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1.
Arthritis Res Ther ; 23(1): 29, 2021 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-33451338

RESUMO

OBJECTIVES: Type I interferons (IFNs) play an important role in the pathophysiology of systemic lupus erythematosus (SLE). While cross-sectional data suggest an association between IFN-induced gene expression and SLE disease activity, interest in this as a biomarker of flare has been tempered by a lack of fluctuation with disease activity in the majority of patients. This led us to question whether IFN-induced gene expression might instead be a biomarker of overall disease severity, with patients with high levels spending more time in an active disease state. METHODS: Levels of five interferon-responsive genes were measured in the whole peripheral blood at baseline visit for 137 SLE patients subsequently followed for 5 years. Log transformed values were summed to yield a composite IFN5 score, and the correlation with various disease outcomes examined. Receiver operator characteristic analyses were performed for outcomes of interest. Kaplan-Meier curves were generated to compare the proportion of flare-free patients with high and low IFN5 scores over time. RESULTS: The baseline IFN5 score was positively correlated with the adjusted mean SLE disease activity index-2000, number of flares, adjusted mean prednisone dose, and number of new immunosuppressive medications over the subsequent 5 years. Optimal cut-offs for the IFN5 score were determined using Youden's index and predicted more severe outcomes with 57-67% accuracy. A high baseline IFN5 level was associated with a significantly increased risk of subsequent flare. CONCLUSIONS: Measurement of the type I IFN signature is a useful tool for predicting the subsequent disease activity course.

2.
J Rheumatol ; 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060314

RESUMO

OBJECTIVE: Specific risk alleles for childhood-onset SLE (cSLE) versus adult-onset SLE (aSLE) patients have not been identified. The aims of this study were to determine if: 1) There is an association between non-HLA-related genetic risk score (GRS) and age of SLE diagnosis; and if 2) There is an association between HLA-related genetic risk score and age of SLE diagnosis. METHODS: Genomic DNA was obtained from 2,001 multi-ethnic patients and genotyped using the Immunochip. Following quality control, genetic risk counting (GRCS), weighted (GRWS) and standardized counting (GRSCS) and standardized weighted (GRSWS) scores were calculated based on independent SNPs from validated SLE-loci. Scores were analyzed in a regression model and adjusted by sex and ancestral population. RESULTS: The analysed cohort consisted of 1,540 patients: 1,351 females and 189 males (675 cSLE and 865 aSLE). There were significant negative associations with age of SLE diagnosis p=0.011 and r2=0.175 for GRWS, p=0.008 and r2=0.178 for GRSCS, p=0.002 and r2=0.176 for GRSWS for all non-HLA genetic risk scores (higher GRS the lower the age of diagnosis.) All HLA genetic risk scores showed significant positive associations with age of diagnosis p=0.049 and r2=0.176 for GRCS, p=0.022 and r2=0.176 for GRWS, p=0.022 and r2=0.176 for GRSCS, p=0.011 and r2=0.177 for GRSWS: higher genetic scores correlated with higher age of diagnosis. CONCLUSION: Our data suggested that there is a linear relationship between genetic risk and age of SLE diagnosis and that HLA and non-HLA genetic risk scores are associated with age of diagnosis in opposite directions.

3.
PLoS One ; 15(7): e0236664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722684

RESUMO

BACKGROUND: Systemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population. METHODS: Flow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy. RESULTS: c1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice. CONCLUSION: Anergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production.


Assuntos
Anticorpos Antinucleares/genética , Anticorpos Antifosfolipídeos/genética , Antígenos/imunologia , Linfócitos B/imunologia , Anergia Clonal , Lúpus Eritematoso Sistêmico/imunologia , Animais , Linfócitos B/citologia , Proliferação de Células , DNA de Cadeia Simples/imunologia , Suscetibilidade a Doenças , Técnicas de Introdução de Genes , Lúpus Eritematoso Sistêmico/genética , Camundongos
4.
Arthritis Care Res (Hoboken) ; 72(12): 1809-1819, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31628718

