The Pennsylvania Department of Health Settlement Funds specifically disclaims responsibility for any analyses, interpretations, or conclusions herein.
To assess defects in expression of critical cell cycle checkpoint genes and proteins in patients with rheumatoid arthritis (RA) relative to presence or absence of methotrexate (MTX) treatment, and to investigate the role of JNK in induction of these genes by MTX.
Flow cytometric analysis was used to quantify changes in levels of intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcription–polymerase chain reaction was used to identify changes in cell cycle checkpoint target genes.
RA patients expressed reduced baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and JNK total protein. The reduction in expression of mRNA for MAPK9, TP53, CDKN1A, and CDKN1B was greater in patients not receiving MTX than in those receiving low-dose MTX, with no difference in expression levels of CHEK2 and RANGAP1 mRNA between MTX-treated and non–MTX-treated patients. Further, JNK levels were inversely correlated with C-reactive protein levels in RA patients. In tissue culture, MTX induced expression of both p53 and p21 by JNK-2– and JNK-1–dependent mechanisms, respectively, while CHEK2 and RANGAP1 were not induced by MTX. MTX also induced ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocked these MTX-mediated effects.
Our findings support the notion that MTX restores some, but not all, of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells, via a JNK-dependent pathway.
Rheumatoid arthritis (RA) is the most common serious autoimmune disease, affecting ∼1.3 million people in the US (1). Often characterized by bone erosion and cartilage destruction due to chronic inflammation, RA is a multisystem disorder affecting synovial spaces between small and large joints. It accounts for ∼250,000 hospitalizations and ∼9 million outpatient visits each year, and its associated costs represent ∼1% of the U.S. gross domestic product (2).
Though initially developed as a chemotherapeutic agent, methotrexate (MTX) has been the mainstay for RA treatment since the 1980s (3–7). Once-weekly administration of MTX at 7.5–25 mg yields optimal clinical outcomes in RA, compared to the 5,000 mg/week dosage used in the treatment of malignancy (7, 8). MTX is a potent, competitive inhibitor of dihydrofolate reductase (DHFR) (9–11), resulting in reduced tetrahydrofolate levels and inhibition of de novo purine and pyrimidine synthesis, leading to cell cycle arrest (8, 12). However, mechanisms of action of low-dose, once-weekly MTX may differ significantly from those of high-dose MTX. Since supplementation with low-dose folic acid (1–5 mg/day) does not attenuate the clinical efficacy of MTX in RA patients, the antiinflammatory actions of low-dose MTX may stem from alternative pathways (8, 13, 14).
Although the etiology of RA is incompletely understood, it is known that T lymphocytes from RA patients exhibit loss of genomic integrity and deficiencies in specific proteins that repair DNA damage and induce cell cycle arrest and apoptosis. Specifically, reduced expression of ATM (a critical component of DNA damage repair and activation of p53-dependent cell cycle arrest and apoptosis), of p53 itself, of checkpoint kinase 2 (CHK-2, which also phosphorylates p53), and of the cyclin-dependent kinase inhibitors p21 and p27 contributes to these defects in RA (15–19).
We recently found that JNK, a MAP kinase, is activated by MTX through production of reactive oxygen species (ROS) due to uncoupling of nitric oxide synthase (NOS) arising from MTX-dependent inhibition of DHFR, which blocks reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4). MTX-mediated JNK activation results in induction of proapoptotic target genes and increased sensitivity to apoptosis (20). Since JNKs, members of the MAP kinase family of proteins, also directly phosphorylate p53, leading to its increased accumulation and activity (21–23), we hypothesized that JNKs, or MAPKs in general, may also be deficient in RA and that MTX therapy may correct not only JNK deficiency, but also deficiencies in critical regulators of cell cycle checkpoints.
