Oligodeoxynucleotide inhibition of Toll‐like receptors 3, 7, 8, and 9 suppresses cytokine production in a human rheumatoid arthritis model

Toll‐like receptors (TLRs) are innate immune receptors that respond to both exogenous and endogenous stimuli and are suggested to contribute to the perpetuation of chronic inflammation associated with rheumatoid arthritis (RA). In particular, the endosomal TLRs 3, 7, 8, and 9 have more recently been postulated to be of importance in RA pathogenesis. In this study, pan inhibition of the endosomal TLRs by a phosphorothioate‐modified inhibitory oligodeoxynucleotide (ODN) is demonstrated in primary human B cells, macrophages, and RA fibroblasts. Inhibition of TLR8 was of particular interest as TLR8 has been associated with RA pathogenesis in both human and murine arthritis models. ODN1411 competitively inhibited TLR8 signaling and was observed to directly bind to a purified TLR8 ectodomain, suggesting inhibition was through a direct interaction with the receptor. Addition of ODN1411 to human RA synovial membrane cultures significantly inhibited spontaneous cytokine production from these cultures, suggesting a potential role for one or more of the endosomal TLRs in inflammatory cytokine production in RA and the potential for inhibitory ODNs as novel therapies.     

Presenilin-1 is expressed in neural progenitor cells in the hippocampus of adult mice.

The functions of the presenilin-1 (PS-1) protein remain largely unknown. In adult brain PS-1 is expressed principally in neurons. However during development PS-1 is expressed more widely including in embryonic neural progenitors. To determine if PS-1 is expressed in neural progenitors in adult hippocampus we used bromodeoxyuridine (BrdU) labeling combined with immunostaining for BrdU, PS-1 and markers of neuronal or glial differentiation. Most BrdU labeled cells also expressed PS-1 at a time when few BrdU labeled cells expressed the early neuronal markers beta-III tubulin or TOAD-64 and none expressed mature neuronal (NeuN or calbindin) or astrocytic (GFAP) markers. Cells expressing PS-1 and the neural progenitor marker nestin were also found. Thus PS-1 is expressed in neural progenitor cells in adult hippocampus implying its possible role in neurogenesis in adult brain.     

Intracellular Abeta is increased by okadaic acid exposure in transfected neuronal and non-neuronal cell lines.

Intracellular Abeta was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Abeta immunostaining was enhanced, as demonstrated by two independent anti-Abeta antibodies. The confocal microscopic study revealed that the immunoreactivity of Abeta was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Abeta sandwich ELISA, showed significantly increased intracellular Abeta. False positive detection of Abeta by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Abeta, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Abeta.     

Construction and characterization of a live, attenuated aroA deletion mutant of Pseudomonas aeruginosa as a candidate intranasal vaccine

Antibodies to the lipopolysaccharide O antigen of Pseudomonas aeruginosa mediate high-level immunity, but protective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protective O-antigen epitopes often elicit the best immune responses. With the goal of developing a broadly protective P. aeruginosa vaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarked aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1. The resultant aroA deletion mutant of PAO1 is designated PAO1 Delta aroA. The aroA deletion was confirmed by both PCR and failure of the mutant to grow on minimal media lacking aromatic amino acids. When evaluated for safety and immunogenicity in mice, PAO1 Delta aroA could be applied either intranasally or intraperitoneally at doses up to 5 x 10(9) CFU per mouse without adverse effects. No dissemination of PAO1 Delta aroA to blood, liver, or spleen was detected after intranasal application, and histological evidence of pneumonia was minimal. Intranasal immunization of mice and rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterial antigens of all seven prototypic P. aeruginosa serogroup O2/O5 strains. The mouse antisera mediated potent phagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediated phagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains. This live, attenuated P. aeruginosa strain PAO1 Delta aroA appears to be safe for potential use as an intranasal vaccine and elicits high titers of opsonic antibodies against multiple strains of the P. aeruginosa O2/O5 serogroup.     

