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1.  Early Diabetic Nephropathy 
Diabetes Care  2013;36(11):3678-3683.
OBJECTIVE
Diabetic nephropathy (DN) is a major cause of mortality in type 1 diabetes. Reduced insulin sensitivity is a well-documented component of type 1 diabetes. We hypothesized that baseline insulin sensitivity would predict development of DN over 6 years.
RESEARCH DESIGN AND METHODS
We assessed the relationship between insulin sensitivity at baseline and development of early phenotypes of DN—microalbuminuria (albumin-creatinine ratio [ACR] ≥30 mg/g) and rapid renal function decline (glomerular filtration rate [GFR] loss >3 mL/min/1.73 m2 per year)—with three Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations over 6 years. Subjects with diabetes (n = 449) and without diabetes (n = 565) in the Coronary Artery Calcification in Type 1 Diabetes study had an estimated insulin sensitivity index (ISI) at baseline and 6-year follow-up.
RESULTS
The ISI was lower in subjects with diabetes than in those without diabetes (P < 0.0001). A higher ISI at baseline predicted a lower odds of developing an ACR ≥30 mg/g (odds ratio 0.65 [95% CI 0.49–0.85], P = 0.003) univariately and after adjusting for HbA1c (0.69 [0.51–0.93], P = 0.01). A higher ISI at baseline conferred protection from a rapid decline of GFR as assessed by CKD-EPI cystatin C (0.77 [0.64–0.92], P = 0.004) and remained significant after adjusting for HbA1c and age (0.80 [0.67–0.97], P = 0.02). We found no relation between ISI and rapid GFR decline estimated by CKD-EPI creatinine (P = 0.38) or CKD-EPI combined cystatin C and creatinine (P = 0.50).
CONCLUSIONS
Over 6 years, a higher ISI independently predicts a lower odds of developing microalbuminuria and rapid GFR decline as estimated with cystatin C, suggesting a relationship between insulin sensitivity and early phenotypes of DN.
doi:10.2337/dc13-0631
PMCID: PMC3816872  PMID: 24026551
2.  DVT Surveillance Program in the ICU: Analysis of Cost-Effectiveness 
PLoS ONE  2014;9(9):e106793.
Background
Venous Thrombo-embolism (VTE – Deep venous thrombosis (DVT) and/or pulmonary embolism (PE) – in traumatized patients causes significant morbidity and mortality. The current study evaluates the effectiveness of DVT surveillance in reducing PE, and performs a cost-effectiveness analysis.
Methods
All traumatized patients admitted to the adult ICU underwent twice weekly DVT surveillance by bilateral lower extremity venous Duplex examination (48-month surveillance period – SP). The rates of DVT and PE were recorded and compared to the rates observed in the 36-month pre-surveillance period (PSP). All patients in both periods received mechanical and pharmacologic prophylaxis unless contraindicated. Total costs – diagnostic, therapeutic and surveillance – for both periods were recorded and the incremental cost for each Quality Adjusted Life Year (QALY) gained was calculated.
Results
4234 patients were eligible (PSP – 1422 and SP – 2812). Rate of DVT in SP (2.8%) was significantly higher than in PSP (1.3%) – p<0.05, and rate of PE in SP (0.7%) was significantly lower than that in PSP (1.5%) – p<0.05. Logistic regression demonstrated that surveillance was an independent predictor of increased DVT detection (OR: 2.53 – CI: 1.462–4.378) and decreased PE incidence (OR: 0.487 – CI: 0.262–0.904). The incremental cost was $509,091/life saved in the base case, translating to $29,102/QALY gained. A sensitivity analysis over four of the parameters used in the model indicated that the incremental cost ranged from $18,661 to $48,821/QALY gained.
Conclusions
Surveillance of traumatized ICU patients increases DVT detection and reduces PE incidence. Costs in terms of QALY gained compares favorably with other interventions accepted by society.
doi:10.1371/journal.pone.0106793
PMCID: PMC4182316  PMID: 25269021
3.  An Assessment of Fixed Interval Timing in Free-Flying Honey Bees (Apis mellifera ligustica): An Analysis of Individual Performance 
PLoS ONE  2014;9(7):e101262.
