Evaluation of N (epsilon)-(3-formyl-3,4-dehydropiperidino)lysine as a novel biomarker for the severity of diabetic retinopathy.

AIMS/HYPOTHESIS: Recent studies suggest that oxidative stress should be monitored alongside HbA(1c) to identify subgroups of diabetic patients at high risk of initiation or progression of retinopathy. The acrolein-derived advanced lipoxidation end-product (ALE), [Formula: see text]-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine), is a useful biomarker that reflects the cumulative burden of oxidative stress over long periods of time. The purpose of the present study was to investigate whether serum and haemoglobin levels of FDP-lysine are associated with the severity of diabetic retinopathy in type 1 and type 2 diabetic patients.

METHODS: Serum and haemoglobin levels of FDP-lysine were measured by competitive ELISA in 59 type 1 and 76 type 2 diabetic patients with no retinopathy, non-proliferative retinopathy or proliferative retinopathy (mean age [+/-SEM] 54.3 +/- 1.3 years), and in 47 non-diabetic control individuals (mean age 51.9 +/- 2.1 years).

RESULTS: Serum and haemoglobin levels of FDP-lysine were significantly increased in diabetic patients compared with control individuals (p = 0.04 and p = 0.002, respectively). However, no significant association was found between levels of serum FDP-lysine and the severity of diabetic retinopathy (p = 0.97). In contrast, increased haemoglobin FDP-lysine levels were observed in patients with proliferative retinopathy compared with patients without retinopathy and with non-proliferative retinopathy (p = 0.04). The relationship of FDP-lysine with proliferative retinopathy was unaltered after adjustment for HbA(1c), or other clinical parameters.

CONCLUSIONS/INTERPRETATION: Our data suggest that haemoglobin FDP-lysine may provide a useful risk marker for the development of proliferative diabetic retinopathy independently of HbA(1c), and that elevated intracellular ALE formation may be involved in the pathogenesis of this sight-threatening complication of diabetes.

Discovery of novel cathepsin S inhibitors by pharmacophore-based virtual high-throughput screening

The cysteine protease cathepsin S (CatS) is involved in the pathogenesis of autoimmune disorders, atherosclerosis, and obesity. Therefore, it represents a promising pharmacological target for drug development. We generated ligand-based and structure-based pharmacophore models for noncovalent and covalent CatS inhibitors to perform virtual high-throughput screening of chemical databases in order to discover novel scaffolds for CatS inhibitors. An in vitro evaluation of the resulting 15 structures revealed seven CatS inhibitors with kinetic constants in the low micromolar range. These compounds can be subjected to further chemical modifications to obtain drugs for the treatment of autoimmune disorders and atherosclerosis.

Anti-infective photodynamic biomaterials for the prevention of intraocular lens-associated infectious endophthalmitis

Bacterial attachment onto intraocular lenses (IOLs) during cataract extraction and IOL implantation is a prominent aetiological factor in the pathogenesis of infectious endophthalmitis. Photodynamic therapy (PDT) and photodynamic antimicrobial chemotherapy (PACT) have shown that photosensitizers are effective treatments for cancer, and in the photoinactivation of bacteria, viruses, fungi and parasites, in the presence of light. To date, no method of localizing the photocytotoxic effect of a photosensitizer at a biomaterial surface has been demonstrated. Here we show a method for concentrating this effect at a material surface to prevent bacterial colonization by attaching a porphyrin photosensitizer at, or near to, that surface, and demonstrate the principle using IOL biomaterials. Anionic hydrogel copolymers were shown to permanently bind a cationic porphyrin through electrostatic interactions as a thin surface layer. The mechanical and thermal properties of the materials showed that the porphyrin acts as a surface cross-linking agent, and renders surfaces more hydrophilic. Importantly, Staphylococcus epidermidis adherence was reduced by up to 99.0 ± 0.42% relative to the control in intense light conditions and 91.7± 5.99% in the dark. The ability to concentrate the photocytotoxic effect at a surface, together with a significant dark effect, provides a platform for a range of light-activated anti-infective biomaterial technologies.

