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1.
Cancer Cell ; 39(11): 1531-1547.e10, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34624218

RESUMO

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-ß signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.


Assuntos
Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Fator 7 de Crescimento de Fibroblastos/genética , Fator de Crescimento de Hepatócito/genética , Neoplasias Pulmonares/patologia , Biópsia , Fibroblastos Associados a Câncer/química , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Medicina de Precisão , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Regulação para Cima
2.
Methods Mol Biol ; 2237: 129-139, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237414

RESUMO

Membrane arrays are a unique array platform option for the detection of multiple analytes or materials simultaneously. Their naturally absorptive properties and near universal use in various laboratory methods make it an excellent source with which to probe multiple factors simultaneously. Any liquid sample type can be probed, from bacterial strains, tissue lysates, secreted proteins, to DNA aptamers. Below, we will describe some considerations in how to print a membrane array and then a specific usage of the membrane arrays as it relates to a sandwich-based antibody array technique for simultaneously detection of secreted proteins in a liquid sample.


Assuntos
Immunoblotting/métodos , Membranas Artificiais , Análise Serial de Proteínas/métodos , Absorção Fisico-Química , Colódio/química , Polivinil/química
3.
J Immunoassay Immunochem ; 37(4): 390-406, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27088654

RESUMO

Golgi membrane protein 1, or GP73, is recently being evaluated as a novel cancer biomarker against prostate cancer, lung adenocarcinoma, and hepatocellular carcinoma (HCC). In the microenvironment of HCC, GP73 expression levels are significantly elevated. It is this elevation that may prove more specific and sensitive for HCC detection than that of the traditional biomarker, alpha-fetoprotein (AFP). This may be especially true if it can be measured and identified earlier in the diagnostic process. We sought to develop a testing platform to measure GP73 levels for the purposes of earlier diagnostic screening of at risk patients. We expressed recombinant GP73 protein to use as an immunogen in order to develop several monoclonal anti-GP73 antibodies. Three clones, 1D7, 2B2, and 5B4, were identified with all three having a higher than 1:5,000,000 titer. These clones were then isotyped and validated to bind the immunogen protein. Different combinations of antibody pairs were then tested in order to create a functional sandwich antibody pair. Using this pair on liver disease patient serum samples, we found that GP73 was significantly elevated when compared to healthy control patient serum (P < 0.0001). Average GP73 levels in HCC patients was 284.0 ng/mL, slightly higher than liver disease patients (265.6 ng/mL), and significantly elevated over normal serum levels is (74.86 ng/mL). The area under the receiver-operating characteristic curve (ROC) for GP73 to detect liver cancer was 0.98 (95% CI, 0.95 to 1.00; P < 0.0001), and GP73 levels had a sensitivity of 97%, a specificity of 87% for detecting liver cancer. By contrast, the sensitivity and specificity of liver disease detection was 76% and 97%, respectively. We then tested detection of 74 serum samples (n control = 46, n liver disease = 7, n liver cancer = 21) by our ELISA testing methodology and commercial kit simultaneously. The results found that our kit and the commercial kit had a good linear correlation coefficient, r(2) = 0.932. Together these clones and our ELISA pair may prove extremely useful in the detection and monitoring of GP73 in HCC and other at risk patients.


Assuntos
Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/imunologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/imunologia , Proteínas de Membrana/imunologia , Anticorpos Monoclonais/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Imuno-Histoquímica , Neoplasias Hepáticas/sangue , Proteínas de Membrana/sangue , Proteínas de Membrana/genética
4.
Monoclon Antib Immunodiagn Immunother ; 34(5): 346-53, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26492623

