RESUMO
Rotavirus (RV), a leading cause of severe diarrhea, primarily infects intestinal epithelial cells (IECs) causing self-limiting illness. To better understand innate immunity to RV, we sought to define the extent to which IEC activation of anti-viral responses required viral replication or could be recapitulated by inactivated RV or its components. Using model human intestinal epithelia, we observed that RV-induced activation of signaling events and gene expression typically associated with viral infection was largely mimicked by administration of ultraviolet (UV)-inactivated RV. Use of anti-interferon (IFN) neutralizing antibodies revealed that such replication-independent anti-viral gene expression required type I IFN signaling. In contrast, RV-induction of nuclear factor-κB-mediated interleukin-8 expression was dependent on viral replication. The anti-viral gene expression induced by UV-RV was not significantly recapitulated by RV RNA or RV virus-like particles although the latter could enter IEC. Together, these results suggest that RV proteins mediate viral entry into epithelial cells leading to intracellular detection of RV RNA that generates an anti-viral response.
Assuntos
Interferon Tipo I/metabolismo , Mucosa Intestinal/metabolismo , NF-kappa B/metabolismo , Infecções por Rotavirus/imunologia , Rotavirus/fisiologia , Anticorpos Bloqueadores/farmacologia , Linhagem Celular , Regulação Viral da Expressão Gênica/imunologia , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Interleucina-8/biossíntese , Interleucina-8/genética , Interleucina-8/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , NF-kappa B/imunologia , RNA Viral/imunologia , Rotavirus/patogenicidade , Infecções por Rotavirus/virologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Raios Ultravioleta , Vírion/imunologia , Inativação de Vírus , Replicação ViralRESUMO
PTLD is an important complication following heart transplantation. To better define the risk factors of PTLD in children, we performed a case-control study. All pediatric cardiac transplant recipients who developed their first episode of PTLD were matched by age (+/-1 yr) and time since transplant (+/-1 yr) with those who did not. PTLD occurred in nine of 95 cardiac transplant recipients (9%), 0.3-7.8 yr following cardiac transplantation (median = 2.5 yr). Patients were 0.1-16.4 yr (median = 3.7) at transplantation. Biopsies revealed polymorphic B cell hyperplasia (three), polymorphic B cell lymphoma (one), monomorphic diffuse large cell B cell lymphoma (three) and monomorphic Burkitt's-like lymphoma (two). Patients who developed PTLD were at no greater risk of death (p = 0.31). Recipient EBV seronegativity at time of transplant (p = 0.08), EBV seroconversion (p = 0.013) and recipient CMV seronegativity (p = 0.015) were associated with the development of PTLD by conditional logistic regression; sex, race, donor age, recipient diagnosis, donor CMV seropositivity, recipient treatment for CMV infection, EBV seropositivity at the time of PTLD diagnosis, and number of rejection episodes, treated rejection episodes, and lympholytics used were not. There was no significant association between PTLD and death in our recipients. EBV seroconversion and recipient CMV seronegativity were associated with the development of PTLD.
Assuntos
Transplante de Coração/efeitos adversos , Transtornos Linfoproliferativos/epidemiologia , Complicações Pós-Operatórias/epidemiologia , Adolescente , Linfócitos B/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Transplante de Coração/mortalidade , Humanos , Hiperplasia , Lactente , Transtornos Linfoproliferativos/patologia , Estudos Retrospectivos , Análise de SobrevidaRESUMO
Rotavirus is a major pathogen of infantile gastroenteritis. It is a large and complex virus with a multilayered capsid organization that integrates the determinants of host specificity, cell entry, and the enzymatic functions necessary for endogenous transcription of the genome that consists of 11 dsRNA segments. These segments encode six structural and six nonstructural proteins. In the last few years, there has been substantial progress in our understanding of both the structural and functional aspects of a variety of molecular processes involved in the replication of this virus. Studies leading to this progress using of a variety of structural and biochemical techniques including the recent application of RNA interference technology have uncovered several unique and intriguing features related to viral morphogenesis. This review focuses on our current understanding of the structural basis of the molecular processes that govern the replication of rotavirus.
Assuntos
Rotavirus/fisiologia , Proteínas Virais/química , Montagem de Vírus , Antígenos Virais/química , Capsídeo/química , Proteínas do Capsídeo/química , Genoma Viral , Concentração de Íons de Hidrogênio , RNA Viral/biossíntese , Proteínas de Ligação a RNA/química , Vírus Reordenados/química , Proteínas não Estruturais Virais/química , Proteínas Virais/fisiologia , Replicação ViralRESUMO
Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.
