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Along with cytokines, extracellular vesicles (EVs) released by immune cells in the joint contribute to osteoarthritis (OA) pathogenesis. By high-resolution flow cytometry, we characterized 18 surface markers and 4 proinflammatory cytokines carried by EVs of various sizes in plasma and synovial fluid (SF) from individuals with knee OA, with a primary focus on immune cells that play a major role in OA pathogenesis. By multiplex immunoassay, we also measured concentrations of cytokines within (endo) and outside (exo) EVs. EVs carrying HLA-DR, -DP and -DQ were the most enriched subpopulations in SF relative to plasma (25-50-fold higher depending on size), suggesting a major contribution to the SF EV pool from infiltrating immune cells in OA joints. In contrast, the CD34 + medium and small EVs, reflecting hematopoietic stem cells, progenitor cells, and endothelial cells, were the most significantly enriched subpopulations in plasma relative to SF (7.3- and 7.7-fold higher). Ratios of EVs derived from neutrophils and lymphocytes were highly correlated between SF and plasma, indicating that plasma EVs could reflect OA severity and serve as systemic biomarkers of OA joint pathogenesis. Select subsets of plasma EVs might also provide next generation autologous biological products for intra-articular therapy of OA joints.

Purpose: Elevated synovial fluid (SF) concentrations of proinflammatory cytokines, degradative enzymes, and cartilage breakdown markers at the time of anterior cruciate ligament (ACL) reconstruction are associated with worse postoperative patient-reported outcomes and cartilage quality. However, it remains unclear if this is due to a more robust or dysregulated inflammatory response or is a function of a more severe injury. The objective of this study was to evaluate the association of the molecular composition of the SF, patient demographics, and injury characteristics to cartilage degradation after acute ACL injury., Methods: We performed a cluster analysis of SF concentrations of proinflammatory and anti-inflammatory cytokines, and biomarkers of cartilage degradation, bony remodeling, and hemarthrosis. We evaluated the association of biomarker clusters with patient demographics, days between injury, Visual Analogue Scale pain, SF aspirate volumes, and bone bruise volumes measured on magnetic resonance imaging., Results: Two clusters were identified from the 35 patients included in this analysis, dysregulated inflammation and low inflammation. The dysregulated inflammation cluster consisted of 10 patients and demonstrated significantly greater concentrations of biomarkers of cartilage degradation (P < 0.05) as well as a lower ratio of anti-inflammatory to proinflammatory cytokines (P = 0.053) when compared with the low inflammation cluster. Patient demographics, bone bruise volumes, SF aspirate volumes, pain, and concomitant injuries did not differ between clusters., Conclusions: A subset of patients exhibited dysregulation of the inflammatory response after acute ACL injury which may increase the risk of posttraumatic osteoarthritis. This response does not appear to be a function of injury severity.

Background: To identify a synovial fluid (SF) biomarker profile characteristic of individuals with an inflammatory osteoarthritis (OA) endotype., Methods: A total of 48 knees (of 25 participants) were characterized for an extensive array of SF biomarkers quantified by Rules Based Medicine using the high-sensitivity multiplex immunoassay, Myriad Human InflammationMAP® 1.0, which included 47 different cytokines, chemokines, and growth factors related to inflammation. Multivariable regression with generalized estimating equations (GEE) and false discovery rate (FDR) correction was used to assess associations of SF RBM biomarkers with etarfolatide imaging scores reflecting synovial inflammation; radiographic knee OA severity (based on Kellgren-Lawrence (KL) grade, joint space narrowing, and osteophyte scores); knee joint symptoms; and SF biomarkers associated with activated macrophages and knee OA progression including CD14 and CD163 (shed by activated macrophages) and elastase (shed by activated neutrophils)., Results: Significant associations of SF biomarkers meeting FDR < 0.05 included soluble (s)VCAM-1 and MMP-3 with synovial inflammation (FDR-adjusted p = 0.025 and 1.06 × 10 -7 ); sVCAM-1, sICAM-1, TIMP-1, and VEGF with radiographic OA severity (p = 1.85 × 10 -5 to 3.97 × 10 -4 ); and VEGF, MMP-3, TIMP-1, sICAM-1, sVCAM-1, and MCP-1 with OA symptoms (p = 2.72 × 10 -5 to 0.050). All these SF biomarkers were highly correlated with macrophage markers CD163 and CD14 in SF (r = 0.43 to 0.90, FDR < 0.05); all but MCP-1 were also highly correlated with neutrophil elastase in SF (r = 0.62 to 0.89, FDR < 0.05)., Conclusions: A subset of six SF biomarkers was related to synovial inflammation in OA, as well as radiographic and symptom severity. These six OA-related SF biomarkers were specifically linked to indicators of activated macrophages and neutrophils. These results attest to an inflammatory OA endotype that may serve as the basis for therapeutic targeting of a subset of individuals at high risk for knee OA progression., Trial Registration: Written informed consent was received from participants prior to inclusion in the study; the study was registered at ClinicalTrials.gov ( NCT01237405 ) on November 9, 2010, prior to enrollment of the first participant.

