bluuebunny/crossref_metadata_2025 huggingface dataset:
Response of the 5′-flanking region of the human 25-hydroxyvitamin D 1α-hydroxylase gene to physiological stimuli using a transgenic mouse model
Authors: Hendrix I , Anderson P H , Omdahl J L , May B K , Morris H A
Published: February 2005

The enzyme 25-hydroxyvitamin D 1α-hydroxylase, or CYP27B1, is the key enzyme in the two-step activation process of vitamin D to 1,25-dihydroxyvitamin D (1,25D). While a number of regulators of the renal CYP27B1 enzyme activity have been recognized for some years, their underlying molecular mechanisms remain largely unknown, and the DNA regions involved in the in vivo regulation of gene expression by these factors have not been delineated. We have generated a transgenic mouse line that expresses 1501 bp of 5′ flanking region together with 44 bp of 5′ untranslated region of the human CYP27B1 gene fused to the firefly luciferase reporter gene. Animals expressing the luciferase gene demonstrated that both luciferase protein and mRNA for CYP27B1 were localized to proximal convoluted tubule cells of the kidney. In 2-week-old animals, the expression of the transgene and the endogenous CYP27B1 mRNA levels in the kidney were highest and fell with increasing age. Both reporter gene expression and CYP27B1 mRNA levels were downregulated in response to increasing amounts of dietary calcium in a dose-dependent manner. Vitamin D deficiency resulted in an increase in both the reporter gene and CYP27B1 expression. Interestingly, the increase in CYP27B1 mRNA levels was substantially higher than the increase in reporter gene expression, suggesting either that there is a post-transcriptional mechanism that increases the amount of CYP27B1 mRNA or that other regulatory elements are required to maximize the effect of vitamin D deficiency. These findings demonstrate that the 1501 bp 5′ flanking region of the CYP27B1 gene directs expression to the proximal convoluted tubules of the kidney and is responsible for increasing transcriptional activity when dietary calcium and vitamin D levels are depleted. It also responds in the kidney to the physiological regulators of development and ageing.
Molecular recognition characteristics in the insulin-like growth factor (IGF)-insulin-like growth factor binding protein -3/5 (IGFBP-3/5) heparin axis
Authors: Beattie James , Phillips Kirsten , Shand John H , Szymanowska Malgorzata , Flint David J , Allan Gordon J
Published: February 2005

Insulin-like growth factor binding proteins (IGFBPs) -3 and -5 are known to interact with various components of the extracellular matrix (ECM; e.g. heparin and heparan sulphate) and this interaction is believed to affect the affinity of both IGFBP species for their cognate ligands – IGF-I and -II. There is little detail on the nature of the molecular complex formed between ECM components, IGFBPs and IGFs although the glycosaminoglycan (GAG) heparin has been reported to reduce the affinity of IGFBP-5 for IGF-I. In order to investigate this phenomenon further, we have undertaken an extensive surface plasmon resonance based biosensor study to report the affinity of IGFBP-3 and -5 for binding heparin (22 and 7 nM respectively). We have also shown that pre-complexation of IGFBP with IGF-I and -II inhibits the subsequent association of IGFBP with heparin and conversely that heparin complexation of IGFBP-3 and -5 inhibits IGFBP binding to biosensor surfaces containing immobilised IGF-I. In addition we have used both IGF-I and heparin coated biosensor surfaces in an attempt to build ternary IGF–IGFBP–heparin complexes in order to gain some insight into the nature of inhibition by heparin of IGFI–IGFBP complex formation. Our data lead us to conclude that the inhibition by heparin is partly competitive in nature, and that ternary complexes of IGF–IGFBP–heparin are either unable to form, or only form unstable transient complexes. The potential biological significance of our data is highlighted by the demonstration that IGF-I and IGF-II can displace endogenous IGFBP-5 from monolayer cultures of the mouse mammary epithelial cell line HC11.
Cloning, pharmacological characterization and tissue distribution of an ORL1 opioid receptor from an amphibian, the rough-skinned newt Taricha granulosa
Authors: Walthers Eliza A , Bradford C Samuel , Moore Frank L
Published: February 2005

We have cloned and characterized an opioid receptor-like (ORL1; also referred to as NOP) receptor from a urodele amphibian, the rough-skinned newt Taricha granulosa The cDNA clone encodes a protein of 368 amino acids that contains the seven hydrophobic domains characteristic of G-protein-coupled receptors, and has the highest sequence identity to the frog (Rana pipiens) nociceptin-like and human ORL1 opioid receptors (79.6 and 68.4%, respectively). Saturation binding assays on membranes from COS-7 cells transiently expressing the newt ORL1 (nORL) receptor revealed a single, high-affinity (estimated Kd, 0.1974 nM) binding site for the ORL1-specific agonist [3H]orphanin FQ analog ([3H]oFQ). In competition binding assays, the [3H]oFQ-binding site, like the mammalian ORL1 receptor, had no affinity for the non-selective opioid receptor antagonist naloxone, the κ-selective agonists U69593 and U50488, or the μ- and δ-selective opioid receptor agonists DAMGO and DPDPE, respectively. However, the nORL receptor displayed higher affinities for the κ-selective agonists dynorphin A (1–13), dynorphin B, and dynorphin A (1–8) (Ki values, 2.8, 151.8, and 183.0 nM, respectively) than its mammalian homologue. The tissue distribution of the nORL receptor, as determined by reverse transcriptase PCR, was also found to differ from reports on the mammalian ORL1 receptor, with mRNA detected in brain, spinal cord, and lung, but not detected in a number of other peripheral tissues reported to express the receptor in mammals. This is the first report describing the expression and characterization of an amphibian ORL1 receptor, and contributes to our understanding of the evolution of the opioid system.
Reconstructing the population history of Puerto Rico by means of mtDNA phylogeographic analysis
Authors: Martínez‐Cruzado Juan C. , Toro‐Labrador Gladys , Viera‐Vera Jorge , Rivera‐Vega Michelle Y. , Startek Jennifer , Latorre‐Esteves Magda , Román‐Colón Alicia , Rivera‐Torres Rebecca , Navarro‐Millán Iris Y. , Gómez‐Sánchez Enid , Caro‐González Héctor Y. , Valencia‐Rivera Patricia
Published: February 2005

