Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Creatine deficiency syndomes : a new model of partial GAMT deficiency affecting brain cell development

Les syndromes de déficiences cérébrales en créatine (CCDS) sont dus à des mutations dans les gènes GATM et G AMT (codant pour les enzymes AGAT et G AMT de la voie de synthèse de créatine) ainsi que SLC6A8 (transporteur de créatine), et génèrent une absence ou une très forte baisse de créatine (Cr) dans le cerveau, mesurée par spectroscopic de résonance magnétique. Les patients CCDS développent des handicaps neurologiques sévères. Les patients AGAT et GAMT peuvent être traités avec des doses importantes de Cr, mais gardent dans la plupart des cas des séquelles neurologiques irréversibles. Aucun traitement efficace n'existe à ce jour pour la déficience en SLC6A8. Bien que de nombreux modèles aient été développés pour comprendre la Cr cérébrale en conditions physiologiques, les pathomécanismes des CCDS ne sont pas encore compris. Des souris transgéniques pour les gènes Gatm, Gamt et Slc6a8 ont été générées, mais elles ne miment que partiellement la pathologie humaine. Parmi les CCDS, la déficience en GAMT est la plus sévère, en raison de l'accumulation cérébrale de l'intermédiaire guanidinoacétate (GAA). Alors que la toxicité cérébrale du GAA a été étudiée par exposition directe au GAA d'animaux adultes sains, les mécanismes de la toxicité du GAA en condition de déficience en GAMT dans le cerveau en développement sont encore inconnus. Le but de ce projet était donc de développer un modèle de déficience en GAMT dans des cultures 3D primaires de cellules nerveuses de rat en agrégats par knock-down du gène GAMT, en utilisant un virus adéno-associé (AAV) induisant le mécanisme d'interférence à l'ARN (RNAi). Le virus scAAV2, à la multiplicité d'infection de 1000, s'est révélé le plus efficace pour transduire tous les types de cellules nerveuses des cultures (neurones, astrocytes, oligodendrocytes), et générer un knock-down maximal de la protéine GAMT de 85% (jour in vitro 18). Cette déficience partielle en GAMT s'est révélée insuffisante pour générer une déficience en Cr, mais a causé l'accumulation attendue de GAA, à des doses comparables aux niveaux observés dans le LCR des patients GAMT. Le GAA a induit une croissance axonale anarchique accompagnée d'une baisse de l'apoptose naturelle, suivis par une induction tardive de mort cellulaire non-apoptotique. Le co-traitement par la Cr a prévenu tous les effets toxiques du GAA. Ce travail montre que l'accumulation de GAA en absence de déficience en Cr est suffisante pour affecter le développement du tissu nerveux, et suggère que des formes de déficiences en GAMT supplémentaires, ne présentant pas de déficiences en Cr, pourraient être découvertes par mesure du GAA, en particulier à travers les programmes récemment proposés de dépistage néonatal de la déficience en GAMT. -- Cerebral creatine deficiency syndromes (CCDS) are caused by mutations in the genes GATM and GAMT (respectively coding for the two enzymes of the creatine synthetic pathway, AGAT and GAMT) as well as SLC6A8 (creatine transporter), and lead to the absence or very strong decrease of creatine (Cr) in the brain when measured by magnetic resonance spectroscopy. Affected patients show severe neurological impairments. While AGAT and GAMT deficient patients can be treated with high dosages of Cr, most remain with irreversible brain sequelae. No treatment has been successful so far for SLC6A8 deficiency. While many models have helped understanding the cerebral Cr pathways in physiological conditions, the pathomechanisms underlying CCDS are yet to be elucidated. Transgenic mice carrying mutations in the Gatm, Gamt and Slc6a8 genes have been developed, but only partially mimic the human pathology. Among CCDS, GAMT deficiency is the most severe, due to the CNS accumulation of the guanidinoacetate (GAA) intermediate. While brain toxicity of GAA has been explored through direct GAA exposure of adult healthy animals, the mechanisms underlying GAA toxicity in GAMT deficiency conditions on the developing CNS are yet unknown. The aim of this project was thus to develop and characterize a GAMT deficiency model in developing brain cells by gene knockdown, by adeno-associated virus (AAV)-driven RNA interference (RNAi) in rat 3D organotypic primary brain cell cultures in aggregates. scAAV2 with a multiplicity of infection of 1000 was shown as the most efficient serotype, was able to transduce all brain cell types (neurons, astrocytes, oligodendrocytes) and to induce a maximal GAMT protein knockdown of 85% (day in vitro 18). Metabolite analysis showed that partial GAMT knockdown was insufficient to induce Cr deficiency but generated the awaited GAA accumulation at concentrations comparable to the levels observed in cerebrospinal fluid of GAMT-deficient patients. Accumulated GAA induced axonal hypersprouting paralleled with inhibition of natural apoptosis, followed by a later induction in non-apoptotic cell death. Cr supplementation led to the prevention of all GAA-induced toxic effects. This work shows that GAA accumulation without Cr deficiency is sufficient to affect CNS development, and suggests that additional partial GAMT deficiencies, which may not show the classical brain Cr deficiency, may be discovered through GAA measurement including by recently proposed neonatal screening programs for GAMT deficiency.

