The role of macrophages in the clearance of inhaled ultrafine titanium dioxide particles

The role of macrophages in the clearance of particles with diameters less than 100 nm (ultrafine or nanoparticles) is not well established, although these particles deposit highly efficiently in peripheral lungs, where particle phagocytosis by macrophages is the primary clearance mechanism. To investigate the uptake of nanoparticles by lung phagocytes, we analyzed the distribution of titanium dioxide particles of 20 nm count median diameter in macrophages obtained by bronchoalveolar lavage at 1 hour and 24 hours after a 1-hour aerosol inhalation. Differential cell counts revealing greater than 96% macrophages and less than 1% neutrophils and lymphocytes excluded inflammatory cell responses. Employing energy-filtering transmission electron microscopy (EFTEM) for elemental microanalysis, we examined 1,594 macrophage profiles in the 1-hour group (n = 6) and 1,609 in the 24-hour group (n = 6). We found 4 particles in 3 macrophage profiles at 1 hour and 47 particles in 27 macrophage profiles at 24 hours. Model-based data analysis revealed an uptake of 0.06 to 0.12% ultrafine titanium-dioxide particles by lung-surface macrophages within 24 hours. Mean (SD) particle diameters were 31 (8) nm at 1 hour and 34 (10) nm at 24 hours. Particles were localized adjacent (within 13-83 nm) to the membrane in vesicles with mean (SD) diameters of 592 (375) nm at 1 hour and 414 (309) nm at 24 hours, containing other material like surfactant. Additional screening of macrophage profiles by conventional TEM revealed no evidence for agglomerated nanoparticles. These results give evidence for a sporadic and rather unspecific uptake of TiO(2)-nanoparticles by lung-surface macrophages within 24 hours after their deposition, and hence for an insufficient role of the key clearance mechanism in peripheral lungs.

Peripheral blood mononuclear cells represent a reservoir of bovine papillomavirus DNA in sarcoid-affected equines

Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from enciphered PBMC DNA derived from 66 horses, revealing E5 in the PBMCs of three individuals with confirmed sarcoids, whereas the remaining 63 sarcoid-free animals were negative for this gene. L1 could not be detected in any PBMC DNA, suggesting either deletion or interruption of this gene in PBMCs of BPV-1/-2-infected equines. These results support the hypothesis that PBMCs may serve as host cells for BPV-1/-2 DNA and contribute to virus latency.

[Pediatric cardiac disease: indications for family screening and risk stratification]

In some forms of cardiac disease in childhood, familial occurrence is frequent. This implies the question whether family members should undergo cardiologic screening examinations. For cardiomyopathies the familiarity is so frequent and morbidity so important that examination by echo of all first degree relatives is recommended. As these cardiomyopathies may develop its phenotype all along a lifetime, repetitive examinations usually are indicated. For the primary electrical diseases the so called channelopathies the same is true, as for the high rate of familial occurrence and the high morbidity. Thus ECG screening of first degree relatives is recommended. In a child with congenital heart disease there are no recommendations with regard to familial screening and cardiological examinations usually are indicated only in case of clinical suspicion for heart disease.

Human basophils and eosinophils are the direct target leukocytes of the novel IL-1 family member IL-33

In mice, interleukin-18 (IL-18) regulates Th1- or Th2-type immune responses depending on the cytokine environment and effector cells involved, and the ST2-ligand, IL-33, primarily promotes an allergic phenotype. Human basophils, major players in allergic inflammation, constitutively express IL-18 receptors, while ST2 surface expression is inducible by IL-3. Unexpectedly, freshly isolated basophils are strongly activated by IL-33, but, in contrast to mouse basophils, do not respond to IL-18. IL-33 promotes IL-4, IL-13 and IL-8 secretion in synergy with IL-3 and/or FcepsilonRI-activation, and enhances FcepsilonRI-induced mediator release. These effects are similar to that of IL-3, but the signaling pathways engaged are distinct because IL-33 strongly activates NF-kappaB and shows a preference for p38 MAP-kinase, while IL-3 acts through Jak/Stat and preferentially activates ERK. Eosinophils are the only other leukocyte-type directly activated by IL-33, as evidenced by screening of p38-activation in peripheral blood cells. Only upon CD3/CD28-ligation, IL-33 weakly enhances Th2 cytokine expression by in vivo polarized Th2 cells. This study on primary human cells demonstrates that basophils and eosinophils are the only direct target leukocytes for IL-33, suggesting that IL-33 promotes allergic inflammation and Th2 polarization mainly by the selective activation of these specialized cells of the innate immune system.

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.