Background: Anterior cruciate ligament tears can lead to posttraumatic osteoarthritis. In addition to biomechanical factors, changes in biochemical profiles within the knee joint after injury and anterior cruciate ligament reconstruction (ACLR) may play a role in accelerating joint degeneration. Hypothesis/Purpose: It was hypothesized that cartilage matrix composition after ACLR is associated with the degree of inflammatory response after initial injury. This study evaluated the association between the inflammatory response after injury-as indicated by cytokine, metalloproteinase, and cartilage degradation marker concentrations in synovial fluid-and articular cartilage degeneration, measured by T1ρ and T2 quantitative magnetic resonance imaging up to 3 years after ACLR., Study Design: Cohort study; Level of evidence, 2., Methods: Twenty-six subjects from a longitudinal cohort study who underwent ACLR at a mean 8.5 weeks after injury (range, 4-19 weeks) had synovial fluid aspirated at the time of surgery. Immunoassays quantified biomarkers in synovial fluid. T1ρ and T2 values of articular cartilage were calculated with magnetic resonance scans acquired prior to surgery and at 6 months and 1, 2, and 3 years after surgery. Pearson correlation coefficients were calculated among the various biomarkers. K-means clustering was used to group subjects with similar biomarker profiles. Generalized estimating equations were used to find the overall differences in T1ρ and T2 values throughout these first 3 years after surgery between the clusters while controlling for other factors., Results: Significant and strong correlations were observed between several cytokines (interleukin 6 [IL-6], IL-8, IL-10, and tumor necrosis factor α) and 2 matrix metalloproteinases (MMP-1 and MMP-3) ( P < .05). Moderate correlations were found among combinations of C-terminal crosslinked telopeptide type II collagen, N-terminal telopeptide, cartilage oligomeric matrix protein, and sulfated glycosaminoglycan ( P < .05). Two clusters were generated, 1 of which was characterized by lower concentrations of cytokines (IL-6, IL-8, IL-10, tumor necrosis factor α) and MMP-1 and MMP-3 and higher sulfated glycosaminoglycan. This cluster was associated with significantly higher T1ρ and T2 values in the medial tibial and patellar cartilage over the first 3 years after ACLR., Conclusion: At the time of ACLR surgery, profiles of synovial fluid inflammatory cytokines, degradative enzymes, and cartilage breakdown products show promise as predictors of abnormal cartilage tissue integrity (increased T1ρ and T2 values) throughout the first 3 years after surgery., Clinical Relevance: The results suggest an intricate relationship between inflammation and cartilage turnover, which can in turn be influenced by timing after injury and patient factors.