Interval timing is a key element of foraging theory, models of predator avoidance, and competitive interactions. Although interval timing is well documented in vertebrate species, it is virtually unstudied in invertebrates. In the present experiment, we used free-flying honey bees (Apis mellifera ligustica) as a model for timing behaviors. Subjects were trained to enter a hole in an automated artificial flower to receive a nectar reinforcer (i.e. reward). Responses were continuously reinforced prior to exposure to either a fixed interval (FI) 15-sec, FI 30-sec, FI 60-sec, or FI 120-sec reinforcement schedule. We measured response rate and post-reinforcement pause within each fixed interval trial between reinforcers. Honey bees responded at higher frequencies earlier in the fixed interval suggesting subject responding did not come under traditional forms of temporal control. Response rates were lower during FI conditions compared to performance on continuous reinforcement schedules, and responding was more resistant to extinction when previously reinforced on FI schedules. However, no “scalloped” or “break-and-run” patterns of group or individual responses reinforced on FI schedules were observed; no traditional evidence of temporal control was found. Finally, longer FI schedules eventually caused all subjects to cease returning to the operant chamber indicating subjects did not tolerate the longer FI schedules.
doi:10.1371/journal.pone.0101262
PMCID: PMC4077790  PMID: 24983960
4.  Loss of sulfiredoxin renders mice resistant to azoxymethane/dextran sulfate sodium-induced colon carcinogenesis 
Carcinogenesis  2013;34(6):1403-1410.
Sulfiredoxin (Srx) is the enzyme that reduces the hyperoxidized inactive form of peroxiredoxins. To study the function of Srx in carcinogenesis in vivo, we tested whether loss of Srx protects mice from cancer development. Srx null mice were generated and colon carcinogenesis was induced by an azoxymethane (AOM) and dextran sulfate sodium (DSS) protocol. Compared with either wild-type (Wt) or heterozygotes, Srx−/− mice had significantly reduced rates in both tumor multiplicity and volume. Mechanistic studies reveal that loss of Srx did not alter tumor cell proliferation; however, increased apoptosis and decreased inflammatory cell infiltration were obvious in tumors from Srx null mice compared with those from Wt control. In addition to the AOM/DSS model, examination of Srx expression in human reveals a tissue-specific expression pattern. Srx expression was also demonstrated in tumors from colorectal cancer patients and the levels of expression were associated with patients’ clinic stages. These data provide the first in vivo evidence that loss of Srx renders mice resistant to AOM/DSS-induced colon carcinogenesis, suggesting that Srx has a critical oncogenic role in cancer development, and Srx may be used as a marker for human colon cancer pathogenicity.
doi:10.1093/carcin/bgt059
PMCID: PMC3670259  PMID: 23393226
5.  Functions and Cellular Compartmentation of the Thioredoxin and Glutathione Pathways in Yeast 
Antioxidants & Redox Signaling  2013;18(13):1699-1711.
Abstract
Significance: The thioredoxin (TRX) and glutathione (GSH) pathways are universally conserved thiol-reductase systems that drive an array of cellular functions involving reversible disulfide formation. Here we consider these pathways in Saccharomyces cerevisiae, focusing on their cell compartment-specific functions, as well as the mechanisms that explain extreme differences of redox states between compartments. Recent Advances: Recent work leads to a model in which the yeast TRX and GSH pathways are not redundant, in contrast to Escherichia coli. The cytosol possesses full sets of both pathways, of which the TRX pathway is dominant, while the GSH pathway acts as back up of the former. The mitochondrial matrix also possesses entire sets of both pathways, in which the GSH pathway has major role in redox control. In both compartments, GSH has also nonredox functions in iron metabolism, essential for viability. The endoplasmic reticulum (ER) and mitochondrial intermembrane space (IMS) are sites of intense thiol oxidation, but except GSH lack thiol-reductase pathways. Critical Issues: What are the thiol-redox links between compartments? Mitochondria are totally independent, and insulated from the other compartments. The cytosol is also totally independent, but also provides reducing power to the ER and IMS, possibly by ways of reduced and oxidized GSH entering and exiting these compartments. Future Directions: Identifying the mechanisms regulating fluxes of GSH and oxidized glutathione between cytosol and ER, IMS, and possibly also peroxisomes, vacuole is needed to establish the proposed model of eukaryotic thiol-redox homeostasis, which should facilitate exploration of this system in mammals and plants. Antioxid. Redox Signal. 18, 1699–1711.
doi:10.1089/ars.2012.5033
PMCID: PMC3771550  PMID: 23198979
6.  Creatine supplementation and oxidative stress in rat liver 
Background
The objective of this study was to determine the effects of creatine supplementation on liver biomarkers of oxidative stress in exercise-trained rats.
Methods
Forty 90-day-old adult male Wistar rats were assigned to four groups for the eight-week experiment. Control group (C) rats received a balanced control diet; creatine control group (CCr) rats received a balanced diet supplemented with 2% creatine; trained group (T) rats received a balanced diet and intense exercise training equivalent to the maximal lactate steady state phase; and supplemented-trained (TCr) rats were given a balanced diet supplemented with 2% creatine and subjected to intense exercise training equivalent to the maximal lactate steady state phase. At the end of the experimental period, concentrations of creatine, hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) were measured as well as the enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-GPx) and catalase (CAT). Liver tissue levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and the GSH/GSSG ratio were also determined.
