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A Pécsi Tudományegyetem Ortopédiai Klinikáján, 2009 márciusától 2012 végéig 62 Bösch műtétet vizsgáltunk, 9 operatőr által elvégezve, az átlagos utánkövetés 66 hónap volt. Mindannyian enyhe vagy középsúlyos hallux valgus deformitással rendelkeztek. A műtét eredményének megítélésére a fenti időszakban műtött betegek adatait a klinikai adatszolgáltató rendszerekből nyertük ki (Medsol, Medview), illetve a betegeket kontrollvizsgálatra visszarendeltük. A betegenként rendelkezésre álló 3 db röntgenfelvételen megmértük a hallux valgus (HV), intermetatarsalis (IM) és distalis metatarsalis ízfelszíni (DMA) szögeket. Továbbá felmértük a szövődményrátát, illetve értékeltük a posztoperatív betegelégedettséget. Mindhárom vizsgált radiológiai szögérték átlagértékei jelentősen csökkentek a preoperatív állapothoz képest, legnagyobb mértékben, szignifikánsan a hallux valgus szög esetében. Az átlag HV szög a preoperatív 37,4 fokról 12,9 fokra javult az utolsó kontrollvizsgálatnál, az átlag IM szög 13,9-ről 6,9 fokra csökkent. A DMA szög átlagértékei preoperatíve 14,2, utolsó kontrollnál 8,2 fok. A vizuális analóg fájdalom skála (VAS) kitöltése alapján, az operált betegek 91%-a nyilatkozott a műtét szubjektív sikerességéről. Szövődményt 9 esetben észleltünk, álízület képződés, korrekcióvesztés, illetve 2 esetben infekció formájában. Összefoglalásként elmondhatjuk a középtávú eredmények tükrében, hogy a Bösch műtét, a hallux valgus deformitás korrekciójára biztonsággal végezhető, megfelelő indikáció mellett jól korrigálja a HV, IM és DMA szögeket.

The aim of the study was to assess the correlation between femoral neck-shaft angles (NSAs) and skeletal maturity in EOS reconstructions from a large population of children. Full-body three-dimensional (3D) reconstructions were generated from 1005 children and young adults (4-24 years old; 449 male, 556 female) using the EOS three-dimensional/3D scanner, with images taken during routine clinical practice. The true NSAs were measured and assessed for correlation with individuals' chronological age and bone age, based on cervical vertebral morphology. Statistical analysis was performed using Spearman correlation, independent t-test and multiple linear regression. NSAs of older and younger individuals within each bone age group and chronological age were further assessed by t-test. NSA values fell from mean 131.89° ± 6.07° at 4 years old to 128.85° ± 4.46° at the age of 16, with only minor decreases thereafter. Significantly higher NSAs (3.16° and 4.45°, respectively) were found in those with a bone age advanced or delayed by more two or more stages compared to their peers of the same chronological age (P < 0.001; P < 0.001). Similarly, within most bone age stages, individuals of advanced or delayed chronological age exhibited elevated values (mean difference ranged from 2.9° to 8.9°, P < 0.05). Incorporation of bone age assessment into proximal femoral evaluation allowed identification of 'fast maturing' and 'slow maturing' sub-categories in developing children, with different expected NSAs. The earlier ossification seen in faster-maturing individuals may lead to the NSA becoming fixed in a more immature valgus conformation.

Absztrakt: Bevezetés: A spasticus cerebralis paresisben szenvedő betegek kezelésének egyik legfontosabb faktora az állást és a járásképességet befolyásoló equinuscontractura műtéti megoldása. Klinikánkon nyílt Z-achillotenotomia mellett az Achilles-ín percutan tripla hemisectióját végezzük rutinszerűen. Célkitűzés: Munkánk célja volt, hogy a klinikánkon spasticus cerebralis paresises betegeken végzett achillotenotomiák hosszú távú eredményeit elemezzük, keressük a fő komplikációk prediszponáló faktorait, illetve összehasonlítsuk a nyílt és percutan végzett műtétek hosszú távú kimenetelét. Módszer: Klinikánkon 1990 és 2006 között 211 betegnél, összesen 347 esetben végeztük el az Achilles-ín megnyújtását. 261 esetben percutan, 86 esetben pedig nyílt feltárásból történtek a műtétek. Munkánk során a betegek átlagosan 15 éves utánkövetésének retrospektív analízisét végeztük el. Az egyes esetek hosszú távú eredményeit a műtéti életkor, az alapbetegség topográfiai megjelenési formája és a cerebralis paresis súlyossága alapján elemeztük. A betegek járóképességét, illetve annak változását a kezelés során az úgynevezett ’Physician Rating Scale’ pontrendszer szerint értékeltük. Eredmények: Recidív equinuscontractura miatt 74 esetben végeztünk reachillotenotomiát (21,3%); 14 esetben (4%) a reachillotenotomiát második alkalommal is el kellett végezni. Túlkorrekcióval 12 esetben (3,5%) találkoztunk. Fiatalabb életkorban (Következtetés: A műtétet követő legfontosabb komplikáció a recidíva volt. Recidíva a legnagyobb arányban a fiatalabb korban operált, az alapbetegség súlyosabb formájában szenvedő betegeknél jelentkezett. Megfigyeltük, hogy a recidíva szoros összefüggést mutat a testnövekedéssel, illetve halmozódást mutat serdülőkorban. Orv Hetil. 2020; 161(8): 306–312.