Background: The study objective was to examine the effect of time and fracture severity on the undiluted synovial fluid (SF) microenvironment during the acute phase following intra-articular fracture (IAF) of the human ankle., Methods: Ankle SF from 54 patients with an acute IAF was analyzed for concentrations of 10 cytokines, 5 matrix metalloproteinases, 2 products of cartilage catabolism, and combined products of heme metabolism. All analytes were correlated with time from fracture and further analyzed for an effect of 3 time subgroups (0-2 days, 3-9 days, and ≥10 days) corresponding to timepoints for clinical ankle fracture interventions. The effect of fracture severity was determined by grouping SF according to the number of radiographic intra-articular fracture lines., Results: Fifteen of 18 analytes were significantly correlated with time. Temporal grouping of SF revealed an initial (0-2 days) spike of pro-inflammatory (IL-12p70, IL-1β, IL-6) and anti-inflammatory (IL-10 and IL-4) cytokines, matrix metalloproteinases (MMP) MMP-9, and sGAG, followed immediately (3-9 days) by products of heme metabolism and an unchallenged surge in mediators and products of cartilage catabolism (MMP-1, MMP-2, MMP-3, MMP-10, and CTX-II). After 10 days, there was a decrease in pro- and anti-inflammatory cytokines but a persistence of mediators of ECM catabolism. There was no clear relationship between the number of fracture lines and SF levels of analytes., Conclusions: This study demonstrated acute temporal fluctuations following ankle IAF resulting in an overall catabolic environment by 10 days post-fracture and supports consideration of an early evacuation of the joint space to reduce the intra-articular inflammatory burden. Clinical Relavence: This study contributes to the understanding of the intra-articular events that potentially contribute to the development of posttraumatic osteoarthritis acutely following IAF in the ankle.

Purpose: Meniscus tears are a common knee injury and are associated with the development of post-traumatic osteoarthritis (OA). The purpose of this study is to evaluate potential OA mediators in the synovial fluid and serum of meniscus tear subjects compared to those in the synovial fluid of radiographic non-OA control knees., Materials and Methods: Sixteen subjects with an isolated unilateral meniscus injury and six subjects who served as reference controls (knee Kellgren-Lawrence grade 0-1) were recruited. Twenty-one biomarkers were measured in serum from meniscus tear subjects and in synovial fluid from both groups. Meniscus tear subjects were further stratified by tear type to assess differences in biomarker levels., Results: Synovial fluid total matrix metalloproteinase (MMP) activity and prostaglandin E2 (PGE2) were increased 25-fold and 290-fold, respectively, in meniscus tear subjects as compared to reference controls (p < 0.05). Synovial fluid MMP activity and PGE2 concentrations were positively correlated in meniscus tear subjects (R = 0.83, p < 0.0001). In meniscus tear subjects, synovial fluid levels of MMP activity, MMP-2, MMP-3, sGAG, COMP, IL-6, and PGE2 were higher than serum levels (p < 0.05). Subjects with complex meniscus tears had higher synovial fluid MMP-10 (p < 0.05) and reduced serum TNFα and IL-8 (p < 0.05) compared to other tear types., Conclusions: Given the degradative and pro-inflammatory roles of MMP activity and PGE2, these molecules may alter the biochemical environment of the joint. Our findings suggest that modulation of PGE2 signaling, MMP activity, or both following a meniscus injury may be targets to promote meniscus repair and prevent OA development.

Background: Posttraumatic osteoarthritis (PTOA) can occur after intra-articular fracture despite anatomic fracture reduction. It has been hypothesized that an early inflammatory response after intra-articular injury could lead to irreversible cartilage damage that progresses to PTOA. Therefore, in addition to meticulous fracture reduction, it would be ideal to prevent this initial inflammatory response but little is known about the composition of the synovial environment after intra-articular fracture. The purpose of this work was to characterize the inflammatory cytokine and matrix metalloproteinase (MMP) composition in the synovial fluid (SF) of patients with acute intra-articular ankle fractures., Methods: Twenty-one patients with an intra-articular ankle fracture were included in this study. All patients had a contralateral ankle joint that was pain free, had no radiographic evidence of arthritis, and no history of trauma. The uninjured ankle served as a matched control. SF was obtained from bilateral ankles at the time of surgery which occurred at a mean of 17 days post-fracture (range 8-40). The SF was analyzed for granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, CTXII, sGAG, and bilirubin/biliverdin (markers of hemearthrosis) using either multiplex assay or ELISA using commercially available kits. Mean concentrations of each factor were compared between SF from fractured and control ankles, and correlation analysis was done to determine potential relationships between levels of cytokines and time from fracture and age at fracture., Results: Twelve of 18 measured factors including GM-CSF, IL-10, IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, and bilirubin/biliverdin were found to be significantly higher in the fractured ankles. Mean concentrations of ECM degradation markers (sGAG and CTXII) were not found to be significatnly different between groups., Conclusion: These data indicate that after intra-articular ankle fracture the SF exhibits a largely pro-inflammatory and extra-cellular matrix degrading environment similar to that described in idiopathic osteoarthritis. IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, and MMP-10 were significantly elevated and may play a role in the development of PTOA., Clinical Relevance: In addition to anatomic fracture reduction, these data lend credence to reducing acute intra-articular inflammation through the development of antagonists to these pro-inflammatory and degrading mediators. Likewise, intra-articular lavage might reduce this inflammatory burden., (© The Author(s) 2015.)

