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.

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.

[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.

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.

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.

Induction of fibrogenic mediators by fine and ultrafine titanium dioxide in rat tracheal explants.

Respirable ambient particles [particulate matter <10 micrometer (PM(10))] are associated with both acute and chronic adverse health effects including chronic airflow obstruction. PM(10) can induce expression of inflammatory and fibrogenic mediators, but there is controversy about the types and/or sizes of particles involved and, in particular, whether ultrafine particles are the major toxic agents. To examine whether particle size affects mediator generation, we exposed rat tracheal explants, an inflammatory cell-free model of the airway wall, to various concentrations up to 500 microgram/cm(2) of fine (0.12 micrometer) or ultrafine (0.021 micrometer) titanium dioxide (anatase), maintained the explants in an organ culture in air for 1-7 days, and used RT-PCR to examine the expression of fibrogenic mediators and procollagen. No increase in gene expression was seen at 1 or 3 days, but at 5 days, ultrafine dust induced a small increase in procollagen. At 7 days, fine titanium dioxide produced significantly greater increases for platelet-derived growth factor (PDGF)-B, transforming growth factor-alpha, and transforming growth factor-beta compared with those by ultrafine dust; both dusts produced similar increases for PDGF-A; and ultrafine dust produced increases in procollagen expression, whereas fine dust had no effect. Expression levels were dose related. Both dusts produced a similar decrease in expression of PDGF receptor-alpha and a similar increase in PDGF receptor-beta. These observations suggest that ultrafine particles are intrinsically able to induce procollagen expression even in the absence of inflammatory cells; that chronic exposure to PM(10) may result in chronic airflow obstruction, in part because of ultrafine particle-mediated increases in airway wall fibrosis; and that chemically identical dusts of differing size can produce quite different patterns of gene expression in the airway wall. Differential upregulation of PDGF receptors does not appear to explain dust-induced fibrosis in this model.

Age-related changes in type-I collagen synthesis in human eyelid skin.

PURPOSE: This study was to determine whether age-related decrements in type I collagen synthesis occur in human eyelid skin. METHODS: Using an antibody to procollagen I, we investigated collagen synthetic activity in skin removed for cosmetic purposes from 10 white patients between the ages of 4 and 77 years. Eleven masked referees graded the immunostaining on a scale of 1 (most intense) to 10 (least intense). RESULTS: The multiple range test for rank by group demonstrated more intense staining in younger patients compared with older patients. An average correlation coefficient of 0.8432 (p < 0.05) existed between each of the referee's rankings. CONCLUSION: Type I collagen synthesis diminishes with age in eyelid skin.

Epitope-specific monoclonal antibodies against human C-terminal procollagen alpha1(III)-propeptide.

We have generated monoclonal antibodies against recombinant C-terminal human procollagen alpha1(III) propeptide (PIIICP), produced in E. coli in high yields. The monoclonal antibodies were screened for epitope specificity using recombinant truncated PIIICP. Several antibodies were identified which recognized different regions of the PIIICP molecule. The ability of the antibodies to detect PIIICP antigens in human cell line lysates and supernatants was demonstrated. As PIIICP antigens are a key marker of extracellular matrix metabolism, the monoclonal antibodies described here should be of value for clinical and basic research.

Epidermolysis bullosa acquisita diagnosed by direct immunoelectron microscopy of the conjunctiva.

OBJECTIVE: To describe for the first time the direct immunoelectron microscopic pattern of immune deposits on the conjunctival basement membrane in epidermolysis bullosa acquisita (EBA). DESIGN: Case reports. PARTICIPANTS: Two patients. INTERVENTION: Epidermolysis bullosa acquisita associated with cicatrizing conjunctivitis. MAIN OUTCOME MEASURES: Direct immunofluorescence and direct immunoelectron microscopy without freezing on conjunctival and skin biopsy specimens, indirect immunofluorescence, Western immunoblot analysis. RESULTS: Results of direct immunoelectron microscopic examination of the conjunctiva showed the presence of immune deposits in the anchoring fibril zone, just beneath the lamina densa, in both patients. This finding was the same as the direct immunoelectron microscopic pattern shown in the skin of these patients, which is known to be very specific for EBA. Direct immunofluorescence was positive in the conjunctiva of only one patient. Indirect immunofluorescence and Western immunoblot analysis failed to detect circulating autoantibodies. CONCLUSIONS: Direct immunoelectron microscopy on the conjunctiva is a useful diagnostic tool to differentiate EBA from other related autoimmune mucocutaneous blistering diseases.

