RESUMO
BACKGROUND: Biliary atresia is the most common cause of obstructive jaundice in infants and conventional cholangiography is the current diagnostic gold standard. Fluorescent cholangiography with indocyanine green can enhance biliary tree visualization during surgery because it is exclusively excreted into the bile ducts and eventually into the intestine. Therefore, we hypothesized that indocyanine green presence in stool could confirm bile duct patency in infants. METHODS: A prospective single center cohort study was performed on infants (age ≤ 12 months) with and without jaundice after obtaining IRB approval. Indocyanine green was administered intravenously (0.1 mg/kg). Soiled diapers collected post-injection were imaged for fluorescence. RESULTS: After indocyanine green administration, fluorescence was detected in soiled diapers for control patients (n = 4, x = 14 h22 m post-injection) and jaundiced patients without biliary atresia (n = 11, x = 13 h28 m post-injection). For biliary atresia patients (n = 7), post-injection soiled diapers before and after Kasai portoenterostomy were collected. Fluorescence was not detected in stool from 6 of 7 biliary atresia patients. As a test, indocyanine green detection in stool was 97% accurate for assessing biliary patency. CONCLUSION: Fluorescent Imaging for Indocyanine Green (FIInd Green) in stool is a fast and accurate approach to assess biliary patency non-invasively in infants. LEVEL OF EVIDENCE: Level III.
Assuntos
Atresia Biliar , Corantes , Fezes , Verde de Indocianina , Humanos , Atresia Biliar/diagnóstico por imagem , Atresia Biliar/cirurgia , Atresia Biliar/complicações , Projetos Piloto , Lactente , Fezes/química , Estudos Prospectivos , Masculino , Feminino , Corantes/administração & dosagem , Colangiografia/métodos , Portoenterostomia Hepática , Imagem Óptica/métodos , Icterícia Obstrutiva/etiologia , Icterícia Obstrutiva/diagnóstico por imagem , Recém-NascidoRESUMO
BACKGROUND & AIMS: Syndromic biliary atresia is a cholangiopathy characterized by fibro-obliterative changes in the extrahepatic bile duct (EHBD) and congenital malformations including laterality defects. The etiology remains elusive and faithful animal models are lacking. Genetic syndromes provide important clues regarding the pathogenic mechanisms underlying the disease. We investigated the role of the gene Pkd1l1 in the pathophysiology of syndromic biliary atresia. METHODS: Constitutive and conditional Pkd1l1 knockout mice were generated to explore genetic pathology as a cause of syndromic biliary atresia. We investigated congenital malformations, EHBD and liver pathology, EHBD gene expression, and biliary epithelial cell turnover. Biliary drainage was functionally assessed with cholangiography. Histology and serum chemistries were assessed after DDC (3,5-diethoxycarbony l-1,4-dihydrocollidine) diet treatment and inhibition of the ciliary signaling effector GLI1. RESULTS: Pkd1l1-deficient mice exhibited congenital anomalies including malrotation and heterotaxy. Pkd1l1-deficient EHBDs were hypertrophic and fibrotic. Pkd1l1-deficient EHBDs were patent but displayed delayed biliary drainage. Pkd1l1-deficient livers exhibited ductular reaction and periportal fibrosis. After DDC treatment, Pkd1l1-deficient mice exhibited EHBD obstruction and advanced liver fibrosis. Pkd1l1-deficient mice had increased expression of fibrosis and extracellular matrix remodeling genes (Tgfα, Cdkn1a, Hb-egf, Fgfr3, Pdgfc, Mmp12, and Mmp15) and decreased expression of genes mediating ciliary signaling (Gli1, Gli2, Ptch1, and Ptch2). Primary cilia were reduced on biliary epithelial cells and altered expression of ciliogenesis genes occurred in Pkd1l1-deficient mice. Small molecule inhibition of the ciliary signaling effector GLI1 with Gant61 recapitulated Pkd1l1-deficiency. CONCLUSIONS: Pkd1l1 loss causes both laterality defects and fibro-proliferative EHBD transformation through disrupted ciliary signaling, phenocopying syndromic biliary atresia. Pkd1l1-deficient mice function as an authentic genetic model for study of the pathogenesis of biliary atresia. IMPACT AND IMPLICATIONS: The syndromic form of biliary atresia is characterized by fibro-obliteration of extrahepatic bile ducts and is often accompanied by laterality defects. The etiology is unknown, but Pkd1l1 was identified as a potential genetic candidate for syndromic biliary atresia. We found that loss of the ciliary gene Pkd1l1 contributes to hepatobiliary pathology in biliary atresia, exhibited by bile duct hypertrophy, reduced biliary drainage, and liver fibrosis in Pkd1l1-deficient mice. Pkd1l1-deficient mice serve as a genetic model of biliary atresia and reveal ciliopathy as an etiology of biliary atresia. This model will help scientists uncover new therapeutic approaches for patients with biliary atresia, while pediatric hepatologists should validate the diagnostic utility of PKD1L1 variants.
