Objective
Rheumatoid arthritis (RA) is characterized by the activation of B cells that produce anti–citrullinated protein antibodies (ACPAs) and rheumatoid factors (RFs), but the mechanisms by which ...tolerance is broken in these B cells remain incompletely understood. We undertook this study to investigate whether ACPA+ and RF+ B cells break tolerance through distinct molecular mechanisms.
Methods
We developed antigen–tetramers to isolate ACPA+ and RF+ B cells and performed single‐cell RNA sequencing on 2,349 B cells from 6 RA patients and 1 healthy donor to analyze their immunoglobulin repertoires and transcriptional programs. Prominent immunoglobulins were expressed as monoclonal antibodies and tested for autoantigen reactivity.
Results
ACPA+ and RF+ B cells were enriched in the peripheral blood of RA patients relative to healthy controls. Characterization of patient‐derived monoclonal antibodies confirmed ACPA and RF targeting of tetramer‐specific B cells at both antigen‐inexperienced and affinity‐matured B cell stages. ACPA+ B cells used more class‐switched isotypes and exhibited more somatic hypermutations relative to RF+ B cells, and these differences were accompanied by down‐regulation of CD72 and up‐regulation of genes that promote class‐switching and T cell–dependent responses. In contrast, RF+ B cells expressed transcriptional programs that stimulate rapid memory reactivation through multiple innate immune pathways. Coexpression analysis revealed that ACPA+ and RF+ B cell–enriched genes belong to distinct transcriptional regulatory networks.
Conclusion
Our findings suggest that ACPA+ and RF+ B cells are imprinted with distinct transcriptional programs, which suggests that these autoantibodies associated with increased inflammation in RA arise from 2 different molecular mechanisms.
Objective
Anti–citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). While epitope spreading of the serum ACPA response is believed to contribute to RA pathogenesis, ...little is understood regarding how this phenomenon occurs. This study was undertaken to analyze the antibody repertoires of individuals with RA to gain insight into the mechanisms leading to epitope spreading of the serum ACPA response in RA.
Methods
Plasmablasts from the blood of 6 RA patients were stained with citrullinated peptide tetramers to identify ACPA‐producing B cells by flow cytometry. Plasmablasts were single‐cell sorted and sequenced to obtain antibody repertoires. Sixty‐nine antibodies were recombinantly expressed, and their anticitrulline reactivities were characterized using a cyclic citrullinated peptide enzyme‐linked immuosorbent assay and synovial antigen arrays. Thirty‐six mutated antibodies designed either to represent ancestral antibodies or to test paratope residues critical for binding, as determined from molecular modeling studies, were also tested for anticitrulline reactivities.
Results
Clonally related monoclonal ACPAs and their shared ancestral antibodies each exhibited differential reactivity against citrullinated antigens. Molecular modeling identified residues within the complementarity‐determining region loops and framework regions predicted to be important for citrullinated antigen binding. Affinity maturation resulted in mutations of these key residues, which conferred binding to different citrullinated epitopes and/or increased polyreactivity to citrullinated epitopes.
Conclusion
These results demonstrate that the different somatic hypermutations accumulated by clonally related B cells during affinity maturation alter the antibody paratope to mediate epitope spreading and polyreactivity of the ACPA response in RA, suggesting that these may be key properties that likely contribute to the pathogenicity of ACPAs.
Objective
A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti–citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these ...autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen(s) are activated and differentiate into plasmablasts, which are released into the blood. We undertook this study to sequence the plasmablast antibody repertoire to define the targets of the active immune response in RA.
Methods
We developed a novel DNA barcoding method to sequence the cognate heavy‐ and light‐chain pairs of antibodies expressed by individual blood plasmablasts in RA. The method uses a universal 5′ adapter that enables full‐length sequencing of the antibodies' variable regions and recombinant expression of the paired antibody chains. The sequence data sets were bioinformatically analyzed to generate phylogenetic trees that identify clonal families of antibodies sharing heavy‐ and light‐chain VJ sequences. Representative antibodies were expressed, and their binding properties were characterized using anti–cyclic citrullinated peptide 2 (anti–CCP‐2) enzyme‐linked immunosorbent assay (ELISA) and antigen microarrays.
Results
We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts from 4 individuals with anti‐CCP+ RA, and recombinantly expressed 14 antibodies that were either “singleton” antibodies or representative of clonal antibody families. Anti–CCP‐2 ELISA identified 4 ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α‐enolase, citrullinated fibrinogen, and citrullinated histone H2B.
Conclusion
Our data provide evidence that autoantibodies targeting α‐enolase, citrullinated fibrinogen, and citrullinated histone H2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA.
