A walking model to assess the onset of analgesia in osteoarthritis knee pain.

OBJECTIVE: To assess a walking model utilizing a set of standardized treadmill walks to measure acute analgesic response in osteoarthritis (OA) of the knee. DESIGN: Randomized, double-blind, placebo-controlled, multiple dose, three-period crossover study. Patients > or =45 years of age (N=22) with symptomatic knee OA were randomized to naproxen 500 mg bid, tramadol/acetaminophen 37.5 mg/325 mg in forced titration, or placebo in each of three periods. Patients performed multiple 20-minute treadmill walks on Day 1 and Day 3 at a consistent self-selected pace predetermined at screening. Pain intensity (PI) during the walks was assessed on an 11-point numerical rating scale at 0, 3, 6, 9, 12, 15, 18, and 20 min. The primary endpoint was the time-weighted average (TWA) change from baseline PI on Day 3 for the two self-paced walks for the active treatments vs placebo. Time to moderate pain (TTMP) was a key secondary endpoint. RESULTS: Compared with placebo, the TWA change from baseline PI on Day 3 was significantly better with tramadol/acetaminophen (P=0.043) but not with naproxen (P=0.089). TWA change from baseline on Day 1 was also significantly better with both tramadol/acetaminophen (P=0.001) and naproxen (P=0.048) compared with placebo. TTMP was significantly better for tramadol/acetaminophen and naproxen than placebo (P<0.001 to P=0.015) for walks on Day 1 after a single dose and on Day 3. CONCLUSIONS: This novel OA pain model was able to discriminate both tramadol/acetaminophen and naproxen from placebo after single and multiple doses. ClinicalTrials.gov identifier: NCT00772967.

Region of interest analysis: by selecting regions with denuded areas can we detect greater amounts of change?

INTRODUCTION: Based on recent analyses, the measures of short-term responsiveness of magnetic resonance imaging (MRI) derived cartilage morphometry may not be as large as earlier studies had suggested. We examined if by selecting regions of interest with denuded cartilage, the remaining cartilage within this region of interest was susceptible to greater rates of cartilage loss. METHODS: Subjects included for this analysis are a subset of the approximately 4700 participants in the Osteoarthritis Initiative (OAI) Study. Bilateral radiographs and 3T MRI (Siemens Trio) of the knees and clinical data are obtained at baseline and annually in all participants. Hundred and fifty subjects from the OAI progression subcohort all of whom had both frequent symptoms and, in the same knee, radiographic osteoarthritis (ROA defined as definite tibio-femoral osteophytes on X-ray) based on a screening reading done at the OAI clinics. One knee from each subject was selected for analysis. Using sagittal 3D DESSwe MR images from the baseline and 12-month follow-up visit, a segmentation algorithm was applied to the cartilage plates of the index knee to compute the cartilage volume, normalized cartilage volume (volume normalized to bone surface interface area), and percent denuded area (Total Cartilage Bone Interface area denuded of cartilage). Summary statistics of the changes (absolute and percentage) from baseline at 1 year and the standardized response mean (SRM), i.e., mean change divided by the standard deviation (SD) of that change were calculated. Analyses are stratified into three groups according to baseline assessment of denuded area: those with no denuded area in the region of interest at baseline, and then two groups (intermediate denuded area (or=median) denuded area) of equal sample size. RESULTS: On average the subjects were 60.9 years of age and obese with a mean body mass index (BMI) of 30.3 kg/m(2). For the combined central medial femur and tibia the mean volume change for the whole sample was -48.2 (SD 159.8) mm(3), which gives an SRM of -0.30. In the subsample of knees with no denuded area the SRM was -0.25, in the knees with intermediate denuded area the SRM was -0.30, and in knees with severe denuded area the SRM was -1.00. For normalized volume of the central medial femur in the subsample of knees with no denuded area the SRM was -0.22, in the knees with intermediate denuded area the SRM was -0.26, and in knees with severe denuded area (n=23) the SRM was -0.71. The magnitude of the SRMs was generally smaller in participants with no denuded area. In contrast, the SRMs in participants with denuded area were larger. CONCLUSION: By selecting participants with the presence of cartilage regions with denuded area the ability to demonstrate change in cartilage loss in that specific location is markedly improved compared to persons without a full thickness lesion in that cartilage plate. This option for screening during recruitment in clinical trials could facilitate the detection of participants at greater risk of subsequent cartilage loss.

