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Native liquid chromatography mass spectrometry (LC-MS) is a commonly used approach for intact analysis of inter-chain cysteine conjugated antibody-drug conjugates (ADCs). Coupling native LC-MS with affinity capture provides a platform for intact ADC analysis from in vivo samples and characterisation of individual drug load species, specifically the impact of drug linker deconjugation, hydrolysis, and differential clearance in a biological system.This manuscript describes data generated from native LC-MS analysis of ADCs from human plasma, both in vitro incubations and clinical samples. It also details the pharmacokinetic (PK) model built to specifically characterise the disposition of individual drug load species from MMAE and MMAF interchain cysteine conjugated ADCs. In vitro deconjugation and hydrolysis rates were similar across both ADCs. Differential clearance of higher loaded species in vivo was pronounced for the MMAE conjugated ADC, while systemic elimination after accounting for deconjugation was similar across drug loads for the MMAF conjugated ADC. This is the first report of affinity capture native LC-MS analysis, and subsequent modelling of deconjugation, hydrolysis and clearance rates of individual drug load species using clinical data from cysteine conjugated ADCs.

A physiologically-based pharmacokinetic (PBPK) model was developed to simulate plasma concentrations of tucatinib (TUKYSA®) after single-dose or multiple-dose administration of 300 mg b.i.d. orally. This PBPK model was subsequently applied to support evaluation of drug-drug interaction (DDI) risk as a perpetrator resulting from tucatinib inhibition of CYP3A4, CYP2C8, CYP2C9, P-gp, or MATE1/2-K. The PBPK model was also applied to support evaluation of DDI risk as a victim resulting from co-administration with CYP3A4 or CYP2C8 inhibitors, or a CYP3A4 inducer. After refinement with clinical DDI data, the final PBPK model was able to recover the clinically observed single and multiple-dose plasma concentrations for tucatinib when tucatinib was administered as a single agent in healthy subjects. In addition, the final model was able to recover clinically observed plasma concentrations of tucatinib when administered in combination with itraconazole, rifampin, or gemfibrozil as well as clinically observed plasma concentrations of probe substrates of CYP3A4, CYP2C8, CYP2C9, P-gp, or MATE1/2-K. The PBPK model was then applied to prospectively predict the potential perpetrator or victim DDIs with other substrates, inducers, or inhibitors. To simulate a potential interaction with a moderate CYP2C8 inhibitor, two novel PBPK models representing a moderate CYP2C8 inhibitor and a sensitive CYP2C8 substrate were developed based on the existing PBPK models for gemfibrozil and rosiglitazone, respectively. The simulated population geometric mean area under the curve ratio of tucatinib with a moderate CYP2C8 inhibitor ranged from 1.98- to 3.08-fold, and based on these results, no dose modifications were proposed for moderate CYP2C8 inhibitors for the tucatinib label., (© 2023 Seagen Inc. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Background and Objective: Tucatinib is a selective tyrosine kinase inhibitor of the human epidermal growth factor receptor 2 (HER2) approved to treat metastatic HER2-positive breast and colorectal cancers. The International Council for Harmonisation of Technical Requirements for Human Use (ICH) E14 guideline mandates that new drugs are assessed for potential effects on cardiac repolarization through electrocardiogram (ECG) evaluation in a QT/corrected QT (TQT) study., Methods: We evaluated the effect of tucatinib on cardiac repolarization in healthy volunteers in a phase I, randomized, partially double-blind, placebo-and positive-controlled three-period crossover study. The primary endpoint was the placebo-corrected change from baseline in QT interval values, corrected for heart rate using Fridericia's method (ΔΔQTcF)., Results: After achieving steady-state tucatinib exposures with 300 mg twice daily, the observed ΔΔQTcF ranged from -2.9 msec at 2 hours post-dose to 0 msec at 4 hours post-dose. The upper bound of the 90% confidence interval (CI) was below 5 ms at all post-dose timepoints. Assay sensitivity was confirmed as the lower bound of the 90% CI and was >5 ms following moxifloxacin dosing. As the mean ΔΔQTcF of tucatinib was predicted to be -  1.80 ms (90% CI -  3.90, 0.30) at clinically relevant tucatinib concentrations (511 ng/mL), an effect of tucatinib on QTcF exceeding 10 ms was excluded within observed ranges of tucatinib (up to ~1000 ng/mL). Tucatinib had no clinically relevant effect on heart rate or cardiac conduction. The safety profile of tucatinib was manageable after multiple doses., Conclusion: Tucatinib had no clinically relevant effects on studied ECG parameters. This study constitutes a clearly negative TQT study per ICH E14 guidance., Clinical Trial Registration: This trial (NCT03777761) was registered on 17 December 2018., (© 2023. The Author(s).)