Results
Hepatic creatine levels were highest in the CCr and TCr groups with increased concentration of H2O2 observed in the T and TCr animal groups. SOD activity was decreased in the TCr group. GSH-GPx activity was increased in the T and TCr groups while CAT was elevated in the CCr and TCr groups. GSH, GGS and the GSH/GSSG ratio did not differ between all animal subsets.
Conclusions
Our results demonstrate that creatine supplementation acts in an additive manner to physical training to raise antioxidant enzymes in rat liver. However, because markers of liver oxidative stress were unchanged, this finding may also indicate that training-induced oxidative stress cannot be ameliorated by creatine supplementation.
doi:10.1186/1550-2783-10-54
PMCID: PMC4029397  PMID: 24325803
Creatine supplementation; Oxidative stress; Enzymes; Treadmill exercise
7.  Insulin Resistance, Cystatin C, and Mortality Among Older Adults 
Diabetes Care  2012;35(6):1355-1360.
OBJECTIVE
Insulin resistance is a risk factor for cardiovascular and noncardiovascular diseases. Impaired kidney function is linked with insulin resistance and may affect relationships of insulin resistance with health outcomes.
RESEARCH DESIGN AND METHODS
We performed a cohort study of 3,138 Cardiovascular Health Study participants (age ≥65 years) without diabetes. Insulin sensitivity index (ISI) was calculated from fasting and 2-h postload insulin and glucose concentrations. Associations of ISI and fasting insulin concentration with all-cause mortality were tested using Cox proportional hazards models, adjusting for demographic variables, prevalent cardiovascular disease, lifestyle variables, waist circumference, and LDL cholesterol. Subsequent models were additionally adjusted for or stratified by glomerular filtration rate estimated using serum cystatin C (eGFR).
RESULTS
A total of 1,810 participants died during the 14.7-year median follow-up. Compared with the highest quartile of ISI, the lowest quartile (most insulin resistant) was associated with 21% (95% CI 6–41) and 11% (−3 to 29) higher risks of death without and with adjustment for eGFR, respectively. Compared with the lowest quartile of fasting insulin concentration, the highest quartile was associated with 22% (4–43) and 4% (−12 to 22) higher risks of death without and with adjustment for eGFR, respectively. Similar attenuation by eGFR was observed when blood pressure, triglycerides, HDL cholesterol, and C-reactive protein were included in models.
CONCLUSIONS
Insulin resistance measured as ISI or fasting insulin concentration is associated with increased risk of death among older adults, adjusting for conventional confounding characteristics. Impaired kidney function may mediate or confound this relationship.
doi:10.2337/dc11-1657
PMCID: PMC3357240  PMID: 22432118
8.  Social Reinforcement Delays in Free-Flying Honey Bees (Apis mellifera L.) 
PLoS ONE  2012;7(10):e46729.
Free-flying honey bees (Apis mellifera L.) reactions were observed when presented with varying schedules of post-reinforcement delays of 0 s, 300 s, or 600 s. We measured inter-visit-interval, response length, inter-response-time, and response rate. Honey bees exposed to these post-reinforcement delay intervals exhibit one of several patterns compared to groups not encountering delays, and had longer inter-visit-intervals. We observed no group differences in inter-response time. Honey bees with higher response rates tended to not finish the experiment. The removal of the delay intervals increased response rates for those subjects that completed the trials.
doi:10.1371/journal.pone.0046729
PMCID: PMC3464271  PMID: 23056425
9.  Muscle protein metabolism in neonatal alloxan-administered rats: effects of continuous and intermittent swimming training 
Background
This study aimed to examine the effects of intermittent and continuous swimming training on muscle protein metabolism in neonatal alloxan-administered rats.
Methods
Wistar rats were used and divided into six groups: sedentary alloxan (SA), sedentary control (SC), continuous trained alloxan (CA), intermittent trained alloxan (IA), continuous trained control (CC) and intermittent trained control (IC). Alloxan (250 mg/kg body weight) was injected into newborn rats at 6 days of age. The continuous training protocol consisted of 12 weeks of swimming training in individual cylinder tanks while supporting a load that was 5% of body weight; uninterrupted swimming for 1 h/day, five days a week. The intermittent training protocol consisted of 12 weeks of swimming training in individual cylinder tanks while supporting a load that was 15% of body weight; 30 s of activity interrupted by 30 s of rest for a total of 20 min/day, five days a week.
