Spontaneous atopic dermatitis due to immune dysregulation in mice lacking Adamts2 and 14.

Since its first description, ADAMTS14 has been considered as an aminoprocollagen peptidase based on its high similarity with ADAMTS3 and ADAMTS2. As its importance for procollagen processing was never experimentally demonstrated in vivo, we generated Adamts14-deficient mice. They are healthy, fertile and display normal aminoprocollagen processing. They were further crossed with Adamts2-deficient mice to evaluate potential functional redundancies between these two highly related enzymes. Initial characterizations made on young Adamts2-Adamts14-deficient animals showed the same phenotype as that of Adamts2-deficient mice, with no further reduction of procollagen processing and no significant aggravation of the structural alterations of collagen fibrils. However, when evaluated at older age, Adamts2-Adamts14-deficient mice surprisingly displayed epidermal lesions, appearing in 2 month-old males and later in some females, and then worsening rapidly. Immunohistological evaluations of skin sections around the lesions revealed thickening of the epidermis, hypercellularity in the dermis and extensive infiltration by immune cells. Additional investigations, performed on young mice before the formation of the initial lesions, revealed that the primary cause of the phenotype was not related to alterations of the epidermal barrier but was rather the result of an abnormal activation and differentiation of T lymphocytes towards a Th1 profile. However, the primary molecular defect probably does not reside in the immune system itself since irradiated Adamts2-Adamts14-deficient mice grafted with WT immune cells still developed lesions. While originally created to better characterize the common and specific functions of ADAMTS2 and ADAMTS14 in extracellular matrix and connective tissues homeostasis, the Adamts2-Adamts14-deficient mice revealed an unexpected but significant role of ADAMTS in the regulation of immune system, possibly through a cross-talk involving mesenchymal cells and the TGFß pathways.

Tocilizumab potentially prevents bone loss in patients with anticitrullinated protein antibody-positive rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with a high risk of osteoporosis and fracture. Interleukin (IL)-6 inhibitors may suppress osteoclast activation. Anticitrullinated protein antibody (ACPA) titers are inversely associated with bone mineral density (BMD). However, the differential effect of ACPA on bone turnover marker (BTM) and BMD changes after IL-6 inhibition remains unclear. This prospective study recruited patients with active RA with inadequate response to methotrexate or biologics. BMD was measured before and after 2-year tocilizumab (TCZ) treatment. Serum osteocalcin, N-terminal propeptide of type I collagen (P1NP), and C-terminal cross-linking telopeptide of type I collagen (CTX) levels were assessed at the baseline and after treatment. We enrolled 76 patients with RA (89.5% women, age: 57.2 ± 13.3 years) receiving TCZ. The 28-joint disease activity score was negatively correlated with BMD and T-scores of the lumbar spine and bilateral femoral neck. ACPA-positive patients had lower lumbar spine and femoral neck T-scores. After 2-year TCZ treatment, CTX levels significantly decreased (0.32 ± 0.21 vs. 0.26 ± 0.17, p = 0.038). Femoral neck BMD increased significantly (0.71 ± 0.22 vs. 0.69 ± 0.55, p = 0.008). Decreased CTX levels and improved BMD were observed only in ACPA-positive patients. After treatment, femoral neck BMD significantly increased only in patients receiving a glucocorticoid dose of ≥5 mg/day. Two-year TCZ treatment reduced bone resorption and increased femoral BMD in ACPA-positive patients. The net effects of glucocorticoids and IL-6 inhibition on BMD imply that strict inflammation control might affect bone metabolism.

Measurement of amino terminal propeptide of type III procollagen (PIIINP) employing the ADVIA Centaur platform. Validation, reference interval and comparison to UniQ RIA.

BACKGROUND: Recently, measurement of amino terminal propeptide of type III procollagen (PIIINP) was introduced as a part of the hepatic cirrhotic marker enhanced liver fibrosis™ test on the automated ADVIA Centaur(®) immunoassay platform (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA). In this article, we show that the Centaur PIIINP may be used in place of the much more labor-intensive RIA method, and we present an age stratified reference interval. METHODS: We analyzed four control samples 20 times over a period of 5 days. Centaur PIIINP assay measurements were compared with the widely used UniQ PIIINP RIA assay (Orion Diagnostica, Espoo, Finland) using 55 patient samples (range=3.7-43.3 µg/L). Furthermore, we established a reference interval based on samples from 287 blood donors. RESULTS: In the concentration range 2.5-11.9 µg/L, the total imprecision was below 8%. Comparison with the UniQ PIIINP RIA assay yielded: Centaur PIIINP µg/L = 1.9 × (UniQ PIIINP RIA) + 0.6 µg/L, r2=0.94. The reference interval for the Centaur PIIINP assay showed no gender difference but was stratified by age [4.0-11.0 µg/L (18-40 years) and 3.5-9.5 µg/L (41-70 years)]. CONCLUSIONS: We conclude that the Centaur PIIINP assay is suitable for routine use with our newly defined reference interval. The results obtained by Centaur correlates well with those obtained by the previously employed RIA, though the absolute values are higher.