Relationship of physical activity with motor skills, aerobic fitness and body fat in preschool children: a cross-sectional and longitudinal study (Ballabeina).

Adiposity, low aerobic fitness and low levels of activity are all associated with clustered cardiovascular disease risk in children and their high prevalence represents a major public health concern. The aim of this study is to investigate the relationship of objectively measured physical activity (PA) with motor skills (agility and balance), aerobic fitness and %body fat in young children. This study is a cross-sectional and longitudinal analyses using mixed linear models. Longitudinal data were adjusted for baseline outcome parameters. In all, 217 healthy preschool children (age 4-6 years, 48% boys) participated in this study. PA (accelerometers), agility (obstacle course), dynamic balance (balance beam), aerobic fitness (20-m shuttle run) and %body fat (bioelectric impedance) at baseline and 9 months later. PA was positively associated with both motor skills and aerobic fitness at baseline as well as with their longitudinal changes. Specifically, only vigorous, but not total or moderate PA, was related to changes in aerobic fitness. Higher PA was associated with less %body fat at baseline, but not with its change. Conversely, baseline motor skills, aerobic fitness or %body fat were not related to changes in PA. In young children, baseline PA was associated with improvements in motor skills and in aerobic fitness, an important determinant of cardiovascular risk.

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Effect of school based physical activity programme (KISS) on fitness and adiposity in primary schoolchildren: cluster randomised controlled trial.

To assess the effectiveness of a school based physical activity programme during one school year on physical and psychological health in young schoolchildren. Cluster randomised controlled trial. 28 classes from 15 elementary schools in Switzerland randomly selected and assigned in a 4:3 ratio to an intervention (n=16) or control arm (n=12) after stratification for grade (first and fifth grade), from August 2005 to June 2006. 540 children, of whom 502 consented and presented at baseline. Children in the intervention arm (n=297) received a multi-component physical activity programme that included structuring the three existing physical education lessons each week and adding two additional lessons a week, daily short activity breaks, and physical activity homework. Children (n=205) and parents in the control group were not informed of an intervention group. For most outcome measures, the assessors were blinded. Primary outcome measures included body fat (sum of four skinfolds), aerobic fitness (shuttle run test), physical activity (accelerometry), and quality of life (questionnaires). Secondary outcome measures included body mass index and cardiovascular risk score (average z score of waist circumference, mean blood pressure, blood glucose, inverted high density lipoprotein cholesterol, and triglycerides). 498 children completed the baseline and follow-up assessments (mean age 6.9 (SD 0.3) years for first grade, 11.1 (0.5) years for fifth grade). After adjustment for grade, sex, baseline values, and clustering within classes, children in the intervention arm compared with controls showed more negative changes in the z score of the sum of four skinfolds (-0.12, 95 % confidence interval -0.21 to -0.03; P=0.009). Likewise, their z scores for aerobic fitness increased more favourably (0.17, 0.01 to 0.32; P=0.04), as did those for moderate-vigorous physical activity in school (1.19, 0.78 to 1.60; P<0.001), all day moderate-vigorous physical activity (0.44, 0.05 to 0.82; P=0.03), and total physical activity in school (0.92, 0.35 to 1.50; P=0.003). Z scores for overall daily physical activity (0.21, -0.21 to 0.63) and physical quality of life (0.42, -1.23 to 2.06) as well as psychological quality of life (0.59, -0.85 to 2.03) did not change significantly. A school based multi-component physical activity intervention including compulsory elements improved physical activity and fitness and reduced adiposity in children. Trial registration Current Controlled Trials ISRCTN15360785.

