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1.
Nat Immunol ; 23(4): 532-542, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35332327

RESUMO

The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here, we show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1ß, which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.


Assuntos
Inflamação , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1 , Animais , COVID-19 , Inflamação/imunologia , Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1/genética , Interleucina-1/imunologia , Lipídeos , Camundongos , RNA , Vacinas Sintéticas , Vacinas de mRNA/efeitos adversos , Vacinas de mRNA/metabolismo
2.
Cell ; 165(1): 165-179, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-26924576

RESUMO

Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput datasets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif datasets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well-defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks.


Assuntos
Redes Reguladoras de Genes , Inflamação/genética , Inflamação/imunologia , Animais , Lipídeo A/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Fator de Resposta Sérica/metabolismo
3.
Cell ; 150(2): 279-90, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22817891

RESUMO

Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.


Assuntos
Cromatina/genética , Perfilação da Expressão Gênica , Inflamação/genética , Macrófagos/metabolismo , Splicing de RNA , Animais , Regulação da Expressão Gênica , Lipídeo A/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Receptor de Interferon alfa e beta/genética , Receptores de Interferon/genética , Análise de Sequência de RNA , Transcrição Gênica
4.
Immunity ; 47(3): 421-434.e3, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28930658

RESUMO

Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.


Assuntos
Regulação da Expressão Gênica/efeitos da radiação , Inflamação/genética , Inflamação/metabolismo , Radiação Ionizante , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Análise por Conglomerados , Proteína Quinase Ativada por DNA/metabolismo , Relação Dose-Resposta à Radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Interferons/metabolismo , Interferons/farmacologia , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Proteínas de Membrana/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Transcrição Gênica/efeitos da radiação , Ativação Transcricional , Regulador Transcricional ERG/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
RNA ; 19(6): 811-27, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23616639

RESUMO

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3' end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3'-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.


Assuntos
Processamento Alternativo , Lipídeo A/farmacologia , Macrófagos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Éxons , Regulação da Expressão Gênica , Inflamação/genética , Íntrons , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Poliadenilação , Clivagem do RNA , Sítios de Splice de RNA , RNA Mensageiro/genética , Fatores de Tempo , Transcrição Gênica
6.
J Exp Med ; 221(2)2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38095631

RESUMO

Toll-like receptors 7 (TLR7) and 8 (TLR8) each sense single-stranded RNA (ssRNA), but their activation results in different immune activation profiles. Attempts to selectively target either TLR7 or TLR8 have been hindered by their high degree of homology. However, recent studies revealed that TLR7 and TLR8 bind different ligands resulting from the processing of ssRNA by endolysosomal RNases. We demonstrate that by introducing precise 2' sugar-modified bases into oligoribonucleotides (ORNs) containing known TLR7 and TLR8 binding motifs, we could prevent RNase-mediated degradation into the monomeric uridine required for TLR8 activation while preserving TLR7 activation. Furthermore, a novel, optimized protocol for CRISPR-Cas9 knockout in primary human plasmacytoid dendritic cells showed that TLR7 activation is dependent on RNase processing of ORNs and revealed a previously undescribed role for RNase 6 in degrading ORNs into TLR ligands. Finally, 2' sugar-modified ORNs demonstrated robust innate immune activation in mice. Altogether, we identified a strategy for creating tunable TLR7-selective agonists.


Assuntos
Ribonucleases , Receptor 7 Toll-Like , Humanos , Camundongos , Animais , Receptor 7 Toll-Like/genética , Nucleotídeos , Receptor 8 Toll-Like/genética , Ligantes , RNA , Adjuvantes Imunológicos , Açúcares
7.
Antimicrob Agents Chemother ; 57(4): 1882-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23380731

RESUMO

Although polymorphonuclear leukocytes (PMNs) are powerfully anti-Aspergillus, transfusion therapy remains controversial, with conflicting results, and experimental support has been lacking. We devised a pulmonary infection model in neutropenic BALB/c mice, used an antibacterial regimen to prevent confounding sepsis, and optimized PMN induction, purifications, and dose. Mice were given 150 mg/kg cyclophosphamide every 4 days and a gentamicin-vancomycin-clindamycin-imipenem regimen daily beginning 4 days before intranasal challenge with 5 × 10(5) Aspergillus conidia. This regimen produced leukopenia (~10% of normal white blood cell [WBC] count; ≤ 10% PMNs) for 10 days, without bacterial superinfection. PMN donors given 100 µg/kg recombinant murine granulocyte colony-stimulating factor (G-CSF) for 10 days yielded 11 × 10(7) to 13.6 × 10(7) WBC/ml (81 to 87% PMNs). Infected mice were given PMN transfusions intravenously. In 2 experiments with up to 70% mortality of neutropenic controls, transfusion of 10(7) PMNs 1 and 4 days after challenge had negligible effects on peripheral WBC counts but improved survival (P = 0.007, 0.02), decreased lung CFU (P = 0.03, 0.005), and cleared infection in 28 to 50% of survivors. Transfusion of 5 × 10(6) PMNs showed partial protection. Transfusions given every other day did not improve protection. Our present results provide an experimental basis for enthusiasm for PMN transfusions in the therapy of aspergillosis in humans.


