Periprosthetic fractures about total knee arthroplasty.

Periprosthetic fracture after total knee arthroplasty presents a difficult complication for many orthopaedic surgeons. These fractures occur most frequently around the distal femur followed by the patella and then tibia. These fractures are frequently complicated by poor bone quality or compromised bone due to the presence of the implants. Surgical treatment is typically necessary and requires varied techniques of open fixation, intramedullary fixation, or revision arthroplasty. Outcomes of these injuries vary widely. This review aims to describe the epidemiology, classification, treatment options and outcomes for periprosthetic fractures following total knee arthroplasty.

A novel chemically modified curcumin reduces inflammation-mediated connective tissue breakdown in a rat model of diabetes: periodontal and systemic effects.

BACKGROUND AND OBJECTIVE: Periodontal disease is the most common chronic inflammatory disease known to mankind (and the major cause of tooth loss in the adult population) and has also been linked to various systemic diseases, particularly diabetes mellitus. Based on the literature linking periodontal disease with diabetes in a "bidirectional manner", the objectives of the current study were to determine: (i) the effect of a model of periodontitis, complicated by diabetes, on mechanisms of tissue breakdown including bone loss; and (ii) the response of the combination of this local and systemic phenotype to a novel pleiotropic matrix metalloproteinase inhibitor, chemically modified curcumin (CMC) 2.24. MATERIAL AND METHODS: Diabetes was induced in adult male rats by intravenous injection of streptozotocin (nondiabetic rats served as controls), and Escherichia coli endotoxin (lipopolysaccharide) was repeatedly injected into the gingiva to induce periodontitis. CMC 2.24 was administered by oral gavage (30 mg/kg) daily; untreated diabetic rats received vehicle alone. After 3 wk of treatment, the rats were killed, and gingiva, jaws, tibia and skin were collected. The maxillary jaws and tibia were dissected and radiographed. The gingival tissues of each experimental group (n = 6 rats/group) were pooled, extracted, partially purified and, together with individual skin samples, analyzed for matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography; MMP-8 was analyzed in gingival and skin tissue extracts, and in serum, by western blotting. The levels of three bone-resorptive cytokines [interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α], were measured in gingival tissue extracts and serum by ELISA. RESULTS: Systemic administration of CMC 2.24 to diabetic rats with endotoxin-induced periodontitis significantly inhibited alveolar bone loss and attenuated the severity of local and systemic inflammation. Moreover, this novel tri-ketonic phenylaminocarbonyl curcumin (CMC 2.24) appeared to reduce the pathologically excessive levels of inducible MMPs to near-normal levels, but appeared to have no significant effect on the constitutive MMPs required for physiologic connective tissue turnover. In addition to the beneficial effects on periodontal disease, induced both locally and systemically, CMC 2.24 also favorably affected extra-oral connective tissues, skin and skeletal bone. CONCLUSION: This study supports our hypothesis that CMC 2.24 is a potential therapeutic pleiotropic MMP inhibitor, with both intracellular and extracellular effects, which reduces local and systemic inflammation and prevents hyperglycemia- and bacteria-induced connective tissue destruction.

Sexually dimorphic regulation and induction of P450s by the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR) is a xenosensing nuclear receptor and regulator of cytochrome P450s (CYPs). However, the role of CAR as a basal regulator of CYP expression nor its role in sexually dimorphic responses have been thoroughly studied. We investigated basal regulation and sexually dimorphic regulation and induction by the potent CAR activator TCPOBOP and the moderate CAR activator Nonylphenol (NP). NP is an environmental estrogen and one of the most commonly found environmental toxicants in Europe and the United States. Previous studies have demonstrated that NP induces several CYPs in a sexually dimorphic manner, however the role of CAR in regulating NP-mediated sexually dimorphic P450 expression and induction has not been elucidated. Therefore, wild-type and CAR-null male and female mice were treated with honey as a carrier, NP, or TCPOBOP and CYP expression monitored by QPCR and Western blotting. CAR basally regulates the expression of Cyp2c29, Cyp2b13, and potentially Cyp2b10 as demonstrated by QPCR. Furthermore, we observed a shift in the testosterone 6alpha/15alpha-hydroxylase ratio in untreated CAR-null female mice to the male pattern, which indicates an alteration in androgen status and suggests a role for androgens as CAR inverse agonists. Xenobiotic-treatments with NP and TCPOBOP induced Cyp2b10, Cyp2c29, and Cyp3a11 in a CAR-mediated fashion; however NP only induced these CYPs in females and TCPOBOP induced these CYPs in both males and females. Interestingly, Cyp2a4, was only induced in wild-type male mice by TCPOBOP suggesting Cyp2a4 induction is not sensitive to CAR-mediated induction in females. Overall, TCPOBOP and NP show similar CYP induction profiles in females, but widely different profiles in males potentially related to lower sensitivity of males to either indirect or moderate CAR activators such as NP. In summary, CAR regulates the basal and chemically inducible expression of several sexually dimorphic xenobiotic metabolizing P450s in a manner that varies depending on the ligand.

