RESUMO
The human gastrointestinal tract is home to hundreds of species of bacteria and the balance between beneficial and pathogenic bacteria plays a critical role in human health and disease. The human infant, however, is born with a sterile gut and the complex gastrointestinal host/bacterial ecosystem is only established after birth by rapid bacterial colonization. Composition of newborn gut flora depends on several factors including type of birth (Ceasarian or natural), manner of early feeding (breast milk or formula), and exposure to local, physical environment. Imbalance in normal, healthy gut flora contributes to several adult human diseases including inflammatory bowel (ulcerative colitis and Crohn's disease) and Clostridium difficile associated disease, and early childhood diseases such as necrotizing enterocolitis. As a first step towards characterization of the role of gut bacteria in human health and disease, we conducted an 850 MHz (1)H nuclear magnetic resonance spectroscopy study to monitor changes in metabolic profiles of urine and fecal extracts of 15 mice following gut sterilization by the broad-spectrum antibiotic enrofloxacin (also known as Baytril). Ten metabolites changed in urine following enrofloxacin treatment including decreased acetate due to loss of microbial catabolism of sugars and polysaccharides, decreased trimethylamine-N-oxide due to loss of microbial catabolism of choline, and increased creatine and creatinine due to loss of microbial enzyme degradation. Eight metabolites changed in fecal extracts of mice treated with enrofloxacin including depletion of amino acids produced by microbial proteases, reduction in metabolites generated by lactate-utilizing bacteria, and increased urea caused by loss of microbial ureases.
Assuntos
Antibacterianos/farmacologia , Fezes/microbiologia , Fluoroquinolonas/farmacologia , Metabolômica , Urina/microbiologia , Administração Oral , Animais , Antibacterianos/administração & dosagem , Bactérias/classificação , Enrofloxacina , Fezes/química , Fluoroquinolonas/administração & dosagem , Trato Gastrointestinal , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise Multivariada , Urina/químicaRESUMO
Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy for which novel therapeutics with improved efficacy are greatly needed. To provide support for clinical immune checkpoint blockade, we comprehensively evaluated the expression of therapeutically targetable immune checkpoint molecules on primary MCL cells. MCL cells showed constitutive expression of Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1), variable CD200, absent PD-L2, Lymphocyte Activation Gene 3 (LAG-3), and Cytotoxic T-cell Associated Protein 4 (CTLA-4). Effector cells from MCL patients expressed PD-1. Co-culture of MCL cells with T-cells induced PD-L1 surface expression, a phenomenon regulated by IFNγ and CD40:CD40L interaction. Induction of PD-L1 was attenuated by concurrent treatment with ibrutinib or duvelisib, suggesting BTK and PI3K are important mediators of PD-L1 expression. Overall, our data provide further insight into the expression of checkpoint molecules in MCL and support the use of PD-L1 blocking antibodies in MCL patients.
Assuntos
Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Linfoma de Célula do Manto/genética , Antígeno B7-H1/genética , Antígeno CTLA-4/genética , Humanos , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/metabolismo , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Mesodermal cells signal to neighboring epithelial cells to modulate their proliferation in both normal and disease states. We adapted a Caenorhabditis elegans organogenesis model to enable a genome-wide mesodermal-specific RNAi screen and discovered 39 factors in mesodermal cells that suppress the proliferation of adjacent Ras pathway-sensitized epithelial cells. These candidates encode components of protein complexes and signaling pathways that converge on the control of chromatin dynamics, cytoplasmic polyadenylation, and translation. Stromal fibroblast-specific deletion of mouse orthologs of several candidates resulted in the hyper-proliferation of mammary gland epithelium. Furthermore, a 33-gene signature of human orthologs was selectively enriched in the tumor stroma of breast cancer patients, and depletion of these factors from normal human breast fibroblasts increased proliferation of co-cultured breast cancer cells. This cross-species approach identified unanticipated regulatory networks in mesodermal cells with growth-suppressive function, exposing the conserved and selective nature of mesodermal-epithelial communication in development and cancer.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Redes Reguladoras de Genes , Proteínas ras/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem da Célula , Proliferação de Células , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Genoma , Humanos , Glândulas Mamárias Animais/citologia , Mesoderma/metabolismo , Camundongos , Mutação/genética , Proteínas Nucleares , Especificidade de Órgãos , Fenótipo , Proteínas Quinases , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Transdução de Sinais/genética , Células Estromais/citologia , Células Estromais/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Beta-ig is a secretory protein embodied by fasciclin I-like repeats containing sequences that might bind integrins and glycosaminoglycans in vivo. Expression of Beta-ig is responsive to Transforming Growth Factor-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating Beta-ig as an ECM adhesive protein of developmental processes. The spatiotemporal distribution of Beta-ig during various stages of murine development was examined and its ability to support adhesion of various cell types assessed. In situ hybridization of mouse embryos (E12.5-E18.5) indicated a prominent, distinct expression pattern for Beta-ig message in connective tissue. Beta-ig transcripts were abundantly expressed during mesenchymal cell condensation in areas of axial, craniofacial and appendicular primordial cartilage from