Aberrant expression of FGFRL1, a novel FGF receptor, in ovarian tumors

FGFRL1 is a novel member of the FGF receptor family. It is expressed at very low levels in a great variety of cell lines and at relatively high levels in SW1353 chondrosarcoma cells, MG63 osteosarcoma cells and A204 rhabdomyosarcoma cells. Screening of 241 different human tumors with the help of a cancer profiling array suggested major alterations in the relative expression of FGFRL1 in ovarian tumors. Five distinct ovary tumors were therefore analyzed by quantitative and competitive PCR. Several tumors were found to exhibit a significant decrease in the expression of FGFRL1 in the tumor tissue relative to the matched control tissue. One ovarian tumor showed a 25-fold increase in the relative expression. Since FGFRL1 appears to be involved in the control of cell proliferation and differentiation, its aberrant expression might contribute to the development and progression of ovarian tumors.

Homeopathic treatment of Arabidopsis thaliana plants infected with Pseudomonas syringae

Homeopathic basic research is still in the screening phase to identify promising model systems that are adapted to the needs and peculiarities of homeopathic medicine and pharmacy. We investigated the potential of a common plant-pathogen system, Arabidopsis thaliana infected with the virulent bacteria Pseudomonas syringae, regarding its response towards a homeopathic treatment. A. thaliana plants were treated with homeopathic preparations before and after infection. Outcome measure was the number of P. syringae bacteria in the leaves of A. thaliana, assessed in randomized and blinded experiments. After a screening of 30 homeopathic preparations, we investigated the effect of Carbo vegetabilis 30x, Magnesium phosphoricum 30x, Nosode 30x, Biplantol (a homeopathic complex remedy), and Biplantol 30x on the infection rate in five or six independent experiments in total. The screening yielded significant effects for four out of 30 tested preparations. In the repeated experimental series, only the homeopathic complex remedy Biplantol induced a significant reduction of the infection rate (p = 0.01; effect size, d = 0.38). None of the other four repeatedly tested preparations (Carbo vegetabilis 30x, Magnesium phosphoricum 30x, Nosode 30x, Biplantol 30x) yielded significant effects in the overall evaluation. This phytopathological model yielded a small to medium effect size and thus might be of interest for homeopathic basic research after further improvement. Compared to Bion (a common SAR inducer used as positive control), the magnitude of the treatment effect of Biplantol was about 50%. Thus, homeopathic formulations might have a potential for the treatment of plant diseases after further optimization. However, the ecological impact should be investigated more closely before widespread application.

Emergence of Financial Intermediaries in Electronic Markets: The Case of Online P2P Lending

We analyze the role of intermediaries in electronic markets using detailed data of more than 14,000 originated loans on an electronic P2P (peer-to-peer) lending platform. In such an electronic credit market, lenders bid to supply a private loan. Screening of potential borrowers and the monitoring of loan repayment can be delegated to designated group leaders. We find that these market participants act as financial intermediaries and significantly improve borrowers' credit conditions by reducing information asymmetries, predominantly for borrowers with less attractive risk characteristics. Our findings may be surprising given the replacement of a bank by an electronic marketplace.

Virulence typing of Escherichia coli using microarrays

We describe a microarray based broad-range screening technique for Escherichia coli virulence typing. Gene probes were amplified by PCR from a plasmid bank of characterised E. coli virulence genes and were spotted onto a glass slide to form an array of capture probes. Genomic DNA from E. coli strains which were to be tested for the presence of these virulence gene sequences was labelled with fluorescent cyanine dyes by random amplification and then hybridised against the array of probes. The hybridisation, washing and data analysis conditions were optimised for glass slides, and the applicability of the method for identifying the presence of the virulence genes was determined using reference strains and clinical isolates. It was found to be a sensitive screening method for detecting virulence genes, and a powerful tool for determining the pathotype of E. coli. It will be possible to expand and automate this microarray technique to make it suitable for rapid and reliable diagnostic screening of bacterial isolates.

Monitoring of Urinary Calcium and Phosphorus Excretion in Preterm Infants - Comparison of two Methods

OBJECTIVES: Premature babies require supplementation with calcium and phosphorus to prevent metabolic bone disease of prematurity. To guide mineral supplementation, two methods of monitoring urinary excretion of calcium and phosphorus are used: urinary calcium or phosphorus concentration and calcium/creatinine or phosphorus/creatinine ratios. We compare these two methods in regards to their agreement on the need for mineral supplementation. METHODS: Retrospective chart review of 230 premature babies with birthweight <1500 g, undergoing screening of urinary spot samples from day 21 of life and fortnightly thereafter. Hypothetical cut-off values for urine calcium or phosphorus concentration (1 mmol/l) and urine calcium/creatinine ratio (0.5 mol/mol) or phosphorus/creatinine ratio (4 mol/mol) were applied to the sample results. The agreement on whether or not to supplement the respective minerals based on the results with the two methods was compared. Multivariate general linear models sought to identify patient characteristic to predict disagreeing results. RESULTS: 24.8% of cases disagreed on the indication for calcium supplementation, 8.8% for phosphorus. Total daily calcium intake was the only patient characteristic associated with discordant results. CONCLUSIONS: With the intention to supplement the respective mineral, comparison of urinary mineral concentration with mineral/creatinine ratio is moderate for Calcium and good for Phosphorus. The results do not allow to identify superiority of either method on the decision which babies require calcium and/or phosphorus supplements.

