Serum total periostin is an independent marker of overall survival in bone metastases of lung adenocarcinoma.

More than 35% of lung adenocarcinoma patients have bone metastases at diagnosis and have a poor survival. Periostin, a carboxylated matrix protein, mediates lung cancer cell dissemination by promoting epithelial-mesenchymal transition, and is involved in bone response to mechanical stress and bone formation regulation. This suggests that periostin may be used as a biomarker to predict survival in lung cancer patients. Serum periostin was assessed at diagnosis in a prospective cohort of 133 patients with lung adenocarcinoma of all stages. Patients were divided into localized and bone metastatic groups. Both groups were matched to healthy controls. Survival analysis and Cox proportional hazards models were conducted in the total population and in bone metastatic group. The median serum periostin level was higher in bone metastatic (n = 67; median: 1752 pmol/L) than in the localized group (n = 66; 861 pmol/L; p < 0.0001). Patients with high periostin (>median) had a poorer overall survival in the whole population (33.3 weeks vs. NR; p < 0.0001) and the bone metastatic group (24.4 vs. 66.1 weeks; p < 0.001). In multivariate analysis, patients with high periostin had increased risk of death (HR = 2.09, 95%CI [1.06-4.13]; p = 0.03). This was also found in the bone metastatic group (HR = 3.62, 95%CI [1.74-7.52]; p = 0.0005). Immunohistochemistry on bone metastasis biopsies showed periostin expression in the bone matrix and nuclear and cytoplasmic staining in cancer cells. Serum periostin was an independent survival biomarker in all-stage and in bone metastatic lung adenocarcinoma patients. IHC data suggest that periostin might be induced in cancer cells in bone metastatic niche in addition to bone microenvironment expression.

Serum periostin is associated with prevalent knee osteoarthritis and disease incidence/progression in women: the OFELY study.

OBJECTIVE: Our aim was to investigate the relationships between serum periostin (POSTN) and both prevalence and incidence/progression of knee osteoarthritis (OA) in women. METHODS: We investigated 594 women (62.7 ± 11.2 yr) from the OFELY cohort. Knee radiographs were scored according to the Kellgren & Lawrence (KL) grading system at baseline and 4 years later. Spine, hip and hand OA were assessed at baseline. Prevalent knee OA was defined by a KL score higher or equal in 2. Progression of KL was defined as an increase of the KL score ≥1 during the 4 years follow-up. Serum POSTN was measured at baseline by ELISA. RESULTS: By non-parametric tests, POSTN was significantly lower in 83 women with a KL score ≥2 at baseline, compared to those with a KL score <2 (n = 511; 1101 ± 300 vs 1181 ± 294 ng/ml, P = 0.002) after adjustment for age, body mass index (BMI), treatments and diseases, prevalent hand OA and prevalent lumbar spine OA. By logistic regression analyses, the odds-ratio of knee OA incidence/progression was significantly reduced by 21% (P = 0.043) for each quartile increase in serum POSTN at baseline, after adjustment for age, BMI, prevalent knee OA, prevalent hand OA and prevalent lumbar spine OA. CONCLUSIONS: We show for the first time that serum POSTN is associated with prevalence and the risk of development/progression of knee OA in women.

Serum periostin is associated with fracture risk in postmenopausal women: a 7-year prospective analysis of the OFELY study.

