ABSTRACT
BACKGROUND: Atopic dermatitis (AD) is among the most common chronic inflammatory skin diseases, usually occurring early in life, and often preceding other atopic diseases such as asthma. TH2 has been believed to play a crucial role in cellular and humoral response in AD, but accumulating evidence has shown that follicular helper T cell (TFH), a critical player in humoral immunity, is associated with disease severity and plays an important role in AD pathogenesis. OBJECTIVES: This study aimed at investigating how TFHs are generated during the pathogenesis of AD, particularly what is the role of keratinocyte-derived cytokine TSLP and Langerhans cells (LCs). METHODS: Two experimental AD mouse models were employed: (1) triggered by the overproduction of TSLP through topical application of MC903, and (2) induced by epicutaneous allergen ovalbumin (OVA) sensitization. RESULTS: This study demonstrated that the development of TFHs and germinal center (GC) response were crucially dependent on TSLP in both the MC903 model and the OVA sensitization model. Moreover, we found that LCs promoted TFH differentiation and GC response in the MC903 model, and the depletion of Langerin+ dendritic cells (DCs) or selective depletion of LCs diminished the TFH/GC response. By contrast, in the model with OVA sensitization, LCs inhibited TFH/GC response and suppressed TH2 skin inflammation and the subsequent asthma. Transcriptomic analysis of Langerin+ and Langerin- migratory DCs revealed that Langerin+ DCs became activated in the MC903 model, whereas these cells remained inactivated in OVA sensitization model. CONCLUSIONS: Together, these studies revealed a dual functionality of LCs in TSLP-promoted TFH and TH2 differentiation in AD pathogenesis.
Subject(s)
Cytokines/immunology , Dermatitis, Atopic/immunology , Langerhans Cells/immunology , Skin/immunology , T-Lymphocytes, Helper-Inducer/immunology , Allergens/immunology , Animals , Calcitriol/analogs & derivatives , Calcitriol/pharmacology , Cell Differentiation , Dermatologic Agents/pharmacology , Gene Expression Profiling , Mice, Inbred BALB C , Mice, Transgenic , Ovalbumin/immunology , Thymic Stromal LymphopoietinABSTRACT
Progesterone receptor (PR) expression is used as a biomarker of oestrogen receptor-α (ERα) function and breast cancer prognosis. Here we show that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behaviour. In the presence of agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast cancer cells, resulting in a unique gene expression programme that is associated with good clinical outcome. Progesterone inhibited oestrogen-mediated growth of ERα(+) cell line xenografts and primary ERα(+) breast tumour explants, and had increased anti-proliferative effects when coupled with an ERα antagonist. Copy number loss of PGR, the gene coding for PR, is a common feature in ERα(+) breast cancers, explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a molecular rheostat to control ERα chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Neoplastic , Receptors, Progesterone/metabolism , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatin/drug effects , Chromatin/genetics , Chromatin/metabolism , DNA Copy Number Variations/genetics , Disease Progression , Estrogen Receptor alpha/antagonists & inhibitors , Estrogens/metabolism , Estrogens/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Ligands , Mice , Progesterone/metabolism , Progesterone/pharmacology , Protein Binding/drug effects , Receptors, Progesterone/genetics , Transcription, Genetic/drug effects , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE AND METHODS: In human basal-like breast cancer, mutations and deletions in TP53 and BRCA1 are frequent oncogenic events. Thus, we interbred mice expressing the CRE-recombinase with mice harboring loxP sites at TP53 and BRCA1 (K14-Cre; p53f/f Brca1f/f) to test the hypothesis that tissue-specific deletion of TP53 and BRCA1 would give rise to tumors reflective of human basal-like breast cancer. RESULTS: In support of our hypothesis, these transgenic mice developed tumors that express basal-like cytokeratins and demonstrated intrinsic gene expression features similar to human basal-like tumors. Array comparative genomic hybridization revealed a striking conservation of copy number alterations between the K14-Cre; p53f/f Brca1f/f mouse model and human basal-like breast cancer. Conserved events included MYC amplification, KRAS amplification, and RB1 loss. Microarray analysis demonstrated that these DNA copy number events also led to corresponding changes in signatures of pathway activation including high proliferation due to RB1 loss. K14-Cre; p53f/f Brca1f/f also matched human basal-like breast cancer for a propensity to have immune cell infiltrates. Given the long latency of K14-Cre; p53f/f Brca1f/f tumors (~ 250 days), we created tumor syngeneic transplant lines, as well as in vitro cell lines, which were tested for sensitivity to carboplatin and paclitaxel. These therapies invoked acute regression, extended overall survival, and resulted in gene expression signatures of an anti-tumor immune response. CONCLUSION: These findings demonstrate that this model is a valuable preclinical resource for the study of human basal-like breast cancer.