RESUMO

OBJECTIVE: Screening for cognitive impairment in systemic lupus erythematosus (SLE) conventionally relies on the American College of Rheumatology (ACR) neuropsychologic battery (NB), which is not universally available. To develop a more accessible screening approach, we assessed validity of the Automated Neuropsychological Assessment Metrics (ANAM). Using the ACR NB as the gold standard for cognitive impairment classification, the objectives were 1) to measure overall discriminative validity of the ANAM for cognitive impairment versus no cognitive impairment, 2) to identify ANAM subtests and scores that best differentiate patients with cognitive impairment from those with no cognitive impairment, and 3) to derive ANAM composite indices and cutoffs. METHODS: A total of 211 consecutive adult patients, female and male, with SLE were administered the ANAM and ACR NB. 1) For overall discriminative validity of the ANAM, we compared patients with cognitive impairment versus those with no cognitive impairment on 4 scores. 2) Six ANAM models using different scores were developed, and the most discriminatory subtests were selected using logistic regression analyses. The area under the receiver operating characteristic curve (AUC) was calculated to establish ANAM validity against the ACR NB. 3) ANAM composite indices and cutoffs were derived for the best models, and sensitivities and specificities were calculated. RESULTS: Patients with no cognitive impairment performed better on most ANAM subtests, supporting ANAM's discriminative validity. Cognitive impairment could be accurately identified by selected ANAM subtests with top models, demonstrating excellent AUCs of 81% and 84%. Derived composite indices and cutoffs demonstrated sensitivity of 78-80% and specificity of 70%. CONCLUSION: This study provides support for ANAM's discriminative validity for cognitive impairment and utility for cognitive screening in adult SLE. Derived composite indices and cutoffs enhance clinical applicability.

5.
Semin Arthritis Rheum ; 50(1): 84-94, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31303437

RESUMO

OBJECTIVES: To systematically review and synthesize literature on 1) the overall prevalence of depression and anxiety in SLE patients in identified studies, and 2) the pooled prevalence per metrics of depression and anxiety in adult SLE patients. METHODS: This review used (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) PRISMA guidelines and in-depth searches in four databases (1954-2016; Ovid-based Medline, Embase, PsycINFO and CINAHL) to identify articles on the prevalence of depression and/or anxiety in adult SLE patients. Included studies were critically appraised and analyzed. The prevalence of depression and anxiety was studied for all included studies, and whenever possible, pooled prevalence (PP) was determined for more commonly used metrics. Statistical and publication bias was assessed using funnel plots. RESULT: A total of 3103 references were identified, 226 were selected for detailed review and 72 were included in the final analysis. OVERALL PREVALENCE: The depression PP, obtained from 69 studies representing 23,386 SLE patients, was 35.0% (95% CI: 29.9%-40.3%). The anxiety PP, obtained from 38 studies representing 4439 SLE patients, was 25.8% (95% CI: 19.2%-32.9%). PREVALENCE PER METRICS USED: The more commonly used instruments included the Centre for Epidemiological Studies - Depression (CES-D), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), Hospital Anxiety and Depression Scales (HADS-A/D), and Hamilton Rating Scales for Depression/Anxiety (HAM-D/A)]. The CES-D was utilized in 13 studies including 1856 SLE patients; depression PP was 41.5% (95% CI: 35.1%-48.1%). The BDI was utilized in 14 studies including 1355 SLE patients and the BAI in 3 studies including 489 patients; depression PP was 39.9% (95% CI: 31.1%-49.1) and anxiety PP was 38.4% (95% CI: 34.2%-42.8%). The HADS-D was utilized in 14 studies including 1238 SLE patients and the HADS-A in 12 studies including 1099 patients respectively; its depression PP was 24.4% (95% CI: 19.1%-30.1%) and anxiety PP was 38.3% (95% CI: 29.1%-47.9%). The HAM-D was utilized in 4 studies including 267 SLE patients and the HAM-A in 4 studies including 213 patients respectively; its depression PP was 40.0% (95% CI: 23.0%-59.0%) and anxiety PP was 39.0% (95% CI: 32.0%-45.0%). CONCLUSION: There was high variability in the prevalence of depression and anxiety, ranging from 8.7%-78.6% and 1.1%-71.4%, respectively. This could be attributed to the lack of consistency in the metrics used and its definition for depression and anxiety in SLE. Studies that used a specific metric, such as the CES-D, BDI or HAM-D, yielded similar depression prevalence. The HADS-D had the lowest prevalence. All metrics of anxiety yielded similar anxiety prevalence.