Checkpoint inhibition targeting programmed cell-death protein 1 has demonstrated efficacy for a wide range of indications including cutaneous malignancy. However, immune-related adverse events (irAEs), including infrequent but visually impactful ocular irAEs, require careful consideration of treatment options, including medication withdrawal, local corticosteroids, or rarely immunomodulation. This case presents a 53-year-old woman who developed uveitis and mucous membrane ulcers after treatment for numerous cutaneous neoplasms, primarily squamous cell carcinoma, with the programmed cell-death protein 1 inhibitor cemiplimab. Ophthalmic examination revealed diffuse choroidal depigmentation consistent with a Vogt-Koyanagi-Harada-like syndrome. Topical and periocular steroids were used to treat the intraocular inflammation, and cemiplimab was discontinued. Because of ongoing severe uveitis, systemic corticosteroids and corticosteroid-sparing immunosuppression were initiated. Specifically, azathioprine and methotrexate were introduced, but each was stopped due to side effects, prompting the initiation of adalimumab (ADA) treatment. While ADA controlled intraocular inflammation, the squamous cell carcinomas were noted to progress, resulting in the discontinuation of ADA. However, a uveitis recurrence was observed. After a discussion of risks and benefits of biologic immunosuppressive therapy, including the risk of vision loss, ADA was restarted with successful disease quiescence at a 16-month follow-up. The cutaneous neoplasms were managed with topical and intralesional therapies, such as 5-fluorouracil. Recent dermatologic examinations suggested no new cutaneous lesions. This scenario presents the effective use of ADA in an ocular irAE that balances the management of sight-threatening ocular inflammation with the risk of promoting recurrent or de novo neoplastic disease., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background and Objective: Tucatinib, a highly selective tyrosine kinase inhibitor of the human epidermal growth factor receptor 2 (HER2) approved for HER2-positive metastatic breast cancer, is cleared by hepatic metabolism and subsequent biliary excretion. Liver disease can alter drug disposition and pharmacokinetics (PK). The objective of this study is to characterize PK and safety of tucatinib in volunteers with hepatic impairment., Methods: This Phase 1 study compared the PK and safety of a single 300-mg oral dose of tucatinib in volunteers with mild, moderate, and severe hepatic impairment (Child-Pugh A/B/C) to healthy volunteers matched for sex, age, and body mass index. Pharmacokinetic parameters were determined for tucatinib and its predominant metabolite ONT-993., Results: Compared with healthy volunteers, tucatinib exposure was similar in volunteers with mild impairment and increased in those with moderate or severe impairment without reaching statistical significance. Respective fold increases in geometric mean ratios for AUC 0-t and AUC 0-∞ were 1.13 and 1.15 in moderate impairment, and 1.43 and 1.61 in severe impairment compared with healthy volunteers. Three treatment-emergent adverse events (nausea, dermatitis, and increased transaminases) were reported in three volunteers and showed no obvious association with hepatic impairment status., Conclusion: The 1.61-fold geometric mean ratio AUC 0-∞ increase in volunteers with severe hepatic impairment supports the recommendation in the tucatinib prescribing information to reduce the dose from 300 mg twice daily to 200 mg twice daily in patients with severe impairment; no dose adjustment is recommended for patients with mild or moderate hepatic impairment. This trial (NCT03722823) was registered on October 29, 2018., (© 2022. The Author(s).)

Competing Interests: The authors declare no conflict of interest.

Tucatinib is a potent tyrosine kinase inhibitor selective for human epidermal growth factor receptor 2 (HER2) approved by the US Food and Drug Administration for the treatment of HER2-positive metastatic breast cancer and in development for other HER2-positive solid tumors. Modest, reversible serum creatinine (SCr) elevations have been observed in tucatinib clinical trials. SCr is conveyed by the renal drug transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) and 2-K (MATE2-K) and can increase in the presence of inhibitors of these transporters. In vitro, tucatinib inhibited OCT2-, MATE1-, and MATE2-K-mediated transport of metformin, with IC 50 values of 14.7, 0.340, and 0.135 µM, respectively. Tucatinib also inhibited OCT2- and MATE1-mediated transport of creatinine, with IC 50 values of 0.107 and 0.0855 µM, respectively. A phase 1 study with metformin administered orally in the absence and presence of tucatinib was conducted in 18 healthy subjects. Renal function was assessed by measuring glomerular filtration rate (GFR; based on iohexol plasma clearance) and endogenous markers (SCr, cystatin C-based estimated glomerular filtration rate [eGFR]) with and without tucatinib. Metformin exposure increased (1.4-fold) and renal clearance decreased (29.99-17.64 L/h) with tucatinib, with no effect on metformin maximum concentration. Creatinine clearance transiently decreased 23% with tucatinib. GFR and eGFR, which are unaffected by OCT2 and/or MATE1/2-K transport, were unchanged with tucatinib. These data demonstrate that tucatinib inhibits OCT2- and MATE1/2-K-mediated tubular secretion of creatinine, which may manifest as mild SCr elevations that are not indicative of renal impairment., (© 2020 Seattle Genetics, Inc. The Journal of Clinical Pharmacology published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