Subject(s)
Disease Models, Animal , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Animals , BRCA1 Protein , Female , Humans , Mice , Mice, TransgenicABSTRACT
PURPOSE: The PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined. METHODS: In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data from human breast tumors, including RNA expression, DNA copy number alterations, and protein expression. In combination with data from a genome-wide RNA-mediated interference screen in human breast cancer cell lines, we identified essential genetic drivers of PI3K/Akt signaling. RESULTS: Our in silico analyses identified SOX4 amplification as a novel modulator of PI3K/Akt signaling in breast cancers and in vitro studies confirmed its role in regulating Akt phosphorylation. CONCLUSIONS: Taken together, these data establish a role for SOX4-mediated PI3K/Akt signaling in breast cancer and suggest that SOX4 may represent a novel therapeutic target and/or biomarker for current PI3K family therapies.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Gene Amplification , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , SOXC Transcription Factors/genetics , Signal Transduction , Breast Neoplasms/pathology , Cell Line, Tumor , Computational Biology/methods , DNA Copy Number Variations , Databases, Genetic , Female , Gene Expression Profiling , Humans , Neoplasms, Basal Cell/genetics , Neoplasms, Basal Cell/metabolism , Neoplasms, Basal Cell/pathology , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , SOXC Transcription Factors/metabolism , TranscriptomeABSTRACT
Pathogens are sensed by innate immune receptors that initiate an efficient adaptive immune response upon activation. The elements of the innate immune recognition process for Paracoccidioides brasiliensis include TLR-2, TLR-4, and dectin-1. However, there are additional receptors necessary for the host immune responses to P. brasiliensis. The nucleotide-binding oligomerization domain-like receptor (NLRs), which activate inflammasomes, are candidate receptors that deserve renewed investigation. After pathogen infection, the NLRs form large signaling platforms called inflammasomes, which lead to caspase-1 activation and maturation of proinflammatory cytokines (IL-18 and IL-1ß). In this study, we showed that NLR family pyrin domain-containing 3 (Nlrp3) is required to induce caspase-1 activation and further secretion of IL-1ß and IL-18 by P. brasiliensis-infected macrophages. Additionally, potassium efflux and lysosomal acidification induced by the fungus were important steps in the caspase-1 activation mechanism. Notably, Nlrp3 and caspase-1 knockout mice were more susceptible to infection than were the wild-type animals, suggesting that the Nlrp3-dependent inflammasomes contribute to host protection against P. brasiliensis. This protective effect occurred owing to the inflammatory response mediated by IL-18, as shown by an augmented fungus burden in IL-18 knockout mice. Taken together, our results show that the Nlrp3 inflammasome is essential for resistance against P. brasiliensis because it orchestrates robust caspase-1 activation and triggers an IL-18-dependent proinflammatory response.
Subject(s)
Carrier Proteins/metabolism , Immunity, Innate , Inflammasomes/metabolism , Interleukin-18/metabolism , Lung Diseases, Fungal/immunology , Lung Diseases, Fungal/metabolism , Animals , Caspase 1/metabolism , Disease Models, Animal , Disease Resistance/genetics , Disease Resistance/immunology , Granuloma/genetics , Granuloma/immunology , Granuloma/metabolism , Inflammasomes/genetics , Interleukin-18/genetics , Interleukin-1beta/biosynthesis , Lung Diseases, Fungal/mortality , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Male , Mice , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Paracoccidioides/immunologyABSTRACT
Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.