PURPOSE: Periostin (POSTN) is a secreted γ-carboxyglutamic acid-containing protein expressed mainly in the periosteum in adult individuals. POSNT deficient mice develop periodontis and osteoporosis with decreased bone strength. The relationship between serum POSTN and bone metabolism and fracture risk in postmenopausal women is unknown. SUBJECTS AND METHODS: Serum POSTN was measured in 607 postmenopausal women (mean age 66.6 ± 8.4 y) from the Os des Femmes de Lyon cohort at the ninth annual follow-up visit (baseline visit of the current analysis). Nonvertebral and clinical vertebral incident fragility fractures were reported annually during 7 years. Areal bone mineral density (BMD; measured by dual energy X-ray absorptiometry) of the hip and bone markers (intact N-terminal propeptide of type I collagen, osteocalcin, and serum type I collagen C-telopeptide) were also measured. RESULTS: At baseline, serum POSTN did not correlate with age, bone markers, and BMD. After a median of 7 years of follow-up, 75 women sustained an incident clinical vertebral or nonvertebral fragility fracture. The proportion of women who had an incident fracture was significantly higher in women with levels of POSTN in the highest quartile than that of women in the three other quartiles (19.5% vs 10.1%, P = .018) after adjustment for age and prevalent fracture. The highest quartile of POSTN was associated with an increased risk of incident fracture with a relative risk (95% confidence interval) of 1.88 (1.1-3.2) after adjustment for age, prevalent fracture, and hip BMD T-score. Patients with both low hip BMD (T-score < -2.5) and high levels of POSTN (fourth quartile) had a relative risk of fracture of 7.1 (95% confidence interval 2.4-21.8) after adjustment for age. CONCLUSION: High serum POSTN levels are independently associated with increased fracture risk in postmenopausal women. These data suggest that serum POSTN could be useful to improve fracture risk assessment.

Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a chronic and slowly progressive disease for which biomarkers may be able to provide a more rapid indication of therapeutic responses to therapy than is currently available; this could accelerate and facilitate OA drug discovery and development programs. The goal of this document is to provide a summary and guide to the application of in vitro (biochemical and other soluble) biomarkers in the development of drugs for OA and to outline and stimulate a research agenda that will further this goal. METHODS: The Biomarkers Working Group representing experts in the field of OA biomarker research from both academia and industry developed this consensus document between 2007 and 2009 at the behest of the Osteoarthritis Research Society International Federal Drug Administration initiative (OARSI FDA initiative). RESULTS: This document summarizes definitions and classification systems for biomarkers, the current outcome measures used in OA clinical trials, applications and potential utility of biomarkers for development of OA therapeutics, the current state of qualification of OA-related biomarkers, pathways for biomarker qualification, critical needs to advance the use of biomarkers for drug development, recommendations regarding practices and clinical trials, and a research agenda to advance the science of OA-related biomarkers. CONCLUSIONS: Although many OA-related biomarkers are currently available they exist in various states of qualification and validation. The biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two general categories, those that will allow targeting of subjects most likely to either respond and/or progress (prognostic value) within a reasonable and manageable time frame for a clinical study (for instance within 1-2 years for an OA trial), and those that provide early feedback for preclinical decision-making and for trial organizers that a drug is having the desired biochemical effect. As in vitro biomarkers are increasingly investigated in the context of specific drug treatments, advances in the field can be expected that will lead to rapid expansion of the list of available biomarkers with increasing understanding of the molecular processes that they represent.

Significance of the serum CTX-II level in an osteoarthritis animal model: a 5-month longitudinal study.

OBJECTIVE: The aim of this study was to investigate the clinical value of serum measurement of C-telopeptide of type II collagen (CTX-II). In correlation with late stages of osteoarthritis (OA) evaluated with histological assessment, the evolution of serum CTX-II concentration was followed during a 20-week longitudinal study in rabbit anterior cruciate ligament transection (ACLT) OA model in adult and growing animals. METHODS: OA was induced in five adult and nine growing rabbits. Four adult and four young rabbits were unoperated. Serum sampling was made at week 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16 and 20 after the surgery in all rabbits. Animals were euthanized 20 weeks after the surgery. Serum CTX-II levels were analyzed with a recently available enzyme-linked immunosorbent assay (ELISA) kit, the protocol of which has been modified to increase the sensitivity of the test. RESULTS: Significant differences for the CTX-II levels at W3, W6, W8, W10, W12, W14, W16 and W20 were observed between the adult ACLT and the control groups. A negative correlation between CTX-II levels and cartilage thickness of the medial compartment of the knee at W8, W10, W12 and a positive correlation between the CTX-II levels and the histomorphological score of the medial compartment of the knee at W3, W6, W8, W10, W12 were noted in adult animals. In young animals, operated or not, we observed high CTX-II levels at the beginning of the study, which decreased until the end. CONCLUSION: Our results suggest the interest of the serum CTX-II monitoring for the OA progression and the relevance of the multiple time point analysis of this biomarker. Moreover, they address the question of the importance of correctly choosing the age of the animals used in the pre-clinical studies of OA.

