Objective
Rheumatoid arthritis (RA) is thought to be a T cell–mediated disease, based on its strong association with HLA class II alleles, clinical responsiveness to T cell–directed therapies, and ...the presence of CD4+ T cells in rheumatoid joints. The presence of anti–citrullinated protein antibodies (ACPAs) in RA serum and the association of these antibodies with HLA–DR4 alleles implicate citrulline‐specific autoreactive T cells in the development and progression of RA. The goal of this study was to determine the characteristics and specificity of autoreactive T cell responses in RA.
Methods
We developed a panel of HLA–DRB1*04:01 tetramers, selecting citrullinated peptides from synovial antigens and verifying their immunogenicity in DRB1*04:01‐transgenic mice. Seven tetramers were used to examine the ex vivo frequency and surface phenotype of citrulline‐specific (Cit‐specific) T cells in patients with RA and healthy subjects with DRB1*04:01 haplotypes, using a magnetic enrichment procedure.
Results
Cit‐specific T cells were detectable in peripheral blood samples from both healthy subjects and RA patients. In comparison to healthy subjects, RA patients had significantly higher frequencies of Cit‐specific T cells, and a greater proportion of these cells displayed a Th1 memory phenotype. Among RA patients, the frequency of Cit‐specific T cells was highest within the first 5 years after diagnosis of RA and was decreased in patients taking biologic agents, irrespective of disease duration.
Conclusion
These findings link the presence of ACPAs in RA with Th1 cells specific for citrullinated epitopes and provide tools for disease‐specific immunomonitoring of autoreactive T cells.
ACPA-positive rheumatoid arthritis (RA) is associated with distinct HLA-DR alleles and immune responses to many citrullinated self-antigens. Herein we investigated the T cell epitope confined within ...α-enolase326-340 in the context of HLA-DRB1*04:01 and assessed the corresponding CD4+ T cells in both the circulation and in the rheumatic joint. Comparative crystallographic analyses were performed for the native and citrullinated α-enolase326-340 peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for ex vivo and in vitro assessment of α−enolase-specific T cells in peripheral blood, synovial fluid and synovial tissue by flow cytometry. The native and modified peptides take a completely conserved structural conformation within the peptide-binding cleft of HLA-DRB1*04:01. The citrulline residue-327 was located N-terminally, protruding towards TCRs. The frequencies of T cells recognizing native eno326-340 were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno326-340 was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, p = 0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, p = 0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within α-enolase326-340 does not appear to lead to complete negative selection of cognate CD4+ T cells. In RA patient samples, only T cells recognizing the citrullinated version of α-enolase326-340 were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance.
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•Antibody responses to citrullinated proteins are common in RA and associate to HLA-DR alleles.•Citrullinated peptides can be presented to T cells on HLA-DR.•Alpha-enolase peptide 326–340 binds HLA-DRB1*04:01 in both its native and citrullinated form.•Crystal structures demonstrate the citrulline/arginine protruding towards the TCR contact area.•HLA-DR tetramer staining showed elevated frequencies of cit-specific T cells in the RA joint.
A pilot-plant scale desymmetrization of the cyclic meso-epoxide 4b, using a chiral lithium amide prepared from symmetrical diamine 17, was designed and implemented to provide allylic alcohol 3b in ...high yield and greater than 99% ee. This chiral alcohol was converted to ketone 2b, a key intermediate in a new asymmetric synthesis of LY459477. Chiral diamine 17 was prepared from a readily available chiral precursor, (R)-α-methylbenzylamine, and could be recovered from the reaction mixture and reused. Studies performed to probe the mechanism of the rearrangement reaction of epoxide 4b showed that diamine 17 provided an optimal combination of selectivity and scaleability for this process.
The shelf‐life of cold and hot water extraction coffees based on sensory and chemical profiles and microbial growth was examined, which also allowed the study of the influence of extraction ...temperature on the chemical and sensorial profiles of coffee. The shelf life of refrigerated cold‐ and hot‐brewed coffee was limited not by microbial stability but rather by deterioration in sensory attributes. Further work is recommended to elucidate the mechanisms of coffee staling in a refrigerated environment, with particular interest in the degradation products of chlorogenic acid, as a significant decline in chlorogenic acid concentration was found over the storage period. Cold‐extracted coffees were found to be chemically and sensorially different beverages from coffees extracted at high temperatures. Additionally, the cold‐brewed coffees had greater sensory flavor stability over the storage time than the hot‐brewed treatment.
Practical application
This study advances the industry's understanding of the shelf life of ready‐to‐drink bottled cold coffees and demonstrates that lower brewing temperatures lead to greater flavor stability over shelf life. The findings also provide brewing parameters that can help guide product developers in modulating the flavor of commercial cold coffees.
The shelf‐life of cold and hot water extraction coffees based on sensory and chemical profiles and microbial growth was examined, which also allowed the study of the influence of extraction temperature on the chemical and sensorial profiles of coffee. The shelf life of refrigerated cold‐ and hot‐brewed coffee was limited not by microbial stability but rather by deterioration in sensory attributes. Additionally, the cold‐brewed coffees had greater sensory flavor stability over the storage time than the hot‐brewed treatment.
