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1.
Nat Immunol ; 20(10): 1299-1310, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31534238

RESUMO

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.


Assuntos
Infecções por Poxviridae/imunologia , Poxviridae/fisiologia , Domínios Proteicos/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Alelos , Animais , Extinção Biológica , Humanos , Imunidade , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Fosforilação
2.
Nature ; 608(7924): 757-765, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35948641

RESUMO

The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago1,2. However, it was not until the advancement of genomic sequencing technologies that the abundance and repertoire of transposable elements were revealed, and they are now known to constitute up to two-thirds of mammalian genomes3,4. The presence of DNA regulatory regions including promoters, enhancers and transcription-factor-binding sites within transposable elements5-8 has led to the hypothesis that transposable elements have been co-opted to regulate mammalian gene expression and cell phenotype8-14. Mammalian transposable elements include recent acquisitions and ancient transposable elements that have been maintained in the genome over evolutionary time. The presence of ancient conserved transposable elements correlates positively with the likelihood of a regulatory function, but functional validation remains an essential step to identify transposable element insertions that have a positive effect on fitness. Here we show that CRISPR-Cas9-mediated deletion of a transposable element-namely the LINE-1 retrotransposon Lx9c11-in mice results in an exaggerated and lethal immune response to virus infection. Lx9c11 is critical for the neogenesis of a non-coding RNA (Lx9c11-RegoS) that regulates genes of the Schlafen family, reduces the hyperinflammatory phenotype and rescues lethality in virus-infected Lx9c11-/- mice. These findings provide evidence that a transposable element can control the immune system to favour host survival during virus infection.


Assuntos
Elementos de DNA Transponíveis , Interações entre Hospedeiro e Microrganismos , Imunidade , Retroelementos , Viroses , Animais , Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/imunologia , Evolução Molecular , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade/genética , Camundongos , RNA não Traduzido/genética , Sequências Reguladoras de Ácido Nucleico/genética , Retroelementos/genética , Retroelementos/imunologia , Viroses/genética , Viroses/imunologia
3.
J Infect Dis ; 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39250505

RESUMO

BACKGROUND: Chlamydia trachomatis (CT) is a globally prevalent sexually transmitted infection (STI) that can result in pelvic inflammatory disease, ectopic pregnancy and infertility in women. Currently, there is no prophylactic vaccine. METHODS: This study examined T cell immunity in a cohort of women recently infected with CT. Participants were screened against peptides spanning 33 of 894 possible CT proteins, either ex vivo or using short-term cell lines (STCL). CT-specific T cells were characterized by IFN-γ ELISpot and flow cytometry. RESULTS: Ex vivo CT-specific T cells were rarely detected; however, following in vitro expanded CT-specific T cells were detected by IFN-γ ELISpot in 90% (27/30) of participants. Notably, over 50% of participants had T cell responses targeting chlamydial protease-like activity factor (CPAF). T cell epitopes were dispersed across the CPAF protein. Flow cytometry analysis of STCL found CT-specific cells, were mainly CD4+, produced IFN-γ and TNF-α and were sustained over 12 months. Ex vivo analysis suggested CT-specific T cells mostly exhibited a central memory phenotype. CONCLUSION: Our results indicate that CT infection elicits low-frequency, persistent CD4 T cell responses in most women and that the secreted protein, CPAF, is an immunoprevalent CT antigen. Altogether, these data support development and testing of CT vaccines that enhance CD4 T cells against CPAF.

4.
Clin Exp Immunol ; 215(3): 279-290, 2024 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-37950348

RESUMO

CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.


Assuntos
Linfócitos T CD8-Positivos , Receptores de Antígenos de Linfócitos T , Humanos , Regulação para Baixo , Receptores de Antígenos de Linfócitos T/genética , Antígenos CD8/metabolismo , Peptídeos/metabolismo , Complexo CD3/metabolismo
5.
Diabetologia ; 66(8): 1516-1531, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37311878

