RESUMO
The integration of basic science into clinical clerkships continues to be a challenge in medical curricula. We developed an integrated session for 3rd year medical students enrolled in OB-Gyn/Pediatric Block. The session focused on transplacental and perinatal infections, and consisted of a student-driven pedagogy activity in which students were required to explain the basic science principles behind the pathophysiology of the clinical presentations, the work-up, and the treatment of the infections. This approach helps students understand how basic science knowledge informs clinical practice and potential increase clerkship-level students' confidence as it makes them serve as leaders of active learning modules. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40670-021-01485-7.
RESUMO
For the last 20 years, undergraduate medical education has seen a major curricular reform movement toward integration of basic and clinical sciences. The rationale for integrated medical school curricula focuses on the application of knowledge in a clinical context and the early ability to practice key skills such as critical thinking and clinical problem-solving. The method and extent of discipline integration can vary widely from single sessions to entire programs. A challenge for integrated curricula is the design of appropriate assessments. The goal of this review is to provide a framework for clinical anatomy educators with definitions of integration, examples of existing integration models, strategies, and instructional methods that promote integration of basic and clinical sciences.
Assuntos
Anatomia/educação , Currículo/tendências , Educação de Graduação em Medicina/métodos , Humanos , Aprendizagem Baseada em ProblemasRESUMO
Introduction: Medical students are introduced to skin rashes during their preclinical years and often express difficulty in differentiating the underlying mechanisms. The preclinical lessons regarding immunologically mediated skin rashes are largely forgotten by the time the students begin diagnosing and treating skin rashes during clinical rotations. This module aims to enhance student understanding of immunologic concepts by integrating material across disciplines, contextualizing within a clinical scenario, and providing opportunity for self-testing. Methods: A diagram illustrating immune responses in allergic contact dermatitis was used in the Texas Tech University Paul L. Foster School of Medicine preclinical curriculum. This diagram was updated as an audiovisual learning module that traced the immune mechanisms and pathogenesis of contact dermatitis from allergen exposure to skin-rash development. A self-assessment quiz and a clinical vignette with questions were included in the module. Student usage was monitored, and an in-class survey evaluating student perception was administered. Results: Sixty-four (58%) first-year medical students used this module. Twenty-eight students completed the in-class survey. Over 95% of respondents felt that the module helped them learn the new material, identify areas of weakness, and both understand the underlying pathology and big picture for this immune response. Discussion: Student survey results indicate the module is clinically relevant and enhances learning. The module may be used as a component of self-directed learning in any immunology curriculum or may be used in any basic immunology course to exemplify the role of the immune system in disease.
Assuntos
Dermatite Alérgica de Contato/imunologia , Currículo/tendências , Dermatite Alérgica de Contato/fisiopatologia , Educação de Graduação em Medicina/métodos , Humanos , Aprendizagem Baseada em Problemas/métodos , Inquéritos e Questionários , TexasRESUMO
Introduction: Medical schools must expand their teaching strategies to address a new generation of medical students and ensure their growth into lifelong, self-directed learners. Integration across basic science disciplines packaged together with clinical medicine produces learning materials that better enable medical students to achieve these goals. Methods: We created a narrated audiovisual learning module illustrating the foundational sciences and clinical presentation surrounding immune responses to viral infections. We integrated immunology, microbiology, histology, pathology, and clinical medicine and included a self-assessment quiz and clinical vignette with questions to test students' understanding of the material. We published the module on our school's learning management system and tracked student usage, which was followed by an in-class survey to assess student perceptions of the usefulness of the module. Results: Sixty-four (59%) of the first-year medical students used the module. Thirty-seven students completed the in-class survey assessing their perceptions of the module. Over 95% of responders reported that the module helped them learn the new material, identify areas of weakness, understand the big picture for this immune response, and apply the material in a clinical context. Discussion: This module illustrates an approach to integrating basic science disciplines in order to facilitate students' understanding of the mechanisms underlying patients' clinical presentations. Survey results indicated that students valued the module as a self-directed learning component that integrated essential clinical concepts. The module was a helpful tool for students to evaluate their comprehension of immunology in a clinical context and can be used as required or optional material.