Assuntos
Atresia Biliar , Cílios , Modelos Animais de Doenças , Células Epiteliais , Camundongos Knockout , Animais , Camundongos , Ductos Biliares Extra-Hepáticos/metabolismo , Ductos Biliares Extra-Hepáticos/patologia , Atresia Biliar/metabolismo , Atresia Biliar/patologia , Atresia Biliar/genética , Cílios/metabolismo , Cílios/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Piridinas , Transdução de SinaisRESUMO
Although midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated an Igfbp2-CreER knockin strain that specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21%-41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration.
Assuntos
Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina , Regeneração Hepática , Fígado , Hepatectomia , Hepatócitos , Homeostase , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismoRESUMO
BACKGROUND: Biliary Atresia (BA), an obstructive cholangiopathy, is the most common cause of end-stage liver disease and liver transplantation in children. Timely differentiation of BA from other causes of neonatal jaundice remains a challenge, yet is critical to improving outcomes. METHODS: Clinical characteristics including demographics, age at jaundice presentation, age at hepatobiliary scintigraphy, age at surgery, severity of liver fibrosis, and native-liver survival were reviewed in infants with hyperbilirubinemia and suspected BA for this single center retrospective cohort study. We investigated the accuracy of hepatobiliary scintigraphy as well as elapsed time from jaundice presentation to diagnostic intervention. RESULTS: BA was suspected in 234 infants. BA was identified in 17% of infants with hepatobiliary scintigraphy and 72% of infants who underwent operative exploration without hepatobiliary scintigraphy. Elapsed time from jaundice presentation to Kasai Portoenterostomy (KPE) for BA patients was 2.1x longer if hepatobiliary scintigraphy was obtained (p = 0.084). The mean age at KPE for this cohort was 66.8 days (n = 54), with a significantly higher mean age at KPE (75.2 days) for infants who were later listed or underwent liver transplantation (p = 0.038). Histologically, the lowest liver fibrosis scores were seen in infants undergoing KPE <30 days old and worsened significantly with increased age (p < 0.001). CONCLUSION: Hepatobiliary scintigraphy compared to operative exploration for the diagnostic evaluation of infants with suspected BA introduces significant time delays to KPE but enables avoidance of surgery in some infants. The temporal pattern of worsening cholestatic liver injury from BA with each day of increased age highlights the importance of intervening as early as possible for the best prognosis. TYPE OF STUDY: Retrospective study, Level of evidence: III.
Assuntos
Atresia Biliar , Icterícia , Recém-Nascido , Lactente , Criança , Humanos , Portoenterostomia Hepática , Estudos Retrospectivos , Atresia Biliar/diagnóstico por imagem , Atresia Biliar/cirurgia , Cintilografia , Icterícia/cirurgia , Cirrose Hepática/cirurgiaRESUMO
BACKGROUND: Wilms tumor (WT) is the most common childhood kidney cancer worldwide, yet its incidence and clinical behavior vary according to race and access to adequate healthcare resources. To guide and streamline therapy in the war-torn and resource-constrained city of Baghdad, Iraq, we conducted a first-ever molecular analysis of 20 WT specimens to characterize the biological features of this lethal disease within this challenged population. METHODS: Next-generation sequencing of ten target genes associated with WT development and treatment resistance (WT1, CTNNB1, WTX, IGF2, CITED1, SIX2, p53, N-MYC, CRABP2, and TOP2A) was completed. Immunohistochemistry was performed for 6 marker proteins of WT (WT1, CTNNB1, NCAM, CITED1, SIX2, and p53). Patient outcomes were compiled. RESULTS: Mutations were detected in previously described WT "hot spots" (e.g., WT1 and CTNNB1) as well as novel loci that may be unique to the Iraqi population. Immunohistochemistry showed expression domains most typical of blastemal-predominant WT. Remarkably, despite the challenges facing families and care providers, only one child, with combined WT1 and CTNNB1 mutations, was confirmed dead from disease. Median clinical follow-up was 40.5 months (range 6-78 months). CONCLUSIONS: These data suggest that WT biology within a population of Iraqi children manifests features both similar to and unique from disease variants in other regions of the world. These observations will help to risk stratify WT patients living in this difficult environment to more or less intensive therapies and to focus treatment on cell-specific targets.