RESUMO

Kallikrein-related peptidase 6 (KLK6) is a serine protease, and one of fifteen kallikrein members located on chromosome 19. KLK6 is associated with the regulation of axonal growth following spinal injury, tumor cell metastasis, and alpha synuclein aggregate pathologies like Parkinson's, making KLK6 a potentially important biomarker. We generated a KLK6 expression vector for large-scale expression in Escherichia coli. After affinity chromatography purification and SDS-PAGE confirmation, mice were immunized with the purified protein for development of monoclonal B cell populations. Our immunizations generated five hybridomas (1D8, 2E3, 3B7, 5G8, and 5H12) against KLK6. Isotyping analysis revealed that clone 2E3 was IgG2b, while the other four clones were IgG1. Further studies found that clone 5G8 could be used to visualize specific KLK6 bands by Western blot analysis from ovarian cancer patient serum and plasma, and also in mouse liver lysates. Finally, we established a sandwich ELISA pair and determined their sensitivity for KLK6 to be 10 ng/mL. These findings establish an essential tool for the detection and analysis of KLK6.


Assuntos
Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/imunologia , Calicreínas/imunologia , Animais , Carcinoma Epitelial do Ovário , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias Ovarianas/imunologia
5.
Int J Inflam ; 2015: 630637, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26346739

RESUMO

Asthma is a chronic inflammatory disease of the airways, resulting in bronchial hyperresponsiveness with every allergen exposure. It is now clear that asthma is not a single disease, but rather a multifaceted syndrome that results from a variety of biologic mechanisms. Asthma is further problematic given that the disease consists of many variants, each with its own etiologic and pathophysiologic factors, including different cellular responses and inflammatory phenotypes. These facets make the rapid and accurate diagnosis (not to mention treatments) of asthma extremely difficult. Protein biomarkers can serve as powerful detection tools in both clinical and basic research applications. Recent endeavors from biomedical researchers have developed technical platforms, such as cytokine antibody arrays, that have been employed and used to further the global analysis of asthma biomarker studies. In this review, we discuss potential asthma biomarkers involved in the pathophysiologic process and eventual pathogenesis of asthma, how these biomarkers are being utilized, and how further testing methods might help improve the diagnosis and treatment strain that current asthma patients suffer.

6.
Adv Clin Chem ; 69: 255-324, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25934364

RESUMO

All of life is regulated by complex and organized chemical reactions that help dictate when to grow, to move, to reproduce, and to die. When these processes go awry, or are interrupted by pathological agents, diseases such as cancer, autoimmunity, or infections can result. Cytokines, chemokines, growth factors, adipokines, and other chemical moieties make up a vast subset of these chemical reactions that are altered in disease states, and monitoring changes in these molecules could provide for the identification of disease biomarkers. From the first identification of carcinoembryonic antigen, to the discovery of prostate-specific antigen, to numerous others described within, biomarkers of disease are detectable in a plethora of sample types. The growing number of biomarkers for infection, autoimmunity, and cancer allow for increasingly early detection, to identification of novel drug targets, to prognostic indicators of disease outcome. However, more and more studies are finding that a single cytokine or growth factor is insufficient as a true disease biomarker and that a more global perspective is needed to understand true disease biology. Such a broad view requires a multiplexed platform for chemical detection, and antibody arrays meet and exceed this need by performing this detection in a high-throughput fashion. Herein, we will discuss how antibody arrays have evolved, and how they have helped direct new drug target design, helped identify therapeutic disease markers, and helped in earlier disease detection. From asthma to renal disease, and neurological dysfunction to immunologic disorders, antibody arrays afford a bright future for new biomarkers discovery.


Assuntos
Anticorpos , Biomarcadores/análise , Diagnóstico Precoce , Análise Serial de Proteínas/métodos , Asma/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/imunologia , Citocinas/sangue , Citocinas/imunologia , Descoberta de Drogas , Humanos , Nefropatias/sangue , Nefropatias/imunologia , Doenças Neurodegenerativas/sangue , Doenças Neurodegenerativas/imunologia , Proteínas/análise , Proteínas/imunologia
7.
J Immunol ; 195(1): 203-9, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26026054