Assuntos
Imunidade Adaptativa/imunologia , Aprendizagem Baseada em Problemas/métodos , Viroses/imunologia , Currículo/tendências , Educação de Graduação em Medicina/métodos , Avaliação Educacional/métodos , Humanos , Estudantes de Medicina/estatística & dados numéricos , Inquéritos e Questionários , Viroses/complicações , Viroses/fisiopatologiaRESUMO
INTRODUCTION: Medical students express frustration that they cannot assemble a comprehensive big picture of how the immune system responds to a microbe and that integration of basic science knowledge, especially across disciplines, with clinical knowledge is difficult. Yet medical student competency requires application of knowledge of immune mechanisms to inform diagnosis and treatment. METHODS: A diagram for immune response to extracellular microbes was previously published by MedEdPORTAL in 2011. This diagram has been updated here as a narrated audiovisual module with integrated histopathology. It contains a self-assessment quiz that tests students' understanding of the module followed by a clinical vignette that tests students' ability to apply the concepts in a clinical context. The module was published and usage was tracked via our learning management system. An in-class survey was conducted to gauge students' perceptions of the module. RESULTS: Eighty-two out of 102 (76%) first-year medical students used the module. Over 85% of survey participants felt that the module was a useful resource for learning and reviewing. More than 90% felt that the module helped them to understand the big picture and identify areas for further study. DISCUSSION: This module assembles a big picture of the immunologic mechanisms involved in a bacterial infection. It was created in response to requests and suggestions by preclinical medical students and used for first-year students during the first few weeks of their training in the basic sciences. This approach integrates multiple disciplines and facilitates students' learning and application of difficult concepts in immunology and pathology.
RESUMO
Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.
Assuntos
Proteína de Ligação a CREB/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Doenças do Esôfago/metabolismo , Esôfago/metabolismo , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Úlcera/metabolismo , Cicatrização , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Doenças do Esôfago/genética , Doenças do Esôfago/patologia , Doenças do Esôfago/fisiopatologia , Esôfago/irrigação sanguínea , Esôfago/efeitos dos fármacos , Esôfago/patologia , Humanos , Masculino , Misoprostol/farmacologia , Mucosa/metabolismo , Mucosa/patologia , Neovascularização Fisiológica , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP2/efeitos dos fármacos , Sistemas do Segundo Mensageiro , Fatores de Tempo , Úlcera/genética , Úlcera/patologia , Úlcera/fisiopatologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
A number of hormones and metabolic mediators signal the brain of changes in the body's energy status and when an imbalance occurs; the brain coordinates the appropriate changes in energy intake and utilization via the control of appetite and food consumption. Under conditions of chronic inflammation and immune activation, there is often a significant loss of body mass and appetite suggesting the presence of shared ligands and signaling pathways mediating "crosstalk" between the immune and neuroendocrine systems. Ghrelin, the endogenous ligand for growth hormone secretagogue receptor (GHS-R), is produced primarily by cells in the stomach and serves as a potent circulating orexigenic hormone controlling food intake, energy expenditure, adiposity and GH secretion. The functional roles of ghrelin and other growth hormone secretagogues (GHS) within the immune system and under states of inflammatory stress and injury are only now coming to light. A number of reports over the past decade have described ghrelin to be a potent anti-inflammatory mediator both in vitro and in vivo and a promising therapeutic agent in the treatment of inflammatory diseases and injury. Moreover, ghrelin has also been shown to promote lymphocyte development in the primary lymphoid organs (bone marrow and thymus) and to ablate age-associated thymic involution. In the current report, we review the literature supporting a role for ghrelin as an anti-inflammatory agent and immunoregulatory hormone/cytokine and its potential use in the treatment of inflammatory diseases and injury.
Assuntos
Grelina/metabolismo , Sistema Imunitário/metabolismo , Inflamação/metabolismo , Animais , Grelina/sangue , Humanos , Inflamação/patologia , Linfócitos/metabolismo , Receptores de Grelina/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Cancer metastasis is a leading cause of cancer morbidity and mortality. More needs to be learned about mechanisms that control this process. In particular, the role of chemokine receptors in metastasis remains controversial. Here, using a highly metastatic breast cancer (4T1) model, we show that lung metastasis is a feature of only a proportion of the tumor cells that express CCR4. Moreover, the primary tumor growing in mammary pads activates remotely the expression of TARC/CCL17 and MDC/CCL22 in the lungs. These chemokines acting through CCR4 attract both tumor and immune cells. However, CCR4-mediated chemotaxis was not sufficient to produce metastasis, as tumor cells in the lung were efficiently eliminated by natural killer (NK) cells. Lung metastasis required CCR4(+) regulatory T cells (Treg), which directly killed NK cells using beta-galactoside-binding protein. Thus, strategies that abrogate any part of this process should improve the outcome through activation of effector cells and prevention of tumor cell migration. We confirm this prediction by killing CCR4(+) cells through delivery of TARC-fused toxins or depleting Tregs and preventing lung metastasis.
