Vanessa G. Cuellar

Diagnostic Utility of Cytokine Biomarkers in the Evaluation of Acute Knee Pain

BACKGROUND The diagnosis of clinically important meniscal tears of the knee remains challenging, and it is unknown why only some injuries become painful. The role of inflammatory cytokines in generating pain following meniscal injury remains unclear. This study aimed to investigate the cytokine profile in patients with acute knee pain believed to be secondary to meniscal damage. METHODS This prospective cohort study included thirty-two patients without rheumatoid arthritis who had knee pain for less than six months, with either an acute or insidious onset, and elected to have arthroscopic treatment after nonoperative management had failed. Twenty-three of these patients elected to have the contralateral, nonoperatively treated knee lavaged at the time of arthroscopy. Fifteen asymptomatic control subjects also contributed samples of knee joint fluid, for a total of seventy samples from forty-seven subjects. Lavage of the operatively treated, contralateral, and control knees was performed with the patient under regional anesthesia prior to arthroscopy, if applicable, by the infusion of sterile saline solution into the knee followed by the immediate withdrawal into a syringe. The concentrations of seventeen inflammatory cytokines and chemokines were measured with use of a multiplexed immunoassay panel. Preoperative magnetic resonance imaging findings and cytokine assay results were compared with intraoperative findings. RESULTS Multivariate analysis of variance detected significantly greater concentrations of interferon gamma (IFN-gamma); interleukins 2, 4, 6, 10, and 13 (IL-2, IL-4, IL-6, IL-10, and IL-13); monocyte chemotactic protein-1 (MCP-1); and macrophage inflammatory protein-1 beta (MIP-1beta) in fluid samples from painful knees than in samples from nonpainful knees. Correlation analysis demonstrated a significant positive correlation between patient-reported pain scores and concentrations of IL-6 (Spearman rho = 0.7), MCP-1 (rho = 0.8), MIP-1beta (rho = 0.6), and IFN-gamma (rho = 0.6). These four cytokines also demonstrated a positive correlation with each other (rho = 0.5 to 0.7). The presence of IFN-gamma, IL-6, MCP-1, or MIP-1beta performed as well as magnetic resonance imaging in the prediction of intraoperative findings. CONCLUSIONS Intra-articular concentrations of four inflammatory cytokines IFN-gamma, IL-6, MCP-1, and MIP-1beta correlated to pain in patients with symptomatic meniscal tears in the knee but were markedly lower in asymptomatic normal knees and in asymptomatic knees with meniscal tears. These cytokines may be involved in the generation of pain following meniscal injury.

Cytokine Profiling in Acute Anterior Cruciate Ligament Injury

PURPOSE To evaluate the presence and relative concentrations of cytokines, known to be involved in the inflammatory cascade, in acute anterior cruciate ligament (ACL) injury. METHODS We evaluated an extensive cytokine profile in synovial fluid from 12 patients with acute ACL injury undergoing arthroscopy compared with 15 control subjects using a BioPlex assay (Bio-Rad Laboratories, Hercules, CA) to measure the concentration of 17 inflammatory cytokines. RESULTS In patients with acute ACL injury compared with asymptomatic control subjects, the following cytokines were identified at significantly increased concentrations (P < .001, Mann-Whitney U test) compared with control samples: interleukin 6 (105 ± 72 v 0 ± 0 pg/ml), interferon γ (1,544 ± 608 v 9 ± 7.5 pg/ml), macrophage inflammatory protein 1β (16 ± 3.8 v 0.3 ± 0.2 pg/ml), and monocyte chemotactic protein 1 (35 ± 13 v 0.5 ± 0.4 pg/ml). There was no case of a cytokine exhibiting increased levels in asymptomatic compared with symptomatic knee samples. CONCLUSIONS This investigation identified 4 specific cytokines (interleukin 6, interferon γ, monocyte chemotactic protein 1, and macrophage inflammatory protein 1β) out of a panel of 17 inflammatory molecules for which the levels were consistently elevated in the context of ACL injury compared with non-painful, non-acutely injured knees in a volunteer population. LEVEL OF EVIDENCE Level IV, prognostic case series.

Cytokine expression in the epidural space: a model of noncompressive disc herniation-induced inflammation.

