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1.
Cell ; 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32059783

RESUMO

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

2.
Nat Immunol ; 21(2): 199-209, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31959979

RESUMO

A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.

3.
J Exp Med ; 217(2)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31841125

RESUMO

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.

4.
Artigo em Inglês | MEDLINE | ID: mdl-31608711

RESUMO

Objective: To conduct a phase-II trial using a ranking and selection paradigm where multiple treatments are compared with limited sample size and the best is chosen for a subsequent efficacy trial versus placebo. This strategy can find an effective treatment faster than traditional strategy of conducting larger trials against placebo. Methods: Sixty amyotrophic lateral sclerosis (ALS) participants were randomized 1:1:1 to creatine 30 g/day (CRE), tamoxifen 40 mg/day (T40), or tamoxifen 80 mg/day (T80), with matching placebo. The primary outcome was 38-week change in ALS Functional Rating Scale-Revised (ALSFRS-R), analyzed in a repeated-measures ANOVA. Secondary outcomes included slow vital capacity (SVC), quantitative muscle strength, early drug discontinuation (EDD), adverse events (AEs), and survival. Results: CRE participants experienced higher rates of drug-related AEs (82% vs. 43% T40, 47% T80) and EDD (50% vs. 24% T40, 29% T80). T80 participants experienced slower adjusted mean decline in ALSFRS-R in points/month (-0.80 vs. -0.84 T40, -0.85 CRE) and quantitative muscle strength but not in SVC and higher rates of mortality. Conclusion: Efficacy of T80 ranked numerically superior to CRE and T40 with respect to ALSFRS-R decline. Following the selection paradigm, T80 would be chosen to test against placebo. The approach was not designed to distinguish among treatments that are nearly equally effective or ineffective. If treatments are equivalent, then under the paradigm, it does not matter which treatment is selected. Newer approaches for increasing trial efficiency, including an adaptive platform trial design, may mitigate limitations of the selection design.

5.
Immunol Cell Biol ; 97(10): 888-901, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31441114

RESUMO

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflects differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ) and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ and PWD/PhJ) and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the international ImMunoGeneTics information system. By contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. By contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand interstrain variation in models of antibody-mediated disease.

6.
Nat Commun ; 10(1): 3120, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311926

RESUMO

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.


Assuntos
Evolução Clonal/genética , Linfócitos/metabolismo , Análise de Célula Única/métodos , Evolução Clonal/imunologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sequência de RNA/métodos
7.
Ann Surg ; 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31356280

RESUMO

BACKGROUND AND OBJECTIVE: The opioid epidemic has stimulated initiatives to reduce the number of unnecessary narcotic prescriptions. We adopted an opt-in prescription system for patients undergoing ambulatory cervical endocrine surgery (CES). We hypothesized that empowering patients to decide whether or not to receive narcotics for pain control would result in fewer unnecessary opioid prescriptions. METHODS: We enrolled all patients scheduled for outpatient CES between July 2017 and June 2018 in a narcotic opt-in program. Patient demographics, procedure characteristics, and postoperative pain scores were collected prospectively. Statistical analyses were performed to correlate clinical predictors with narcotic request. Results were compared against a historical control group. The study was approved by the University IRB. RESULTS: A total of 216 consecutive patients underwent outpatient CES following implementation of the program. Only nine (4%) requested prescription narcotic medication at discharge, and no patient called after discharge to request analgesic medications. Compared with our prior treatment paradigm, we achieved a 96.6% reduction in the number of narcotic tablets prescribed, and a 98% reduction in unconsumed tablets. Univariate analysis suggested history of substance abuse (P < 0.001), anxiety (P = 0.01), depression (P < 0.001), baseline narcotic use (P = 0.004), highest pain postoperatively (P = 0.004), and incision length (P = 0.007) as predictive for narcotic request. Multivariate analysis retained significance with incision length and history of substance abuse. CONCLUSION: By empowering patients undergoing ambulatory CES to accept or decline a prescription, we reduced the number of prescribed narcotic tablets by 96.6%. Although longer incisions and prior substance abuse predict higher likelihood of requesting pain medication on discharge, 207 of 216 patients were treated with acetaminophen alone.

