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.

Heterogeneity of human bone.

Matched samples of bone from the lumbar spine and tibia were obtained at autopsy from three adult males who had no known evidence of metabolic bone disease at the time of their demise. The soluble noncollagenous bone proteins were quantitatively extracted from these samples and assayed for the relative content of two bone-associated proteins, osteocalcin and osteonectin. When compared to trabecular bone, cortical bone had higher levels of osteocalcin and much lower levels of osteonectin. When concentration is expressed per gram of dried bone, the osteocalcin excess in cortical bone ranged from 30- to 32-fold, and the osteonectin excess in trabecular bone ranged from 21- to 47-fold. These differences were significant (P less than 0.01) using analysis of variance. We conclude that the human skeleton is not homogeneous with regard to these biochemical markers and that cortical and trabecular bone are biochemically quite distinct. This implies that these two types of bone may be subject to distinct regulatory mechanisms and that global assessments of skeletal function and bone quality based upon soluble markers should be applied with caution. The data also imply that a differential assessment of skeletal performance may be possible using biochemical serum markers.

p56lyn catalyzes a reversible autophosphorylation reaction and a nucleoside diphosphate kinase reaction.

The reversible autophosphorylation of the pp60c-src family tyrosine kinase, p56lyn has been characterized by a simple procedure that involves the examination of the enzyme catalyzed radioisotope exchange between ATP and ADP. The equilibrium constant of the reaction was determined to be 3.31 and corresponded to a standard free energy of hydrolysis of the phosphotyrosine bond in p56lyn of -8.08 kcal/mol. GDP was capable of substituting for ADP as phosphate acceptor so that p56lyn displayed a nucleoside diphosphate kinase activity.

Synthesis of peptides by the solid-phase method. 7. Substance P and analogues.

Substance P and 21 related peptides containing isosteric or isofunctional groups were prepared by the solid-phase method. After purification by gel filtration and ion-exchange chromatography, the compounds were characterized by thin-layer chromatography, paper electrophoresis, and amino acid and elemental analysis. The biological activities of the peptides were evaluated in vitro on the guinea pig ileum, the rabbit mesenteric vein, and the dog common carotid artery and in vivo on the rat blood pressure. It is shown that the replacement of some residues in the undecapeptide substance P causes variable losses of apparent affinity with a little or no change in the intrinsic activity. All the analogues used in the present study were found to be inactive as antagonists.

Synthesis of peptides by the solid-phase method. 6. Neurotensin, fragments, and analogues.

Neurotensin (NT) and 24 related compounds, including fragments or analogues modified at the C-terminal end of the parent molecule, have been prepared by the solid-phase method. After purification by cation-exchange chromatography, the compounds were characterized by thin-layer chromatography, amino acid analysis, elemental analysis, and high-pressure liquid chromatography. The stimulating effects of the peptides were evaluated in rat stomach strips, in isolated spontaneously beating atria of guinea pigs, and in the coronaries of perfused rat hearts. The differences between the biological activities of these compounds are discussed.

Serial transfer of GVH-R splenomegaly in chicken embryos.

Using histocompatible chicken strains B14 and B19, we demonstrate that the capacity to induce a GVH-R in an embryo could be induced precociously by the reaction itself. While naive chickens display this capacity around 3 to 4 weeks posthatching, embryos engrafted with adult allogeneic cells at E13 or E8 became endowed with this capacity at E20 and E15, respectively. Furthermore, this acceleration could be obtained by serial transfer of splenic cells through a sequence of three embryos undergoing the GVH-R. The highest efficiency in transfer was realized by regularly alternating the MHC haplotype at each transfer. It is concluded that the original cells from the adult donor may be partially responsible for the transfer, but that cells from the successive embryos are also certainly involved. Thus, maturation of the embryonic immune system appears accelerated by a GVH-R.

Ontogeny of the GVH-R inducing capacity, in conventional and germ-free chickens.