6.
Rheumatology (Oxford) ; 59(1): 90-98, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31236574

RESUMO

OBJECTIVE: LN is one of the most common and severe manifestations of SLE. Our aim was to test the association of SLE risk loci with LN risk in childhood-onset SLE (cSLE) and adult-onset SLE (aSLE). METHODS: Two Toronto-based tertiary care SLE cohorts included cSLE (diagnosed <18 years) and aSLE patients (diagnosed ⩾18 years). Patients met ACR and/or SLICC SLE criteria and were genotyped on the Illumina Multi-Ethnic Global Array or Omni1-Quad arrays. We identified those with and without biopsy-confirmed LN. HLA and non-HLA additive SLE risk-weighted genetic risk scores (GRSs) were tested for association with LN risk in logistic models, stratified by cSLE/aSLE and ancestry. Stratified effect estimates were meta-analysed. RESULTS: Of 1237 participants, 572 had cSLE (41% with LN) and 665 had aSLE (30% with LN). Increasing non-HLA GRS was significantly associated with increased LN risk [odds ratio (OR) = 1.26; 95% CI 1.09, 1.46; P = 0.0006], as was increasing HLA GRS in Europeans (OR = 1.55; 95% CI 1.07, 2.25; P = 0.03). There was a trend for stronger associations between both GRSs and LN risk in Europeans with cSLE compared with aSLE. When restricting cases to proliferative LN, the magnitude of these associations increased for both the non-HLA (OR = 1.30; 95% CI 1.10, 1.52; P = 0.002) and HLA GRS (OR = 1.99; 95% CI 1.29, 3.08; P = 0.002). CONCLUSION: We observed an association between known SLE risk loci and LN risk in children and adults with SLE, with the strongest effect observed among Europeans with cSLE. Future studies will include SLE-risk single nucleotide polymorphisms specific to non-European ancestral groups and validate findings in an independent cohort.


Assuntos
Idade de Início , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Lúpus Eritematoso Sistêmico/genética , Nefrite Lúpica/genética , Adolescente , Adulto , Criança , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Predisposição Genética para Doença/etnologia , Genótipo , Humanos , Modelos Logísticos , Lúpus Eritematoso Sistêmico/etnologia , Nefrite Lúpica/etnologia , Masculino , Razão de Chances , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Adulto Jovem
7.
Arthritis Res Ther ; 21(1): 223, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685018

RESUMO

BACKGROUND: Fatigue is a common symptom of systemic autoimmune rheumatic disease (SARD). Patients with SARD have a protracted pre-clinical phase during which progressive immunologic derangements occur culminating in disease. In this study, we sought to determine when fatigue develops and whether its presence correlates with inflammatory factors or predicts disease progression. METHODS: Anti-nuclear antibody (ANA)-negative healthy controls (HCs) and ANA-positive participants with no criteria, at least one clinical criteria (undifferentiated connective tissue disease, UCTD), or meeting SARD classification criteria were recruited. Fatigue was assessed using a modified version of the FACIT-F questionnaire and the presence of fibromyalgia determined using a questionnaire based on the modified 2010 ACR criteria. Peripheral blood expression of five IFN-induced genes was quantified by NanoString and the levels of IL-1ß, IL-6, or TNF-α by ELISA. RESULTS: Fatigue was as prevalent and severe in individuals lacking SARD criteria as it was in UCTD and SARD. Overall, ~ 1/3 of ANA+ subjects met fibromyalgia criteria, with no differences between sub-groups. Although fatigue was more severe in these individuals, those lacking fibromyalgia remained significantly more fatigued than ANA- HC. However, even in these subjects, fatigue correlated with the widespread pain index and symptom severity scores on the fibromyalgia questionnaire. Fatigue was not associated with elevated cytokine levels in any of the ANA+ sub-groups and did not predict imminent disease progression. CONCLUSIONS: Fatigue is common in ANA+ individuals lacking sufficient criteria for a SARD diagnosis, correlates with fibromyalgia-related symptoms, and is not associated with inflammation or predictive of disease progression.