Background/objective: New classification criteria for SLE have recently been developed. How these criteria affect the classification of patients with the SLE-mimicking condition UCTD is poorly understood. This study investigated the reclassification of UCTD patients using newly derived SLE criteria., Methods: Patients with UCTD were identified within a single academic medical center using ICD9/10 codes. Medical record review was performed to confirm UCTD diagnosis and identify disease features present at diagnosis. The SLICC and ACR/EULAR criteria were applied, after which we compared the proportion of patients reclassified as SLE and determined which disease features were associated with reclassification., Results: A total of 129 patients were included in the study. When applying the SLICC and ACR/EULAR criteria, 18 (14.0%) and 26 patients (20.2%) were reclassified as SLE. Comparison with McNemar's test trended toward statistical significance (p = 0.057). Cohen's kappa coefficient was 0.62 (p < 0.001), indicating substantial agreement between these criteria. Disease features associated with reclassification as SLE were renal involvement, leukopenia, thrombocytopenia, anti- dsDNA antibody, hypocomplementemia, non-scarring alopecia (SLICC), and arthritis (ACR/EULAR)., Conclusions: Both the SLICC and ACR/EULAR criteria exhibit increased SLE classification. These newer classification criteria could be used to increase the number of SLE patients in future clinical studies.

Treatment of cardiac sarcoidosis is challenging, as the disease can be refractory to traditional treatment with steroids. Infliximab, a tumor necrosis factor-α inhibitor, has been reportedly used in cardiac sarcoidosis, but published evidence is limited. The potential cardiotoxicity of infliximab and the Food and Drug Administration black-box warning for patients with heart failure have hindered the use of this agent in cardiac sarcoidosis. Here, we report a case of refractory cardiac sarcoidosis successfully treated with infliximab and discuss the important role of fluorine-18-fluorodeoxyglucose positron emission tomography in prognostication and guidance of therapy. ( Level of Difficulty: Intermediate. )., (© 2020 The Authors.)

Objectives: The aim was to estimate the impact of TNF inhibitor (TNFi) exposure on radiographic disease progression in US Veterans with RA during the first year after initiating therapy., Methods: This historical longitudinal cohort design used clinical and claims data to evaluate radiographic progression after initiation of TNFi. US Veterans with RA initiating TNFi treatment (index date), ≥ 6 months pre-index and ≥ 12 months post-index VA enrolment/activity, and initial (6 months pre-index to 30 days post-index) and follow-up (10-18 months post-index) bilateral hand radiographs were eligible. The cumulative TNFi exposure and change in modified Sharp score (MSS) between initial and follow-up radiographs were calculated. The percentage of patients with clinically meaningful change in MSS (≥ 5) for each month of exposure was assessed using a longitudinal marginal structural model with inverse probability of treatment weights. Mean values and CIs were generated using 1000 bootstrapped samples., Results: For 246 eligible patients, the mean (s.d.) age was 58 (11) years; 81% were male. The mean (s.d.) initial MSS was 19.6 (33.4) (range 0-214). The mean change (s.d.) in MSS was 0.3 (3.6) (median 0, range -19 to 22). Patients with the greatest exposure had the least radiographic progression for both crude and adjusted model analyses. Adjusted rates of MSS change ≥ 5 points (95% CI) were 10.6% (9.8%, 11.4%) for patients with 3 months of exposure compared with 5.4% (5.1%, 5.7%) for patients with 12 months of exposure., Conclusion: One-year changes in radiographic progression were small. Patients with the greatest cumulative TNFi exposure experienced the least progression., (Published by Oxford University Press on behalf of the British Society for Rheumatology 2019. This work is written by US Government employees and is in the public domain in the US.)