Influence of a lifestyle intervention in preschool children on physiological and psychological parameters (Ballabeina): study design of a cluster randomized controlled trial.

Childhood obesity and physical inactivity are increasing dramatically worldwide. Children of low socioeconomic status and/or children of migrant background are especially at risk. In general, the overall effectiveness of school-based programs on health-related outcomes has been disappointing. A special gap exists for younger children and in high risk groups. This paper describes the rationale, design, curriculum, and evaluation of a multicenter preschool randomized intervention study conducted in areas with a high migrant population in two out of 26 Swiss cantons. Twenty preschool classes in the German (canton St. Gallen) and another 20 in the French (canton Vaud) part of Switzerland were separately selected and randomized to an intervention and a control arm by the use of opaque envelopes. The multidisciplinary lifestyle intervention aimed to increase physical activity and sleep duration, to reinforce healthy nutrition and eating behaviour, and to reduce media use. According to the ecological model, it included children, their parents and the teachers. The regular teachers performed the majority of the intervention and were supported by a local health promoter. The intervention included physical activity lessons, adaptation of the built infrastructure; promotion of regional extracurricular physical activity; playful lessons about nutrition, media use and sleep, funny homework cards and information materials for teachers and parents. It lasted one school year. Baseline and post-intervention evaluations were performed in both arms. Primary outcome measures included BMI and aerobic fitness (20 m shuttle run test). Secondary outcomes included total (skinfolds, bioelectrical impedance) and central (waist circumference) body fat, motor abilities (obstacle course, static and dynamic balance), physical activity and sleep duration (accelerometry and questionnaires), nutritional behaviour and food intake, media use, quality of life and signs of hyperactivity (questionnaires), attention and spatial working memory ability (two validated tests). Researchers were blinded to group allocation. The purpose of this paper is to outline the design of a school-based multicenter cluster randomized, controlled trial aiming to reduce body mass index and to increase aerobic fitness in preschool children in culturally different parts of Switzerland with a high migrant population. Trial Registration: (clinicaltrials.gov) NCT00674544.

Sweet sixteen for ANLS.

Since its introduction 16 years ago, the astrocyte-neuron lactate shuttle (ANLS) model has profoundly modified our understanding of neuroenergetics by bringing a cellular and molecular resolution. Praised or disputed, the concept has never ceased to attract attention, leading to critical advances and unexpected insights. Here, we summarize recent experimental evidence further supporting the main tenets of the model. Thus, evidence for distinct metabolic phenotypes between neurons (mainly oxidative) and astrocytes (mainly glycolytic) have been provided by genomics and classical metabolic approaches. Moreover, it has become clear that astrocytes act as a syncytium to distribute energy substrates such as lactate to active neurones. Glycogen, the main energy reserve located in astrocytes, is used as a lactate source to sustain glutamatergic neurotransmission and synaptic plasticity. Lactate is also emerging as a neuroprotective agent as well as a key signal to regulate blood flow. Characterization of monocarboxylate transporter regulation indicates a possible involvement in synaptic plasticity and memory. Finally, several modeling studies captured the implications of such findings for many brain functions. The ANLS model now represents a useful, experimentally based framework to better understand the coupling between neuronal activity and energetics as it relates to neuronal plasticity, neurodegeneration, and functional brain imaging.