Subject(s)
Breast Neoplasms/genetics , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Mutation/genetics , Paracrine Communication , Phosphatidylinositol 3-Kinases/genetics , Signal Transduction , Amphiregulin/metabolism , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatography, Liquid , Class I Phosphatidylinositol 3-Kinases , Disease-Free Survival , Down-Regulation/drug effects , Epidermal Growth Factor/pharmacology , ErbB Receptors/antagonists & inhibitors , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Humans , Mice, Nude , Neoplasm Proteins/metabolism , Paracrine Communication/drug effects , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proteomics , Signal Transduction/drug effects , Tandem Mass Spectrometry , Up-Regulation/drug effectsABSTRACT
Cutibacterium (formerly Propionibacterium) acnes is involved in chronic/low-grade pathologies such as sarcoidosis or prosthetic joint infection (PJI). In these diseases, granulomatous structures are frequently observed. In this study, we induced a physiological granulomatous reaction in response to different well-characterized clinical C. acnes isolates in order to investigate the cellular process during granuloma formation. Three C. acnes isolates selected according to their origin (PJI, sarcoidosis and acne) were typed by MLST. All C. acnes isolates generated granulomatous structures in our experimental conditions. The bacterial burden was better controlled by granulomas induced by the sarcoidosis C. acnes isolate. The PJI C. acnes isolate, belonging to CC36, promoted the recruitment of CD8+ lymphocytes inside the granuloma. In contrast, the acne and sarcoidosis C. acnes isolates, belonging to phylotypes IA1/CC18 and IA2/CC28, respectively, generated a higher number of granulomas and promoted the recruitment of CD4+ lymphocytes inside the granuloma. Our results provide new evidence supporting the role of C. acnes in the development of sarcoidosis and new explanations concerning the mechanisms underlying PJI due to C. acnes.
Subject(s)
Gram-Positive Bacterial Infections/complications , Gram-Positive Bacterial Infections/immunology , Granuloma/etiology , Immunity , Propionibacterium acnes/immunology , Disease Susceptibility , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/microbiology , Multilocus Sequence Typing , Propionibacterium acnes/classification , Propionibacterium acnes/geneticsABSTRACT
A large number of DNA copy number alterations (CNAs) exist in human breast cancers, and thus characterizing the most frequent CNAs is key to advancing therapeutics because it is likely that these regions contain breast tumor 'drivers' (i.e., cancer causal genes). This study aims to characterize the genomic landscape of breast cancer CNAs and identify potential subtype-specific drivers using a large set of human breast tumors and genetically engineered mouse (GEM) mammary tumors. Using a novel method called SWITCHplus, we identified subtype-specific DNA CNAs occurring at a 15% or greater frequency, which excluded many well-known breast cancer-related drivers such as amplification of ERBB2, and deletions of TP53 and RB1. A comparison of CNAs between mouse and human breast tumors identified regions with shared subtype-specific CNAs. Additional criteria that included gene expression-to-copy number correlation, a DawnRank network analysis, and RNA interference functional studies highlighted candidate driver genes that fulfilled these multiple criteria. Numerous regions of shared CNAs were observed between human breast tumors and GEM mammary tumor models that shared similar gene expression features. Specifically, we identified chromosome 1q21-23 as a Basal-like subtype-enriched region with multiple potential driver genes including PI4KB, SHC1, and NCSTN. This step-wise computational approach based on a cross-species comparison is applicable to any tumor type for which sufficient human and model system DNA copy number data exist, and in this instance, highlights that a single region of amplification may in fact harbor multiple driver genes.
