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1.
J Fungi (Basel) ; 8(10)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36294555

RESUMO

The majority of human coccidioidomycosis infections are asymptomatic or self-limited but may have sequestered spherules in highly structured granulomas. Under immunosuppression, reactivation of fungal growth can result in severe disease. B6D2F1 mice asymptomatically infected with C. posadasii strain 1038 were immunosuppressed with dexamethasone (DXM) in drinking water. Treated mice died 16−25 days later, while untreated mice survived (p < 0.001). Flow cytometry of lung granulomas on days 5, 10, 15, and 20 of DXM treatment showed immune cell populations decreased 0.5−1 log compared with untreated mice though neutrophils and CD19+IgD−IgM− cells rebounded by day 20. Histopathology demonstrated loss of granuloma structure by day 5 and increasing spherules through day 20. On day 20, T-cells were nearly absent and disorganized pyogranulomatous lesions included sheets of plasma cells and innumerable spherules. Mice given DXM for 14 days then stopped (DXM stop) survived 6 weeks (9/10). Lung fungal burdens were significantly lower (p = 0.0447) than mice that continued treatment (DXM cont) but higher than untreated mice. Histopathologically, DXM stop mice did not redevelop controlled granulomas by sacrifice, though T-cells were densely scattered throughout the lesions. This demonstrates a mouse model suitable for further study to understand the immunologic components responsible for maintenance control of coccidioidomycosis.

2.
Curr Protoc ; 2(9): e547, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36066328

RESUMO

The Collaborative Cross (CC) and the Diversity Outbred (DO) stock mouse panels are the most powerful murine genetics tools available to the genetics community. Together, they combine the strength of inbred animal models with the diversity of outbred populations. Using the 63 CC strains or a panel of DO mice, each derived from the same 8 parental mouse strains, researchers can map genetic contributions to exceptionally complex immunological and infectious disease traits that would require far greater powering if performed by genome-wide association studies (GWAS) in human populations. These tools allow genes to be studied in heterozygous and homozygous states and provide a platform to study epistasis between interacting loci. Most importantly, once a quantitative phenotype is investigated and quantitative trait loci are identified, confirmatory genetic studies can be performed, which is often problematic using the GWAS approach. In addition, novel stable mouse models for immune phenotypes are often derived from studies utilizing the DO and CC mice that can serve as stronger model systems than existing ones in the field. The CC/DO systems have contributed to the fields of cancer immunology, autoimmunity, vaccinology, infectious disease, allergy, tissue rejection, and tolerance but have thus far been greatly underutilized. In this article, we present a recent review of the field and point out key areas of immunology that are ripe for further investigation and awaiting new CC/DO research projects. We also highlight some of the strong computational tools that have been developed for analyzing CC/DO genetic and phenotypic data. Additionally, we have formed a centralized community on the CyVerse infrastructure where immunogeneticists can utilize those software tools, collaborate with groups across the world, and expand the use of the CC and DO systems for investigating immunogenetic phenomena. © 2022 Wiley Periodicals LLC.


Assuntos
Camundongos de Cruzamento Colaborativo , Doenças Transmissíveis , Animais , Camundongos de Cruzamento Colaborativo/genética , Doenças Transmissíveis/genética , Cruzamentos Genéticos , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Locos de Características Quantitativas
3.
mSphere ; 5(5)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32878927

RESUMO

Toxoplasma gondii's tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo This predilection for neurons suggests that T. gondii's persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells.IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii's persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/química , Animais , Perfilação da Expressão Gênica , Microdissecção e Captura a Laser , Camundongos , Monócitos/imunologia , Análise de Sequência de RNA , Toxoplasmose/parasitologia
4.
Proc Natl Acad Sci U S A ; 115(29): E6817-E6825, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29967140