Tyrosine kinase inhibitors (TKIs) are widely used to treat solid tumors but can be cardiotoxic. The molecular basis for this toxicity and its relationship to therapeutic mechanisms remain unclear; we therefore undertook a systems-level analysis of human cardiomyocytes (CMs) exposed to four TKIs. CMs differentiated from human induced pluripotent stem cells (hiPSCs) were exposed to sunitinib, sorafenib, lapatinib, or erlotinib, and responses were assessed by functional assays, microscopy, RNA sequencing, and mass spectrometry (GEO: GSE114686; PRIDE: PXD012043). TKIs have diverse effects on hiPSC-CMs distinct from inhibition of tyrosine-kinase-mediated signal transduction; cardiac metabolism is particularly sensitive. Following sorafenib treatment, oxidative phosphorylation is downregulated, resulting in a profound defect in mitochondrial energetics. Cells adapt by upregulating aerobic glycolysis. Adaptation makes cells less acutely sensitive to sorafenib but may have long-term negative consequences. Thus, CMs exhibit adaptive responses to anti-cancer drugs conceptually similar to those previously shown in tumors to mediate drug resistance., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Idiopathic aortitis is an inflammatory disease of the aorta that is diagnosed after the less frequent infectious and rheumatologic variants are excluded. The etiology and natural history of the disease are poorly understood, and its presentation is variable; the need for exclusion of infectious, malignant, and rheumatologic causes can make its evaluation and diagnosis challenging. Treatment is tailored to the diagnosis and may include observation, antimicrobial therapies, and immunosuppressive agents when appropriate. Operative therapy is rarely needed and reserved for symptomatic patients or instances in which infection cannot be excluded. We present a case of idiopathic aortitis that resolved spontaneously with expectant management and discuss the pitfalls in the diagnosis and care of the disease.

Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS 3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS 3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS 3 approach provides dramatic performance improvements for quantitative multiplexed experiments.

Background: The gut microbiome plays a fundamental role in the human host's overall health by contributing key biological functions such as expanded metabolism and pathogen defense/immune control. In a healthy individual, the gut microbiome co-exists within the human host in a symbiotic, non-inflammatory relationship that enables mutual benefits, such as microbial degradation of indigestible food products into small molecules that the host can utilize, and enhanced pathogen defense. In abnormal conditions, such as Crohn's disease, this favorable metabolic relationship breaks down and a variety of undesirable activities result, including chronic inflammation and other health-related issues. It has been difficult, however, to elucidate the overall functional characteristics of this relationship because the microbiota can vary substantially in composition for healthy humans and possibly even more in individuals with gut disease conditions such as Crohn's disease. Overall, this suggests that microbial membership composition may not be the best way to characterize a phenotype. Alternatively, it seems to be more informative to examine and characterize the functional composition of a gut microbiome. Towards that end, this study examines 25 metaproteomes measured in several Crohn's disease patients' post-resection surgery across the course of 1 year, in order to examine persistence of microbial taxa, genes, proteins, and metabolic functional distributions across time in individuals whose microbiome might be more variable due to the gut disease condition., Results: The measured metaproteomes were highly personalized, with all the temporally-related metaproteomes clustering most closely by individual. In general, the metaproteomes were remarkably distinct between individuals and to a lesser extent within individuals. This prompted a need to characterize the metaproteome at a higher functional level, which was achieved by annotating identified protein groups with KEGG orthologous groups to infer metabolic modules. At this level, similar and redundant metabolic functions across multiple phyla were observed across time and between individuals. Tracking through these various metabolic modules revealed a clear path from carbohydrate, lipid, and amino acid degradation to central metabolism and finally the production of fermentation products., Conclusions: The human gut metaproteome can vary quite substantially across time and individuals. However, despite substantial intra-individual variation in the metaproteomes, there is a clear persistence of conserved metabolic functions across time and individuals. Additionally, the persistence of these core functions is redundant across multiple phyla but is not always observable in the same sample. Finally, the gut microbiome's metabolism is not driven by a set of discrete linear pathways but a web of interconnected reactions facilitated by a network of enzymes that connect multiple molecules across multiple pathways.

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

Phosphorylation-mediated signaling pathways have major implications in cellular regulation and disease. However, proteins with roles in these pathways are frequently less abundant and phosphorylation is often sub-stoichiometric. As such, the efficient enrichment, and subsequent recovery of phosphorylated peptides, is vital. Mass spectrometry-based proteomics is a well-established approach for quantifying thousands of phosphorylation events in a single experiment. We designed a peptide internal standard-based assay directed toward sample preparation strategies for mass spectrometry analysis to understand better phosphopeptide recovery from enrichment strategies. We coupled mass-differential tandem mass tag (mTMT) reagents (specifically, TMTzero and TMTsuper-heavy), nine mass spectrometry-amenable phosphopeptides (phos9), and peak area measurements from extracted ion chromatograms to determine phosphopeptide recovery. We showcase this mTMT/phos9 recovery assay by evaluating three phosphopeptide enrichment workflows. Our assay provides data on the recovery of phosphopeptides, which complement other metrics, namely the number of identified phosphopeptides and enrichment specificity. Our mTMT/phos9 assay is applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy. Graphical Abstract ᅟ.

IgG4-related disease (IgG-RD) describes a group of fibroinflammatory diseases that affect a variety of tissues resulting in tumor-like effect and/or organ dysfunction. Clinical presentation varies according to the tissue(s) involved, and diagnosis relies on tissue findings of dense infiltration of IgG4-positive plasma cells and a characteristic storiform fibrosis. Treatment is mainly with glucocorticoids, while multiple immunosuppressive medications can be used as adjuvant agents. Rituximab has showed promising results, but further studies are needed.