Characterization of a murine type IIB procollagen-specific antibody.

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1-3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1(+ex2) knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1(+ex2) homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.

Characterization of a novel mouse monoclonal antibody, clone 1E8.33, highly specific for human procollagen 11A1, a tumor-associated stromal component.

A novel IgG1, κ mouse monoclonal antibody (clone 1E8.33) to human procollagen 11A1 has been generated. This antibody is poorly mutated, essentially in germ line configuration; its complementarity determining regions (CDRs) are especially rich in tyrosine and serine residues. The epitope recognized is encompassed in the YNYGTMESYQTEAPR amino acid stretch within the variable region of human procollagen 11A1. Human procollagens 5A1 and 11A1 are very similar. However, this antibody does not cross-react with human procollagen 5A1. In human breast tumors, only the activated peritumoral myofibroblasts show a strong intracytoplasmic staining with this antibody. As procollagen 11A1 is overexpressed in the stroma of human tumors with desmoplastic reaction, this antibody represents a valuable tool for diagnostic purposes.

Enzyme-linked immunosorbent serum assays (ELISAs) for rat and human N-terminal pro-peptide of collagen type I (PINP)--assessment of corresponding epitopes.

OBJECTIVES: The present study describes two newly developed N-terminal pro-peptides of collagen type I (PINP) competitive enzyme-linked immunosorbent assays (ELISAs) for the assessment of corresponding PINP epitopes in the rat- and human species. METHODS: Monoclonal antibodies were raised against corresponding rat and human PINP sequences and competitive assays were developed for each species. They were evaluated in relevant pre-clinical or clinical studies. RESULTS: The antibody characterizations indicated that PINP indeed was recognized. Technical robust assays were obtained. Rat PINP and tALP showed similar patterns in the gold standard osteoporosis rat ovariectomized (OVX) model. No liver contribution was observed in the liver fibrosis rat bile duct ligation model (BDL). In an osteoporosis study, the human serum PINP levels were significantly decreased after ibandronate treatment compared to placebo. CONCLUSIONS: The two corresponding PINP assays were specific and these bone turnover markers may improve translational science for the evaluation for bone-related diseases.

[Correlation of lymphocytes and PIIINP with the combined pulmonary fibrosis and emphysema].

AIM: To approach the relationships among inflammation, immune response, and fibrosis in combined pulmonary fibrosis and emphysema (CPFE) through the observation of distributions of lymphocyte subtypes, the variation of Pro-collagen III N-terminal peptide (PIIINP) expression and the severity of pulmonary fibrosis (PF) in CPFE. METHODS: From March 2005 to March 2007, 21 diagnosed cases of CPFE, 25 diagnosed cases of idiopathic pulmonary fibrosis (IPF) and 19 cases of controls were involved in the study from the First Affiliated Hospital of Xi'an Jiaotong University. The patients were subjected to the following investigations including pathological changes in lung tissue biopsy specimens by light microscopy, counting and classification of inflammatory cells out of bronchoalveolar lavage fluids (BALF), determination of T-lymphocyte subtypes by flow cytometry (FCM), and detection of PIIINP level in BALF and blood serum by radioimmunoassay. RESULTS: The pathological data showed higher degree of fibrosis in IPF group than that in CPFE group (P<0.01), but the level of fibrosis in the two Zgroups had nothing to do with smoking status (P>0.05). The inflammatory cells and lymphocyte cells in BALF were more in CPFE group than those in IPF and control groups (P<0.05, P<0.01 respectively). The FCM showed CPFE group had more CD8+ T-lymphocytes than IPF and control groups (P<0.05), whereas CPFE and IPF groups showed significantly lower CD4+/CD8+ ratio than the control group (P<0.01). There was no significantly statistical difference in the percentage of CD4+ T-lymphocytes among the three groups (P>0.05). CPFE and IPF groups exhibited significantly higher level of blood PIIINP than the control group (P<0.01), while IPF group showed markedly higher level of blood PIIINP than CPFE group (P<0.01). BALF and blood level of PIIINP were positively correlated (gamma=0.82). CONCLUSION: The pulmonary fibrosis in CPFE shows intrinsic characteristics, with smoking not being the major or direct PF-driven factor. The CPFE group showed significant inflammation predominated by T-lymphocytes, especially CD8+; T-lymphocyte, as compared with the IPF and control groups, hence pointing to the fact that a novel anti-lymphocytes and immune regulation strategy may be useful for disease intervention. Blood serum PIIINP may be used as a marker for early detection of CPFE and also as a monitor for efficacy of treatments.