RESUMO

Lifelong interactions between host and the ubiquitous and persistent cytomegalovirus (CMV) have been proposed to contribute to the age-related decline in immunity. Prior work from us and others found some support for that idea, yet evidence that this led to increased vulnerability to other infections was not obtained. Moreover, evidence has accumulated that CMV infection can be beneficial to immune defense in young/adult mice and humans, dominantly via enhanced innate immunity. Here, we describe an unexpected impact of murine CMV (MCMV) upon the T cell response of old mice to Listeria monocytogenes expressing the model antigen, OVA (Lm-OVA). Single-cell sequencing of the OVA-specific CD8 T cell receptor ß (TCRß) repertoire of old mice demonstrated that old MCMV-infected mice recruited many diverse clonotypes that afforded broad and often more efficient recognition of antigenic peptide variants. This stood in contrast to old control mice, which exhibited strong narrowing and homogenization of the elicited repertoire. High-throughput sequencing of the total naïve CD8 TCRß repertoire showed that many of these diverse OVA-specific clonotypes were present in the naïve CD8 repertoire of mice in all groups (adult, old control, and old MCMV+) yet were only recruited into the Lm-OVA response in MCMV+ old mice. These results have profound implications for our understanding of T cell immunity over a life span and suggest that our coevolution with CMV may include surprising, potentially positive impacts on adaptive heterologous immunity in late life.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Imunidade Celular , Listeria monocytogenes/imunologia , Listeriose/imunologia , Muromegalovirus/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Infecções por Citomegalovirus/patologia , Listeriose/patologia , Masculino , Camundongos
5.
Front Immunol ; 9: 976, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867956

RESUMO

Background: Recent technological advances in immune repertoire sequencing have created tremendous potential for advancing our understanding of adaptive immune response dynamics in various states of health and disease. Immune repertoire sequencing produces large, highly complex data sets, however, which require specialized methods and software tools for their effective analysis and interpretation. Results: VDJServer is a cloud-based analysis portal for immune repertoire sequence data that provide access to a suite of tools for a complete analysis workflow, including modules for preprocessing and quality control of sequence reads, V(D)J gene segment assignment, repertoire characterization, and repertoire comparison. VDJServer also provides sophisticated visualizations for exploratory analysis. It is accessible through a standard web browser via a graphical user interface designed for use by immunologists, clinicians, and bioinformatics researchers. VDJServer provides a data commons for public sharing of repertoire sequencing data, as well as private sharing of data between users. We describe the main functionality and architecture of VDJServer and demonstrate its capabilities with use cases from cancer immunology and autoimmunity. Conclusion: VDJServer provides a complete analysis suite for human and mouse T-cell and B-cell receptor repertoire sequencing data. The combination of its user-friendly interface and high-performance computing allows large immune repertoire sequencing projects to be analyzed with no programming or software installation required. VDJServer is a web-accessible cloud platform that provides access through a graphical user interface to a data management infrastructure, a collection of analysis tools covering all steps in an analysis, and an infrastructure for sharing data along with workflows, results, and computational provenance. VDJServer is a free, publicly available, and open-source licensed resource.


Assuntos
Computação em Nuvem , Biologia Computacional/métodos , Genômica/métodos , Éxons VDJ/imunologia , Animais , Metodologias Computacionais , Humanos , Disseminação de Informação , Camundongos , Software , Interface Usuário-Computador , Navegador , Fluxo de Trabalho
6.
Vet Immunol Immunopathol ; 197: 76-86, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29475511

RESUMO

Ideally, CD8+ T-cell responses against virally infected or malignant cells are defined at the level of the specific peptide and restricting MHC class I element, a determination not yet made in the dog. To advance the discovery of canine CTL epitopes, we sought to determine whether a putative classical MHC class Ia gene, Dog Leukocyte Antigen (DLA)-88, presents peptides from a viral pathogen, canine distemper virus (CDV). To investigate this possibility, DLA-88*508:01, an allele prevalent in Golden Retrievers, was expressed as a FLAG-tagged construct in canine histiocytic cells to allow affinity purification of peptide-DLA-88 complexes and subsequent elution of bound peptides. Pattern analysis of self peptide sequences, which were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), permitted binding preferences to be inferred. DLA-88*508:01 binds peptides that are 9-to-12 amino acids in length, with a modest preference for 9- and 11-mers. Hydrophobic residues are favored at positions 2 and 3, as are K, R or F residues at the C-terminus. Testing motif-matched and -unmatched synthetic peptides via peptide-MHC surface stabilization assay using a DLA-88*508:01-transfected, TAP-deficient RMA-S line supported these conclusions. With CDV infection, 22 viral peptides ranging from 9-to-12 residues in length were identified in DLA-88*508:01 eluates by LC-MS/MS. Combined motif analysis and surface stabilization assay data suggested that 11 of these 22 peptides, derived from CDV hemagglutinin, large polymerase, matrix, nucleocapsid, and V proteins, were processed and presented, and thus, potential targets of anti-viral CTL in DLA-88*508:01-bearing dogs. The presentation of diverse self and viral peptides indicates that DLA-88 is a classical MHC class Ia gene.