Herein we report that lymphocytes from patients with RA exhibit a selective deficiency in MAPK9 (JNK-2), but not other MAPK transcripts. MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 transcript levels, along with JNK total protein levels, are significantly lower in RA patients compared to healthy control subjects. Further, MAPK9, TP53, CDKN1A, and CDKN1B transcript levels are higher in RA patients who are taking MTX compared to those who are not, whereas similar increases in CHEK2 and RANGAP1 transcript do not occur with MTX treatment. In cell culture models, MTX directly induces increased expression of p53, p21, and p27, but not CHEK2 or RANGAP1. We hypothesize that the therapeutic activity of MTX may arise in part from its ability to restore expression levels of key proteins required for cell cycle checkpoint arrest and that defects in the cell cycle and DNA damage response pathway may not only contribute to disease pathogenesis but may also serve as important markers of RA disease progression. These defects might represent novel therapeutic targets for disease management.
PATIENTS AND METHODS
The study group consisted of 43 control subjects with no current chronic or acute infection and no family history of autoimmune disease, and 36 patients meeting the American College of Rheumatology/European League Against Rheumatism classification criteria for RA (24). Demographic characteristics of the control subjects and the RA patients with or without MTX treatment did not differ significantly (Table 1). Blood samples were also obtained from patients with other autoimmune diseases, i.e., multiple sclerosis (MS), ulcerative colitis, Crohn's disease, and systemic lupus erythematosus. These samples were collected at 3 different sites in the US. There were no significant differences between study groups in terms of age, race, and sex composition. Relevant institutional review board approval was obtained at all participating sites.
Table 1. Demographic characteristics of the RA patients and healthy controls and clinical characteristics of the RA patients*
Controls (n = 43)
RA, no MTX treatment (n = 18)
RA, MTX treatment (n = 18)
Except where indicated otherwise, values are the percent. RA = rheumatoid arthritis; MTX = methotrexate; HCQ = hydroxychloroquine; TNF = tumor necrosis factor.
Defined as the presence of at least 3 of the following: morning stiffness >45 minutes, >3 swollen joints, >6 tender joints, and erythrocyte sedimentation rate >28 mm/hour.
MTX, BH4, caffeine, theophylline, folic acid, and N-acetyl-L-cysteine (NAC) were obtained from Sigma; 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM–H2DCF-DA) was obtained from Invitrogen.
Cells were cultured in RPMI 1640 medium (1 μg/ml folic acid) supplemented with 10% (volume/volume) fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin, and 1% (v/v) L-glutamine at 37°C in 5% CO2. The Jurkat human T cell line was obtained from ATCC. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Hypaque centrifugation or cell preparation tube with sodium heparin according to the recommendations of the manufacturer (BD Biosciences). For T cell activation, PBMCs were cultured for 72 hours with anti-CD3 antibody (OKT3 Clone; ATCC) in complete medium containing 30 units/ml interleukin-2. MTX was used in concentrations of 0.1 μM or 1 μM, and culture periods ranged from 24 hours to 48 hours of continuous exposure to MTX. Pharmacokinetic analysis indicates that ingestion of a 20-mg tablet of MTX produces plasma MTX concentrations of ∼0.5 μM after 1 hour and ∼0.1 μM after 10 hours (25).
RNA isolation and real-time quantitative polymerase chain reaction (qPCR).
Total RNA was purified from blood collected in PAXgene tubes according to the instructions of the manufacturer (Qiagen) or from cell cultures using Tri-Reagent (Molecular Research Center) and quantified using a NanoDrop 1000 spectrophotometer. Complementary DNA (cDNA) was synthesized from 5 μg total RNA (SuperScript III First-Strand Synthesis Kit; Invitrogen) with oligo(dT) as the primer. Real-time qPCR (ABI-7300 Real Time PCR System; Applied Biosystems) was performed in duplicate with a TaqMan gene expression assay, in volumes of 25 μl with 50 ng cDNA and TaqMan assay mix; GAPDH was used as a housekeeping gene and control.
C-reactive protein (CRP) and JNK assay.
Whole blood samples were obtained from RA patients, and PBMCs were isolated via Ficoll-Hypaque centrifugation. Isolated PBMCs were fixed and permeabilized prior to flow cytometric analysis to quantify JNK total protein levels ex vivo. Plasma from each blood sample was retained for analysis of CRP by enzyme linked immunosorbent assay (ELISA) according to the instructions of the manufacturer (R&D Systems).