Results
At 28 days, the alloxan animals displayed higher glycemia after glucose overload than the control animals. No differences in insulinemia among the groups were detected. At 120 days, no differences in serum albumin and total protein among the groups were observed. Compared to the other groups, DNA concentrations were higher in the alloxan animals that were subjected to continuous training, whereas the DNA/protein ratio was higher in the alloxan animals that were subjected to intermittent training.
Conclusion
It was concluded that continuous and intermittent training sessions were effective in altering muscle growth by hyperplasia and hypertrophy, respectively, in alloxan-administered animals.
doi:10.1186/1758-5996-4-5
PMCID: PMC3292999  PMID: 22309804
DMT2; exercise training; protein synthesis; hypertrophy muscle
10.  Effects of cholinesterase inhibition in supraspinal and spinal neural pathways on the micturition reflex in rats 
BJU international  2009;104(8):1163-1169.
Objective
To investigate whether activation of brain and spinal cholinergic pathways affects the micturition reflex in rats.
Materials and Methods
The effects of intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of neostigmine as a cholinesterase inhibitor and oxotremorine-M (OXO-M) as a muscarinic acetylcholine receptor (mAChRs) agonist, on the micturition reflex were evaluated by infusion cystometrography (CMG) in urethane-anaesthetized untreated rats or rats pretreated with capsaicin.
Results
Neostigmine injected i.c.v increased bladder capacity (BC) and pressure threshold (PT) dose-dependently, with an increase in maximum voiding pressure (MVP) and a decrease in voiding efficiency (VE) at higher doses. Also, neostigmine injected i.t. increased the BC and PT dose-dependently without changing MVP or VE, and these effects were not apparent in capsaicin-pretreated rats. In both routes, atropine as an antagonist of mAChRs, but not mecamylamine as a nicotinic-AChR antagonist, almost completely antagonized the effects of neostigmine. The rank order of potencies of the antagonists for increasing effects of BC induced by 1 nmol of neostigmine was: pirenzepine (an M1 mAChR antagonist) = atropine > 4-DAMP (an M3 mAChR antagonist) ≫ methoctramine (an M2 mAChR antagonist) and tropicamide (an M4 mAChR antagonist) via the i.c.v route; and atropine > methoctramine > pirenzepine > tropicamide and 4-DAMP via the i.t. route, respectively. OXO-M injected via i.c.v and i.t. had the same effects on BC, PT, MVP and VE as neostigmine by i.c.v and i.t., respectively.
Conclusions
These results indicate that activation of muscarinic cholinergic mechanisms by the cholinesterase inhibitor in the brain and spinal cord can inhibit the micturition reflex, mainly by affecting afferent pathways. These mAChR-induced inhibitory effects seem to be mediated through M1/M3 receptor subtypes in the brain, while in the spinal cord, the M1/M2 receptor subtypes might be involved in inhibitory effects, which are mediated via inhibition of mechanoceptive C-fibre afferent pathways.
doi:10.1111/j.1464-410X.2009.08515.x
PMCID: PMC2863018  PMID: 19338542
neostigmine; brain; spinal cord; micturition reflex; muscarinic receptors
11.  A PARADIGM FOR OPERANT CONDITIONING IN BLOW FLIES (PHORMIA TERRAE NOVAE ROBINEAU-DESVOIDY, 1830) 
An operant conditioning situation for the blow fly (Protophormia terrae novae) is described. Individual flies are trained to enter and reenter a hole as the operant response. Only a few sessions of contingent reinforcement are required to increase response rates. When the response is no longer followed by food, the rate of entering the hole decreases. Control procedures revealed that rate of responding is not a simple overall result of feeding or of aging. The flies entered into the hole only if the response was required to obtain the food.
doi:10.1901/jeab.2010.93-81
PMCID: PMC2801537  PMID: 20676269
associative learning; operant conditioning; blow flies; apparatus
12.  H2O2 Activates the Nuclear Localization of Msn2 and Maf1 through Thioredoxins in Saccharomyces cerevisiae▿  
Eukaryotic Cell  2009;8(9):1429-1438.