Assuntos
Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose , Pré-Escolar , DNA Topoisomerases Tipo II/genética , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Fator de Crescimento Insulin-Like II/genética , Iraque , Neoplasias Renais/patologia , Masculino , Reação em Cadeia da Polimerase Multiplex , Mutação , Proteína Proto-Oncogênica N-Myc/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores do Ácido Retinoico/genética , Análise de Sequência de DNA/métodos , Transativadores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas WT1/genética , Proteínas WT1/metabolismo , Tumor de Wilms/patologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
UNLABELLED: STAT3-driven expression of small proline rich protein 2a (SPRR2a), which acts as an src homology 3 (SH3) domain ligand, induces biliary epithelial cell (BEC) epithelial-mesenchymal transition (EMT), which, in turn, promotes wound healing. SPRR2a also quenches free radicals and protects against oxidative stress and DNA damage in nonneoplastic BEC. Sprr2a-induced EMT also increases local invasiveness of cholangiocarcinomas (CC), but prevents metastases. Understanding SPRR2a regulation of EMT has potential for therapeutic targeting in both benign and malignant liver disease. Molecular mechanisms responsible for SPRR2a-induced EMT were characterized, in vitro, and then evidence for utilization of these pathways was sought in human intrahepatic CC, in vivo, using multiplex labeling and software-assisted morphometric analysis. SPRR2a complexes with ZEB1 and CtBP on the microRNA (miR)-200c/141 promoter resulting in synergic suppression of miR-200c/141 transcription, which is required for maintenance of the BEC epithelial phenotype. SPRR2a induction promotes dephosphorylation and nuclear translocation of the SH3-domain containing protein GRB2 and an SH3-domain ligand in ZEB1 is required for SPRR2a-induced synergic suppression of miR-200c/141. Multiplex protein labeling of CC and morphometric analyses showed: 1) up-regulation of ZEB-1, and 2) down-regulation of CK19 in intrahepatic CC compared to nonneoplastic BEC, consistent with previous CC proteomic studies showing EMT during cholangiocarcinogenesis. CONCLUSION: SPRR2a modulates ZEB-1 signaling by way of miR-200c/141-associated EMT through SH3-domain networks and contributes to benign and malignant BEC wound-healing responses.
Assuntos
Neoplasias dos Ductos Biliares/fisiopatologia , Ductos Biliares Intra-Hepáticos/fisiopatologia , Colangiocarcinoma/fisiopatologia , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Hepatopatias/fisiopatologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Proteínas Ricas em Prolina do Estrato Córneo/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hepatopatias/genética , Hepatopatias/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cicatrização/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Domínios de Homologia de src/fisiologiaRESUMO
Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.
Assuntos
Bactérias/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Intestinos/microbiologia , Células de Kupffer/fisiologia , Transplante de Fígado/efeitos adversos , Traumatismo por Reperfusão/etiologia , Animais , Bactérias/isolamento & purificação , Translocação Bacteriana/fisiologia , Ceco/microbiologia , Ceco/patologia , Endotélio Vascular/metabolismo , Vida Livre de Germes , Glicoproteínas/biossíntese , Imunofenotipagem , Células de Kupffer/imunologia , Fígado/imunologia , Fígado/metabolismo , Masculino , Proteínas de Membrana Transportadoras , Metagenoma , Camundongos , Fagocitose , Receptores de Reconhecimento de Padrão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
BACKGROUND: Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions. RESULTS: Using a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA. CONCLUSION: Up-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Histona Desacetilase 1/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , DNA/metabolismo , Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/metabolismo , Células Hep G2 , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Ubiquitinação , Regulação para CimaRESUMO
Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.
