A2M inhibits inflammatory mediators of chondrocytes by blocking IL-1ß/NF-κB pathway.

A hallmark of osteoarthritis (OA) is cartilage degeneration, which has been previously correlated with dramatic increases in inflammatory enzymes. Specifically, interleukin-1ß (IL-1ß) and subsequent upregulation of nuclear factor kappa B (NF-κB) is implicated as an important player in the development of posttraumatic osteoarthritis (PTOA). Alpha 2-macroglobulin (A2M) can inhibit this inflammatory pathway, making it a promising therapy for PTOA. Herein, we demonstrate that A2M binds and neutralizes IL-1ß, blocking downstream NF-κB-induced catabolism seen in in vitro. Human chondrocytes (cell line C28) were incubated with A2M protein and then treated with IL-1ß. A2M was labeled with VivoTag™ 680 to localize the protein postincubation. The degree of binding between A2M and IL-1ß was evaluated through immunoprecipitation (IP). Catabolic proteins, including IL-1ß and NF-kB, were detected by Western blot. Pro-inflammatory and chondrocyte-related gene expression was examined by qRT-PCR. VivoTag™ 680-labeled A2M was observed in the cytoplasm of C28 human chondrocytes by fluorescence microscopy. IP experiments demonstrated that A2M could bind IL-1ß. Additionally, western blot analysis revealed that A2M neutralized IL-1ß and NF-κB in a dose-dependent manner. Moreover, A2M decreased levels of MMPs and TNF-α and increased the expression of cartilage protective genes Col2, Type2, Smad4, and aggrecan. Mostly importantly, A2M was shown to directly neutralize IL-1ß to downregulate the pro-inflammatory responses mediated by the NF-kB pathway. These results demonstrate a mechanism by which A2M reduces inflammatory catabolic activity and protects cartilage after joint injury. Further in vivo studies are needed to fully understand the potential of A2M as a novel PTOA therapy.

A novel large animal model of posttraumatic osteoarthritis induced by inflammation with mechanical stability.

OBJECTIVES: Animal models are needed to reliably separate the effects of mechanical joint instability and inflammation on posttraumatic osteoarthritis (PTOA) pathogenesis. We hypothesized that our modified intra-articular drilling (mIAD) procedure induces cartilage damage and synovial changes through increased inflammation without causing changes in gait. METHODS: Twenty-four Yucatan minipigs were randomized into the mIAD (n=12) or sham control group (n=12). mIAD animals had two osseous tunnels drilled into each of the tibia and femur adjacent to the anterior cruciate ligament (ACL) attachment sites on the left hind knee. Surgical and contralateral limbs were harvested 15 weeks post-surgery. Cartilage degeneration was evaluated macroscopically and histologically. Synovial changes were evaluated histologically. Interleukin-1 beta (IL-1ß), nuclear factor kappa B (NF-κB), and tumor necrosis factor alpha (TNF-α) mRNA expression levels in the synovial membrane were measured using quantitative real-time polymerase chain reaction. IL-1ß and NF-κB levels in chondrocytes were assessed using immunohistochemistry. Load asymmetry during gait was recorded by a pressure-sensing walkway system before and after surgery. RESULTS: The mIAD surgical knees demonstrated greater gross and histological cartilage damage than contralateral (P<.01) and sham knees (P<.05). Synovitis was present only in the mIAD surgical knee. Synovial inflammatory marker (IL-1ß, NF-κB, and TNF-α) expression was three times higher in the mIAD surgical knee than the contralateral (P<.05). Chondrocyte IL-1ß and NF-κB levels were highest in the mIAD surgical knee. In general, there were no significant changes in gait. CONCLUSIONS: The mIAD model induced PTOA through inflammation without affecting gait mechanics. This large animal model has significant applications for evaluating the role of inflammation in PTOA and for developing therapies aimed at reducing inflammation following joint injury.

Enhanced Bounding Techniques to Reduce the Protein Conformational Search Space.

The complexity and enormous size of the conformational space that must be explored for the protein tertiary structure prediction problem has led to the development of a wide assortment of algorithmic approaches. In this study, we apply state-of-the-art tertiary structure prediction algorithms and instead focus on the development of bounding techniques to reduce the conformational search space. Dihedral angle bounds on the ϕ and ψ angles are established based on the predicted secondary structure and studies of the allowed regions of ϕ/ψ space. Distance bounds are developed based on predicted secondary structure information (including ß-sheet topology predictions) to further reduce the search space. This bounding strategy is entirely independent of the degree of homology between the target protein and the database of proteins with experimentally-determined structures. The proposed approach is applied to the structure prediction of protein G as an illustrative example, yielding a significantly higher number of near-native protein tertiary structure predictions.