Objective: To describe the character and composition of the 2015 US adult rheumatology workforce, evaluate workforce trends, and project supply and demand for clinical rheumatology care for 2015-2030., Methods: The 2015 Workforce Study of Rheumatology Specialists in the US used primary and secondary data sources to estimate the baseline adult rheumatology workforce and determine demographic and geographic factors relevant to workforce modeling. Supply and demand was projected through 2030, utilizing data-driven estimations regarding the proportion and clinical full-time equivalent (FTE) of academic versus nonacademic practitioners., Results: The 2015 adult workforce (physicians, nurse practitioners, and physician assistants) was estimated to be 6,013 providers (5,415 clinical FTE). At baseline, the estimated demand exceeded the supply of clinical FTE by 700 (12.9%). By 2030, the supply of rheumatology clinical providers is projected to fall to 4,882 providers, or 4,051 clinical FTE (a 25.2% decrease in supply from 2015 baseline levels). Demand in 2030 is projected to exceed supply by 4,133 clinical FTE (102%)., Conclusion: The adult rheumatology workforce projections reflect a major demographic and geographic shift that will significantly impact the supply of the future workforce by 2030. These shifts include baby-boomer retirements, a millennial predominance, and an increase of female and part-time providers, in parallel with an increased demand for adult rheumatology care due to the growing and aging US population. Regional and innovative strategies will be necessary to manage access to care and reduce barriers to care for rheumatology patients., (© 2018, American College of Rheumatology.)

While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.

Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Should biologic therapy be added first in patients with active rheumatoid arthritis or should clinicians first add the less costly but effective combination of conventional therapies?, Competing Interests: Author disclosures The authors report no actual or potential conflicts of interest with regard to this article.

Objective: To examine whether vascular calcifications on hand films in RA might aid in determining mortality risk., Methods: Hand radiographs from 906 RA patients were scored as positive or negative for vascular calcifications. Patient characteristics associated with vascular calcifications were assessed using multivariable logistic regression, and associations with mortality were examined using Cox proportional hazards regression. Cytokines and multiplex ACPA were measured in both groups., Results: A total of 99 patients (11%) demonstrated radiographic vascular calcifications. Factors independently associated with vascular calcifications included diabetes [odds ratio (OR) 2.85; 95% CI 1.43, 5.66], cardiovascular disease at enrolment (OR 2.48; 95% CI 1.01, 6.09), prednisone use (OR 1.90; 95% CI 1.25, 2.91), current smoking (OR 0.06; 95% CI 0.01, 0.23) and former smoking (OR 0.36; 95% CI 0.27, 0.48) vs never smoking. In cytokine and ACPA subtype analysis, IL-4 and anti-citrullinated apolipoprotein E were significantly increased in patients with vascular calcifications in fully adjusted multivariable models. After multivariable adjustment, vascular calcifications were associated with an increase in all-cause mortality (hazard ratio 1.41; 95% CI 1.12, 1.78; P = 0.004)., Conclusion: Vascular calcifications on hand radiographs were independently associated with increased all-cause mortality in RA. Mechanisms underpinning the associations of IL-4 and select ACPA with vascular calcifications and their utility as biomarkers predictive of cardiovascular disease risk in RA merit further study., (Published by Oxford University Press on behalf of the British Society for Rheumatology 2015. This work is written by US Government employees and is in the public domain in the US.)

Background: This study examines: 1) alveolar bone loss (ABL), a hallmark of periodontitis, in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) patients versus control patients with osteoarthritis (OA); and 2) the association of ABL with RA disease activity and ACPA concentrations, including multiple antigen-specific ACPA., Methods: This multicenter case-control study includes 617 patients diagnosed with RA (n = 287) or OA (n = 330). Panoramic radiographs were taken; patients were categorized into low, moderate, or high tertiles based on mean percentage ABL. Serum ACPA was measured using second-generation anticyclic citrullinated peptide enzyme-linked immunosorbent assay and a multiplex platform to assess distinct antigen-specific ACPA. A generalized linear mixed model for binary data was used to compare stratified ABL in RA versus OA patients. Associations of moderate and high ABL (versus low) with RA disease activity and severity measures were examined using multivariate regression. Antigen-specific ACPA responses were compared among ABL tertiles using significance analysis of microarrays., Results: ACPA-positive patients with RA had a significantly higher mean percentage of sites with ABL >20% compared with patients with OA (P = 0.03). After multivariate adjustment, greater ABL was significantly associated with higher serum ACPA concentration (P = 0.004), 28-joint Disease Activity Score (P = 0.023), health assessment questionnaire disability (P = 0.05), tender joint count (P = 0.02) and joint space narrowing scores (P = 0.05) among patients with RA. ACPAs targeting citrullinated vimentin and histone were significantly higher in moderate and high ABL groups versus low, regardless of smoking status (q <0.1%)., Conclusions: Greater ABL was associated with higher ACPA, consistent with findings at articular sites. ACPA targeting could provide novel insight into important linkages between RA and periodontitis.