Uncoupling of collagen II metabolism in newly diagnosed, untreated rheumatoid arthritis is linked to inflammation and antibodies against cyclic citrullinated peptides.

OBJECTIVE: To investigate the relationship between markers of collagen II synthesis and degradation with disease activity measures, autoantibodies, and radiographic outcomes in a 4-year protocol on patients with early rheumatoid arthritis (RA) who are naïve to disease-modifying antirheumatic drugs. METHODS: One hundred sixty patients with newly diagnosed, untreated RA entered the Cyclosporine, Methotrexate, Steroid in RA (CIMESTRA) trial. Disease activity and radiograph status were measured at baseline and 4 years. The N-terminal propeptide of collagen IIA (PIIANP) and the cross-linked C-telopeptide of collagen II (CTX-II) were quantified at baseline by ELISA. PIIANP was also assayed at 2 and 4 years. Anticyclic citrullinated peptide (anti-CCP) was recorded at baseline. An uncoupling index for cartilage collagen metabolism was calculated from PIIANP and CTX-II measurements. RESULTS: PIIANP was low at diagnosis and 4 years on (p < 0.001), irrespective of treatment and disease activity. PIIANP was lowest in anti-CCP positive patients (p = 0.006), and there was a negative correlation between PIIANP and anti-CCP titers (rho = -0.25, p 0.002). CTX-II was increased (p < 0.001) and correlated positively with disease activity and radiographic progression, but not with anti-CCP (p = 0.93). The uncoupling index was not superior to CTX-II in predicting radiographic changes. CONCLUSION: These results suggest that cartilage collagen formation and degradation are unbalanced when RA is diagnosed. The different associations of collagen II anabolism (PIIANP) and collagen II degradation (CTX-II) with anti-CCP, synovitis, and radiographic progression indicate that at this early stage of RA, cartilage collagen degradation is mainly driven by synovitis, while anti-CCP antibodies may interfere with cartilage regeneration by inhibiting collagen IIA formation. Trial registration j.nr NCT00209859.

Allergic airway inflammation initiates long-term vascular remodeling of the pulmonary circulation.

BACKGROUND: Asthma and allergic airway inflammation are associated with persistent structural alterations in the bronchi, i.e. airway remodeling. Previous studies have shown that during allergic airway inflammation, similar structural alterations may also be evoked in the pulmonary circulation. However, it remained unknown whether remodeling of the pulmonary circulation is as persistent as airway remodeling. The aim of this study is to investigate the reversibility and resolution of vascular remodeling, induced by allergic airway inflammation. METHODS: A validated mouse model of allergic airway inflammation, utilizing ovalbumin as allergen, was employed. Animals were sacrificed 1 day, 1 week or 1 month after the last allergen exposure, and different parameters of remodeling (smooth muscle mass, proliferation of smooth muscle cells and endothelial cells as well as number of myofibroblasts and procollagen-I-producing cells) were investigated and quantified histologically. RESULTS: Allergen exposure resulted in allergic airway inflammation characterized by a transient leukocyte infiltration and in structural alterations in both airway and vascular compartments. The increase in vascular smooth muscle mass and endothelial proliferation persisted at 1 month after the last allergen exposure. The other parameters and cellular inflammatory response returned to baseline within 1 month after the last allergen challenge. CONCLUSIONS: Based on the findings in this study, we conclude that acute allergic airway inflammation, although being initiated from the airways, is able to evoke similar long-term structural alterations in pulmonary vessels as described for bronchi.

Transforming growth factor beta1 T29C gene polymorphism and hypertension: relationship with cardiovascular and renal damage.