Assuntos
Granulócitos/transplante , Transfusão de Leucócitos/métodos , Aspergilose Pulmonar/terapia , Animais , Aspergillus fumigatus/patogenicidade , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutropenia/terapia , Neutrófilos/citologia , Neutrófilos/fisiologia
8.
Nat Biotechnol ; 2023 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-37857725

RESUMO

The broad application of precision cancer immunotherapies is limited by the number of validated neoepitopes that are common among patients or tumor types. To expand the known repertoire of shared neoantigen-human leukocyte antigen (HLA) complexes, we developed a high-throughput platform that coupled an in vitro peptide-HLA binding assay with engineered cellular models expressing individual HLA alleles in combination with a concatenated transgene harboring 47 common cancer neoantigens. From more than 24,000 possible neoepitope-HLA combinations, biochemical and computational assessment yielded 844 unique candidates, of which 86 were verified after immunoprecipitation mass spectrometry analyses of engineered, monoallelic cell lines. To evaluate the potential for immunogenicity, we identified T cell receptors that recognized select neoepitope-HLA pairs and elicited a response after introduction into human T cells. These cellular systems and our data on therapeutically relevant neoepitopes in their HLA contexts will aid researchers studying antigen processing as well as neoepitope targeting therapies.

9.
Acta Pharm Sin B ; 12(4): 1624-1635, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35251918

RESUMO

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

10.
Antimicrob Agents Chemother ; 53(5): 1858-62, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19273680

RESUMO

In separate previous studies, we have shown that lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious against coccidioidal meningitis in rabbits. Here, we compared ABLC and AmBi directly in a coccidioidal meningitis model. Male New Zealand White rabbits were infected with 5 x 10(4) Coccidioides posadasii arthroconidia by direct cisternal puncture. Therapy with intravenous ABLC or AmBi at 7.5 or 15 mg/kg of body weight or sterile 5% dextrose water (D5W) began 5 days later. Clinical assessments were done daily; cerebrospinal fluid and blood samples were obtained on day 15 and upon euthanasia. Survivors to day 25 were euthanatized, the numbers of CFU in their tissues were determined, and histology analyses of the brains and spinal cords were done. Controls showed progressive disease, whereas animals treated with either dose of either drug showed few clinical signs of infection. All ABLC- or AmBi-treated rabbits survived, whereas eight of nine D5W-treated rabbits were euthanatized before day 25 (P < 0.0001). Numbers of CFU in the brains and spinal cords of ABLC- or AmBi-treated animals were 100- to 10,000-fold lower than those in the corresponding tissues of D5W-treated animals (P < 0.0006 to 0.0001). However, only two or fewer given a regimen of ABLC or AmBi were cured of infection in both tissues. Fewer ABLC-treated rabbits (four of eight treated with 7.5 mg/kg and five of eight treated with 15 mg/kg) than controls (nine of nine) had meningitis at any level of severity (P, 0.015 or 0.043 for animals treated with ABLC at 7.5 or 15 mg/kg, respectively). Although groups of rabbits treated with AmBi regimens did not have significantly fewer animals with meningitis than the control group (P > 0.05), ABLC and AmBi were not significantly different. In this model, intravenous ABLC and AmBi were similarly highly effective, with few clinical signs of infection, 100% survival, and significantly reduced fungal burdens among treated animals. There appeared to be little benefit in using the 15-mg/kg dosage of either formulation. There was no significant advantage of one drug over the other for this indication. Further studies are required to determine the lowest effective doses of these formulations.