The onset of pancreas development in the foregut endoderm is marked by activation of the homeobox gene Pdx1 (IPF1). Pdx1 is essential for the expansion of the pancreatic primordium and the ...development of endocrine islets. The control of Pdx1 expression has been only partially elucidated. We demonstrate here that the winged-helix transcription factors Foxa1 and Foxa2 co-occupy multiple regulatory domains in the Pdx1 gene. Compound conditional ablation of both Foxa1 and Foxa2 in the pancreatic primordium results in complete loss of Pdx1 expression and severe pancreatic hypoplasia. Mutant mice exhibit hyperglycemia with severely disrupted acinar and islet development, and die shortly after birth. Assessment of developmental markers in the mutant pancreas revealed a failure in the expansion of the pancreatic anlage, a blockage of exocrine and endocrine cell differentiation, and an arrest at the primitive duct stage. Comparing their relative developmental activity, we find that Foxa2 is the major regulator in promoting pancreas development and cell differentiation. Using chromatin immunoprecipitations (ChIP) and ChIP sequencing (ChIPSeq) of fetal pancreas and islet chromatin, we demonstrate that Foxa1 and Foxa2 predominantly occupy a distal enhancer at -6.4 kb relative to the transcriptional start site in the Pdx1 gene. In addition, occupancy of the well-characterized proximal Pdx1 enhancer by Foxa1 and Foxa2 is developmental stage-dependent. Thus, the regulation of Pdx1 expression by Foxa1 and Foxa2 is a key early event controlling the expansion and differentiation of the pancreatic primordia.
► We provide the first standardized chemical durability test on tellurite glasses. ► The glasses we studied showed a wide variety of chemical durability. ► The best-performing glass showed good ...halide retention following melting and durability testing. ► These glasses have very high densities resulting in high volumetric waste loading ability.
Tellurite glasses have historically been shown to host large concentrations of halides. They are here considered for the first time as a waste form for immobilizing chloride wastes, such as may be generated in the proposed molten alkali salt electrochemical separations step in nuclear fuel reprocessing. Key properties of several tellurite glasses are determined to assess acceptability as a chloride waste form. TeO2 glasses with other oxides (PbO, Al2O3+B2O3, WO3, P2O5, or ZnO) were fabricated with and without 10mass% of a simulated (non-radioactive) mixed alkali, alkaline-earth, and rare earth chloride waste. Measured chemical durability is compared for the glasses, as determined by the product consistency test (PCT), a common standardized chemical durability test often used to validate borosilicate glass waste forms. The glass with the most promise as a waste form is the TeO2–PbO system, as it offers good halide retention, a low sodium release (by PCT) comparable with high-level waste silicate glass waste forms, and a high storage density.
Objective
Antibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA–DRB1*0401. This suggests that T cells ...specific for peptides derived from citrullinated vimentin presented in the context of HLA–DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA–DRB1*0401 and to characterize the resulting T cell responses.
Methods
We first predicted an HLA‐binding T cell epitope from citrullinated vimentin based on the binding motif of HLA–DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59–78 (cit‐vimentin aa 59–78) was constructed and used to screen for specific T cells in HLA–DRB1*0401–transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit‐vimentin aa 59–78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex‐based analysis.
Results
The citrullinated form of vimentin aa 59–78 bound to HLA–DRB1*0401, but the native form could not. Subsequently, cit‐vimentin aa 59–78–specific T cells were detected in immunized mice and in the periphery of both HLA–DR*0401–positive healthy control subjects and HLA–DR*0401–positive patients with RA, using class II MHC tetramers, CD154 up‐regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon‐γ) in response to cit‐vimentin aa 59–78 was significantly higher in patients compared with controls.
Conclusion
Here, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA–DRB1*0401–restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.
To evaluate the frequency and appropriateness of nil per os (nothing by mouth) (NPO) orders and determine the number of meals missed because of these orders among hospitalized patients.
We ...retrospectively analyzed inpatient NPO orders at an academic institution in the United States. The frequency and duration of NPO orders and the number of meals missed because of these orders were assessed for adult patients admitted to the hospital medicine services from January 1, 2013, through December 31, 2013, with a hospital stay of 2 or more and 30 or fewer days. Two blinded reviewers assessed if the order could be avoided or the period shortened for a random sample of NPO orders of 120 or more minutes' duration that were written for patients on the general medicine ward.
A total of 3641 NPO orders were identified. At least one NPO order was placed in 46.6% of the admissions (2211 of 4743). The median duration of NPO orders was 12.8 hours (interquartile range, 9.2-17.3 hours), resulting in 2 (interquartile range, 1-4) missed meals. Of 1130 NPO orders reviewed, 263 (23.3%; 95% CI, 20.9%-25.8%) were deemed avoidable (κ statistic, 0.68), and 482 (42.7%) were unavoidable but led to more missed meals than needed. Taken together, patients could have had 44.8% of the meals (1085 of 2424; 95% CI, 42.8%-46.7%) missed due to NPO orders.
Approximately half of the patients admitted to the hospital medicine services experienced a period of fasting. One in 4 NPO orders and nearly half of missed meals could have been avoided. Further study is warranted to assess the generalizability of our findings.
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•FRET was used to monitor Ca2+-dependent N-cTnC opening in skinned myocardial fibers.•Inhibition of strong cross-bridges reduced the maximal extent of N-cTnC opening.•Cycling ...cross-bridges enhanced the overall exposure of the N-cTnC hydrophobic patch.•Non-cycling strong cross-bridges stimulated N-cTnC opening under nanomolar Ca2+.•The overall stability of N-cTnC opening correlated positively with Ca2+ sensitivity.
The in situ structural coupling between the cardiac troponin (cTn) Ca2+-sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using Förster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca2+ titrations of cTnC(T13C/N51C)AEDENS/DDPM-reconstituted fibers showed that the Ca2+-dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force−Ca2+ relationship. N-cTnC opening still occurred steeply during Ca2+ titrations in the presence of 1mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca2+-sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca2+ levels, treatment with ADP·Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca2+ titrations in the presence of ADP·Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca2+ with increased Ca2+-sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca2+-sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI.
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