Subject(s)
Breast Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Chromosome Mapping , Chromosomes, Human, Pair 1 , Oncogenes , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Computational Biology , DNA Copy Number Variations , Databases, Nucleic Acid , Female , Gene Dosage , Gene Regulatory Networks , Humans , Mice , Neoplasms, Basal Cell/genetics , Neoplasms, Basal Cell/metabolism , Neoplasms, Basal Cell/pathology , Receptors, Notch/genetics , Receptors, Notch/metabolism , Signal Transduction , Species SpecificityABSTRACT
The innate immune response to Trypanosoma cruzi infection comprises several pattern recognition receptors (PRRs), including TLR-2, -4, -7, and -9, as well as the cytosolic receptor Nod1. However, there are additional PRRs that account for the host immune responses to T. cruzi. In this context, the nucleotide-binding oligomerization domain-like receptors (NLRs) that activate the inflammasomes are candidate receptors that deserve renewed investigation. Following pathogen infection, NLRs form large molecular platforms, termed inflammasomes, which activate caspase-1 and induce the production of active IL-1ß and IL-18. In this study, we evaluated the involvement of inflammasomes in T. cruzi infection and demonstrated that apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasomes, including NLR family, pyrin domain-containing 3 (NLRP3), but not NLR family, caspase recruitment domain-containing 4 or NLR family, pyrin domain-containing 6, are required for triggering the activation of caspase-1 and the secretion of IL-1ß. The mechanism by which T. cruzi mediates the activation of the ASC/NLRP3 pathway involves K⺠efflux, lysosomal acidification, reactive oxygen species generation, and lysosomal damage. We also demonstrate that despite normal IFN-γ production in the heart, ASCâ»/â» and caspase-1â»/â» infected mice exhibit a higher incidence of mortality, cardiac parasitism, and heart inflammation. These data suggest that ASC inflammasomes are critical determinants of host resistance to infection with T. cruzi.
Subject(s)
Chagas Disease/immunology , Cytoskeletal Proteins/immunology , Disease Resistance/immunology , Inflammasomes/immunology , Interleukin-1beta/immunology , Animals , Apoptosis Regulatory Proteins , CARD Signaling Adaptor Proteins , Carrier Proteins/immunology , Caspase 1/immunology , Flow Cytometry , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Oligonucleotide Array Sequence Analysis , Trypanosoma cruzi/immunologyABSTRACT
Coxsackievirus B (CVB) is a common cause of acute and chronic infectious myocarditis and pancreatitis. Th1 cells producing IFN-γ and TNF-α are important for CVB clearance, but they are also associated with the pathogenesis of inflammatory lesions, suggesting that the modulation of Th1 and Th2 balance is likely important in controlling CVB-induced pancreatitis. We investigated the role of IL-33, which is an important recently discovered cytokine for induction of Th2-associated responses, in experimental CVB5 infection. We found that mice deficient in IL-33R, T1/ST2, significantly developed more severe pancreatitis, had greater weight loss, and contained higher viral load compared with wild-type (WT) mice when infected with CVB5. Conversely, WT mice treated with rIL-33 developed significantly lower viral titers, and pancreatitis was attenuated. Mechanistic studies demonstrated that IL-33 enhances the degranulation and production of IFN-γ and TNF-α by CD8(+) T and NK cells, which is associated with viral clearance. Furthermore, IL-33 triggers the production of IL-4 from mast cells, which results in enhanced differentiation of M2 macrophages and regulatory T cells, leading to the attenuation of inflammatory pancreatitis. Adoptively transferred mast cells or M2 macrophages reversed the heightened pancreatitis in the T1/ST2(-/-) mice. In contrast, inhibition of regulatory T cells exacerbated the disease in WT mice. Together, our findings reveal an unrecognized IL-33/ST2 functional pathway and a key mechanism for CVB5-induced pancreatitis. These data further suggest a novel approach in treating virus-induced pancreatitis, which is a major medical condition with unmet clinical needs.