Assuntos
Cálcio/fisiologia , Compartimento Celular , Glicoproteínas/fisiologia , Toxinas Biológicas/fisiologia , Proteínas não Estruturais Virais/fisiologia , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi , Proteínas de Fluorescência Verde/genética , Humanos , Transporte Proteico , Rotavirus , Infecções por Rotavirus , Replicação ViralRESUMO
Angiogenesis has been shown to be dysregulated in coronary artery (CA) aneurysms in the chronic phase of Kawasaki disease (KD). Neovascularization may occur in inflammatory-related vascular diseases because many angiogenesis mediators are secreted by inflammatory cells. We hypothesized that inflammation of the acute KD CA aneurysm could lead to dysregulation of angiogenesis mediators and subsequent neovascularization. To investigate this hypothesis, acute fatal KD cardiac tissues were immunostained for angiogenic inducers and inhibitors. Microvessel density was determined and the degree of inflammation assessed. Marked inflammation and angiogenesis were found in acute KD CA aneurysms and myocardium, with the highest microvessel density seen in patients who died 2-3 weeks after onset of the disease. Expression of proangiogenic proteins was higher than expression of inhibitors in KD CA aneurysms and myocardium. Angiogenesis mediators were localized to inflammatory cells in the myointima, adventitia, and myocardium. We conclude that significant neovascularization occurs in acute KD CA aneurysms and myocardium much sooner after onset of the disease than has been previously reported, that multiple angiogenesis factors are involved, and that dysregulation of angiogenesis likely contributes to KD vasculopathy.
Assuntos
Aneurisma Coronário/mortalidade , Vasos Coronários/patologia , Síndrome de Linfonodos Mucocutâneos/mortalidade , Síndrome de Linfonodos Mucocutâneos/patologia , Miocárdio/patologia , Neovascularização Patológica/mortalidade , Doença Aguda , Aneurisma Roto/mortalidade , Angiostatinas/metabolismo , Estudos de Casos e Controles , Criança , Aneurisma Coronário/metabolismo , Aneurisma Coronário/patologia , Vasos Coronários/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Mastócitos/metabolismo , Microcirculação , Síndrome de Linfonodos Mucocutâneos/metabolismo , Miocardite/metabolismo , Miocardite/mortalidade , Miocardite/patologia , Miocárdio/metabolismo , Neovascularização Patológica/metabolismo , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Trombospondinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Plasma cell granulomas are lesions of uncertain histogenesis arising in a variety of locations, most commonly the lung. Treatment for these lesions is complete surgical excision if possible. Unresectable pulmonary lesions respond to oral corticosteroids and radiation therapy. We report the long-term outcome of two unusual pediatric cases of cardiac plasma cell granulomas originating within the right ventricle and posterior aspect of the left ventricle. The limited literature reports advocate surgical resection for this entity, with no discussion of alternative treatment strategies for unresectable lesions. We prospectively evaluated the response to postoperative oral steroid therapy, as complete surgical excision was not possible in either case. Sequential echocardiography demonstrated additional significant reduction in the size of the masses and the patients remain asymptomatic at 9 and 5.5 years follow-up, without evidence of obstruction or recurrence. Oral corticosteroids should be considered as a treatment option for any unresectable plasma cell granuloma.
