The role of cell-substrate interaction in regulating osteoclast activation: potential implications in targeting bone loss in rheumatoid arthritis.

Analysis of tissues retrieved from the bone-pannus interface from patients with rheumatoid arthritis (RA) and studies in animal models of inflammatory arthritis provide strong evidence that osteoclasts, the cells that are essential for physiological bone resorption, are responsible for articular bone destruction in RA. However, current treatments that specifically target osteoclast-mediated bone resorption in RA have not been successful in preventing bone erosions, and new therapeutic strategies are needed. It has been noted that, although osteoclast precursors are present within the bone microenvironment at sites of pathological bone resorption, cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface and adjacent calcified cartilage. These findings provide evidence that, in addition to requirements for specific cytokines, interaction of osteoclast precursors with these mineralised matrices results in activation of specific signal pathways and the induction of unique gene products that are essential for terminal osteoclast differentiation and activation. These studies are designed to define the gene products and signalling pathways regulated by bone and calcified cartilage, to identify new molecular targets and novel therapeutic approaches for preventing osteoclast-mediated joint destruction in RA and related forms of pathological bone loss.

Role of cell-matrix interactions in osteoclast differentiation.

Osteoclast and their mononuclear cell precursors are present within the bone microenvironment at sites of physiologic and pathologic bone resorption. Analysis of tissues from sites of bone resorption reveal that cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface. We hypothesize that in addition to cytokines, components of the bone matrix and specific cell surface receptors on osteoclasts and their precursors play an essential role in determining the genetic profile and functional properties of fully differentiated resorbing osteoclasts. We have employed expression profiling, with an in vitro model of matrix-dependent osteoclast differentiation, to identify the molecular pathways by which bone matrix-interactions induce terminal osteoclast differentiation and activation. In preliminary studies, we have identified unique genes and transcriptional pathways that are induced by interaction of osteoclast precursors with specific components of the mineralized bone matrix. The authenticity of the gene profiles, as markers of osteoclast differentiation and activation, have been provisionally validated using an in vivo animal bone implantation model and by examination of tissues from patients with specific forms of pathologic osteoclast-mediated bone resorption. The ultimate goal of our studies is to identify new molecular targets for inhibiting osteoclast-mediated bone loss in disorders of pathologic bone loss. The early work of Walker et al. (Walker 1972) in parabiotic animals, and the subsequent studies of Burger et al. (Burger, Van der Meer, van de Gevel, et al. 1982) using a co-culture model with fetal bone rudiments and bone marrow-derived cells, have helped to establish that osteoclasts are derived from macrophage precursors of colony forming unit-macrophage (CFU-M lineage). As such, they share a common hematopoietic origin with other CFU-M lineage cells, including tissue macrophages that populate the lung (alveolar macrophages), liver (Kupfer cells), synovium (synovial macrophages) and other organs. They also share a common lineage

Controlled clinical trial of a novel hemostatic agent in cardiac surgery. The Fusion Matrix Study Group.

BACKGROUND: We performed a prospective randomized trial to compare FloSeal Matrix (Fusion Medical Technologies, Inc, Mountain View, CA), a gelatin-based hemostatic sealant, with Gelfoam-Thrombin (Gelfoam, Pharmacia and Upjohn, Kalamazoo, MI; Thrombin, Gentrac Inc, Middeton, WI) (control group) to control perioperative bleeding. METHODS: A total of 93 patients undergoing cardiac operations were randomized into the FloSeal or control group after standard surgical means to control bleeding had failed. The bleeding site was evaluated at 1, 2, 3, 6, and 10 minutes after applying the hemostatic agent. If bleeding stopped within 10 minutes, the application was considered to be successful. In the case of a failure, the surgeon could use any means preferred (except FloSeal) to achieve hemostasis. All bleeding sites in a patient were treated with the hemostatic agent to which the patient was randomized. Follow-up evaluation was performed at 12 to 36 hours and 6 to 8 weeks after operation. RESULTS: FloSeal stopped bleeding in 94% of the patients (first bleeding site only) within 10 minutes, compared to 60% in the control group (p = 0.001). At 3 minutes, successful hemostasis was achieved in 72% of the FloSeal group compared with 23% in the control group (p = 0.0001). There was no difference in the adverse event profile between the two groups. CONCLUSIONS: FloSeal Matrix demonstrated efficacy superior to that of Gelfoam-Thrombin and had a safety profile similar to that of Gelfoam-Thrombin when used as a topical hemostatic agent during cardiac surgery procedures.