RESUMO

AIMS/HYPOTHESIS: NF-κB activation unites metabolic and inflammatory responses in many diseases yet less is known about the role that NF-κB plays in normal metabolism. In this study we investigated how RELA impacts the beta cell transcriptional landscape and provides network control over glucoregulation. METHODS: We generated novel mouse lines harbouring beta cell-specific deletion of either the Rela gene, encoding the canonical NF-κB transcription factor p65 (ßp65KO mice), or the Ikbkg gene, encoding the NF-κB essential modulator NEMO (ßNEMOKO mice), as well as ßA20Tg mice that carry beta cell-specific and forced transgenic expression of the NF-κB-negative regulator gene Tnfaip3, which encodes the A20 protein. Mouse studies were complemented by bioinformatics analysis of human islet chromatin accessibility (assay for transposase-accessible chromatin with sequencing [ATAC-seq]), promoter capture Hi-C (pcHi-C) and p65 binding (chromatin immunoprecipitation-sequencing [ChIP-seq]) data to investigate genome-wide control of the human beta cell metabolic programme. RESULTS: Rela deficiency resulted in complete loss of stimulus-dependent inflammatory gene upregulation, consistent with its known role in governing inflammation. However, Rela deletion also rendered mice glucose intolerant because of functional loss of insulin secretion. Glucose intolerance was intrinsic to beta cells as ßp65KO islets failed to secrete insulin ex vivo in response to a glucose challenge and were unable to restore metabolic control when transplanted into secondary chemical-induced hyperglycaemic recipients. Maintenance of glucose tolerance required Rela but was independent of classical NF-κB inflammatory cascades, as blocking NF-κB signalling in vivo by beta cell knockout of Ikbkg (NEMO), or beta cell overexpression of Tnfaip3 (A20), did not cause severe glucose intolerance. Thus, basal p65 activity has an essential and islet-intrinsic role in maintaining normal glucose homeostasis. Genome-wide bioinformatic mapping revealed the presence of p65 binding sites in the promoter regions of specific metabolic genes and in the majority of islet enhancer hubs (~70% of ~1300 hubs), which are responsible for shaping beta cell type-specific gene expression programmes. Indeed, the islet-specific metabolic genes Slc2a2, Capn9 and Pfkm identified within the large network of islet enhancer hub genes showed dysregulated expression in ßp65KO islets. CONCLUSIONS/INTERPRETATION: These data demonstrate an unappreciated role for RELA as a regulator of islet-specific transcriptional programmes necessary for the maintenance of healthy glucose metabolism. These findings have clinical implications for the use of anti-inflammatories, which influence NF-κB activation and are associated with diabetes.


Assuntos
Intolerância à Glucose , Fator de Transcrição RelA , Animais , Humanos , Camundongos , Cromatina , Glucose , NF-kappa B/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
6.
Kidney Int ; 103(6): 1105-1119, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37097268

RESUMO

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.


Assuntos
Injúria Renal Aguda , NF-kappa B , Humanos , Camundongos , Animais , NF-kappa B/metabolismo , Fatores de Transcrição/genética , Ubiquitina , Estudo de Associação Genômica Ampla , Ligases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Injúria Renal Aguda/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética
7.
Proc Natl Acad Sci U S A ; 116(27): 13508-13516, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31196957

RESUMO

Injection of Interleukin-2 (IL-2) complexed with a particular anti-IL-2 monoclonal antibody (mab) JES6-1 has been shown to selectively expand CD4+Foxp3+ T regulatory T cells (Tregs) in vivo. Although the potency of this approach with regard to transplantation has already been proven in an islet transplantation model, skin graft survival could not be prolonged. Since the latter is relevant to human allograft survival, we sought to improve the efficiency of IL-2 complex (cplx) treatment for skin allograft survival in a stringent murine skin graft model. Here, we show that combining low doses of IL-2 cplxs with rapamycin and blockade of the inflammatory cytokine IL-6 leads to long-term (>75 d) survival of major histocompatibility complex-different skin allografts without the need for immunosuppression. Allograft survival was critically dependent on CD25+FoxP3+ Tregs and was not accompanied by impaired responsiveness toward donor alloantigens in vitro after IL-2 cplx treatment was stopped. Furthermore, second donor-type skin grafts were rejected and provoked rejection of the primary graft, suggesting that operational tolerance is not systemic but restricted to the graft. These findings plus the lack of donor-specific antibody formation imply that prolonged graft survival was largely a reflection of immunological ignorance. The results may represent a potentially clinically translatable strategy for the development of protocols for tolerance induction.