Subject(s)
Coxsackievirus Infections/immunology , Interleukins/physiology , Pancreatitis/immunology , Receptors, Interleukin/physiology , Signal Transduction/immunology , Animals , Cells, Cultured , Coxsackievirus Infections/metabolism , Coxsackievirus Infections/pathology , Disease Models, Animal , Interleukin-1 Receptor-Like 1 Protein , Interleukin-33 , Interleukins/administration & dosage , Interleukins/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Knockout , Pancreatitis/pathology , Pancreatitis/virology , Receptors, Interleukin/biosynthesis , Viral Load/immunology , Weight Loss/immunologyABSTRACT
Background and Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P = .003 and P = .0005, respectively). Conclusion: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.
ABSTRACT
An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)-like receptor proteins in host response to T. cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappaB-dependent products in response to infection and failed to restrict T. cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T. cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T. cruzi infection by mechanisms independent of cytokine production.
Subject(s)
Chagas Disease/immunology , Immunity, Innate , Nod1 Signaling Adaptor Protein/physiology , Trypanosoma cruzi/immunology , Animals , Chagas Disease/genetics , Chagas Disease/metabolism , Genetic Predisposition to Disease , Immunity, Innate/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/physiology , Nod1 Signaling Adaptor Protein/deficiency , Nod1 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/deficiency , Nod2 Signaling Adaptor Protein/genetics , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptors/physiologyABSTRACT
Studies on the breeding of vulnerable and endangered bird species are hindered by low numbers of individuals, inaccessible location of nests, unfavourable environmental conditions, and complex behavioural patterns. In addition, intraspecific variation may emerge only following long-term, systematic observations of little-known patterns and processes. Here, data collected over 30 years were used to determine growth model of hyacinth macaw (Anodorhynchus hyacinthinus) chicks in the Pantanal biome of Brazil. During this period, the speed of growth and body mass of chicks varied widely. Four growth models were tested: logistic, Gompertz, Richards, and cubic polynomial. They were fitted using three biometric measurements: body mass, total length, and tail length. The best-fitting growth curves were identified using Akaike's information criterion. The best models were the cubic polynomial for body mass, Richards for total length, and Gompertz for tail length. We confirmed the occurrence of dwarf individuals, whose body mass, total length, and tail length were 20%, 22%, and 70% smaller, respectively, than in the overall population. The dwarfs remain small in size after having fledged and are easily identified as adults. We discuss the importance of long-term studies to identify windows of opportunity for further research that will help in the conservation of endangered macaw species.
Subject(s)
Hyacinthus , Parrots , Animals , Brazil/epidemiology , Endangered Species , Humans , Plant BreedingABSTRACT
Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas' disease.
Subject(s)
Antigens, Surface/immunology , Apoptosis Regulatory Proteins/immunology , Chagas Cardiomyopathy/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Trypanosoma cruzi/immunology , Animals , Antigens, Surface/metabolism , Apoptosis Regulatory Proteins/metabolism , Cell Separation , Chagas Cardiomyopathy/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorescent Antibody Technique , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Programmed Cell Death 1 Receptor , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Development of simple and fully characterized immunomodulatory molecules is an active area of research to enhance current immunotherapies. Monophosphoryl lipid A (MPL), a nontoxic lipidic derivative from bacteria, is the first and currently only adjuvant approved in humans. However, its capacity to induce a potent response against weak immunogenic tumoral-associated antigens remains limited. Herein, a new generation of lipidic immunomodulators to conduct a structure-activity relationship study to determine the minimal structural elements conferring immunomodulatory properties is introduced. Two lead molecules characterized by a short succinyl linker between two oleyl chains and a polar headgroup consisting of either naturally occurring tobramycin (DOST) or kanamycin (DOSK) are identified. These two lipoaminoglycosides self-assemble in very small vesicles. In a wide variety of cells including 3D human cell culture, DOST and DOSK induce the upregulation of proinflammatory cytokines and interferon-inducible proteins in a dose and time-dependent manner via a caveolae-dependent proinflammatory mechanism and phosphatidylinositol phospholipase C activation. Furthermore, after intratumoral administration, these lipoaminoglycosides induce an efficient immune response leading to significant antitumor activity in a mouse breast cancer model. Altogether, these findings indicate that DOST and DOSK are two groundbreaking synthetic lipid immunostimulators that can be used as adjuvants to enhance current immunotherapeutic treatments.