Assuntos
Anticorpos Antinucleares/sangue , Citocinas/sangue , Progressão da Doença , Fadiga/sangue , Doenças Reumáticas/sangue , Índice de Gravidade de Doença , Adulto , Idoso , Anticorpos Antinucleares/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/imunologia , Citocinas/imunologia , Fadiga/diagnóstico , Fadiga/imunologia , Feminino , Fibromialgia/sangue , Fibromialgia/diagnóstico , Fibromialgia/imunologia , Previsões , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/imunologia , Masculino , Pessoa de Meia-Idade , Doenças Reumáticas/diagnóstico , Doenças Reumáticas/imunologia , Comportamento de Redução do Risco , Adulto Jovem
8.
J Clin Med ; 8(10)2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547518

RESUMO

The International Society of Nephrology/Renal Pathology Society (ISN/RPS) lupus nephritis (LN) classification is under reconsideration, given challenges with inter-rater reliability and resultant inconsistent relationship with treatment response. Integration of molecular classifiers into histologic evaluation can improve diagnostic precision and identify therapeutic targets. This study described the relationship between histological and molecular phenotypes and clinical responses in LN. Renal compartmental mRNA abundance was measured in 54 biopsy specimens from LN patients and correlated to ISN/RPS classification and individual histologic lesions. A subset of transcripts was also evaluated in sequential biopsies of a separate longitudinal cohort of 36 patients with paired samples obtained at the time of flare and at follow up. Unsupervised clustering based on mRNA abundance did not demonstrate a relationship with the (ISN/RPS) classification, nor did univariate statistical analysis. Exploratory analyses suggested a correlation with individual histologic lesions. Glomerular FN1 (fibronectin), SPP1 (secreted phosphoprotein 1), and LGALS3 (galectin 3) abundance correlated with disease activity and changed following treatment. Exploratory analyses suggested relationships between specific transcripts and individual histologic lesions, with the important representation of interferon-regulated genes. Our findings suggested that the current LN classification could be refined by the inclusion of molecular descriptors. Combining molecular and pathologic kidney biopsy phenotypes may hold promise to better classify disease and identify actionable treatment targets and merits further exploration in larger cohorts.

9.
Arthritis Res Ther ; 20(1): 264, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30486869

RESUMO

BACKGROUND: Diagnosis of systemic autoimmune rheumatic diseases (SARD) relies on the presence of hallmark anti-nuclear antibodies (ANA), many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals, most of whom will not develop SARD. Here, we examined a unique cohort of asymptomatic ANA+ individuals to determine whether they share any of the cellular immunologic features seen in SARD. METHODS: Healthy ANA- controls and ANA+ (ANA ≥1:160 by immunofluorescence) participants with no SARD criteria, with at least one criterion (undifferentiated connective tissue disease (UCTD)), or meeting SARD classification criteria were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry and transcript levels of BAFF, interferon (IFN)-induced and plasma cell-expressed genes were quantified by NanoString. RESULTS: A number of the immunologic abnormalities seen in SARD, including changes in peripheral B (switched memory) and T (iNKT, T regulatory, activated memory T follicular helper) subsets and B cell activation, were also seen in asymptomatic ANA+ subjects and those with UCTD. The extent of these immunologic changes correlated with ANA titer or the number of different specific ANAs produced. Principal component analysis of the cellular data indicated that a significant proportion of asymptomatic ANA+ subjects and subjects with UCTD clustered  with patients with early SARD, rather than ANA- healthy controls. CONCLUSIONS: ANA production is associated with altered T and B cell activation even in asymptomatic individuals. Some of the currently accepted cellular features of SARD may be associated with ANA production rather than the immunologic events that cause symptoms in SARD.


Assuntos
Anticorpos Antinucleares/imunologia , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Doenças Reumáticas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Idoso , Anticorpos Antinucleares/análise , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/metabolismo , Linfócitos B/metabolismo , Estudos de Coortes , Feminino , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Plasmócitos/metabolismo , Doenças Reumáticas/diagnóstico , Doenças Reumáticas/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo
10.
Sci Adv ; 4(11): eaar7653, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30417091

RESUMO

Memory B cells and plasma cells are antigen-experienced cells tasked with the maintenance of humoral protection. Despite these prominent functions, definitive cell surface markers have not been identified for these cells. We report here the isolation and characterization of the monoclonal variable lymphocyte receptor B (VLRB) N8 antibody from the evolutionarily distant sea lamprey that specifically recognizes memory B cells and plasma cells in humans. Unexpectedly, we determined that VLRB N8 recognizes the human leukocyte antigen-I (HLA-I) antigen in a tyrosine sulfation-dependent manner. Furthermore, we observed increased binding of VLRB N8 to memory B cells in individuals with autoimmune disorders multiple sclerosis and systemic lupus erythematosus. Our study indicates that lamprey VLR antibodies uniquely recognize a memory B cell- and plasma cell-specific posttranslational modification of HLA-I, the expression of which is up-regulated during B cell activation.


Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Memória Imunológica/imunologia , Plasmócitos/imunologia , Receptores de Antígenos/imunologia , Tirosina/análogos & derivados , Animais , Anticorpos Monoclonais/sangue , Linfócitos B/metabolismo , Células Cultivadas , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Lampreias/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Plasmócitos/metabolismo , Receptores de Antígenos/metabolismo , Tirosina/química
11.
Nat Immunol ; 19(6): 571-582, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29760532

RESUMO

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.


Assuntos
Apoptose/imunologia , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Animais , Humanos , Camundongos , Transdução de Sinais/imunologia , Receptor Toll-Like 9/imunologia
12.
PLoS One ; 13(5): e0196117, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29742110

RESUMO

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.


Assuntos
Perfilação da Expressão Gênica , Nefrite Lúpica/genética , Nefrite Lúpica/imunologia , Neutrófilos/metabolismo , Adulto , Contagem de Células , Feminino , Humanos , Interferons/farmacologia , Nefrite Lúpica/etiologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Adulto Jovem
13.
J Immunol Methods ; 457: 33-40, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29614266

RESUMO

Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.


Assuntos
Linfócitos B/citologia , Sistemas CRISPR-Cas , Engenharia Genética , Ribonucleoproteínas/genética , Adolescente , Adulto , Linfócitos B/imunologia , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Mutação , Tonsila Palatina/citologia , Reparo de DNA por Recombinação , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Adulto Jovem
14.
PLoS One ; 12(6): e0179506, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28628673

RESUMO

Lupus is characterized by a loss of B cell tolerance leading to autoantibody production. In this study, we explored the mechanisms underlying this loss of tolerance using B6 congenic mice with an interval from New Zealand Black chromosome 1 (denoted c1(96-100)) sufficient for anti-nuclear antibody production. Transgenes for soluble hen egg white lysozyme (sHEL) and anti-HEL immunoglobulin were crossed onto this background and various tolerance mechanisms examined. We found that c1(96-100) mice produced increased levels of IgM and IgG anti-HEL antibodies compared to B6 mice and had higher proportions of germinal center B cells and long-lived plasma cells, suggesting a germinal center-dependent breach of B cell anergy. Consistent with impaired anergy induction, c1(96-100) double transgenic B cells showed enhanced survival and CD86 upregulation. Hematopoietic chimeric sHEL mice with a mixture of B6 and c1(96-100) HEL transgenic B cells recapitulated these results, suggesting the presence of a B cell autonomous defect. Surprisingly, however, there was equivalent recruitment of B6 and c1(96-100) B cells into germinal centers and differentiation to splenic plasmablasts in these mice. In contrast, there were increased proportions of c1(96-100) T follicular helper cells and long-lived plasma cells as compared to their B6 counterparts, suggesting that both B and T cell defects are required to breach germinal center tolerance in this model. This possibility was further supported by experiments showing an enhanced breach of anergy in double transgenic mice with a longer chromosome 1 interval with additional T cell defects.


Assuntos
Linfócitos B/metabolismo , Cromossomos/genética , Tolerância Imunológica , Animais , Apoptose , Linfócitos B/citologia , Linfócitos B/imunologia , Antígeno B7-2/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Galinhas , Cromossomos/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Muramidase/genética , Muramidase/metabolismo , Nova Zelândia , Fosfatidilinositol 3-Quinases/metabolismo , Baço/metabolismo , Baço/patologia , Regulação para Cima
15.
J Immunol ; 198(10): 3949-3962, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28373584

RESUMO

Invariant NKT (iNKT) cells are innate lymphocytes that respond to glycolipids presented by the MHC class Ib molecule CD1d and are rapidly activated to produce large quantities of cytokines and chemokines. iNKT cell development uniquely depends on interactions between double-positive thymocytes that provide key homotypic interactions between signaling lymphocyte activation molecule (SLAM) family members. However, the role of SLAM receptors in the differentiation of iNKT cell effector subsets and activation has not been explored. In this article, we show that C57BL/6 mice containing the New Zealand Black Slam locus have profound alterations in Ly108, CD150, and Ly9 expression that is associated with iNKT cell hyporesponsiveness. This loss of function was only apparent when dendritic cells and iNKT cells had a loss of SLAM receptor expression. Using small interfering RNA knockdowns and peptide-blocking strategies, we demonstrated that trans-Ly108 interactions between dendritic cells and iNKT cells are critical for robust activation. LY108 costimulation similarly increased human iNKT cell activation. Thus, in addition to its established role in iNKT cell ontogeny, Ly108 regulates iNKT cell function in mice and humans.