trans-Stilbene oxide induces expression of genes involved in metabolism and transport in mouse liver via CAR and Nrf2 transcription factors.

trans-Stilbene oxide (TSO) induces drug metabolizing enzymes in rat and mouse liver. TSO is considered a phenobarbital-like compound because it induces Cyp2B mRNA expression in liver. Phenobarbital increases Cyp2B expression in liver via activation of the constitutive androstane receptor (CAR). The purpose of this study was to determine whether TSO induces gene expression in mouse liver via CAR activation. TSO increased CAR nuclear localization in mouse liver, activated the human Cyp2B6 promoter in liver in vivo, and activated a reporter plasmid that contains five nuclear receptor 1 (NR1) binding sites in HepG2 cells. TSO administration increased expression of Cyp2b10, NAD(P)H:quinone oxidoreductase (Nqo1), epoxide hydrolase, heme oxygenase-1, UDP-glucuronosyl-transferase (Ugt) 1a6 and 2b5, and multidrug resistance-associated proteins (Mrp) 2 and 3 mRNA in livers from male mice. Cyp2b10 and epoxide hydrolase induction by TSO was decreased in livers from CAR-null mice, compared with wild-type mice, suggesting CAR involvement. In contrast, TSO administration induced Nqo1 and Mrp3 mRNA expression equally in livers from wild-type and CAR-null mice, suggesting that TSO induces expression of some genes through a mechanism independent of CAR. TSO increased nuclear staining of the transcription factor Nrf2 in liver, and activated an antioxidant/electrophile response element luciferase reporter construct that was transfected into HepG2 cells. In summary, in mice, TSO increases Cyp2b10 and epoxide hydrolase expression in mice via CAR, and potentially induces Nqo1 and Mrp3 expression via Nrf2. Moreover, our data demonstrate that a single compound can activate both CAR and Nrf2 transcription factors in liver.

CAR, the continuously advancing receptor, in drug metabolism and disease.

The detoxification and elimination of potentially toxic foreign and endogenous compounds depends on the concerted action of xenobiotic metabolizing enzymes. Nuclear hormone receptors (NHRs) have emerged as key regulators of the expression of these enzymes and his review focuses on the xenosenor CAR (Constitutive Androstane Receptor, NR1I3). CAR is highly expressed in the liver and the small intestine, two key tissues expressing xenobiotic metabolizing enzymes, and mediates the induction of their expression by the widely used antiepileptic drug, phenobarbital (PB) and the potent synthetic inducer 1, 4-bis-(2-(3, 5, -dichloropyridyloxy)) benzene (TCPOBOP). TCPOBOP is an agonist ligand for CAR. PB induces its nuclear translocation, which results in increased expression of CAR target genes since, unlike the classical, ligand-dependent nuclear receptors, CAR is an apparently constitutive transactivator. This constitutive activity is inhibited by the inverse agonist ligands androstanol and androstenol. The CAR mediated induction of the expression of xenobiotic metabolizing enzymes is generally protective, but can be deleterious if toxic metabolites are produced. CAR also has a protective role in the stress response elicited by hyperbilirubinemia, as well as lithocholic acid induced cholestasis. In addition, recent studies show that CAR activation disrupts thyroid hormone homeostasis. Finally, CAR activation promotes hepatocyte proliferation and blocks apoptosis, and is essential for the tumorigenesis induced by its activators PB and TCPOBOP. The role of CAR in endobiotic and xenobiotics metabolism has clinical implications in disease prevention, drug-drug interactions, and the development of better drug treatments.

CAR/PXR provide directives for Cyp3a41 gene regulation differently from Cyp3a11.