Assuntos
Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Receptores CCR4/metabolismo , Linfócitos T Reguladores/patologia , Animais , Linhagem Celular Tumoral , Quimiocina CCL17/metabolismo , Quimiocina CCL22/metabolismo , Citotoxicidade Imunológica/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Linfócitos T Reguladores/imunologiaRESUMO
Regulatory T cells (Tregs) and beta-galactoside-binding protein (betaGBP), a regulatory protein often found expressed at sites of immunological privilege, have similar functions. Their presence affects the outcome of harmful autoimmunity and cancers, including experimental autoimmune encephalomyelitis and malignant gliomas. Here we report a novel pathway by which Tregs express and utilize betaGBP to control CD8(+) T cell responses partially activating TCR signaling but blocking PI3K activity. As a result, this leads to a loss of p21(ras), ERK and Akt activities despite activation of TCR proximal signals, such as phosphorylation of CD3zeta, Zap70, Lat and PKCtheta. Although non-processive TCR signaling often leads to cell anergy, Tregs/betaGBP did not affect cell viability. Instead, betaGBP/Tregs transiently prevented activation of CD8(+) T cells with self-antigens, while keeping their responses to xenogeneic antigens unaffected.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Galectina 1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Autoantígenos/imunologia , Autoantígenos/metabolismo , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Ensaio de Imunoadsorção Enzimática , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Citometria de Fluxo , Galectina 1/imunologia , Humanos , Microscopia Confocal , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Proteínas ras/imunologia , Proteínas ras/metabolismoRESUMO
PURPOSE: The sperm-derived SPANX family proteins can be found expressed in human tumors. Here, we aimed to perform a comprehensive study to evaluate immunotherapeutic relevance of one of its members, SPANX-B. We wanted to test its expression pattern in human tumors and to evaluate CD4(+) and CD8(+) T-cell responses in healthy humans after in vitro immunizations. EXPERIMENTAL DESIGN: Expression of SPANX-B in human malignancies, including a multitumor tissue array of 145 primary tumors, was assessed using reverse transcription-PCR, Western blotting, and immunohistochemical analysis. T-cell immunogenicity and immunodominant epitopes of SPANX-B were studied using in vitro immunizations of healthy human donor-derived leukocytes. RESULTS: SPANX-B was abundantly expressed in melanoma and carcinomas of lung, ovary, colon, and breast. In melanoma, tissue array data indicated that it was expressed in advanced and metastatic disease. Unlike most tumor-associated antigens, SPANX-B was an immunogenic antigen that was recognized by circulating T-cell precursors in healthy humans. Importantly, these T cells were readily expanded to generate SPANX-B-specific helper CD4(+) and cytolytic CD8(+) T cells that recognized unique immunodominant epitopes: at least one HLA-DR-restricted Pep-9 epitope (SPANX-B(12-23)) and two HLA-A2-restricted Pep-2 and Pep-4 epitopes (SPANX-B(23-31) and SPANX-B(57-65), respectively). CD8(+) T cells were fully functional to recognize and lyse HLA-A2-expressing tumors, including primary human melanomas. CONCLUSIONS: SPANX-B is an immunogenic sperm-derived antigen that is expressed in several human tumors. SPANX-B is also efficiently recognized by the human T-cell immune arm, indicating its significant value for the development of protective and therapeutic cancer vaccines.