Alpha-helical topology prediction and generation of distance restraints in membrane proteins.

The field of protein structure prediction has seen significant advances in recent years. Researchers have followed a multitude of approaches, including methods based on comparative modeling, fold recognition and threading, and first-principles techniques. It is noteworthy that the structure prediction of membrane proteins is comparatively less studied by researchers in the field. A membrane protein is characterized by a protein structure that extends into or through the lipid-lipid bilayer of a cell. The structure is influenced by the combination of the hydrophobic bilayer region, the direct interaction with the bilayer, and the aqueous external environment. Due to the difficulty in obtaining reliable experimental structures, accurate computational prediction of membrane proteins is of paramount importance. An optimization model has been developed to predict the interhelical interactions in alpha-helical membrane proteins. A database of alpha-helical membrane proteins of known structure and limited sequence identity can be constructed to develop interaction probabilities. By then maximizing the occurrence of highly probable pairwise or three-residue interactions, realistic contacts can be predicted by imposing a number of geometrical constraints. The development of these low distance contacts can provide additional distance restraints for first principles-based approaches to the tertiary structure prediction problem. The proposed approach is shown to successfully predict interhelical contacts in several membrane protein systems, including bovine rhodopsin and the recently released human beta2 adrenergic receptor protein structure.

Biclustering via optimal re-ordering of data matrices in systems biology: rigorous methods and comparative studies.

BACKGROUND: The analysis of large-scale data sets via clustering techniques is utilized in a number of applications. Biclustering in particular has emerged as an important problem in the analysis of gene expression data since genes may only jointly respond over a subset of conditions. Biclustering algorithms also have important applications in sample classification where, for instance, tissue samples can be classified as cancerous or normal. Many of the methods for biclustering, and clustering algorithms in general, utilize simplified models or heuristic strategies for identifying the "best" grouping of elements according to some metric and cluster definition and thus result in suboptimal clusters. RESULTS: In this article, we present a rigorous approach to biclustering, OREO, which is based on the Optimal RE-Ordering of the rows and columns of a data matrix so as to globally minimize the dissimilarity metric. The physical permutations of the rows and columns of the data matrix can be modeled as either a network flow problem or a traveling salesman problem. Cluster boundaries in one dimension are used to partition and re-order the other dimensions of the corresponding submatrices to generate biclusters. The performance of OREO is tested on (a) metabolite concentration data, (b) an image reconstruction matrix, (c) synthetic data with implanted biclusters, and gene expression data for (d) colon cancer data, (e) breast cancer data, as well as (f) yeast segregant data to validate the ability of the proposed method and compare it to existing biclustering and clustering methods. CONCLUSION: We demonstrate that this rigorous global optimization method for biclustering produces clusters with more insightful groupings of similar entities, such as genes or metabolites sharing common functions, than other clustering and biclustering algorithms and can reconstruct underlying fundamental patterns in the data for several distinct sets of data matrices arising in important biological applications.

Descriptor-free molecular discovery in large libraries by adaptive substituent reordering.

Molecular discovery often involves identification of the best functional groups (substituents) on a scaffold. When multiple substitution sites are present, the number of possible substituent combinations can be very large. This article introduces a strategy for efficiently optimizing the substituent combinations by iterative rounds of compound sampling, substituent reordering to produce the most regular property landscape, and property estimation over the landscape. Application of this approach to a large pharmaceutical compound library demonstrates its ability to find active compounds with a threefold reduction in synthetic and assaying effort, even without knowing the molecular identity of any compound.

Intra-articular Recombinant Human Proteoglycan 4 Mitigates Cartilage Damage After Destabilization of the Medial Meniscus in the Yucatan Minipig.

BACKGROUND: Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. OBJECTIVE: To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. STUDY DESIGN: Controlled laboratory study. METHODS: Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O-fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1ß (IL-1ß) levels in synovial fluid and serum. RESULTS: The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group ( P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups ( P = .23). However, no differences in microscopic damage scores were observed between the 3 groups ( P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group ( P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS ( P = .013) and rhPRG4+hyaluronan ( P = .011) groups. In serum and synovial fluid, both rhPRG4 ( P = .006; P = .017) and rhPRG4+hyaluronan groups ( P = .009; P = .03) presented decreased IL-1ß levels. CONCLUSION: All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. CLINICAL RELEVANCE: Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.

Posterolateral intertransverse lumbar arthrodesis in the New Zealand White rabbit model: II. Operative technique.