Assuntos
Epitélio/metabolismo , MicroRNAs/biossíntese , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Modelos Genéticos , Fenótipo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Transcrição Gênica , Regulação para CimaRESUMO
DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding suggests that such synthetic immunogens could be important for examination of their potential as part of an efficient HIV DNA vaccine.
Assuntos
Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Sequência Consenso , Reações Cruzadas , Feminino , Anticorpos Anti-HIV/sangue , HIV-1/genética , HIV-1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagemRESUMO
Various technologies including nucleic acid, protein, and metabolic array analyses of blood, liver tissue, and bile are emerging as powerful tools in the study of hepatic pathophysiology. The entire lexicon of liver disease, however, has been written using classical hematoxylin-eosin staining and light microscopic examination. The authors' goal is to develop new tools to enhance histopathologic examination of liver tissue that would enrich the information gained from liver biopsy analysis, enable quantitative analysis, and bridge the gap between various "-omics" tools and interpretation of routine liver biopsy results. This article describes the progress achieved during the past 2 years in developing multiplex quantum dot (nanoparticle) staining and combining it with high-resolution whole-slide imaging using a slide scanner equipped with filters to capture 9 distinct fluorescent signals for multiple antigens. The authors first focused on precise characterization of leukocyte subsets, but soon realized that the data generated were beyond the practical limits that could be properly evaluated, analyzed, and interpreted visually by a pathologist. Therefore, the authors collaborated with the open source FARSIGHT image analysis project (http://www.farsight-toolkit.org). FARSIGHT's goal is to develop and disseminate the next-generation toolkit of automated image analysis methods to enable quantification of molecular biomarkers on a cell-by-cell basis from multiparameter images. The resulting data can be used for histocytometric studies of the complex and dynamic tissue microenvironments that are of biomedical interest. The authors envisage that these tools will eventually be incorporated into the routine practice of surgical pathology and precipitate a revolution in the specialty.
Assuntos
Biópsia/métodos , Imuno-Histoquímica/métodos , Hepatopatias/patologia , Processamento Eletrônico de Dados/instrumentação , Processamento Eletrônico de Dados/métodos , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Hepatopatias/diagnóstico , Pontos QuânticosRESUMO
Minimization of immunosuppression and donor-specific tolerance to MHC-mismatched organ grafts are important clinical goals. The therapeutic potential of regulatory T cells (Tregs) has been demonstrated, but conditions for optimizing their in vivo function posttransplant in nonlymphocyte-depleted hosts remain undefined. In this study, we address mechanisms through which inhibition of the mammalian target of rapamycin (Rapa) synergizes with alloantigen-specific Treg (AAsTreg) to permit long-term, donor-specific heart graft survival in immunocompetent hosts. Crucially, immature allogeneic dendritic cells allowed AAsTreg selection in vitro, with minimal expansion of unwanted (Th17) cells. The rendered Treg potently inhibited T cell proliferation in an Ag-specific manner. However, these AAsTreg remained unable to control T cells stimulated by allogeneic mature dendritic cells, a phenomenon dependent on the release of proinflammatory cytokines. In vivo, Rapa administration reduced danger-associated IL-6 production, T cell proliferation, and graft infiltration. Based on these observations, AAsTreg were administered posttransplant (day 7) in combination with a short course of Rapa and rendered >80% long-term (>150 d) graft survival, a result superior to that achieved with polyclonal Treg. Moreover, graft protection was alloantigen-specific. Significantly, long-term graft survival was associated with alloreactive T cell anergy. These findings delineate combination of transient mammalian target of Rapa inhibition with appropriate AAsTreg selection as an effective approach to promote long-term organ graft survival.
