Alternative methods for skeletal maturity estimation with the EOS scanner-Experience from 934 patients.

BACKGROUND: Hand-wrist bone age assessment methods are not possible on typical EOS 2D/3D images without body position modifications that may affect spinal position. We aimed to identify and assess lesser known bone age assessment alternatives that may be applied retrospectively and without the need for extra imaging. MATERIALS AND METHODS: After review of 2857 articles, nine bone age methods were selected and applied retrospectively in pilot study (thirteen individuals), followed by evaluation of EOS images of 934 4-24-year-olds. Difficulty of assessment and time taken were recorded, and reliability calculated. RESULTS: Five methods proved promising after pilot study. Risser 'plus' could be applied with no difficulty in 89.5% of scans (836/934) followed by the Oxford hip method (78.6%, 734/934), cervical (79.0%, 738/934), calcaneus (70.8%, 669/934) and the knee (68.2%, 667/934). Calcaneus and cervical methods proved to be fastest at 17.7s (95% confidence interval, 16.0s to 19.38s & 26.5s (95% CI, 22.16s to 30.75s), respectively, with Oxford hip the slowest at 82.0 s (95% CI, 76.12 to 87.88s). Difficulties included: regions lying outside of the image-assessment was difficult or impossible in upper cervical vertebrae (46/934 images 4.9%) and calcaneus methods (144/934 images, 15.4%); position: lower step length was associated with difficult lateral knee assessment & head/hand position with cervical evaluation; and resolution: in the higher stages of the hip, calcaneal and knee methods. CONCLUSIONS: Hip, iliac crest and cervical regions can be assessed on the majority of EOS scans and may be useful for retrospective application. Calcaneus evaluation is a simple and rapidly applicable method that may be appropriate if consideration is given to include full imaging of the foot.

Femoral neck-shaft angle and bone age in 4- to 24-year-olds based on 1005 EOS three-dimensional reconstructions.

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.

[Long-term follow-up of achillotenotomy in patients with cerebral palsy]. / Achillotenotomiák hosszú távú eredményei spasticus cerebralis paresisben szenvedo betegekben.

Introduction: The surgical solution of equinus deformity is one of the most important factors in the treatment of patients with cerebral palsy. We perform open Z achillotenotomy and percutaneus triple hemisection routinely in our department. Aim: The goal of our work was to analyze the long-term results of achillotenotomies in patients with cerebral palsy, to look for predisposing factors of major complications, and to compare the results of the performed operative methods. Method: Between 1990 and 2006, we performed 347 surgical Achilles tendon lengthenings. In 261 cases, the operations were performed percutaneusly, and in 86 cases we performed open Z achillotenotomy. The average follow-up time was 15 years. The long-term outcomes were analyzed based on the age at surgery, the topographic appearance and the severity of cerebral palsy. Analysis regarding functional outcome was based on the widely known Physician Rating Scale system. Results: Due to recurrent equinus deformity, re-achillotenotomy was performed in 74 cases (21.3%), and in 14 cases (4%) the re-achillotenotomy needed to be performed a second time. We encountered overcorrection and calcaneus deformity in 12 cases (3.5%). Recurrence rate was higher in patients operated at a younger age (<7 years) and in patients with a more severe cerebral palsy (GMFCS II-III, ~26%). Recurrence showed accumulation in patients 9-14 years old. Conclusion: The major complication we encountered was recurrence of the equinus deformity. The majority of relapses occured in patients who were operated at a younger age and suffered from a more severe form of cerebral palsy. We observed that recurrence showed an association with growth and accumulated in aldolescence. Orv Hetil. 2020; 161(8): 306-312.

[Bone age - alternatives for skeletal maturity assessment for the EOS scanner]. / Csontkor ­ a csontrendszeri érettség mérésének lehetosége EOS készülékkel.

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<0.05). Difficulties were principally absent (12%) or obscured (23%) landmarks. CONCLUSION: 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.

