Host resistance and pathogen infectivity in host populations with varying connectivity

Theory predicts that hosts and pathogens will evolve higher resistance and aggressiveness in systems where populations are spatially connected than in situations in which populations are isolated and dispersal is more local. In a large cross‐inoculation experiment we surveyed patterns of host resistance and pathogen infectivity in anther‐smut diseased Viscaria alpina populations from three contrasting areas where populations range from continuous, through patchy but spatially connected to highly isolated demes. In agreement with theory, isolated populations of V. alpina were more susceptible on average than either patchily distributed or continuous populations. While increased dispersal in connected systems increases disease spread, it may also increase host gene flow and the potential for greater host resistance to evolve. In the Viscaria–Microbotryum system, pathogen infectivity mirrored patterns of host resistance with strains from the isolated populations being the least infective and strains from the more resistant continuous populations being the most infective on average, suggesting that high resistance selects for high infectivity. To our knowledge this study is the first to characterize the impacts of varying spatial connectivity on patterns of host resistance and pathogen infectivity in a natural system.     

Stochastic Dynamic Model for Estimation of Rate Constants and Their Variances from Noisy and Heterogeneous PET Measurements

Tissue heterogeneity, radioactive decay and measurement noise are the main error sources in compartmental modeling used to estimate the physiologic rate constants of various radiopharmaceuticals from a dynamic PET study. We introduce a new approach to this problem by modeling the tissue heterogeneity with random rate constants in compartment models. In addition, the Poisson nature of the radioactive decay is included as a Poisson random variable in the measurement equations. The estimation problem will be carried out using the maximum likelihood estimation. With this approach, we do not only get accurate mean estimates for the rate constants, but also estimates for tissue heterogeneity within the region of interest and other possibly unknown model parameters, e.g. instrument noise variance, as well. We also avoid the problem of the optimal weighting of the data related to the conventionally used weighted least-squares method. The new approach was tested with simulated time–activity curves from the conventional three compartment – three rate constants model with normally distributed rate constants and with a noise mixture of Poisson and normally distributed random variables. Our simulation results showed that this new model gave accurate estimates for the mean of the rate constants, the measurement noise parameter and also for the tissue heterogeneity, i.e. for the variance of the rate constants within the region of interest.     

MEK-ERK-mediated phosphorylation of Mdm2 at Ser-166 in hepatocytes. Mdm2 is activated in response to inhibited Akt signaling

Mdm2 inactivates the tumor suppressor p53 and Akt has been shown to be a major activator of Mdm2 in many cell types. We have investigated the regulation of Mdm2 in hepatocytes. We found that growth factor-induced Ser-166 phosphorylation of Mdm2 was inhibited by the MEK inhibitors U0126 and PD98059 in HepG2 cells and in a rat liver cell line, TRL 1215. Also, bile acids and oxidative stress induced phosphorylation of Mdm2 at Ser-166 by an apparently MEK-ERK-dependent mechanism. In contrast, Ser-166 phosphorylation of Mdm2 in lung cells was mediated by Akt. Further studies revealed that phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin induced phosphorylated ERK Tyr-204 and pMdm2 Ser-166 phosphorylations in hepatocytes in culture and in rat hepatocytes in vivo. In HepG2 cells, this effect was inhibited by U0126 and PD98059. LY294002 also reduced the level of pRaf Ser-259. Furthermore, we have shown that myr-Akt-induced overexpression of pAkt suppressed the levels of pMdm2 Ser-166 in hepatocytes. These data indicate a reversed relationship between Akt and Mdm2 in hepatocytes and suggest that Akt is a negative regulator of Raf-MEK-ERK-Mdm2 in this cell type. Ser-166 phosphorylation of Mdm2 has been shown to increase its ubiquitin ligase activity and increase p53 degradation, and our data indicated an attenuated p53 response to DNA damage in hepatocytes exhibiting high levels of pMdm2 Ser-166. Taken together, our data indicate that Mdm2 phosphorylation is regulated via MEK-ERK in hepatocytes. This Mdm2 signaling might be important for the regeneration of hepatocytes after centrilobular cell death.     

Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding

Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3zeta protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172) and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.     

The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter

The late-infantile-onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile-onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide-polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted "MGC33302"), which encodes a novel polytopic 518-amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs.     

Novel route for elimination of brain oxysterols across the blood-brain barrier: conversion into 7alpha-hydroxy-3-oxo-4-cholestenoic acid

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mug/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C(27) steroidal acid 7alpha-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7alpha-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7alpha-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.     