100 long-term implantable left ventricular assist devices: the Columbia Presbyterian interim experience.

BACKGROUND: The use of left ventricular assist devices (LVADs) as bridge to transplantation is now accepted as a standard of care for a subset of end-stage heart failure patients. Our interim experience with both pneumatically and electrically powered ThermoCardiosystems LVADs is presented to outline the benefits and limitations of device support as well as discuss its potential role as bridge to recovery and as destination therapy. METHODS AND RESULTS: Detailed records were kept prospectively for all patients undergoing LVAD insertion. One hundred LVADs were inserted over 7 years into 95 patients, with an overall survival rate of 75% and a transplantation rate of 70%. Four patients underwent device explant for recovered myocardial function. Three patients received LVADs as destination therapy in the ongoing REMATCH (Randomized Evaluation of Mechanical Assist Treatment for Congestive Heart failure) trial. Overall mean patient age was 51 years, and mean duration of support was 108 days. There were 25 device-related infections including the drive line, device pocket, and blood-contacting surfaces. Cerebral vascular accidents and other embolic events occurred in 7 patients with six deaths. There were four device malfunctions and nine graft-related hemorrhages, resulting in six reoperations and three deaths. CONCLUSIONS: The use of long-term implantable LVADs will likely not be limited to bridge to transplantation. The REMATCH trial has commenced to study the role LVADs may have as an alternative to medical management. Furthermore, as the issues of myocardial recovery are examined, the "bridge to recovery" may be an important additional role for these assist devices.

Full activation of chimeric receptors by hybrids between parathyroid hormone and calcitonin. Evidence for a common pattern of ligand-receptor interaction.

Calcitonin (CT) and parathyroid hormone (PTH), whose receptors belong to the same family of G protein-coupled receptors, share no amino acid sequence homology and selectively activate either CT or PTH receptors. We now show, however, that reciprocal hybrid ligands (CT/PTH and PTH/CT), which do not activate the "wild-type" receptors, activate PTH/CT and CT/PTH receptor chimeras, respectively. Our findings indicate that PTH and CT share a similar architecture with at least two functional, receptor-specific domains. These domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate appropriate receptor chimeras. Therefore, both ligands follow, despite their very different primary sequences, a common pattern of ligand-receptor interaction.

Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction.

Two distinct calcitonin (CT) receptor (CTR)-encoding cDNAs (designated GC-2 and GC-10) were cloned and characterized from giant cell tumor of bone (GCT). Both GC-2 and GC-10 differ structurally from the human ovarian cell CTR (o-hCTR) that we cloned previously, but differ from each other only by the presence (GC-10) or absence (GC-2) of a predicted 16-amino acid insert in the putative first intracellular domain. Expression of all three CTR isoforms in COS cells demonstrated that GC-2 has a lower binding affinity for salmon (s) CT (Kd approximately 15 nM) than GC-10 or o-hCTR (Kd approximately 1.5 nM). Maximal stimulatory concentrations of CT resulted in a mean accumulation of cAMP in GC-2 transfected cells that was greater than eight times higher than in cells transfected with GC-10 after normalizing for the number of receptor-expressing cells. The marked difference in maximal cAMP response was also apparent after normalizing for receptor number. GC-2 also demonstrated a more potent ligand-mediated cAMP response compared with GC-10 for both human (h) and sCT (the EC50 values for GC-2 were approximately 0.2 nM for sCT and approximately 2 nM for hCT; EC50 values for GC-10 were approximately 6 nM for sCT and approximately 25 nM for hCT). Reverse transcriptase PCR of GCT RNA indicated that GC-2 transcripts are more abundant than those encoding for GC-10. In situ hybridization on GCT tissue sections demonstrated CTR mRNA expression in osteoclast-like cells. We localized the human CTR gene to chromosome 7 in band q22. The distinct functional characteristics of GC-2 and GC-10, which differ in structure only in the first intracellular domain, indicate that the first intracellular domain of the CTR plays a previously unidentified role in modulating ligand binding and signal transduction via the G protein/adenylate cyclase system.

Cataloging Internet resources.

The number of resources available on the Internet continues to expand exponentially, but finding appropriate resources is still a fragmented, hit-or-miss operation. Traditional library expertise in bibliographic description and access should be applied to the management of this emerging body of material. In the process, catalogers will be able to assess the adequacy of current tools (e.g., cataloging codes, machine-readable cataloging formats, integrated library systems) for providing access to Internet resources and will contribute credibly to design or redesign of access tools. This paper outlines the major issues that must be considered in cataloging electronic resources.