Distribution of T29C TGFbeta1 gene polymorphism was analysed in 260 hypertensive and 134 normotensive subjects. Circulating TGFbeta1 and procollagen type III levels, microalbuminuria, left ventricular geometry and function were evaluated in all the hypertensives subgrouped according to T29C TGFbeta1 gene polymorphism. Circulating TGFbeta1 by ELISA technique, procollagen type III by a specific radioimmunoassay, microalbuminuria by radioimmunoassay, left ventricular geometry and function by echocardiography were determined. All groups were comparable for gender, age and sex. Regarding T29C TGFbeta1 gene polymorphism, prevalence of TC or CC genotypes was significantly (p<0.05) higher in hypertensives than normotensives. TC and CC hypertensives were characterized by a higher prevalence of subjects with microalbuminuria (p<0.001 TC vs TT; p<0.05 CC vs TT), left ventricular hypertrophy (p<0.01 TC and CC vs TT), and by increased levels of procollagen type III (p<0.05 TC and CC vs TT). TC hypertensives were also characterized by a significant increase (p<0.05) of LVM and LVM/h(2.7 )and of urinary albumin excretion (p<0.05) values than those detectable in TT hypertensives. Our data suggest that T29C TGFbeta1 gene polymorphism was associated to clinical characteristics suitable to recognize hypertensives with a higher severity of hypertension.

FACIT collagen (1alpha-chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian Ciona intestinalis.

Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1alpha-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60 kDa band identifiable as 1alpha-chain-Ci-type IX-Col. Observations of body wall sections (immunohistochemistry method) supported the role of hemocytes and showed that epidermis expressed Ci-type IX-Col 1alpha-chain in the time course of the inflammatory reaction (within 24h). Transcript and protein were mainly found in the epidermis that outlined the proximal side of the tunic matrix (at 24h after LPS injection), in cells associated with the epidermis at 4 and 192 h. In conclusion, the C. intestinalis inflammatory response to LPS challenge appeared to be composed of a complex reaction set, and for the first time we showed in ascidians a granulation tissue with FACIT-collagen production that could participate in inflammation and wound healing. Like in vertebrates, C. intestinalis acute inflammatory reactions result in a regulated pattern of tissue repair with collagen expression during remodelling. Ci-type IX-Col could be involved in a network of non-fibril-forming collagens that participates in the organization of extracellular matrix and defense responses.

Antibody isotype responses to paramyosin, a vaccine candidate for schistosomiasis, and their correlations with resistance and fibrosis in patients infected with Schistosoma japonicum in Leyte, The Philippines.

We examined whether antibody isotype responses to paramyosin (PM), a vaccine candidate for schistosomiasis, are associated with age-dependent resistance and pathology in liver fibrosis using human sera collected from 139 individuals infected with Schistosoma japonicum in Leyte, The Philippines. We report that IgA and IgG3 responses to PM showed a positive correlation with age and that the epitopes responsible were localized predominantly within the N-terminal half of PM. In addition, the IgG3 response to PM was associated with serum level of procollagen-III-peptide (P-III-P), an indicator of progression of liver fibrosis. These results imply that IgG3 against PM may not only provoke age-dependent resistance to S. japonicum infection but also enhance liver fibrosis. In contrast, levels of IgE to PM and to multiple PM fragments showed a negative correlation with P-III-P level. Thus, in contrast to IgG3, increases in PM-specific IgE may contribute to suppression of liver pathogenesis in schistosomiasis.

Development of an assay for the quantification of type I collagen synthesis in the guinea pig.

There is a need for a reliable assay for the quantification of collagen type I synthesis in the guinea pig, an important model for many connective tissue diseases. Procollagen type I C-terminal propeptide (PICP) is the established marker of type I collagen synthesis but, to date, no assay has been developed to measure PICP in guinea pig tissue extracts. A monoclonal antibody, known to cross-react with intact guinea pig procollagen type I (anti-PICP), was tested for its ability to bind soluble guinea pig PICP in crude skin extracts using a biosensor. Anti-PICP was immobilised to the surface of a sensor chip and antibody-antigen binding was detected using the phenomenon of surface plasmon resonance (SPR). The binding component in the SPR-immunoassay was identified as PICP by purification and N-terminal sequencing. Guinea pig PICP was purified from skin by gel filtration, ion exchange chromatography and lectin affinity chromatography. Purified PICP was then biotinylated and used with anti-PICP to develop a competition ELISA that was able to selectively and sensitively measure PICP in extracts of guinea pig connective tissue.

Development and clinical application in arthritis of a new immunoassay for serum type IIA procollagen NH2 propeptide.