Abstract: Introduction: Hand and wrist bone age assessment methods cannot be performed when using the recommended patient position within the EOS scanner. Aim: We aimed to assess alternative methods for use with the EOS. Method: After investigating 9 alternatives, five methods were selected – cervical vertebra (Hassel–Farman), iliac crest (Risser ‘plus’), hip (Oxford), knee (O’Connor), calcaneus (Nicholson) – and applied to EOS scans of 114, 2–21-year-old normal individuals. Intraclass correlation coefficient tests for reliability and Spearman correlation with calendar age were assessed. Results: Intra- and interobserver reliabilities were all excellent, except with the knee method (0.865 – ‘good’). Calcaneal and cervical methods were the fastest to apply (mean 17.5 s, 33.4 s per evaluation), however, calcanei were unassessable in 14% of scans (versus 1% of cervical). All methods correlated significantly with calendar age (r>0.829, p

Background Scoliosis is a complex three-dimensional deformity. While the frontal profile is well understood, increasing attention has turned to balance in the sagittal plane. The present study evaluated changes in sagittal spino-pelvic parameters in a large Hungarian population with adolescent idiopathic scoliosis. Methods EOS 2D/3D images of 458 scoliotic and 69 control cases were analyzed. After performing 3D reconstructions, the sagittal parameters were assessed as a whole and by curve type using independent sample t test and linear regression analysis. Results Patients with scoliosis had significantly decreased thoracic kyphosis (p

Absztrakt

Hand and wrist bone age assessment methods cannot be performed when using the recommended patient position within the EOS scanner.We aimed to assess alternative methods for use with the EOS.After investigating 9 alternatives, five methods were selected - cervical vertebra (Hassel-Farman), iliac crest (Risser 'plus'), hip (Oxford), knee (O'Connor), calcaneus (Nicholson) - and applied to EOS scans of 114, 2-21-year-old normal individuals. Intraclass correlation coefficient tests for reliability and Spearman correlation with calendar age were assessed.Intra- and interobserver reliabilities were all excellent, except with the knee method (0.865 - 'good'). Calcaneal and cervical methods were the fastest to apply (mean 17.5 s, 33.4 s per evaluation), however, calcanei were unassessable in 14% of scans (versus 1% of cervical). All methods correlated significantly with calendar age (r0.829, p0.05). Difficulties were principally absent (12%) or obscured (23%) landmarks.Bone age assessment is possible with all 5 methods, however, the Hassel-Farman method proved to be easily useable, fast and reliable. Orv Hetil. 2019; 160(16): 619-628.Absztrakt: Bevezetés: Az EOS 2D/3D rendszerrel készült felvételeken nem ábrázolódik megfelelően a csontkor megállapítására leggyakrabban használt kéz és csukló. Célkitűzés: Kutatásunk célja, hogy alternatív csontkormérési lehetőségeket keressünk EOS-felvételeken való alkalmazásra. Módszer: 9 mérési módszer bevonásával pilotvizsgálatot végeztünk, amely alapján 5 módszert válogattunk be: nyaki csigolyát (Hassel–Farman), csípőlapátot (Risser ’plus’), térdet (O’Connor), sarokcsontot (Nicholson), csípőt (Oxford) értékelve. 114 egészséges, 2–21 éves eset EOS-felvételein intra- és interobszerver megbízhatósági vizsgálatot végeztünk, valamint Spearman-korrelációval összevetettük a csont- és kronológiai kort. Eredmények: A megbízhatósági vizsgálatok minden módszer esetében kiváló eredményt adtak (csoporton belüli korreláció0,9), kivéve az O’Connor-módszert (0,865 – jó). A Nicholson- és a Hassel–Farman-módszer bizonyult a leggyorsabbnak (átlag: 17,5 mp és 33,4 mp), viszont a sarokcsontok 14%-a nem volt vizsgálható (a cervicalis esetén 1%). Minden módszer szignifikáns összefüggést mutatott a korral (korrelációs koefficiens0,829). Az értékelésnél nehézséget jelentettek a nem ábrázolódó (12%) vagy egymásra vetülő (23%) csontrészek. Következtetés: Csontkor-megállapítás mind az 5 módszer alkalmazásával lehetséges, de kiemelkedett a nagy megbízhatósággal, gyorsan, közel az összes felvételen alkalmazható Hassel–Farman-módszer. Orv Hetil. 2019; 160(16): 619–628.

The aim of this study was to evaluate bone age and its correlation with the lower limbs' developing skeletal anatomy during growth. 1005 children and young adults were evaluated for bone age and 14 different parameters measured on lower-limb reconstructions from radiological examinations carried out with an EOS 2D/3D system in the course of routine orthopedic indicated diagnostic practice. Cervical vertebral morphology evaluation for bone age using the Hassel-Farman method, which describes six stages of maturity, was selected. Intra- and inter-observer reliability tests for this method, and for the EOS 3D reconstructions were performed. Statistical analysis were performed using Spearman correlation, multiple linear regression, and t-test. The intra- and inter-observer reliability of the Hassel-Farman method and the EOS 3D lower-limb reconstruction were found to be excellent. Interestingly one bone age stage could include individuals across a 12.1 year range, and conversely individuals of the same calendar age could be of one of 3.2 different bone age stages. In the prepubertal age groups all six bone stages could be observed. Bone age revealed a stronger relationship, lower standard deviations with groups and proved to be a better discriminating variable than the calendar age by collodiaphyseal angle, femoral, and tibial torsion, femorotibial rotation, and mechanical tibiofemoral angle. Bone age is an indicator of skeletal maturity and may more accurately describe the growth of some lower limb parameters. As a result we suggest the consideration of bone age when evaluating lower-limb biomechanic-anatomical parameters. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1431-1441, 2017.

A veleszületett csípőízületi dysplasiánál az acetabulum és a femur normálistól eltérő anatómiai megjelenése a primer csípőprotézis beültetést megnehezíti. Míg a vápánál az acetabulum hypoplasiája jelenti a fő problémát, addig a femurnál számítanunk kell a fokozott antetorzióra, a szűk velőűrre, a nyak fokozott valgus állására. Az anatómiai szituációt tovább bonyolítják a korábban elvégzett műtétek. A problémák megoldására két lehetőség közül választhatunk, használhatunk kisméretű hagyományos protézisszárat, vagy annak dysplasiás változatát. A szerzők közleményükben esetismertetések formájában mutatják be az általuk leggyakrabban használt dysplasiás szártípusokat, valamint áttekintik a témában megjelent nemzetközi irodalmi ajánlásokat. Hangsúlyozzák, hogy valamennyi esetben a különböző szártípusok ellenére is jó funkcionális és radiológiai eredményt értek el.

