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| Jon Giles, M.D.
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Abstract 524 Engagement of Toll-like receptor 2 is the Central Trigger of Acute Neutrophilic Gouty Inflammation in Vivo Background: The robust inflammatory reaction to intra-articular crystals of monosodium urate (MSU) in gout is mediated through the elaboration of inflammatory cytokines and chemokines that recruit neutrophils to the joint space. These effects are mediated through the NF-kB and AP-1 nuclear transcription pathways. However, the upstream extracellular signaling by which MSU crystals activate these intracellular effectors has not been previously known. Here, Liu-Bryan et al investigate the role of Toll-like receptor 2 (TLR2) in the process of acute gouty inflammation. Methods/Results: Experiment 1 - In articular chondrocytes, the engagement and uptake of MSU crystals was assessed via RT-PCR. Inhibition of uptake was observed with the addition of neutralizing antibodies to TLR2 or tollip (a negative regulator of TLR2). Enhanced expression of IL-1 was observed when TLR2 was overexpressed. These effects were not observed in parallel constructs of other TLRs. Experiment 2 (in vitro) - MSU crystals were added to bone marrow-derived macrophages from wild-type (WT), TLR2 knock-out (KO), and MyD88 KO mice (MyD88 is a component of the intracellular portion of the TLR2 receptor signaling complex). Decreased production of IL-1, TNF-a, and KC/GRO-a(a neutrophil chemotactant) was observed in TLR2 KO and MyD88 KO mice compared to WT mice. Experiment 3 (in vivo) - Subcutaneous air pouches were constructed in WT, TLR2 KO, and MyD88 KO mice to mimic articular synovium. Six hours post-injection of MSU crystals, TLR2 KO and MyD88 KO mice demonstrated significantly less neutrophilic inflammation and IL-1 production compared to WT mice. Conclusion: MSU crystal engagement of TLR2 and the subsequent signaling cascade are key events in the initiation of acute gouty inflammation. Editorial Comments: This elegant series of experiments demonstrates a link between components of the innate immune system and the intracellular signaling pathways that lead to the elaboration of gouty inflammation. Whether these mechanisms can be shown to be operative in human joints remains to be answered. It is also challenging to consider how a huge MSU crystal interacts physically with a relatively small Toll receptor. Further work will be required to determine whether TLR2 or components of the TLR2 signaling complex can serve as targets for effective therapy of gout. | ||
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Abstract 525: ANKH Mutations Cause Both Familial and Sporadic Calcium Pyrophosphate Dihydrate Chondrocalcinosis and Increase ANKH Transcription/Translation Calcium pyrophosphate deposition disease (CPPD) usually occurs sporadically, although isolated families in which the disease has been inherited in an autosomal dominant pattern have been identified and are well characterized. Genetic defects in some of these families have been mapped to a chromosomal area which includes a gene termed ANKH, the products of which play a role in trafficking inorganic pyrophosphates through cell membranes. Here Zhang et al explore the function of ANKH mutations in sporadic and familial CPPD. Methods: DNA was obtained from all four affected family members of an Argentinean family known to have familial CPPD, 128 patients with sporadic CPPD, and 500 ethnically matched healthy controls. Genomic sequencing of the ANKH gene was conducted on all four familial CPPD samples, 24 of the 128 cases of sporadic CPPD, and 24 of the 500 controls. Variants identified from complete sequencing were then used to screen the remaining samples. These genetic variants of ANKH were generated by site directed mutagenesis and transfected into immortalized human articular chondrocytes with minimal basal ANKH expression. Extracellular inorganic pyrophosphate was used to monitor ANKH expression. Results: A significant association with sporadic CPPD and a specific G to A mutation in the ANKH gene 5-UTR was identified (4% of sporadic CPPD cases with homozygous alleles vs. approximately 0.5% of healthy controls with homozygous alleles, p = 0.0006). This mutation was different from several previously described mutations in familial cases of CPPD (+14 bp C to T, C-terminal GAG deletion, and 143 bp T to C). In vitro transfections of the identified 5-UTR G to A mutation, as well as the +14 bp C to T, and the C-terminal GAG deletion (but not the 143 bp T to C mutation) were associated with an increase in the transcription and translation of ANKH protein and in increased extracellular inorganic pyrophosphate levels compared to wild type ANKH. This effect was abrogated by the administration of anti-ANKH antibodies. Other