Sagittal plane assessment of spino-pelvic complex in a Central European population with adolescent idiopathic scoliosis: a case control study.

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 < 0.001) with values T1-T12, 34.1 ± 17.1o vs. 43.4 ± 12.7o in control; T4-T12, 27.1 ± 18.8o vs. 37.7 ± 15.1o in control; and T5-T12, 24.9 ± 15.8o vs. 32.9 ± 15.0o in control. Changes in thoracic kyphosis correlated with magnitude of the Cobb angle (p < 0.001). No significant change was found in lumbar lordosis and the pelvic parameters. After substratification according to the Lenke classification and individually evaluating subgroups, results were similar with a significant decrease in only the thoracic kyphosis. A strong correlation was seen between sacral slope, pelvic incidence, and lumbar lordosis, and between pelvic version and thoracic kyphosis in control and scoliotic groups, whereas pelvic incidence was also seen to be correlated with thoracic kyphosis in scoliosis patients. CONCLUSION: Adolescent idiopathic scoliosis patients showed a significant decrease in thoracic kyphosis, and the magnitude of the decrease was directly related to the Cobb angle. Changes in pelvic incidence were minimal but were also significantly correlated with thoracic changes. Changes were similar though not identical to those seen in other Caucasian studies and differed from those in other ethnicities. Scoliotic curves and their effect on pelvic balance must still be regarded as individual to each patient, necessitating individual assessment, although changes perhaps can be predicted by patient ethnicity.

Determination and correlation of lower limb anatomical parameters and bone age during skeletal growth (based on 1005 cases).

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.

Pilot Study of Cartilage Repair in the Knee Joint with Multiply Incised Chondral Allograft.

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 cm(2) 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.

Three-dimensional quantitative analysis of the proximal femur and the pelvis in children and adolescents using an upright biplanar slot-scanning X-ray system.

BACKGROUND: The anatomy and biomechanics of the pelvis and lower limbs play a key role in the development of orthopaedic disorders. OBJECTIVE: This study aimed to establish normal reference standards for the measurement of gender-specific pelvic and femoral parameters in children and adolescents with the EOS 2-D/3-D system. MATERIALS AND METHODS: EOS 2-D images of 508 individuals (ages 4-16 years) were obtained as part of routine diagnostics. Patients with lower limb abnormalities were excluded. Pelvic and femoral surface 3-D models were generated and clinical parameters calculated by sterEOS 3-D reconstruction software. Data were evaluated using Spearman correlation, paired-samples and independent-samples t-test and linear regression analysis. RESULTS: Changes in anatomical parameters were found to correlate with age and gender in 1) femoral mechanical axis length: 27.3-43.7 cm (males), 25.5-41.2 cm (females), 2) femoral head diameter: 29.4-46.1 mm (males), 27.7-41.3 mm (females), 3) femoral offset: 26.8-42.4 mm (males), 25.5-37.9 mm (females) and 4) femoral neck length: 35.1-52.9 mm (males), 32.8-48.1 mm (females). There was no gender-specific correlation for the neck shaft angle with values from 130.4° to 129.3°, for femoral torsion (22.5°-19.4°), for sacral slope (39.0°-44.4°) and for lateral pelvic tilt (5.1 mm-6.2 mm). Sagittal pelvic tilt exhibited no significant correlation with age showing average values of 6.5°. CONCLUSIONS: The EOS 2-D/3-D system proved to be a valuable method in the evaluation of female and male developmental changes in pelvic and lower limb anatomical parameters, in normal individuals younger than 16 years of age.

Prospective analysis of human leukocyte functional tests reveals metal sensitivity in patients with hip implant.