The haplogroup identities of 800 mtDNAs randomly and systematically selected to be representative of the population of Puerto Rico were determined by restriction fragment length polymorphism (RFLP), revealing maternal ancestries in this highly mixed population of 61.3% Amerindian, 27.2% sub‐Saharan African, and 11.5% West Eurasian. West Eurasian frequencies were low in all 28 municipalities sampled, and displayed no geographic patterns. Thus, a statistically significant negative correlation was observed between the Amerindian and African frequencies of the municipalities. In addition, a statistically highly significant geographic pattern was observed for Amerindian and African mtDNAs. In a scenario in which Amerindian mtDNAs prevailed on either side of longitude 66°16′ West, Amerindian mtDNAs were more frequent west of longitude 66°16′ West than east of it, and the opposite was true for African mtDNAs. Haplogroup A had the highest frequency among Amerindian samples (52.4%), suggesting its predominance among the native Taínos. Principal component analysis showed that the sub‐Saharan African fraction had a strong affinity to West Africans. In addition, the magnitudes of the Senegambian and Gulf of Guinea components in Puerto Rico were between those of Cape Verde and São Tomé. Furthermore, the West Eurasian component did not conform to European haplogroup frequencies. HVR‐I sequences of haplogroup U samples revealed a strong North African influence among West Eurasian mtDNAs and a new sub‐Saharan African clade. Am J Phys Anthropol 128:131‐155, 2005. © 2005 Wiley‐Liss, Inc.
Genetic origin of lupus in NZB/SWR hybrids: Lessons from an intercross study
Authors: Xie Shangkui , Li Li , Chang SoogHee , Sharma Ruchi , Kaliyaperumal Arunan , Datta Syamal K. , Mohan Chandra
Published: February 2005

Objective

(SWR × NZB)F1 (or SNF1) hybrid mice succumb to lupus nephritis. A previous analysis of SNF1 × NZB backcross mice revealed the existence of 4 SWR loci (H2 on chromosome 17, Swrl‐1 on chromosome 1, Swrl‐2 on chromosome 14, and Swrl‐3 on chromosome 18) and 2 NZB loci (Nba1 and Lbw2/Sbw2, both on chromosome 4). A second study focusing on SNF1 × SWR backcross offspring uncovered 5 suggestive loci for antinuclear antibody formation, consisting of 3 dominant NZB contributions (Nba4 on chromosome 5, Lbw4 on chromosome 6, and Nba5 on chromosome 7) and 2 recessive SWR contributions (Swrl‐1 on chromosome 1 and Swrl‐4 on chromosome 10). The present intercross study was executed to replicate the earlier findings, using an independent panel of (SWR × NZB)F2 offspring.

Methods

A panel of (NZB × SWR)F2 hybrids were phenotyped (for renal disease, early mortality, and a variety of autoantibodies) and genotyped (using 95 microsatellite primers positioned across all 19 autosomes and the X chromosome). Linkage analysis was conducted using the derived phenotype and genotype data, with the interval‐mapping program MapManager.

Results

Four suggestive loci were mapped: Swrl‐5 on chromosome 1 (peak at 106 cM), linked to hypergammaglobulinemia; an NZB locus on chromosome 5 (Nba4; peak at 15 cM), linked to IgG anti–single‐stranded DNA (anti‐ssDNA) antibodies, IgG anti–doubled‐stranded DNA (anti‐dsDNA) antibodies, and glomerulonephritis; an NZB locus on chromosome 13 (Nba6; peak at 28 cM), linked to IgG anti‐dsDNA antibodies; and an SWR locus on chromosome 14 (Swrl‐2; peak at 30 cM), linked to IgG anti‐ssDNA antibodies. Eight additional loci revealed linkage at P < 0.01, of which 7 co‐mapped with lupus susceptibility loci previously identified in other models.

Conclusion

Considering all 3 mapping studies together, lupus in SWR/NZB hybrids appears to be the epistatic end product of several distinct loci, of which 3 SWR‐derived loci (Swrl‐1, Swrl‐2, and Swrl‐3) and 5 NZB‐derived loci (Nba1, Nba3, Nba4, Nba5, and Lbw4) have been independently confirmed. The immunologic functions and molecular identities of these loci await elucidation.
Novel role of CD40 in Fas‐dependent apoptosis of cultured salivary epithelial cells from patients with Sjögren's syndrome
Authors: Ping Li , Ogawa Noriyoshi , Sugai Susumu
Published: February 2005

Objective

To determine the role of Fas and CD40 in the molecular mechanism of salivary epithelial cell death in Sjögren's syndrome (SS).

Methods

The expression of Fas and CD40 in SS salivary epithelial cells was analyzed by flow cytometry. Induction of apoptosis with anti‐Fas and/or anti‐CD40 monoclonal antibodies (mAb) was examined by morphologic analysis, DNA fragmentation, and TUNEL assay. Expression of c‐FLIP, Fas‐associated phosphatase 1, FADD, Bcl‐2, Bcl‐xL, and Bcl‐xS was determined by Western blot analysis.