Late infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutations

We examined 26 individuals with clinical and electron microscopic signs of late infantile neuronal ceroid lipofuscinosis (LINCL). In 22 cases, we found both pathogenic alleles. Sixteen patients exclusively carried either one or a combination of the two common mutations R208X and IVS5-1G > C. In the remaining cases, four missense mutations could be detected, of which R127Q, N286S, and T353P represent novel, previously not described alleles. A clinical performance score was developed by rating motor, visual, and verbal functions and the incidence of cerebral seizures in 3-month intervals during the course of the disease. A Total Disability Score was derived by summing up the single scores for motor, visual, and verbal functions. The 16 individuals with the two common mutations were grouped together (referred to as standard patients), and the 5th, 50th, and 95th centiles were calculated and graphically depicted over time. The scores for motor function and language ability dropped earliest and progressed very similarly in the standard patients. The performance curves of two children with the N286S mutation slightly diverged from the 95th centile. However, the performance curves of one patient with atypical LINCL carrying the R127Q mutation fell far beyond the 95th centile. The presented performance rating clearly and quantitatively delineates the disease course of the LINCL patients and hence offers a useful tool for clinical evaluation of future therapeutic interventions. In addition, the described performance score system can be applied to other types of neuronal ceroid lipofuscinoses and could be adapted to various other neurodegenerative diseases of childhood.     

QT interval prolongation as a biomarker for torsades de pointes and sudden death in drug development

Prolongation of the QT interval on the surface 12-lead electrocardiogram is widely accepted as a biomarker for the potential of a drug to produce torsades de pointes and/or sudden death. Detection of drug-induced prolongation of the QT interval in animals and man is frequently confounded by extrinsic and intrinsic factors that limit the ability to detect a true drug effect. In particular drugs that increase heart rate show an apparent increase in QT interval that confounds assessment of a true drug effect on cardiac ventricular repolarization. The basis for the use of the QT interval as a biomarker will be examined.     

Fibroblast growth factor-2 in remodeling of the developing basement membrane zone in the trachea of infant rhesus monkeys sensitized and challenged with allergen

SUMMARY: Remodeling of the epithelial basement membrane zone (BMZ) involves increased deposition of collagen, resulting in thickening of the BMZ. The current study focuses on fibroblast growth factor-2 (FGF-2) in the tracheal BMZ in house dust mite allergen (HDMA)-sensitized infant rhesus monkeys, challenged with HDMA at a time when the BMZ is undergoing active postnatal development. To detect structural changes in the BMZ, we measured collagens I, III, and V. To detect changes in the function of the BMZ, we measured immunoreactivity of the heparan sulfate proteoglycan, perlecan, and FGF-2. We found significant thickening of the tracheal BMZ (p < 0.05) with each of these parameters. We also found that all HDMA tracheal samples expressed thin focal areas of the BMZ associated with leukocyte trafficking. These areas were depleted of perlecan and FGF-2; however, increased FGF-2 immunoreactivity was present in the adjacent basal cells. We conclude that basal cells and FGF-2 are involved with significant remodeling of the BMZ in the developing trachea of infant rhesus monkeys exposed to HDMA.     

The red blood cell in vascular occlusion

Red blood cells (RBC) have unique flow-affecting properties--namely, aggregability, deformability and adherence to endothelial cells (EC)--which play major roles in blood flow. Under normal flow-induced shear stress RBC are dispersed, their adherence to EC is insignificant, and they are sufficiently deformable to enable tissue perfusion. However, in pathological conditions that are associated with low-flow states (e.g., trauma, ischemia), elevated plasma components (mainly fibrinogen), or altered RBC properties (e.g., hemoglobinopathies, oxidative stress, inflammation, diabetes), RBC flow properties are altered and present a circulatory risk.     

Hyperpolarized shifts in the voltage dependence of fast inactivation of Nav1.4 and Nav1.5 in a rat model of critical illness myopathy

Critical illness myopathy is a disorder in which skeletal muscle becomes electrically inexcitable. We previously demonstrated that a shift in the voltage dependence of fast inactivation of sodium currents contributes to inexcitability of affected fibres in an animal model of critical illness myopathy in which denervated rat skeletal muscle is treated with corticosteroids (steroid-denervated; SD). In the current study we examined whether expression of Na_v1.5 contributes to the altered voltage dependence of sodium channel inactivation in SD muscle. We used TTX and μ-conotoxin GIIIB to selectively block Na_v1.4 in SD muscle and found that the level of Na_v1.5 did not correlate closely with the shift in fast inactivation. Surprisingly, we found that the voltage dependence of inactivation of Na_v1.4 was similar to that of Na_v1.5 in skeletal muscle in vivo . In severely affected fibres, inactivation of both Na_v1.4 and Na_v1.5 was shifted towards hyperpolarized potentials. We examined the role of denervation and steroid treatment in the shift of the voltage dependence of inactivation and found that both denervation and steroid treatment contribute to the shift in inactivation. Our results suggest that modulation of the voltage dependence of inactivation of both Na_v1.4 and Na_v1.5 in vivo contributes to loss of electrical excitability in SD muscle.