Assuntos
Apresentação de Antígeno , Vírus da Cinomose Canina/química , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/química , Proteínas Virais/química , Alelos , Motivos de Aminoácidos , Animais , Vírus da Cinomose Canina/imunologia , Cães/genética , Epitopos/química , Epitopos/imunologia , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/imunologia , Ligação Proteica , Linfócitos T/imunologia , Proteínas Virais/imunologia
7.
BMC Bioinformatics ; 17(Suppl 13): 333, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27766961

RESUMO

BACKGROUND: The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses. RESULTS: To help address this problem, we propose a standardized file format for representing V(D)J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D)J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format. CONCLUSIONS: The VDJML suite will allow users to streamline their V(D)J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://vdjserver.org/vdjml/ . We welcome participation from the community in developing the file format standard, as well as code contributions.


Assuntos
Genômica/métodos , Receptores Imunológicos/genética , Software , Recombinação V(D)J , Humanos , Disseminação de Informação
8.
Infect Immun ; 84(10): 3007-16, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27481239

RESUMO

The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of <1,000 CFU from an otherwise lethal C. posadasii intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to C. posadasii infection when used as a vaccine, the Δcps1 strain is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals.


Assuntos
Coccidioides/fisiologia , Coccidioidomicose/genética , Deleção de Sequência , Fatores de Virulência/genética , Virulência/fisiologia , Animais , Coccidioides/genética , Coccidioidomicose/prevenção & controle , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Vacinação/métodos
9.
Cell Immunol ; 299: 6-13, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26547365

RESUMO

In this communication we describe a novel way to use Next Generation Sequence from the receptors expressed on T and B cells. This informatics methodology is named iWAS, for immunonome Wide Association Study, where we use the immune receptor sequences derived from T and B cells and the features of those receptors (sequences themselves, V/J gene usage, length and character each of the CDR3 sub-regions) to define biomarkers of health and disease, as well as responses to therapies. Unlike GWAS, which do not provide immediate access to mechanism, the associations with immune receptors immediately suggest possible and plausible entrée's into disease pathogenesis and treatment.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Imunológicas/métodos , Informática , Análise de Sequência de DNA/métodos , Linfócitos B/imunologia , Estudo de Associação Genômica Ampla , Receptores de Superfície Celular/genética , Linfócitos T/imunologia , Recombinação V(D)J/genética
10.
Cancer Immunol Res ; 3(3): 228-35, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25576336

RESUMO

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8⁺ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer⁺ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor ß (TCRß) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRß clonotypes in a patient with a UNC-CDK4-1 tetramer⁺ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRß repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRß clonotypes represented >17% of the entire TCRß repertoire-far in excess of the UNC-CDK4-1 tetramer⁺ frequency-indicating that the recurrent TCRß clonotypes identified from UNC-CDK-4-1 tetramer⁺ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCRß clonotype sequences onto TCRß repertoires can help confirm or refute antigen-specific T-cell expansion in vivo.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Leucemia/imunologia , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Células U937
11.
J Am Assoc Lab Anim Sci ; 53(5): 517-22, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25255075