Results

Expression of Fas and CD40 was significantly higher in SS salivary epithelial cells than in normal cells after interferon‐γ (IFNγ) stimulation (P < 0.001 for both Fas and CD40). Although neither anti‐Fas (CH11) nor anti‐CD40 mAb alone could induce typical apoptosis, the two together and preincubation with IFNγ efficiently induced apoptosis in SS salivary epithelial cells. This apoptosis was almost completely blocked by neutralizing anti‐Fas mAb (ZB4), whereas an antagonistic mAb to CD40 (ch5D12) partially inhibited anti‐Fas/anti‐CD40–induced apoptosis. Also, c‐FLIP, an important inhibitory molecule in the Fas death pathway, was strongly expressed in SS salivary epithelial cells, but its expression was down‐regulated at the protein level by anti‐CD40 mAb.

Conclusion

CD40 signals promote Fas‐dependent death of SS salivary epithelial cells by down‐regulating c‐FLIP expression. The presence of c‐FLIP in these cells may explain their resistance to undergoing apoptosis in response to either anti‐Fas or anti‐CD40 mAb, despite their surface expression of both proteins. These findings suggest that SS salivary epithelial cell death requires the cooperation of both Fas and CD40.
The relationship between body composition and knee cartilage volume in healthy, middle‐aged subjects
Authors: Cicuttini Flavia M. , Teichtahl Andrew J. , Wluka Anita E. , Davis Susan , Strauss Boyd J. G. , Ebeling Peter R.
Published: February 2005

Objective

Although obesity is widely accepted as a risk factor for knee osteoarthritis, it is not clear whether individual components of body composition, such as the mass and distribution of muscle and fat, are associated with development of the disease. This study examined the effect of measures of body composition on the longitudinal change in tibial cartilage volume.

Methods

Body composition, assessed via dual x‐ray absorptiometry, and tibial cartilage volume, assessed via magnetic resonance imaging, were measured in 86 healthy men and women who were mid‐life in age. Change in tibial cartilage volume was assessed by imaging each knee 2 years after the baseline measurement and determining the difference from baseline in tibial cartilage volume. Correlations were determined between the muscle and fat mass of the arm, leg, and total body and the volume of the lateral‐ and medial‐tibial cartilage, as well as the change in tibial cartilage volume over 2 years, after adjusting for confounders.

Results

There was a significant association between muscle mass and the medial‐tibial cartilage volume, independent of age, sex, body mass index, tibial bone area, and level of physical activity. Although there was a positive association between muscle mass and the lateral‐tibial cartilage volume, this did not persist after adjustment for confounders. Loss of muscle mass was associated with an increased loss of medial‐ and lateral‐tibial cartilage over 2 years, after adjusting for confounders. No relationship was apparent between fat mass and either medial‐ or lateral‐tibial cartilage volume, or between fat mass and change in either medial‐ or lateral‐tibial cartilage volume over 2 years, after adjusting for confounders.

Conclusion

Muscle mass is an independent predictor of medial‐tibial cartilage volume in healthy people in mid‐life and is associated with a reduction in the rate of loss of tibial cartilage. This suggests that increased muscle mass may be protective against the onset of osteoarthritis.
Pioglitazone, a peroxisome proliferator–activated receptor γ agonist, reduces the progression of experimental osteoarthritis in guinea pigs
Authors: Kobayashi Tetsuya , Notoya Kohei , Naito Takako , Unno Satoko , Nakamura Akihiro , Martel‐Pelletier Johanne , Pelletier Jean‐Pierre
Published: February 2005

Objective

To evaluate the in vivo therapeutic effect of pioglitazone, a peroxisome proliferator–activated receptor γ (PPARγ) agonist, on the development of lesions in a guinea pig model of osteoarthritis (OA), and to determine the influence of pioglitazone on the synthesis of matrix metalloproteinase 13 (MMP‐13) and interleukin‐1β (IL‐1β) in articular cartilage.

Methods

The OA model was created by partial medial meniscectomy of the right knee joint. The guinea pigs were divided into 4 treatment groups: unoperated animals that received no treatment (normal), operated animals (OA guinea pigs) that received placebo, OA guinea pigs that received oral pioglitazone at 2 mg/kg/day, and OA guinea pigs that received oral pioglitazone at 20 mg/kg/day. The animals began receiving medication 1 day after surgery and were killed 4 weeks later. Macroscopic and histologic analyses were performed on the cartilage. The levels of MMP‐13 and IL‐1β in OA cartilage chondrocytes were evaluated by immunohistochemistry.

Results

OA guinea pigs treated with the highest dosages of pioglitazone showed a significant decrease, compared with the OA placebo group, in the surface area (size) and grade (depth) of cartilage macroscopic lesions on the tibial plateaus. The histologic severity of cartilage lesions was also reduced. A significantly higher percentage of chondrocytes in the middle and deep layers stained positive for MMP‐13 and IL‐1β in cartilage from placebo‐treated OA guinea pigs compared with normal controls. Guinea pigs treated with the highest dosage of pioglitazone demonstrated a significant reduction in the levels of both MMP‐13 and IL‐1β in OA cartilage.

Conclusion

This is the first in vivo study demonstrating that a PPARγ agonist, pioglitazone, could reduce the severity of experimental OA. This effect was associated with a reduction in the levels of MMP‐13 and IL‐1β, which are known to play an important role in the pathophysiology of OA lesions.
Efficacy of repeated intravenous infusions of an anti–tumor necrosis factor α monoclonal antibody, infliximab, in persistently active, refractory juvenile idiopathic arthritis: Results of an open‐label prospective study
Authors: Gerloni Valeria , Pontikaki Irene , Gattinara Maurizio , Desiati Francesca , Lupi Elide , Lurati Alfredomaria , Salmaso Alessandra , Fantini Flavio
Published: February 2005

Objective

To evaluate the efficacy and safety of a chimeric monoclonal anti–tumor necrosis factor α antibody (infliximab) with methotrexate (MTX) in juvenile idiopathic arthritis (JIA) with an active polyarticular course that is not responsive to MTX.