E12.5-E14.5. Beginning at E15.5, Beta-ig transcripts appeared in collagen-rich tissues, including dura mater and corneal stroma. During E16.5-E18.5, Beta-ig transcripts were observed in proliferating chondrocytes and areas of endochondral ossification in joint and articular cartilage formation. Connective tissues expressed Beta-ig transcripts within the nasal septum and surrounding cartilage primordia, and in the pericardium, optic cup, kidney, ovary, esophagus, diaphragm, bronchi, trachea and corneal epithelium, and during cardiac valve formation. These patterns of expression indicate that Beta-ig may be involved in tissue morphogenesis. Cells derived from mesenchyme attached onto a substratum comprised of purified recombinant Beta-ig. Taken together, the results indicate that Beta-ig is expressed principally in collagen-rich tissues where it may interact with cells and ECM molecules, perhaps playing a role in tissue morphogenesis.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Sequência de Aminoácidos , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/fisiologia , Desenvolvimento Embrionário e Fetal , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Camundongos , Dados de Sequência Molecular , Especificidade de ÓrgãosRESUMO
The dental pulp is a favorable model for studies of interactions between nociceptive sensory neurons and their peripheral target tissues. In the present study, we retrogradely labeled pulpal afferent neurons with an improved method that permits monitoring of changes in neuronal phenotype in response to controlled tooth injuries. The capacity of retrograde neuronal tracers to diffuse through dentinal tubules was exploited, thereby avoiding the severe injury to the pulp associated with previous tracer application methods. The strategy was to apply the durable fluorescent tracer, Fluoro-gold (FG), to exposed dentin in the floor of shallow cavities in molars, in order to pre-label pulpal neurons in trigeminal ganglia of young adult Sprague-Dawley rats. A high percentage of pupal afferent neurons were retrogradely labeled by application of FG to exposed dentin and the FG fluorescent signal persisted in most labeled neurons for at least 8 weeks. Following tracer application to dentin, the pulp tissue appeared normal histologically, with the exception that a layer of reactive dentin was deposited at the pulp-dentin border beneath the shallow cavities. Assessment of expression of calcitonin gene-related peptide (CGRP) and brain derived neurotrophic factor (BDNF) indicated that pulpal neurons remained in a quiescent, baseline condition cytochemically following application of tracer to cavities in dentin and upregulation of these markers could be detected in neurons that projected to teeth that received a test injury subsequent to tracer application. Thus, labeling of trigeminal neurons via dentinal tubules provides the basis for a useful model for precisely assessing properties of pulpal afferents in both quiescent and activated states.
Assuntos
Polpa Dentária/inervação , Polpa Dentária/metabolismo , Neurônios Aferentes/patologia , Estilbamidinas , Traumatismos Dentários/metabolismo , Administração Tópica , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Peptídeo Relacionado com Gene de Calcitonina/genética , Polpa Dentária/lesões , Extravasamento de Materiais Terapêuticos e Diagnósticos , Feminino , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/farmacocinética , Dente Molar/lesões , Dente Molar/inervação , Dente Molar/metabolismo , Neurônios Aferentes/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem/métodos , Traumatismos Dentários/fisiopatologia , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/patologia , Gânglio Trigeminal/fisiopatologiaRESUMO
During muscle development, the p75(NTR) is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75(NTR)-deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75(NTR) was detected on myoblasts but the high affinity NGF receptor, trk A, was not co-expressed with p75(NTR). In vitro, proliferating C2C12 and primary myoblasts co-expressed the p75(NTR) and MyoD, but immunofluorescent analysis of primary myoblasts and RT-PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75(NTR) in neurons, p75(NTR)-positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I-kappaB and AKT, the cells contained increased levels of both phospho-proteins, both of which promote cell survival. By contrast, neurotrophin-treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75(NTR) mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle.
Assuntos
Músculos/fisiologia , Mioblastos/citologia , Mioblastos/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , DNA/biossíntese , Proteínas I-kappa B/metabolismo , Camundongos , Mioblastos/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/deficiência , Receptores de Fator de Crescimento Neural/genéticaRESUMO
This study demonstrates that innervation dependent on two different neurotrophin tyrosine kinase (trk) receptors can form the same types of sensory endings (Merkel endings) in the same target (Merkel cells of vibrissa follicles). Some endings transiently express trkA during their initial development, whereas others express trkC throughout their development. Consequently, elimination of kinase domains of either trkA or trkC each result in a partial loss of Merkel endings, whereas absence of kinase domains of both receptors results in a total loss. At the onset of Merkel ending development, at least one kinase-lacking trkC isoform is transiently expressed on all the follicle cells, while neurotrophin 3 is transiently expressed only in the cells at the middle third of the follicle where the Merkel endings and cells develop. This transient non-neuronal expression of truncated trkC is essential for development of any Merkel endings, whereas some Merkel endings and cells still begin to develop in the absence of neurotrophin 3. Therefore, truncated trkC plays a more important role in the development of this innervation than kinase forms of trkA or trkC or of NT3, the only known ligand for trkC receptors.