasing mortality and major disabilities in infants with moderate-severe hypoxic-ischaemic encephalopathy (HIE). Objectives: To describe our experience within the first year of implementation, and to evaluate the feasibility and safety of this intervention. Methods: Retrospective study of 20 patients with moderate-severe HIE treated with whole body hypothermia in the Agrupación Sanitaria Hospital Sant Joan de Déu-Hospital Clínic, between January 2009 and June 2010.ResultsDuring this period, 50 patients with perinatal HIE, 26 of them moderate- severe, were admitted to our units. Twenty patients received hypothermia (13 with severe and 7 with moderate HIE). All of them had at least one risk factor for perinatal hypoxia-ischaemia, and clinical signs of HIE. Fifteen had clinical and/or EEG seizures. Core temperature was maintained at 33.5±0.5°C in 76.5% of determinations for infants cooled with a manual control device, and in 93.6% for those cooled with a servo-controlled device (P<0,0001). Re-warming took a median time of 10.5hours. No potentially severe complications related to hypothermia were observed. Seven patients (35%) died, all of them with severe HIE. Conclusions: There were no difficulties in any of the steps of this intervention, and no potentially severe complications related to it were recorded. Both manual and servo-control methods are equally effective on maintaining the target temperature, although temperature shows less variability using the servo-controlled equipment(AU)

[Moderate cerebral hypothermia in hypoxic-ischaemic encephalopathy: Experience after one year]. / Hipotermia cerebral moderada en la encefalopatía hipóxico-isquémica. Experiencia en el primer año de su puesta en marcha.

INTRODUCTION: Moderate cerebral hypothermia has been shown to be an effective intervention in decreasing mortality and major disabilities in infants with moderate-severe hypoxic-ischaemic encephalopathy (HIE). OBJECTIVES: To describe our experience within the first year of implementation, and to evaluate the feasibility and safety of this intervention. METHODS: Retrospective study of 20 patients with moderate-severe HIE treated with whole body hypothermia in the Agrupación Sanitaria Hospital Sant Joan de Déu-Hospital Clínic, between January 2009 and June 2010. RESULTS: During this period, 50 patients with perinatal HIE, 26 of them moderate- severe, were admitted to our units. Twenty patients received hypothermia (13 with severe and 7 with moderate HIE). All of them had at least one risk factor for perinatal hypoxia-ischaemia, and clinical signs of HIE. Fifteen had clinical and/or EEG seizures. Core temperature was maintained at 33.5 ± 0.5°C in 76.5% of determinations for infants cooled with a manual control device, and in 93.6% for those cooled with a servo-controlled device (P<.0001). Re-warming took a median time of 10.5 hours. No potentially severe complications related to hypothermia were observed. Seven patients (35%) died, all of them with severe HIE. CONCLUSIONS: There were no difficulties in any of the steps of this intervention, and no potentially severe complications related to it were recorded. Both manual and servo-control methods are equally effective on maintaining the target temperature, although temperature shows less variability using the servo-controlled equipment.

Neuroprotección con hipotermia en el recién nacido con encefalopatía hipóxico-isquémica. Guía de estándares para su aplicación clínica / Neuroprotection with hypothermia in the newborn with hypoxic-ischaemic encephalopathy. standard guidelines for its clinical application

La estandarización de la hipotermia como tratamiento de la encefalopatía hipóxico-isquémica perinatal se apoya en la evidencia científica actual. El documento que se presenta a continuación ha sido realizado por los autores a petición de la Sociedad Española de Neonatología y pretende ser una «guía de estándares» para su aplicación clínica. Se discuten los interrogantes que pueden surgir al pasar del estricto marco de los ensayos clínicos a la práctica clínica cotidiana: reconocimiento precoz de la encefalopatía clínica, criterios de inclusión y exclusión, hipotermia durante el transporte, modalidades de hipotermia (cerebral selectiva o sistémica) y efectos secundarios del tratamiento. El tratamiento con hipotermia ha cambiado el pronóstico de los niños con encefalopatía hipóxico-isquémica y nuestras decisiones de esfuerzo terapéutico. En este sentido, es de especial relevancia conocer el cambio del valor predictivo de la exploración clínica y de la electroencefalografía en los niños tratados con hipotermia. Para mejorar la eficacia de este tratamiento neuroprotector, es necesaria una mejor identificación de los pacientes con daño cerebral potencialmente reversible. Nuevos biomarcadores de daño cerebral facilitarán esta tarea. Todo niño tratado con hipotermia debe ser incluido en un programa de seguimiento para evaluar su neurodesarrollo (AU)