Methods

Twenty‐four young adults with long‐lasting, refractory JIA were enrolled in an open, prospective, 2‐year pilot study. Patients received intravenous infliximab at 3 mg/kg of body weight at weeks 0, 2, and 6 and every 8 weeks thereafter, with weekly subcutaneous MTX.

Results

The median duration of therapy was 9.1 months. Significant improvements were observed in the number of joints (28‐joint count) with active disease (median 6 at baseline, 2 at 2 weeks, 0 at 6 months, 0 at 1 year; P < 0.05). Pain as well as patient's and physician's global assessments of disease status were assessed on 0–100‐mm (0 = best; 100 = worst) visual analog scales (VAS). There were significant improvements in VAS pain scores (45 at baseline, 25 at 2 weeks, 8.5 at 6 months, 10 at 1 year; P < 0.05), patient's global assessment of disease status (50 at baseline, 22 at 2 weeks, 11.5 at 6 months, 18 at 1 year; P < 0.05), and physician's global assessment of disease status (50.5 at baseline, 22.5 at 2 weeks, 6.5 at 6 months, 10 at 1 year; P < 0.01). In addition, there were significant improvements in the erythrocyte sedimentation rate (64 mm/hour at baseline, 36 mm/hour at 2 weeks, 23.5 mm/hour at 6 months, 35 mm/hour at 1 year; P < 0.01) and C‐reactive protein level (4.9 mg/dl at baseline, 2.8 mg/dl at 2 weeks, 3.1 mg/dl at 6 months, 3.2 mg/dl at 1 year; P < 0.005). The percentage of patients meeting the American College of Rheumatology 20% improvement criteria at each assessment ranged from 54.2% to 86.7%. Of the responses on the Disease Activity Score in 28 joints, 37.5–63.6% were classified as “good,” 14.3–33.3% were classified as “moderate,” and 18–37.5% were classified as “no response.” Twelve patients (50%) had adverse events, and 5 patients (20.8%) withdrew.

Conclusion

Infliximab plus MTX showed high effectiveness and safety in short‐ and medium‐term treatment of long‐lasting refractory JIA. A controlled multicenter clinical trial is needed.
Use of local electroporation enhances methotrexate effects with minimum dose in adjuvant‐induced arthritis
Authors: Tada Masahiro , Inui Kentaro , Koike Tatsuya , Takaoka Kunio
Published: February 2005

Objective

To investigate the effects of electrical pulses on the ability of methotrexate (MTX) to attenuate inflammation and subsequent joint destruction in rats with adjuvant‐induced arthritis (AIA).

Methods

Rats in the experimental group received an intraperitoneal injection of MTX (0.125 mg/kg body weight), followed 30 minutes later by application of direct electrical pulses (50V, 8 Hz) to their left hind paws with an electroporation apparatus (M+/E+ group; n = 8). The procedure was repeated twice weekly for 3 weeks. Three control groups received the following treatments, respectively: MTX without electrical treatment (M+/E− group; n = 9), electrical treatment but no MTX (M−/E+ group; n = 10), or no electrical treatment and no MTX (M−/E− group; n = 9). Progression of AIA was monitored by joint swelling and radiologic and histologic changes in the ankle joint.

Results

Three weeks after injection of the adjuvant, and at the height of the arthritic reaction, the swelling and radiologic and histologic changes in the left hind paws in the M+/E+ rats were significantly reduced, as compared with changes observed in the control groups.

Conclusion

These results demonstrate that application of electrical pulses in combination with use of systemic low‐dose MTX can ameliorate local arthritic reactions. This response probably occurs because electrical stimulation promotes transient passage of MTX through pores in the cell membranes, with a resultant local increase in the concentration of the drug within the cells. These results point to a potential use of electrochemotherapy to increase the efficacy of MTX or other drugs in an arthritic joint that is refractory to treatment, without increasing the dose of the drug.
Two single‐nucleotide polymorphisms in the 5′ and 3′ ends of the osteopontin gene contribute to susceptibility to systemic lupus erythematosus
Authors: D'Alfonso S. , Barizzone N. , Giordano M. , Chiocchetti A. , Magnani C. , Castelli L. , Indelicato M. , Giacopelli F. , Marchini M. , Scorza R. , Danieli M. G. , Cappelli M. , Migliaresi S. , Bigliardo B. , Sabbadini M. G. , Baldissera E. , Galeazzi M. , Sebastiani G. D. , Minisola G. , Ravazzolo R. , Dianzani U. , Momigliano‐Richiardi P.
Published: February 2005

Objective

To test the association of osteopontin (OPN) polymorphisms with systemic lupus erythematosus (SLE).

Methods

The coding 5′ and 3′ flanking regions of the OPN gene were scanned for polymorphisms by denaturing high‐performance liquid chromatography. A case–control association study was performed in 394 Italian SLE patients and 479 matched controls. OPN serum levels were determined by enzyme‐linked immunosorbent assay in 40 patients and 124 controls, and the mean levels were compared between the different OPN genotypes.