RESUMO

Multiple NOD. Cg-Prkdc(scid)Il2rg(tm1Wjl)Tg(HLA-A2.1)Enge/Sz (NSG/A2) transgenic mice maintained in a mouse barrier facility were submitted for necropsy to determine the cause of facial alopecia, tachypnea, dyspnea, and sudden death. Pneumonia and soft-tissue abscesses were observed, and Pasteurella pneumotropica biotype Jawetz was consistently isolated from the upper respiratory tract, lung, and abscesses. Epidemiologic investigation within the facility revealed presence of this pathogen in mice generated or rederived by the intramural Genetically Engineered Mouse Model (GEMM) Core but not in mice procured from several approved commercial vendors. Epidemiologic data suggested the infection originated from female or vasectomized male ND4 mice obtained from a commercial vendor and then comingled by the GEMM Core to induce pseudopregnancy in female mice for embryo implantation. Enrofloxacin delivered in drinking water (85 mg/kg body weight daily) for 14 d was sufficient to clear bacterial infection in normal, breeding, and immune-deficient mice without the need to change the antibiotic water source. This modified treatment regimen was administered to 2400 cages of mice to eradicate Pasteurella pneumotropica from the facility. Follow-up PCR testing for P. pneumotropica biotype Jawetz remained uniformly negative at 2, 6, 12, and 52 wk after treatment in multiple strains of mice that were originally infected. Together, these data indicate that enrofloxacin can eradicate P. pneumotropica from infected mice in a less labor-intensive approach that does not require breeding cessation and that is easily adaptable to the standard biweekly cage change schedule for individually ventilated cages.


Assuntos
Antibacterianos/administração & dosagem , Fluoroquinolonas/administração & dosagem , Camundongos Endogâmicos NOD , Infecções por Pasteurella/veterinária , Pasteurella pneumotropica/isolamento & purificação , Doenças dos Roedores/tratamento farmacológico , Criação de Animais Domésticos , Animais , Animais de Laboratório , Enrofloxacina , Feminino , Masculino , Infecções por Pasteurella/tratamento farmacológico , Infecções por Pasteurella/imunologia , Infecções por Pasteurella/microbiologia , Doenças dos Roedores/imunologia , Doenças dos Roedores/microbiologia
12.
Transpl Immunol ; 29(1-4): 138-45, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24161680

RESUMO

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno H-Y/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunotoxinas/imunologia , Depleção Linfocítica/métodos , Peptídeos/imunologia , Aloenxertos , Animais , Transplante de Medula Óssea , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Antígeno H-Y/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/farmacologia , Imunotoxinas/genética , Imunotoxinas/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Peptídeos/genética , Peptídeos/farmacologia
13.
Immunogenetics ; 65(9): 675-89, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23812210

RESUMO

Cytotoxic CD8+ T-cell immunosurveillance for intracellular pathogens, such as viruses, is controlled by classical major histocompatibility complex (MHC) class Ia molecules, and ideally, these antiviral T-cell populations are defined by the specific peptide and restricting MHC allele. Surprisingly, despite the utility of the cat in modeling human viral immunity, little is known about the feline leukocyte antigen class I complex (FLAI). Only a few coding sequences with uncertain locus origin and expression patterns have been reported. Of 19 class I genes, three loci--FLAI-E, FLAI-H, and FLAI-K--are predicted to encode classical molecules, and our objective was to evaluate their status by analyzing polymorphisms and tissue expression. Using locus-specific, PCR-based genotyping, we amplified 33 FLAI-E, FLAI-H, and FLAI-K alleles from 12 cats of various breeds, identifying, for the first time, alleles across three distinct loci in a feline species. Alleles shared the expected polymorphic and invariant sites in the α1/α2 domains, and full-length cDNA clones possessed all characteristic class Ia exons. Alleles could be assigned to a specific locus with reasonable confidence, although there was evidence of potentially confounding interlocus recombination between FLAI-E and FLAI-K. Only FLAI-E, FLAI-H, and FLAI-K origin alleles were amplified from cDNAs of multiple tissue types. We also defined hypervariable regions across these genes, which permitted the assignment of names to both novel and established alleles. As predicted, FLAI-E, FLAI-H, and FLAI-K fulfill the major criteria of class Ia genes. These data represent a necessary prerequisite for studying epitope-specific antiviral CD8+ T-cell responses in cats.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Gatos/genética , Antígenos de Histocompatibilidade Classe I/genética , Vírus/imunologia , Sequência de Aminoácidos , Animais , Gatos/imunologia , Clonagem Molecular , Regiões Determinantes de Complementaridade/genética , Genótipo , Antígenos de Histocompatibilidade Classe I/química , Modelos Animais , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência
14.
Front Microbiol ; 4: 16, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23403609

RESUMO

Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E(2) (PGE(2)). Synthesis of PGE(2) by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE(2) synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial) that result in up-regulation of the PGE(2) biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE(2) synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE(2) in primary macrophages, we infected cells with individual mutants from the closely related strain F. tularensis subspecies novicida U112 (U112) two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE(2) by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI). Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE(2). This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE(2). We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE(2), while U112 pdpA::Tn does not grow yet does induce PGE(2). We also found that U112 iglC::Tn neither grows nor induces PGE(2). These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE(2) synthesis. These mutants provide a critical entrée into the pathways used in the host for PGE(2) induction.