Standardisation of hypothermia as a treatment for perinatal hypoxic-ischaemic encephalopathy is supported by current scientific evidence. The following document was prepared by the authors on request of the Spanish Society of Neonatology and is intended to be a guide for the proper implementation of this therapy. We discuss the difficulties that may arise when moving from the strict framework of clinical trials to clinical daily care: early recognition of clinical encephalopathy, inclusion and exclusion criteria, hypothermia during transport, type of hypothermia (selective head or systemic cooling) and side effects of therapy. The availability of hypothermia therapy has changed the prognosis of children with hypoxic-ischaemic encephalopathy and our choices of therapeutic support. In this sense, it is especially important to be aware of the changes in the predictive value of the neurological examination and the electroencephalographic recording in cooled infants. In order to improve neuroprotection with hypothermia we need earlier recognition of to recognise earlier the infants that may benefit from cooling. Biomarkers of brain injury could help us in the selection of these patients. Every single infant treated with hypothermia must be included in a follow up program in order to assess neurodevelopmental outcome (AU)

[Neuroprotection with hypothermia in the newborn with hypoxic-ischaemic encephalopathy. Standard guidelines for its clinical application]. / Neuroprotección con hipotermia en el recién nacido con encefalopatía hipóxico-isquémica. Guía de estándares para su aplicación clínica.

Standardisation of hypothermia as a treatment for perinatal hypoxic-ischaemic encephalopathy is supported by current scientific evidence. The following document was prepared by the authors on request of the Spanish Society of Neonatology and is intended to be a guide for the proper implementation of this therapy. We discuss the difficulties that may arise when moving from the strict framework of clinical trials to clinical daily care: early recognition of clinical encephalopathy, inclusion and exclusion criteria, hypothermia during transport, type of hypothermia (selective head or systemic cooling) and side effects of therapy. The availability of hypothermia therapy has changed the prognosis of children with hypoxic-ischaemic encephalopathy and our choices of therapeutic support. In this sense, it is especially important to be aware of the changes in the predictive value of the neurological examination and the electroencephalographic recording in cooled infants. In order to improve neuroprotection with hypothermia we need earlier recognition of to recognise earlier the infants that may benefit from cooling. Biomarkers of brain injury could help us in the selection of these patients. Every single infant treated with hypothermia must be included in a follow up program in order to assess neurodevelopmental outcome.

Identification of iron-acquisition proteins of Avibacterium paragallinarum.

When Avibacterium paragallinarum reference strain 0083 (serovar A) was grown in an iron-restricted culture medium, the expression of the 60, 68 and 93 kDa outer membrane proteins increased as compared with normal media. Sera of chickens experimentally infected with Av. paragallinarum recognized these iron-restriction induced proteins, suggesting their expression in vivo. The three outer membrane proteins were identified as transferrin receptor and iron transport proteins by mass spectroscopy and a search in sequence databases. As these proteins have been reported to be regulated by the Fur protein in many bacteria, we investigated, through molecular methods, the presence of the fur gene in Av. paragallinarum. A candidate fur gene of Av. paragallinarum was amplified by polymerase chain reaction using complementary primers to conserved regions of fur gene sequences from members of the Pasteurellaceae family. The nucleotide sequence of the cloned gene, from ATG to TAA stop codon, was 453 base pairs in length and the deduced amino acid sequence showed 94% identity with Fur sequences of Actinobacillus pleuropneumoniae and Haemophilus ducreyi. The Av. paragallinarum deduced Fur protein (17.8 kDa) amino acid sequence contains the N-terminal helix-turn-helix DNA-binding domain and the two iron-binding sites in the C-terminal end, typical of other described Fur proteins. The study of iron-restriction-induced proteins and the mechanism regulating their expression could lead to an understanding of the responses of Av. paragallinarum to survive in an iron-restricted environment on host mucosal surfaces.

Gallibacterium anatis-secreted metalloproteases degrade chicken IgG.

Gallibacterium anatis (previously named Pasteurella haemolytica-like) is considered a normal inhabitant of genital and upper respiratory tracts of healthy chickens, but it is also associated with different pathological conditions. Secreted metalloproteases from field and reference G. anatis cultures were obtained by methanol precipitation and were characterized. Proteins of molecular mass higher than 100 kDa showing proteolytic activity were observed in 10% polyacrylamide gels copolymerized with 1% bovine casein. They were active at alkaline pH, and inhibited by ethylenediamine tetraacetic acid. Their activity was stable at 50 degrees C, but partially inhibited at 60 degrees C, and totally inhibited at higher temperatures. Secreted proteins were able to degrade chicken IgG after 24 h of incubation, and cross-reacted with a polyclonal antibody against purified protease from Actinobacillus pleuropneumoniae. Secreted metalloproteases could play a role in infections caused by G. anatis.