Results

Among the 13 detected single‐nucleotide polymorphisms (SNPs), alleles −156G (frequency 0.714 versus 0.651; P = 0.006, corrected P [Pcorr] = 0.036) and +1239C (0.377 versus 0.297; P = 0.00094, Pcorr = 0.0056) were significantly increased in the SLE patients compared with the controls. The presence of the associated allele in single or double dose conferred an odds ratio (OR) of 2.35 (95% confidence interval [95% CI] 1.38–4.02) for SNP −156 and an OR of 1.57 (95% CI 1.16–2.13) for SNP +1239. These effects were independent of each other, i.e., not a consequence of linkage disequilibrium between the 2 alleles. The risk associated with a double dose of susceptibility alleles at both SNPs was 3.8‐fold higher (95% CI 2.0–7.4) relative to the complete absence of susceptibility alleles. With regard to individual clinical and immunologic features, a significant association was seen between lymphadenopathy and −156 genotypes (overall P = 0.0011, Pcorr = 0.046). A significantly increased OPN serum level was detected in healthy individuals carrying +1239C (P = 0.002), which is indicative of an association between the SLE susceptibility allele and OPN levels.

Conclusion

These data suggest the independent effect of a promoter (−156) and a 3′‐untranslated region (+1239) SNP in SLE susceptibility. We can speculate that these sequence variants (or others in perfect linkage disequilibrium) create a predisposition to high production of OPN, and that this in turn may confer susceptibility to SLE.
Oral treatment with PD‐0200347, an α2δ ligand, reduces the development of experimental osteoarthritis by inhibiting metalloproteinases and inducible nitric oxide synthase gene expression and synthesis in cartilage chondrocytes
Authors: Boileau Christelle , Martel‐Pelletier Johanne , Brunet Julie , Tardif Ginette , Schrier Denis , Flory Craig , El‐Kattan Ayman , Boily Martin , Pelletier Jean‐Pierre
Published: February 2005

Objective

To examine the in vivo effects of PD‐0200347, an α2δ ligand of voltage‐activated Ca2+ channels and a compound chemically related to pregabalin and gabapentin, on the development of cartilage structural changes in an experimental dog model of osteoarthritis (OA). The effects of PD‐0200347 on the major pathways involved in OA cartilage degradation, including matrix metalloproteinases (MMPs) and the inducible form of nitric oxide synthase (iNOS), were also studied.

Methods

OA was surgically induced in dogs by sectioning the anterior cruciate ligament. OA dogs were randomly distributed into 3 groups and treated orally with either 1) placebo, 2) 15 mg/kg/day of PD‐0200347, or 3) 90 mg/kg/day of PD‐0200347. Dogs were killed 12 weeks after surgery. The severity of the lesions was scored macroscopically and histologically. Cartilage specimens from the femoral condyles and tibial plateaus were processed for RNA extraction and quantitative reverse transcription–polymerase chain reaction (RT‐PCR) or immunohistochemistry. Specific probes and antibodies were used to study the messenger RNA and protein levels of iNOS, MMP‐1, MMP‐3, and MMP‐13.

Results

No clinical signs of drug toxicity were noted in the treated animals. Treatment with PD‐0200347 at both dosages tested (15 and 90 mg/kg/day) reduced the development of cartilage lesions. There was a reduction in the score of lesions, with a statistically significant (P = 0.01) difference when the highest dosage of the drug was administered. The reduction in the score was mainly related to a decrease in the surface size of the lesions. Quantitative RT‐PCR showed that PD‐0200347 significantly reduced the expression of MMP‐13, a key mediator in OA. Immunohistochemical analyses showed that treatment with PD‐0200347 significantly reduced the synthesis of all key OA mediators studied.

Conclusion

This study demonstrated the efficacy of PD‐0200347 in reducing the progression of cartilage structural changes in a dog model of OA. It also showed that this effect is linked to the inhibition of the major pathophysiologic mediators responsible for cartilage degradation.
Fibroblast‐like synoviocytes derived from patients with rheumatoid arthritis show the imprint of synovial tissue heterogeneity: Evidence of a link between an increased myofibroblast‐like phenotype and high‐inflammation synovitis
Authors: Kasperkovitz Pia V. , Timmer Trieneke C. G. , Smeets Tom J. , Verbeet Nicolette L. , Tak Paul P. , van baarsen Lisa G. M. , Baltus Belinda , Huizinga Tom W. J. , Pieterman Elsbet , Fero Mike , Firestein Gary S. , van der Pouw Kraan Tineke C. T. M. , Verweij Cornelis L.
Published: February 2005

Objective

Given the heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues, we sought to determine whether this variability was also reflected at the level of the fibroblast‐like synoviocyte (FLS) cultured from RA synovial tissues.

Methods

Gene expression profiles in FLS cultured from synovial tissues obtained from 19 RA patients were analyzed using complementary DNA microarrays and hierarchical cluster analysis. To validate the subclassification, we performed prediction analysis and principal components analysis. Genes that differed significantly in their expression between FLS cultures were selected using Statistical Analysis of Microarrays software. Real‐time quantitative polymerase chain reaction was performed to validate the microarray data. Immunocytochemistry was applied to study the expression of the genes of interest in FLS and synovial tissues.

Results

Hierarchical clustering identified 2 main groups of FLS characterized by distinctive gene expression profiles. FLS from high‐inflammation synovial tissues revealed increased expression of a transforming growth factor β/activin A–inducible gene profile that is characteristic of myofibroblasts, a cell type considered to be involved in wound healing, whereas increased production of growth factor (insulin‐like growth factor 2/insulin‐like growth factor binding protein 5) appeared to constitute a characteristic feature of FLS derived from low‐inflammation synovial tissues. The molecular feature that defines the myofibroblast‐like phenotype was reflected as an increased proportion of myofibroblast‐like cells in the heterogeneous FLS population. Myofibroblast‐like cells were also found upon immunohistochemical analysis of synovial tissue.