Assuntos
Antígenos de Neoplasias/análise , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Proteínas Nucleares/análise , Sequência de Aminoácidos , Vacinas Anticâncer/imunologia , Humanos , Epitopos Imunodominantes , Dados de Sequência Molecular , Neoplasias/terapia , Proteínas Nucleares/imunologiaRESUMO
Dexamethasone (DM) is a synthetic member of the glucocorticoid (GC) class of hormones that possesses anti-inflammatory and immunosuppressant activity and is commonly used to treat chronic inflammatory disorders, severe allergies, and other disease states. Although GCs are known to mediate well-defined transcriptional effects via GC receptors (GCR), there is increasing evidence that GCs also initiate rapid nongenomic signaling events in a variety of cell types. Here, we report that DM induces the phosphorylation of Lck and the activation of other downstream mediators, including p59Fyn, Zap70, Rac1, and Vav in resting but not activated human T cells. DM treatment also augments CXCL12-mediated signaling in resting T cells through its cell surface receptor, CXCR4 resulting in the enhanced actin polymerization, Rac activation, and cell migration on ligand exposure. Lck was found to be a critical intermediate in these DM-induced signaling activities. Moreover, DM-mediated Lck phosphorylation in T cells was dependent on the presence of both the GCR and the CD45 molecule. Overall, these results elucidate additional nongenomic effects of DM and the GCR on resting human T cells, inducing Lck and downstream kinase activation and augmenting chemokine signaling and function.
Assuntos
Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/efeitos dos fármacos , Receptores CXCR4/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Citometria de Fluxo , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosforilação/efeitos dos fármacos , Receptores CXCR4/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Despite its potency, the wider use of immunotherapy for B cell malignancies is hampered by the lack of well-defined tumor-specific Ags. In this study, we demonstrate that an evolutionarily conserved 37-kDa immature laminin receptor protein (OFA-iLRP), a nonimmunogenic embryonic Ag expressed by a variety of tumors, is rendered immunogenic if targeted to the APCs using the CCR6 ligands MIP3alpha/CCL20 and mDF2beta. The CCR6 targeting facilitated efficient Ag cross-presentation and induction of tumor-neutralizing CTLs. Although the Ag targeting alone, without activation of dendritic cells (DCs), is proposed to induce tolerance, and MIP3alpha does not directly activate DCs, the MIP3alpha-based vaccine efficiently induced protective and therapeutic antitumor responses. The responses were as strong as those elicited by the OFA-iLRP fusions with moieties that activated DCs and Th1-type cytokine responses, mDF2beta, or mycobacterial Hsp70 Ag. Although the same cDNA encodes the dimerized high-affinity mature 67-kDa mLRP that is expressed in normal tissues to stabilize the binding of laminin to cell surface integrins, the vaccines expressing OFA-iLRP elicited long-term protective CD8(+) T cell-mediated memory responses against syngeneic B cell lymphoma, indicating the potential application of these simple vaccines as preventive and therapeutic formulations for human use.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Linfoma de Células B/terapia , Receptores de Quimiocinas/imunologia , Receptores de Laminina/imunologia , Animais , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/genética , Vacinas Anticâncer/imunologia , Quimiocina CCL20 , Quimiocinas CC/imunologia , Clonagem Molecular , Citotoxicidade Imunológica , Feminino , Linfoma de Células B/imunologia , Proteínas Inflamatórias de Macrófagos/imunologia , Camundongos , Microscopia Confocal , Receptores CCR6 , Receptores de Laminina/genética , VacinaçãoRESUMO
Expression of chemokine receptors by tumors, specifically CCR4 on cutaneous T cell lymphomas, is often associated with a poor disease outcome. To test the hypothesis that chemokine receptor-expressing tumors can be successfully controlled by delivering toxins through their chemokine receptors, we have generated fusion proteins designated chemotoxins: chemokines fused with toxic moieties that are nontoxic unless delivered into the cell cytosol. We demonstrate that chemokines fused with human RNase eosinophil-derived neurotoxin or with a truncated fragment of Pseudomonas exotoxin 38 are able to specifically kill tumors in vitro upon internalization through their respective chemokine receptors. Moreover, treatment with the thymus and activation-regulated chemokine (CCL17)-expressing chemotoxin efficiently eradicated CCR4-expressing cutaneous T cell lymphoma/leukemia established in NOD-SCID mice. Taken together, this work represents a novel concept that may allow control of growth and dissemination of tumors that use chemokine receptors to metastasize and circumvent immunosurveillance.