Systemic therapy with estrogen or estrogen with progestin has no effect on skin collagen in postmenopausal women.

OBJECTIVES: To investigate the effect of estrogen alone or combined with progestin on the amount and synthesis of skin collagen in postmenopausal women. METHODS: Forty-three early postmenopausal women were enrolled into this open, non-randomized parallel-groups study. Fifteen women received a continuous oral dose of 2 mg of 17 beta-estradiol and 1 mg of norethisterone acetate daily and 14 women an oral dose of 2 mg estradiol valerate daily. Fourteen subjects served as controls. The histology and type I and III procollagen immunohistochemistry of the skin, skin thickness, the amount of total collagen determined by a colorimetric method and the synthesis of type I and III collagens determined by analysing procollagen propeptides in the suction blister fluid were studied before the treatment and at 6 and 12 months. The proportional area of elastic fibers and the thickness of the epidermis were assessed from the sections obtained before the treatment and at 12 months, with computerized image analysis. RESULTS: Skin thickness, the amount and rate of collagen synthesis, the proportional area of elastic fibers and the thickness of the epidermis were not affected by either 17 beta-estradiol and 1 mg of norethisterone acetate or 2 mg of estradiol valerate. No histological or immunohistological changes were detected in the skin specimens during the 12-month treatment period compared to the baseline or to the skin specimens of the control group. CONCLUSIONS: A 1-year treatment with systemic estrogen alone or combined with progestin does not change the amount of collagen or the rate of collagen synthesis in postmenopausal women.

Human osteoclastoma-derived stromal cells: correlation of the ability to form mineralized nodules in vitro with formation of bone in vivo.

It has been suggested that the stromal element of human osteoclastomas contains osteoblastic cells. In this study, we demonstrate that osteoclast-depleted, passaged stromal cells express alkaline phosphatase and osteocalcin in vitro and form mineralized nodules under appropriate culture conditions. In addition, we describe a model in which severe combined immunodeficient (SCID) mice were used to support the differentiation of these putative human osteoblast progenitors in vivo. Lesions formed from human stromal cells were identified using the OKa blood group antigen and human procollagen type I antibodies. By 21 days, the lesion was a complete bone unit: a fully mineralized cortex, remodeling trabeculae, and a highly cellular marrow space. Stromal cells derived from six out of seven osteoclastomas produced identical lesions. Further studies have demonstrated that the capacity of the osteoclastoma-derived stromal cells to form bone in vivo and in vitro is passage dependent; early passages were osteogenic in both model systems, while later passages were not. In conclusion, we have developed a model in which the osteogenic nature of cells can be confirmed in vivo. Furthermore, human osteoclastoma-derived stromal cells provide a source of these osteogenic cells to study human osteoblast differentiation, both in vivo and in vitro.

Fetal antigen 2 (FA2): the aminopropeptide of the alpha 1 chain of human type I procollagen. A study on skin, tumour biology and bone metabolism.