Protein kinase B/Akt activity is involved in renal TGF-beta 1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

Co-regulation of Gremlin and Notch signalling in diabetic nephropathy

Diabetic nephropathy is currently the leading cause of end-stage renal disease worldwide, and occurs in approximately one third of all diabetic patients. The molecular pathogenesis of diabetic nephropathy has not been fully characterized and novel mediators and drivers of the disease are still being described. Previous data from our laboratory has identified the developmentally regulated gene Gremlin as a novel target implicated in diabetic nephropathy in vitro and in vivo. We used bioinformatic analysis to examine whether Gremlin gene sequence and structure could be used to identify other genes implicated in diabetic nephropathy. The Notch ligand Jagged1 and its downstream effector, hairy enhancer of split-1 (Hes1), were identified as genes with significant similarity to Gremlin in terms of promoter structure and predicted microRNA binding elements. This led us to discover that transforming growth factor-beta (TGFß1), a primary driver of cellular changes in the kidney during nephropathy, increased Gremlin, Jagged1 and Hes1 expression in human kidney epithelial cells. Elevated levels of Gremlin, Jagged1 and Hes1 were also detected in extracts from renal biopsies from diabetic nephropathy patients, but not in control living donors. In situ hybridization identified specific upregulation and co-expression of Gremlin, Jagged1 and Hes1 in the same tubuli of kidneys from diabetic nephropathy patients, but not controls. Finally, Notch pathway gene clustering showed that samples from diabetic nephropathy patients grouped together, distinct from both control living donors and patients with minimal change disease. Together, these data suggest that Notch pathway gene expression is elevated in diabetic nephropathy, co-incident with Gremlin, and may contribute to the pathogenesis of this disease.

Bimanual coordination in chronic schizophrenia

Anomalies of movement are observed both clinically and experimentally in schizophrenia. While the basal ganglia have been implicated in its pathogenesis, the nature of such involvement is equivocal. The basal ganglia may be involved in bimanual coordination through their input to the supplementary motor area (SMA). While a neglected area of study in schizophrenia. a bimanual movement task may provide a means of assessing the functional integrity of the motor circuit. Twelve patients with chronic schizophrenia and 12 matched control participants performed a bimanual movement task on a set of vertically mounted cranks at different speeds (1 and 2 Hz) and phase relationships. Participants performed in-phase movements (hands separated by 0 degrees) and out-of-phase movements (hands separated by 180 degrees) at both speeds with an external cue on or off. All participants performed the in-phase movements well. irrespective of speed or cueing conditions. Patients with schizophrenia were unable to perform the out-of-phase movements, particularly at the faster speed, reverting instead to the in-phase movement. There was no effect of external cueing on any of the movement conditions. These results suggest a specific problem of bimanual coordination indicative of SMA dysfunction per se and/or faulty callosal integration. A disturbance in the ability to switch attention during the out-of-phase task may also be involved. (C) 2001 Academic Press.

Neuropeptide Y (NPY) and NPY Y1 receptor in periodontal health and disease

Abstract
OBJECTIVES:
Neuropeptide Y (NPY) coordinates inflammation and bone metabolism which are central to the pathogenesis of periodontitis. The present study was designed to determine whether NPY was quantifiable in human gingival crevicular fluid (GCF) and to test the null hypothesis that GCF levels of NPY were the same in periodontal health and disease. A subsidiary aim was to determine the potential functionality of released NPY by detecting the presence of NPY Y1 receptors in gingival tissue.
DESIGN:
The periodontitis group consisted of 20 subjects (10 females and 10 males) mean age 41.4 (S.D. 9.6 years). The control group comprised 20 subjects (10 females and 10 males) mean age 37.4 (S.D. 11.7 years). NPY levels in GCF were measured in periodontal health and disease by radioimmunoassay. NPY Y1 receptor expression in gingival tissue was determined by Western blotting of membrane protein extracts from healthy and inflamed gum.
RESULTS:
Healthy sites from control subjects had significantly higher levels of NPY than diseased sites from periodontitis subjects. NPY Y1 receptor protein was detected in both healthy and inflamed gingival tissue by Western blotting.
CONCLUSIONS:
The significantly elevated levels of NPY in GCF from healthy compared with periodontitis sites suggests a tonic role for NPY, the functionality of which is indicated by the presence of NPY Y1 receptors in local gingival tissue.

Proteomic analysis of circulating immune complexes in juvenile idiopathic arthritis reveals disease-associated proteins

Juvenile idiopathic arthritis reflects a group of clinically heterogeneous arthritides hallmarked by elevated concentrations of circulating immune complexes. In this study, the circulating immune complex proteome was examined to elucidate disease-associated proteins that are overexpressed in patients with an aggressive, and at times destructive, disease phenotype. To solve this proteome, circulating immune complexes were isolated from the sera of patients with chronic, erosive or early-onset, aggressive disease and from patients in medical remission or healthy controls subsequent to protein separation by 2-DE. Thirty-seven protein spots were overexpressed in the circulating immune complexes of the aggressive disease groups as compared to controls, 28 of which have been confidently identified to date. Proteolytic fragments of glyceraldehyde-3-phosphate dehydrogenase, serotransferrin, and a-1-antitrypsin have been identified among others. In total, these 28 putative disease-associated proteins most definitely contribute to immune complex formation and likely have a significant role in disease etiology and pathogenesis. Moreover, these proteins represent markers of aggressive disease, which could aid in diagnosis and management strategies, and potential therapeutic targets to prevent or control disease outcome. This is the first in-depth analysis of the circulating immune complex proteome in juvenile idiopathic arthritis.