Assuntos
Glucocorticoides/administração & dosagem , Granuloma de Células Plasmáticas/terapia , Prednisona/administração & dosagem , Administração Oral , Biópsia , Procedimentos Cirúrgicos Cardíacos , Ecocardiografia , Feminino , Seguimentos , Granuloma de Células Plasmáticas/diagnóstico , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Microscopia Eletrônica , Estudos Prospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Prostate cells secrete many molecules capable of regulating angiogenesis; however, which of these actually function as essential regulators of neovascularization is not yet clear. METHODS: Functional angiogenic mediators secreted by normal and diseased prostate cells were identified using an in vitro angiogenesis assay. These factors were quantified by immunoblot or ELISA and localized in tissue by immunohistochemistry. RESULTS: Normal prostate epithelial cell secretions were anti-angiogenic due to inhibitory thrombospondin-1 (TSP-1) whereas this inhibitor was decreased in the pro-angiogenic secretions derived from benign prostatic hyperplasia (BPH) and cancer cells. This pro-angiogenic activity depended primarily on fibroblast growth factor-2 (FGF-2) and/or vascular endothelial growth factor (VEGF) whose secretion was increased. Immunolocalization studies confirmed that the changes detected in vitro also occurred in vivo. CONCLUSIONS: During disease progression in the prostate, production of TSP-1, the major inhibitor, is down-regulated while that of stimulatory FGF-2 and/or VEGF rise, leading to the induction of the new vessels necessary to support tumor growth.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Linfocinas/fisiologia , Neovascularização Patológica/fisiopatologia , Próstata/irrigação sanguínea , Neoplasias da Próstata/irrigação sanguínea , Trombospondina 1/fisiologia , Adolescente , Adulto , Western Blotting , Fatores de Crescimento Endotelial/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Linfocinas/metabolismo , Masculino , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/fisiologia , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Trombospondina 1/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence of N-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-microm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [(3)H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium, but SA-dependent and SA-independent rotavirus strains infect polarized epithelial cells by different mechanisms, which may be relevant for pathogenesis and selection of vaccine strains. Finally, rotavirus-induced alterations of the epithelial barrier and paracellular permeability suggest that common mechanisms of pathogenesis may exist between viral and bacterial pathogens of the intestinal tract.
Assuntos
Polaridade Celular , Células Epiteliais/virologia , Ácido N-Acetilneuramínico/metabolismo , Rotavirus/fisiologia , Animais , Linhagem Celular , Movimento Celular , Cães , Células Epiteliais/ultraestrutura , Humanos , Macaca mulatta , Membranas Artificiais , Microscopia Eletrônica , Rotavirus/metabolismo , Rotavirus/ultraestruturaRESUMO
OBJECTIVE: In 1996, we introduced the free tracheal autograft technique for repair of congenital tracheal stenosis from complete tracheal rings in infants and children. Sources of possible concern with this procedure include the potential for autograft ischemia, patch dehiscence, and recurrent stenosis. Vascular endothelial growth factor is a potent angiogenic inducer (particularly in the setting of ischemia, hypoxia, or both) and is postulated to promote tissue healing. The purpose of this study was to test the hypothesis that pretreatment of tracheal autografts with topical vascular endothelial growth factor would enhance tracheal healing. METHODS: In a rabbit model of tracheal reconstruction (n = 32), an elliptically shaped portion of the anterior tracheal wall was excised. The excised portion of trachea was one third of the tracheal circumference and 2 cm in length (6 tracheal rings). This portion of trachea (the autograft) was soaked in either vascular endothelial growth factor (5 microg/mL, n = 16) or normal saline solution (n = 16) for 15 minutes before being reimplanted in the resultant tracheal opening. Animals were killed and autografts were examined at 2 weeks, 1 month, and 2 months postoperatively for gross and microscopic characteristics. RESULTS: By 2 weeks, and progressing through 1 and 2 months, autografts treated with vascular endothelial growth factor, as compared with control autografts, had reduced luminal stenosis, submucosal fibrosis, and inflammatory infiltrate (P <.05). The autografts tended to become malaligned in control animals, whereas the tracheal architecture was preserved in rabbits treated with vascular endothelial growth factor. Microvascular vessel density was significantly greater in all vascular endothelial growth factor groups (P <.05) at all time intervals. CONCLUSIONS: Topical treatment of free tracheal autografts with vascular endothelial growth factor in a rabbit tracheal reconstruction model enhanced healing, as evidenced by accelerated autograft revascularization, reduced submucosal fibrosis and inflammation, and preservation of the normal tracheal architecture. Topical vascular endothelial growth factor may improve future results of tracheal reconstruction.