Assuntos
Sobrevivência de Enxerto/imunologia , Transplante de Coração/métodos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T Reguladores/transplante , Adjuvantes Imunológicos , Animais , Sobrevivência de Enxerto/efeitos dos fármacos , Humanos , Imunocompetência , Isoantígenos/imunologia , Transfusão de Linfócitos/métodos , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/citologia , Serina-Treonina Quinases TOR , Resultado do TratamentoRESUMO
Cellular immunity plays an important role in controlling HIV-1 replication. One of the major challenges in developing an HIV-1 DNA vaccine is to generate broader and more potent cellular responses. In this study, we constructed three novel constructs expressing SIVmac antigens Env, Pol and Gag, respectively, with the goal of increasing anti-SIV cellular immunity. The results demonstrate that these constructs can induce strong cellular immune responses in a murine model. Moreover, when applied to cynomolgus macaques, these constructs are not only able to elicit robust IFN-gamma effector responses, but also induce SIV antigen-specific CD8(+) T cells that have high proliferative capacity. These data suggest that such DNA immunogens deserve further examination for their potential to control viral replication.
Assuntos
Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Produtos do Gene pol/imunologia , Vacinas contra a SAIDS/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Feminino , Produtos do Gene env/genética , Produtos do Gene gag/genética , Produtos do Gene pol/genética , Imunização , Interferon gama/biossíntese , Ativação Linfocitária , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
Human papillomavirus type 16 (HPV-16) infection is associated with a majority of cervical cancers and a significant proportion of head and neck cancers. Here, we describe a novel-engineered DNA vaccine that encodes a HPV-16 consensus E6/E7 fusion gene (pConE6E7) with the goal of increasing its antitumor cellular immunity. Compared to an early stage HPV-16 E7 DNA vaccine (pE7), this construct was up to five times more potent in driving E7-specific cellular immune responses. Prophylactic administration of this vaccine resulted in 100% protection against HPV E6 and E7-expressing tumors. Therapeutic studies indicated that vaccination with pConE6E7 prevented or delayed the growth of tumors. Moreover, immunization with pConE6E7 could also partially overcome immune tolerance in E6/E7 transgenic mice. Such DNA immunogens are interesting candidates for further study to investigate mechanisms of tumor immune rejection in vivo.
Assuntos
Antígenos Virais/imunologia , Papillomavirus Humano 16/imunologia , Proteínas Oncogênicas Virais/imunologia , Vacinas contra Papillomavirus/imunologia , Proteínas Repressoras/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Sequência de Bases , DNA Viral/química , DNA Viral/genética , Feminino , Papillomavirus Humano 16/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Neoplasias/patologia , Neoplasias/prevenção & controle , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Vacinas contra Papillomavirus/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Repressoras/genética , Análise de Sequência de DNA , Vacinas de DNA/genéticaRESUMO
The frequency of H5N1 avian influenza outbreaks in China and Eastern Europe has raised concern in the world health community regarding the potential for an influenza pandemic. Efforts to monitor the disease will only provide minimal warning in a global society, and steps must be taken to prevent the morbidity and mortality associated with past pandemics. The current stockpiling of antibody-inducing "bird flu" vaccines assumes the strain that emerges will be the same as strains currently circulating. We propose a novel consensus-based approach to vaccine development, employing a DNA vaccine strategy that can provide more highly cross-reactive cellular immunity against lethal influenza infection. We show such constructs can induce strong cellular immunity against H5 influenza antigens.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/prevenção & controle , Vacinação/veterinária , Vacinas de DNA/administração & dosagem , Animais , Aves , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/imunologiaRESUMO
An important goal for human immunodeficiency virus (HIV) vaccines is to develop immunogens that induce broader and more potent cellular immune responses. In this study of DNA vaccine potency, we constructed a novel subtype B env gene (EY2E1-B) with the goal of increasing vaccine antigen immune potency. The vaccine cassette was designed based on subtype B-specific consensus sequence with several modifications, including codon optimization, RNA optimization, the addition of a Kozak sequence, and a substituted immunoglobulin E leader sequence. The V1 and V2 loops were shortened and the cytoplasmic tail was truncated to prevent envelope recycling. Three different strains of mice (BALB/c, C57BL/6, and HLA-A2 transgenic mice) were immunized three times with pEY2E1-B or the primary DNA immunogen pEK2P-B alone. The analysis of specific antibody responses suggested that EY2E1-B could induce a moderate subtype B-specific antibody response. Moreover, this construct was up to four times more potent at driving cellular immune responses. Epitope mapping results indicated that there is an increase in the breadth and magnitude of cross-reactive cellular responses induced by the EY2E1-B immunogen. These properties suggest that such a synthetic immunogen deserves further examination for its potential to serve as a component antigen in an HIV vaccine cocktail.