Idiopathic pulmonary arterial hypertension (IPAH) is a devastating pulmonary vascular disease in which autoimmune and inflammatory phenomena are implicated. B cells and autoantibodies have been ...associated with IPAH and identified as potential therapeutic targets. However, the specific populations of B cells involved and their roles in disease pathogenesis are not clearly defined. We aimed to assess the levels of activated B cells (plasmablasts) in IPAH, and to characterize recombinant antibodies derived from these plasmablasts. Blood plasmablasts are elevated in IPAH, remain elevated over time, and produce IgA autoantibodies. Single‐cell sequencing of plasmablasts in IPAH revealed repertoires of affinity‐matured antibodies with small clonal expansions, consistent with an ongoing autoimmune response. Recombinant antibodies representative of these clonal lineages bound known autoantigen targets and displayed an unexpectedly high degree of polyreactivity. Representative IPAH plasmablast recombinant antibodies stimulated human umbilical vein endothelial cells to produce cytokines and overexpress the adhesion molecule ICAM‐1. Together, our results demonstrate an ongoing adaptive autoimmune response involving IgA plasmablasts that produce anti‐endothelial cell autoantibodies in IPAH. These antibodies stimulate endothelial cell production of cytokines and adhesion molecules, which may contribute to disease pathogenesis. These findings suggest a role for mucosally‐driven autoimmunity and autoimmune injury in the pathogenesis of IPAH.
Idiopathic pulmonary arterial hypertension (IPAH) is a rare but deadly pulmonary vascular disease. We report that IgA‐producing blood plasmablasts are elevated in IPAH, and that antibodies derived from sequencing these plasmablasts stimulate endothelial cell production of inflammatory mediators. Our findings suggest that mucosally‐driven autoimmunity contributes to the pathogenesis of IPAH.
NK cells directly kill mycobacteria through a contactdependent mechanism leading to the release of perforin and granulysin.
Although the mechanisms underlying the cytotoxic effect of NK cells on ...tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell‐surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.
Abstract We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach ...to elucidate the plasmablast antibody response to influenza vaccination. We show that > 75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average > 20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that ‘original antigenic sin’ shapes the antibody response to influenza vaccination.
Background/purpose Carbapenem-resistant Pseudomonas aeruginosa infections have been a challenge and issue in hospital settings. However, the clinical impact of P. aeruginosa blood isolates resistant ...only to carbapenems has never been discussed previously. Methods To assess the risk factors and clinical significance of bacteremia caused by carbapenem resistance only P. aeruginosa (CROPA), a 6-year retrospective case–control study was conducted. The CROPA strains were defined as isolates susceptible to ciprofloxacin, antipseudomonal penicillins and cephalosporins, and aminoglycosides but resistant to one antipseudomonal carbapenem (imipenem or meropenem) or both. The controls were selected among patients with bacteremia due to P. aeruginosa susceptible to all above classes of antipseudomonal antibiotics, which was defined as all-susceptible P. aeruginosa. Results Twenty-five patients had at least one blood culture positive for CROPA, and 50 controls had all-susceptible P. aeruginosa bacteremia. CROPA bacteremia had a high 30-day mortality rate (72.0%), as compared to 26.0% for the controls ( p < 0.001). Through multivariate analysis, carbapenem exposure was the only risk factor for developing CROPA bacteremia ( p = 0.002). A comparison between the surviving and deceased patients with CROPA bacteremia showed that nine (50%) of those who died, but none of the survivors, received carbapenems as the initial empirical therapy ( p = 0.027). Conclusion Carbapenem exposure was associated with emergence of CROPA infections. Repeated carbapenem use in such patients might increase rates of inappropriate initial empirical treatment and mortality. Prudent carbapenem use is important to reduce the emergence of CROPA.
Abstract Background Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in microbial identification. This study evaluated the performance ...of MALDI-TOF MS and investigated the economic and medical impact of MALDI-TOF MS implementation. Methods A total of 12,202 clinical isolates collected from April to September 2013 were identified using MALDI-TOF MS, and the success rates in identifying isolates were analyzed. The differences in the processing time, cost of consumables, weight of waste, and clinical impact between MALDI-TOF MS and biochemical reaction were compared. Results MALDI-TOF MS successfully identified 96% of 12,202 isolates, including 96.8% of 10,502 aerobes, 90.5% of 1481 anaerobes, 93.8% of 81 yeasts, and 90.6% of 138 nontuberculous mycobacteria at the genus level. By using MALDI-TOF MS, the processing time for aerobes decreased from 32.5 hours to 4.1 hours, and that for anaerobes decreased from 71.5 hours to 46 hours. For detection of aerobes and anaerobes, the cost of consumables was estimated to decrease by US$0.9 per isolate, thus saving US$94,500 in total annual isolation. Furthermore, the weight of waste decreased six-fold, resulting in a reduction of 350 kg/month or 4.2 tons/year. MALDI-TOF MS also increased the percentage of correct antibiotics treatment for Escherichia coli and Klebsiella pneumonia from 56.1% to 75% and shortened the initiation time of the correct antibiotic action from 3.3 hours to 2.5 hours. Conclusions MALDI-TOF MS is a rapid, reliable, economical, and environmentally friendly method for routine microbial identification and may contribute to early appropriate antibiotic treatment in clinical settings.
Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the ...nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαβ pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9–23 and proinsulin. Consistently, insulin B:9–23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28−/− recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.
Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated ...epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (n = 48, 35.8%), ST414 (n = 22, 16.4%), and ST78 (n = 16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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