Assuntos
Modelos Animais de Doenças , Fatores de Crescimento Endotelial/uso terapêutico , Linfocinas/uso terapêutico , Pré-Medicação/métodos , Traqueia/transplante , Estenose Traqueal/congênito , Estenose Traqueal/cirurgia , Cicatrização/efeitos dos fármacos , Administração Cutânea , Animais , Avaliação Pré-Clínica de Medicamentos , Fatores de Crescimento Endotelial/farmacologia , Fatores de Crescimento Endotelial/fisiologia , Feminino , Fibrose , Inflamação , Linfocinas/farmacologia , Linfocinas/fisiologia , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Coelhos , Distribuição Aleatória , Recidiva , Índice de Gravidade de Doença , Deiscência da Ferida Operatória/etiologia , Deiscência da Ferida Operatória/psicologia , Fatores de Tempo , Estenose Traqueal/classificação , Estenose Traqueal/patologia , Transplante Autólogo/efeitos adversos , Transplante Autólogo/métodos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The pathogenesis of coronary arterial inflammation in acute Kawasaki disease (KD) is unclear. To test the hypothesis that the KD vascular lesion is an activated T lymphocyte-dependent process, immunohistochemical studies were done on coronary artery aneurysms from 8 fatal acute KD cases by using antibodies to CD45RO (activated or memory T lymphocyte), CD8 (cytotoxic T lymphocyte), CD4 (helper T lymphocyte), HAM56 (macrophage), and CD20 (B lymphocyte). Acute KD coronary arteritis was characterized by transmural infiltration of CD45RO T lymphocytes with CD8 T lymphocytes predominating over CD4 T lymphocytes. Macrophages were present primarily in the adventitial layer; B lymphocytes were notably absent. These data lend support to the hypotheses that KD results from infection with an intracellular pathogen, such as a virus, whose antigens are presented by major histocompatibility complex class I molecules, and that CD8 T lymphocytes and macrophages are important in the pathogenesis of KD coronary aneurysms.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Aneurisma Coronário/imunologia , Macrófagos/imunologia , Síndrome de Linfonodos Mucocutâneos/imunologia , Antígenos CD20/análise , Antígenos CD8/análise , Criança , Pré-Escolar , Aneurisma Coronário/complicações , Vasos Coronários/imunologia , Endotélio Vascular/imunologia , Feminino , Humanos , Imuno-Histoquímica , Lactente , Antígenos Comuns de Leucócito/análise , Masculino , Síndrome de Linfonodos Mucocutâneos/complicaçõesRESUMO
OBJECTIVES: The objective was to study the impact of nonadherence on late rejection after pediatric heart transplantation. STUDY DESIGN: This was a retrospective cohort study of cardiac transplant recipients surviving >6 months (n = 50). Patients were stratified by episodes of late rejection. End points were defined by cyclosporin A (CSA) level, CSA level variability, and patient admission of nonadherence. RESULTS: In 15 patients there were 49 episodes of late rejection, and 37 (76%) were associated with nonadherence. Of these patients, 7 of 15 died, and 3 of 15 had transplant coronary artery disease. Risk factors for the rejection were single-parent home, non-white, older age, and higher CSA level variability. In 35 nonrejectors there were 4 deaths from sepsis, post-transplant lymphoproliferative disease, renal failure, and encephalomyelitis. CONCLUSION: Late rejection after pediatric heart transplantation is associated with nonadherence, is common during adolescence, and is associated with poor outcome.
Assuntos
Rejeição de Enxerto/etiologia , Transplante de Coração/imunologia , Imunossupressores/uso terapêutico , Recusa do Paciente ao Tratamento , Adolescente , Criança , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Ciclosporina/sangue , Ciclosporina/uso terapêutico , Feminino , Seguimentos , Rejeição de Enxerto/mortalidade , Transplante de Coração/mortalidade , Humanos , Imunossupressores/sangue , Masculino , Avaliação de Resultados em Cuidados de Saúde , Estudos Retrospectivos , Fatores de Risco , Fatores de TempoRESUMO
The outcome of intestinal infection with rotaviruses is more complex than initially appreciated, and it is affected by a complex interplay of host and viral factors. Rotaviruses infect intestinal enterocytes, and the early events in infection are mediated by virus-epithelial cell interactions. Diarrhoea may be caused by several mechanisms including (i) malabsorption that occurs secondary to the destruction of enterocytes, (ii) villus ischaemia and activation of the enteric nervous system that may be evoked by release of a vasoactive agent from infected epithelial cells in the absence of significant pathologic lesions or enterocyte damage, and (iii) intestinal secretion stimulated by the intracellular or extracellular action of the rotavirus non-structural protein, NSP4, a novel enterotoxin and secretory agonist with pleiotropic properties. New studies of rotavirus infection of polarized intestinal epithelial cells show that rotaviruses infect cells differently depending on whether or not they require sialic acid for initial binding, and infection alters epithelial cell functions. NSP4 also affects epithelial cell function and interactions. NSP4 (i) induces an age- and dose-dependent diarrhoeal response in young rodents that is similar to virus-induced disease, (ii) stimulates a Ca(2+)-dependent cell permeability where the secretory response is age-dependent, and (iii) alters epithelial cell integrity. Antibody to NSP4 protects mouse pups from diarrhoea induced by homotypic and heterotypic viruses. These data support a new mechanism of rotavirus-induced diarrhoea whereby a viral enterotoxin triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. This new information about how a gastrointestinal virus causes disease demonstrates common pathogenic mechanisms for viral and bacterial pathogens not previously appreciated. These results also suggest new approaches to prevent or treat rotavirus-induced diarrhoea.