8.
Cell ; 177(6): 1566-1582.e17, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31104840

RESUMO

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

9.
Front Immunol ; 10: 435, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30936866

RESUMO

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT®, the international ImMunoGeneTics information system® (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT®. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC-focusing, to begin with, on human IGHV genes-with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.

10.
Sci Transl Med ; 11(481)2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814336

RESUMO

Antigenic exposures at epithelial sites in infancy and early childhood are thought to influence the maturation of humoral immunity and modulate the risk of developing immunoglobulin E (IgE)-mediated allergic disease. How different kinds of environmental exposures influence B cell isotype switching to IgE, IgG, or IgA, and the somatic mutation maturation of these antibody pools, is not fully understood. We sequenced antibody repertoires in longitudinal blood samples in a birth cohort from infancy through the first 3 years of life and found that, whereas IgG and IgA show linear increases in mutational maturation with age, IgM and IgD mutations are more closely tied to pathogen exposure. IgE mutation frequencies are primarily increased in children with impaired skin barrier conditions such as eczema, suggesting that IgE affinity maturation could provide a mechanistic link between epithelial barrier failure and allergy development.

11.
Radiology ; 290(1): 254-261, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30299233

RESUMO

Purpose To develop and validate a predictive model for postembolization syndrome (PES) following transarterial hepatic chemoembolization (TACE) for hepatocellular carcinoma. Materials and Methods In this single-center, retrospective study, 370 patients underwent 513 TACE procedures between October 2014 and September 2016. Seventy percent of the patients were randomly assigned to a training data set and the remaining 30% were assigned to a testing data set. Variables included demographic, laboratory, clinical, and procedural details. PES was defined as pain and/or nausea beyond 6 hours after TACE that required intravenous medication for symptom control. The predictive model was developed by using conditional inference trees and Lasso regression. Results Demographics, laboratory data, performance, tumor characteristics, and procedural details were statistically similar for the training and testing data sets. Overall, 83 of 370 patients (22.4%) after 107 of 513 TACE procedures (20.8%) met the predefined criteria. Factors identified at univariable analysis included large tumor burden (P = .004), drug-eluting embolic TACE (P = .03), doxorubicin dose (P = .003), history of PES (P < .001) and chronic pain (P < .001), of which history of PES, tumor burden, and drug-eluting embolic TACE were identified as the strongest predictors by the multivariable analysis and were used to develop the predictive model. When applied to the testing data set, the model demonstrated an area under the curve of 0.62, sensitivity of 79% (22 of 28), specificity of 44.2% (53 of 120), and a negative predictive value of 90% (53 of 59). Conclusion The model identified history of postembolization syndrome, tumor burden, and drug-eluting embolic chemoembolization as predictors of protracted recovery because of postembolization syndrome. © RSNA, 2018.


Assuntos
Carcinoma Hepatocelular/terapia , Quimioembolização Terapêutica/efeitos adversos , Neoplasias Hepáticas/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimioembolização Terapêutica/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Estudos Retrospectivos , Síndrome
12.
Disabil Rehabil ; 41(23): 2731-2749, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29911901

RESUMO

Objectives: The objective of this study is to identify patient reported experiences of using community rehabilitation and/or support services whilst living with a long-term neurological condition, and perceptions of their impact on quality of life.Methods: Nine electronic databases were searched for peer-reviewed qualitative studies from 2005 to 2016, which met the inclusion criteria. Critical appraisal, data extraction, and quality assessment of 37 included papers were performed by three reviewers. One hundred and one findings were extracted. Meta-aggregation was used to synthesize findings.Findings: Seven 'synthesized findings' [SF] were produced: Interactions with some professionals provide active participation, choice, confidence and autonomy [SF1]; Interactions with some professionals are disempowering and depersonalized [SF2]; Effective communication, specialist knowledge and an individualized approach to information provision is needed [SF3]; Indicators of success vary and may not be clear [SF4]; Informal support from family/friends is valued [SF5]; Opportunities for peer support/social interaction is valued [SF6]; Coordination required to ensure continuity during transition to community [SF7].Conclusion: Patient reported experiences identified common factors associated with process quality (respect, choice, autonomy, information provision, communication) and activities of patient centered care (personalized care, shared decision-making, self-management support) despite heterogeneity of neurological conditions, service configurations, and geographical location. These factors impact quality of life.Implications for RehabilitationPatient reported experiences provide useful information about courtesy, respect, choice, autonomy, information provision, and communication.Outcomes of self-efficacy and self-management are important for people with stable and progressive long-term neurological conditions.Interactions with individual professionals influence engagement, self-efficacy, and self-management for people with long-term neurological conditions.Training for health and social care professionals should develop the advanced communication skills and behavior required to facilitate self-efficacy and self-management.