Cells were suspended in phosphate buffered saline with 10% FBS and 0.1% sodium azide. For intracellular protein determinations, cells were fixed with paraformaldehyde, permeabilized (Triton X-100 and Nonidet P40) using Perm/Wash Buffer (BD Biosciences), and labeled with primary antibodies for 24 hours at 0–4°C followed by incubation with fluorescence-labeled secondary antibodies for 1 hour at 0–4°C. The following primary antibodies were used: rabbit anti-JNK (sc-571; Santa Cruz Biotechnology), polyclonal rabbit anti–p-JNK (pT183/pY185) (558268; BD PharMingen), polyclonal rabbit anti-p38 (9212; Cell Signaling Technology), polyclonal rabbit anti-p53 (NB200-171; Novus Biologicals), and polyclonal rabbit anti-p21 (ab7960; Abcam). Fluorescein isothiocyanate–labeled goat anti-rabbit IgG (554020; BD PharMingen) and phycoerythrin-labeled goat anti-rabbit IgG (4050-09; Southern Biotech) were used as secondary antibodies. The following surface stains were used: Pacific Blue–labeled mouse anti-human CD4, Alexa Fluor 700–labeled mouse anti-human CD8 (BD PharMingen), and allophycocyanin–labeled mouse anti-human CD19 (558116, 557945, and 555335, respectively; all from BD PharMingen). Apoptosis was assessed using a PE Annexin-V Apoptosis Detection Kit I (BD PharMingen). Cells were analyzed using a 3-laser BD LSRII flow cytometer at the Vanderbilt Medical Center Flow Core or a BD FACSCanto II at the Hershey Medical Center Flow Cytometry Core.
Plasmids and cell transfection.
JNK-1 (MAPK-8) and JNK-2 (MAPK-9) dominant-negative (DN) mutants were from the laboratory of Dr. Roger J. Davis (University of Massachusetts Medical School, Worcester, MA). Plasmids (obtained through the Addgene repository) were transfected into Jurkat T cells using a Cell Line Nucleofector Kit V according to the instructions of the manufacturer (Amaxa).
The significance of the differences between groups was determined by Student's t-test. P values less than 0.05 were considered significant.
Reduced MAPK9 expression in vivo in RA.
Previously, we demonstrated that MTX stimulates increased JNK activity and expression levels in tissue culture models (20). Therefore, we sought to determine transcript levels of MAPK family members in RA and control subjects by analyzing gene expression levels in blood samples collected in PAXgene tubes. Expression levels, determined by real-time qPCR, were calculated as the ratio to GAPDH expression levels, for normalization. The genes MAPK8 and MAPK9 correspond to JNK-1 and JNK-2 proteins, respectively. Compared with controls, RA patients exhibited significantly lower levels of MAPK9 messenger RNA (mRNA). Transcript levels of other MAPKs, including genes encoding p38 and ERK proteins, were not significantly different between the RA and control cohorts (data available from the corresponding author upon request).
We next measured JNK expression levels by flow cytometry. JNK total protein levels were diminished in CD4+ and CD8+ T cells, as well as CD19+ B cells, from RA patients not receiving MTX, relative to levels in control subjects (Figure 1A). In contrast, levels of p38 protein, another member of the MAPK family that is often activated by a variety of different cellular stresses including inflammatory cytokines, ultraviolet exposure, and growth factors (26, 27), were not significantly different between the RA and control groups in any of the lymphocyte subsets examined.
Inverse correlation between JNK expression levels and CRP levels in vivo.
CRP, found in plasma, is a common marker of inflammation that is often used as a marker to gauge disease activity or therapeutic efficacy in RA (28, 29). Building upon our finding that JNK levels were diminished in RA patients not taking MTX, we sought to better understand the relationship between JNK and inflammation. Plasma and PBMCs were isolated from RA patients not currently receiving MTX therapy. JNK levels were determined by flow cytometry, and CRP levels were measured by ELISA. We found an inverse correlation between lymphocyte JNK levels and CRP concentrations in vivo (Figure 1B). Thus, independent of MTX therapy, low JNK levels were associated with higher levels of inflammation and high JNK levels were associated with lower levels of inflammation in RA.
MTX therapy restores MAPK9 expression in vivo.