Cloning and characterization of a mouse brain calcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene.

We have identified and characterized a mouse brain calcitonin receptor (CTR) complementary DNA (cDNA). This cDNA encodes a receptor protein that, after expression, has high affinity binding for salmon calcitonin (Kd approximately, 12.5 nM) and is coupled to adenylate cyclase. The binding affinity of this expressed receptor for salmon calcitonin is lower than that described for the previously cloned porcine renal and human ovarian CTRs, but is similar to that of the recently described rat brain CTR, designated the C1b form of the receptor. Analysis of the deduced structure of the mouse brain CTR reveals that it is highly related to the other CTR cDNAs that belong to a distinct family of G-protein-coupled receptors with seven transmembrane-spanning domains. The major structural feature that distinguishes the mouse cDNA clone from the other CTRs is the presence of a consecutive 111-basepair nucleotide sequence that encodes a 37-amino acid sequence which is predicted to localize to the first extracellular loop between the second and third transmembrane-spanning domains. We have mapped the CTR gene in the mouse to the proximal region of chromosome 6, which is homologous to the 7q region of human chromosome 7; only a single CTR gene was identified. Preliminary analysis of the mouse CTR gene reveals that it is complex, consisting of multiple exons separated by lengthy introns that would allow for splice variants consistent with the existence of multiple CTR isoforms predicted from the CTR cDNA clones. The differential cellular and tissue distribution of these functionally distinct CTR isoforms provides the molecular basis for the previously reported widespread distribution and functional heterogeneity of the CTR.

Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line.

A human ovarian small cell carcinoma line (BIN-67) expresses abundant calcitonin (CT) receptors (CTR) (143,000 per cell) that are coupled, to adenylate cyclase. The dissociation constants (Kd) for the CTRs on these BIN-67 cells is approximately 0.42 nM for salmon CT and approximately 4.6 nM for human CT. To clone a human CTR (hCTR), a BIN-67 cDNA library was screened using a cDNA probe from a porcine renal CTR (pCTR) that we recently cloned. One positive clone of 3,588 bp was identified. Transfection of this cDNA into COS cells resulted in expression of receptors with high affinity for salmon CT (Kd = approximately 0.44 nM) and for human CT (Kd = approximately 5.4 nM). The expressed hCTR was coupled to adenylate cyclase. Northern analysis with the hCTR cDNA probe indicated a single transcript of approximately 4.2 kb. The cloned cDNA encodes a putative peptide of 490 amino acids with seven potential transmembrane domains. The amino acid sequence of the hCTR is 73% identical to the pCTR, although the hCTR contains an insert of 16 amino acids between transmembrane domain I and II. The structural differences may account for observed differences in binding affinity between the porcine renal and human ovarian CTRs. The CTRs are closely related to the receptors for parathyroid hormone-parathyroid hormone-related peptide and secretin; these receptors comprise a distinct family of G protein-coupled seven transmembrane domain receptors. Interestingly, the hCTR sequence is remotely related to the cAMP receptor of Dictyostelium discoideum (21% identical), but is not significantly related to other G protein-coupled receptor sequences now in the data bases.

A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C.

The signal transduction pathways of the recently cloned porcine kidney calcitonin (CT) receptor were evaluated. This receptor, when stably transfected into MC-3T3 cells, avidly bound salmon CT (SCT) [dissociation constant (Kd) = 4 nM]. Incubation with SCT resulted in a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) [50% effective concentration (EC50) = 0.02 nM] in transfected cells (referred to as PC-1 cells). Binding kinetics and cAMP dose response relationships were similar to those of the native receptor in LLC-PK1 cells. PC-1 cells also responded to calcitonin gene-related peptide (CGRP), but the EC50 value for cAMP accumulation was more than three orders of magnitude higher than for SCT. Exposure of PC-1 cells to SCT (5 nM to 1 microM) produced a dose-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]i), whereas CGRP did not. The initial rise in [Ca2+]i was not dependent on extracellular Ca2+, suggesting that SCT induced release of Ca2+ from intracellular stores. SCT also increased inositol trisphosphate production in PC-1 cells. In conclusion, the cloned, transfected porcine CT receptor functionally couples to and activates both adenylyl cyclase and phospholipase C. This dual coupling is also a characteristic of the parathyroid hormone receptor, which has significant homology in amino acid sequence with the CT receptor.