This study reports that Cyp3a41 gene contains 13 exons and is localized on the chromosome 5. CYP3A41 is a female-specific isoform that is predominantly expressed in the liver. Estrogen signaling is not responsible for its female specificity. CYP3A41 expression in kidney and brain is observed only in 50% of mice examined. PXR mediates dexamethasone-dependent suppression of CYP3A41. In contrast to CYP3A11, CYP3A41 expression is not induced by pregnenolone-16alpha-carbonitrile (PCN) in wild-type mice, but is significantly suppressed by PCN in PXR(-/-) mice. Phenobarbital and TCPOBOP induce CYP3A11 expression only in the presence of CAR, but have no effect on CYP3A41 expression. Immunoblot and erythromycin demethylase activity analysis reveal robust CYP3A induction after PCN treatment, which is poorly correlated to CYP3A41. These findings suggest a differential role for CAR/PXR in regulating individual CYP3A isoforms by previously characterized CYP3A inducers.

Specific and overlapping functions of the nuclear hormone receptors CAR and PXR in xenobiotic response.

The products of the cytochrome P450 (CYP) genes play an important role in the detoxification of xenobiotics and environmental contaminants, and many foreign chemicals or xenobiotics can induce their expression. We have previously shown that the nuclear hormone receptor CAR (Constitutive Androstane Receptor, NR113) mediates the well studied induction of CYP2B10 gene expression by phenobarbital (PB) and 1, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene (TCPOBOP). We have used the CAR knockout mouse model to explore the broader functions of this xenobiotic receptor. In addition to the liver, CAR is expressed in the epithelial cells of the villi in the small intestine, and this expression is required for CYP2B10 induction in response to PB and TCPOBOP in those cells. In agreement with previous observations that CAR can bind to regulatory elements in CYP3A genes, CAR is also required for induction of expression of CYP3A11 in response to both PB and TCPOBOP in liver. In males, CAR is also required for induction of liver CYP2A4 expression. In wild type animals, pretreatment with the CAR inverse agonist androstenol blocks the response of both the CYP2B10 and CYP3A11 genes to PB and TCPOBOP, and decreases basal CYP3A11 expression. CAR is also required for the response of CYP2B10 to several additional xenobiotic inducers, including chlorpromazine, clotrimazole and dieldrin, but not dexamethasone, an agonist for both the xenobiotic receptor PXR (Pregnane X Receptor NR112) and the glucocorticoid receptor. Chlorpromazine induction of CYP3A11 is also absent in CAR-deficient animals, but the responses to clotrimazole and dieldrin are retained, indicating that both of these inducers can also activate PXR (Pregnane X Receptor NR112). We conclude that CAR has broad functions in xenobiotic responses. Some are specific to CAR but others, including induction of the important drug metabolizing enzyme CYP3A, overlap with those of PXR.

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

New insights into receptor ligand binding domains from a novel assembly assay.

Previous studies have demonstrated that hormone binding stabilizes the ligand binding domain (LBD) of the nuclear hormone receptors against proteolysis. We have confirmed and extended this observation using a newly developed assembly assay. In this assay, the LBD is divided into two parts, of which one includes the first helix of this domain and the other corresponds to the remainder of the LBD. Several independent criteria demonstrate that these two fragments can assemble into a functional LBD in the presence of a ligand, but not in its absence, and that this is a reflection of the stabilizing effect of ligand. We have also used this assay to demonstrate that binding of the nuclear receptor corepressor NCoR can directly stabilize the LBD. Overall, these results highlight the dynamic nature of the LBD and suggest that current models for activation based solely on allosteric effects on the C-terminal helix may be too limited.

Role reversal: new insights from new ligands for the xenobiotic receptor CAR.

In the classic model of nuclear receptor signaling, specific hormone binding results in the recruitment of coactivator proteins and transcriptional activation. Recent results with newly characterized nuclear receptors have expanded this model to include new types of ligands and novel transcriptional responses. Both inverse agonists and conventional agonist ligands have been identified for the xenobiotic receptor constitutive androstane receptor (CAR), a constitutive activator of transcription in the absence of ligands.

Mutations in the small heterodimer partner gene are associated with mild obesity in Japanese subjects.