We next determined levels of expression of MAPK9 in RA patients who were and those who were not currently receiving MTX, compared with control subjects. MAPK9 levels were significantly higher in patients taking MTX than in those not taking MTX (Figure 1C). However, the mean level of MAPK9 expression was still lower in the MTX-treated RA group than in the control group. Given these results, we analyzed blood samples from patients with other autoimmune diseases to determine if MAPK9 deficiency was unique to RA. Blood from patients with MS, a neurologic autoimmune disease, also exhibited decreased transcript levels of MAPK9 relative to controls (Figure 1D), whereas MAPK9 transcript levels were not reduced in samples from patients with ulcerative colitis, Crohn's disease, or systemic lupus erythematosus. Thus, we conclude that MAPK9 deficiency exists in MS but is not a general feature of all human autoimmune diseases.
MTX restores TP53, CDKN1A, and CDKN1B levels in vivo.
We next examined expression of genes encoding proteins necessary for cell cycle checkpoint arrest, including TP53 (p53), CDKN1B (p27), CDKN1A (p21), CHEK2 (CHK-2), and RANGAP1 Ran GTPase-activating protein 1 [RANGAP-1]). Transcript levels of each gene were significantly underexpressed in lymphocytes from RA patients not currently taking MTX compared with controls (Figure 2). We then compared expression levels of these additional genes in RA patients who were versus those who were not currently receiving MTX, in relation to levels in controls. As with MAPK9 deficiency, we found that TP53, CDKN1A, and CDKN1B levels were significantly higher in patients who were currently receiving MTX compared with patients who were not, but the mean level was not restored to that in the control population. In contrast to expression levels of TP53, CDKN1A, and CDKN1B, there were no differences in transcript levels of either CHEK2 or RANGAP1 in the RA group with versus the RA group without MTX therapy. These results suggest that MTX may restore deficiencies in expression of cell cycle checkpoint proteins, p53, p21, and p27, but not deficiencies in expression of cell cycle checkpoint proteins CHK-2 and RANGAP-1.
Induction of TP53 and CDKN1A by MTX in Jurkat T cells.
Given the finding that expression levels of TP53, CDKN1A, and CDKN1B were elevated in RA patients taking MTX compared to those not taking MTX but levels of CHEK2 and RANGAP1 were not, we sought to better understand the mechanistic basis of these differences. Previously, we showed that sub-micromolar concentrations of MTX induce transcription of jun in a homogeneous T cell population (Jurkat T cells) by DHFR-dependent depletion of BH4, resulting in uncoupling of NOS and corresponding increased ROS production and JNK activation (20). Therefore, we used this model system to investigate MTX induction of TP53, CDKN1A, RANGAP1, and CHEK2. In these expression studies, we found that MTX directly stimulated an increase in levels of TP53 and CDKN1A transcript (Figure 3A). In contrast, expression levels of CHEK2 and RANGAP1 were not significantly altered by MTX.
Since MTX stimulates adenosine release and activation of adenosine receptors, we investigated whether the broad-spectrum adenosine receptor antagonists caffeine and theophylline, at pharmacologic concentrations, significantly altered the transcriptional profile of these genes. Treatment with caffeine, theophylline, or the combination did not alter induction of TP53 or CDKN1A transcripts by MTX (Figure 3B). In contrast, supplementation with either the free radical scavenger NAC or folic acid prevented induction of TP53 and CDKN1A by MTX (Figure 3C). NAC supplementation of MTX-treated cultures prevents production of ROS, activation of JNK, and induction of jun, a JNK target gene (20). Folic acid at a very high concentration (100 μM) also increases intracellular BH4 levels through its active form, 5-methyltetrahydrofolate, and restores the amount of available nitric oxide in the cell (30, 31).
In addition, we treated Jurkat cells with MTX and assayed intracellular protein concentrations by flow cytometry. MTX treatment significantly increased levels of JNK, p-JNK, p53, and p21, and supplementation with BH4 significantly reduced expression of these proteins (Figure 3D).