Effects of intrahippocampal NAC(61-95) injections on memory in the rat and attenuation with vitamin E

Parkinson's disease (PD)-related dementia affects approximately 40% of PD patients and the severity of this dementia correlates significantly with the density of Lewy body (LB) deposition in the PD brain. Aggregated alpha-synuclein protein is the major component of LB's and the non-amyloid component (NAC) region of alpha-synuclein, residues 61-95, is essential for the aggregation and toxicity of this protein. The current study evaluated the effect of pre-aggregated NAC(61-95) injected into the CA3 area of the dorsal hippocampus of the brain on memory in the rat. Previous research has suggested that oxidative stress processes may play a role in the neuropathology of PD, therefore the effect of treatment with vitamin E, an antioxidant, was also evaluated. Male Sprague-Dawley rats were trained in two-lever operant chambers under an alternating-lever cyclic-ratio (ALCR) schedule of food reinforcement. When responding showed no trends, subjects were divided into four groups. Two groups were injected bilaterally into the dorsal hippocampus with aggregated NAC(61-95) (5 mu l suspension), and two groups were injected bilaterally into the dorsal hippocampus with sterile water (5 mu l). Subgroups were treated with either vitamin E (150 mg/kg in Soya oil) or vehicle (Soya oil) daily. Injection of NAC(61-95) induced memory deficits and vitamin E treatment alleviated these. In addition, NAC(61-95) injections induced activated astrocytes and chronic treatment with vitamin E reduced the numbers of activated astrocytes. These results suggest that aggregated NAC(61-95) and associated oxidative stress, may play a role in the pathogenesis of cognitive deficits seen in PD-induced dementia. (C) 2009 Elsevier Inc. All rights reserved.

Functional Interleukin-17 Receptor A is Expressed in Central Nervous System Glia and Upregulated in Experimental Autoimmune Encephalomyelitis

Background: Interleukin-17A (IL-17A) is the founding member of a novel family of inflammatory cytokines that plays a critical role in the pathogenesis of many autoimmune diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). IL-17A signals through its receptor, IL-17RA, which is expressed in many peripheral tissues; however, expression of IL-17RA in the central nervous system (CNS) and its role in CNS inflammation are not well understood. Methods: EAE was induced in C57Bl/6 mice by immunization with myelin oligodendroglial glycoprotein. IL-17RA expression in the CNS was compared between control and EAE mice using RT-PCR, in situ hybridization, and immunohistochemistry. Cell-type specific expression was examined in isolated astrocytic and microglial cell cultures. Cytokine and chemokine production was measured in IL-17A treated cultures to evaluate the functional status of IL-17RA. Results: Here we report increased IL-17RA expression in the CNS of mice with EAE, and constitutive expression of functional IL-17RA in mouse CNS tissue. Specifically, astrocytes and microglia express IL-17RA in vitro, and IL-17A treatment induces biological responses in these cells, including significant upregulation of MCP-1, MCP-5, MIP-2 and KC chemokine secretion. Exogenous IL-17A does not significantly alter the expression of IL-17RA in glial cells, suggesting that upregulation of chemokines by glial cells is due to IL-17A signaling through constitutively expressed IL-17RA. Conclusion: IL-17RA expression is significantly increased in the CNS of mice with EAE compared to healthy mice, suggesting that IL-17RA signaling in glial cells can play an important role in autoimmune inflammation of the CNS and may be a potential pathway to target for therapeutic interventions. © 2009 Sarma et al; licensee BioMed Central Ltd.

Induction of Distinct Neurologic Disease Manifestations during Relapsing Fever Requires T Lymphocytes.

Relapsing fever borreliosis is a multisystemic infection characterized primarily by bacteremia but can extend to the CNS. The incidence of CNS disease manifestations in humans depends on the infecting relapsing fever Borrelia species. In the murine model of Borrelia hermsii infection we found high incidence of distinct signs of CNS disease that ranged from a flaccid tail to complete paralysis of hind limbs. Infiltration of large number of T cells into the spinal cord of B. hermsii-infected mice and the upregulation of MHC class II and CD80 on infiltrating macrophages and on microglial cells suggested a role for T cell and Ag-presenting cell interactions in this pathogenesis. Indeed, B. hermsii infection did not induce CNS disease manifestations in T cell-deficient mice (TCR-ß × d-/-), although it resulted in bacteremia comparable to wild-type (Wt) level. Moreover, the infiltration of immune cells into the spinal cord of TCR-ß × d-/- mice was reduced and the resident microglial cells were not activated. Histopathological analysis of lumbar sections of the spinal cord confirmed severe inflammation in Wt but not in TCR-ß × d-/- mice. Induction of CNS disease was dependent on the B. hermsii strain as well as on the ability of the host to control bacteremia. Mice that are impaired in controlling B. hermsii, such as CD14-/- mice, exhibited more severe CNS disease than Wt mice. This study demonstrates that distinct neurologic disease manifestations develop during relapsing fever and that T cells play a critical role in the induction of neuropathogenesis.