Mutations in several genes encoding transcription factors of the hepatocyte nuclear factor (HNF) cascade are associated with maturity-onset diabetes of the young (MODY), a monogenic form of early-onset diabetes mellitus. The ability of the orphan nuclear receptor small heterodimer partner (SHP, NR0B2) to modulate the transcriptional activity of MODY1 protein, the nuclear receptor HNF-4alpha, suggested SHP as a candidate MODY gene. We screened 173 unrelated Japanese subjects with early-onset diabetes for mutations in this gene and found five different mutations (H53fsdel10, L98fsdel9insAC, R34X, A195S, and R213C) in 6 subjects as well as one apparent polymorphism (R216H), all present in the heterozygous state. Interestingly, all of the subjects with the mutations were mildly or moderately obese at onset of diabetes, and analysis of the lineages of these individuals indicated that the SHP mutations were associated with obesity rather than with diabetes. Therefore, an additional group of 101 unrelated nondiabetic subjects with early-onset obesity was screened for mutations in the SHP gene. Two of the previously observed mutations (R34X and A195S) and two additional mutations (R57W and G189E) were identified in 6 subjects, whereas no mutations were identified in 116 young nondiabetic lean controls (P = 0.0094). Functional studies of the mutant proteins show that the mutations result in the loss of SHP activity. These results suggest that genetic variation in the SHP gene contributes to increased body weight and reveal a pathway leading to this common metabolic disorder in Japanese.

Commonly used reagents and equipment.

This appendix lists recipes for common reagents and also describes equipment that is typically found in a molecular biology laboratory.

Genomic DNA libraries.

Genomic DNA libraries are almost always screened by hybridization using a radioactive nucleic acid probe. Since this approach is essentially independent of a particular vector or type of target DNA, the main problem faced when considering creation of a genomic DNA library is simply generating a large enough number of recombinant DNA clones. The basic strategies used to address this problem have included both minimizing the number of clones necessary by incorporating large fragments of genomic DNA, and maximizing cloning efficiency by using vectors based on bacteriophage lambda. This unit discusses the appropriate numerical considerations for both ordinary genomic DNA libraries and subgenomic DNA libraries, and then describes a limited number of appropriate vectors.

cDNA libraries.

This unit provides a basic description of issues that require careful consideration before undertaking construction of cDNA libraries. Topics include: generating a double-stranded DNA copy of the mRNA, the quality of the mRNA, the relative abundance of the clone of interest, the choice of screening method, the size of the library that will be necessary to include the clone of interest, and choices of linkers or adaptors for insertion into the vector.

Gene synthesis: assembly of target sequences using mutually priming long oligonucleotides.

This protocol uses pairs of oligonucleotides annealed at a short duplex segment at their 3' ends as both templates and primers (mutually primed synthesis) to generate desired sequences up to 400 bp in a single step. The procedure is divided into three sections: design of the oligonucleotides, extension by mutually primed synthesis, and cloning of the extension products. The strategy for design of oligonucleotides is discussed in the commentary.

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.

Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding.

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.

The farnesoid X-activated receptor mediates bile acid activation of phospholipid transfer protein gene expression.

Bile acids facilitate the absorption of dietary lipids and fat-soluble vitamins and are physiological ligands for the farnesoid X-activated receptor (FXR), a member of the nuclear hormone receptor superfamily. FXR functions as a heterodimer with the retinoid X receptor and in the presence of ligand, the heterodimer binds to specific DNA sequences in the promoters of target genes to regulate gene transcription. Phospholipid transfer protein (PLTP) has been identified as a possible target gene for FXR because the human promoter contains a potential FXR response element, an inverted repeat in which consensus receptor-binding hexamers are separated by one nucleotide (inverted repeat-1). PLTP is essential in the transfer of very low density lipoprotein phospholipids into high density lipoprotein (Jiang, X. C., Bruce, C., Mar, J., Lin, M., Ji, Y., Francone, O. L., and Tall, A. R. (1999) J. Clin. Invest. 103, 907-914). Here we report the regulation of PLTP gene expression by FXR and bile acids. In CV-1 cells, cotransfection of FXR and the retinoid X receptor resulted in bile acid-dependent transactivation of a luciferase reporter construct containing the human PLTP promoter. Mutation analysis demonstrated that the inverted repeat-1 (IR-1) in the PLTP promoter is required for this transactivation. Finally, we demonstrate that bile acids are able to regulate PLTP gene expression in vivo. Mice fed a chow diet supplemented with bile acid showed increased hepatic PLTP mRNA levels. These results suggest that FXR may play a role in high density lipoprotein metabolism via the regulation of PLTP gene expression.