The aim of this study was to evaluate bone age and its correlation with the lower limbs' developing skeletal anatomy during growth. 1005 children and young adults were evaluated for bone age and 14 different parameters measured on lower-limb reconstructions from radiological examinations carried out with an EOS 2D/3D system in the course of routine orthopedic indicated diagnostic practice. Cervical vertebral morphology evaluation for bone age using the Hassel-Farman method, which describes six stages of maturity, was selected. Intra- and inter-observer reliability tests for this method, and for the EOS 3D reconstructions were performed. Statistical analysis were performed using Spearman correlation, multiple linear regression, and t-test. The intra- and inter-observer reliability of the Hassel-Farman method and the EOS 3D lower-limb reconstruction were found to be excellent. Interestingly one bone age stage could include individuals across a 12.1 year range, and conversely individuals of the same calendar age could be of one of 3.2 different bone age stages. In the prepubertal age groups all six bone stages could be observed. Bone age revealed a stronger relationship, lower standard deviations with groups and proved to be a better discriminating variable than the calendar age by collodiaphyseal angle, femoral, and tibial torsion, femorotibial rotation, and mechanical tibiofemoral angle. Bone age is an indicator of skeletal maturity and may more accurately describe the growth of some lower limb parameters. As a result we suggest the consideration of bone age when evaluating lower-limb biomechanic-anatomical parameters. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1431-1441, 2017.

The periprosthetic granulomatous soft tissue [designated iterfacial membrane (IFM) in this study] exhibits heterogeneous histopathological features, in which highly vascularized areas with dense cellularity alternate with fibrotic and pseudocapsule-like tissue structures. Although macrophage/monocyte activation is a prominent event in the periprosthetic environment, fibroblasts also phagocytose particulate wear debris both in vivo and in vitro. Particulate wear debris and/or cytokines/growth factors alone or in combination (e.g., in conditioned media of explant cultures of IFMs) stimulated normal synovial and IFM fibroblasts to express inflammatory mediators and growth factors such as interleukin (IL)-1β, IL-6, IL-8, three isoforms of vascular endothelial growth factor (VEGF), monocyte/macrophage chemoattractant protein-1 (MCP-1), macrophage-colony-stimulating factor (M-CSF), cycloxygenases (Cox-1 and Cox-2), acid- and basic-fibroblast growth factors (FGF-1 and FGF-2), leukemia inhibitory factor-1 (LIF-1), transforming growth factor β-1 (TGF-β1), receptor activator of nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG). Thus, the fibroblast is capable of expressing a wide array of angiogenic and osteoclastogenic factors which are involved in the detrimental processes of the periprosthetic osteolysis. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1378–1388, 2007

Background Focal cartilage lesions in the knee joint have limited capacity to heal. Current animal experiments show that incisions of the deep zone of a cartilage allograft allow acceptable integration for the graft. Questions/Purposes We performed this clinical study to determine (1) if the multiply incised cartilage graft is surgically applicable for focal cartilage lesions, (2) whether this allograft has a potential to integrate to the repair site, and (3) if patients show clinical improvement. Patients and Methods Seven patients with 8 chondral lesions were enrolled into the study. Symptomatic lesions between 2 and 8 cm2 were accepted. Additional injuries were allowed but were addressed simultaneously. Grafts were tailored to match and the deep zone of the cartilage was multiply incised to augment the basal integration before securing in place. Rigorous postoperative physiotherapy followed. At 12 and 24 months the patients’ satisfaction were measured and serial magnetic resonance imaging (MRI) was performed in 6 patients. Results Following the implantations no adverse reaction occurred. MRI evaluation postoperatively showed the graft in place in 5 out of 6 patients. In 1 patient, MRI suggested partial delamination at 1 year and graft degeneration at 2 years. Short Form–36 health survey and the Lysholm knee score demonstrated a significant improvement in the first year; however, by 2 years there was a noticeable drop in the scores. Conclusions. Multiply incised pure chondral allograft used for cartilage repair appears to be a relatively safe method. Further studies are necessary to assess its potential in cartilage repair before its clinical use.

Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes in various cell types of the periprosthetic region. We have previously reported that titanium particles stimulate the selective induction of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human osteoblast-like osteosarcoma cells. In this study, we characterize the human bone marrow-derived osteoblast chemokine response to titanium particles. We demonstrate that titanium particles result in enhanced IL-8 and MCP-1 protein secretion as well as differential chemokine gene activation. Osteoblast chemokine expression was regulated at the level of gene transcription, with a time-dependent induction of NF-kappaB activation. Inhibition studies with N-acetyl-L-cysteine (Nac) and MG-132 suggest that titanium particle activation of NF-kappaB activity and IL-8 chemokine expression involves oxidant signaling and IkappaBalpha-proteasomal degradation. Activation of the NF-kappaB transcription factor, as well as the IL-8 gene, are redox-regulated. We also demonstrate that while cytochalasin D, a potent inhibitor of phagocytosis, suppressed the titanium particle effect on IL-8 protein release in human bone marrow-derived osteoblasts, the inhibitor had no effect on IL-8 expression in MG-63 osteoblast-like cells. Collectively, these results provide insight into the potential mechanisms responsible for the particulate activation of osteoblast chemokine expression and suggest an important role for the osteoblast in the pathogenesis of periprosthetic osteolysis.