Change in cartilage morphometry: a sample of the progression cohort of the Osteoarthritis Initiative.

OBJECTIVE: The performance characteristics of hyaline articular cartilage measurement on magnetic resonance imaging (MRI) need to be accurately delineated before widespread application of this technology. Our objective was to assess the rate of natural disease progression of cartilage morphometry measures from baseline to 1 year in knees with osteoarthritis (OA) from a subset of participants from the Osteoarthritis Initiative (OAI). METHODS: Subjects included for this exploratory analysis are a subset of the approximately 4700 participants in the OAI Study. Bilateral radiographs and 3T MRI (Siemans Trio) of the knees and clinical data were obtained at baseline and annually in all participants. 160 subjects from the OAI Progression subcohort all of whom had both frequent symptoms and, in the same knee, radiographic OA based on a screening reading done at the OAI clinics were eligible for this exploratory analysis. One knee from each subject was selected for analysis. 150 participants were included. Using sagittal 3D DESSwe (double echo, steady-state sequence with water excitation) MR images from the baseline and 12 follow-up month visit, a segmentation algorithm was applied to the cartilage plates of the index knee to compute the cartilage volume, normalised cartilage volume (volume normalised to bone surface interface area), and percentage denuded area (total cartilage bone interface area denuded of cartilage). RESULTS: Summary statistics of the changes (absolute and percentage) from baseline at 1 year and the standardised response mean (SRM), ie, mean change divided by the SD change were calculated. On average the subjects were 60.9 years of age and obese, with a mean body mass index of 30.3 kg/m2. The SRMs for cartilage volume of various locations are: central medial tibia -0.096; central medial femur -0.394; and patella -0.198. The SRMs for normalised cartilage volume of the various locations are central medial tibia -0.044, central medial femur -0.338 and patella -0.193. The majority of participants had a denuded area at baseline in the central medial femur (62%) and central medial tibia (60%). In general, the SRMs were small. CONCLUSIONS: These descriptive results of cartilage morphometry and its change at the 1-year time point from the first substantive MRI data release from the OAI Progression subcohort indicate that the annualised rates of change are small with the central medial femur showing the greatest consistent change.

CD18 activation epitopes induced by leukocyte activation.

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.

Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site.

The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the K(d) of ICAM-1 binding. The K(d) of wild-type LFA-1 (1.2 x 10(-1) s(-1)) differed with that of the activating IDAS mutant (1.9 x 10(-2) s(-1)), but their K(a) values were identical (2.2 x 10(5) M(-1)s(-1)). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.

Leupaxin is a novel LIM domain protein that forms a complex with PYK2.

We have identified a novel cytoplasmic protein, leupaxin, that is preferentially expressed in hematopoietic cells and is most homologous to the focal adhesion protein, paxillin. Leupaxin possesses two types of protein interaction domains. There are four carboxyl-terminal LIM domains in leupaxin that share 70% amino acid identity and 80% similarity with those in paxillin. Paxillin LIM domains mediate localization to focal contacts. In the amino-terminal region of leupaxin there are three short stretches of approximately 13 amino acids that share 70-90% similarity with paxillin LD motifs. Paxillin LD motifs have been implicated in focal adhesion kinase (FAK) and vinculin binding resulting in the localization of FAK to focal adhesions. Leupaxin is expressed in cell types, such as macrophage, that lack FAK. We demonstrate here that leupaxin associates with a second FAK family member, PYK2. As leupaxin and PYK2 are both preferentially expressed in leukocytes they may therefore form a cell type-specific signaling complex. We also demonstrate that leupaxin is a substrate for a tyrosine kinase in lymphoid cells and thus may function in and be regulated by tyrosine kinase activity. Leupaxin is thus a phosphotyrosine protein with LD and LIM binding motifs most homologous to paxillin that may assemble and regulate PYK2 signaling complexes in leukocytes.

Rapid targeting of nuclear proteins to the cytoplasm.

