Risk factors for infection with coagulase-negative staphylococci in newborns from the neonatal unit of a brazilian university hospital

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Perfil fenotípico de potenciais células iniciadoras tumorais no tumor venéreo transmissível canino ex vivo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES: FTY720 modulates CD4(+)T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4(+) graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

Immune response and tolerability of varicella vaccine in children and adolescents with systemic lupus erythematosus previously exposed to varicella-zoster virus

Objectives The aim of the present paper is to evaluate the immune response and tolerability of varicella vaccine in children and adolescents with systemic lupus erythematosus previously exposed to varicella-zoster virus. Methods We performed a prospective and controlled study on a group of 54 SLE patients that were chosen at random to be or not to be vaccinated (28 were vaccinated and 26 were not). Twenty-eight healthy controls, of matching age and sex were also vaccinated. All were submitted to a questionnaire, physical evaluation and laboratory assays: lymphocyte immuno-phenotyping by flow cytometry, plasma varicella zoster virus (VZV) serology by ELISA and in vitro interferon gamma (IFN gamma) production by T-cells after stimulus with VZV antigen. They were evaluated before vaccination and at 30, 45, 180 and 360 days afterwards. Results We did not observe any differences in the frequency of adverse events in both vaccinated groups. At study entry, all individuals were seropositive for VZV antibodies. The serum VZV antibody titres similarly increased after vaccination. The frequency of flares and the SLEDAI score were also similar among the patients. Thirty days after vaccination the production of IFN gamma specific to VZV was lower in the SLE group compared to healthy, controls. In the follow-up we observed 4 cases of herpes zoster in the SLE unvaccinated group, but no zoster in the vaccinated group. Conclusion The varicella vaccine was well tolerated in SLE group, who had pre-existing immunity to varicella. The varicella vaccine immunogenicity measurement by serum antibody titres was appropriate. The incidence of HZ was lower in the vaccinated lupus group.

Period determination in the food-entrainable and methamphetamine-sensitive circadian oscillator(s)

Daily rhythmic processes are coordinated by circadian clocks, which are present in numerous central and peripheral tissues. In mammals, two circadian clocks, the food-entrainable oscillator (FEO) and methamphetamine-sensitive circadian oscillator (MASCO), are "black box" mysteries because their anatomical loci are unknown and their outputs are not expressed under normal physiological conditions. In the current study, the investigation of the timekeeping mechanisms of the FEO and MASCO in mice with disruption of all three paralogs of the canonical clock gene, Period, revealed unique and convergent findings. We found that both the MASCO and FEO in Per1(-/-)/Per2(-/-)/Per3(-/-) mice are circadian oscillators with unusually short (similar to 21 h) periods. These data demonstrate that the canonical Period genes are involved in period determination in the FEO and MASCO, and computational modeling supports the hypothesis that the FEO and MASCO use the same timekeeping mechanism or are the same circadian oscillator. Finally, these studies identify Per1(-/-)/Per2(-/-)/Per3(-/-) mice as a unique tool critical to the search for the elusive anatomical location(s) of the FEO and MASCO.

CD44(+)/CD24(-) cells and lymph node metastasis in stage I and II invasive ductal carcinoma of the breast

The presence of tumor-initiating cells (CD44(+)/CD24(-)) in solid tumors has been reported as a possible cause of cancer metastasis and treatment failure. Nevertheless, little is know about the presence of CD44(+)/CD24(-) cells within the primary tumor and metastasis. The proportion of CD44(+)/CD24(-) cells was analyzed in 40 samples and in 10 lymph node metastases using flow cytometry phenotyping. Anti-human CD326 (EpCam; FITC), antihuman CD227 (MUC-1; FITC), anti-human CD44 (APC), and anti-human CD24 (PE), anti-ABCG2 (PE), and anti-CXCR4 (PeCy7) were used for phenotype analysis. The mean patient age was 60.5 years (range, 33-87 years); mean primary tumor size (pT) was 1.8 cm (0.5-3.5 cm). The Wilcoxon or Kruskal-Wallis test was used for univariate analyses. Logistic regression was used for multivariate analysis. The median percentage of CD44(+)/CD24(-) cells within primary invasive ductal carcinomas (IDC) was 2.7% (range, 0.2-71.2). In lymph node metastases, we observed a mean of 6.1% (range, 0.07-53.7). The percentage of CD44(+)/CD24(-) cells in IDCs was not associated with age, pT, tumor grade and HER2. We observed a significantly enrichment of CD44(+)/CD24(-) and ABCG2(+) cells in ESA(+) cell population in patients with positive lymph nodes (P = 0.02 and P = 0.04, respectively). Our data suggest that metastatic dissemination is associated with an increase in tumorinitiating cells in stage I and II breast cancer.