13.
Front Immunol ; 10: 2961, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921202

RESUMO

Mammalian immunoglobulin (IG) genes are found in complex loci that contain hundreds of highly similar pseudogenes, functional genes and repetitive elements, which has made their investigation particularly challenging. High-throughput sequencing has provided new avenues for the investigation of these loci, and has recently been applied to study the IG genes of important inbred mouse strains, revealing unexpected differences between their IG loci. This demonstrated that the structural differences are of such magnitude that they call into question the merits of the current mouse IG gene nomenclatures. Three nomenclatures for the mouse IG heavy chain locus (Igh) are presently in use, and they are all positional nomenclatures using the C57BL/6 genome reference sequence as their template. The continued use of these nomenclatures requires that genes of other inbred strains be confidently identified as allelic variants of C57BL/6 genes, but this is clearly impossible. The unusual breeding histories of inbred mouse strains mean that, regardless of the genetics of wild mice, no single ancestral origin for the IG loci exists for laboratory mice. Here we present a general discussion of the challenges this presents for any IG nomenclature. Furthermore, we describe principles that could be followed in the formulation of a solution to these challenges. Finally, we propose a non-positional nomenclature that accords with the guidelines of the International Mouse Nomenclature Committee, and outline strategies that can be adopted to meet the nomenclature challenges if three systems are to give way to a new one.

14.
BMC Womens Health ; 18(1): 137, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30180846

RESUMO

BACKGROUND: In the last thirty years there has been a rise in harmful alcohol use amongst White British women. Approaches to alcohol harm reduction typically position drinking as an individual behaviour, with an emphasis on people to make changes to and by themselves. Moving away from an individual approach, this paper works with a relational framework to develop understanding of non-dependent women's drinking in the context of their everyday lives. It draws on Feminist Ethics of Care theory, to consider the importance of care in women's lives and alcohol as an element of their 'practices of care' in different relationships. METHODS: The study adopted an interpretive approach and drew on feminist principles of practice. Qualitative one-to-one face-to-face interviews were undertaken with twenty-six White women living in the North East of England. Participants were aged between 24 and 67 years. Thematic analysis of the data was carried out. RESULTS: Participants' relationships came through the analysis as central to understanding the way alcohol did and not feature in care practices. In couple relationships drinking offered a way of doing 'care' together, yet when it was used too often it no longer became appropriate as a form of care. In non-family relationships alcohol enabled care giving and receiving, while disguising that care was being received. In relationships with mothers the use of alcohol was relatively absent in the care practices described. Participants' relationship to alcohol as a form of care of self, particularly when drinking alone, was closely related to their roles and responsibilities to others. CONCLUSIONS: Overall the data suggests that interventions targeting women's drinking should start from a position that women are relational. Moreover that when care by others is lacking or unavailable, alcohol can increasingly be introduced into care practices, and the reproduction of these practices may be leading to an increase in heavy drinking. By seeing alcohol use in the context of wider familial and non-familial relationships, this work has important implications for future interventions.


Assuntos
Consumo de Bebidas Alcoólicas/psicologia , Feminismo , Relações Interpessoais , Adulto , Idoso , Inglaterra , Grupo com Ancestrais do Continente Europeu , Feminino , Humanos , Entrevistas como Assunto , Pessoa de Meia-Idade , Relações Mãe-Filho , Pesquisa Qualitativa , Apoio Social , Adulto Jovem
15.
Surgery ; 164(6): 1372-1376, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30149938