Assuntos
Gastroenterite/patologia , Gastroenterite/virologia , Infecções por Rotavirus/patologia , Infecções por Rotavirus/virologia , Rotavirus/patogenicidade , Animais , Diarreia/patologia , Diarreia/prevenção & controle , Diarreia/terapia , Diarreia/virologia , Enterotoxinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Gastroenterite/metabolismo , Gastroenterite/terapia , Glicoproteínas/metabolismo , Humanos , Microvilosidades/patologia , Microvilosidades/virologia , Rotavirus/fisiologia , Infecções por Rotavirus/metabolismo , Infecções por Rotavirus/terapia , Toxinas Biológicas , Proteínas não Estruturais Virais/metabolismoRESUMO
Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5(*) and VP8(*) in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8(*) portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5(*), at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.
Assuntos
Proteínas do Capsídeo , Capsídeo/química , Rotavirus/química , Tripsina/farmacologia , Sequência de Aminoácidos , Animais , Capsídeo/metabolismo , Chlorocebus aethiops , Microscopia EletrônicaRESUMO
Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.
Assuntos
Apoptose , Criopreservação , Hepatócitos/citologia , Animais , Adesão Celular , Separação Celular , Criopreservação/métodos , Feminino , Glucose , Hepatócitos/fisiologia , Hepatócitos/transplante , Humanos , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , SoluçõesRESUMO
Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.
Assuntos
Inibidores da Angiogênese/farmacologia , Retinopatia Diabética/prevenção & controle , Proteínas do Olho , Isquemia/complicações , Fatores de Crescimento Neural , Proteínas/farmacologia , Vasos Retinianos/efeitos dos fármacos , Serpinas/farmacologia , Animais , Apoptose , Células Cultivadas , Retinopatia Diabética/etiologia , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologiaRESUMO
Neuroblastoma is notable for its cellular heterogeneity and unpredictable outcome. Tumors are a variable mixture of primitive malignant neuroblasts, more differentiated ganglionic cells, Schwann and endothelial cells. Although often fatal, neuroblastomas can spontaneously regress, possibly due to favorable autocrine and paracrine interactions among these cells. Here, pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis and inducer of neural differentiation, is shown to be produced by ganglionic cells and Schwann cells, but not by more primitive tumor cells. Although undifferentiated neuroblastoma tumor cell secretions were angiogenic primarily due to vascular endothelial growth factor, secretions of Schwann cells were anti-angiogenic due to PEDF. In addition, PEDF was the major factor responsible for Schwann cell's ability to induce tumor cell differentiation in vitro and recombinant PEDF had the same effect in vitro and in vivo. Both the growth and the survival of Schwann cells were enhanced by PEDF. Thus PEDF may serve as a multifunctional antitumor agent in neuroblastomas, inhibiting angiogenesis while promoting the numbers of Schwann cells and differentiated tumor cells that in turn produce PEDF, suggesting that its clinical administration could stimulate a multifaceted antitumor feedback loop with the potential to limit and possibly regress tumor growth.