Study Design. Animal study. Objective. Development of an animal model for the study of biochemical changes that occur in the epidural space after intervertebral disc herniation. Summary of Background Data. Although strong evidence for an inflammatory component exists, the biochemical processes underlying pain after disc herniation remain unknown. Methods. Epidural lavage was performed in 48 rats after L5 dorsal root ganglion exposure at baseline and 3, 6, or 24 hours after placement of autologous nucleus pulposus (NP) (N = 15), saline (N = 15), or NP + an interferon-&ggr; antibody (anti-IFN-&ggr;; N = 18) directly onto the dorsal root ganglion. Multiplex assays quantifying interleukin (IL)-1&agr;, IL-1&bgr;, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor &agr; (TNF-&agr;), IFN-&ggr;, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were performed. NP (N = 7) was also analyzed for these cytokines by placing NP into saline and measuring the relative concentration. Results. Cytokines measured low at baseline (0–100 pg/mL) in all groups. Compared with saline, NP application caused IL-6 elevation, peaking at T = 3 hours, that was prevented by anti-IFN-&ggr;. NP induced elevation of TNF-&agr;, peaking at T = 24 hours and was prevented by anti-IFN-&ggr;. IFN-&ggr; was elevated after NP at T = 3 hours and T = 24 hours. IL-1&agr; was similar after saline versus NP. The concentrations of IL-1&bgr; and IL-10 were elevated at T = 3 hours, 6 hours, and 24 hours in all groups without between-groups difference. The level of IL-4 peaked at T = 3 hours in the NP group and was different than saline and NP + anti-IFN-&ggr; groups, but the time effect was insignificant. There was no change for GM-CSF. The concentration of cytokines measured in normal NP was less than 2 pg/mL for all cytokines except TNF-&agr;. Conclusion. In this model of acute noncompressive disc herniation, NP caused the elevation of epidural IL-6, TNF-&agr;, and IFN-&ggr;—all attenuated by IFN-&ggr; blockade. IL-1&bgr; and IL-10 were both significantly elevated by saline alone and their response was not prevented by IFN-&ggr; blockade. This model may prove useful for the study of the biochemical processes by which NP induces inflammation-induced nerve root irritation and radiculopathic pain.

Epidural Interferon Gamma-Immunoreactivity: A Biomarker for Lumbar Nerve Root Irritation

Study Design. Prospective observational cohort. Objective. Correlate epidural inflammatory cytokines with the clinical response to epidural steroid injection in patients with lumbar nerve root irritation. Summary of Background Data. Some back pain syndromes are thought to be associated with activation of inflammatory pathways and others may be associated with primary mechanical derangements. Human studies providing detailed evidence for the primary inflammatory causation, which may be best treated with anti-inflammatory strategies, are lacking. There are currently no accurate diagnostic tests to predict the response to epidural steroid injection or surgical intervention in back pain and sciatica syndromes. Methods. Forty-seven consecutive patients with lumbar degenerative changes and low back and/or leg pain were prospectively enrolled. An epidural lavage was performed, followed by injection of marcaine/depo-medrol. Subjects scored their pain before and 3 months after the procedure. The immunoreactivity of an array of cytokines was measured in lavage samples and compared with clinical response to the therapeutic injection. Ten subjects underwent repeat epidural lavage sampling 3 months after the steroid injection. Results. Interferon gamma (IFN&ggr;) was the most consistently detected cytokine. IFN&ggr;-immunoreactivity also highly correlated with reported reduction of pain 3-months after the epidural steroid injection. In subjects reporting significant pain relief (>50%) from the injection, mean [IFN&ggr;] was significantly greater compared with patients experiencing no significant relief. The IFN&ggr;-immunoreactivity in repeat lavage samples decreased to trace residual concentrations in patients who reported pain relief from the steroid injection. Conclusion. The presence of epidural IFN&ggr;-immunoreactivity corresponding to >10 pg/mL predicted significant pain relief after epidural steroid injection with >95% accuracy. These results suggest that IFN&ggr; may be part of a biochemical cascade triggering pain in sciatica; IFN&ggr;-immunoreactivity may aid as a biomarker for predicting the response to steroid therapy and/or surgical intervention, and may serve as a future therapeutic target.