Assuntos
Antineoplásicos/toxicidade , Citotoxicidade Imunológica , Imunotoxinas/toxicidade , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/terapia , Receptores de Quimiocinas/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/toxicidade , Animais , Antineoplásicos/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Quimiocina CCL17 , Quimiocinas CC/administração & dosagem , Quimiocinas CC/genética , Quimiocinas CC/toxicidade , Citotoxicidade Imunológica/genética , Neurotoxina Derivada de Eosinófilo/genética , Neurotoxina Derivada de Eosinófilo/toxicidade , Exotoxinas/genética , Exotoxinas/toxicidade , Feminino , Humanos , Imunotoxinas/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia , Receptores CCR4 , Receptores de Quimiocinas/biossíntese , Proteínas Virais/genética , Proteínas Virais/toxicidade , Fatores de Virulência/genética , Fatores de Virulência/toxicidade , Exotoxina A de Pseudomonas aeruginosaRESUMO
Regulatory CD25(+)CD4(+) T cells (Tregs) play an important role in the control of peripheral tolerance. In this study we demonstrate that human peripheral blood Tregs can be divided into two distinct populations based on the expression of CCR4. The majority ( approximately 75%) of freshly isolated Tregs express CCR4 and presumably represent memory-type Tregs. Interestingly, CCR4(-) Tregs require anti-CD3 Ab-mediated activation to acquire a regulatory activity, while CCR4(+) Tregs appear to be already primed to suppress the proliferation of CD8(+) T cells. CCR4 is also expressed on CD25(low)CD4(+) T cells (CCR4(+) non-Tregs) that mostly suppress Th1-type polarization without affecting T cell proliferation, presumably via the production of immunomodulatory cytokines like IL-10. In contrast, CCR4(+) Tregs express FasL to primarily regulate T cell proliferation via a contact-mediated process involving FasL/Fas signaling, a major regulatory pathway of T cell homeostasis. Finally, we also demonstrate that the depletion of CCR4(+) T cells leads to Th1-type polarization of CD4(+) T cells and augmentation of CD8(+) T cell responses to tumor Ags.
Assuntos
Proteína Ligante Fas/fisiologia , Receptores de Quimiocinas/fisiologia , Linfócitos T Reguladores/fisiologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/imunologia , Polaridade Celular , Humanos , Interleucina-10/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptores CCR4 , Receptores de Quimiocinas/análise , Células Th1/fisiologiaRESUMO
Chemokines are key controllers of cell trafficking and are involved in numerous pathologic and inflammatory conditions. However, the fate of a chemokine ligand, once it is endocytosed with its receptor, remains obscure. Here, using chemokine-tumor antigen fusion constructs, we demonstrate for the first time that chemokines are internalized to early/late endosomal and lysosomal compartments through a clathrin-dependent process and subsequently delivered to the cytosol for proteasomal processing, facilitating efficient cross-presentation to the TAP-1-dependent MHC class I processing pathway. These data not only elucidate the intracellular fate of chemokine ligands upon receptor uptake, but also demonstrate the superior carrier potency of chemokines for delivering self-antigens to both class I and II processing pathways to induce CD8(+) and CD4(+) T-cell responses.
Assuntos
Apresentação de Antígeno/imunologia , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores de Quimiocinas/imunologia , Transdução de Sinais/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Apresentação de Antígeno/genética , Autoantígenos/genética , Linfócitos T CD4-Positivos , Quimiocinas/genética , Quimiocinas/imunologia , Clatrina/genética , Clatrina/imunologia , Endocitose/genética , Endocitose/imunologia , Endossomos/genética , Endossomos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II , Humanos , Ligantes , Lisossomos/genética , Lisossomos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptores de Quimiocinas/genética , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Platelet-activating factor (PAF) and PAF-like phospholipids are inactivated by PAF-acetylhydrolase (PAF-AH). Using nonhyperlipidemic animals, we tested whether local expression of