Inflammatory markers and incident heart failure risk in older adults: the Health ABC (Health, Aging, and Body Composition) study.

OBJECTIVES: The purpose of this study was to evaluate the association between inflammation and heart failure (HF) risk in older adults. BACKGROUND: Inflammation is associated with HF risk factors and also directly affects myocardial function. METHODS: The association of baseline serum concentrations of interleukin (IL)-6, tumor necrosis factor-alpha, and C-reactive protein (CRP) with incident HF was assessed with Cox models among 2,610 older persons without prevalent HF enrolled in the Health ABC (Health, Aging, and Body Composition) study (age 73.6 +/- 2.9 years; 48.3% men; 59.6% white). RESULTS: During follow-up (median 9.4 years), HF developed in 311 (11.9%) participants. In models controlling for clinical characteristics, ankle-arm index, and incident coronary heart disease, doubling of IL-6, tumor necrosis factor-alpha, and CRP concentrations was associated with 29% (95% confidence interval: 13% to 47%; p < 0.001), 46% (95% confidence interval: 17% to 84%; p = 0.001), and 9% (95% confidence interval: -1% to 24%; p = 0.087) increase in HF risk, respectively. In models including all 3 markers, IL-6, and tumor necrosis factor-alpha, but not CRP, remained significant. These associations were similar across sex and race and persisted in models accounting for death as a competing event. Post-HF ejection fraction was available in 239 (76.8%) cases; inflammatory markers had stronger association with HF with preserved ejection fraction. Repeat IL-6 and CRP determinations at 1-year follow-up did not provide incremental information. Addition of IL-6 to the clinical Health ABC HF model improved model discrimination (C index from 0.717 to 0.734; p = 0.001) and fit (decreased Bayes information criterion by 17.8; p < 0.001). CONCLUSIONS: Inflammatory markers are associated with HF risk among older adults and may improve HF risk stratification.

Obese and fit adolescents have lower blood pressure levels than obese and unfit counterparts.

The aim of the study was to assess the effects of physical fitness on the relationships between body mass index (BMI) and body fat (BF) on blood pressure (BP) levels. Cross-sectional study conducted in 25 schools of Lisbon (Portugal), including 2041 boys and 1995 girls aged 10-18. BF was assessed by bioimpedance. Cardiovascular fitness was assessed by the 20-meter shuttle run and classified as fit/unfit. Obesity (BMI or BF defined) was defined according to international criteria. In both sexes, BMI was positively related with systolic and diastolic BP, while BF was only positively related with diastolic BP z-scores. No interaction was found between fitness and BMI categories regarding BP levels, while for BF a significant interaction was found. Being fit reduced the BF-induced increase in the Odds ratio (OR) of presenting with high BP: OR (95% confidence interval) 1.01 (0.73-1.40) and 0.99 (0.70-1.38) for overweight and obese fit boys, respectively, the corresponding values for unfit overweight and obese boys being 1.33 (0.94-1.90) and 1.75 (1.34-2.28), respectively. The values were 0.88 (0.57-1.35) and 1.66 (0.98-2.80) for overweight and obese fit girls, respectively, the corresponding values for unfit overweight and obese being 1.63 (1.12-2.37) and 1.90 (1.32-2.73) respectively. No interaction was found between fitness and BMI-defined overweight and obesity. Being fit reduces the negative impact of BF on BP levels and high BP status in adolescents. This protective effect was not found with BMI.

Increased body fat is independently and negatively related with cardiorespiratory fitness levels in children and adolescents with normal weight.