BACKGROUND: The transcription factor NF-ATc plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-ATc translocates from the cytoplasm to the nucleus in response to a rise in intracellular calcium, and immediately returns to the cytoplasm when intracellular calcium levels fall. The rapid nuclear exit of NF-ATc is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals. RESULTS: To study the nuclear export of NF-ATc, we have developed a general, non-invasive assay for the identification and study of nuclear export signals (NESs). The NES is defined by its ability to translocate a protein from the nucleus to the cytoplasm when the two are tethered by a membrane-permeable ligand. This procedure has allowed us to identify a NES within NF-ATc that functions in concert with a glycogen synthase kinase-regulated process to direct the rapid nuclear exit of NF-ATc. CONCLUSIONS: The rapid nuclear export of NF-ATc via its NES and a glycogen synthase kinase-regulated event may be an important mechanism for insulating cells from transient spikes in intracellular calcium which might otherwise lead to inappropriate activation. The assay we have developed allows the rapid identification of NESs and can be used as a general method for the inducible cytoplasmic export of nuclear proteins.

Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction.

The NF-AT family of transcription factors participates in the regulation of early immune response genes such as IL-2, IL-4, CD40 ligand, and Fas ligand in response to Ca2+/calcineurin signals initiated at the antigen receptor. Calcineurin activation leads to the rapid translocation of NF-AT family members from cytoplasm to nucleus, an event that is blocked by the immunosuppressive drugs cyclosporin A and FK506. We show that translocation requires two redundant nuclear localization sequences and that one sequence is in an intramolecular association with phosphorserines in a conserved motif located at the amino terminus of each NF-AT protein. Mutation of serines in this motif in NF-ATc both disrupts this intramolecular interaction and leads to nuclear localization, suggesting a model of NF-AT nuclear import in which dephosphorylation by calcineurin causes exposure of two nuclear localization sequences.

Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3.

The transcription factor NF-AT responds to Ca2+-calcineurin signals by translocating to the nucleus, where it participates in the activation of early immune response genes. Calcineurin dephosphorylates conserved serine residues in the amino terminus of NF-AT, resulting in nuclear import. Purification of the NF-AT kinase revealed that it is composed of a priming kinase activity and glycogen synthase kinase-3 (GSK-3). GSK-3 phosphorylates conserved serines necessary for nuclear export, promotes nuclear exit, and thereby opposes Ca2+-calcineurin signaling. Because GSK-3 responds to signals initiated by Wnt and other ligands, NF-AT family members could be effectors of these pathways.

Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase.

Stimulation of the T lymphocyte antigen receptor-CD3 complex (TCR-CD3) causes T cell activation by a process associated with increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Evidence exists suggesting that GTP-binding (G) proteins, particularly the pertussis toxin (PT)-sensitive Gi proteins, participate in this signal transduction pathway. To clarify the role of Gi proteins in TCR-CD3 signaling, and to investigate other possible functions of Gi molecules in T cells, we expressed the S1 subunit of PT in the thymocytes of transgenic mice using the lymphocyte-specific lck promoter. Transgenic thymocytes contained S1 activity and exhibited profound depletion of Gi protein PT substrates in a manner suggesting their inactivation by S1 in vivo. Nevertheless, treatment of transgenic thymocytes with mitogenic stimuli provoked normal increases in intracellular free Ca2+ concentrations and IL-2 secretion, indicating that Gi proteins are not required for T cell activation. These normal signaling responses notwithstanding, mature thymocytes accumulated in lck-PT mice and did not appear in secondary lymphoid organs or in the circulation. Viewed in the context of the known features of Bordetella pertussis infection, our results suggest that a PT-sensitive signaling process, probably involving Gi proteins, regulates thymocyte emigration.

A small multigene family encodes Gi signal-transduction proteins.

The guanine nucleotide-binding regulatory proteins known as G proteins are receptor-associated signal-transduction molecules that are implicated in the control of a variety of metabolic processes. Recent evidence suggests that G proteins may mediate B-lymphocyte responses to bacterial lipopolysaccharide and may also transduce signals from the T-cell antigen receptor. Since these receptors are uniquely expressed on lymphoid cells, we used molecular cloning strategies to ask whether lymphocytes contain specialized G-protein alpha subunits to assist in signal transduction. Comparison of our two deduced human alpha i amino acid sequences with those previously determined for bovine and rodent G proteins permits the identification of three closely related but distinct types of alpha i molecules that comprise a small multigene family. Using gene-specific probes, we found that both of our alpha i genes are expressed in most cell types but in differing ratios. Our data support the view that a modest repertoire of extremely closely related G proteins mediates the transduction of signals derived from multiple different receptor molecules.