Methods and strategies to identify the mode of action of phytoactive compounds

Small molecules affecting biological processes in plants are widely used in agricultural practice as herbicides or plant growth regulators and in basic plant sciences as probes to study the physiology of plants. Most of the compounds were identified in large screens by the agrochemical industry, as phytoactive natural products and more recently, novel phytoactive compounds originated from academic research by chemical screens performed to induce specific phenotypes of interest. The aim of the present PhD thesis is to evaluate different approaches used for the identification of the primary mode of action (MoA) of a phytoactive compound. Based on the methodologies used for MoA identification, three approaches are discerned: a phenotyping approach, an approach based on a genetic screen and a biochemical screening approach.rnFour scientific publications resulting from my work are presented as examples of how a phenotyping approach can successfully be applied to describe the plant MoA of different compounds in detail.rnI. A subgroup of cyanoacrylates has been discovered as plant growth inhibitors. A set of bioassays indicated a specific effect on cell division. Cytological investigations of the cell division process in plant cell cultures, studies of microtubule assembly with green fluorescent protein marker lines in vivo and cross resistant studies with Eleusine indica plants harbouring a mutation in alpha-tubulin, led to the description of alpha-tubulin as a target site of cyanoacrylates (Tresch et al., 2005).rnII. The MoA of the herbicide flamprop-m-methyl was not known so far. The studies described in Tresch et al. (2008) indicate a primary effect on cell division. Detailed studies unravelled a specific effect on mitotic microtubule figures, causing a block in cell division. In contrast to other inhibitors of microtubule rearrangement such as dinitroanilines, flamprop-m-methyl did not influence microtubule assembly in vitro. An influence of flamprop-m-methyl on a target within the cytoskeleton signalling network could be proposed (Tresch et al., 2008).rnIII. The herbicide endothall is a protein phosphatase inhibitor structurally related to the natural product cantharidin. Bioassay studies indicated a dominant effect on dark-growing cells that was unrelated to effects observed in the light. Cytological characterisation of the microtubule cytoskeleton in corn tissue and heterotrophic tobacco cells showed a specific effect of endothall on mitotic spindle formation and ultrastructure of the nucleus in combination with a decrease of the proliferation index. The observed effects are similar to those of other protein phosphatase inhibitors such as cantharidin and the structurally different okadaic acid. Additionally, the observed effects show similarities to knock-out lines of the TON1 pathway, a protein phosphatase-regulated signalling pathway. The data presented in Tresch et al. (2011) associate endothall’s known in vitro inhibition of protein phosphatases with in vivo-effects and suggest an interaction between endothall and the TON1 pathway.rnIV. Mefluidide as a plant growth regulator induces growth retardation and a specific phenotype indicating an inhibition of fatty acid biosynthesis. A test of the cuticle functionality suggested a defect in the biosynthesis of very-long-chain fatty acids (VLCFA) or waxes. Metabolic profiling studies showed similarities with different groups of VLCFA synthesis inhibitors. Detailed analyses of VLCFA composition in tissues of duckweed (Lemna paucicostata) indicated a specific inhibition of the known herbicide target 3 ketoacyl-CoA synthase (KCS). Inhibitor studies using a yeast expression system established for plant KCS proteins verified the potency of mefluidide as an inhibitor of plant KCS enzymes. It could be shown that the strength of inhibition varied for different KCS homologues. The Arabidopsis Cer6 protein, which induces a plant growth phenotype similar to mefluidide when knocked out, was one of the most sensitive KCS enzymes (Tresch et al., 2012).rnThe findings of my own work were combined with other publications reporting a successful identification of the MoA and primary target proteins of different compounds or compound classes.rnA revised three-tier approach for the MoA identification of phytoactive compounds is proposed. The approach consists of a 1st level aiming to address compound stability, uniformity of effects in different species, general cytotoxicity and the effect on common processes like transcription and translation. Based on these findings advanced studies can be defined to start the 2nd level of MoA characterisation, either with further phenotypic characterisation, starting a genetic screen or establishing a biochemical screen. At the 3rd level, enzyme assays or protein affinity studies should show the activity of the compound on the hypothesized target and should associate the in vitro effects with the in vivo profile of the compound.