To explore early signature genes playing critical roles in the initial steps in an autoimmune murine model of rheumatoid arthritis (RA) (proteoglycan (PG)-induced arthritis; PGIA), we performed gene expression profiling of "arthritogenic" spleen cells stimulated with cartilage PG, and compared them to differentially expressed genes, identified in joints prior to the onset of arthritis, and then in the acute and chronic phases of the disease. A total of 280 genes were up-regulated and 226 genes were suppressed in in vitro PG-stimulated lymphocytes at a minimum of 2-fold expression change. Functional gene classification identified several major clusters of biological activity. Expression of immunoglobulin genes (66 transcripts) was downregulated by approximately 3.7-fold, whereas most of the other genes with immune/inflammation-associated functions such as interleukins (IL-1, -2, -4, -6, -10, -12, -16, -17), chemokine receptors and their ligands (Cxcl1, Ccl2, 7, 8, 9, 10, 22, Ccr2, Ccr5), and major components of the complement cascade were upregulated. Using adoptive disease transfer with stimulated lymphocytes into SCID mice, followed by gene expression profiling of SCID paws, indicated that 37 genes were differentially expressed in yet non-inflamed (pre-arthritic) paws; these genes were related mostly to chemokine, IFN-gamma and TNF-alpha signaling. However, the majority of differentially expressed immune response-related genes were silent in pre-arthritic joints, and only 12 genes were found differentially expressed both in antigen (PG)-stimulated lymphocytes and in the synovium prior to the onset of arthritis. Most of these "arthritis-initiation" genes belonged to chemokine mediated cell motility. Transcripts of chemokine receptor 5 (Ccr5), chemokine ligand 7 (Ccl7) and IFN-gamma-inducible proteins (Ifi47) and GTP-ase 1 were expressed at the highest levels in both antigen-stimulated lymphocytes and pre-inflamed synovium, which suggests a key role of these genes in both lymphocyte maturation and arthritis initiation.

Periprosthetic bone loss after total joint arthroplasty is a major clinical problem resulting in aseptic loosening of the implant. Among many cell types, osteoblasts play a crucial role in the development of peri-implant osteolysis. In this study, we tested the effects of calcitriol (1α,25-dihydroxy-vitamin-D3) and the bisphosphonate pamidronate on titanium-particle- and TNF-α-induced release of interleukin-6 and suppression of osteoblast-specific gene expressions in bone-marrow-derived stromal cells with an osteoblastic phenotype. We monitored the expression of procollagen α1[1], osteocalcin, osteonectin and alkaline phosphatase mRNAs by Northern blots and real-time reverse transcription and polymerase chain reaction analyses. The release of various cytokines was also analysed by ELISA. We found that calcitriol or pamidronate could only partially recover the altered functions of osteoblasts when added alone. Only a combination of these compounds restored all the tested functions of osteoblasts. The local delivery of these drugs may have therapeutic potential to prevent or to treat periprosthetic osteolysis and aseptic loosening of implants.

Objective. To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex–matched (H-2d) F2 hybrid mice with proteoglycan (PG)–induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome. Methods. (BALB/c DBA/2)F2 hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor , interleukin-1 [IL-1], IL-6, interferon-, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F2 hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed. Results. Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus–locus interaction. Conclusion. Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex.

SUMMARYMucosal tolerance is a natural mechanism that prevents immunological reactions to antigens by altering the activity of immune cells of pathogenic clones without modulating the entire immune system. This ‘natural immune suppression’ can be exploited when antigen(s) of the target organ in an autoimmune disease is used for mucosal treatment. Being inspired by the experimental results in animal models, clinical trials using type II collagen for mucosal treatment have been conducted in rheumatoid arthritis. High-density proteoglycan (aggrecan) is another major macromolecular component in articular cartilage, and may be a candidate autoantigen for provoking immune reactions in patients with rheumatoid arthritis. Indeed, like type II collagen, systemic immunization of genetically susceptible mice with proteoglycan (PG) aggrecan induces progressive autoimmune polyarthritis. Here, we investigated whether intranasally applied PG can be effective in suppressing PG-induced arthritis (PGIA) in BALB/c mice. We found that nasal administration of 100μg PG exerted a strong suppressive effect on both the incidence and severity of the disease, most probably by reducing responsiveness towards the immunizing PG antigen. When we transferred PGIA into genetically matched but immunodeficient SCID mice, we were able to establish a tolerized state, but only if the recipient SCID mice received lymphocytes from tolerized animals and intranasal treatment with PG was continued. Without nasally administered antigen, the transferred anergic cells recovered and arthritis rapidly developed in a severe form. Intranasal PG treatment of recipient SCID mice was ineffective when cells from non-tolerized arthritic donors were transferred, in which case the regular weekly ‘tolerizing’ dose of PG made the disease worse. Our results suggest that mucosal treatment in an already existing disease may result in paradoxical outcomes.

Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor kappaB (NF-kappaB), and causes an approximately 50% decrease in the steady-state messenger RNA (mRNA) level of procollagen alpha1[I]. In this study, we identify three NF-kappaB binding sites within the human procollagen alpha1[I] gene promoter, show that titanium particles stimulate their binding of the NF-kappaB subunits Rel A (p65) and NF-kappaB1 (p50), and find NF-kappaB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF-kappaB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF-kappaB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF-kappaB, suggesting the involvement of redox signals in NF-kappaB-mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen alpha1[I] mRNA expression and effectively blocked the titanium-induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen alpha1[I] mRNA. Collectively, these results show that titanium particles can activate NF-kappaB signaling in osteoblasts and suggest that NF-kappaB binding to the collagen gene promoter has a functional role in the down-regulation of procollagen alpha1[I] gene transcription.