15.
PLoS One ; 7(8): e43333, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916243

RESUMO

Plexins are a family of genes (A,B,C, and D) that are expressed in many organ systems. Plexins expressed in the immune system have been implicated in cell movement and cell-cell interaction during the course of an immune response. In this study, the expression pattern of Plexin-B2 and Plexin-D1 in dendritic cells (DCs), which are central in immune activation, was investigated. Plexin-B2 and Plexin-D1 are reciprocally expressed in myeloid and plasmacytoid DC populations. Plasmacytoid DCs have high Plexin-B2 but low Plexin-D1, while the opposite is true of myeloid DCs. Expression of Plexin-B2 and Plexin-D1 is modulated upon activation of DCs by TLR ligands, TNFα, and anti-CD40, again in a reciprocal fashion. Semaphorin3E, a ligand for Plexin-D1 and Plexin-B2, is expressed by T cells, and interestingly, is dramatically higher on Th2 cells and on DCs. The expression of Plexins and their ligands on DCs and T cells suggest functional relevance. To explore this, we utilized chimeric mice lacking Plxnb2 or Plxnd1. Absence of Plexin-B2 and Plexin-D1 on DCs did not affect the ability of these cells to upregulate costimulatory molecules or the ability of these cells to activate antigen specific T cells. Additionally, Plexin-B2 and Plexin-D1 were dispensable for chemokine-directed in-vitro migration of DCs towards key DC chemokines, CXCL12 and CCL19. However, the absence of either Plexin-B2 or Plexin-D1 on DCs leads to constitutive expression of IL-12/IL-23p40. This is the first report to show an association between Plexin-B2 and Plexin-D1 with the negative regulation of IL-12/IL-23p40 in DCs. This work also shows the presence of Plexin-B2 and Plexin-D1 on mouse DC subpopulations, and indicates that these two proteins play a role in IL-12/IL-23p40 production that is likely to impact the immune response.


Assuntos
Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
J Diabetes Sci Technol ; 6(3): 515-24, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22768881

RESUMO

Major histocompatibility complex (MHC) class I and MHC class II molecules present short peptides that are derived from endogenous and exogenous proteins, respectively, to cognate T-cell receptors (TCRs) on the surface of T cells. The exquisite specificity with which T cells recognize particular peptide-major-histocompatibility-complex (pMHC) combinations has permitted development of soluble pMHC multimers that bind exclusively to selected T-cell populations. Because the pathogenesis of type 1 diabetes mellitus (T1DM) is driven largely by islet-reactive T-cell activity that causes ß-cell death, these reagents are useful tools for studying and, potentially, for treating this disease. When coupled to fluorophores or paramagnetic nanoparticles, pMHC multimers have been used to visualize the expansion and islet invasion of T-cell effectors during diabetogenesis. Administration of pMHC multimers to mice has been shown to modulate T-cell responses by signaling through the TCR or by delivering a toxic moiety that deletes the targeted T cell. In the nonobese diabetic mouse model of T1DM, a pMHC-I tetramer coupled to a potent ribosome-inactivating toxin caused long-term elimination of a specific diabetogenic cluster of differentiation 8+ T-cell population from the pancreatic islets and delayed the onset of diabetes. This review will provide an overview of the development and use of pMHC multimers, particularly in T1DM, and describe the therapeutic promise these reagents have as an antigen-specific means of ameliorating deleterious T-cell responses in this autoimmune disease.