Fine Mapping of qroot-yield-1.06, a QTL for Root, Plant Vigor and Yield in Maize

Root-yield-1.06 is a major QTL affecting root system architecture (RSA) and other agronomic traits in maize. The effect of this QTL has been evaluated with the development of near isogenic lines (NILs) differing at the QTL position. The objective of this study was to fine map qroot-yield-1.06 by marker-assisted searching for chromosome recombinants in the QTL interval and concurrent root phenotyping in both controlled and field conditions, through successive generations. Complementary approaches such as QTL meta-analysis and RNA-seq were deployed in order to help prioritizing candidate genes within the QTL target region. Using a selected group of genotypes, field based root analysis by ‘shovelomics’ enabled to accurately collect RSA information of adult maize plants. Shovelomics combined with software-assisted root imaging analysis proved to be an informative and relatively highly automated phenotyping protocol. A QTL interval mapping was conducted using a segregating population at the seedling stage grown in controlled environment. Results enabled to narrow down the QTL interval and to identify new polymorphic markers for MAS in field experiments. A collection of homozygous recombinant NILs was developed by screening segregating populations with markers flanking qroot-yield-1.06. A first set of lines from this collection was phenotyped based on the adapted shovelomics protocol. QTL analysis based on these data highlighted an interval of 1.3 Mb as completely linked with the target QTL but, a larger safer interval of 4.1 Mb was selected for further investigations. QTL meta-analysis allows to synthetize information on root QTLs and two mQTLs were identified in the qroot-yield-1.06 interval. Trascriptomics analysis based on RNA-seq data of the two contrasting QTL-NILs, confirmed alternative haplotypes at chromosome bin 1.06. qroot-yield-1.06 has now been delimited to a 4.1-Mb interval, and thanks to the availability of additional untested homozygous recombinant NILs, the potentially achievable mapping resolution at qroot-yield-1.06 is c. 50 kb.

In-frame triplet deletions in RHD alter the D antigen phenotype

BACKGROUND: The deletion of three adjacent nucleotides in an exon may cause the lack of a single amino acid, while the protein sequence remains otherwise unchanged. Only one such in-frame deletion is known in the two RH genes, represented by the RHCE allele ceBP expressing a "very weak e antigen." STUDY DESIGN AND METHODS: Blood donor samples were recognized because of discrepant results of D phenotyping. Six samples came from Switzerland and one from Northern Germany. The molecular structures were determined by genomic DNA nucleotide sequencing of RHD. RESULTS: Two different variant D antigens were explained by RHD alleles harboring one in-frame triplet deletion each. Both single-amino-acid deletions led to partial D phenotypes with weak D antigen expression. Because of their D category V-like phenotypes, the RHD(Arg229del) allele was dubbed DVL-1 and the RHD(Lys235del) allele DVL-2. These in-frame triplet deletions are located in GAGAA or GAAGA repeats of the RHD exon 5. CONCLUSION: Partial D may be caused by a single-amino-acid deletion in RhD. The altered RhD protein segments in DVL types are adjacent to the extracellular loop 4, which constitutes one of the most immunogenic parts of the D antigen. These RhD protein segments are also altered in all DV, which may explain the similarity in phenotype. At the nucleotide level, the triplet deletions may have resulted from replication slippage. A total of nine amino acid positions in an Rhesus protein may be affected by this mechanism.