Assuntos
Anfotericina B , Antifúngicos , Coccidioides/efeitos dos fármacos , Coccidioidomicose/tratamento farmacológico , Meningite Fúngica/tratamento farmacológico , Anfotericina B/administração & dosagem , Anfotericina B/uso terapêutico , Animais , Antifúngicos/administração & dosagem , Antifúngicos/uso terapêutico , Encéfalo/microbiologia , Encéfalo/patologia , Líquido Cefalorraquidiano/microbiologia , Coccidioides/virologia , Coccidioidomicose/microbiologia , Coccidioidomicose/mortalidade , Coccidioidomicose/patologia , Modelos Animais de Doenças , Humanos , Masculino , Meningite Fúngica/microbiologia , Meningite Fúngica/mortalidade , Meningite Fúngica/patologia , Coelhos , Índice de Gravidade de Doença , Medula Espinal/microbiologia , Medula Espinal/patologia , Resultado do Tratamento
11.
Nat Commun ; 10(1): 5228, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31745090

RESUMO

Profound global loss of DNA methylation is a hallmark of many cancers. One potential consequence of this is the reactivation of transposable elements (TEs) which could stimulate the immune system via cell-intrinsic antiviral responses. Here, we develop REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observe increased expression of over 400 TE subfamilies, of which 262 appear to result from a proximal loss of DNA methylation. The most recurrent TEs are among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent results in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing inflammation and the display of potentially immunogenic neoantigens.


Assuntos
Antígenos de Neoplasias/imunologia , Biologia Computacional/métodos , Elementos de DNA Transponíveis/imunologia , Neoplasias/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/genética , Metilação de DNA/imunologia , Elementos de DNA Transponíveis/genética , Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Humanos , Imunoterapia/métodos , Neoplasias/genética , Neoplasias/terapia , Análise de Sequência de RNA
12.
Diagn Microbiol Infect Dis ; 62(1): 106-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18555634

RESUMO

Candida parapsilosis family has 3 proposed species: C. parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. C. parapsilosis sensu stricto had significantly higher caspofungin (CAS) and anidulafungin MICs than C. orthopsilosis or C. metapsilosis; C. metapsilosis was least susceptible to fluconazole. C. parapsilosis sensu stricto more frequently displayed (37%) paradoxical growth in CAS (P < or = 0.02). These species susceptibility differences could affect therapeutic choices.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Equinocandinas/farmacologia , Fluconazol/farmacologia , Anidulafungina , Candida/classificação , Candida/genética , Candidíase/microbiologia , Caspofungina , Humanos , Lipopeptídeos , Testes de Sensibilidade Microbiana , Técnica de Amplificação ao Acaso de DNA Polimórfico
13.
PLoS One ; 10(7): e0132061, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26147648

RESUMO

A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development.


Assuntos
Envelhecimento/imunologia , Regulação da Expressão Gênica/imunologia , Monócitos/imunologia , Adulto , Idoso , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Recém-Nascido , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/citologia
14.
Artigo em Inglês | MEDLINE | ID: mdl-24747344

RESUMO

Virtually all living organisms have evolved mechanisms to adapt to their environment by sensing environmental stresses and inducing the transcription of appropriate sets of response genes in a coordinated fashion. In the vertebrate immune system, the highly selective response to an environmental stimulus, often an invading microorganism, plays an especially important role in regulating the activities of, and interactions among, the many cell types involved in innate and adaptive immunity. It is now widely appreciated that the selective response to a stimulus requires the concerted action of signal transduction pathways, transcription factors, and chromatin structure. Many proteins and pathways that help to regulate a response have been characterized. However, our understanding of the gene-specific and global logic through which a highly selective response is elicited has only recently begun to emerge.


Assuntos
Regulação da Expressão Gênica , Transcrição Gênica , Animais , Cromatina/química , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Redes Reguladoras de Genes , Humanos , Subunidade p40 da Interleucina-12/metabolismo , Camundongos , Nucleossomos/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Fatores de Transcrição/metabolismo
15.
Immunol Lett ; 128(2): 105-7, 2010 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-20064561

RESUMO

Mannose binding lectin (MBL) is a protein of the collectin family that appears important in resistance to invasive pulmonary aspergillosis. We assessed the role of MBL in experimental systemic aspergillosis. MBL-sufficient C57BL/6 (WT) mice and B6.129S4--Mb11(tm1Kata) Mb12(tm1Kata)/J MBL A and C gene-knockout (KO) mice were infected intravenously with different inocula of Aspergillus fumigatus conidia. WT and KO mice were dose-responsively susceptible. In no instance were the KO mice more susceptible than WT. At the highest inoculum, all WT and 90% of KO mice died on day 4 (P>0.05). Reduction of the inoculum to 5.5 x 10(6) conidia was lethal, but comparison showed KO mice less susceptible to lethal infection (P<0.015). At the lowest inoculum used, deaths of KO mice were delayed, but survival was not significantly different than WT (P>0.05). These results suggest MBL may play a deleterious role in systemic aspergillosis.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/patogenicidade , Lectina de Ligação a Manose/genética , Animais , Aspergilose/genética , Aspergilose/microbiologia , Aspergilose/mortalidade , Modelos Animais de Doenças , Feminino , Humanos , Lectina de Ligação a Manose/metabolismo , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sobrevida
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