The lymphocyte response to serum protein complexed with metal from implant alloy degradation was investigated in this in vitro study using primary human lymphocytes from healthy volunteers (n = 10). Cobalt chromium molybdenum alloy (Co-Cr-Mo, ASTM F-75) and titanium alloy (Ti-6Al-4V, ASTM F-136) beads (70 microm) were incubated in agitated human serum at 37 degrees C to simulate naturally occurring metal implant alloy degradation processes. Particulate free serum samples that had been incubated with metal were then separated into molecular weight based fractions. The amounts of soluble Cr and Ti within each serum fraction were measured and correlated with lymphocyte proliferation response to the individual serum fractions. Lymphocytes from each subject were cultured with 11 autologous molecular weight based serum fractions either with or without added metal. Two molecular weight ranges of human serum proteins were associated with the binding of Cr and Ti from Co-Cr-Mo and Ti implant alloy degradation (at

Metal toxicity from sources such as orthopaedic implants was investigated in terms of immune system hyper-reactivity to metal implant alloy degradation products. Lymphocyte response to serum protein complexed with metal from implant alloy degradation was investigated in thisin vitrostudy using primary human lymphocytes from healthy volunteers (n = 10). Cobalt chromium molybdenum alloy (Co-Cr-Mo, ASTM F-75) and titanium allay (Ti-6A1-4V, ASTM F-136) beads (70 um) were incubated in agitated human serum at 37 degrees Celsius to simulate naturally occurring metal implant alloy degradation processes. Particulate free serum samples, which were incubated with metal, were then separated into molecular weight based fractions. The amounts of soluble Cr and Ti within each serum fraction were measured and correlated with lymphocyte proliferation response to the individual serum fractions. Lymphocytes from each subject were cultured with 11 autologaus molecular weight based serum fractions either with or without added metal. Two molecular weight ranges of human serum proteins were associated with the binding of Cr and Ti from Co-Cr-Mo and Ti implant alloy degradation (at < 30 and 180-330 kDa). High molecular weight serum proteins 180 kDa) demonstrated greater lymphocyte reactivity when complexed with metal released from Co-Cr-Mo alloy and Ti alloy than with low (5-30 kDa) and midrange (30-77 kDa) serum proteins. When the amount of lymphocyte stimulation was normalized to both the moles of metal and the moles of protein within each fraction (Metal-Protein Complex Reactivity Index, MPCRI), Cr from Co-Cr-Mo alloy degradation demonstrated approximately 10 fold greater reactivity than Ti in the higher molecular weight serum proteins (~ 180-250 kDa). Thisin vitrostudy demonstrated a lymphocyte proliferative response to both Co-Cr-Mo and Ti alloy metalloprotein degradation products. This response was greatest when the metals were complexed with high molecular weight proteins, and with metal-protein complexes formed from Co-Cr-Mo alloy degradation. (Mol Cell Siochem222:127-136, 2001)

Proteoglycan-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA) both in terms of its pathology and its genetics. PGIA can only be induced in susceptible mouse strains and their F2 progeny. Using the F2 hybrids resulting from an F1 intercross of a newly identified susceptible (C3H/HeJCr) and an established resistant (C57BL/6) strain of mouse, our goals were to: 1) identify the strain-specific loci that confer PGIA susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze the effect of the MHC haplotype on quantitative trait loci (QTL) detection. To identify QTLs, we performed a genome scan on the F2 hybrids. For pathophysiological analyses, we measured pro- and antiinflammatory cytokines such as IL-1, IL-6, IFN-γ, IL-4, IL-10, IL-12, Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. We have identified four new PGIA-linked QTLs (Pgia13 through Pgia16) and confirmed two (Pgia5, Pgia10) from our previous study. All new MHC-independent QTLs were associated with either disease onset or severity. Comprehensive statistical analysis demonstrated that while soluble CD44, IL-6, and IgG1 vs IgG2 heteroantibody levels differed significantly between the arthritic and nonarthritic groups, only Ab-related parameters colocalized with the QTLs. Importantly, the mixed haplotype (H-2b and H-2k) of the C3H × C57BL/6 F2 intercross reduced the detection of several previously identified QTLs to suggestive levels, indicating a masking effect of unmatched MHCs.

To explore early signature genes playing critical roles in the initial steps in an autoimmune murine model of rheumatoid arthritis (RA) (proteoglycan (PG)-induced arthritis; PGIA), we performed gene expression profiling of "arthritogenic" spleen cells stimulated with cartilage PG, and compared them to differentially expressed genes, identified in joints prior to the onset of arthritis, and then in the acute and chronic phases of the disease. A total of 280 genes were up-regulated and 226 genes were suppressed in in vitro PG-stimulated lymphocytes at a minimum of 2-fold expression change. Functional gene classification identified several major clusters of biological activity. Expression of immunoglobulin genes (66 transcripts) was downregulated by approximately 3.7-fold, whereas most of the other genes with immune/inflammation-associated functions such as interleukins (IL-1, -2, -4, -6, -10, -12, -16, -17), chemokine receptors and their ligands (Cxcl1, Ccl2, 7, 8, 9, 10, 22, Ccr2, Ccr5), and major components of the complement cascade were upregulated. Using adoptive disease transfer with stimulated lymphocytes into SCID mice, followed by gene expression profiling of SCID paws, indicated that 37 genes were differentially expressed in yet non-inflamed (pre-arthritic) paws; these genes were related mostly to chemokine, IFN-gamma and TNF-alpha signaling. However, the majority of differentially expressed immune response-related genes were silent in pre-arthritic joints, and only 12 genes were found differentially expressed both in antigen (PG)-stimulated lymphocytes and in the synovium prior to the onset of arthritis. Most of these "arthritis-initiation" genes belonged to chemokine mediated cell motility. Transcripts of chemokine receptor 5 (Ccr5), chemokine ligand 7 (Ccl7) and IFN-gamma-inducible proteins (Ifi47) and GTP-ase 1 were expressed at the highest levels in both antigen-stimulated lymphocytes and pre-inflamed synovium, which suggests a key role of these genes in both lymphocyte maturation and arthritis initiation.