Objective: To examine the degree to which shared risk factors explain the relationship of periodontitis (PD) to rheumatoid arthritis (RA) and to determine the associations of PD and Porphyromonas gingivalis with pathologic and clinical features of RA., Methods: Patients with RA (n = 287) and patients with osteoarthritis as disease controls (n = 330) underwent a standardized periodontal examination. The HLA-DRB1 status of all participants was imputed using single-nucleotide polymorphisms from the extended major histocompatibility complex. Circulating anti-P gingivalis antibodies were measured using an enzyme-linked immunosorbent assay, and subgingival plaque was assessed for the presence of P gingivalis using polymerase chain reaction (PCR). Associations of PD with RA were examined using multivariable regression., Results: Presence of PD was more common in patients with RA and patients with anti-citrullinated protein antibody (ACPA)-positive RA (n = 240; determined using the anti-cyclic citrullinated peptide 2 [anti-CCP-2] test) than in controls (35% and 37%, respectively, versus 26%; P = 0.022 and P = 0.006, respectively). There were no differences between RA patients and controls in the levels of anti-P gingivalis or the frequency of P gingivalis positivity by PCR. The anti-P gingivalis findings showed a weak, but statistically significant, association with the findings for both anti-CCP-2 (r = 0.14, P = 0.022) and rheumatoid factor (RF) (r = 0.19, P = 0.001). Presence of PD was associated with increased swollen joint counts (P = 0.004), greater disease activity according to the 28-joint Disease Activity Score using C-reactive protein level (P = 0.045), and higher total Sharp scores of radiographic damage (P = 0.015), as well as with the presence and levels of anti-CCP-2 (P = 0.011) and RF (P < 0.001). The expression levels of select ACPAs (including antibodies to citrullinated filaggrin) were higher in patients with subgingival P gingivalis and in those with higher levels of anti-P gingivalis antibodies, irrespective of smoking status. Associations of PD with established seropositive RA were independent of all covariates examined, including evidence of P gingivalis infection., Conclusion: Both PD and P gingivalis appear to shape the autoreactivity of RA. In addition, these results demonstrate an independent relationship between PD and established seropositive RA., (Copyright © 2014 by the American College of Rheumatology.)

The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities., Competing Interests: The authors have declared that no competing interests exist.

Primary human somatic cells grown in culture divide a finite number of times, exhibiting progressive changes in metabolism and morphology before cessation of cycling. This telomere-initiated cellular senescence occurs because cells have halted production of telomerase, a DNA polymerase required for stabilization of chromosome ends. Telomerase-deficient Saccharomyces cerevisiae cells undergo a similar process, with most cells arresting growth after approximately 60 generations. In the current study we demonstrate that senescence is largely reversible. Reactivation of telomerase (EST2) expression in the growth-arrested cells led to resumption of cycling and reversal of senescent cell characteristics. Rescue was also observed after mating of senescent haploid cells with telomerase-proficient cells to form stable diploids. Although senescence was reversible in DNA damage checkpoint response mutants (mec3 and/or rad24 cells), survival of recombination-defective rad52 mutants remained low after telomerase reactivation. Telomere lengths in rescued est2 cells were initially half those of wildtype cells, but could be restored to normal by propagation for ∼70 generations in the presence of telomerase. These results place limitations on possible models for senescence and indicate that most cells, despite gross morphological changes and short, resected telomeres, do not experience lethal DNA damage and become irreversibly committed to death., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers.

Accurate protein identification in large-scale proteomics experiments relies upon a detailed, accurate protein catalogue, which is derived from predictions of open reading frames based on genome sequence data. Integration of mass spectrometry-based proteomics data with computational proteome predictions from environmental metagenomic sequences has been challenging because of the variable overlap between proteomic datasets and corresponding short-read nucleotide sequence data. In this study, we have benchmarked several strategies for increasing microbial peptide spectral matching in metaproteomic datasets using protein predictions generated from matched metagenomic sequences from the same human fecal samples. Additionally, we investigated the impact of mass spectrometry-based filters (high mass accuracy, delta correlation), and de novo peptide sequencing on the number and robustness of peptide-spectrum assignments in these complex datasets. In summary, we find that high mass accuracy peptide measurements searched against non-assembled reads from DNA sequencing of the same samples significantly increased identifiable proteins without sacrificing accuracy.