Objective: The inflammatory response following an articular fracture is thought to play a role in the development of posttraumatic arthritis (PTA) but has not been well characterized. The objective of this study was to characterize the acute inflammatory response, both locally and systemically, in joint synovium, synovial fluid (SF), and serum following articular fracture of the ankle. We hypothesized that intraarticular fracture would alter the synovial environment and lead to increased local and systemic inflammation., Methods: Synovial tissue biopsy specimens, SF samples, and serum samples were collected from patients with an acute articular ankle fracture (n = 6). Additional samples (normal, ankle osteoarthritis [OA], and knee OA [n = 6 per group]) were included for comparative analyses. Synovial tissue was assessed for synovitis and macrophage count. SF and serum were assessed for cytokines (interferon-γ [IFNγ], interleukin-1β [IL-1β], IL-6, IL-8, IL-10, IL-12p70, and tumor necrosis factor α) and chemokines (eotaxin, eotaxin 3, IFNγ-inducible 10-kd protein, monocyte chemotactic protein 1 [MCP-1], MCP-4, macrophage-derived chemokine, macrophage inflammatory protein 1β, and thymus and activation-regulated chemokine)., Results: Synovitis scores were significantly higher in ankle fracture tissue compared with normal ankle tissue (P = 0.007), and there was a trend toward an increased abundance of CD68+ macrophages in ankle fracture synovium compared with normal knee synovium (P = 0.06). The concentrations of all cytokines and chemokines were elevated in the SF of patients with ankle fracture compared with those in SF from OA patients with no history of trauma. Only the concentration of IL-6 was significantly increased in the serum of patients with ankle fracture compared with normal serum (P = 0.027)., Conclusion: Articular fracture of the ankle increased acute local inflammation, as indicated by increased synovitis, increased macrophage infiltration into synovial tissue, and increased SF concentrations of biomarkers of inflammation. Characterizing the acute response to articular fracture provides insight into the healing process and may help to identify patients who may be at greater risk of PTA., (© 2015, American College of Rheumatology.)

Objective: To develop a method to correct for the unknown dilution of synovial fluid that occurs during lavage of a joint, we evaluated the utility of urea, a molecule that is neither synthesized nor metabolized by joint tissues, as a means of correcting for the dilutional effects of lavage procedures and effusions., Methods: Joint fluids were obtained from normal canine joints by direct aspiration (n = 41) and lavage (n = 10). Acute joint injury was induced in 4 joints by intraarticular injection of chymopapain. Serum and joint fluid levels of urea and joint fluid concentrations of glucose, lactate, cartilage oligomeric matrix protein (COMP), and keratan sulfate (KS) were measured in these 55 joints., Results: Urea concentrations in joint fluid were directly proportional to those in serum throughout a wide range of concentrations in normal joints. From this relationship, the dilution factor introduced by joint lavage was determined. This method was applied to quantify biomarker concentrations in synovial lavage fluid and was found to successfully correct for lavage-induced dilution of glucose, lactate, COMP, and KS to levels equivalent to those in samples aspirated directly. In the context of chymopapain-induced joint effusion, urea concentrations continued to be proportional to serum concentrations, but were much lower, enabling an estimation of the change in the volume of distribution (V(d)) of a marker due to a change in joint water content in the setting of inflammation characterized by effusion. Lactate and KS levels rose markedly in response to chymopapain. After adjustment for the V(d), the glucose concentration in the chymopapain-injected joints did not change., Conclusion: Urea provides a robust method of quantifying and correcting for the dilution of synovial fluid due to joint lavage or inflammation. This method is potentially applicable to surrogate marker studies in human arthritis.