Conclusion

Our findings support the notion that heterogeneity between synovial tissues is reflected in FLS as a stable trait, and provide evidence of a possible link between the behavior of FLS and the inflammation status of RA synovium.
Rituximab in the treatment of dermatomyositis: An open‐label pilot study
Authors: Levine Todd D.
Published: February 2005

Objective

To test the hypothesis that B cells play a role in the pathophysiology of dermatomyositis (DM) by examining the effect of B cell depletion in patients with symptomatic DM. Patients were treated with rituximab, a CD20+ B cell–depleting monoclonal antibody.

Methods

This was an open‐label uncontrolled pilot trial in 7 adult patients with DM, 6 of whom had longstanding illness that was responding inadequately to a number of currently available immunosuppressive agents. All patients received 4 intravenous infusions of rituximab given at weekly intervals. Patients were followed up for up to 1 year without further treatment with rituximab. One patient was lost to followup. The principal efficacy outcome was muscle strength, measured by quantitative dynomometry.

Results

All 6 evaluable patients exhibited major clinical improvement, with muscle strength increasing over baseline by 36–113%. Maximal improvements in muscle strength occurred as early as 12 weeks after the initial infusion of rituximab. CD20+ B cells were effectively depleted in all patients by 12 weeks. Four patients experienced a return of symptoms that coincided with the return of B cells before the 52‐week end point. Two patients maintained their increased muscle strength at 52 weeks, and 1 of these patients maintained this strength even after the return of B cells. Other symptoms of DM, including rash, alopecia, and reduced forced vital capacity, improved markedly in patients with these symptoms. Rituximab was well tolerated, with no treatment‐related severe or serious adverse events during the observation period of this study.

Conclusion

This small open‐label study of DM patients treated with rituximab provided sufficiently encouraging results to justify a more formal evaluation of the value of B cell depletion therapy in the treatment of DM.
Inhibition of the development of collagen‐induced arthritis in rhesus monkeys by a small molecular weight antagonist of CCR5
Authors: Vierboom Michel P. M. , Zavodny Paul J. , Chou Chuan‐Chu , Tagat Jayaram R. , Pugliese‐Sivo Catherine , Strizki Julie , Steensma Ruo W. , McCombie Stuart W. , Çelebi‐Paul Liesbeth , Remarque Ed , Jonker Margreet , Narula Satwant K. , Hart Bert't
Published: February 2005

Objective

Collagen‐induced arthritis (CIA) in the rhesus monkey is a nonhuman primate model of rheumatoid arthritis (RA). The close phylogenetic relationship between humans and the rhesus monkey makes this model useful for the preclinical safety and efficacy testing of new therapies that are inactive in animals more distinctly related to humans. In this study, we tested the therapeutic potential of a novel, small molecular weight antagonist of CCR5, SCH‐X, in this model.

Methods

CIA was induced in 10 rhesus monkeys. The animals were allocated to receive SCH‐X or saline as the control (n = 5 in each group). Treatment was initiated on the day of CIA induction and continued for 45 days. Monkeys were monitored before and 63 days after CIA induction for macroscopic signs of clinical arthritis, such as soft‐tissue swelling and body weight. Furthermore, markers of inflammation and joint degradation were monitored to follow the disease course.

Results

Only 2 of 5 animals in the SCH‐X–treated group displayed prominent soft‐tissue swelling, compared with all 5 saline‐treated monkeys. In addition to the suppression of joint inflammation, treatment with SCH‐X resulted in a reduction in joint destruction, as demonstrated by lower rates of urinary excretion of collagen crosslinks, with confirmation by histology. Whereas in all saline‐treated monkeys, marked erosion of joint cartilage was observed, this was absent in 4 of the 5 SCH‐X–treated monkeys.

Conclusion

The systemic effects of treatment with SCH‐X were a suppressed acute‐phase reaction (reduction in C‐reactive protein level) in the 3 treated monkeys with CIA that remained asymptomatic, and an altered antibody response toward type II collagen. The results suggest that the CCR5 antagonist SCH‐X might have a strong clinical potential for treatment during periods of active inflammation, as seen in RA.
The intracellular 52‐kd Ro/SSA autoantigen in keratinocytes is up‐regulated by tumor necrosis factor α via tumor necrosis factor receptor I
Authors: Gerl Velia , Hostmann Björn , Johnen Christa , Waka Aderajew , Gerl Markus , Schumann Frank , Klein Rolf , Radbruch Andreas , Hiepe Falk
Published: February 2005

Objective

Previous studies have shown that the nuclear Ro/SSA autoantigens involved in photosensitive cutaneous lupus manifestations are regulated by ultraviolet B (UVB) irradiation. UVB exposure triggers the release of tumor necrosis factor α (TNFα) from keratinocytes in the epidermis and from mast cells in the dermis. The present study aimed to characterize the effect of TNFα on messenger RNA (mRNA) and protein expression of the intracellular 52‐kd Ro/SSA autoantigen in primary human keratinocytes and to elucidate the TNFα receptor (TNFR) signaling pathways mediating this effect.

Methods

Expression of 52‐kd Ro/SSA mRNA in primary human keratinocytes was investigated by quantitative real‐time polymerase chain reaction (LightCycler system) using GAPDH as the housekeeping gene. Expression of 52‐kd Ro/SSA protein was studied by flow cytometry after staining intracellular protein with IgG purified from an anti–52‐kd Ro/SSA–positive serum. TNFR function was assessed by culturing cells in the presence and absence of neutralizing antibodies directed against the TNFR subunits TNFRI and TNFRII.