Assuntos
Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Transplante de Pele , Linfócitos T Reguladores/imunologia , Aloenxertos , Animais , Anticorpos Monoclonais/imunologia , Feminino , Citometria de Fluxo , Sobrevivência de Enxerto/imunologia , Imunossupressores/uso terapêutico , Interleucina-2/imunologia , Interleucina-6/antagonistas & inibidores , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Sirolimo/uso terapêutico
8.
PLoS Pathog ; 13(7): e1006510, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28759651

RESUMO

In order to inform the rational design of HIV-1 preventive and cure interventions it is critical to understand the events occurring during acute HIV-1 infection (AHI). Using viral deep sequencing on six participants from the early capture acute infection RV217 cohort, we have studied HIV-1 evolution in plasma collected twice weekly during the first weeks following the advent of viremia. The analysis of infections established by multiple transmitted/founder (T/F) viruses revealed novel viral profiles that included: a) the low-level persistence of minor T/F variants, b) the rapid replacement of the major T/F by a minor T/F, and c) an initial expansion of the minor T/F followed by a quick collapse of the same minor T/F to low frequency. In most participants, cytotoxic T-lymphocyte (CTL) escape was first detected at the end of peak viremia downslope, proceeded at higher rates than previously measured in HIV-1 infection, and usually occurred through the exploration of multiple mutational pathways within an epitope. The rapid emergence of CTL escape variants suggests a strong and early CTL response. Minor T/F viral strains can contribute to rapid and varied profiles of HIV-1 quasispecies evolution during AHI. Overall, our results demonstrate that early, deep, and frequent sampling is needed to investigate viral/host interaction during AHI, which could help identify prerequisites for prevention and cure of HIV-1 infection.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Adolescente , Adulto , Estudos de Coortes , Feminino , Infecções por HIV/imunologia , Infecções por HIV/transmissão , HIV-1/classificação , HIV-1/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Evasão da Resposta Imune , Masculino , Pessoa de Meia-Idade , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Adulto Jovem
10.
J Immunol ; 199(7): 2366-2376, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28835458

RESUMO

IL-17-producing γδ T (γδT-17) cells have proved to be an important early source of IL-17 in many inflammatory settings and are emerging as an important participant in protumor immune responses. Considering that their peripheral activation depends largely on innate signals rather than TCR ligation, it is important to understand what mechanisms exist to curb unwanted activation. Expression of the high-affinity IL-2R on γδT-17 cells prompted us to investigate a role for this cytokine. We found γδT-17 cells to be enriched, not depleted, in IL-2-deficient mice. The absence of IL-2 also resulted in higher IL-17 production and the emergence of IL-17+IFN-γ+ double producers. Furthermore, the addition of IL-2 to in vitro cultures of sorted γδT-17 cells was able to moderate IL-17 and affect differentiation into polyfunctional cytokine-producing cells. Interestingly, the Vγ6+ subset was more susceptible to the effects of IL-2 than Vγ4+ γδT-17 cells. We also found that unlike other γδ T cells, γδT-17 cells do not produce IL-2, but express Blimp-1, a known transcriptional repressor of IL-2. Although IL-2 was able to induce robust proliferation of γδT-17 cells, it did not sustain viability, negatively impacting their survival via downregulation of the IL-7R. Taken together, these data indicate that IL-2 can augment the γδT-17 response in favor of short-lived effectors with limited plasticity, particularly in the presence of IL-1ß and IL-23. In this way, IL-2 may act to curtail the innate-like response of γδT-17 cells upon arrival of IL-2-producing adaptive immune cells at the site of inflammation.


Assuntos
Interleucina-17/biossíntese , Interleucina-2/imunologia , Interleucina-2/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Citometria de Fluxo , Inflamação , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-17/imunologia , Interleucina-2/deficiência , Interleucina-2/genética , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptores de Interleucina-7/genética , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
J Immunol ; 195(11): 5123-35, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26491200