ABSTRACT
BACKGROUND: The dissatisfaction of health professionals in emergency services has a negative influence on both the quality of care provided for acute myocardial infarction (AMI) patients and the retention of those professionals. OBJECTIVE: To assess physicians' satisfaction with the structure of care and diagnosis at the emergency services in the Northern Region of Minas Gerais before the implementation of the AMI system of care. METHODS: This cross-sectional study included physicians from the emergency units of the ambulance service (SAMU) and level II, III and IV regional hospitals. Satisfaction was assessed by using the CARDIOSATIS-Team scale. The median score for each item, the overall scale and the domains were calculated and then compared by groups using the non-parametric Mann-Whitney test. Correlation between time since graduation and satisfaction level was assessed using Spearman correlation. A p value < 0.05 was considered significant. RESULTS: Of the 137 physicians included in the study, 46% worked at SAMU. Most of the interviewees showed overall dissatisfaction with the structure of care, and the median score for the overall scale was 2.0 [interquartile range (IQR) 2.0-4.0]. Most SAMU physicians expressed their dissatisfaction with the care provided (54%), the structure for managing cardiovascular diseases (52%), and the technology available for diagnosis (54%). The evaluation of the overall satisfaction evidenced that the dissatisfaction of SAMU physicians was lower when compared to that of hospital emergency physicians. Level III/IV hospital physicians expressed greater overall satisfaction when compared to level II hospital physicians. CONCLUSION: This study showed the overall dissatisfaction of the emergency physicians in the region assessed with the structure of care for cardiovascular emergencies.
Subject(s)
Emergency Medical Services , Emergency Medicine , Job Satisfaction , Medical Staff, Hospital/statistics & numerical data , Myocardial Infarction/therapy , Cross-Sectional Studies , Emergency Medical Services/statistics & numerical data , Emergency Medicine/statistics & numerical data , Female , Humans , Male , Surveys and QuestionnairesABSTRACT
Altered myocardial perfusion is a common finding in chronic Chagas cardiomyopathy (CCC), but its underlying histologic changes have not been elucidated. We investigated the occurrence of myocardial perfusion defects (MPDs) and the correlated regional changes to histology in an experimental model of CCC in hamsters. Methods: Female Syrian hamsters (n = 34) were infected with 3.5 × 104 to 105 trypomastigote forms of Trypanosoma cruzi, Y strain, and 6-10 mo afterward underwent in vivo imaging including resting 99mTc-sestamibi SPECT, segmental and global left ventricular function assessment using 2-dimensional echocardiography, and 18F-FDG PET for evaluation of myocardial viability. Histologic analysis included quantification of fibrosis, inflammatory infiltration, and the diameter and density of myocardial microcirculation. Results: MPDs were present in 17 (50%) of the infected animals. Histologic analysis revealed no transmural scar in segments with an MPD, and normal or mildly reduced 18F-FDG uptake, indicating viable myocardium. Infected animals with an MPD, in comparison to infected animals without an MPD and control animals, showed a lower left ventricular ejection fraction (P = 0.012), a higher wall motion score index (P = 0.004), and a higher extent of inflammatory infiltration (P = 0.018) but a similar extent of fibrosis (P = 0.15) and similar microvascular diameter and density (P > 0.05). Segments with an MPD (n = 65), as compared with normally perfused regions in the same animal (n = 156), showed a higher wall motion score index (P = 0.005) but a similar extent of inflammatory infiltration, a similar extent of fibrosis, and a similar microvascular diameter and density. Conclusion: Resting MPDs are frequent in experimental CCC and are associated with myocardial inflammation but do not designate scar tissue, corresponding to regions with metabolically viable myocardium.