The cellular response to hydrogen peroxide (H2O2) is characterized by a repression of growth-related processes and an enhanced expression of genes important for cell defense. In budding yeast, this response requires the activation of a set of transcriptional effectors. Some of them, such as the transcriptional activator Yap1, are specific to oxidative stress, and others, such as the transcriptional activators Msn2/4 and the negative regulator Maf1, are activated by a wide spectrum of stress conditions. How these general effectors are activated in response to oxidative stress remains an open question. In this study, we demonstrate that the two cytoplasmic thioredoxins, Trx1 and Trx2, are essential to trigger the nuclear accumulation of Msn2/4 and Maf1, specifically under H2O2 treatment. Contrary to the case with many stress conditions previously described for yeast, the H2O2-induced nuclear accumulation of Msn2 and Maf1 does not correlate with the downregulation of PKA kinase activity. Nevertheless, we show that PP2A phosphatase activity is essential for driving Maf1 dephosphorylation and its subsequent nuclear accumulation in response to H2O2 treatment. Interestingly, under this condition, the lack of PP2A activity has no impact on the subcellular localization of Msn2, demonstrating that the H2O2 signaling pathways share a common route through the thioredoxin system and then diverge to activate Msn2 and Maf1, the final integrators of these pathways.
doi:10.1128/EC.00106-09
PMCID: PMC2747830  PMID: 19581440
14.  S100A4 is expressed at site of invasion in rheumatoid arthritis synovium and modulates production of matrix metalloproteinases 
Annals of the Rheumatic Diseases  2006;65(12):1645-1648.
The metastasis‐associated protein S100A4 promotes the progression of cancer by regulating the remodelling of the extracellular matrix. The expression of S100A4 in vivo is shown and the functional role of S100A4 in the pathogenesis of osteoarthritis and rheumatoid arthritisis is explored. The expression of S100A4 in rheumatoid arthritis, osteoarthritis and normal synovial tissues was determined by immunohistochemistry. The expression of matrix metalloproteinase (MMP) mRNA was measured in rheumatoid arthritis and osteoarthritis synovial fibroblasts treated and untreated with S100A4 oligomer by real‐time polymerase chain reaction. Levels of released MMPs were confirmed by ELISA in cell culture supernatants. S100A4 protein was expressed in rheumatoid arthritis and osteoarthritis synovial tissues, in contrast with normal synovium. S100A4 up regulated MMP‐3 mRNA in rheumatoid arthritis synovial fluid, with a peak after 6 h. This resulted in release of MMP‐3 protein. MMP‐1, MMP‐9 and MMP‐13 mRNA were also up regulated in synovial fluid, but with different kinetics. MMP‐14 mRNA showed no change. Thus, S100A4 protein is expressed in synovial tissues of patients with rheumatoid arthritis and osteoarthritis in contrast with healthy people. It induces the expression and release of MMP‐3 and other MMPs from synovial fluid. The data suggest that S100A4‐producing cells could be involved in the pathogenesis of osteoarthritis and rheumatoid arthritis, including pannus formation and joint destruction.
doi:10.1136/ard.2005.047704
PMCID: PMC1798462  PMID: 17105852
15.  The Major Portal of Entry of Koi Herpesvirus in Cyprinus carpio Is the Skin▿  
Journal of Virology  2009;83(7):2819-2830.
Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed “U-tube,” to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.
doi:10.1128/JVI.02305-08
PMCID: PMC2655586  PMID: 19153228
16.  Trichostatin A sensitises rheumatoid arthritis synovial fibroblasts for TRAIL‐induced apoptosis 
Annals of the Rheumatic Diseases  2005;65(7):910-912.
Background
Histone acetylation/deacetylation has a critical role in the regulation of transcription by altering the chromatin structure.
Objective
To analyse the effect of trichostatin A (TSA), a streptomyces metabolite which specifically inhibits mammalian histone deacetylases, on TRAIL‐induced apoptosis of rheumatoid arthritis synovial fibroblasts (RASF).
Methods
Apoptotic cells were detected after co‐treatment of RASF with TRAIL (200 ng/ml) and TSA (0.5, 1, and 2 μmol/l) by flow cytometry using propidium iodide/annexin‐V‐FITC staining. Cell proliferation was assessed using the MTS proliferation test. Induction of the cell cycle inhibitor p21Waf/Cip1 by TSA was analysed by western blot. Expression of the TRAIL receptor‐2 (DR5) on the cell surface of RASF was analysed by flow cytometry. Levels of soluble TRAIL were measured in synovial fluid of patients with RA and osteoarthritis (OA) by ELISA.
Results
Co‐treatment of the cells with TSA and TRAIL induced cell death in a synergistic and dose dependent manner, whereas TRAIL and TSA alone had no effect or only a modest effect. RASF express DR5 (TRAIL receptor 2), but treatment of the cells with TSA for 24 hours did not change the expression level of DR5, as it is shown for cancer cells. TSA induced cell cycle arrest in RASF through up regulation of p21Waf1/Cip1. Levels of soluble TRAIL were significantly higher in RA than in OA synovial fluids.