In vitro effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites.

Upon the screening of 16 antiproliferative compounds against Toxoplasma gondii and Neospora caninum, two hydrolytically stable ruthenium complexes (compounds 16 and 18) exhibited 50% inhibitory concentrations of 18.7 and 41.1 nM (T. gondii) and 6.7 and 11.3 nM (N. caninum). To achieve parasiticidal activity with compound 16, long-term treatment (22 to 27 days at 80 to 160 nM) was required. Transmission electron microscopy demonstrated the rapid impact on and ultrastructural alterations in both parasites. These preliminary findings suggest that the potential of ruthenium-based compounds should thus be further exploited.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate.

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.

Isolation and {\it in vitro}-immunosuppressive characterization of a novel cyclosporine metabolite

Cyclosporine (CsA) has shown great benefit to organ transplant recipients, as an immunosuppressive drug. To optimize CsA immunosuppressive therapy, pharmacodynamic evaluation of serial patient serum samples after CsA administration, using mixed lymphocyte culture (MLC) assays, revealed in vitro serum immunosuppressive activity of a CsA-like, ether-extractable component, associated with good clinical outcome in vivo. Since the in vitro immunosuppressive CsA metabolites, M-17 and M-1, are erythrocyte-bound, the immunosuppressive activity demonstrated in patient serum suggests that other immunosuppressive metabolites need exist. To test this hypothesis and obtain CsA metabolites for study, ether-extracted bile from tritiated and nonradioactive CsA-treated pigs was processed by novel high performance liquid and thin-layer chromatography (HPLC and HPTLC) techniques. Initial MLC screening of potential metabolites revealed a component, designated M-E, to have immunosuppressive activity. Pig bile-derived M-E was characterized as a CsA metabolite, by radioactive CsA tracer studies, by 56% crossreactivity in CsA radioimmunoassay, and by mass spectrometric (MS) analysis. MS revealed a CsA ring structure, hydroxylated at a site other than at amino acid one. M-E was different than M-1 and M-17, as demonstrated by different retention properties for each metabolite, using HPTLC and a novel rhodamine B/ $\alpha$-cyclodextrin stain, and using HPLC, performed by Sandoz, that revealed M-E to be different than previously characterized metabolites. The immunosuppressive activity of M-E was quantified by determination of mean metabolite potency ratio in human MLC assays, which was found to be 0.79 $\pm$ 0.23 (CsA, 1.0). Similar to parent drug, M-E revealed inter-individual differences in its immunosuppressive activity. M-E demonstrates inhibition of IL-2 production by concanavalin A stimulated C3H mouse spleen cells, similar to CsA, as determined with an IL-2 dependent mouse cytotoxic T-cell line. ^

Differential expression of urate oxidase in rat liver

An affinity-purified monospecific antibody was prepared to study the differential expression of the peroxisomal enzyme urate oxidase in rat liver during development and in various metabolic states. Monospecific antibody for urate oxidase was affinity purified from a pool of antibodies initially produced against a mixture of proteins from a Percoll density gradient fraction. Immunogold staining of samples of the gradient fraction and rat liver tissue with the affinity-purified antibody demonstrated labelling of peroxisomal core structures. Screening of liver homogenates from rats at different developmental stages using immunoblot analysis demonstrated low levels of urate oxidase prior to 20 days of age; at 20 days of age, urate oxidase levels are 2-fold greater than the 15-day old levels and approximate adult levels. Catalase expression during rat development mimicked the differential expression pattern of urate oxidase. The increase between days 15 and 20 was determined to be independent of the process of weaning. Administration of exogenous glucocorticoid hormone to 10-day old rats resulted in a precocious rise (2.5-fold) in urate oxidase levels, but adrenalectomy at 10 days of age did not cause decreased expression in the fourth week of life. In adult animals, exogenous glucocorticoid did not influence urate oxidase levels, but adrenalectomized rats had urate oxidase levels that were 40 percent of control expression 4 days post-surgery. Catalase expression was not influenced by glucocorticoid status in these studies. Glucocorticoid regulation of urate oxidase expression appears to be one part of a more complex mechanism controlling levels of the enzyme. Exogenous glucocorticoid administration influenced urate oxidase levels in an age-dependent manner; in addition, it is possible that the control mechanism for urate oxidase may include factors which can modulate expression in the absence of glucocorticoids. The effect of glucocorticoids on urate oxidase expression can not be extended to include all peroxisomal proteins, since catalase is unaffected. Glucocorticoids appear to participate in the complex regulation of urate oxidase expression; glucocorticoids influence urate oxidase specifically and do not modulate all peroxisomal proteins. ^