Accelerated Degeneration After Failed Cervical and Lumbar Nucleoplasty

Study Design Observational cohort study. Summary of Background Data Studies evaluating the treatment of presumed discogenic spine pain using nucleoplasty have reported variable success rates. It has been suggested that these procedures lower the intradiscal pressure, reduce disk protrusion, improve disk hydration, and restore disk height. It is proposed that such structural changes in treated disks correspond to the clinical improvement in patients. Radiographic and clinical evidence showing the efficacy of nucleoplasty remains inadequate. Objective To document the comparative changes in magnetic resonance imaging (MRI) the appearance of disk morphology as reflected by Pfirrmann classification scores before and after the nucleoplasty treatment in patients with continued symptoms. Methods Twenty-eight consecutive patients with persistent symptoms after nucleoplasty within 1 year of treatment were evaluated. Prenucleoplasty and postnucleoplasty MRIs were evaluated and Pfirrmann scores of the symptomatic level were determined. Results In all the treated patients, comparison between the prenucleoplasty and the postnucleoplasty MRI of the targeted disks failed to show increased signal hydration, disk space height improvement, or shrinkage of the preoperative disk bulge at a mean time of 6 months after the procedure. Of the 17 cervical levels treated in 12 patients, 5 seemed to show progressive degeneration at treated levels (42% of the patients). Of the 17 lumbar procedures in 16 patients, 4 seemed to show progressive degeneration (25% of the patients) and 1 developed a new spondylolisthesis (6.3%). Thus, 32% of the patients in our cohort showed progressive degeneration at the treated level. The median Pfirrmann score in both prenucleoplasty and postnucleoplasty was 2, and the mean Pfirrmann classification prenucleoplasty and postnucleoplasty was 1.8 and 2.1, respectively (P<0.05, 2-tailed t test). Conclusion This study failed to detect any morphologic improvement of disk abnormalities by MRI evaluation in patients with persistent pain, who then underwent nucleoplasty. Thirty-two percent showed progressive degeneration in less than 1 year after nucleoplasty, a rate greater than expected by natural progression during the interval of examination.

Identification of a complex between fibronectin and aggrecan G3 domain in synovial fluid of patients with painful meniscal pathology

OBJECTIVES We previously described a panel of four cytokines biomarkers in knee synovial fluid for acute knee pain associated with meniscal pathology. The cytokine biomarkers included interferon gamma (IFN-gamma), interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1beta). Validation studies using other immunologic techniques confirmed the presence of IL-6, MCP-1 and MIP-1beta, but not IFN-gamma. Therefore we sought the identity of the IFN-gamma signal in synovial fluid. METHODS Knee synovial fluid was collected from patients with an acute, painful meniscal injury, as well as asymptomatic volunteers. A combination of high-pressure chromatography, mass spectrometry and immunological techniques were used to enrich and identify the protein components representing the IFN-gamma signal. RESULTS A protein complex of fibronectin and the aggrecan G3 domain was identified in the synovial fluid of patients with a meniscal tear and pain that was absent in asymptomatic controls. This protein complex correlated to the IFN-gamma signal. A novel enzyme-linked immunosorbent assay (ELISA) was developed to specifically identify the complex in synovial fluid. CONCLUSIONS We have identified a protein complex of fibronectin and aggrecan G3 domain that is a candidate biomarker for pain associated with meniscal injury.

α2-Macroglobulin: Autologous Protease Inhibition Technology

α2-Macroglobulin (A2M) is a plasma glycoprotein best known for its ability to inhibit a broad spectrum of serine, threonine, and metalloproteases as well as inflammatory cytokines by a unique bait-and-trap method. A2M has emerged as a unique potential treatment of cartilage-based pathology and inflammatory arthritides. This article describes the unique method by which A2M not only inhibits the associated inflammatory cascade but also disrupts the catabolic process of cartilage degeneration. Autologous concentrated A2M from plasma is currently in use to successfully treat various painful arthritides. Future directions will focus on recombinant variants that enhance its anti-inflammatory and disease-modifying potential.

Poster 10 Is There a Chondroprotective Effect of Autologous Platelet Integrated Concentrate (APIC) on an Osteoarthritis (OA) Rabbit Model? A Pilot Study

exam, was otherwise normal. He was subsequently sent for radiographs of the pelvis and bilateral (B/L) hips which revealed advanced degenerative joint disease (DJD). On AP view, radiographic measurements of the neck shaft angles were found to be greater than 142°, while lateral radiographs showed an anteverted neck in relation to the femoral shaft. Setting: Outpatient physiatry office. Results or Clinical Course: The radiographic findings, in combination with clinical presentation, helped confirm the diagnosis of B/L pistol grip hip deformity (PGD). Such deformity led to severe progressive DJD, which limited both ROM and the performance of activities of daily living. As a result, the patient underwent B/L total hip arthroplasty (THA) without complication. Discussion: PGD is a pre-arthritic condition with a greater prevalence in adults who are younger than 50 years old and are very physically active. Such patients presenting with groin pain and decreased hip ROM should be evaluated for PGD. The triad of Cam type anterior femoral acetabular impingement, DJD, and a femoral neck-shaft angle 140° or 115° with radiographic evidence of an abnormally shaped femoral head is suggestive of PGD. Significant benefit may arise from early recognition and orthopedic evaluation in terms of diagnosis and treatment. Conclusions: PGD left untreated is progressive and ultimately leads to THA. With early recognition, there is potential for successful treatment via arthroscopy. It is therefore imperative that the rehabilitation physician be familiar with this condition so that such a condition does not go unnoticed.