Conclusion
Because TSA sensitises RASF for TRAIL‐induced apoptosis, it is concluded that TSA discloses sensitive sites in the cascade of TRAIL signalling and may represent a new principle for the treatment of RA.
doi:10.1136/ard.2005.044065
PMCID: PMC1798225  PMID: 16284094
trichostatin A; TRAIL; apoptosis; synovial fibroblasts; rheumatoid arthritis
17.  Soluble ErbB3 Levels in Bone Marrow and Plasma of Men with Prostate Cancer 
Purpose
Prostate cancer (PCa) tends to metastasize to bone and induce osteoblastic lesions. We identified a soluble form of ErbB3, p45-sErbB3, in bone marrow supernatant from men with PCa bone metastasis and showed that p45-sErbB3 forms bone. We aimed to understand clinical implications of soluble ErbB3 (sErbB3) by establishing an ELISA to detect sErbB3 levels in bone marrow and plasma samples.
Experimental Design
We performed ELISAs on marrow from 108 men (34 with androgen-dependent disease, 30 with androgen-independent disease (AI) but negative bone scan [AI/BS-], and 44 with AI and positive bone scan [AI/BS+]); sequential marrow from 5 men during treatment; plasma from 52 men before and after docetaxel treatment and from 95 men ≥70 years old without PCa.
Results
Some men with clinically detectable bone metastasis had high sErbB3 levels. Within the AI/BS- group, higher sErbB3 levels seemed to yield lower rates of bone metastasis. In the AI/BS+ group, detectable bone metastases took longer to appear in men with higher sErbB3 levels than in men with lower sErbB3 levels (median, 82 vs. 41 months). However, high sErbB3 levels did not confer survival benefit after metastasis development. Among men with metastatic progression in bone, docetaxel treatment reduced plasma sErbB3 (P < 0.0001) but did not affect bone-specific AP (P = 0.206) or prostate-specific antigen (P = 0.906). sErbB3 was also detected in men without PCa.
Conclusions
The apparent correlation between higher sErbB3 levels and longer time to bone metastasis suggests that sErbB3 participates in prostate cancer’s progression in bone.
doi:10.1158/1078-0432.CCR-08-0472
PMCID: PMC2562877  PMID: 18559590
soluble ErbB3; prostate cancer; bone metastasis
18.  Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi▿  
Journal of Virology  2008;82(10):4955-4964.
Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.
doi:10.1128/JVI.00211-08
PMCID: PMC2346741  PMID: 18337580
19.  Galectin-3 is induced in rheumatoid arthritis synovial fibroblasts after adhesion to cartilage oligomeric matrix protein 
Annals of the Rheumatic Diseases  2004;64(3):419-424.
Background: Galectin-3 is expressed in the synovial tissue of patients with rheumatoid arthritis (RA), particularly at sites of joint destruction.
Objective: To explore the possibilities that galectin-3 is induced either by proinflammatory cytokines or by adhesion to cartilage components.
Methods: Cell culture plates were coated with fibronectin, collagens I–VI, or cartilage oligomeric matrix protein (COMP), and the suspended cells were then added. The medium was changed after 1 hour at 37°C. Adherent cells were further incubated for 18 hours in the presence or absence of tumour necrosis factor α (TNFα) or interleukin 1ß. Cells were pretreated with murine IgG1, anti-CD29, -CD51, -CD61 (integrins), or -CD3 monoclonal antibodies and transferred to culture plates coated with COMP. Adherent cells were counted by light microscopy. The expression of intracellular galectin-3, or cell surface CD29, CD51, and CD61 was determined by flow cytometry before and after adhesion.
Results: Four times more RA synovial fibroblasts (SF) than osteoarthritis SF adhered to COMP. RA SF presented more cell surface integrins, and monoclonal antibodies against CD51 inhibited the adhesion to COMP by 80%. TNFα reduced the expression of CD61 and the adhesion to COMP, but did not reverse the adhesion once it had taken place. The adhesion of RA SF to COMP was found to increase the intracellular level of galectin-3. In contrast, intracellular galectin-3 decreased after exposure to TNFα.
Conclusion: The increase of galectin-3 occurs after adhesion to COMP, and the αVß3 receptor (CD51/CD61) has a pivotal role in this process.
doi:10.1136/ard.2004.023135
PMCID: PMC1755412  PMID: 15345499
20.  Disulfide Bond-mediated Multimerization of Ask1 and Its Reduction by Thioredoxin-1 Regulate H2O2-induced c-Jun NH2-terminal Kinase Activation and Apoptosis 
Molecular Biology of the Cell  2007;18(10):3903-3913.