RESUMO

BACKGROUND: Surgical techniques for adrenalectomy have evolved substantially over the last century. Although minimally invasive approaches are favored for benign disease, open adrenalectomy remains the gold standard for large tumors and those concerning for malignancy. Most reports describe the use of midline, subcostal, or thoracoabdominal incisions for open adrenalectomy. We studied our experience with the Makuuchi incision, designed to optimize exposure and minimize denervation of the abdominal wall. METHODS: All open adrenalectomies at the University of Rochester from 2009 to 2017 were retrospectively reviewed. Patient demographic characteristics, intraoperative details, and postoperative complications were investigated. Surgical site infection and hernia rates of Makuuchi incision were compared with non-Makuuchi incision patients and with published standards. The study was approved by the university Institutional Review Board. RESULTS: A total of 41 adrenalectomies were performed via Makuuchi incision. Population statistics included a mean age of 51.7 (19-86) years, a mean body mass index of 29.7 (17.3-45.8), and a mean tumor diameter of 8 cm (3.1-26 cm). Fourteen (34%) required multivisceral resection. Twenty-one (51%) were previous or current smokers, and 9 (22%) had hypercortisolemia. Median duration of stay was 6 days (4-73). Incisional hernia occurred in 5 patients (12%) and surgical site infection in 3 patients (7%), 2 patients had Cushing syndrome and 1 was immunosuppressed. Pain was managed with patient-controlled epidural anesthesia or patient-controlled anesthesia with postoperative day 1 daily morphine equivalents equating to 0.5 mg of hydromorphone q2h. Among 15 non-Makuuchi incision patients, there were 2 hernias (13%), 2 surgical site infections (13%), and 1 case of postoperative pneumonia. CONCLUSION: The Makuuchi incision is well tolerated and affords outstanding exposure of the adrenals and adjacent viscera. Incisional hernia and surgical site infection rates were favorable compared with published rates for midline or subcostal incisions, despite an obese population with a high incidence of hypercortisolism and immunosuppression.


Assuntos
Neoplasias das Glândulas Suprarrenais/cirurgia , Adrenalectomia/métodos , Parede Abdominal/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
16.
Arthritis Rheumatol ; 70(10): 1617-1625, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29697211

RESUMO

OBJECTIVE: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. METHODS: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. RESULTS: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. CONCLUSION: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.


Assuntos
Cadeias Pesadas de Imunoglobulinas/sangue , Leucócitos Mononucleares/fisiologia , Fator Reumatoide/sangue , Síndrome de Sjogren/sangue , Adulto , Linfócitos B/metabolismo , Compostos de Boro/metabolismo , Rastreamento de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica , Fator Reumatoide/imunologia , Síndrome de Sjogren/imunologia
17.
Front Immunol ; 9: 118, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29472918

RESUMO

In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.


Assuntos
Anticorpos , Sequenciamento de Nucleotídeos em Larga Escala , Afinidade de Anticorpos , Bacteriófagos , Mapeamento de Epitopos , Humanos
18.
Immunogenetics ; 70(3): 143-158, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29260260

RESUMO

The immune systems of all mammals include populations of B cells producing antibodies with incredibly diverse specificities. Repertoire diversity has been described as the "miracle of immunology," and it was long thought to be the result of essentially stochastic processes. Recently, however, analysis of high throughput gene sequencing data has shown that hard-wired biases in these processes result in antibody repertoires that are broadly predictable. The repertoires of mice and humans are both predictable, but they are strikingly different. In this review, features of the naïve antibody repertoires of the two species are contrasted. We show that the mouse repertoire includes a conspicuous population of public clonotypes that are shared by different individuals of an inbred strain. These clonotypes are the result of gene rearrangements that involve little gene processing. By skewing repertoire formation toward such sequences, which probably target commonly encountered pathogens, it may be that the relatively small mouse repertoire is appropriate and effective despite its size. Species like the mouse face challenges that are a direct consequence of their small body sizes and the limitations this places on the antibody arsenal-particularly early in ontogeny. We propose that it is the differences in the naïve repertoires of mice and humans, and the differences in the ways these repertoires are used, which ensure that the very different biological needs of the two species are met. The processes that contribute to repertoire formation may appear to be stochastic, but in both species, evolution has left little to chance.


Assuntos
Anticorpos/imunologia , Linfócitos B/imunologia , Evolução Molecular , Sistema Imunitário , Animais , Anticorpos/genética , Humanos , Camundongos
20.
Nat Microbiol ; 2: 17031, 2017 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-28288098

RESUMO

Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Šcryo-electron microscopy structure of the Plasmodium falciparum 80S ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated centre. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-electron microscopy to guide the development of mefloquine derivatives to inhibit parasite protein synthesis.


Assuntos
Antimaláricos/farmacologia , Mefloquina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/efeitos dos fármacos
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