Type II collagen, the most abundant protein of cartilage matrix, is synthesized as a procollagen molecule including the N-(PIINP) and C-(PIICP) propeptides at each end. Type II procollagen is produced in two forms as the result of alternative RNA splicing. One form (IIA) includes and the other form (IIB) excludes a 69-amino acid cysteine-rich globular domain encoded by exon 2 in PIINP. During the process of synthesis, these N-propeptides are removed by specific proteases and released in the circulation, and their levels are believed to reflect type II collagen synthesis. In this chapter we describe the development of a specific enzyme-linked immunosorbent assay (ELISA) for the measurement of the IIA form of PIINP (PIIANP) in serum based on a polyclonal antibody raised against recombinant human exon 2 fusion protein of type II procollagen. We show that this ELISA is highly specific for circulating PIIANP and has adequate technical precision. In patients with knee osteoarthritis and rheumatoid arthritis, serum PIIANP was decreased by 53% (p < 0.0001) and 35% (p < 0.001), respectively, suggesting that type IIA collagen synthesis is altered in these arthritic diseases. The measurement of serum PIIANP may be useful for the clinical investigation of patients with joint diseases.

Serum levels of type IIA procollagen amino terminal propeptide (PIIANP) are decreased in patients with knee osteoarthritis and rheumatoid arthritis.

OBJECTIVE: The aim of this study was to develop a specific immunoassay for PIIANP and measure its serum concentration in healthy controls and in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, we investigated circulating forms recognized by antiserum IIA in pools of serum from healthy adults, patients with OA and patients with RA. DESIGN: Using as immunogen and standard the recombinant human Glutathione S-Transferase (GST)-exon 2 fusion protein of type II collagen, we developed a competitive polyclonal antibody-based ELISA. We compare serum PIIANP levels in 43 patients with knee OA (23 women and 20 men; mean age: 62.6+/-9.6 yr), 63 women with RA (mean age: 54+/-16 yr) and 88 healthy controls (67 women, mean age: 53+/-13 yr and 21 men, mean age: 63+/-7 yr). We randomly selected serum in each group for analyze circulating forms. RESULTS: The immunoassay we developed demonstrated adequate intra and inter-assay precision (CV<10%) and dilution recovery (mean: 96%), allowing accurate measurements of serum PIIANP from 1.13 to 40 ng/ml. No significant cross-reactivity of the ELISA was observed with purified intact human procollagen type I N-propeptide, circulating thrombospondin and von Willebrand factor, proteins which exhibit significant sequence homology with PIIANP. Western blot analysis showed that antiserum IIA recognized two circulating immunoreactive forms of approximately 80 and 100 KDa respectively in serum from healthy adults, patients with OA and RA but also in a pool of synovial fluids from patients with OA. Serum PIIANP levels were markedly decreased in patients with knee OA (12.0+/-3.2 vs 25.8+/-7.5 ng/ml for OA and controls respectively, P<0.0001) and RA (14.1+/-2.5 ng/ml vs 21.7+/-7.6 ng/ml for RA and controls respectively, P<0.0001). In patients with RA, serum PIIANP levels were higher in those taking low-dose prednisone compared to non-users (15.0+/-2.4 vs 13.5+/-2.4 ng/ml, P<0.05). CONCLUSIONS: We have developed the first specific immunoassay for serum PIIANP which exhibits adequate technical performances. This assay detects specifically two immunoreactive forms both in healthy adults and patients with arthritis and does not cross react with other proteins with sequence homology with PIIANP. Levels of PIIANP were significantly decreased in patients with knee OA and RA suggesting that type IIA collagen synthesis may be altered in these arthritic diseases. The measurement of type IIA collagen synthesis with this new molecular marker may be useful for the clinical investigation of patients with joint diseases.

Immunolocalization of carboxy-terminal type II procollagen peptide in regenerated articular cartilage of osteoarthritic knees after reduction of mechanical stress.