Body mass index (BMI) is related with cardiorespiratory fitness (CRF), but less is known regarding the combined relationships between BMI and body fat (BF) on CRF. Cross-sectional study included 2361 girls and 2328 boys aged 10–18 years living in the area of Lisbon, Portugal. BMI was calculated by measuring height and weight, and obesity was assessed by international criteria. BF was assessed by bioimpedance. CRF was assessed by the 20-m shuttle run and the participants were classified as normal-to-high or low-CRF level according to Fitness gram criterion-referenced standards. The prevalence of low CRF was 47 and 39% in girls and boys, respectively. The corresponding values for the prevalence of obesity were 4.8 and 5.6% (not significant) and of excess BF of 12.1 and 25.1% (P <0.001), respectively. In both sexes, BMI and BF were inversely related with CRF: r = – 0.53 and – 0.45 for BMI and % BF, respectively, in boys and the corresponding values in girls were – 0.50 and – 0.33 (all P <0.01). When compared with a participant with normal BMI and BF, the odds ratios (95% confidence interval) for low CRF were 1.94 (1.46–2.58) for a participant with normal BMI and high BF, and 6.19 (5.02–7.63) for a participant with high BMI and high BF. The prevalence of low-CRF levels is high in Portuguese youths. BF negatively influences CRF levels among children/adolescents with normal BMI.

Reduction in the use of diagnostic tests in infants with risk factors for early-onset neonatal sepsis does not delay antibiotic treatment.

Despite a low positive predictive value, diagnostic tests such as complete blood count (CBC) and C-reactive protein (CRP) are commonly used to evaluate whether infants with risk factors for early-onset neonatal sepsis (EOS) should be treated with antibiotics. We investigated the impact of implementing a protocol aiming at reducing the number of diagnostic tests in infants with risk factors for EOS in order to compare the diagnostic performance of repeated clinical examination with CBC and CRP measurement. The primary outcome was the time between birth and the first dose of antibiotics in infants treated for suspected EOS. Among the 11,503 infants born at ≥35 weeks during the study period, 222 were treated with antibiotics for suspected EOS. The proportion of infants receiving antibiotics for suspected EOS was 2.1% and 1.7% before and after the change of protocol (p = 0.09). Reduction of diagnostic tests was associated with earlier antibiotic treatment in infants treated for suspected EOS (hazard ratio 1.58; 95% confidence interval [CI] 1.20-2.07; p <0.001), and in infants with neonatal infection (hazard ratio 2.20; 95% CI 1.19-4.06; p = 0.01). There was no difference in the duration of hospital stay nor in the proportion of infants requiring respiratory or cardiovascular support before and after the change of protocol. Reduction of diagnostic tests such as CBC and CRP does not delay initiation of antibiotic treatment in infants with suspected EOS. The importance of clinical examination in infants with risk factors for EOS should be emphasised.

Association of socioeconomic status with inflammatory markers: a two cohort comparison.

OBJECTIVE: To assess the association between socioeconomic status (SES) and inflammatory markers using two different European population samples. METHODS: We used data from the CoLaus (N=6412, Lausanne, Switzerland) and EPIPorto (N=1205, Porto, Portugal) studies. Education and occupational position were used as indicators of socioeconomic status (SES). High-sensitivity C-reactive protein (hs-CRP) was available for both cohorts. Interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were available in CoLaus; leukocyte count and fibrinogen in EPIPorto. RESULTS: We showed that low SES was significantly associated with high inflammation in both studies. We also showed that behavioural factors contributed the most to SES differences in inflammation. In both studies the larger difference between the lowest and the highest SES was observed for hs-CRP. In the Swiss sample, a linear association between education and hs-CRP persisted after adjustment for all mediating factors and confounders considered (p for linear trend <0.001). CONCLUSION: Large social differences exist in inflammatory activity, in part independently from demographic and behavioural factors, chronic conditions and medication use. SES differences in inflammation are also similar in countries with different underlying socioeconomic conditions.