RESUMO

CD8 airway resident memory T (TRM) cells are a distinctive TRM population with a high turnover rate and a unique phenotype influenced by their localization within the airways. Their role in mediating protective immunity to respiratory pathogens, although suggested by many studies, has not been directly proven. This study provides definitive evidence that airway CD8 TRM cells are sufficient to mediate protection against respiratory virus challenge. Despite being poorly cytolytic in vivo and failing to expand after encountering Ag, airway CD8 TRM cells rapidly express effector cytokines, with IFN-γ being produced most robustly. Notably, established airway CD8 TRM cells possess the ability to produce IFN-γ faster than systemic effector memory CD8 T cells. Furthermore, naive mice receiving intratracheal transfer of airway CD8 TRM cells lacking the ability to produce IFN-γ were less effective at controlling pathogen load upon heterologous challenge. This direct evidence of airway CD8 TRM cell-mediated protection demonstrates the importance of these cells as a first line of defense for optimal immunity against respiratory pathogens and suggests they should be considered in the development of future cell-mediated vaccines.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon gama/biossíntese , Pulmão/imunologia , Infecções por Orthomyxoviridae/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/transplante , Terapia Baseada em Transplante de Células e Tecidos , Interações Hospedeiro-Patógeno , Memória Imunológica , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon gama/metabolismo , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/terapia , Infecções por Orthomyxoviridae/virologia , Carga Viral , Replicação Viral
8.
J Immunol ; 188(9): 4340-8, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22447978

RESUMO

Repetitive Ag encounter, coupled with dynamic changes in Ag density and inflammation, imparts phenotypic and functional heterogeneity to memory virus-specific CD8 T cells in persistently infected hosts. For herpesvirus infections, which cycle between latency and reactivation, recent studies demonstrate that virus-specific T cell memory is predominantly derived from naive precursors recruited during acute infection. Whether functional memory T cells to viruses that persist in a nonlatent, low-level infectious state (smoldering infection) originate from acute infection-recruited naive T cells is not known. Using mouse polyomavirus (MPyV) infection, we previously showed that virus-specific CD8 T cells in persistently infected mice are stably maintained and functionally competent; however, a sizeable fraction of these memory T cells are short-lived. Further, we found that naive anti-MPyV CD8 T cells are primed de novo during persistent infection and contribute to maintenance of the virus-specific CD8 T cell population and its phenotypic heterogeneity. Using a new MPyV-specific TCR-transgenic system, we now demonstrate that virus-specific CD8 T cells recruited during persistent infection possess multicytokine effector function, have strong replication potential, express a phenotype profile indicative of authentic memory capability, and are stably maintained. In contrast, CD8 T cells recruited early in MPyV infection express phenotypic and functional attributes of clonal exhaustion, including attrition from the memory pool. These findings indicate that naive virus-specific CD8 T cells recruited during persistent infection contribute to preservation of functional memory against a smoldering viral infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Infecções por Polyomavirus/imunologia , Polyomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Camundongos , Camundongos Knockout , Infecções por Polyomavirus/genética , Infecções Tumorais por Vírus/genética
9.
J Immunol ; 188(7): 3071-9, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22393155

RESUMO

We recently identified a protective MHC class Ib-restricted CD8 T cell response to infection with mouse polyomavirus. These CD8 T cells recognize a peptide from aa 139-147 of the VP2 viral capsid protein bound to the nonpolymorphic H-2Q9 molecule, a member of the Qa-2 family of ß(2)m-associated MHC class Ib molecules. Q9:VP2.139-specific CD8 T cells exhibit an unusual inflationary response characterized by a gradual expansion over 3 mo followed by a stable maintenance phase. We previously demonstrated that Q9:VP2.139-specific CD8 T cells are dependent on Ag for expansion, but not for long-term maintenance. In this study, we tested the hypothesis that the expansion and maintenance components of the Q9:VP2.139-specific T cell response are differentially dependent on CD4 T cell help and CD28 costimulation. Depletion of CD4(+) cells and CD28/CD40L blockade impaired expansion of Q9:VP2.139-specific CD8 T cells, and intrinsic CD28 signaling was sufficient for expansion. In contrast, CD4 T cell insufficiency, but not CD28/CD40L blockade, resulted in a decline in frequency of Q9:VP2.139-specific CD8 T cells during the maintenance phase. These results indicate that the Q9:VP2.139-specific CD8 T cell response to mouse polyomavirus infection depends on CD4 T cell help and CD28 costimulation for inflationary expansion, but only on CD4 T cell help for maintenance.


