Attenuated Listeria monocytogenes vaccine vectors expressing influenza A nucleoprotein: preclinical evaluation and oral inoculation of volunteers

Listeria monocytogenes vectors have shown promise for delivery of viral and tumor antigens in animals. We used two mutant vector strains deleted for actA/plcB (BMB72) and actA/inlB (BMB54), and engineered both strains to secrete a heterologous nucleoprotein antigen from the Influenza A virus. Strains were evaluated in vitro and in mice. Twenty-two healthy volunteers received single oral doses of either strain in a physiological study of safety, shedding, and immunogenicity. Volunteers were observed in the hospital for seven days and had daily blood cultures, routine safety blood tests (complete blood count with differential; hepatic and renal function), and fecal cultures; none had fever, positive blood cultures, prolonged shedding, or serious or unexpected events. Four of 12 volunteers who received the actA/plcB-deleted strain had minor, transient, asymptomatic serum transaminase elevations (maximum increase 1.4× upper normal). Six of six volunteers who received ≥4 × 10(9) colony forming units had detectable mucosal immune responses to listerial antigens, but not to the vectored influenza antigen. Approximately half the volunteers had modest interferon-γ ELISpot responses to a complex listerial antigen, but none had increases over their baseline responses to the influenza antigen. Comparison with prior work suggests that foreign antigen expression, and perhaps also freezing, may adversely affect the organisms' immunogenicity.     

Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis

Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling.     

Specific nucleoprotein complexes within adenovirus capsids.

Adenoviral DNA was examined within capsids by dimethyl sulfate footprinting. Protein-DNA interactions were visualized through ligation-mediated PCR (LM-PCR). Signals for protein binding were found adjacent to both inverted terminal repeats (ITR). There were no indications of close protein binding at several other loci of the viral genome. Therefore, adenovirus type 5 seems to contain sequence- or locus-specific DNA binding proteins within the virion.     

Pravastatin reverses the membrane cholesterol reorganization induced by myocardial infarction within lipid rafts in CD14/CD16 circulating monocytes

Large numbers of monocytes are recruited in the infarcted myocardium. Their cell membranes contain cholesterol-rich microdomains called lipids rafts, which participate in numerous signaling cascades. In addition to its cholesterol-lowering effect, pravastatin has several pleiotropic effects and is widely used as secondary prevention treatment after myocardial infarction (MI). The aim of this study was to investigate the effects of pravastatin on the organization of cholesterol within monocyte membrane rafts from patients who had suffered myocardial infarction. Monocytes from healthy donors and acute MI patients were cultured with or without 4 μM pravastatin. Lipid rafts were extracted by Lubrol WX, caveolae and flat rafts were separated using a modified sucrose gradient. Cholesterol level and caveolin-1 expression in lipid rafts were determined. In healthy donors, cholesterol was concentrated in flat rafts (63 ± 3 vs 13 ± 1%, p < 0.001). While monocytes from MI patients presented similar cholesterol distribution in both caveolae and flat rafts. Cholesterol distribution was higher in flat rafts in healthy donors, compared to MI patients (63 ± 3 vs 41 ± 2%, p < 0.001), with less distribution in caveolae (13 ± 1 vs 34 ± 2%, p < 0.001). Pravastatin reversed the cholesterol distribution in MI patients cells between flat rafts (41 ± 2 vs 66 ± 3%, p < 0.001) and caveolae (34 ± 2 vs 18 ± 1%, p < 0.001). In conclusion, MI redistributes cholesterol from flat rafts to caveolae indicating monocyte membrane reorganization. In vitro pravastatin treatment restored basal conditions in MI monocytes, suggesting another effect of statins.     

GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis

Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G(1)-S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation.     