Assuntos
Antineoplásicos/metabolismo , Proteínas do Olho , Fatores de Crescimento Neural , Neuroblastoma/metabolismo , Neuroblastoma/prevenção & controle , Epitélio Pigmentado Ocular/fisiologia , Proteínas/fisiologia , Células de Schwann/fisiologia , Serpinas/fisiologia , Inibidores da Angiogênese/metabolismo , Animais , Antineoplásicos/farmacologia , Bovinos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Feminino , Substâncias de Crescimento/metabolismo , Humanos , Injeções Subcutâneas , Linfocinas/metabolismo , Camundongos , Camundongos Nus , Neuroblastoma/irrigação sanguínea , Neuroblastoma/patologia , Proteínas/administração & dosagem , Proteínas/metabolismo , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/administração & dosagem , Células de Schwann/metabolismo , Serpinas/administração & dosagem , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We investigated the immunogenicity of recombinant double-layered rotavirus-like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) Wa and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT), as mucosal adjuvant. Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (two-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activated antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-reactivated ASC (derived from memory B cells) from intestinal and systemic lymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, peripheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavirus-specific immunoglobulin M (IgM), IgA, and IgG ASC and memory B-cell responses were detected by PID 21 or 28 in intestinal and systemic lymphoid tissues after i.n. inoculation with two or three doses of 2/6-VLPs with or without mLT. Greater mean numbers of virus-specific ASC and memory B cells in all tissues prechallenge were induced in pigs inoculated with two doses of SA11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT. After challenge, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-neutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after challenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mLT). Three doses of SA 11 2/6-VLP plus mLT induced the highest mean numbers of IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge. However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6-VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculation of gnotobiotic pigs with 2/6-VLPs did not confer protection against human rotavirus challenge.
Assuntos
Linfócitos B/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli , Vida Livre de Germes/imunologia , Imunidade Inata , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Animais , Formação de Anticorpos , Bovinos , Infecções por Escherichia coli/imunologia , Humanos , Mutação , Suínos , Vírion/imunologiaRESUMO
The etiology and pathogenesis of Kawasaki disease (KD) remain unknown. As previously reported, in US patients with acute KD, IgA plasma cells (PCs) infiltrate the vascular wall. To determine whether IgA PCs are increased at mucosal sites in KD and to determine whether other nonvascular KD tissues are infiltrated by IgA PCs, the cells were immunolocalized and quantitated in tissue sections taken from 18 US and Japanese patients who died of acute KD and from 10 age-matched controls. IgA PCs were significantly increased in the trachea of patients who died of acute KD, compared with controls (P<.01), a finding that was similar to findings in children with fatal respiratory viral infection. IgA PCs also infiltrated coronary artery, pancreas, and kidney in all KD patients. These findings strongly support entry of the KD etiologic agent through the upper respiratory tract, resulting in an IgA immune response, with systemic spread to vascular tissue, pancreas, and kidney.
Assuntos
Imunoglobulina A/análise , Síndrome de Linfonodos Mucocutâneos/imunologia , Síndrome de Linfonodos Mucocutâneos/patologia , Plasmócitos/patologia , Doença Aguda , Causas de Morte , Criança , Pré-Escolar , Vasos Coronários/imunologia , Vasos Coronários/patologia , Etnicidade , Feminino , Humanos , Lactente , Japão , Rim/imunologia , Rim/patologia , Masculino , Pâncreas/imunologia , Pâncreas/patologia , Plasmócitos/imunologia , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Estados UnidosRESUMO
BACKGROUND: A randomized, prospective, double-blind study of 29 children was performed to evaluate the hypothesis that dexamethasone administration prior to cardiopulmonary bypass would decrease the inflammatory mediator release and improve the postoperative clinical course. METHODS: Fifteen children received dexamethasone (1 mg/kg intravenously) and 14 (controls) received saline solution 1 hour prior to CPB. Serial blood analyses for interleukin-6, tumor necrosis factor-alpha, complement component C3a, and absolute neutrophil count were performed. Postoperative variables evaluated included temperature, supplemental fluids, alveolar-arterial oxygen gradient, and days of mechanical ventilation. RESULTS: Dexamethasone caused an eightfold decrease in interleukin-6 levels and a greater than threefold decrease in tumor necrosis factor-alpha levels after CPB (p < 0.05). Complement component C3a and absolute neutrophil count were not affected by dexamethasone. The mean rectal temperature for the first 24 hours postoperatively was significantly lower in the group given dexamethasone than in the controls (37.2 degrees +/- 0.4 degrees C versus 37.7 degrees +/- 4 degrees C; p = 0.007). Dexamethasone-treated patients required less supplemental fluid during the first 48 hours (22 +/- 28 mL/kg versus 47 +/- 34 mL/kg; p = 0.04). Compared with controls, dexamethasone-treated children had significantly lower alveolar-arterial oxygen gradients during the first 24 hours (144 +/- 108 mm Hg versus 214 +/- 118 mm Hg; p = 0.02) and required less mechanical ventilation (median duration, 3 days versus 5 days; p = 0.02). CONCLUSIONS: Dexamethasone administration prior to CPB in children leads to a reduction in the postbypass inflammatory response as assessed by cytokine levels and clinical course.