RESUMO

The selection of affinity-matured Ab-producing B cells is supported by interactions with T follicular helper (Tfh) cells. In addition to cell surface-expressed molecules, cytokines produced by Tfh cells, such as IL-21 and IL-4, provide B cell helper signals. In this study, we analyze how the fitness of Th cells can influence Ab responses. To do this, we used a model in which IL-21R-sufficient (wild-type [WT]) and -deficient (Il21r(-/-)) Ag-specific Tfh cells were used to help immunodeficient Il21r(-/-) B cells following T-dependent immunization. Il21r(-/-) B cells that had received help from WT Tfh cells, but not from Il21r(-/-) Tfh cells, generated affinity-matured Ab upon recall immunization. This effect was dependent on IL-4 produced in the primary response and associated with an increased fraction of memory B cells. Il21r(-/-) Tfh cells were distinguished from WT Tfh cells by a decreased frequency, reduced conjugate formation with B cells, increased expression of programmed cell death 1, and reduced production of IL-4. IL-21 also influenced responsiveness to IL-4 because expression of both membrane IL-4R and the IL-4-neutralizing soluble (s)IL-4R were reduced in Il21r(-/-) mice. Furthermore, the concentration of sIL-4R was found to correlate inversely with the amount of IgE in sera, such that the highest IgE levels were observed in Il21r(-/-) mice with the least sIL-4R. Taken together, these findings underscore the important collaboration between IL-4 and IL-21 in shaping T-dependent Ab responses.


Assuntos
Linfócitos B/imunologia , Subunidade alfa de Receptor de Interleucina-21/genética , Interleucina-4/imunologia , Interleucinas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Formação de Anticorpos/imunologia , Imunoglobulina E/sangue , Subunidade alfa de Receptor de Interleucina-21/imunologia , Interleucina-4/biossíntese , Interleucinas/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Receptores de Superfície Celular/biossíntese , Transdução de Sinais/imunologia
12.
Open Forum Infect Dis ; 11(5): ofae212, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38756763

RESUMO

Background: Persistence of HIV-1 in reservoirs necessitates life-long antiretroviral therapy (ART). There are conflicting data using genetic analysis on whether persistence includes an actively replicating reservoir with strong evidence arguing against replication. Methods: We investigated the possibility of ongoing viral evolution during suppressive therapy by comparing near full-length viral genomic sequences using phylogenetic analysis of viral RNA in plasma before therapy initiation early after infection and from virus induced to grow from the latent reservoir after a period of suppressive ART. We also focused our analysis on evidence of selective pressure by drugs in the treatment regimen and at sites of selective pressure by the adaptive immune response. Results: Viral genomes induced to grow from the latent reservoir from 10 participants with up to 9 years on suppressive ART were highly similar to the nearly homogeneous sequences in plasma taken early after infection at ART initiation. This finding was consistent across the entire genome and when the analysis focused on sites targeted by the drug regimen and by host selective pressure of antibody and cytotoxic T cells. The lack of viral evolution away from pretherapy sequences in spite of demonstrated selective pressure is most consistent with a lack of viral replication during reservoir persistence. Conclusions: These results do not support ongoing viral replication as a mechanism of HIV-1 persistence during suppressive ART.

13.
Commun Biol ; 7(1): 1340, 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39414943

RESUMO

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization.


Assuntos
Inclusão em Parafina , Análise de Sequência de RNA , Análise de Célula Única , Fixação de Tecidos , Inclusão em Parafina/métodos , Humanos , Análise de Sequência de RNA/métodos , Fixação de Tecidos/métodos , Análise de Célula Única/métodos , Formaldeído/química , Transcriptoma , Perfilação da Expressão Gênica/métodos , Fluxo de Trabalho
14.
J Pathol ; 227(3): 306-14, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22294324

RESUMO

Micro-RNAs (miRNAs) are frequently dysregulated in a range of human malignancies, many have been shown to act either as tumour supressors or oncogenes and several have been implicated in breast cancer. However, breast cancer is a diverse disease and little is known about the relationships between miRNA expression, clinical outcome and tumour subtype. We used locked nucleic acid probe in situ hybridization (LNA-ISH) to visualize, in tissue micro-arrays (TMAs) of 2919 formalin-fixed paraffin-embedded (FFPE) archival breast tumours, the expression of two key miRNAs that are frequently lost in a range of solid malignancies, let-7b and miR-205. These miRNAs were also quantified by quantitative reverse transcription PCR in cores of FFPE tissue from 40 of these cases, demonstrating that LNA-ISH is semi-quantitative. The tumours in the TMAs were assigned to subtypes based on their immunohistochemical (IHC) staining with ER, PR, HER2, CK5/6 and EGFR. let-7b expression was shown to be associated with luminal tumours and to have an independent significant positive prognostic value in this group. miR-205 is associated with tumours of ductal morphology and is of significant positive prognostic value within these tumours. We propose that the expression of miR-205 may contribute to ductal tumour morphology.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Perfilação da Expressão Gênica/métodos , Hibridização In Situ , MicroRNAs/análise , Análise de Sequência com Séries de Oligonucleotídeos , Biomarcadores Tumorais/análise , Biópsia , Neoplasias da Mama/química , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/secundário , Carcinoma Lobular/química , Carcinoma Lobular/mortalidade , Carcinoma Lobular/secundário , Distribuição de Qui-Quadrado , Feminino , Fixadores , Formaldeído , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Inclusão em Parafina , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fixação de Tecidos/métodos
15.
Nat Genet ; 31(4): 424-8, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12091909