Three strains of MHC homozygous chickens were used to study the ontogeny of the GVH-R. It was found that this function of the immune system was acquired with a delay in animals raised in germ-free conditions. In our previous work, we had shown that, contrary to expectation, bursal cells were capable of mounting a GVH-R against embryos, and actually were particularly efficient in this regard when compared to spleen cells. The present experiments have disclosed that splenomegaly induction may be dissociated from the toxic effect that bursal cells also exert on recipient embryos.

Brain-derived neurotrophic factor stimulates survival and neuronal differentiation in cultured avian neural crest.

The response of trunk neural crest cells taken from precise levels of the neural axis and cultured together with adjacent somites to Brain-derived neurotrophic factor (BDNF), was examined in cultures grown in a chemically defined medium. In control cultures, the number of neural crest-derived neurons expressing the HNK-1 epitope, increased as a function of somitic level in a caudorostral direction. Treatment of cultures with increasing concentrations of BDNF (50 pg/ml to 1 ng/ml) resulted in a 1.5- to 6-fold stimulation in the number of neurons developing from crest cells excised at advanced and post-migratory stages, whereas early migrating crest cells were responsive only to concentrations equal to or higher than 1 ng/ml of BDNF. Nerve growth factor used at 5 and 30 ng/ml had no effect on survival of HNK-1-positive cells at any of the somitic levels tested. In an attempt to identify the subpopulation of HNK-1-immunoreactive neurons responding to BDNF, control and treated cultures were stained for the HNK-1 antibody in combination with substance P (SP) antibodies (as a marker for sensory neurons). SP immunoreactivity localized to a subpopulation of phase-bright, HNK-1-positive neurons. The absolute number of SP-positive neurons increased 2- to 4-fold upon BDNF treatment; however, their relative proportion within the population expressing the HNK-1 epitope remained essentially unchanged from control to treated cultures (on day 1, 20% as compared to 23.3% and on day 2, 44.6% compared to 49.7% for control and treated cultures, respectively). Taken together, these data suggest that BDNF stimulates primary neuronal differentiation of SP expressing neurons, and/or their survival.

Conservative management of carpal tunnel syndrome: a reexamination of steroid injection and splinting.

The awareness of carpal tunnel syndrome by the lay public has increased dramatically in recent years, with an apparent shift in patient-population presentation. We prospectively studied steroid injection and wrist splinting in 76 hands in 57 patients, presenting without advanced disease or associated medical conditions, by standard evaluation and protocol of treatment. The average age of the patients was 38 years; 50 women and 7 men were included. Follow-up examination after simultaneous steroid injection and splinting averaged 11 months. Ten hands were noted to be symptom-free at the final evaluation. Women were noted to have a significant decrease in the rate of symptom resolution when compared to men. Patients, 40 years of age or younger, were also noted to have a significant decrease in the rate of symptom resolution when compared to patients over 40 years of age. No significant differences were noted when comparing symptom duration prior to treatment or workers' compensation insurance status to final symptom resolution. Young women are the least likely to have resolution of carpal tunnel syndrome symptoms when treated conservatively.

Tumors of the upper urinary tract: 10 years of experience.

We reviewed 108 patients with upper urinary tract tumors who underwent surgical treatment during a 10-year period (87 men and 21 women with a mean age of 63.5 years). Of the tumors 97% were unilateral and only 3 patients had bilateral tumors. Two-thirds of the patients had a single tumor focus and a third had 2 or more tumor foci. Additionally, there were 31 patients (28.7%) with previous and/or simultaneous bladder tumors. Nephroureterectomy was performed in 92 cases, nephrectomy in 6 and a conservative operation in 13. In 65 cases lymphadenectomy was added. The survival rates at 5 and 10 years were 67 and 65%, respectively. Of the patients 90% with cancer-related deaths had high grade tumors. Of the 15 patients with positive lymph nodes 87% died of metastasis compared to 8% of the 50 patients with negative lymph nodes. Nine patients (8.7%) had relapse in the upper urinary tract, 6 (5.8%) in the ipsilateral ureter and 3 (2.9%) in the contralateral ureter. Of these 3 patients 2 had recurrent multifocal bladder tumors. For patients who present with an upper urinary tract tumor the risk of a bladder cancer was approximately 9% and that of a contralateral urothelial tumor was 1%.

