The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis

Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1β, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.

Optimization of a label-free biosensor vertically characterized based on a periodic lattice of high aspect ratio SU-8 nano-pillars with a simplified 2D theoretical model

We have recently demonstrated a biosensor based on a lattice of SU8 pillars on a 1 μm SiO2/Si wafer by measuring vertically reflectivity as a function of wavelength. The biodetection has been proven with the combination of Bovine Serum Albumin (BSA) protein and its antibody (antiBSA). A BSA layer is attached to the pillars; the biorecognition of antiBSA involves a shift in the reflectivity curve, related with the concentration of antiBSA. A detection limit in the order of 2 ng/ml is achieved for a rhombic lattice of pillars with a lattice parameter (a) of 800 nm, a height (h) of 420 nm and a diameter(d) of 200 nm. These results correlate with calculations using 3D-finite difference time domain method. A 2D simplified model is proposed, consisting of a multilayer model where the pillars are turned into a 420 nm layer with an effective refractive index obtained by using Beam Propagation Method (BPM) algorithm. Results provided by this model are in good correlation with experimental data, reaching a reduction in time from one day to 15 minutes, giving a fast but accurate tool to optimize the design and maximizing sensitivity, and allows analyzing the influence of different variables (diameter, height and lattice parameter). Sensitivity is obtained for a variety of configurations, reaching a limit of detection under 1 ng/ml. Optimum design is not only chosen because of its sensitivity but also its feasibility, both from fabrication (limited by aspect ratio and proximity of the pillars) and fluidic point of view. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Cell surface expression of the Alzheimer disease-related presenilin proteins

The presenilin proteins PS-1 and PS-2 are crucially involved in Alzheimer disease (AD), but their molecular functions are not known. They are integral membrane proteins, but whether they can be expressed at the surface of cells has been in dispute. Here we show by immunofluorescence experiments, using anti-peptide antibodies specific for either PS-1 or PS-2, that live cultured DAMI cells and differentiated human NT2N neuronal cells are specifically immunolabeled for their endogenous as well as transfected presenilins, although the cells cannot be immunolabeled for their intracellular tubulin, unless they are first fixed and permeabilized. These and other results establish that portions of the presenilins are indeed expressed at the surfaces of these cells. These findings support our previous proposal that the presenilins on the surface of a cell engage in intercellular interactions with the β-amyloid precursor protein on the surface of a neighboring cell, as a critical step in the molecular and cellular mechanisms that lead to AD.

A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity

We have characterized a nontoxic mutant of cholera toxin (CT) as a mucosal adjuvant in mice. The mutant CT was made by substitution of serine with phenylalanine at position 61 of the A subunit (S61F), which resulted in loss of ADP ribosyltransferase activity and toxicity. Mice were intranasally immunized with ovalbumin, tetanus toxoid, or influenza virus either alone or together with mutant CT S61F, native CT, or recombinant CT-B. Mice immunized with these proteins plus S61F showed high serum titers of protein-specific IgG and IgA antibodies that were comparable to those induced by native CT. Further, high protein-specific IgA antibody responses were observed in nasal and vaginal washes, saliva, and fecal extracts as well as increased numbers of IgG and IgA antibody forming cells in cervical lymph nodes and lung tissues of mice intranasally immunized with these proteins and S61F or native CT, but not with recombinant CT-B or protein alone. Both S61F and native CT enhanced the induction of ovalbumin-specific CD4+ T cells in lung and splenic tissues, and these T cells produced a Th2-type cytokine pattern of interleukin 4 (IL-4), IL-5, IL-6, and IL-10 as determined by analysis of secreted proteins and by quantitation of cytokine-specific mRNA. These results have shown that mutant CT S61F is an effective mucosal adjuvant when administrated intranasally and induces mucosal and systemic antibody responses which are mediated by CD4+ Th2-type cells.

Human interleukin-10-related T cell-derived inducible factor: Molecular cloning and functional characterization as an hepatocyte-stimulating factor

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, α1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the β chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.

The effects of aging on gene expression in the hypothalamus and cortex of mice

A better understanding of the molecular effects of aging in the brain may help to reveal important aspects of organismal aging, as well as processes that lead to age-related brain dysfunction. In this study, we have examined differences in gene expression in the hypothalamus and cortex of young and aged mice by using high-density oligonucleotide arrays. A number of key genes involved in neuronal structure and signaling are differentially expressed in both the aged hypothalamus and cortex, including synaptotagmin I, cAMP-dependent protein kinase C β, apolipoprotein E, protein phosphatase 2A, and prostaglandin D. Misregulation of these proteins may contribute to age-related memory deficits and neurodegenerative diseases. In addition, many proteases that play essential roles in regulating neuropeptide metabolism, amyloid precursor protein processing, and neuronal apoptosis are up-regulated in the aged brain and likely contribute significantly to brain aging. Finally, a subset of these genes whose expression is affected by aging are oppositely affected by exposure of mice to an enriched environment, suggesting that these genes may play important roles in learning and memory.

Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the α-secretase ADAM 10

Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer's disease. In the present report, we identify the α-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neural cell lines with either the cholesterol-extracting agent methyl-β-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted α-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of α-secretase activity resulted in a decreased secretion of Aβ peptides. Several mechanisms were elucidated as being the basis of enhanced α-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-β-cyclodextrin; treatment with lovastatin resulted in higher expression of the α-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic α-secretase pathway and the formation of neuroprotective α-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer's disease.

Kinesin molecular motors: Transport pathways, receptors, and human disease

Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.

Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

Genetic dissection of Alzheimer disease, a heterogeneous disorder.