RESUMO

The alternative pathway of complement is activated continuously in vivo through the C3 'tick-over' pathway. This pathway is triggered by the hydrolysis of C3, resulting in the formation of C3 convertase. This, in turn, generates C3b, which mediates many of the biological functions of complement. Factor H, the main regulator of this activation, prevents formation and promotes dissociation of the C3 convertase enzyme, and, together with factor I, mediates the proteolytic inactivation of C3b. Factor H deficiency, described in 29 individuals from 12 families and in pigs, allows unhindered activation of fluid-phase C3 and severe depletion of plasma C3 (ref. 11). Membranoproliferative glomerulonephritis (MPGN) occurs in factor H-deficient humans and pigs. Although MPGN has been reported in other conditions in which uncontrolled activation of C3 occurs, the role of C3 dysregulation in the pathogenesis of MPGN is not understood. Here we show that mice deficient in factor H (Cfh(-/-) mice) develop MPGN spontaneously and are hypersensitive to developing renal injury caused by immune complexes. Introducing a second mutation in the gene encoding complement factor B, which prevents C3 turnover in vivo, obviates the phenotype of Cfh(-/-) mice. Thus, uncontrolled C3 activation in vivo is essential for the development of MPGN associated with deficiency of factor H.


Assuntos
Ativação do Complemento/genética , Complemento C3/imunologia , Fator H do Complemento/genética , Glomerulonefrite/genética , Glomerulonefrite/fisiopatologia , Animais , Complemento C3/metabolismo , Complemento C9/metabolismo , Fator H do Complemento/metabolismo , Modelos Animais de Doenças , Feminino , Rim/patologia , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Mutação
16.
Bio Protoc ; 12(6): e4354, 2022 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-35434196

RESUMO

The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) measures CD8+ T cell-mediated inhibition of HIV replication in CD4+ T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. Different VIAs that differ in length of CD8:CD4 T cell culture periods (6-13 days), purity of CD4 cultures [isolated CD4+ T cells or CD8+ depleted peripheral blood mononuclear cells (PBMCs)], HIV strains (laboratory strains, isolates, reporter viruses) and read-outs of virus inhibition (p24 ELISA, intracellular measurement of p24, luciferase reporter expression, and viral gag RNA) have been reported. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence intervals. We identify methodological and analysis changes that could be incorporated into other protocols to improve assay reproducibility. We found that in people living with HIV (PLWH) on antiretroviral therapy (ART), CD8 T cell virus inhibition was largely stable over time, supporting the use of this assay and/or analysis methods to examine therapeutic interventions. Graphic abstract.

17.
Front Immunol ; 13: 811525, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35464428

RESUMO

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.


Assuntos
Infertilidade Feminina , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Infertilidade Feminina/genética , Inflamação/genética , Camundongos , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética
19.
Front Immunol ; 12: 666991, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34276657

RESUMO

The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Soropositividade para HIV/imunologia , HIV-1/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Replicação Viral/imunologia , Antivirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Estudos de Casos e Controles , Células Cultivadas , Técnicas de Cocultura , Estudos Transversais , Proteína do Núcleo p24 do HIV/imunologia , Soropositividade para HIV/sangue , Soropositividade para HIV/tratamento farmacológico , Soropositividade para HIV/virologia , Humanos , Resultado do Tratamento
20.
Sci Rep ; 10(1): 19085, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154446

RESUMO

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.


Assuntos
Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/genética , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Doenças Autoimunes/etiologia , Doenças Autoimunes/genética , Sequência Conservada , Evolução Molecular , Variação Genética , Humanos , Inflamação/etiologia , Inflamação/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Modelos Animais , Modelos Genéticos , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/deficiência , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/deficiência
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