[Tumors of the upper excretory urinary tract. A homogeneous series of 108 patients]. / Tumeurs de la voie excrétrice urinaire haute. Une série homogène de 108 patients.

In their study of a homogeneous series of 108 patients operated for a transitional cell tumor of the upper urinary tract, the authors emphasize the following points: --every third tumor has several foci, and it is very difficult to make a complete inventory of all tumoral foci before surgery; --the great majority of these tumors are unilateral. Bilateral recurrence following radical unilateral excision is quite exceptional; --low-grade superficial tumors virtually never invade the lymph nodes, and their prognosis is excellent as a rule; --grade III carcinomas area almost always infiltrating and present high risks of lymph node invasion. Lymph node involvement means that the evolution will be lethal in all cases. None of the patients having invaded lymph nodes survived; --metastases appear soon, usually within the first 24 months, but mainly between the 6th and 12th months following surgery; --20% of the patients in this series died of metastases; --the actuarial survival rate after 5 years is 68%.

Complications of transesophageal echocardiography in the ED.

The complication rate of transesophageal echocardiography (TEE) performed in clinical settings outside the emergency department (ED) has been reported to be 1% to 3%. The rate of complications of performing TEE in the ED has not been established. The purpose of this study was to determine the rate of complications associated TEE with carried out on ED patients, and to investigate parameters that might predict complications. A retrospective chart review was carried out on consecutive ED patients undergoing TEE at a major referral center. Complications were abstracted. Parameters to predict complications were assessed, including age, gender, vital signs, pulse oximetry values, serum bicarbonate level, and hematocrit level. A total of 142 patients underwent TEE in the ED during the study period; 88 of these were trauma patients. There were 18 (12.6%) complications: death (1), respiratory insufficiency/failure (7), hypotension (3), emesis (4), agitation (2), and cardiac dysrhythmia (1). None of the tested variables predicted a complication. TEE carried out in the ED has a higher complication rate than has been reported in other clinical settings.

[Separation of the virotoxins of the mushroom Amanita virosa and comparative study of their interaction on actin in vitro]. / Séparation des virotoxines du champignon Amanita virosa et étude comparative de leur interaction sur l'actine in vitro.

Cyclic peptides have been extracted with methanol from Amanita virosa and separated by preparative HPLC. Six peptides were obtained: phalloidin and five new peptides recently identified by Faulstich as virotoxins. We have compared the interaction of these six peptides with actin in vitro. They increased the rate of polymerization of actin and protected F-actin against several denaturating agents: proteases, heat, chaotropic ions, cytochalasin B, and DNAse I. The five virotoxins have therefore the same biological properties as phalloidin. However, the differential spectra of interaction between actin and the five virotoxins are different than the differential spectra between actin and phalloidin, thus it appears that the molecular interaction of actin with virotoxins is different than with phalloidin. The five virotoxins have the same activity. Although these virotoxins have different functional groups on amino acids 1 and 7, it is concluded that these two amino acids are of minor importance in the interaction of these peptides with actin.

The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles.

The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.

Rapid assay of human plasma carboxypeptidase N by high-performance liquid chromatographic separation of hippuryl-lysine and its product.

A rapid and sensitive method for measuring carboxypeptidase N (CPN) activity in human plasma is described. The procedure is based on the hydrolysis of a high-specificity/low-affinity substrate, hippuryl-L-lysine, to its products hippuric acid and lysine. The substrate and product are separated quantitatively by high-performance liquid chromatography in less than 10 min following a minimal sample preparation time. The advantages of this method over previous ones are discussed and data are presented demonstrating the reliability of this method for the routine clinical determination of CPN activity.