The genetics of Alzheimer disease (AD) are complex and not completely understood. Mutations in the amyloid precursor protein gene (APP) can cause early-onset autosomal dominant AD. In vitro studies indicate that cells expressing mutant APPs overproduce pathogenic forms of the A beta peptide, the major component of AD amyloid. However, mutations in the APP gene are responsible for 5% or less of all early-onset familial AD. A locus on chromosome 14 is responsible for AD in other early-onset AD families and represents the most severe form of the disease in terms of age of onset and rate of decline. Attempts to identify the AD3 gene by positional cloning methods are underway. At least one additional early-onset AD locus remains to be located. In late-onset AD, the apolipoprotein E gene allele epsilon 4 is a risk factor for AD. This allele appears to act as a dose-dependent age-of-onset modifier. The epsilon 2 allele of this gene may be protective. Other late-onset susceptibility factors remain to be identified.

Early Alzheimer disease-like histopathology increases in frequency with age in mice transgenic for beta-APP751.

beta-Amyloid deposition and neurofibrillary tangle formation are two histopathological features of Alzheimer disease. We have previously reported that beta-amyloid immunoreactive deposits form in the brains of transgenic mice programmed for neuronal expression of the 751-amino acid isoform of human beta-amyloid precursor protein (beta-APP751) and now describe that these animals also display Alz50 intraneuronal immunoreactivity similar to that seen in early Alzheimer disease. This suggests that abnormal beta-APP expression and/or beta-amyloid deposition promotes pathogenic alterations in tau protein. The frequency of both beta-amyloid deposition and Alz50-positive neurons was twice as prevalent in brains from old (22 months) as compared to young (2-3 months) beta-APP751 transgenic mice. This increase in histopathology with age in beta-APP751 transgenic mice parallels the time-dependent progression seen in the human disease.

Air pollution exposure and novel biomarkers of inflammation and cardiac stress in the Multi-Ethnic Study of Atherosclerosis and Air Pollution (MESA Air)

Thesis (Master's)--University of Washington, 2016-06

Psychiatric genetics in Australia

Australian research in psychiatric genetics covers molecular genetic studies of depression, anxiety, alcohol dependence, Alzheimer's disease, bipolar disorder, schizophrenia, autism, and attention deficit hyperactivity disorder. For each disorder, a variety of clinical cohorts have been recruited including affected sib pair families, trios, case/controls, and twins from a large population-based twin registry. These studies are taking place both independently and in collaboration with international groups. Microarray studies now complement DNA investigations, while animal models are in development An Australian government genome facility provides a high throughput genotyping and mutation detection service to the Australian scientific community, enhancing the contribution of Australian psychiatric genetics groups to gene discovery. (C) 2003 Lippincott Williams Wilkins.

Treatment of dementia with neurotransmission modulation

The prevalence of dementia is growing in developed countries where elderly patients are increasing in numbers. Neurotransmission modulation is one approach to the treatment of dementia. Cholinergic precursors, anticholinesterases, nicotine receptor agonists and muscarinic M-2 receptor antagonists are agents that enhance cholinergic neurotransmission and that depend on having some intact cholinergic innervation to be effective in the treatment of dementia. The cholinergic precursor choline alfoscerate may be emerging as a potential useful drug in the treatment of dementia, with few adverse effects. Of the anticholinesterases, donepezil, in addition to having a similar efficacy to tacrine in mild-to-moderate Alzheimer's disease (AD), appears to have major advantages; its use is associated with lower drop-out rates in clinical trials, a lower incidence of cholinergic-like side effects and no liver toxicity. Rivastigmine is efficacious in the treatment in dementia with Lewy bodies, a condition in which the other anticholinesterases have not been tested extensively to date. Galantamine is an anticholinesterase and also acts as an allosteric potentiating modulator at nicotinic receptors to increase the release of acetylcholine. Pooled data from clinical trials of patients with mild-to-moderate AD suggest that the benefits and safety profile of galantamine are similar to those of the anticholinesterases. Selective nicotine receptor agonists are being developed that enhance cognitive performance without influencing autonomic and skeletal muscle function, but these have not yet entered clinical trial for dementia. Unlike the cholinergic enhancers, the M, receptor agonists do not depend upon intact cholinergic nerves but on intact M, receptors for their action, which are mainly preserved in AD and dementia with Lewy bodies. The M, receptor-selective agonists developed to date have shown limited efficacy in clinical trials and have a high incidence of side effects. A major recent advancement in the treatment of dementia is memantine, a non-competitive antagonist at NMDA receptors. Memantine is beneficial in the treatment of severe and moderate to-severe AD and may also be of some benefit in the treatment of mild-to-moderate vascular dementia. Drugs that modulate 5-HT, somatostatin and noradrenergic neurotransmission are also being considered for the treatment of dementia.

Physical activity, exercise, and inflammatory markers in older adults: Findings from the health, aging and body composition study

OBJECTIVES: To examine the association between physical activity and inflammatory markers, with consideration for body fatness and antioxidant use. DESIGN: Cross-sectional study, using baseline data from the Health, Aging and Body Composition Study. SETTING: Metropolitan areas surrounding Pittsburgh, Pennsylvania, and Memphis, Tennessee. PARTICIPANTS: Black and white, well-functioning men and women (N=3,075), aged 70 to 79. MEASUREMENTS: Interviewer-administered questionnaires of previous-week household, walking, exercise, and occupational/volunteer physical activities. Analysis of covariance was used to examine the association between activity level and serum C-reactive protein (CRP), interleukin-6 (IL-6), and plasma tumor necrosis factor alpha (TNFalpha) with covariate adjustment. Antioxidant supplement use (multivitamin, vitamins E or C, beta carotene) was evaluated as an effect modifier of the association. RESULTS: Higher levels of exercise were associated with lower levels of CRP (P