PAF-AH into injured arteries might induce antithrombotic and antiinflammatory effects.Method and Results- Balloon-injured rabbit carotid arteries were infected at the time of injury with an adenovirus expressing either human plasma PAF-AH (AdPAF-AH) or bacterial beta-galactosidase (AdLacZ) or infused with saline. Seven days later, shear stress-induced thrombosis was observed in all AdLacZ-infected and saline-infused arteries (controls) but eliminated in AdPAF-AH-treated contralateral arteries, even in the presence of epinephrine or an inhibitor of NO production. Injury-induced expression of tissue factor was also significantly suppressed. In AdPAF-AH-treated arteries compared with controls, the expressions of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and macrophage infiltration were decreased by 66%, 66%, and 71%, respectively (P<0.01), and intimal area and intima/media ratio were decreased on day 21 by 43% and 52%, respectively (P<0.05). Within 1 week after injury, oxidized lipoproteins (OxLDL) had readily accumulated in the arterial wall. However, this was markedly reduced in the AdPAF-AH-treated arteries. No differences in the titers of autoantibodies to OxLDL or total cholesterol in blood were found between controls and AdPAF-AH-treated rabbits. CONCLUSIONS: Our results show for the first time that OxLDL accumulates in arteries in nonhyperlipidemic animals within 1 week after injury and that local expression of PAF-AH reduces this accumulation and exerts antiinflammatory, antithrombotic, and antiproliferative effects without changing the plasma levels of PAF-AH activity or titers of autoantibodies to OxLDL.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Lesões das Artérias Carótidas/terapia , Inflamação/prevenção & controle , Lipoproteínas LDL/metabolismo , Trombose/prevenção & controle , Túnica Íntima/crescimento & desenvolvimento , 1-Alquil-2-acetilglicerofosfocolina Esterase/administração & dosagem , 1-Alquil-2-acetilglicerofosfocolina Esterase/farmacologia , Animais , Animais Geneticamente Modificados , Anti-Inflamatórios/administração & dosagem , Autoanticorpos/análise , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/complicações , Cateterismo/efeitos adversos , Moléculas de Adesão Celular/análise , Fibrinolíticos/administração & dosagem , Humanos , Inflamação/terapia , Lipoproteínas LDL/efeitos dos fármacos , Macrófagos/fisiologia , Coelhos , Estresse Mecânico , Trombose/etiologia , Trombose/terapia , Transdução GenéticaRESUMO
BACKGROUND & AIMS: Serum response factor (SRF) regulates transcription of immediate early genes and muscle genes. In this study, we examined the role of SRF in gastric ulcer healing and the mechanisms involved. METHODS: Gastric ulcers were induced in rats by serosal application of acetic acid. Gastric specimens were obtained sequentially after ulcer induction for analyses of SRF messenger RNA (mRNA), protein expression, and for immunohistochemistry. We examined the role of SRF in ulcer healing by local injection of an SRF expression plasmid into ulcers (gene therapy). To elucidate the cellular mechanisms of the action of SRF, we examined the effect of SRF overexpression on actin dynamics, cell migration, and proliferation in rat gastric epithelial cell (RGM1) and smooth muscle cell (A7R5). To determine the clinical relevance, we examined SRF expression in human gastric ulcer specimens. RESULTS: Gastric ulceration activated SRF expression in epithelial cells lining regenerating glands and in myofibroblasts and smooth muscle cells of granulation tissue. SRF up-regulation in human gastric ulcers was similar to that found in rat gastric ulcers. Gene therapy with SRF significantly accelerated experimental gastric ulcer healing and promoted re-epithelialization and muscle restoration. Overexpression of SRF in RGM1 and A7R5 cells accelerates migration and proliferation of these cells by promoting actin polymerization and activation of immediately early genes. CONCLUSIONS: Activation of SRF is an important component of ulcer healing. SRF promotes migration and proliferation of gastric epithelial and smooth muscle cells, which are essential for re-epithelialization and restoration of muscular structures.