Fetal antigen 2 (FA2) was purified from second trimester human amniotic fluid (AF) using immunospecific chromatography, gel filtration and reversed phase chromatography. Amino acid composition and sequence analyses of purified FA2 revealed identity to the amino acid sequence of the aminopropeptide of the alpha 1 chain of human type I procollagen. Gel filtration of AF as well as purified FA2 demonstrated two molecular forms of FA2, which during gel filtration were elucidated corresponding to MW of approximately 100 kDa and 30 kDa. The molecular forms were referred to as the high molecular component (FA2-HM) and low molecular component (FA2-LM), respectively. SDS-PAGE analysis (reducing and nonreducing conditions) showed a MW of 27 kDa for both FA2-HM and FA2-LM. Mass spectrometry analysis however revealed a MW of 14.343 +/- 3Da. Proteins with collagenous domains often reveal aberrant behaviour both in gel filtration and SDS-PAGE and this phenomenon may account for the apparent higher MW of FA2 observed using these techniques compared to mass spectrometry and amino acid composition/sequence analyses. A polyclonal, monospecific rabbit antibody raised against FA2 was used in immunohistochemical localization studies. Due to the obvious association of FA2 to the BM structure in adult and fetal skin, it was initially referred to as a BM associated component. However, FA2 was immunologically distinct from the well established BM components and moreover, the diffuse distribution of FA2 along the BM differed from linear localization of BM components (e.g. collagen type IV and laminin). The identification of FA2 as the aminopropeptide of the alpha 1 chain of human procollagen type I has the implication that in the immunohistochemical investigations the anti-FA2 may not distinguish between the free propeptide and procollagen type I. In the papillary dermal region of human adult skin type I collagen are arranged in loose fibrils to which the aminoterminal propeptide is still attached. Anti-FA2 may recognize epitopes in the type I procollagen present in this region, giving rise to the diffuse FA2 distribution along the BM. However, whether FA2 also appear as a free component in this region is unknown. In fetal skin where collagen is vividly layed down in the mesenchymal compartment staining with anti-FA2 revealed a diffuse mesenchymal distribution. Demonstration of FA2 within the cytoplasm of proliferating fibroblasts and diffusely in granulation tissue during wound healing suggest that FA2 was involved in the reparative processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Endothelial and fibroblastic activation in scleroderma. The myth of the "uninvolved skin".

We studied the immunohistochemistry of the skin of scleroderma patients to determine the differences (if any) between clinically "affected" and "nonaffected" areas. We examined paired skin biopsy samples from clinically involved forearm skin ("affected") and clinically uninvolved proximal skin ("nonaffected") taken from 19 patients with diffuse scleroderma and from 15 normal control subjects. We stained the sections with antibodies to endothelial leukocyte-adherence molecule type 1 (ELAM-1; to detect endothelial activation) and to procollagen-1 (PC-1; to detect newly formed, unprocessed collagen). There was increased expression of ELAM-1 and PC-1 in sclerodermatous skin as compared with the controls, but there was no difference between clinically affected and nonaffected skin samples. In 10 of 11 patients whose condition was getting worse, endothelial and fibroblast activation preceded fibrosis. Endothelial and fibroblast activation are more widespread in the skin of scleroderma patients than is evident by inspection on physical examination. What appears to be "normal" skin in diffuse scleroderma is already pathologic, as shown by abnormal endothelial activation and procollagen production.

Feedback regulation of collagen gene expression: a Trojan horse approach.

The mechanisms involved in feedback regulation of type I procollagen synthesis by the N-terminal propeptide of the pro alpha 1(I) chain, termed Col 1, are poorly understood. We have constructed a metallothionein-human collagen chimeric minigene (pMTCol) that codes for a Col 1 fusion protein but lacks a signal peptide sequence and, therefore, would be expected to direct the synthesis of the fusion protein to the cytosol. Baby hamster kidney cells and fetal calf ligament cells, transfected with pMTCol, transcribed the gene and synthesized an intracellular antigen that was identified as the fusion protein with a monospecific antibody. Transfected fetal calf ligament fibroblasts showed significantly reduced levels of endogenously produced type I collagen, as determined by imaging and digital quantitation of immunofluorescence by confocal microscopy; synthesis of fibronectin, thrombospondin, and SPARC (secreted protein, acidic and rich in cysteine) was unchanged or increased in these cells. This recombinant approach offers the potential for a systematic analysis of feedback regulation of collagen synthesis.

Detection of procollagen biosynthesis using peptide-specific antibodies.