Evaluación de la actividad leishmanicida de los extractos etanolicos de rollinia rufinervis sobre Leishmania chagasi / Evaluation of the leishmanicidal activity of the ethanolyc extractys of Rollinia rufinervis on Leishmania chagasi

En este estudio se evalúa la bioactividad de las fracciones obtenidas de los extractos etanólicos de las hojas, de la corteza del tronco y de la corteza de la raíz de Rollinia rufinervis (Annonaceae), las cuales presentan actividad citotóxica y se detecta la presencia de las acetogeninas, que pueden causar la actividad antiparasitaria. Las fracciones son probadas a concentraciones entre 0.095µg/mL y 100 µg/mL contra promastigotes de Leishmania chagasi, evaluando la movilidad de los parásitos a las 72 horas. El efecto de las fracciones es contrastado con el medio Schneider-Drosófila sin extracto y con Glucantime, utilizados como control. Las fracciones de Rollinia rufinervis de: acetato de etilo de raíz, éter de petróleo de corteza, acetato de etilo de corteza y acetato de etilo de hojas, presentan actividad leishmanicida. La última fracción es la de mayor actividad causando inmovilidad apreciable o total de los parásitos a lo largo del experimento. De ella se aisló posiblemente el ß-sitosteroL

Subcellular fractions of Brucella ovis distinctively induce the production of interleukin-2, interleukin-4, and interferon-gamma in mice.

The aim of this study was to evaluate the effect of 3 Brucella ovis subcellular protein fractions: Outer membrane (OMP), inner membrane (IMP), and cytoplasm (CP), on cellular immune response by in vitro production of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. Each fraction was inoculated 3 times into Balb/c mice, primary cultures of mice spleen cells were done, and these were then stimulated with the fractions. Culture supernatants were collected at 24, 48, 72, 96, and 120 h postinoculation. Cytokine concentration was measured by Duoset-enzyme-linked immunosorbent assay (ELISA). The OMP fraction induced highest cellular immune response of 1000 pg/mL of IL-2 at 24 h, which decreased to < 100 pg/mL by 96 h. The IL-2 response for the IMP fraction was low at 24 h, but exceeded that of the OMP fraction at 72, 96, and 120 h. The CP showed a poor IL response. Regarding the IFN-gamma production, OMP and IMP induced a high response at 120 h. These results open the possibility for the use of B. ovis outer and inner membrane proteins as a subcellular vaccine.

A 24-kDa cloned zinc metalloprotease from Actinobacillus pleuropneumoniae is common to all serotypes and cleaves actin in vitro.

Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence that.codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease.

Secretion of proteases from Pasteurella multocida isolates.

The capability of Pasteurella multocida to secrete proteases to the culture medium and their characterization were studied in five animal isolates (bovine, chicken, sheep, and two from pig). All the isolates produced proteases in a wide range of molecular mass. It is suggested that they are neutral metalloproteases, since they were optimally active between pH 6 and 7, inhibited by chelating agents but not by other protease inhibitors, and reactivated by calcium. Proteases from isolates were able to degrade IgG. Several proteins from supernatants of cultures precipitated with 70% (NH4)2SO4 of all the P. multocida isolates were recognized by a polyclonal antiserum raised against a purified protease from Actinobacillus pleuropneumoniae. Protease production might play an important role during tissue colonization and in P. multocida diseases.

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.

Secreted proteases from Actinobacillus pleuropneumoniae serotype 1 degrade porcine gelatin, hemoglobin and immunoglobulin A.

It was found that 48 hour cultures of Actinobacillus pleuropneumoniae secreted proteases into the medium. Electrophoresis in polyacrylamide gels (10%) copolymerized with porcine gelatin (0.1%), of the 70% (NH4)2SO4 precipitate from the culture supernatants, displayed protease activities of different molecular weights: > 200, 200, 90, 80, 70 and 50 kDa. They had activity over a broad range of pHs (4-8), with an optimal pH of 6-7. All were inhibited by 10 mM EDTA, and reactivated by 10 mM calcium. They were stable at -20 degrees C for more than a month. The proteases also degraded porcine IgA and porcine, human, and bovine hemoglobin, although they appeared to be less active against the hemoglobins. The IgA was totally cleaved in 48 h, using supernatants concentrated with polyvinyl pyrrolidone or the 70% (NH4)2SO4. Extracellular proteases could play a role in virulence.

Actinobacillus pleuropneumoniae: virulence and gene cloning.

Actinobacillus pleuropneumoniae is the causal agent of porcine contagious pleuropneumonia (PCP). The infection produces important economic losses in porciculture due to its high morbidity and mortality. Survivors are asymptomatic carriers infectious to other pigs and have low alimentary conversion. The causative agent possesses several virulence factors: adhesion fimbriae, lipopolysaccharide of the outer membrane, capsule, and cytolysins. In addition, our group has reported secretion proteases of a wide pH range of activity. These proteases degrade different substrates such as porcine gelatin, hemoglobin and IgA, and bovine or human hemoglobin. To control PCP dissemination, farmers require serodiagnostic tests which detect carriers and discriminate between vaccinated and infected animals. Bacterines used as immunogens are serotype specific and do not prevent the infection. Genes have been cloned that codify a cohemolysin, cytolysins, and an iron-binding protein. We have cloned A. pleuropneumoniae genes using the expression plasmids pUC19 and Bluescript, in Escherichia coli Q358 and DH5 alpha; the screening for antigen production was made in four groups of pigs (vaccinated, experimentally infected, naturally infected, and from slaughterhouses); two E. coli clones expressed polypeptides recognized by sera from all the groups.