XPA A23G, XPC Lys939Gln, XPD Lys751Gln and XPD Asp312Asn polymorphisms, interactions with smoking, alcohol and dietary factors, and risk of colorectal cancer

Polymorphisms in the XPD and the XPC gene have been associated with a lower DNA repair capacity. We determined the risk of colorectal cancer in association with the four polymorphisms XPA A23G, XPC Lys939Gln, XPD Lys751Gln and XPD Asp312Asn, and interactions between the polymorphisms and the environmental factors: smoking intensity, intake of alcohol, red meat, processed meat, fish and poultry, fruits and vegetables and dietary fibres, in relation to development of colorectal cancer in a study population of 405 colorectal cancer cases and a comparison group of 810 persons, nested within the Danish prospective cohort, Diet, Cancer and Health, of 57053 cohort members. No association was found between the XPC Lys939Gln, XPA A23G, XPD Lys751Gln, and XPD Asp312Asn polymorphisms and risk of colorectal cancer. The association of the XPD Lys751Gln polymorphism was statistically significantly different between genders, with a lower risk of colorectal cancer among women carrying the variant allele. We observed a statistically significant interaction between the XPC Lys939Gln polymorphism and consumption of red meat, with a 3.7-fold increase in colorectal cancer risk per 100g red meat intake per day among carriers of the homozygous variant, but virtually no effect of red meat intake among carriers of the wild type allele. In the light of the multiple comparisons being made, this result may be a chance finding. The results showed no interaction between the XPD Lys751Gln, XPA A23G, and XPD Asp312Asn polymorphisms and the environmental factors for the development of colorectal cancer. Overall, the results of the present study indicate that the four polymorphisms are not of major importance in colorectal cancer carcinogenesis.     

Genotoxicity, inflammation and physico-chemical properties of fine particle samples from an incineration energy plant and urban air

Airborne particulate matter (PM) was sampled by use of an electrostatic sampler in an oven hall and a receiving hall in a waste-incineration energy plant, and from urban air in a heavy-traffic street and from background air in Copenhagen. PM was sampled for 1-2 weeks, four samples at each site. The samples were extracted and examined for mutagenicity in Salmonella typhimurium strains TA98, YG1041 and YG5161, for content of inorganic elements and for the presence of eight polycyclic aromatic hydrocarbons. The induction of IL-6 and IL-8 mRNA expression and the presence of DNA damage - tested by the comet assay - were determined after 24-h incubations with human A549 lung epithelial cells. The PM(2.5) concentration was about twofold greater in the oven hall than in the receiving hall. The particle size distribution in the receiving hall was similar to that in street air (maximum mode at about 25nm), but the distribution was completely different in the oven hall (maximum mode at about 150nm). Also chemically, the samples from the oven hall were highly different from the other samples. PM extracts from the receiving hall, street and background air were more mutagenic than the PM extracts from the oven hall. PM from all four sites caused similar levels of DNA damage in A549 cells; only the oven hall samples gave results that were statistically significantly different from those obtained with street-air samples. The receiving hall and the urban air samples were similarly inflammatory (relative IL-8 mRNA expression), whereas the oven hall did not cause a statistically significant increase in IL-8 mRNA expression. A principal component analysis separated the oven hall and the receiving hall by the first principal component. These two sites were separated from street and background air with the second principal component. Several clusters of constituents were identified. One cluster consisted of all the polycyclic aromatic hydrocarbons (PAH), several groups of metals and one group of the biological endpoints (DNA damage, IL-6 and IL-8 mRNA expression). The PAH and the inorganic content of the air in the receiving hall may be due to vehicle emissions and suspended waste particles. The inorganic content in the street and background air may have been influenced by break wear, road emissions and long-range transport. The results from a partial least-square regression analysis predicted that both PAHs and a group of metals including Fe and Mn contributed to IL-6 and IL-8 induction. Only Mn and Sr were predicted to influence DNA damage statistically significantly. Among the PAHs only chrysene had influence on DNA damage. The PM from the oven hall was markedly different from the PM at other locations in particle size distribution, chemical composition and the resulting biological effects when A549 cells were incubated with the PM. These characteristics and observations in the oven hall indicated that the PM source was oven exhaust, which was well combusted.     

Elevated Membrane and Soluble CD64: A Novel Marker Reflecting Altered FcγR Function and Disease in Early Rheumatoid Arthritis That Can Be Regulated by Anti-Rheumatic Treatment

Objectives

Fc receptors (FcR) interacting with immune complexes (ICs) is a central event in the immune pathogenesis of rheumatoid arthritis (RA). Here we asked if a specific FcR is linked to RA pathogenesis and if FcR activities relate to disease and treatment outcome in early RA.

Material and Methods

Twenty autoantibody-positive RA patients and 33 HC were included. The patients were evaluated before and after treatment with methotrexate and prednisolone. At follow-up, the EULAR response criteria were applied to determine the individual treatment outcomes. Serum immunoglobulin levels were measured and the expression of FcR for IgG (FcγR) and IgA (FcαR) on peripheral blood monocytes were determined by flow cytometry. The monocytic FcγR function was evaluated by human IgG1 and IgG3 IC-binding and TNFα stimulated release. Plasma levels of soluble FcRs (sFcRs) were determined with ELISA.

Results

The IgG1 and IgG3 levels were elevated in the RA sera. The RA monocytes expressed more CD64 and cell surface-bound IgG than HC monocytes, and showed an impaired FcγR function as reflected by changes in IC-binding and decreased IC-stimulated TNFα secretion. These findings correlated significantly with different disease activity markers. Furthermore, sFcRs were elevated in the patient plasma, and sCD64 was specific for RA (compared with a reference group of patients with active psoriatic arthritis). Following treatment, immunoglobulins and sFcR levels were reduced, whereas membrane CD64 was only decreased in patients with good response to treatment.

Conclusions

Early RA patients display increased membrane and soluble CD64 and an impaired FcγR function correlating with joint disease activity. Beneficial responses of anti-rheumatic treatment in patients reduce CD64. These data suggest sCD64 as an important objective biomarker in RA.