Subject(s)
Chagas Cardiomyopathy/physiopathology , Coronary Circulation , Animals , Chagas Cardiomyopathy/diagnostic imaging , Chagas Cardiomyopathy/pathology , Chronic Disease , Cricetinae , Disease Models, Animal , Female , Fluorodeoxyglucose F18 , Microvessels/diagnostic imaging , Microvessels/physiopathology , Myocardial Perfusion Imaging , Myocardium/pathology , Positron-Emission Tomography , Systole/physiology , Tissue Survival , Ventricular Dysfunction, Left/physiopathologyABSTRACT
Chagas disease is caused by infection with the protozoan Trypanosoma cruzi (T. cruzi) and is an important cause of severe inflammatory heart disease. However, the mechanisms driving Chagas disease cardiomyopathy have not been completely elucidated. Here, we show that the canonical PI3Kγ pathway is upregulated in both human chagasic hearts and hearts of acutely infected mice. PI3Kγ-deficient mice and mutant mice carrying catalytically inactive PI3Kγ are more susceptible to T. cruzi infection. The canonical PI3Kγ signaling in myeloid cells is essential to restrict T. cruzi heart parasitism and ultimately to avoid myocarditis, heart damage, and death of mice. Furthermore, high PIK3CG expression correlates with low parasitism in human Chagas' hearts. In conclusion, these results indicate an essential role of the canonical PI3Kγ signaling pathway in the control of T. cruzi infection, providing further insight into the molecular mechanisms involved in the pathophysiology of chagasic heart disease.
Subject(s)
Chagas Cardiomyopathy/immunology , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Signal Transduction/immunology , Trypanosoma cruzi/immunology , Adult , Animals , Biopsy , Cell Line , Chagas Cardiomyopathy/parasitology , Chagas Cardiomyopathy/pathology , Class Ib Phosphatidylinositol 3-Kinase/genetics , Disease Models, Animal , Female , Heart/parasitology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Myeloid Cells/immunology , Myeloid Cells/metabolism , Myocardium/immunology , Myocardium/pathology , Phosphoinositide-3 Kinase Inhibitors , Quinoxalines/pharmacology , Thiazolidinediones/pharmacology , Trypanosoma cruzi/pathogenicity , Up-RegulationABSTRACT
Type B coxsackievirus (CVB) is a common cause of acute and chronic myocarditis, meningitis and pancreatitis, often leading to heart failure and pancreatic deficiency. The polarization of CD4+ T lymphocytes and their cytokine milieu are key factors in the outcome of CVB-induced diseases. Thus, sensing the virus and driving the adaptive immune response are essential for the establishment of a protective immune response. TLR3 is a crucial virus recognition receptor that confers the host with resistance to CVB infection. In the current study, we found that TLR3 expression in dendritic cells plays a role in their activation upon CVB3 infection in vitro, as TLR3-deficient dendritic cells up-regulate CD80 and CD86 to a less degree than WT cells. Instead, they up-regulated the inhibitory molecule PD-L1 and secreted considerably lower levels of TNF-α and IL-10 and a higher level of IL-23. T lymphocyte proliferation in co-culture with CVB3-infected dendritic cells was increased by TLR3-expressing DCs and other cells. Furthermore, in the absence of TLR3, the T lymphocyte response was shifted toward a Th17 profile, which was previously reported to be deleterious for the host. TLR3-deficient mice were very susceptible to CVB3 infection, with increased pancreatic injury and extensive inflammatory infiltrate in the heart that was associated with uncontrolled viral replication. Adoptive transfer of TLR3+ dendritic cells slightly improved the survival of TLR-deficient mice following CVB3 infection. Therefore, our findings highlight the importance of TLR3 signaling in DCs and in other cells to induce activation and polarization of the CD4+ T lymphocyte response toward a Th1 profile and consequently for a better outcome of CVB3 infection. These data provide new insight into the immune-mediated mechanisms by which CVBs are recognized and cleared in order to prevent the development of myocarditis and pancreatitis and may contribute to the design of therapies for enteroviral infections.