Results

TNFα‐induced up‐regulation of 52‐kd Ro/SSA mRNA expression peaked at 4 hours, followed by up‐regulation of intracellular 52‐kd Ro/SSA protein expression at 24 hours, independently of apoptosis. Between different donors, a high variability of both constitutive expression levels and TNFα‐induced changes in 52‐kd Ro/SSA mRNA and protein expression was observed. The up‐regulatory effect of TNFα on 52‐kd Ro/SSA mRNA and protein expression was inhibited by anti‐TNFRI antibodies but enhanced by anti‐TNFRII antibodies.

Conclusion

The finding that TNFα up‐regulates 52‐kd Ro/SSA expression in keratinocytes via TNFRI suggests that it may play a role in the pathogenesis of anti‐Ro/SSA–associated cutaneous lupus erythematosus.
Increased unrecognized coronary heart disease and sudden deaths in rheumatoid arthritis: A population‐based cohort study
Authors: Maradit‐Kremers Hilal , Crowson Cynthia S. , Nicola Paulo J. , Ballman Karla V. , Roger Véronique L. , Jacobsen Steve J. , Gabriel Sherine E.
Published: February 2005

Objective

To examine the risk of clinical coronary heart disease (CHD) in patients with rheumatoid arthritis (RA) compared with age‐ and sex‐matched non‐RA subjects, and to determine whether RA is a risk factor for CHD after accounting for traditional CHD risk factors.

Methods

We assembled a population‐based incidence cohort of 603 Rochester, Minnesota residents ages ≥18 years who first fulfilled the American College of Rheumatology (ACR) 1987 criteria for RA between January 1, 1955 and January 1, 1995, and 603 age‐ and sex‐matched non‐RA subjects. All subjects were followed up through their complete inpatient and outpatient medical records, beginning at age 18 years until death, migration, or January 1, 2001. Data were collected on CHD events and traditional CHD risk factors (diabetes mellitus, hypertension, dyslipidemia, body mass index, smoking) using established diagnostic criteria. CHD events included hospitalized myocardial infarction (MI), unrecognized MI, coronary revascularization procedures, angina pectoris, and sudden CHD deaths. Conditional logistic regression and Cox regression models were used to estimate the risk of CHD associated with RA, both prior to and following RA diagnosis, after adjusting for CHD risk factors.

Results

During the 2‐year period immediately prior to fulfillment of the ACR criteria, RA patients were significantly more likely to have been hospitalized for acute MI (odds ratio [OR] 3.17, 95% confidence interval [95% CI] 1.16–8.68) or to have experienced unrecognized MIs (OR 5.86, 95% CI 1.29–26.64), and less likely to have a history of angina pectoris (OR 0.58, 95% CI 0.34–0.99) compared with non‐RA subjects. After the RA incidence date, RA patients were twice as likely to experience unrecognized MIs (hazard ratio [HR] 2.13, 95% CI 1.13–4.03) and sudden deaths (HR 1.94, 95% CI 1.06–3.55) and less likely to undergo coronary artery bypass grafting (HR 0.36, 95% CI 0.16–0.80) compared with non‐RA subjects. Adjustment for the CHD risk factors did not substantially change the risk estimates.

Conclusion

Patients with RA have a significantly higher risk of CHD when compared with non‐RA subjects. RA patients are less likely to report symptoms of angina and more likely to experience unrecognized MI and sudden cardiac death. The risk of CHD in RA patients precedes the ACR criteria–based diagnosis of RA, and the risk cannot be explained by an increased incidence of traditional CHD risk factors in RA patients.
The risk of congestive heart failure in rheumatoid arthritis: A population‐based study over 46 years
Authors: Nicola Paulo J. , Maradit‐Kremers Hilal , Roger Véronique L. , Jacobsen Steven J. , Crowson Cynthia S. , Ballman Karla V. , Gabriel Sherine E.
Published: February 2005

Objective

It is hypothesized that the systemic inflammation associated with rheumatoid arthritis (RA) promotes an increased risk of cardiovascular (CV) morbidity and mortality. We examined the risk and determinants of congestive heart failure (CHF) in patients with RA.

Methods

We assembled a population‐based, retrospective incidence cohort from among all individuals living in Rochester, Minnesota, in whom RA (defined according to the American College of Rheumatology 1987 criteria) was first diagnosed between 1955 and 1995, and an age‐ and sex‐matched non‐RA cohort. After excluding patients in whom CHF occurred before the RA index date, all subjects were followed up until either death, incident CHF (defined according to the Framingham Heart Study criteria), migration from the county, or until January 1, 2001. Detailed information from the complete medical records (including all inpatient and outpatient care provided by all local providers) regarding RA, ischemic heart disease, and traditional CV risk factors was collected. Cox models were used to estimate the effect of RA on the development of CHF, adjusting for CV risk factors and/or ischemic heart disease.

Results

The study population included 575 patients with RA and 583 subjects without RA. The CHF incidence rates were 1.99 and 1.16 cases per 100 person‐years in patients with RA and in non‐RA subjects, respectively (rate ratio 1.7, 95% confidence interval [95% CI] 1.3–2.1). After 30 years of followup, the cumulative incidence of CHF was 34.0% in patients with RA and 25.2% in non‐RA subjects (P< 0.001). RA conferred a significant excess risk of CHF (hazard ratio [HR] 1.87, 95% CI 1.47–2.39) after adjusting for demographics, ischemic heart disease, and CV risk factors. The risk was higher among patients with RA who were rheumatoid factor (RF) positive (HR 2.59, 95% CI 1.95–3.43) than among those who were RF negative (HR 1.28, 95% CI 0.93–1.78).

Conclusion

Compared with persons without RA, patients with RA have twice the risk of developing CHF. This excess risk is not explained by traditional CV risk factors and/or clinical ischemic heart disease.
Antisense inhibition of osteogenic protein 1 disturbs human articular cartilage integrity
Authors: Söder Stephan , Hakimiyan Arnavaz , Rueger David C. , Kuettner Klaus E. , Aigner Thomas , Chubinskaya Susan
Published: February 2005

Objective

To delineate the role of endogenous osteogenic protein 1 (OP‐1) in human articular cartilage homeostasis via the inhibition of OP‐1 gene expression by antisense oligonucleotides.