OBJECTIVE: The purpose of this study was to investigate the immunolocalization of carboxy-terminal type II procollagen peptide (pCOL-II-C) in the regenerated articular cartilage grown 1-2 years after reduction of mechanical stress by correction of varus deformity with high tibial osteotomy (HTO) for knees with medial compartmental osteoarthritis. DESIGN: The series included 24 knees of 16 patients with a mean age of 70 (56-79) years. Synovial fluid and tissue specimens of the regenerated articular cartilage were obtained at the time of plate removal with arthrotomy. Tissue specimens were decalcified and stained with toluidine blue, safranin O, anti-type I and type II collagen and anti-pCOL-II-C. Pineda's histological grading of articular cartilage repair and Okada's grade of immunostaining were employed to assess the regenerated articular cartilage. RESULTS: In knees with regeneration of articular cartilage, there was a positive linear correlation between the grade of immunostaining and the concentration of synovial fluid pCOL-II-C (r=0.652; P< 0.001). Similarly, a positive linear correlation was observed between the grade of immunostaining and the histological grading score (r=0.683; P< 0.001). CONCLUSIONS: The immunostaining and synovial fluid concentration of pCOL-II-C decreased in accordance with the progression of articular cartilage regeneration observed after reduction of mechanical stress by correction of deformity with HTO.

Amelioration of relapsing polychondritis in a child treated with oral collagen.

Relapsing polychondritis (RP) is a disease characterized by inflammation and the destruction of cartilage. The detection of antibodies to native type II collagen (CII) in the sera of some patients with relapsing polychondritis suggests that autoimmunity to this cartilage specific protein plays a role in the pathogenesis of the disease. RP is so rare that controlled therapeutic trials have not been carried out. We describe herein a child with RP who had amelioration of symptoms and a deviation in the cellular immune response to CII after being treated with daily oral CII as a toleragen.

The carboxyl terminus of type VII collagen mediates antiparallel dimer formation and constitutes a new antigenic epitope for epidermolysis Bullosa acquisita autoantibodies.

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC2 domain has been implicated in catalyzing the antiparallel dimer formation of type VII procollagen. In this study, we produced the entire 161 amino acids of the NC2 domain plus 186 amino acids of adjacent collagenous domain (NC2/COL) and purified large quantities of the recombinant NC2/COL protein. Recombinant NC2/COL readily formed disulfide-bonded hexamers, each representing one antiparallel dimer of collagen VII. Removal of the collagenous helical domain from NC2/COL by collagenase digestion abolished the antiparallel dimer formation. Using site-directed mutagenesis, we found that mutation of either cysteine 2802 or cysteine 2804 alone within the NC2 domain blocked antiparallel dimer formation. In contrast, a single cysteine mutation, 2634, within the collagenous helical domain had no effect. A generated methionine to lysine substitution, M2798K, that is associated with recessive dystrophic epidermolysis bullosa, was unable to form antiparallel dimers. Furthermore, autoantibodies from epidermolysis bullosa acquisita patients also reacted with NC2/COL. We conclude that NC2 and its adjacent collagenous segment mediate antiparallel dimer formation of collagen VII. Epidermolysis bullosa acquisita autoantibodies bound to this domain may destabilize anchoring fibrils by interfering with antiparallel dimer assembly leading to epidermal-dermal disadherence.

Increased amount of type III pN-collagen in human abdominal aortic aneurysms: evidence for impaired type III collagen fibrillogenesis.

PURPOSE: This study aimed to characterize the distribution of structural domains of type I and III collagens in the wall of abdominal aortic aneurysms (AAAs), by the use of undilated atherosclerotic aortas (aortoiliac occlusive disease [AOD]) and healthy abdominal aortas as controls. METHODS: Immunohistochemical staining was applied with antibodies for the aminoterminal propeptides of type I (PINP) and type III (PIIINP) procollagens, which represent newly synthesized type I and III pN-collagens. In addition, an antibody against the aminoterminal telopeptide of type III collagen (IIINTP) was used as a means of detecting maturely cross-linked type III collagen fibrils. RESULTS: The newly synthesized type III procollagen detected by means of PIIINP staining was concentrated in the media in aneurysmal aortas, whereas type I pN-collagen was localized in the intima in both AAAs and AODs. The healthy aortas showed no immunoreactivity for either PIIINP or PINP. The cross-linked type III collagen, detected by means of IIINTP staining, stained transmurally in all study groups, but appeared more abundant in the media in AAAs. CONCLUSION: Our results strongly suggest that the metabolism of type III collagen is enhanced in AAAs. Intensive type III pN-collagen staining was present mainly in the media layer in AAAs, suggesting a role of type III collagen in aneurysm formation, whereas type I pN-collagen was present in the intima in both AAAs and AODs, suggesting that type I collagen synthesis is a fibroproliferative response related to the atherosclerotic process. The increased type III pN-collagen in AAAs may result in impaired fibril formation and, thus, in decreased tensile strength of aneurysmal tissue.