Angiofil-mediated visualization of the vascular system by microcomputed tomography: a feasibility study

Visualization of the vascular systems of organs or of small animals is important for an assessment of basic physiological conditions, especially in studies that involve genetically manipulated mice. For a detailed morphological analysis of the vascular tree, it is necessary to demonstrate the system in its entirety. In this study, we present a new lipophilic contrast agent, Angiofil, for performing postmortem microangiography by using microcomputed tomography. The new contrast agent was tested in 10 wild-type mice. Imaging of the vascular system revealed vessels down to the caliber of capillaries, and the digital three-dimensional data obtained from the scans allowed for virtual cutting, amplification, and scaling without destroying the sample. By use of computer software, parameters such as vessel length and caliber could be quantified and remapped by color coding onto the surface of the vascular system. The liquid Angiofil is easy to handle and highly radio-opaque. Because of its lipophilic abilities, it is retained intravascularly, hence it facilitates virtual vessel segmentation, and yields an enduring signal which is advantageous during repetitive investigations, or if samples need to be transported from the site of preparation to the place of actual analysis, respectively. These characteristics make Angiofil a promising novel contrast agent; when combined with microcomputed tomography, it has the potential to turn into a powerful method for rapid vascular phenotyping.

Phenotypic and genetic characterization of Pasteurella multocida and related isolates from rabbits in Switzerland

Several bacteria belonging to the family Pasteurellaceae are potential pathogens in rabbits. In particular, Pasteurella multocida is considered to be important, and outbreaks caused by this species result in considerable economic losses in rabbitries. However, Pasteurellaceae spp. isolated from rabbits are poorly characterized, and thus, proper identification of P. multocida isolates from these animals is problematic and often unsatisfactory, thereby hampering epidemiological investigations. Therefore, 228 isolates from rabbit populations originating from a breeding and fattening organization with group management and postmortem cases with pasteurellosis from individual owners were phenotypically and genotypically analyzed using biochemical tests and repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Furthermore, 41 samples representing observed phenotypes were selected for phylogenetic analysis using 16S ribosomal RNA and rpoB genes. The REP-PCR typing and phylogenetic analyses correlated well and appeared to be distinct molecular methods for characterization of rabbit isolates. Phenotyping, however, diverged from molecular recognition, reflecting the problematic conventional diagnosis of these strains. The fermentation of sorbitol appeared to be an imprecise indicator for P. multocida subspecies classification. According to REP-PCR and sequencing results, 82% of the isolates were characterized as P. multocida subsp. multocida, 3% as P. multocida subsp. septica, and 5% as P. multocida. Further, 5% were identified as Pasteurella canis. The other 5% represented a homogeneous group of unknown species belonging to the Pasteurellaceae. Samples obtained from individual postmortem cases demonstrated a higher phenotypic and genetic heterogeneity than samples from group management rabbits.

Neural reprogramming in retinal degeneration.

PURPOSE: Early visual defects in degenerative diseases such as retinitis pigmentosa (RP) may arise from phased remodeling of the neural retina. The authors sought to explore the functional expression of ionotropic (iGluR) and group 3, type 6 metabotropic (mGluR6) glutamate receptors in late-stage photoreceptor degeneration. METHODS: Excitation mapping with organic cations and computational molecular phenotyping were used to determine whether retinal neurons displayed functional glutamate receptor signaling in rodent models of retinal degeneration and a sample of human RP. RESULTS: After photoreceptor loss in rodent models of RP, bipolar cells lose mGluR6 and iGluR glutamate-activated currents, whereas amacrine and ganglion cells retain iGluR-mediated responsivity. Paradoxically, amacrine and ganglion cells show spontaneous iGluR signals in vivo even though bipolar cells lack glutamate-coupled depolarization mechanisms. Cone survival can rescue iGluR expression by OFF bipolar cells. In a case of human RP with cone sparing, iGluR signaling appeared intact, but the number of bipolar cells expressing functional iGluRs was double that of normal retina. CONCLUSIONS: RP triggers permanent loss of bipolar cell glutamate receptor expression, though spontaneous iGluR-mediated signaling by amacrine and ganglion cells implies that such truncated bipolar cells still release glutamate in response to some nonglutamatergic depolarization. Focal cone-sparing can preserve iGluR display by nearby bipolar cells, which may facilitate late RP photoreceptor transplantation attempts. An instance of human RP provides evidence that rod bipolar cell dendrite switching likely triggers new gene expression patterns and may impair cone pathway function.