BACKGROUND: The aim of the study was to examine the reactivity of peripheral human leukocytes to various metal ions prior and following hip replacement in order to investigate implant-induced metal sensitivity. METHODS: Three patient groups were set up: (1) individuals without implants and no history of metal allergy (7 cases), (2) individuals without implants and known history of metal allergy (7 cases), and (3) patients undergoing cementless hip replacement (40 cases). Blood samples were taken in groups 1 and 2 at three different occasions; in group 3, prior and 3, 6, 12, 24, and 36 months after surgery. Peripheral leukocytes were separated and left either untreated or challenged with Ti, NiCl2, CoCl2, CrCl3, and phytohemagglutinin. Cell proliferation, cytokine release, and leukocyte migration inhibition assays were performed. Metal-induced reactivity was considered when all three assays showed significant change. Skin patch tests were also carried out. RESULTS: Both skin patch tests and leukocyte functional tests were negative in group 1, and both were positive in group 2. In group 3, after 6 months, 12% of the patients showed reactivity to the tested metals except for NiCl2. Following the 36-month period, 18% of group three became sensitive to metals (including all the earlier 12%). In contrast, patch tests were negative at each time point in group 3. CONCLUSIONS: Orthopedic implant material may induce metal reactivity after implantation in a manner where susceptibility is yet to be elucidated. Leukocyte triple assay technique might be a useful tool to test implant material-related sensitivity.

Role of fibroblasts and fibroblast-derived growth factors in periprosthetic angiogenesis.

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)-1beta, 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 beta-1 (TGF-beta1), 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.

Antigen-induced differential gene expression in lymphocytes and gene expression profile in synovium prior to the onset of arthritis.

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.

The role of fibroblasts and fibroblast-derived factors in periprosthetic osteolysis.

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 alpha (TNFalpha), monocyte chemoattractant protein 1 (MCP-1), interleukin-1beta (IL-1beta), 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-1beta, IL-6, IL-8, cyclooxygenase 1 (COX-1), COX-2, leukemia inhibitory factor 1, transforming growth factor beta1 (TGFbeta1), and TGFbeta receptor type I. In addition, interface membrane fibroblasts expressed RANKL and osteoprotegerin in response to stimulation with conditioned media, TNFalpha, or IL-1beta. 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.

Chemokine gene activation in human bone marrow-derived osteoblasts following exposure to particulate wear debris.

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.

Gene expression profiling in murine autoimmune arthritis during the initiation and progression of joint inflammation.

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.

Cutting edge: Cbl-b: one of the key molecules tuning CD28- and CTLA-4-mediated T cell costimulation.

Cbl-b negatively regulates CD28-dependent T cell activation. In this report, we tested the hypothesis that CD28 and CTLA-4 have opposite roles in tuning T cell activation threshold by controlling the levels of Cbl-b protein expression. We demonstrate that CD28 costimulation potentiates TCR-induced Cbl-b degradation, whereas CTLA-4-B7 interaction is required for Cbl-b re-expression. In support of this finding, Cbl-b expression in CTLA-4 knockout (KO) T cells is significantly reduced, and treating CTLA-4KO mice with human CTLA-4Ig to block CD28-B7 interaction restores Cbl-b expression on T cells. Furthermore, CD28 and CTLA-4 costimulatory effects are compromised in Cbl-bKO T cells. These observations indicate that CD28 and CTLA-4 tightly regulate Cbl-b expression which is critical for establishing the threshold for T cell activation.

Identification and quantification of disease-related gene clusters.

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 approximately 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.

Sex effect on clinical and immunologic quantitative trait loci in a murine model of rheumatoid arthritis.

OBJECTIVE: 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. METHODS: (BALB/c x DBA/2)F(2) 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 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. 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.

Genetic rescue of chondrodysplasia and the perinatal lethal effect of cartilage link protein deficiency.

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.

CD4+CD25+ immunoregulatory T cells may not be involved in controlling autoimmune arthritis.

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.

Cutting edge: regulation of T cell activation threshold by CD28 costimulation through targeting Cbl-b for ubiquitination.

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.