Apoptosis signal-regulated kinase-1 (Ask1) lies upstream of a major redox-sensitive pathway leading to the activation of Jun NH2-terminal kinase (JNK) and the induction of apoptosis. We found that cell exposure to H2O2 caused the rapid oxidation of Ask1, leading to its multimerization through the formation of interchain disulfide bonds. Oxidized Ask1 was fully reduced within minutes after induction by H2O2. During this reduction, the thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) became covalently associated with Ask1. Overexpression of Trx1 accelerated the reduction of Ask1, and a redox-inactive mutant of Trx1 (C35S) remained trapped with Ask1, blocking its reduction. Preventing the oxidation of Ask1 by either overexpressing Trx1 or using an Ask1 mutant in which the sensitive cysteines were mutated (Ask1-ΔCys) impaired the activation of JNK and the induction of apoptosis while having little effect on Ask1 activation. These results indicate that Ask1 oxidation is required at a step subsequent to activation for signaling downstream of Ask1 after H2O2 treatment.
doi:10.1091/mbc.E07-05-0491
PMCID: PMC1995733  PMID: 17652454
21.  Discrepancy between mRNA and protein expression of tumour suppressor maspin in synovial tissue may contribute to synovial hyperplasia in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2004;63(10):1205-1211.
Objective: To investigate the expression of maspin in RA synovial tissue and compare it with the expression in osteoarthritis (OA) and normal synovial tissue (NS).
Methods: Using specific primers for maspin, a 237 bp fragment was amplified from cDNA obtained from cultured RA, OA, and normal synovial fibroblasts (SF) by RT-PCR. Additionally, mRNA expression levels were determined quantitatively by real time PCR. mRNA expression of maspin was investigated on snap frozen and paraffin embedded synovial tissue sections by in situ hybridisation. Immunohistochemistry was used to identify the cell type expressing maspin. SDS-PAGE and western blotting were performed to evaluate the protein expression in cultured SF. To confirm protein synthesis in situ, immunohistochemistry with specific anti-maspin antibodies was performed in synovial tissue sections of patients with RA.
Results: RT-PCR showed expression of maspin in all cDNA samples from cultured SF. Maspin mRNA was found to be decreased in RA SF twofold and 70-fold compared with OA SF and NS SF, respectively. Maspin mRNA was expressed in RA, OA, and normal synovial tissue. Importantly, maspin transcripts were also found at sites of invasion into cartilage and bone. At the protein level, maspin could be detected in RA and, less prominently, OA SF. In RA synovial tissue, maspin protein was detected in only a few synovial lining cells.
Conclusion: Maspin is expressed intensively in RA SF at the mRNA level, but only slightly at the protein level, possibly owing to down regulation of maspin; this may contribute to the hyperplasia of synovial tissue in RA.
doi:10.1136/ard.2003.006312
PMCID: PMC1754744  PMID: 15361372
22.  Increased DNA fragmentation and ultrastructural changes in fibromyalgic muscle fibres 
Annals of the Rheumatic Diseases  2004;63(3):245-251.
Objective: To determine whether there is evidence of increased DNA fragmentation and ultrastructural changes in muscle tissue of patients with fibromyalgia (FM) compared with healthy controls.
Methods: Muscle tissues from 10 community residents with FM and 10 age and sex matched healthy controls were examined "blindly" for the presence of DNA fragmentation by two different methods: terminal deoxynucleotidyl transferase (TdT) staining (TUNEL) and the FragEL-Klenow DNA fragmentation detection kit. Ultrastructural analysis of tissue was performed by electron microscopy.
Results: DNA fragmentation was detected by both methods in 55.4 (SEM 2.5)% of the nuclei in muscle tissue of patients with FM compared with 16.1 (4.1)% (p<0.001) of the nuclei in healthy controls. Contrary to expectation, no typical features of apoptosis could be detected by electron microscopy. The myofibres and actin filaments were disorganised and lipofuscin bodies were seen; glycogen and lipid accumulation were also found. The number of mitochondria was significantly lower in patients with FM than in controls and seemed to be morphologically altered.
Conclusion: The ultrastructural changes described suggest that patients with FM are characterised by abnormalities in muscle tissue that include increased DNA fragmentation and changes in the number and size of mitochondria. These cellular changes are not signs of apoptosis. Persistent focal contractions in muscle may contribute to ultrastructural tissue abnormalities as well as to the induction and/or chronicity of nociceptive transmission from muscle to the central nervous system.
doi:10.1136/ard.2002.004762
PMCID: PMC1754917  PMID: 14962957
23.  p53 in rheumatoid arthritis synovial fibroblasts at sites of invasion 
Annals of the Rheumatic Diseases  2003;62(12):1139-1144.