The availability of biologic agents targeting tumor necrosis factor (TNF)-alpha represents a significant advance in the management of rheumatoid arthritis. Anti-TNF-alpha therapy has been associated with dramatic improvements in the clinical signs and symptoms of rheumatoid arthritis and has been shown to greatly retard the destructive process that too often characterizes this condition. Although effective and well-tolerated in a substantial proportion of patients, primary and secondary failures of anti-TNF-alpha strategies have been well described, affecting up to one-third to one-half of subjects treated with these agents. Switching from one anti-TNF-alpha agent to a second (or even third) anti-TNF-alpha therapy has emerged as a means of addressing treatment failures with this drug class. This review examines data addressing the practice of switching anti-TNF-alpha agents in the context of initial treatment failure, with a focus on data from peer-reviewed reports.

Objective: To prospectively assess the efficacy of intramuscular (i.m.) triamcinolone acetonide in the treatment of pseudogout., Methods: Fourteen patients with crystal proven pseudogout presenting with an acute attack within 5 days of onset were treated with intramuscular triamcinolone acetonide 60 mg and followed for 30 days. Patients with inadequate response were eligible for a 2nd triamcinolone acetonide injection on Day 1-2., Results: Twelve patients had contraindication to nonsteroidal antiinflammatory agents (NSAID). Acute arthritis was monoarticular in 10 patients, and involved 2 or more joints in 4 patients. All patients had good clinical response to triamcinolone acetonide based on restoration of near baseline joint range of motion and joint circumference, and at least 50% improvement in patient and physician global assessment. Major clinical improvement occurred by Day 1-2 (2 patients), Day 3-4 (11 patients), and Day 10-14 (one patient). Six patients required a 2nd triamcinolone acetonide injection on Day 1-2. Toxicities were not observed., Conclusion: I.m. triamcinolone acetonide appears to be safe, well tolerated, and effective in the treatment of pseudogout. It may be a reasonable alternative therapy when NSAID are contraindicated, and for polyarticular attacks where intraarticular corticosteroids are impractical.

We describe a case of a young woman with a magnesium renal wasting syndrome leading to severe hypomagnesemia and a chronic pseudo-osteoaritis calcium pyrophosphate crystal deposition disease (CPDD) arthropathy. A chronic CPDD arthropathy secondary to hypomagnesemia has not been previously reported. The identification of CPDD, particularly in the young, can be a clue to the presence of a potentially treatable underlying metabolic disorder, such as hypomagnesemia.

Wide variability exists in the treatment of gout. We compared the treatment practices of rheumatologists with those of primary care physicians (PCPs) in the management of gout. Pharmacy records were reviewed to identify patients treated with colchicine, allopurinol, probenecid, or sulfinpyrazone. Forty PCP patients were compared with 33 patients followed by rheumatologists. Rheumatologists were three times more likely to confirm the diagnosis with joint aspiration and guide therapy with 24-h urine uric acid collections than were PCPs. Rheumatologists were more likely to use prophylaxis in acute gout before initiating uric acid-lowering therapy than were PCPs. All PCP patients were treated with allopurinol compared with 65% of rheumatology patients. Mean posttreatment uric acid levels were lower for rheumatology patients (5.0 mg/dL) compared with PCP patients (6.0 mg/dL). Previous studies have reported poor symptom control and increased toxicity in gouty patients with suboptimal treatment. With the vast majority of patients being treated by PCPs in a man-aged care setting, further studies will be necessary to determine whether treatment variability affects outcome between the two groups.

We present a 42-year-old woman with primary Sjögren's syndrome and a polyclonal gammopathy who presented with pretibial petechiae, purpura, and tender indurated plaques. The indurated plaques revealed a lobular plasma cell panniculitis, and thus Sjögren's syndrome should be added to the short list of collagen vascular diseases that can present as plasma cell panniculitis. Her biopsies also demonstrated intense perieccrine plasma cell infiltrates that may account for Sjögren's syndrome-associated hypohidrosis. We also observed occasional vascular occlusion of vessels with an amorphous, eosinophilic material possibly related to her hypergammaglobulinemic purpura.

Objective: To test the usefulness and cost savings resulting from application of the new American College of Rheumatology (ACR) guidelines for assessing the risk for the development of clinically significant liver disease in rheumatoid arthritis (RA) patients treated with methotrexate (MTX)., Methods: One-hundred twelve MTX-treated RA patients were prospectively followed up for MTX hepatotoxicity and underwent liver biopsies according to modified guidelines of the Psoriatic Task Force (PTF). All biopsies were graded according to the Roenigk classification. The new ACR recommendations were then retrospectively applied to test their usefulness and cost-effectiveness in this cohort., Results: Based on the PTF guidelines, 66 patients underwent liver biopsies; a total of 110 liver biopsies were performed. Two patients had biopsy-related complications. Five patients were found to have Roenigk grade IIIB or IV histologic abnormalities. The total cost for this group was $111,380. Applying the new ACR criteria, only 15 patients would have undergone liver biopsies; there would have been a total of 18 biopsies, with no complications. Four of the 5 patients with Roenigk grade IIIB or IV liver abnormalities would have been identified. One patient with insulin-dependent diabetes mellitus (IDDM) who was found to have cirrhosis (Roenigk grade IV) on liver biopsy as a result of use of the PTF guidelines would have been missed with use of the ACR guidelines. The total cost for the group receiving biopsies based on the ACR guidelines would have been $16,956. Overall, the new ACR guidelines had 80% sensitivity and 82% specificity and resulted in a cost savings of $1,430 per patient., Conclusion: The new ACR guidelines on MTX monitoring and biopsy surveillance appear to be clinically useful and result in considerable cost savings. However, 1 IDDM patient with significant liver histologic abnormalities would have been missed. We suggest that IDDM be added to the ACR guidelines as a risk factor for MTX hepatotoxicity.