Novel Ru(II) oximato complexes with silent oxygen atom: Synthesis,chemistry and biological activities

A series of octahedral complexes, [Ru(CO)(EPh₃)₂(bhmh)] (E = P or As; H₂bhmh = benzoic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide), [Ru(CO)(EPh₃)₂(ihmh)] (H₂ihmh = isonicotinic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide), [Ru(CO)(EPh₃)₂(hhmh)] (H₂hhmh = 2-hydroxy-benzoic acid (2-hydroxyimino-1-methyl-propylidene)-hydrazide) have been prepared by a facile procedure. X-ray structure determination of three of the complexes revealed that the hydrazone ligand coordinates through the imine and the oxime nitrogen and the amide oxygen atoms. In all the complexes, the N–OH moiety of the oxime is deprotonated to give an N–O^? species and this oxygen atom did not coordinate to the central metal atom. The oxidation–reduction processes for each of these complexes have been determined in CH₃CN by cyclic voltammetry. The complexes displayed two oxidation couples and one irreversible reduction response between +1.6 and ?1.6 V. The trend in the half wave potentials reflects the electronic nature of the hydrazone ligand. Antibacterial activity of the ligands and the complexes has been evaluated against five pathogenic bacteria. The binding of the complexes with herring sperm DNA has also been investigated by UV–Vis spectroscopy and cyclic voltammetry.     

Validation of a parent-proxy, obesity-specific quality-of-life measure: sizing them up

The aims of the present study were to develop and validate a new obesity-specific, parent-proxy measure of health-related quality of life (HRQOL), Sizing Them Up. Participants included 220 obese youth (M(age) = 11.6 years, 68% female, 53% African American, M(BMI) = 36.7) and their primary caregivers (88% mothers). Primary caregivers completed a demographics questionnaire and two HRQOL measures: Sizing Them Up (obesity-specific) and PedsQL (generic). Youth height and weight were measured. Psychometric evaluation of Sizing Them Up was completed by conducting a factor analysis and determining internal consistency coefficients, test-retest reliability, convergent and discriminant validity, predictive validity, responsiveness to change, and minimal clinically important difference (MCID) scores. Sizing Them Up is a 22-item measure with six scales (i.e., Emotional Functioning, Physical Functioning, Teasing/Marginalization, Positive Social Attributes, Mealtime Challenges, and School Functioning) that account for 66% of the variance. The measure also includes an Adolescent Developmental Adaptation module. Sizing Them Up had internal consistency coefficients ranging from 0.59 to 0.91 and test-retest reliabilities ranging from 0.57 to 0.80. Validity was demonstrated by significant relations between a majority of Sizing Them Up scales and BMI z-scores. Sizing Them Up also demonstrated good convergent validity with other HRQOL measures and responsiveness to change related to weight loss for adolescents who had undergone bariatric surgery. Overall, Sizing Them Up is a reliable and valid parent-proxy measure of obesity-specific HRQOL that can be used in both clinical and research settings.     

Negative peer perceptions of obese children in the classroom environment

Objective: It is asserted that the more immediate and observable consequences of pediatric obesity are psychosocial in nature. This study examines the peer relations of clinically referred obese youth compared to demographically comparable nonoverweight peers within the classroom environment. Methods and Procedures: Peer‐, teacher‐, and self‐reports of behavioral reputation (Revised Class Play (RCP)), and peer reports of social acceptance, nonsocial attributes (attractiveness, athleticism, academic competence), and health interference (school absence, illness, fatigue) were obtained regarding 90 obese youth (BMI > 95th percentile; 8–16 years, 57% girls, 50% African American) and 76 nonoverweight demographically similar comparison classmates. Results: Relative to comparison peers, obese children were nominated significantly less often as a best friend and rated lower in peer acceptance, although the two groups did not differ in the number of reciprocated friendships. Obese youth were described by peer, teacher, and self‐report as more socially withdrawn and by peers as displaying less leadership and greater aggressive‐disruptive behavior. Peers also described obese youth as less physically attractive, less athletic, more sick, tired, and absent from school. Being seen as less attractive and less athletic by peers helped to explain differences in obese and nonoverweight youth's levels of peer acceptance. Discussion: Clinically referred obese youth are characterized by peer relations that differ from those of nonoverweight youth. The peer environment provides a rich context to understand the social consequences of pediatric obesity as well as factors that could be targeted in intervention to promote more positive health and psychosocial outcomes.