Synthesis of peptides by the solid-phase method. V. Substance P and analogs.

We have synthesized a series of 12 analogs of the undecapeptide substance P in order to perform a structure-activity study of this peptide. In the present work, each residue was substituted by L-alanine, and the C-terminal amide was replaced by the free carboxyl in order to pinpoint biologically important side chains and functional groups. The synthesis of the analogs was carried out by the automatic solid-phase method. Couplings were performed by the symmetrical anhydride procedure. After cleavage with liquid HF, the peptides were purified by gel filtration and ion-exchange chromatography. Their purity was assessed by thin-layer chromatography, paper electrophoresis, amino acid and elemental analyses, and high pressure liquid chromatography. They were tested for biological activity in vitro on the ileum of the guinea pig, the mesenteric vein of the rabbit, and the vas deferens of the rat, and in vivo by measuring their effect on the blood pressure of the rat.

[Extracorporeal shockwave lithotripsy in children]. / La litotricia extracorpórea por ondas de choque en el niño.

Upper urinary tract lithiasis is a rare condition in children in comparison to its incidence in the adult population. The distribution of lithiasis in children has been reported to be 56% for idiopathic lithiasis, 20% for metabolic lithiasis, and 25% for lithiasis associated with uropathy. Extracorporeal shock wave lithotripsy (ESWL) has become the treatment of choice for upper urinary tract calculi in the adult patient as well as in children after minor changes and complementary material had permitted the utilization of the Dornier HM-3 lithotripter in these small patients. The reported 3-month success rates range from 47% to 83%, according to the number of patients or kidneys free from calculi. The series reported in the literature have a mean of about 60% completely stone-free kidneys evidenced on the X-ray of the abdomen without preparation at 3 months. Its indications and results are comparable with those of ESWL in the adult. However, the long-term results have as yet not been evaluated and, to date, its hypothetical effects on renal growth or arterial pressure have not yet been elucidated.

[Value of extracorporeal shockwave lithotripsy in the treatment of urinary lithiasis in children]. / Intérêt de la lithotriptie extra-corporelle par ondes de choc pour le traitement de la lithiase urinaire de l'enfant.

We report our experience with extracorporeal lithotripsy (Dornier HM3) in a series of 26 children with a mean age of 11.6 years, treated for 3 1/2 years. 12 children (46%) had a previous history of calculi and 7 (27%) had already undergone surgery on the same side. Treatment requires two transducer investigations before lithotripsy. With the exception of minor modifications the technique is the same as in the adult. Three (9.7%) postlithotripsy complications were noted, requiring 2 drainage procedures to be carried out on the urinary tract. At 3 months, the success rate (no residual calculi on the plain abdominal film) was 60.7%. This study confirms the efficacy of lithotripsy in the treatment of urinary lithiasis in children.

T-lymphocyte subsets in the embryonic spleen undergoing a graft-versus-host reaction.

Allogeneic immunocompetent T cells injected into chicken embryos induce a graft-versus-host reaction (GVHR) whose most prominent manifestation is splenic hyperplasia. The highly inbred CC and CB strains of chickens used here are, respectively, homozygous for the B4 or B12 MHC haplotypes. By means of a panel of immunological reagents, including alloantisera and monoclonal antibodies against public domains of the T-cell receptor, CD4, CD8, and the inducible interleukin-2-receptor light chain (CD25), it is shown that the bulk of cells in the enlarged spleen are of host origin and do not express markers typical of mature T or B lymphocytes. Among recipient splenocytes, the quantitatively most important population consists of TCR alpha beta-TCR gamma delta- CD4-CD8+CD25+ (TCR0) lymphocytes. Donor cells encountered in the spleen prevalently exhibit a TCR alpha beta+CD4+CD8-CD25+ phenotype and proliferate in vivo. The data demonstrate that nonspecific host and potentially specific donor-derived cellular elements contribute to splenomegaly.