Purpose: To examine the potential relationship between gout and hypothyroidism., Patients and Methods: Fifty-four consecutive patients with a diagnosis of monosodium urate crystal-proven gouty arthritis on joint aspiration were prospectively evaluated for hypothyroidism with an ultrasensitive thyroid-stimulating hormone (TSH) assay. Twenty-five patients with a diagnosis of monosodium urate crystal-proven gout were retrospectively identified from a population of 137 patients receiving uric acid-lowering medications. These patients were also screened for hypothyroidism. Age, race, sex, and weight matched patients with noninflammatory rheumatic diseases and no history of gout served as controls. Hypothyroidism was diagnosed when a TSH was greater than 6.0 microU/mL or if a history of hypothyroidism requiring replacement therapy was documented., Results: The prevalence of hypothyroidism in the prospective group was significantly increased compared to controls (P < 0.05). Overall 15% of these patients, 25% of women and 12% of the men, had hypothyroidism. These rates were 2.5 times greater in women and 6 times greater in men than found in the controls. The mean TSH of the prospective gouty patients was also significantly greater than those levels found in control patients (5.2 +/- 12 versus 1.8 +/- 1.1 microU/mL, P < 0.05, chi-square), even when all abnormally elevated TSH values were excluded from analysis. The prevalence of hypothyroidism in the retrospective group was even higher: 20% overall, 40% in women and 15% in men., Conclusions: The prevalence of hypothyroidism is significantly increased in patients with aspirate-proven gouty arthritis. Screening for hypothyroidism with an ultrasensitive thyroid-stimulating hormone assay should be considered in all patients presenting with gouty arthritis and those with a history of recurrent gouty flares.

The objectives of this experimental study were to determine (a) how quickly the medial collateral ligament (MCL) and the anterior cruciate ligament (ACL) elongate when a lateral impact force is imparted to the knee and if a person can react rapidly enough to provide protective muscle forces in the case of such an impact, (b) if the MCL and the ACL elongate simultaneously during a lateral impact, and (c) if resection of the ACL affects elongation of the MCL during a lateral impact. Eight whole-leg cadaver specimens were used. Each leg was mounted vertically in a testing-frame with the knee in 0 and 30 degrees of flexion. A submaximal impact was delivered from the lateral side by a pendulum instrumented with a force transducer. Elongation of the midsubstance of the MCL and the ACL was measured with Hall-effect displacement transducers. The ACL was resected and the entire test sequence was repeated. Following a lateral impact, elongation of the MCL and ACL reached peak values by 70 ms. This study indicated that contraction of the leg musculature would not protect the MCL and ACL from injury when a lateral impact load is applied to the knee. The MCL and the ACL never elongated simultaneously during a lateral impact. After lateral impact loading, the time required to reach maximum elongation (peak delay) averaged 52 ms in the anterior MCL fibers and 61 ms in the ACL when the knee was in 0 degrees of flexion. At 30 degrees of flexion, the peak delay averaged 38 ms in the anterior MCL fibers and 22 ms in the ACL.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the ability of prophylactic knee braces to reduce or limit medial collateral and anterior cruciate ligament elongation under dynamic loading conditions, we used cadaveric specimens that had a surrogate soft tissue material that matched the tissue compliance of in vivo contracted muscles. Eight cadaveric specimens were fitted with four prophylactic knee braces and instrumented with Hall Effect Strain Transducers on both the medial collateral and anterior cruciate ligament. Each specimen was mounted in a testing frame while a lateral impact was applied to the knee joint by a pendulum at levels below the injury threshold. Legs were tested at 0 degrees and 30 degrees of knee flexion, both with and without an intact anterior cruciate ligament. The maximum elongation for each ligament was calculated as a percentage of the initial measured length. The addition of a prophylactic knee brace significantly reduced the level of impact force at the point of impact, but this did not result in a significant reduction of anterior cruciate ligament elongation for any test. Although not significant, all braces tested were more effective at reducing medial collateral ligament elongation during a lateral impact with the knee flexion at 30 degrees than at 0 degrees.