Assuntos
Antígenos Ly/metabolismo , Células Dendríticas/metabolismo , Ativação Linfocitária , Células T Matadoras Naturais/imunologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Animais , Antígenos CD1d/imunologia , Antígenos Ly/genética , Antígenos Ly/imunologia , Diferenciação Celular , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , RNA Interferente Pequeno , Família de Moléculas de Sinalização da Ativação Linfocitária/deficiência , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia
16.
Arthritis Res Ther ; 19(1): 41, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28245862

RESUMO

BACKGROUND: Elevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody-positive (ANA+) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA+ individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question. METHODS: Healthy ANA- control subjects and ANA+ titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score. RESULTS: The mean IFN5 scores were increased in all ANA+ participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA+ and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA+ subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA+ subset, this score also correlated with the ANA titre, whereas in the other ANA+ subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores. CONCLUSIONS: An IFN signature is seen in a significant proportion of ANA+ individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms.


Assuntos
Anticorpos Antinucleares/imunologia , Doenças Autoimunes/imunologia , Interferon Tipo I/imunologia , Doenças Reumáticas/imunologia , Adulto , Idoso , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linhagem Celular Tumoral , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/imunologia , Doenças Reumáticas/diagnóstico , Doenças Reumáticas/genética , eIF-2 Quinase/genética , eIF-2 Quinase/imunologia
17.
J Rheumatol ; 44(1): 18-23, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803141

RESUMO

OBJECTIVE: Case ascertainment through self-report is a convenient but often inaccurate method to collect information. The purposes of this study were to develop, assess the sensibility, and validate a tool to identify cases of systemic autoimmune rheumatic diseases (SARD) in the outpatient setting. METHODS: The SARD tool was administered to subjects sampled from specialty clinics. Determinants of sensibility - comprehensibility, feasibility, validity, and acceptability - were evaluated using a numeric rating scale from 1-7. Comprehensibility was evaluated using the Flesch Reading Ease and the Flesch-Kincaid Grade Level. Self-reported diagnoses were validated against medical records using Cohen's κ statistic. RESULTS: There were 141 participants [systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis, Sjögren syndrome (SS), inflammatory myositis (polymyositis/dermatomyositis; PM/DM), and controls] who completed the questionnaire. The Flesch Reading Ease score was 77.1 and the Flesch-Kincaid Grade Level was 4.4. Respondents endorsed (mean ± SD) comprehensibility (6.12 ± 0.92), feasibility (5.94 ± 0.81), validity (5.35 ± 1.10), and acceptability (3.10 ± 2.03). The SARD tool had a sensitivity of 0.91 (95% CI 0.88-0.94) and a specificity of 0.99 (95% CI 0.96-1.00). The agreement between the SARD tool and medical record was κ = 0.82 (95% CI 0.77-0.88). Subgroup analysis by SARD found κ coefficients for SLE to be κ = 0.88 (95% CI 0.79-0.97), SSc κ = 1.0 (95% CI 1.0-1.0), PM/DM κ = 0.72 (95% CI 0.49-0.95), and SS κ = 0.85 (95% CI 0.71-0.99). The screening questions had sensitivity ranging from 0.96 to 1.0 and specificity ranging from 0.88 to 1.0. CONCLUSION: This SARD case ascertainment tool has demonstrable sensibility and validity. The use of both screening and confirmatory questions confers added accuracy.