genetic variants were not associated with these effects on inorganic pyrophosphate production or markers of chondrocyte hypertrophy. Conclusion: Specific genetic mutations of ANKH may account for increased extracellular pyrophosphate levels, a putative step in the pathogenesis of CPPD. Familial and sporadic cases of CPPD do not appear to share the same functional genetic mutations. Editorial Comment: This elegant series of experiments illustrate a role of gain of function polymorphisms of ANKH in the pathogenesis of CPPD, a common, poorly understood disorder with few specific therapies. Whether ANKH directed therapies will prove to be effective in preventing or treating disease is speculative, as a host of additional non-genetic factors may play a role in the phenotypic expression of CPPD. | ||
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Late Breaking Abstract 18: A Phase 3 Study Comparing the Safety and Efficacy of Oral Febuxostat and Allopurinol in Subjects with Hyperuricemia and Gout Surprisingly few new pharmacotherapies for gout have been introduced since the introduction of allopurinol in the 1960s. As many patients will experience dose limiting toxicities or incomplete responses to currently available therapies, new therapeutic modalities are clearly needed. Here, Becker et al examine the safety and efficacy of febuxostat, a non-purine selective inhibitor of xanthine oxidase/xanthine dehydrogenase on serum uric acid levels. Methods: Subjects with gout and serum uric acid level (sUA) > 8.0 mg/dL were randomized to receive oral febuxostat (80 or 120 mg per day) or oral allopurinol (300 mg per day) and followed for 52 weeks. The primary efficacy endpoint was the proportion of patients achieving sUA < 6.0 mg/dL for three successive months. Secondary efficacy outcomes included the sUA and reduction in tophus area at week 52. Results: 760 subjects (96% male, mean age 52 years) were randomized. Mean sUA at entry was 9.84 mg/dL. 24% of subjects enrolled had tophi. Efficacy 756 subjects were included in the primary intent-to-treat analysis. 53% of subjects in the febuxostat 80mg group and 62% of subjects in the febuxostat 120 mg group achieved the primary efficacy endpoint compared with 21% of subjects in the allopurinol group (p < 0.05 for each febuxostat treatment group compared to the allopurinol treatment group). Of the 482 subjects remaining in the study at week 52, 81% of subjects in the febuxostat 80mg group and 82% of subjects in the febuxostat 120 mg group demonstrated a sUA < 6.0 mg/dL compared with 39% of subjects in the allopurinol group (p < 0.05 for each febuxostat treatment group compared to the allopurinol treatment group). Subjects achieving sUA < 6.0 mg/dL had fewer gout flares requiring treatment compared to subjects not achieving this level of sUA reduction (6% vs. 14% (raw data not provided)). Safety Overall numbers of adverse events were high, but similar across treatment groups (80%, 75%, and 85% for the febuxostat 80mg, febuxostat 120mg, and allopurinol treatment groups respectively). Most treatment related adverse events were reported as mild to moderate and included liver function abnormalities, diarrhea, headache, and joint related symptoms. 47 patients reported serious adverse events, similar in frequency across treatment groups. 4 deaths occurred, none of which was attributed to active treatment. Conclusion: Daily oral febuxostat (80 or 120 mg) is effective in reducing sUA in subjects with gout and hyperuricemia compared to allopurinol 300 mg per day. Safety of both doses of febuxostat was comparable to allopurinol. Editorial Comment: This investigation shows great promise for a new and effective therapy for hyperuricemia and gout. As 300 mg per day is routinely the starting dose of allopurinol, with most patients who tolerate the medication requiring higher doses to achieve normalization of uric acid levels, further study is required to establish equivalence of febuxostat against larger doses of allopurinol. As newer agents are needed for patients with contraindications to allopurinol (i.e. renal insufficiency), recent publication of a Phase I study of febuxostat (ucleosides Nucleotides Nucleic Acids 23(8-9):1117, 2004) suggested that febuxostat may be of use in patients with renal impairment without adverse consequences. This will require additional study. The differences in the numbers of patients randomized compared to those completing week 52 (756 vs. 456) is skewed toward the patients with favorable outcomes, and thus some of the results presented here should be interpreted with caution. Particularly, the secondary efficacy endpoints at week 52, presented here as completers-only rather than intent-to-treat, is an analysis strategy sure to inflate the magnitude of the efficacy results.
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