The inflammatory response to joint injury, specifically intra‐articular fracture, has been implicated in posttraumatic arthritis development. However, the role of T cells in regulating the development of posttraumatic arthritis is unclear. We hypothesized that the absence of T cells would lead to less severe posttraumatic arthritis following intra‐articular fracture. T cell‐deficient, athymic nude, and wild‐type C57BL/6NJ mice were assessed at 8 weeks following closed articular fracture. Joints were assessed using histologic scores of arthritis, synovitis, and bone morphology via micro computed tomography. Cells were profiled in whole blood via flow cytometry, and plasma and synovial fluid derived cytokines were quantified by multiplex analysis. Compared to C57BL/6NJ mice, nude mice had significantly greater histologic evidence of arthritis and synovitis. Whole blood immune cell profiling revealed a lower percentage of dendritic cells but increased natural killer (NK) cells in nude mice. Concurrently, nude mice had significantly higher levels of NK cells in synovial tissue. Concentrations of plasma interleukin 1β (IL‐1β) and tumor necrosis factor α, and synovial fluid IL‐12, IL‐17, and IL‐6 in both knees were greater in nude mice. Outcomes of this study suggest that T cells may play a protective regulatory role against the development of posttraumatic arthritis. Clinical significance: Lack of functional T cells exacerbated the development of posttraumatic arthritis following intra‐articular fracture suggesting that critical regulators of the immune responses, contained within the T cell population, are required for protection. Future research identifying the specific T cell subsets responsible for modulating disease immunopathogenesis will lead to new therapeutic targets to mitigate posttraumatic arthritis. [ABSTRACT FROM AUTHOR]

Background.Themajority of patients develop posttraumatic osteoarthritiswithin 15 years of anterior cruciate ligament (ACL) injury. Inflammatory and chondrodegenerative biomarkers have been associated with both pain and the progression of osteoarthritis; however, it remains unclear if preoperative biomarkers differ for patientswith inferior postoperative outcomes.Hypothesis/Purpose. Thepurpose of this pilot studywas to compare biomarkers collected on the day ofACL reconstruction between patientswith "good" or "poor" 2-year postoperative outcomes. We hypothesized that inflammatory cytokines and chondrodegenerative biomarker concentrations would be significantly greater in patients with poorer outcomes. Study Design. Prospective cohort design. Methods. 22 patients (9 females, 13males; age = 19.5 ±4.1 years; BMI = 24.1±3.6 kg/m²) previously enrolled in a randomized trial evaluating early anti-inflammatory treatment after ACL injury. Biomarkers of chondrodegeneration and inflammation were assessed from synovial fluid (sf) samples collected on the day of ACL reconstruction. Participants completed Knee Injury and Osteoarthritis Outcome Score (KOOS) and International Knee Documentation Committee (IKDC) questionnaires two years following surgery. Patients were then categorized based on whether their KOOS Quality of Life (QOL) score surpassed the Patient Acceptable Symptom State (PASS) threshold of 62.5 points or the IKDC PASS threshold of 75.9 points. Results. Patients that failed to reach the QOL PASS threshold after surgery (n = 6, 27%) had significantly greater sf interleukin-1 alpha (IL-1α; p = 0.004), IL-1 receptor antagonist (IL-1ra; p = 0.03), and matrix metalloproteinase-9 (MMP-9; p = 0.01) concentrations on the day of surgery. Patients that failed to reach the IKDC PASS threshold (n = 9, 41%) had significantly greater sf IL-1α (p = 0.02). Conclusion. These pilot data suggest that initial biochemical changes after injury may be an indicator of poor outcomes that are not mitigated by surgical stabilization alone. Biological adjuvant treatment in addition to ACL reconstruction may be beneficial; however, these data should be used for hypothesis generation and more definitive randomized clinical trials are necessary. [ABSTRACT FROM AUTHOR]