Methods

Human adult normal articular cartilage was obtained from the knee and ankle joints of 34 organ donors. Chondrocytes were cultured as tissue explants or isolated cells in alginate or high‐density monolayers for 48 hours in the presence of OP‐1 antisense or sense oligonucleotides. The effect of OP‐1 antisense inhibition was evaluated by reverse transcription–polymerase chain reaction, 35S incorporation, dimethylmethylene blue assay, histology with Safranin O staining, and immunohistochemistry with anti–proOP‐1, anti–mature OP‐1, and anti‐aggrecan antibodies.

Results

Antisense treatment inhibited OP‐1 gene expression by a mean ± SD of 34 ± 12% (P < 0.01) in chondrocytes cultured in monolayers and by 77 ± 27% (P < 0.03) in alginate beads. The inhibition of autocrine OP‐1 caused a striking decrease in aggrecan gene expression, in total proteoglycan content accumulated in cartilage matrix, and in the ability of chondrocytes to newly synthesize proteoglycans. OP‐1 antisense reduced aggrecan messenger RNA expression by 42 ± 17% (P < 0.05) and proteoglycan synthesis by 48 ± 23% (P < 0.01). Histology and immunohistochemistry revealed a dramatic decrease in Safranin O staining and reduced anti‐aggrecan staining (primarily in the superficial and middle cartilage layers) with OP‐1 antisense treatment.

Conclusion

Our results suggest that OP‐1 is an important endogenous cartilage factor that regulates matrix integrity and possibly needs to be induced or up‐regulated to maintain normal cartilage homeostasis. These findings confirm our hypothesis that a lack of autocrine OP‐1 may lead to an elevated susceptibility of chondrocytes to the catabolic processes, thus contributing/promoting cartilage degeneration.
Remission of proliferative lupus nephritis following B cell depletion therapy is preceded by down‐regulation of the T cell costimulatory molecule CD40 ligand: An open‐label trial
Authors: Sfikakis P. P. , Boletis J. N. , Lionaki S. , Vigklis V. , Fragiadaki K. G. , Iniotaki A. , Moutsopoulos H. M.
Published: February 2005

Objective

Autoreactive B cells play a key role in tissue injury in systemic autoimmune disease, and therefore a treatment resulting in B cell depletion could have benefit. This open‐label study was undertaken to evaluate the efficacy of the anti‐CD20 monoclonal antibody rituximab in the treatment of lupus nephritis.

Methods

Lupus patients with active proliferative nephritis (4 with focal disease and 6 with diffuse disease) received rituximab (4 weekly infusions of 375 mg/m2) combined with oral prednisolone. Clinical, laboratory, and immunologic responses, including peripheral lymphocyte subsets measured by flow cytometry, were prospectively assessed at monthly intervals for 12 months. Complete remission of nephritis was defined as normal serum creatinine and albumin levels, inactive urine sediment, and 24‐hour urinary protein <500 mg. Partial remission was defined as >50% improvement in all renal parameters that were abnormal at baseline.

Results

B cell depletion lasted from 1 month to 7 months and was well tolerated. Partial remission was achieved in 8 of 10 patients within a median of 2 months (range 1–4 months); in 5 of them, complete remission was subsequently established (at a median of 3 months from baseline), and it was sustained at 12 months in 4. As early as 1 month from baseline, the expression of the costimulatory molecule CD40 ligand on CD4+ T cells was decreased by 4‐fold, and it was almost blocked when partial remission was clinically evident. The expression of T cell activation markers CD69 and HLA–DR was significantly decreased at time points when partial remission was observed, and was further decreased during complete remission. In contrast, in patients who did not exhibit a response or when relapse was detected in patients in whom an initial remission had been achieved, such decreases were not prominent. Serum concentrations of double‐stranded DNA autoantibodies were decreased in all patients, regardless of clinical outcome.

Conclusion

Following B cell depletion, clinical remission of lupus nephritis is associated with a decrease in T helper cell activation, suggesting an additional role for B cells, independent of autoantibody production, in promoting disease. A controlled trial to confirm these promising clinical results is warranted.
Reduction of joint damage in severe rheumatoid arthritis by high‐dose chemotherapy and autologous stem cell transplantation
Authors: Verburg Robert J. , Sont Jacob K. , van Laar Jacob M.
Published: February 2005

Objective

To examine the influence of high‐dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT) on joint damage in patients with rheumatoid arthritis.

Methods

Eight patients with active, refractory, progressively erosive RA were treated. The conditioning regimen consisted of intravenous administration of high doses of cyclophosphamide (totaling 200 mg/kg), with subsequent reinfusion of the positively selected graft. Radiographs of hands and feet were obtained before, and at 1 and 2 years after transplantation. All radiographs of hands and feet obtained up to 6 years before transplantation were also collected to compare radiographic progression before and after HDC + ASCT. Scoring of all radiographs was performed according to the Larsen scale by a trained investigator who was blinded with regard to the clinical data.

Results

Radiographic assessment by the Larsen scale showed a decreased progression of joint damage. Before transplantation, the mean Larsen score increased at a rate of 8.9 points per year. During the 2 years after transplantation, the mean rate of progression in the Larsen score decreased to 2.7 points per year (P = 0.023 by paired t‐test).

Conclusion

The results of the present analysis demonstrate major beneficial effects of HDC + ASCT on the rate of joint destruction during the first 2 years of followup after treatment.