MTX alters the transcriptional profile of cells by increasing expression of a number of genes that encode proteins with proapoptotic function (20). This shift depends upon ROS-stimulated phosphorylation of JNK. Therefore, we performed transient transfection experiments with JNK-1– and JNK-2–DN mutants to determine if either JNK-1 or JNK-2 activity was necessary for MTX-mediated induction of p53 or p21. We found that the JNK-1–DN mutant reduced MTX-dependent p21 expression while the JNK-2–DN mutant reduced MTX-dependent p53 expression (Figure 3E). We therefore conclude that induction of p53 by MTX is mediated by JNK-2, whereas induction of p21 by MTX is mediated by JNK-1.
BH4 supplementation blocks MTX-mediated JNK induction, ROS production, and apoptosis priming in activated T cells.
MTX increases the sensitivity of Jurkat cells to apoptosis via a JNK-dependent pathway (20). In the present study, isolated PBMCs were stimulated with anti-CD3 for 72 hours and treated with MTX (0.1 μM) for an additional 24 hours. Cultures were supplemented with interleukin-2 to promote T cell proliferation. Changes in apoptosis were determined by flow cytometry after cells were labeled with annexin V. Mitogen-activated T cells exhibited slightly increased sensitivity to apoptosis in response to MTX alone, but this was enhanced by exposure to anti-Fas, similar to observations in Jurkat cells (Figure 4A). Supplementation of cell cultures with BH4 (30 μM) prevented MTX plus anti-Fas–mediated increases in sensitivity to apoptosis.
Another form of apoptosis induced in activated T cells occurs via T cell receptor stimulation and is often referred to as activation-induced cell death (AICD) (32). We cultured activated T cells with MTX for 24 hours, followed by stimulation with anti-CD3. Apoptosis was measured by annexin V labeling after an additional 24 hours of incubation. Treatment with MTX markedly increased the level of AICD in activated T cells (Figure 4B). We also measured JNK expression levels in MTX-treated activated T cells by flow cytometry. As in Jurkat cells, JNK expression levels were increased following stimulation with MTX (Figure 4C). The increase in JNK levels was prevented by supplementation with BH4. We next investigated MTX-stimulated ROS production in activated T cells, by flow cytometry using CM–H2DCF-DA dye. MTX induced production of ROS in activated T cells (Figure 4D), and ROS production by activated T cells was also inhibited by supplementation with BH4.
Our results support the notion that MTX inhibition of DHFR depletes intracellular stores of BH4 in activated T cells, increasing ROS production and leading to JNK activation and alteration in sensitivity to apoptosis. Further, our findings suggest that addition of BH4 reverses these MTX-mediated effects.
In RA, T cells exhibit functional defects in cell survival, DNA damage responses, and apoptosis (15, 33–35). MAPKs play key roles in these fundamental cellular processes (36), and our recent work demonstrates that MTX, one of the most frequently prescribed pharmacologic agents in RA, activates JNK and the prototypical downstream targets c-Jun and c-Fos (components of the activator protein 1 complex) and increases sensitivity of cells to apoptosis, raising the question of whether RA T cells may have deficiencies in JNK or other MAPKs (20). Our present results demonstrate that levels of MAPK9 transcript, but not transcript for other MAPK genes, and of JNK protein in lymphocytes are markedly reduced in RA patients not receiving MTX therapy. MAPK9 transcript levels in RA patients treated with once-weekly MTX are increased compared with those in non–MTX-treated patients.
Transcript levels of other genes encoding proteins critical to DNA damage–induced cell cycle checkpoint arrest, i.e., TP53, CDKN1A, CDKN1B, RANGAP1, and CHEK2, were also diminished in RA patients. Expression levels of TP53, CDKN1A, and, CDKN1B, but not of CHEK2 or RANGAP1, were significantly lower in non–MTX-treated patients than in those receiving MTX. Our results are consistent with a model whereby defects are present at each checkpoint along the cell cycle, i.e., G1: CDKN1B (p27), S: CDKN1A (p21), G2: CHEK2 (CHK-2), and M: RANGAP1 (RANGAP1) (37–40) and RA patient T cells respond to MTX therapy by restoring a portion, but not all, of these cell cycle checkpoints (Figure 5). In a cell model, MTX induced expression of JNK protein and subsequent induction of p21 and p53 transcripts and protein through JNK-1– and JNK-2–mediated pathways, respectively. MTX failed to increase RANGAP1 and CHEK2 transcripts. Further, MTX stimulated activated T cells to increase ROS production and JNK protein levels, and to increase apoptosis sensitivity via a mechanism that could be reversed by BH4.