Background: Post-traumatic osteoarthritis (PTOA) is responsible for the majority of cases of ankle arthritis. While acute and end-stage intra-articular inflammation has previously been described, the state of the joint between fracture healing and end-stage PTOA remains undefined. This study characterized synovial fluid (SF) composition of ankles after bone healing of an intra-articular fracture to identify factors that may contribute to the development of PTOA. Methods: Of an original 21 patients whose SF was characterized acutely following intra-articular ankle fractures, 7 returned for planned hardware (syndesmotic screw) removal after bone healing (approximately 6 months) and consented to a second bilateral SF collection. SF concentrations of 15 cytokines and matrix metalloproteinases (MMPs) and 2 markers each of cartilage catabolism (CTXII and glycosaminoglycan) and hemarthrosis (biliverdin and bilirubin) were compared for previously fractured and contralateral, uninjured ankles from the same patient. Analysis was also performed to determine the effect of the number of fracture lines and involvement of soft tissue on SF composition. Results: Interleukin (IL)-6, IL-8, MMP-1, MMP-2, and MMP-3 were significantly elevated in the SF from healed ankles compared to matched contralateral uninjured ankles at approximately 6 months after fracture. There were no differences in markers of cartilage catabolism or hemarthrosis. Only IL-1α was affected by the number of fracture lines while differences were not detected for other analytes or with respect to the involvment of soft tissue. Conclusions: Sustained intra-articular inflammation, even after complete bone healing, was suggested by elevations of pro-inflammatory cytokines (IL-6 and IL-8). In addition, elevated concentrations of MMPs were also noted and were consistent with a persistent inflammatory environment. This study suggests new evidence of persistent intra-articular inflammation after intra-articular ankle fracture healing and suggests potential mediators for PTOA development. Clinical Relevance: This work may be relevant to the clinical diagnosis and treatment of post-traumatic osteoarthritis. [ABSTRACT FROM AUTHOR]

Background: It is increasingly recognized that biochemical abnormalities of the joint precede radiographic abnormalities of posttraumatic osteoarthritis (PTOA) by as much as decades. A growing body of evidence strongly suggests that the progression from anterior cruciate ligament (ACL) injury to PTOA is multifactorial, involving the interplay between biomechanical disturbances and biochemical homeostasis of articular cartilage. Purpose: The purposes of this randomized study using an acute ACL injury model were to (1) evaluate the natural progression of inflammatory and chondrodegenerative biomarkers, (2) evaluate the relationship between subjective reports of pain and inflammatory and chondrodegenerative biomarkers, and (3) determine if postinjury arthrocentesis and corticosteroid injection offer the ability to alter this biochemical cascade. Study Design: Randomized controlled trial; Level of evidence, 2. Methods: A total of 49 patients were randomized to 4 groups: group 1 (corticosteroid at 4 days after ACL injury, placebo injection of saline at 2 weeks), group 2 (placebo at 4 days after ACL injury, corticosteroid at 2 weeks), group 3 (corticosteroid at both time intervals), or a placebo group (saline injections at both time intervals). Patient-reported outcome measures and synovial biomarkers were collected at approximately 4 days, 11 days, and 5 weeks after injury. The change between the time points was assessed for all variables using Wilcoxon tests, and the relationship between changes in outcome scores and biomarkers were assessed by calculating Spearman ρ. Outcomes and biomarkers were also compared between the 4 groups using Kruskal-Wallis tests. Results: No adverse events or infections were observed in any study patients. With the exception of matrix metalloproteinase 1 (MMP-1) and tumor necrosis factor-inducible gene 6 (TSG-6), chondrodegenerative markers worsened over the first 5 weeks while all patient-reported outcomes improved during this time, regardless of treatment group. Patient-reported outcomes did not differ between patients receiving corticosteroid injections and the placebo group. However, increases in C-telopeptide of type II collagen (CTX-II), associated with collagen type II breakdown, were significantly greater in the placebo group (1.32 ± 1.10 ng/mL) than in either of the groups that received the corticosteroid injection within the first several days after injury (group 1: 0.23 ± 0.27 ng/mL [P = .01]; group 3: 0.19 ± 0.34 ng/mL [P = .01]). Conclusion: PTOA begins at the time of injury and results early on in dramatic matrix changes in the knee. However, it is encouraging that early intervention with an anti-inflammatory agent was able to affect biomarkers of chondral degeneration. Should early intervention lead to meaningful changes in either the onset or severity of symptomatic PTOA, the current treatment paradigm for patients with ACL injury may have to be restructured to include early aspiration and intra-articular intervention. Trial Registration: ClinicalTrials.gov identifier: NCT01692756. [ABSTRACT FROM AUTHOR]