Objective: To analyse the functional response of p53 in rheumatoid arthritis synovial fibroblasts (RASF) in vitro and in vivo and to investigate whether activation of p53 modulates the destructive process of RASF.
Methods: RASF and controls grown on chamber slides were either directly examined with DO7 anti-p53 antibodies by immunofluorescence or irradiated with 10 Gy x rays and analysed time dependently for the expression of p53. The percentage of positive cells was evaluated by a quantitative scoring system. RASF and normal (N) SF cultured in vitro were co-implanted with human cartilage in SCID mice for 60 days. Consecutively, the invasion score was evaluated, and the number of p53 positive cells was determined at the sites of invasion by immunohistochemistry. In addition, synovial tissues from RA, osteoarthritis, and normal synovia were stained with DO7 antibodies.
Results: In vitro the rate of expression of p53 in RASF was low (<5%), but transiently inducible by ionising irradiation (50%). In vitro low p53 expressing RASF disclosed, when invading articular cartilage, a nuclear p53 signal in 20% of the cells, indicating the induction of p53 in a distinct population of RASF during the invasive process.
Conclusions: These data suggest an inductive p53 response at sites of cartilage invasion during the destructive process driven by activated RASF.
doi:10.1136/ard.2003.007401
PMCID: PMC1754413  PMID: 14644850
24.  Effectiveness of a measurement feedback system on outcome in rheumatoid arthritis: a controlled clinical trial 
Annals of the Rheumatic Diseases  2003;62(7):624-629.
Background: With the help of a measurement feedback system, the treatment strategy for individual patients with rheumatoid arthritis (RA) can be adjusted to achieve optimal control of disease activity.
Objective: To study whether a measurement feedback system is effective in reducing disease activity in patients with RA.
Methods: Forty eight rheumatologists and 264 patients participated in a controlled clinical trial. A three month control period was followed by a 12 month period, where feedback on disease activity, disability, and damage was provided to the rheumatologist. The primary outcome measure was the rheumatoid arthritis disease activity index (RADAI).
Results: The feedback system was used for 142/228 (62%) patients. Disease modifying antirheumatic drug changes occurred in 69/169 (41%) patients. In patients with high disease activity and feedback use (n=70), the RADAI decreased in the feedback period by –0.27 points per 30 days (p<0.05), as compared with the control period. Patients for whom the feedback system was used had a better outcome than non-users.
Conclusion: Much more training on the use of a feedback system and outcome measures, as well as the inclusion of explicit treatment guidelines will be necessary to increase the clinical use of measurement feedback and, possibly, to reduce disease activity for a larger number of patients with RA.
doi:10.1136/ard.62.7.624
PMCID: PMC1754606  PMID: 12810423
25.  The ICIDH-2 as a framework for the assessment of functioning and disability in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2002;61(3):225-231.
Objective: To investigate by a cross sectional study in patients with rheumatoid arthritis (RA) the relationship between measures of impairment, activity limitation, and participation of the model of functioning and disability (ICIDH-2).
Methods: Inclusion data of patients with RA (n=803) from the Swiss Clinical Quality Management Group were used. Impairments were measured by the Short Form-36 (SF-36) bodily pain scale, rheumatoid arthritis disease activity index (RADAI), disease activity score (DAS28), and radiographic scoring (x ray). Activity limitation was measured with the Health Assessment Questionnaire (HAQ) and the SF-36 physical functioning scale. Participation was measured with the SF-36 role and social functioning scales. Spearman (partial) correlations were used for analysis.
Results: Impairment and activity limitation dimensions of the ICIDH-2 model are related; correlations with the HAQ were: SF-36 bodily pain (rs=-0.61), RADAI (rs=0.58), DAS28 (rs=0.49), and x ray (rs=0.35). Similar correlations were found for SF-36 physical functioning. Activity limitation and participation restriction dimensions are also related: the HAQ correlates well with SF-36 role-physical (rs=-0.53) and SF-36 social functioning (rs=-0.43); SF-36 physical functioning correlates similarly. For impairment and participation restriction dimensions only SF-36 bodily pain is substantially correlated (rs=0.47 and 0.48) with SF-36 role-physical, after correcting for the influence of the activity limitation dimension (HAQ and SF-36 physical functioning).
Conclusions: In this cross sectional study of patients with RA, impairments are associated with activity limitations, and activity limitations are associated with participation restrictions. Pain is the only impairment directly associated with participation restrictions. Based on the results of this study, it is strongly recommended that the ICIDH-2 framework is used in clinical trials and observational studies including the assessment of disease consequences in RA.
doi:10.1136/ard.61.3.225
PMCID: PMC1754033  PMID: 11830427

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