MMP and non-MMP-mediated release of aggrecan and its fragments from articular cartilage: a comparative study of three different aggrecan and glycosaminoglycan assays.

OBJECTIVE: Aggrecan is the major proteoglycan in articular cartilage and is known to be degraded by various proteases, including matrix metalloproteinases (MMPs). The present study was undertaken to develop immunoassays detecting aggrecan and its fragments generated by MMP and non-MMP-mediated proteolysis. METHODS: Two immunoassays were developed: (1) the G1/G2 sandwich assay employing a monoclonal antibody (F-78) both as a capturing and a detecting antibody, and (2) the 342-G2 sandwich assay substituting the capturing antibody in the G1/G2 test with a monoclonal antibody, AF-28 recognizing the 342FFGVG neo-epitope generated by MMP cleavage. These assays were compared to the commercially available glycosaminoglycan (GAG) assay. RESULTS: In supernatants of Oncostatin M and Tumor Necrosis Factor alpha (OSM/TNFalpha) stimulated explants, high levels of G1/G2 fragments and GAGs were released in the initial phase (days 2-5), followed by low levels in the intermediate (days 9-12) and late phase (days 12-21). MMP-generated fragments were detected in the late phase only. In the presence of the general MMP inhibitor GM6001, 342-G2 was not detected, whereas the G1/G2 profile remained virtually unchanged. In patients with rheumatoid arthritis (RA), the release of G1/G2 molecules was decreased (27.3%), and that of the 342-G2 fragments increased compared to healthy controls (33.3%). CONCLUSION: The stimulation of bovine articular cartilage explants with OSM/TNFalpha released aggrecan fragments both in an MMP and non-MMP-mediated route. These immunoassays carry a potential as diagnostic tools for the quantitative assessment of the cartilage turnover in RA patients in addition to their utility in ex vivo explant cultures.

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.

Microfibrillar composition of umbilical cord matrix: characterization of fibrillin, collagen VI and intact collagen V.

Ultrastructural studies made on human umbilical cord revealed that the striated collagen fibrils of the Wharton's jelly matrix are mixed with many microfibrillar structures. Microfibrils were found with a tubular cross-section of 10-12 nm diameter and were organized as beaded filaments characteristic of fibrillin-rich microfibrils. Beads had an average diameter of 25 nm and were spaced at about 50-80 nm. This ultrastructural observation was confirmed by indirect immunofluorescent staining of the jelly matrix using monoclonal antibody to fibrillin. Another constituent of the microfibrillar network was present as typical 100-nm periodic filaments of type VI collagen. Indirect immunofluorescent staining using antibodies to collagen VI showed for the first time that this collagen appeared to be distributed largely in the jelly matrix. In addition, other microfibrils with no specific banding pattern were observed. These microfibrils may constitute an organization of type V collagen different from the one which is generally assembled in heterotypic fibrils with collagen I. Among the latter heterotypic fibrils, type V collagen was studied using an anti-peptide antibody to the most N-terminal non-collagenous region of its alpha 2(V) chain. This antibody recognized a filamentous mesh decorating the bundles of collagen fibrils by immunofluorescent staining. This indicates that at least this part of alpha 2(V) chain may be accessible to the antibody at the surface of the fibrils.

Processing of type XI collagen. Determination of the matrix forms of the alpha1(XI) chain.