Peptides corresponding to selected sequences of the alpha 1 chain of the COOH propeptide of type I and type III human procollagen were synthesized and used as antigens to develop polyclonal and monoclonal antibodies. The antibodies were shown to be epitope specific using a peptide-based solid phase enzyme-linked immunoadsorbent assay. The antibodies were specific for the appropriate procollagens and the COOH propeptides isolated from serum-free culture supernatants of human skin fibroblasts. The rabbit antisera directed to the type I synthetic peptide bound the intact procollagen molecule and both the procollagen alpha 1(I) and alpha 2(I) chains after the reduction of the disulfide bonds. In addition, the antisera bound intact type I COOH propeptide, generated by bacterial collagenase treatment of procollagen, and the individual chains of the propeptide after reduction. In contrast, a monoclonal antibody to the type I peptide was able to bind only to the reduced form of the COOH propeptide. Both rabbit polyclonal and murine monoclonal antibodies directed to the type III synthetic peptide bound the intact and the individual chains of type III procollagen as well as the intact and reduced forms of the type III COOH propeptide. The antibodies have been used to detect procollagen synthesis in two human osteosarcoma cell lines and the differential expression of procollagen in the culture medium of rat lung fibroblasts grown in the presence or absence of glucocorticoids.

Fibroblast markers in labial salivary gland biopsies in progressive systemic sclerosis.

Labial salivary gland (LSG) biopsies from 13 patients were studied. Three were normal glands, five showed fibrosis induced by progressive systemic sclerosis (PSS) and five more had PSS-induced fibrosis combined with and focal sialadenitis compatible with Sjögren's syndrome (SS). Monoclonal antibodies to proline-4-hydroxylase (alpha PH or 5B5-A) and the carboxyterminal domain of human type I procollagen (alpha pC or M-38) were used as fibroblast markers. Immunostaining was done with avidin-biotin-peroxidase complex (ABC). Using various sample controls (including cultured fibroblasts and specimens enriched for lymphocytes, plasma cells, granulocytes, monocytes and dendritic cells) as well as analysis of various LSG resident cells, the specificity of the alpha PH and alpha pC markers for fibroblasts was established. Cross reactions were only seen with plasma cells and acinar cells containing the beta subunit of PH or disulfide isomerase involved in SS-SH interchange reactions in these secretory cells. All fibroblasts in fibroblast monolayer cultures at their logarithmic phase of growth stained with the fibroblast markers studied, but false negative staining was seen with resting, mature fibroblasts in dense connective tissue in LSG sections. Therefore, it can be concluded that proline 4-hydroxylase and the COOH-terminal domain of type-I procollagen both indicate fibroblast involvement in collagen (type l) synthesis and thus identify active but not resting fibroblasts. PH+ fibroblast-like cells and pC+ fibroblasts were both more frequent in PSS LSGs than in normal glands, suggesting active local fibroblast involvement in PSS.(ABSTRACT TRUNCATED AT 250 WORDS)

The aminoterminal propeptide of type III procollagen and basement membrane components in serum during wound healing in man.

The aim of this study was to investigate if the serum concentrations of antigens related to basement membrane components and to the interstitial type III collagen reflect the sequential and transitional synthesis of basement membranes and of type III collagen in wound granulation tissue. The aminoterminal propeptide of type III collagen, the 7S domain of type IV collagen and the P1 fragment of laminin were determined in 11, 10 and 6 patients, respectively, during wound healing following intraabdominal surgery. No change was observed in the type III propeptide level at the first postoperative day. During further follow-up (mean 71 days, range 21-155 days), the propeptide levels showed a transitional increase, with a maximum at day 10. A sequential pattern was observed in the increase of the serum concentrations, as the maximum increase of the basement related antigens in serum occurred within the first 7 postoperative days. This is in accordance with observations of an early formation of basement membranes in blood vessels, preceding deposition of interstitial collagens in granulation tissue. The results support the assumption that the serum concentrations of connective tissue related antigens may be valuable markers of wound healing in man.