BACKGROUND CONTEXT: Although the New Zealand White (NZW) rabbit model has been increasingly used for spinal fusion research, reported complication rates have been as high as 23%. The substantial animal morbidity and mortality associated with the model makes experimentation inefficient and can lead to faulty data analysis. Operative complications are in part the result of suboptimal execution of the experimental protocol. PURPOSE: To describe an experimental protocol for posterolateral intertransverse lumbar arthrodesis in the NZW rabbit and to analyze the effect of the protocol on the complication rate of this model. STUDY DESIGN/SETTING: This is a descriptive analysis of the surgical technique (and related complications) used for posterolateral spinal arthrodesis in the NZW rabbit model. This study was performed at a university research facility. METHODS: The complications of posterolateral intertransverse lumbar arthrodesis in the NZW rabbit were analyzed before and after instituting a refined experimental protocol. RESULTS: A total of 77 NZW rabbits underwent a posterolateral lumbar arthrodesis. In the first 48 animals (Group A) complications occurred at a rate of 26% and included anesthetic demise, nerve palsies, wound infections or seromas and fusion of the wrong level. After the institution of a refined experimental protocol, there were no complications in a second group of 29 rabbits (Group B). CONCLUSIONS: Adherence to a precise experimental protocol for posterolateral lumbar arthrodesis can minimize complications, thereby enhancing the NZW rabbit as a cost-effective and accurate animal model for spinal fusion research.

An improved hybrid global optimization method for protein tertiary structure prediction.

First principles approaches to the protein structure prediction problem must search through an enormous conformational space to identify low-energy, near-native structures. In this paper, we describe the formulation of the tertiary structure prediction problem as a nonlinear constrained minimization problem, where the goal is to minimize the energy of a protein conformation subject to constraints on torsion angles and interatomic distances. The core of the proposed algorithm is a hybrid global optimization method that combines the benefits of the αBB deterministic global optimization approach with conformational space annealing. These global optimization techniques employ a local minimization strategy that combines torsion angle dynamics and rotamer optimization to identify and improve the selection of initial conformations and then applies a sequential quadratic programming approach to further minimize the energy of the protein conformations subject to constraints. The proposed algorithm demonstrates the ability to identify both lower energy protein structures, as well as larger ensembles of low-energy conformations.

Recombinant human platelet-derived growth factor-BB augmentation of new-bone formation in a rat model of distraction osteogenesis.

BACKGROUND: Distraction osteogenesis creates a challenging bone-healing environment with protracted demand for cells of the osteoblast lineage. Platelet-derived growth factor-BB (PDGF-BB) is an osteoblast mitogen and chemotaxin that has been shown to accelerate and/or enhance bone-healing in several preclinical studies. The purpose of the present study was to determine whether recombinant human platelet-derived growth factor-BB (rhPDGF-BB) would have a similar effect on regenerate healing after distraction osteogenesis. METHODS: Unilateral 7-mm mid-diaphyseal femoral lengthening procedures were performed in eighty-three male Sprague-Dawley rats that were separated into five experimental groups. During the distraction period (Days 7 to 28), each animal received a weekly 50-microL injection of either sodium acetate buffer, bovine collagen dissolved in sodium acetate buffer, or one of three concentrations of rhPDGF-BB (100, 300, or 1000 microg/mL) into the distraction site. Animals from each group were killed on Days 35, 42, 49, 56, and 63. Healing was assessed with biweekly serial radiographs, micro-computed tomography of the explanted bones, and histologic analysis. RESULTS: rhPDGF-BB treatment significantly increased new-bone formation at the midconsolidation time points (Days 42, 49, and 56) as well as the union rate. On Day 49 regenerate bone volume was significantly greater in each of the three rhPDGF-BB-treated groups than in the controls (p < 0.05, p = 0.0002, and p < 0.05 for the 100, 300, and 1000 microg/mL rhPDGF-BB groups, respectively), whereas on Day 42 regenerate bone volume was significantly greater in the 300 and 1000 microg/mL rhPDGF-BB groups than in the controls (p = 0.0002 and p < 0.05, respectively) and on Day 56 regenerate bone volume was significantly greater in the 100 and 300 microg/mL rhPDGF-BB groups than in the controls (p < 0.05 and p < 0.0001, respectively). The overall union rate was 40.4% (nineteen of forty-seven) in the rhPDGF-BB-treated animals, compared with 4.5% (one of twenty-two) in the controls (p = 0.01). The radiographic and histologic results were consistent with new-bone formation as quantified by micro-computed tomography, although they were less definitive. CONCLUSIONS: The administration of exogenous rhPDGF-BB into the distraction site during diaphyseal distraction enhanced bone-healing in a rat model of distraction osteogenesis as evidenced by both increased regenerate new-bone formation and a higher union rate.