Objective This study was undertaken to investigate how fibroblasts respond to stimulation with particulate wear debris and/or conditioned media obtained from pathologic tissue, and whether these activated fibroblasts express compounds that are involved in bone resorption. Methods Conditioned media from explant cultures of synovial tissue, periprosthetic soft tissue (interface membranes), titanium particles, and proinflammatory cytokines were used to stimulate fibroblasts. RNase protection assay was used to measure altered gene expression, and enzyme-linked immunosorbent assay, Western blot hybridization, and flow cytometry were used to determine fibroblast protein expression. Tartrate-resistant acid phosphatase staining was used to identify multinucleated osteoclast-like cells. Results The most dominant compounds measured in the conditioned media from interface membranes were tumor necrosis factor α (TNFα), monocyte chemoattractant protein 1 (MCP-1), interleukin-1β (IL-1β), IL-6, IL-8, and vascular endothelial growth factor. Fibroblasts phagocytosed particulate wear debris and responded to cytokine/chemokine stimulation. The most prominent up-regulated genes and proteins secreted by fibroblasts in response to stimulation were matrix metalloproteinase 1, MCP-1, IL-1β, IL-6, IL-8, cyclooxygenase 1 (COX-1), COX-2, leukemia inhibitory factor 1, transforming growth factor β1 (TGFβ1), and TGFβ receptor type I. In addition, interface membrane fibroblasts expressed RANKL and osteoprotegerin in response to stimulation with conditioned media, TNFα, or IL-1β. Stimulated fibroblasts cocultured with bone marrow cells in the presence of macrophage colony-stimulating factor induced osteoclastogenesis. Conclusion Interface membrane fibroblasts respond directly to particulate wear debris, possibly via phagocytosis, expressing proinflammatory cytokines and RANKL. Thus, these cells may be actively involved in osteoclastogenesis and pathologic (periprosthetic) bone resorption.

Background: Proteoglycan aggrecan (PG)-induced arthritis (PGIA) is the only systemic autoimmune murine model which affects the axial skeleton, but no studies have been performed characterising the progression of spine involvement. Objectives: To follow pathological events in experimental spondylitis, and underline its clinical, radiographic, and histological similarities to human ankylosing spondylitis (AS); and to determine whether the spondyloarthropathy is a shared phenomenon with PGIA, or an "independent" disease. Methods: Arthritis/spondylitis susceptible BALB/c and resistant DBA/2 mice, and their F1 and F2 hybrids were immunised with cartilage PG, and radiographic and histological studies were performed before onset and weekly during the progression of spondylitis. Results: About 70% of the PG immunised BALB/c mice develop spondyloarthropathy (proteoglycan-induced spondylitis (PGISp), and the progression of the disease is very similar to human AS. It begins with inflammation in the sacroiliac joints and with enthesitis, and then progresses upwards, affecting multiple intervertebral disks. In F2 hybrids of arthritis/spondylitis susceptible BALB/c and resistant DBA/2 mice the incidence of arthritis was 43.5%, whereas the incidence of spondylitis was >60%. Some arthritic F2 hybrid mice had no spondylitis, whereas others developed spondylitis in the absence of peripheral arthritis. Conclusions: The PGISp model provides a valuable tool for studying autoimmune reactions in spondylitis, and identifying genetic loci associated with spondyloarthropathy.

We present here an extensive study of differential gene expression in the initiation, acute and chronic phases of murine autoimmune arthritis with the use of high-density oligonucleotide arrays interrogating the entire mouse genome. Arthritis was induced in severe combined immunodeficient mice by using adoptive transfer of lymphocytes from proteoglycan-immunized arthritic BALB/c mice. In this unique system only proteoglycan-specific lymphocytes are transferred from arthritic mice into syngeneic immunodeficient recipients that lack adaptive immunity but have intact innate immunity on an identical (BALB/c) genetic background.Differential gene expression in response to donor lymphocytes that migrated into the joint can therefore be monitored in a precisely timed manner, even before the onset of inflammation. The initiation phase of adoptively transferred disease (several days before the onset of joint swelling) was characterized by differential expression of 37 genes, mostly related to chemokines, interferon-gamma and tumor necrosis factor-alpha signaling, and T cell functions. These were designated early arthritis 'signature' genes because they could distinguish between the naive and the pre-arthritic state. Acute joint inflammation was characterized by at least twofold overexpression of 256 genes and the downregulation of 21 genes, whereas in chronic arthritis a total of 418 genes with an equal proportion of upregulated and downregulated transcripts were expressed differentially. Hierarchical clustering and functional classification of inflammation-related and arthritis-related genes indicated that the most common biological activities were represented by genes encoding interleukins, chemokine receptors and ligands, and by those involved in antigen recognition and processing.

Motivation: DNA microarray technology and the completion of human and mouse genome sequencing programs are now offering new avenues for the investigation of complex genetic diseases. In particular, this makes possible the study of the spatial distribution of disease-related genes within the genome. We report on the first systematic search for clustering of genes associated with a polygenic autoimmune disease. Results: Using a set of cDNA microarray chip experiments in two mouse models of rheumatoid arthritis, we have identified ∼200 genes based on their expression in inflamed joints and mapped them into the genome. We compute the spatial autocorrelation function of the selected genes and find that they tend to cluster over scales of a few megabase pairs. We then identify significant gene clusters using a friends-of-friends algorithm. This approach should aid in discovering functionally related gene clusters in the mammalian genome.

To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F(2) hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome.(BALB/c x DBA/2)F(2) hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor alpha, interleukin-1 [IL-1], IL-6, interferon-gamma, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F(2) hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed.Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction.Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex.