Assuntos
Autoimunidade , Diabetes Mellitus Tipo 1/terapia , Antígenos de Histocompatibilidade/uso terapêutico , Imunoterapia/métodos , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/uso terapêutico , Linfócitos T/imunologia , Animais , Diabetes Mellitus Tipo 1/imunologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Imagem Molecular/métodos , Peptídeos/imunologia , Multimerização Proteica , Transdução de Sinais
17.
Gastroenterology ; 140(4): 1334-44, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21237170

RESUMO

BACKGROUND & AIMS: Studies of hepatitis C virus (HCV) infection, immunopathogenesis, and resulting liver diseases have been hampered by the lack of a small animal model. We developed humanized mice with human immune system and liver tissues to improve the studies of hepatitis C virus pathogenesis and treatment. METHODS: To promote engraftment of human hepatocytes, we expressed a fusion protein of the FK506 binding protein (FKBP) and caspase 8 under control of the albumin promoter (AFC8), which induces liver cell death, in Balb/C Rag2(-/-) γC-null mice. Cotransplantation of human CD34(+) human hematopoietic stem cells (HSC) and hepatocyte progenitors into the transgenic mice led to efficient engraftment of human leukocytes and hepatocytes. We then infected these humanized mice (AFC8-hu HSC/Hep) with primary HCV isolates and studied HCV-induced immune responses and liver diseases. RESULTS: AFC8-hu HSC/Hep mice supported HCV infection in the liver and generated a human immune T-cell response against HCV. HCV infection induced liver inflammation, hepatitis, and fibrosis, which correlated with activation of stellate cells and expression of human fibrogenic genes. CONCLUSIONS: AFC8-hu HSC/Hep mice are a useful model of HCV infection, the immune response, and liver disease because they contain human immune system and liver cells. These mice become infected with HCV, generate a specific immune response against the virus, and develop liver diseases that include hepatitis and fibrosis. This model might also be used to develop therapeutics for HCV infection.


Assuntos
Modelos Animais de Doenças , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Hepatócitos/transplante , Camundongos Transgênicos , Animais , Caspase 8/genética , Caspase 8/imunologia , Proteínas de Ligação a DNA/genética , Feminino , Hepatite C Crônica/genética , Hepatite C Crônica/patologia , Hepatócitos/imunologia , Hepatócitos/patologia , Humanos , Cirrose Hepática/genética , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco/imunologia , Células-Tronco/patologia , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/imunologia , Transplante Heterólogo
18.
J Immunol ; 184(8): 4196-204, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20220085

RESUMO

There is compelling evidence that self-reactive CD8(+) T cells are a major factor in development and progression of type 1 diabetes in animals and humans. Hence, great effort has been expended to define the specificity of autoimmune CD8(+) T cells and to alter their responses. Much work has focused on tolerization of T cells using proteins or peptides. A weakness in this approach is that residual autoreactive T cells may be activated and exacerbate disease. In this report, we use a novel approach, toxin-coupled MHC class I tetramers. Used for some time to identify Ag-specific cells, in this study, we use that same property to delete the Ag-specific cells. We show that saporin-coupled tetramers can delete islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive T cells in vitro and in vivo. Sequence analysis of TCRbeta-chains of IGRP(+) cells reveals the repertoire complexity in the islets is markedly decreased as NOD mice age and significantly altered in toxic tetramer-treated NOD mice. Further tetramer(+) T cells in the islets are almost completely deleted, and, surprisingly, loss of tetramer(+) T cells in the islets is long lasting. Finally, we show deletion at 8 wk of age of IGRP(+) CD8(+) T cells, but not dystophia myotonica kinase- or insulin B-reactive cells, significantly delays diabetes in NOD mice.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/prevenção & controle , Antígenos H-2/administração & dosagem , Imunotoxinas/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Microglobulina beta-2/administração & dosagem , Animais , Autoantígenos/imunologia , Autoantígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Morte Celular/imunologia , Movimento Celular/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Progressão da Doença , Epitopos de Linfócito T/imunologia , Feminino , Glucose-6-Fosfatase/administração & dosagem , Glucose-6-Fosfatase/biossíntese , Glucose-6-Fosfatase/imunologia , Antígenos H-2/toxicidade , Antígeno de Histocompatibilidade H-2D , Imunotoxinas/toxicidade , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Mimetismo Molecular/imunologia , Proteínas/administração & dosagem , Proteínas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Saporinas , Microglobulina beta-2/toxicidade
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