Cost and power analysis of linkage disequilibrium methods for locating genes influencing quantitative traits in humans

Linkage disequilibrium methods can be used to find genes influencing quantitative trait variation in humans. Linkage disequilibrium methods can require smaller sample sizes than linkage equilibrium methods, such as the variance component approach to find loci with a specific effect size. The increase in power is at the expense of requiring more markers to be typed to scan the entire genome. This thesis compares different linkage disequilibrium methods to determine which factors influence the power to detect disequilibrium. The costs of disequilibrium and equilibrium tests were compared to determine whether the savings in phenotyping costs when using disequilibrium methods outweigh the additional genotyping costs.^ Nine linkage disequilibrium tests were examined by simulation. Five tests involve selecting isolated unrelated individuals while four involved the selection of parent child trios (TDT). All nine tests were found to be able to identify disequilibrium with the correct significance level in Hardy-Weinberg populations. Increasing linked genetic variance and trait allele frequency were found to increase the power to detect disequilibrium, while increasing the number of generations and distance between marker and trait loci decreased the power to detect disequilibrium. Discordant sampling was used for several of the tests. It was found that the more stringent the sampling, the greater the power to detect disequilibrium in a sample of given size. The power to detect disequilibrium was not affected by the presence of polygenic effects.^ When the trait locus had more than two trait alleles, the power of the tests maximized to less than one. For the simulation methods used here, when there were more than two-trait alleles there was a probability equal to 1-heterozygosity of the marker locus that both trait alleles were in disequilibrium with the same marker allele, resulting in the marker being uninformative for disequilibrium.^ The five tests using isolated unrelated individuals were found to have excess error rates when there was disequilibrium due to population admixture. Increased error rates also resulted from increased unlinked major gene effects, discordant trait allele frequency, and increased disequilibrium. Polygenic effects did not affect the error rates. The TDT, Transmission Disequilibrium Test, based tests were not liable to any increase in error rates.^ For all sample ascertainment costs, for recent mutations ($<$100 generations) linkage disequilibrium tests were less expensive than the variance component test to carry out. Candidate gene scans saved even more money. The use of recently admixed populations also decreased the cost of performing a linkage disequilibrium test. ^

Clinical and biochemical profiles suggest fibromuscular dysplasia is a systemic disease with altered TGF-β expression and connective tissue features.

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor β (TGF-β) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P<0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF-β1 (P=0.009), TGF-β2 (P=0.004) and additional inflammatory markers, and increased TGF-β1 (P=0.0009) and TGF-β2 (P=0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age- and gender-matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF-β signaling and offers TGF-β as a marker of FMD.

Implementation of a mandatory donor RHD screening in Switzerland

Starting in 2013, blood donors must be tested at least using: (1) one monoclonal anti-D and one anti-CDE (alternatively full RhCcEe phenotyping), and (2) all RhD negative donors must be tested for RHD exons 5 and 10 plus one further exonic, or intronic RHD specificity, according to the guidelines of the Blood Transfusion Service of the Swiss Red Cross (BTS SRC). In 2012 an adequate stock of RHD screened donors was built. Of all 25,370 RhD negative Swiss donors tested in 2012, 20,015 tested at BTS Berne and 5355 at BTS Zürich, showed 120 (0.47%) RHD positivity. Thirty-seven (0.15%) had to be redefined as RhD positive. Routine molecular RHD screening is reliable, rapid and cost-effective and provides safer RBC units in Switzerland.