Assuntos
Células Epiteliais/fisiologia , Miócitos de Músculo Liso/fisiologia , Fator de Resposta Sérica/genética , Úlcera Gástrica/fisiopatologia , Cicatrização/fisiologia , Animais , Divisão Celular , Movimento Celular , DNA Complementar/farmacologia , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Células Epiteliais/citologia , Expressão Gênica , Terapia Genética , Proteínas Imediatamente Precoces/genética , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Miócitos de Músculo Liso/citologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Fator de Resposta Sérica/metabolismo , Úlcera Gástrica/patologia , Úlcera Gástrica/terapia , Fibras de Estresse/metabolismo , Fatores de Transcrição/genéticaRESUMO
Regulation of angiogenesis by nitric oxide (NO) is controversial since NO has been shown to have both pro- and anti-angiogenic effects. In this study, we examined the effect of the NO donor, S-nitro-N-acetyl-penicillamine (SNAP), on in vitro angiogenesis, and the mechanisms involved: PKC activity, ERK and c-Jun phosphorylation, and AP-1 DNA binding activity, in microvascular endothelial cells. SNAP, at 0.5-4 mM, significantly and dose-dependently inhibited angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 80%, 83%, and 63% and 73%, respectively. SNAP at concentrations > 2mM also abolished AP-1 binding activity. Lower concentrations of SNAP (0.1-0.3 mM) significantly increased angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 46%, 60%, and 61% and 180%, respectively. These findings indicate that the dual pro- and anti-angiogenic actions of NO are dose-dependent and suggest that they are mediated by PKC and ERK acting on AP-1.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Fisiológica/fisiologia , Óxido Nítrico/farmacologia , Proteína Quinase C/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , DNA/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Ativação Enzimática/efeitos dos fármacos , Microcirculação , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/biossíntese , Doadores de Óxido Nítrico/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , S-Nitroso-N-Acetilpenicilamina/farmacologia , Estômago/irrigação sanguínea , Estômago/citologiaRESUMO
Our previous studies demonstrated that enhanced epithelial cell proliferation is important for healing of experimental esophageal ulcers. However, the roles of angiogenesis, its major mediator, vascular endothelial growth factor (VEGF), and the mechanism(s) regulating VEGF expression during esophageal ulcer healing remain unknown. Esophageal ulcers were induced in rats by focal application of acetic acid. We studied expressions of hypoxia-inducible transcription factor-1 alpha (HIF-1 alpha), an activator of the VEGF gene, and VEGF by reverse transcriptase-polymerase chain reaction, Western blotting, and immunostaining. To determine the efficacy of VEGF gene therapy in esophageal ulcer healing, we studied whether a single local injection of plasmid cDNA encoding recombinant human VEGF(165) affects ulcer healing and angiogenesis. Esophageal ulceration induced HIF-1 alpha protein expression and VEGF gene activation reflected by increased VEGF mRNA (240%) and VEGF protein (310%) levels. HIF-1 alpha protein was expressed in microvessels bordering necrosis where it co-localized with VEGF. Injection of cDNA encoding VEGF(165) significantly enhanced angiogenesis and accelerated esophageal ulcer healing. These results: 1) suggest that HIF-1 alpha may mediate esophageal ulceration-triggered VEGF gene activation, 2) indicate an essential role of VEGF and angiogenesis in esophageal ulcer healing, and 3) demonstrate the feasibility of gene therapy for the treatment of esophageal ulcers.
Assuntos
Fatores de Crescimento Endotelial/genética , Doenças do Esôfago/genética , Esôfago/irrigação sanguínea , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfocinas/genética , Úlcera Gástrica/genética , Fatores de Transcrição/genética , Cicatrização/fisiologia , Animais , Sequência de Bases , Primers do DNA , Modelos Animais de Doenças , Doenças do Esôfago/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Masculino , Neovascularização Patológica , Plasmídeos , Biossíntese de Proteínas , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Úlcera Gástrica/patologia , Transcrição Gênica , Ativação Transcricional , Úlcera/genética , Úlcera/patologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Cicatrização/genéticaRESUMO
Prostaglandins (PGs), bioactive lipid molecules produced by cyclooxygenase enzymes (COX-1 and COX-2), have diverse biological activities, including growth-promoting actions on gastrointestinal mucosa. They are also implicated in the growth of colonic polyps and cancers. However, the precise mechanisms of these trophic actions of PGs remain unclear. As activation of the epidermal growth factor receptor (EGFR) triggers mitogenic signaling in gastrointestinal mucosa, and its expression is also upregulated in colonic cancers and most neoplasms, we investigated whether PGs transactivate EGFR. Here we provide evidence that prostaglandin E2 (PGE2) rapidly phosphorylates EGFR and triggers the extracellular signal-regulated kinase 2 (ERK2)--mitogenic signaling pathway in normal gastric epithelial (RGM1) and colon cancer (Caco-2, LoVo and HT-29) cell lines. Inactivation of EGFR kinase with selective inhibitors significantly reduces PGE2-induced ERK2 activation, c-fos mRNA expression and cell proliferation. Inhibition of matrix metalloproteinases (MMPs), transforming growth factor-alpha (TGF-alpha) or c-Src blocked PGE2-mediated EGFR transactivation and downstream signaling indicating that PGE2-induced EGFR transactivation involves signaling transduced via TGF-alpha, an EGFR ligand, likely released by c-Src-activated MMP(s). Our findings that PGE2 transactivates EGFR reveal a previously unknown mechanism by which PGE2 mediates trophic actions resulting in gastric and intestinal hypertrophy as well as growth of colonic polyps and cancers.