Assuntos
Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Infecções por Polyomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Ligante de CD40/imunologia , Divisão Celular , Seleção Clonal Mediada por Antígeno , Feminino , Interleucina-2/farmacologia , Rim/virologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Polyomavirus/isolamento & purificação , Infecções por Polyomavirus/virologia , Glândulas Salivares/virologia , Infecções Tumorais por Vírus/virologia
10.
J Virol ; 85(19): 10126-34, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21775464

RESUMO

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.


Assuntos
Interferon gama/imunologia , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/patologia , Polyomavirus/imunologia , Polyomavirus/patogenicidade , Animais , Animais Recém-Nascidos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Interferon gama/administração & dosagem , Rim/imunologia , Rim/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças dos Roedores/imunologia , Doenças dos Roedores/patologia , Doenças dos Roedores/virologia , Carga Viral , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos
11.
J Immunol ; 185(3): 1692-700, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20622115

RESUMO

Virus-specific CD4(+) T cells optimize antiviral responses by providing help for antiviral humoral responses and CD8(+) T cell differentiation. Although CD4(+) T cell responses to viral infections that undergo complete clearance have been studied extensively, less is known about virus-specific CD4(+) T cell responses to viruses that persistently infect their hosts. Using a mouse polyomavirus (MPyV) infection model, we previously demonstrated that CD4(+) T cells are essential for recruiting naive MPyV-specific CD8(+) T cells in persistently infected mice. In this study, we defined two dominant MPyV-specific CD4(+) T cell populations, one directed toward an epitope derived from the nonstructural large T Ag and the other from the major viral capsid protein of MPyV. These MPyV-specific CD4(+) T cells vary in terms of their magnitude, functional profile, and phenotype during acute and persistent phases of infection. Using a minimally myeloablative-mixed bone marrow chimerism approach, we further show that naive virus-specific CD4(+) T cells, like anti-MPyV CD8(+) T cells, are primed de novo during persistent virus infection. In summary, these findings reveal quantitative and qualitative differences in the CD4(+) T cell response to a persistent virus infection and demonstrate that naive antiviral CD4(+) T cells are recruited during chronic polyomavirus infection.


Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Linfócitos T CD4-Positivos/imunologia , Proteínas do Capsídeo/imunologia , Movimento Celular/imunologia , Epitopos de Linfócito T/imunologia , Infecções por Polyomavirus/imunologia , Animais , Antígenos Virais de Tumores/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Doença Crônica , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Polyomavirus/crescimento & desenvolvimento , Polyomavirus/imunologia , Infecções por Polyomavirus/patologia , Infecções por Polyomavirus/virologia
12.
J Immunol ; 182(9): 5198-202, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19380764

RESUMO

The requirement for Ag in maintaining memory CD8 T cells often differs between infections that are acutely resolved and those that persist. Using the mouse polyoma virus (PyV) persistent infection model, we recently described a novel CD8 T cell response directed to a PyV peptide presented by Q9, an MHC class Ib molecule. This antiviral Q9-restricted CD8 T cell response is characterized by a 3-mo expansion phase followed by a long-term plateau phase. In this study, we demonstrate that viral Ag is required for this protracted inflation phase but is dispensable for the maintenance of this Q9-restricted CD8 T cell response. Moreover, proliferation by memory T cells, not recruitment of naive PyV-specific T cells, is primarily responsible for Q9-restricted, anti-PyV CD8 T cell inflation. These data reveal a dynamic shift in Ag dependence by an MHC class Ib-restricted memory CD8 T cell response during a persistent viral infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígenos H-2/imunologia , Infecções por Polyomavirus/imunologia , Polyomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/virologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Proliferação de Células , Feminino , Antígenos H-2/genética , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Polyomavirus/patologia , Infecções por Polyomavirus/virologia , Fatores de Tempo , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia
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