The chronic fatigue syndrome: A comparative pathway analysis

In this paper, we introduce a method to detect pathological pathways of a disease. We aim to identify biological processes rather than single genes affected by the chronic fatigue syndrome (CFS). So far, CFS has neither diagnostic clinical signals nor abnormalities that could be diagnosed by laboratory examinations. It is also unclear if the CFS represents one disease or can be subdivided in different categories. We use information from clinical trials, the gene ontology (GO) database as well as gene expression data to identify undirected dependency graphs (UDGs) representing biological processes according to the GO database. The structural comparison of UDGs of sick versus non-sick patients allows us to make predictions about the modification of pathways due to pathogenesis.

Salbutamol up-regulates matrix metalloproteinase-9 in the alveolar space in the acute respiratory distress syndrome.

Objectives: Acute respiratory distress syndrome (ARDS) is characterized by alveolar-capillary barrier damage. Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of ARDS. In the Beta Agonists in Acute Lung Injury Trial, intravenous salbutamol reduced extravascular lung water (EVLW) in patients with ARDS at day 4 but not inflammatory cytokines or neutrophil recruitment. We hypothesized that salbutamol reduces MMP activity in ARDS.

Methods: MMP-1/-2/-3/-7/-8/-9/-12/-13 was measured in supernatants of distal lung epithelial cells, type II alveolar cells, and bronchoalveolar lavage (BAL) fluid from patients in the Beta Agonists in Acute Lung Injury study by multiplex bead array and tissue inhibitors of metalloproteinases (TIMPs)-1/-2 by enzyme-linked immunosorbent assay. MMP-9 protein and activity levels were further measured by gelatin zymography and fluorokine assay.

Measurements and Main Results: BAL fluid MMP-1/-2/-3 declined by day 4, whereas total MMP-9 tended to increase. Unexpectedly, salbutamol augmented MMP-9 activity. Salbutamol induced 33.7- and 13.2-fold upregulation in total and lipocalin-associated MMP-9, respectively at day 4, compared with 2.0- and 1.3-fold increase in the placebo group, p < 0.03. Salbutamol did not affect BAL fluid TIMP-1/-2. Net active MMP-9 was higher in the salbutamol group (4222 pg/mL, interquartile range: 513-7551) at day 4 compared with placebo (151 pg/mL, 124-2108), p = 0.012. Subjects with an increase in BAL fluid MMP-9 during the 4-day period had lower EVLW measurements than those in whom MMP-9 fell (10 vs. 17 mL/kg, p = 0.004): change in lung water correlated inversely with change in MMP-9, r = -.54, p = 0.0296. Salbutamol up-regulated MMP-9 and down-regulated TIMP-1/-2 secretion in vitro by distal lung epithelial cells. Inhibition of MMP-9 activity in cultures of type II alveolar epithelial cells reduced wound healing.

Conclusions: Salbutamol specifically up-regulates MMP-9 in vitro and in vivo in patients with ARDS. Up-regulated MMP-9 is associated with a reduction in EVLW. MMP-9 activity is required for alveolar epithelial wound healing in vitro. Data suggest MMP-9 may have a previously unrecognized beneficial role in reducing pulmonary edema in ARDS by improving alveolar epithelial healing.

Investigation of the association of BMP gene variants with nephropathy in Type 1 diabetes mellitus

Aims Diabetic nephropathy is a leading cause of end-stage renal disease. The transforming growth factor beta-bone morphogenic protein (BMP) pathway is implicated in the pathogenesis of diabetic nephropathy. The BMP2, BMP4 and BMP7 genes are located near linkage peaks for renal dysfunction, and we hypothesize that genetic polymorphisms in these biological and positional candidate genes may be risk factors for diabetic kidney disease.

Statins for the treatment of dementia

The use of statin therapy in established Alzheimer's disease (AD) or vascular dementia (VaD) is a relatively unexplored area. In AD ss-amyloid protein (Ass) is deposited in the form of extracellular plaques and previous studies have determined Ass generation is cholesterol dependent. Hypercholesterolaemia has also been implicated in the pathogenesis of VaD. Due to the role of statins in cholesterol reduction it is biologically plausible they may be efficacious in the treatment of AD and dementia.