The general view is that checkpoints at each stage of the cell cycle exist to maintain fidelity of the genome through the process of DNA replication and cell division. Loss of genomic integrity in hematopoietic cells in RA may arise from combined defects in DNA repair machinery, e.g., ATM, MRE11, NBS1, and RAD50, and defects in expression of proteins required to establish each cell cycle checkpoint. One hypothesis is that deficient DNA repair machinery exerts replicative stress upon T cells in RA, increasing apoptosis and promoting proliferation and selection of autoreactive T cells in response to lymphopenia (33). Failure of cell cycle checkpoints may also contribute to RA via multiple mechanisms, including loss of immunologic tolerance to self antigen, significant enhancement of T cell activation in response to external stimuli, or maintenance of effector cells in a proliferative state for excessive periods of time. Defects in cell cycle arrest may also produce a proinflammatory state. For example, loss of genomic integrity leads to activation of the transcription factor NF-κB via an ATM-dependent mechanism (41). Deficiencies in p53 may also contribute to NF-κB activation (42). Thus, these defects in cell cycle checkpoints and repair of DNA damage may be critical for establishment and maintenance of the chronic inflammation that is responsible for much of the pathogenesis of RA.
The therapeutic efficacy of MTX may arise, in part, via its ability to restore JNK and p53 pathways, leading to apoptosis of activated RA T cells in the setting of pervasive DNA damage. In addition, MTX may promote autoreactive T cells to undergo apoptosis upon secondary stimulation with self antigen. Further, MTX treatment shifts the cytokine balance from a proinflammatory state to an antiinflammatory state. Depletion of BH4 in activated T cells via inhibition of guanosine triphosphate cyclohydrolase 1, the rate-limiting enzyme in the synthesis of BH4, similarly shifts the cytokine balance from a proinflammatory to an antiinflammatory state by uncoupling inducible NOS (43). Thus, MTX may exert multiple effects on T cells to reduce the inflammatory state in RA.
Synovial fibroblast-like cells, or synoviocytes, in RA also exhibit defects in p53 function and in cell cycle control. Additionally, synoviocytes contribute to extracellular matrix destruction and joint and cartilage destruction in RA (44, 45). Thus, MTX may also act on RA synoviocytes to restore p53 function and cell cycle control, thus reducing synovial hyperplasia and its manifestations. Future studies are planned to test this hypothesis.
Defects in cell cycle control and apoptosis play well-established roles in malignancy. An emerging view is that defects in these pathways have broader ramifications for human disease. For example, in lymphocytes from patients with MS, defects in ATM–CHK-2–p53 that confer an inability to properly undergo apoptosis due to underexpression of ATM and impaired ability to stabilize p53 have been identified; these may contribute to perpetuation and progression of this disease (46). Our finding of decreased MAPK9 mRNA expression in MS represents an additional connection between RA and MS. In the broad sense, defects in DNA damage responses have been linked to infertility, cardiovascular disease, and metabolic syndrome (47), and indeed, patients with RA frequently also have inflammation-associated coronary artery atherosclerosis and increased insulin resistance, which may lead to increased mortality (48, 49).
MTX, the standard of care in the treatment of RA, restores a portion of these defects in cell cycle checkpoints and DNA damage responses in RA. However, as our data show, it still falls short of correcting the DNA damage response pathway and normalizing cell cycle checkpoint deficiencies. These results highlight the need for additional pharmacologic agents to specifically target these cell cycle checkpoints.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Aune had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Spurlock, Tossberg, Olsen, Aune.
Acquisition of data. Spurlock, Tossberg, Fuchs, Olsen.
Analysis and interpretation of data. Spurlock, Tossberg, Olsen, Aune.
Author Tossberg is an employee of ArthroChip.
We thank Carl McAloose (Penn State Milton S. Hershey Medical Center) for technical assistance, and personnel at the Clinical Trials Center at Vanderbilt University Medical Center for assistance with collection of patient samples.