The targeted disruption of cartilage link protein gene (Crtl1) in homozygous mice resulted in a severe chondrodysplasia and perinatal lethality. This raised the question of whether the abnormalities seen in Crtl1 null mice are all caused by the absence of link protein in cartilage or whether the deficiency of the protein in other tissues and organs contributed to the phenotype. To address this question we have generated transgenic mice overexpressing cartilage link protein under the control of a cartilage-specific promoter, and then these transgenic mice were used for a genetic rescue of abnormalities in Crtl1 null mice. While the overexpression of cartilage link protein resulted in no abnormal phenotype, the cartilage-specific transgene expression of link protein could completely prevent the perinatal mortality of link protein-deficient mice and, depending on the level of the link protein expression, rescue skeletal abnormalities. Although link protein was originally isolated from cartilage, we found and determined Crtl1 transcripts and corresponding proteins in every organ tested from mouse embryos to aging animals. We also identified three additional members of the link protein family, all co-localized with hyaluronic acid-binding proteoglycans in the mouse genome. The ubiquitous presence of link protein suggests a general and systemic function of link protein in the organization of extracellular matrix in a number of tissues, possibly interacting with other proteoglycans, such as versican, brevican, and neurocan.

Optimal T cell activation requires signaling through the TCR and CD28 costimulatory receptor. CD28 costimulation is believed to set the threshold for T cell activation. Recently, Cbl-b, a ubiquitin ligase, has been shown to negatively regulate CD28-dependent T cell activation. In this report, we show that CD28 costimulation selectively induces greater ubiquitination and degradation of Cbl-b in wild-type T cells than CD3 stimulation alone, and TCR-induced Cbl-b ubiquitination and degradation are significantly reduced in CD28-deficient T cells. Stimulation of CD28-deficient T cells with higher doses of anti-CD3 results in increased ubiquitination of Cbl-b, which correlates with enhanced T cell responses. Our results demonstrate that CD28 costimulation regulates the threshold for T cell activation, at least in part, by promoting Cbl-b ubiquitination and degradation.

Exposure of human osteoblasts to ultrafine titanium (Ti) particles has been shown to alter osteoblast gene expression. We previously reported that Ti particles can increase IL-6 release and suppress the gene expression of procollagens alpha1[I] and alpha1[III] in human osteoblasts. In this study, we now demonstrate that Ti particles can rapidly induce the chemotactic cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), two immediate early stress responsive chemokines important for the activation and chemotaxis of neutrophils and macrophages, respectively. In MG-63 osteosarcoma cells and bone marrow derived primary osteoblasts Ti particles selectively increased the steady state levels of IL-8 and MCP-1 mRNA in a time and concentration dependent manner. The increased chemokine mRNA correlated with increased secretion of IL-8 and MCP-1 protein. Actinomycin D, a potent RNA polymerase II inhibitor, blocked the Ti particle induction of IL-8 and MCP-1 mRNA expression, whereas cycloheximide, which inhibits protein synthesis, failed to inhibit chemokine gene expression suggesting Ti particles directly target activation of chemokine gene transcription. Consistent with a transcriptional mechanism not involving new protein synthesis, we demonstrate that Ti particles induce the binding of the p65 and p50 subunits of the latent transcription factor NF-kappaB to the IL-8 gene promoter. Taken together, these data demonstrate that Ti particles can activate transcription of the stress responsive chemokine genes IL-8 and MCP-1 in human osteoblasts.

Metal debris from implants has been shown to alter the function of osteoblasts in cell cultures. Its remains unclear, however, if specific forms of released ionic metals are involved in the pathogenesis of periprosthetic osteolysis. We evaluated the relative effects of ionic forms of implant metals by treating human osteoblast-like MG-63 osteosarcoma cells with eight concentrations (0.001-10.0 mM) of Cr(+3), Mo(+5), Al(+3), Ta(+5), Co(+2), Ni(+2), Fe(+3), Cu(+2), Mn(+2), Mg(+2), Na(+2), and V(+3) chloride solutions. The results demonstrated that the metal ions differentially affected osteoblast proliferation, viability, type-I collagen gene expression, and cytokine release. The metal ions were ranked in order from least to most toxic (based on a 50% reduction in viability) as follows: Na < Cr < Mg < Mo < Al < Ta < Co < Ni < Fe < Cu < Mn < V. Metal-induced decreases in osteoblast proliferation were similar in ranking. Nontoxic concentrations of metals had no effect on procollagen alpha1[I] gene expression; only at toxic concentrations did metals produce a decrease in gene expression. The most toxic metals (V, Mn, Fe, and Ni) were also the only metals found to induce IL-6 secretion on a per cell basis (of the cytokines tested, interleukin 6 (IL-6), interleukin beta 1 (IL-1beta), transforming growth factor beta 1 (TGF-beta1), and tumor necrosis factor alpha (TNF-alpha), only IL-6 was detectable in the culture medium after 48 h for any metal at any concentration). Less toxic metals (e.g., Co and Cr) had little effect on IL-6 release, even at high concentrations. In general, metal ions reduced osteoblast function (i.e., proliferation and collagen gene expression) in proportion to the degree of toxicity. These results support the hypothesis that adverse local cellular responses (particularly necrotic responses) associated with metal debris from implanted metallic devices may be due in part to metal ions released from implants or from particulate debris.

Limited information is available on the responses of osteoblasts to wear debris, corrosion products, and cytokines and on the roles of altered osteoblast functions in the development of periprosthetic bone loss. Wear debris-challenged osteoblasts exhibit altered functions resulting in the loss of their capacity to produce bone matrix and to replace the resorbed bone. Also, osteoblasts may secrete cytokines, which act in a paracrine fashion to recruit inflammatory cells into the periprosthetic space and to stimulate osteoclastic bone resorption. These effects may be mediated in part by ionic metal dissolution products. We review the mechanisms by which altered osteoblast functions, in response to particulate wear debris, corrosion products, and cytokines and growth factors, may contribute to the development and the progression of periprosthetic osteolysis.