Type XI collagen is mainly found as a minor constituent in type II-containing fibrils and presents a alpha1(XI)alpha2(XI)alpha3(XI) stoichiometry. This molecule was shown to be partially processed in its intact tissue form. Moreover, alternative splicing has been demonstrated in the variable region of the N-terminal domain of alpha1(XI) and alpha2(XI) chains. In this work, the processing of a major intact form of alpha1(XI) from matrix laid down by chick chondrocytes in culture was identified using N-terminal sequencing and antibodies to synthetic peptides corresponding to the N-terminal propeptide cDNA-derived sequence. The results show that the fully processed form of alpha1(XI) begins at Gln254 of the N-terminal propeptide, seven residues before the end of the proline/arginine-rich protein region encoded by exon I (Zhidkova, N. I., Justice, S. K., and Mayne, R. (1995) J. Biol. Chem. 270, 9486-9493). This sequence is immediately followed by a sequence encoded by exon III. The processing takes place at an Ala-Gln sequence that corresponds to a consensus sequence for procollagen N-proteinase. The antibody raised against a sequence located within the region corresponding to exon IV (anti-P8) fails to recognize this fully processed form of the alpha1(XI) chain. It recognizes, however, two minor bands of high molecular mass. These results suggest that a major cartilage form of alpha1(XI) is the product of alternative splicing in which sequences encoded by both exons II and IV are skipped. The presence of a highly acidic subdomain encoded by exon III at the N terminus of the major form of the alpha1(XI) chain, as predicted by these data, provides potential sites for interaction of collagen XI with other molecules.

Diversity in the processing events at the N-terminus of type-V collagen.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

Sleep and wake after benzodiazepine hypnotics: a 20-hour EEG comparison of lormetazepam and flunitrazepam.

In an electropharmacokinetic study, the effects of lormetazepam and flunitrazepam were compared by the means of a sleep EEG and waking EEG during the following daytime. At a 1-week interval, 6 normal subjects received at random either 2 mg lormetazepam or 2 mg flunitrazepam in a double-blind, crossover fashion. Sleep EEG was recorded throughout the night; 6-min EEG samples were recorded every hour during 10 hours on the following daytime for spectral analysis. Night sleep after flunitrazepam showed lower Stage IV sleep than after lormetazepam. During daytime, only flunitrazepam induced an increased percentage of beta 2 frequencies, which remained above baseline up to 10 hours after awakening, indicating a prolonged impregnation time. This study permitted comparison of the relative intensity and duration of these two benzodiazepines: lormetazepam appeared to be a short-acting hypnotic while flunitrazepam displayed longer modification of the brain electrical activity.

Self-reports of anxiety level and EEG changes after a single dose of benzodiazepines. Double-blind comparison of two forms of oxazepam.

A new formulation of oxazepam especially designed to increase the speed of absorption and eliminate the need to use water (freeze-dried dosage formulation; FDDF) was compared in double-blind and crossover conditions with the standard tablets of the same compound. 5 inpatients with generalized anxiety disorder received at 1-week intervals a single 30 mg dose of one of the compounds. Every 8 min for 96 min after drug intake, they completed a battery of visual analogue scales and had an EEG recording with computerized spectral analysis. Results showed a significantly more rapid onset of activity of FDDF oxazepam for both the self-reports of anxiety level (p less than 0.005) and the specific beta 2 EEG changes (p less than 0.0001), which were significantly correlated (r = -0.73; p less than 0.01). Moreover, all patients rated FDDF oxazepam as having faster onset of action in clinical change than regular tablets (p less than 0.05). This study shows the value of visual analogue scales, pharmaco-EEG, and crossover design in well-selected anxious inpatients in substantiating clinical differences between anxiolytic pharmacotherapies.

[Comparative study of the CNV and the quantitative EEG during a simple motor task (author's transl)]. / Etude comparée de la V.C.N. et de l'E.EG.G. quantitatif au cours d'une tache motrice simple.

The purpose of this study was to evaluate CNV amplitude variability and its degree of covariance with the spontaneous EEG (quantified by FFT algorithm) and the reaction time. 14 healthy male subjects made from atypical for one hour, and they had to keep their eyes closed. Recordings were during this period. CNV experimental paradigm was performed (S1--S2 = 1500 msec). The major findings of this study were that: 1) the CNV amplitude progressively decreased during the first part of the test (habituation) and then tended to stabilize. While not correlated with the vigilance index of spontaneous EEG (alpha/theta + delta index) the CNV amplitude was significantly related to the alpha reactivity index (% alpha of spontaneous EEG--% alpha of the S1--S2 EEG), 2) the CNV slope (calculated by drawing two points situated at 600 msec and 1400 msec after S1) showed significant relationship to both these EEG indices and the reaction time. These data are discussed in terms of Cooper's distinction between "scopeutic" and "categoric" processes (1978) and in terms of the Tecce model of the CNV (1972).