Assuntos
Doenças Autoimunes/diagnóstico , Doenças Reumáticas/diagnóstico , Inquéritos e Questionários , Adulto , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade
18.
Arthritis Res Ther ; 18(1): 218, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27716443

RESUMO

BACKGROUND: Management of lupus nephritis (LN) would be greatly aided by the discovery of biomarkers that accurately reflect changes in disease activity. Here, we used a proteomics approach to identify potential urinary biomarkers associated with LN. METHODS: Urine was obtained from 60 LN patients with paired renal biopsies, 25 active non-LN SLE patients, and 24 healthy controls. Using Luminex, 128 analytes were quantified and normalized to urinary creatinine levels. Data were analyzed by linear modeling and non-parametric statistics, with corrections for multiple comparisons. A second cohort of 33 active LN, 16 active non-LN, and 30 remission LN SLE patients was used to validate the results. RESULTS: Forty-four analytes were identified that were significantly increased in active LN as compared to active non-LN. This included a number of unique proteins (e.g., TIMP-1, PAI-1, PF4, vWF, and IL-15) as well as known candidate LN biomarkers (e.g., adiponectin, sVCAM-1, and IL-6), that differed markedly (>4-fold) between active LN and non-LN, all of which were confirmed in the validation cohort and normalized in remission LN patients. These proteins demonstrated an enhanced ability to discriminate between active LN and non-LN patients over several previously reported biomarkers. Ten proteins were found to significantly correlate with the activity score on renal biopsy, eight of which strongly discriminated between active proliferative and non-proliferative/chronic renal lesions. CONCLUSIONS: A number of promising urinary biomarkers that correlate with the presence of active renal disease and/or renal biopsy changes were identified and appear to outperform many of the existing proposed biomarkers.


Assuntos
Biomarcadores/urina , Lúpus Eritematoso Sistêmico/urina , Nefrite Lúpica/urina , Adolescente , Adulto , Idoso , Área Sob a Curva , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Curva ROC , Adulto Jovem
19.
PLoS One ; 11(3): e0150515, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26964093

RESUMO

The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5+ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5+ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5+ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations.


Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Antígenos CD5 , Interleucina-10 , Lúpus Eritematoso Sistêmico , Células T Matadoras Naturais/imunologia , Animais , Linfócitos B/patologia , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos Endogâmicos NZB , Camundongos Knockout , Células T Matadoras Naturais/patologia
20.
J Rheumatol ; 42(12): 2318-26, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26568589

RESUMO

OBJECTIVE: Serologically active clinically quiescent (SACQ) patients with systemic lupus erythematosus (SLE) remain clinically quiescent for prolonged periods despite anti-dsDNA antibodies and/or low complements, indicating the presence of immune complexes. The immune mechanisms leading to this quiescence are unknown. However, in addition to activating complement, immune complex uptake by various cells leads to the production of interferon (IFN)-α and other proinflammatory factors that are also involved in tissue damage. Here we investigate whether production of these factors is reduced in SACQ patients. METHODS: The levels of 5 IFN-induced genes and 19 cyto/chemokines were measured in SACQ patients and were compared with those in serologically and clinically active (SACA) and serologically and clinically quiescent (SQCQ) patients. SACQ and SQCQ were defined as ≥ 2 years without clinical activity, with/without persistent serologic activity, respectively, and off corticosteroids/immunosuppressives. SACA was defined as disease activity compelling immunosuppression. Levels of OAS1, IFIT1, MX1, LY6E, and ISG15 were measured by quantitative real-time polymerase chain reaction (PCR) and a composite score (IFN-5) derived from this. Plasma cyto/chemokines were measured by Luminex assay. Nonparametric univariate and logistic regression analyses were conducted. RESULTS: There were no differences in gene expression or cyto/chemokine levels between SACQ and SQCQ patients. The SACQ IFN-5 score was significantly lower than that of SACA (p = 0.003) and was driven by SACQ status, not by autoantibody profile or disease duration. Levels of granulocyte-macrophage colony-stimulating factor, interleukin (IL) 6, IL-10, IFN-γ-inducible protein 10, monocyte chemoattractant protein 1, and tumor necrosis factor-α were significantly lower in SACQ than SACA. CONCLUSION: The levels of proinflammatory factors in SACQ mirror those of SQCQ patients, indicating reduced production of these factors despite the presence of immune complexes.


Assuntos
Citocinas/sangue , Interferons/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/fisiopatologia , Adolescente , Adulto , Biomarcadores/sangue , Quimiocinas/sangue , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Modelos Logísticos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Masculino , Monitorização Fisiológica , Análise Multivariada , Pacientes Ambulatoriais/estatística & dados numéricos , Prognóstico , Estudos Prospectivos , Medição de Risco , Testes Sorológicos , Índice de Gravidade de Doença , Estatísticas não Paramétricas , Adulto Jovem
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