Background: Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contribute to periprosthetic osteolysis. The particle-induced activation of macrophages and monocytes has been extensively studied, but only limited information is available on the response of osteoblasts to particulate wear debris. This study examines the effects of particulate wear debris, proinflammatory cytokines, and growth factors on osteoblast functions. Methods: MG-63 osteoblasts were treated with metal particles (titanium, titanium alloy, and chromium orthophosphate) or polymeric particles (polyethylene and polystyrene) of phagocytosable sizes or were treated with exogenous cytokines and growth factors. The kinetics of particle phagocytosis and the number of engulfed particles were assessed with use of fluoresceinated particles. Cell proliferation was determined according to [3H]-thymidine incorporation, and cell viability was determined by either fluorescein diacetate uptake or trypan blue exclusion. Expressions of osteoblast-specific genes were quantified with Northern blot hybridization, and the secretions of osteoblast-specific proteins and cytokines were analyzed by enzyme-linked immunosorbent assays. Results: MG-63 osteoblasts phagocytosed particles and became saturated after twenty-four hours. A maximum of forty to sixty particles per cell were phagocytosed. Each type of particle significantly suppressed procollagen a1[I] gene expression (p < 0.05), whereas other osteoblast-specific genes (osteonectin, osteocalcin, and alkaline phosphatase) did not show significant changes. Particle-stimulated osteoblasts released interleukin-6 (p < 0.05) and a smaller amount of transforming growth factor-b1. Particles reduced cell proliferation in a dose-dependent manner without affecting cell viability (p < 0.05). Exogenous tumor necrosis factor-a also enhanced the release of interleukin-6 (p < 0.01) and transforming growth factor-b1 (p < 0.05), whereas the secretion of transforming growth factor-b1 was increased by insulin-like growth factor-I and prostaglandin E2 as well. Insulin-like growth factor-I and transforming growth factor-b1 significantly increased procollagen a1[I] gene expression in osteoblasts (p < 0.05), while tumor necrosis factor-a and prostaglandin E2 significantly suppressed procollagen a1[I] gene expression (p < 0.01). In contrast, neither exogenous nor endogenous interleukin-6 had any effect on other cytokine secretion, on proliferation, or on procollagen a1[I] gene expression. The transcription inhibitor actinomycin D reduced both procollagen a1[I] transcription and interleukin-6 production. Inhibitors of protein synthesis (cyclohexamide) and intracellular protein transport (brefeldin A and monensin) blocked the release of interleukin-6, but none of these compounds influenced the suppressive effect of titanium on procollagen a1[I] gene expression. Conclusions: MG-63 osteoblasts phagocytose particulate wear debris, and this process induces interleukin-6 production and suppresses type-I collagen synthesis. Osteoblast-derived interleukin-6 may induce osteoclast differentiation and/or activation, but the resorbed bone cannot be replaced by new bone because of diminished osteoblast function (reduced type-I collagen synthesis). Exogenous cytokines (tumor necrosis factor-a and interleukin-1b), growth factors (insulin-like growth factor-I and transforming growth factor-b1), and prostaglandin E2 can modify particulate-induced alterations of osteoblast functions. Clinical Relevance: Altered osteoblast functions probably contribute to the progression of periprosthetic osteolysis. Suppressed osteoblast functions, however, could be compensated for by certain growth factors, such as insulin-like growth factor-I or transforming growth factor-b1. These growth factors, if delivered locally, may have therapeutic potential to prevent or reverse periprosthetic osteolysis.

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

We present here an extensive study of differential gene expression in the initiation, acute and chronic phases of murine autoimmune arthritis with the use of high-density oligonucleotide arrays interrogating the entire mouse genome. Arthritis was induced in severe combined immunodeficient mice by using adoptive transfer of lymphocytes from proteoglycan-immunized arthritic BALB/c mice. In this unique system only proteoglycan-specific lymphocytes are transferred from arthritic mice into syngeneic immunodeficient recipients that lack adaptive immunity but have intact innate immunity on an identical (BALB/c) genetic background. Differential gene expression in response to donor lymphocytes that migrated into the joint can therefore be monitored in a precisely timed manner, even before the onset of inflammation. The initiation phase of adoptively transferred disease (several days before the onset of joint swelling) was characterized by differential expression of 37 genes, mostly related to chemokines, interferon-γ and tumor necrosis factor-α signaling, and T cell functions. These were designated early arthritis 'signature' genes because they could distinguish between the naive and the pre-arthritic state. Acute joint inflammation was characterized by at least twofold overexpression of 256 genes and the downregulation of 21 genes, whereas in chronic arthritis a total of 418 genes with an equal proportion of upregulated and downregulated transcripts were expressed differentially. Hierarchical clustering and functional classification of inflammation-related and arthritis-related genes indicated that the most common biological activities were represented by genes encoding interleukins, chemokine receptors and ligands, and by those involved in antigen recognition and processing.

Accumulating evidence suggests that regulatory T cells play a crucial role in preventing autoimmunity. Recently, a naturally occurring CD4+CD25+ T-cell subset that is anergic and also suppressive has been shown to suppress autoimmunity in several animal models. We used proteoglycan-induced arthritis (PGIA) as a study model to investigate the role of the CD4+CD25+ regulatory T cells in autoimmune arthritis. There was no significant change in the percentage of CD4+CD25+ T cells during the immunization period when proteoglycan- or ovalbumin-immunized BALB/c and C57BL/6 mice were compared. An adoptive transfer study showed that the CD4+CD25+ T cells did not protect severe combined immunodeficient mice from arthritis when they were cotransferred with splenocytes from arthritic animals. Similarly, depletion of the CD4+CD25+ T cells did not enhance the onset of the disease or disease severity in severe combined immunodeficient mice. Moreover, CD28-deficient mice, which have very few CD4+CD25+ T cells, were highly resistant to PGIA. These findings indicate that the CD4+CD25+ regulatory T cells may not play a critical role in controlling PGIA.