RESUMO
Understanding the bioaerosol dynamics of droplets and droplet nuclei emitted during respiratory activities is important for understanding how infectious diseases are transmitted and potentially controlled. To this end, we conducted experiments to quantify the size-resolved dynamics of indoor bioaerosol transport and control in an unoccupied apartment unit operating under four different HVAC particle filtration conditions. Two model organisms (Escherichia coli K12 and bacteriophage T4) were aerosolized under alternating low and high flow rates to roughly represent constant breathing and periodic coughing. Size-resolved aerosol sampling and settle plate swabbing were conducted in multiple locations. Samples were analyzed by DNA extraction and quantitative polymerase chain reaction (qPCR). DNA from both organisms was detected during all test conditions in all air samples up to 7 m away from the source, but decreased in magnitude with the distance from the source. A greater fraction of T4 DNA was recovered from the aerosol size fractions smaller than 1 µm than E. coli K12 at all air sampling locations. Higher efficiency HVAC filtration also reduced the amount of DNA recovered in air samples and on settle plates located 3-7 m from the source.
Assuntos
Aerossóis/análise , Microbiologia do Ar , Poluição do Ar em Ambientes Fechados/análise , Doenças Transmissíveis/transmissão , Monitoramento Ambiental , Ventilação , Ar Condicionado , Bacteriófago T4 , Tosse , Escherichia coli , Humanos , Umidade , Respiração , TemperaturaRESUMO
Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a 6-8-kD, lipopolysaccharide-inducible monocyte and neutrophil chemotactic protein that may be important in acute and chronic inflammation. The present study determined the sequential production, source, and in vivo contribution of murine MIP-1 alpha in synchronized Schistosoma mansoni egg pulmonary granuloma formation. Granulomas were examined under conditions of primary, secondary vigorous, and secondary immunomodulated immunity. Secreted MIP-1 alpha was measured in 24-h supernatants from intact granulomas (700/ml) cultured with or without soluble egg antigen (SEA). Primary granulomas isolated from naive mice over a 16-d period showed low spontaneous MIP-1 alpha production (< 1 ng/ml). However, when primary granulomas were challenged with SEA, significant MIP-1 alpha production was observed beginning at day 4 and peaking at day 16. Intact vigorous (isolated from 8-wk-infected mice) and modulated (isolated from 20-wk-infected mice) secondary pulmonary granulomas demonstrated comparable spontaneous MIP-1 alpha production. Addition of SEA to vigorous stage granulomas augmented expression of MIP-1 alpha at all time points, whereas stimulated modulated stage granulomas did not increase production. The latter observation is likely related to endogenous immunoregulatory mechanisms reported for modulated stage animals. Immunohistochemical localization of MIP-1 alpha in granuloma sections and cytocentrifuge preparations from vigorous lesions localized MIP-1 alpha production to macrophages within granulomas. Treatment of mice with rabbit anti-mouse MIP-1 alpha antibodies significantly decreased 8-d primary granuloma formation (> 40%) when compared with control mice. Anti-MIP-1 alpha sera also decreased vigorous (> 20%), but not modulated granuloma formation. These findings demonstrate that MIP-1 alpha contributes to cellular recruitment during schistosome egg granuloma formation.
Assuntos
Citocinas/fisiologia , Granuloma/imunologia , Pneumopatias Parasitárias/imunologia , Monocinas/fisiologia , Esquistossomose mansoni/imunologia , Animais , Sequência de Bases , Quimiocina CCL3 , Quimiocina CCL4 , Citocinas/genética , Feminino , Proteínas Inflamatórias de Macrófagos , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Monocinas/genética , Óvulo , Reação em Cadeia da Polimerase , CoelhosRESUMO
A new neutrophil-activating peptide, termed ENA-78, was identified in the conditioned media of stimulated human type II epithelial cell line A549. In response to stimulation with either interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha), ENA-78 was produced and secreted concomitantly with IL-8, GRO alpha, and GRO gamma. ENA-78 consists of 78 amino acids [sequence; see text] and has a molecular weight of 8,357. It has four cysteines positioned identically to those of IL-8 and analogues, and thus belongs to the CXC family of peptides. ENA-78 is related to neutrophil-activating peptide 2 (NAP-2) and GRO alpha (sequence identity, 53% and 52%, respectively) and IL-8 (22% identity). Like NAP-2 and GRO alpha, ENA-78 stimulates neutrophils, inducing chemotaxis, a rise in intracellular free calcium and exocytosis. Cross-desensitization experiments indicate that ENA-78 acts through the same type of receptors as IL-8, NAP-2, and GRO alpha.
Assuntos
Quimiocinas CXC , Interleucina-8/análogos & derivados , Interleucina-8/farmacologia , Neutrófilos/efeitos dos fármacos , Peptídeos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Quimiocina CXCL5 , Humanos , Interleucina-8/química , Dados de Sequência Molecular , Peptídeos/química , beta-TromboglobulinaRESUMO
Neutrophil (polymorphonuclear leukocyte [PMN]) sequestration is one of the histologic hallmarks of an acute inflammatory response. During the natural evolution of an inflammatory response, PMNs are often replaced by mononuclear cells. This shift in the elicitation of specific leukocyte populations usually occurs as the inflammatory lesion enters either the repair/resolution stage or progresses to a chronic inflammation. To elucidate a potential mechanism for the temporal change from predominantly PMN recruitment to the presence of monocytes, we postulated that PMNs could be a rich source of monocyte chemotactic factors. In our studies, we have identified a dose-dependent induction of monocyte chemotactic activity by PMNs treated with lipopolysaccharide (LPS; 1-100 ng/ml). Interestingly, this monocyte chemotactic activity was significantly attenuated in the presence of neutralizing anti-human macrophage inflammatory protein 1 alpha (MIP-1 alpha) antibodies. Moreover, immunolocalization studies demonstrated the expression of MIP-1 alpha by stimulated PMNs. These findings showed that a significant amount of PMN-derived monocyte chemotactic activity was attributable to MIP-1 alpha. Subsequent characterization of MIP-1 alpha steady-state mRNA and antigen expression demonstrated both a dose- and time-dependent production by LPS-treated PMNs. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a potent PMN activator, failed to induce the expression of MIP-1 alpha over a wide range of concentrations. However, PMNs stimulated in the presence of both LPS and GM-CSF resulted in a synergistic expression pattern for MIP-1 alpha. PMNs stimulated in the presence of both GM-CSF and LPS demonstrated an enhanced and prolonged expression for both MIP-1 alpha mRNA and antigen, as compared with LPS alone. Messenger RNA stabilization analyses demonstrated that MIP-1 alpha mRNA isolated from PMNs stimulated in the presence of GM-CSF and LPS had a prolonged mRNA t1/2, as compared with LPS alone. These findings support the notion that PMNs are capable of producing MIP-1 alpha in the presence of LPS, and that GM-CSF can influence this production through prolongation of MIP-1 alpha mRNA t1/2. The production of PMN-derived MIP-1 alpha, in association with the expression of appropriate adhesion molecules at a site of inflammation, may be one of the central events that contributes to the temporal shift from predominantly PMNs to monocytes during the evolution of inflammation.
Assuntos
Citocinas/biossíntese , Monocinas/biossíntese , Neutrófilos/metabolismo , Sequência de Bases , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Quimiotaxia de Leucócito , Meios de Cultivo Condicionados , Citocinas/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Lipopolissacarídeos/farmacologia , Proteínas Inflamatórias de Macrófagos , Dados de Sequência Molecular , Monócitos/imunologia , Monocinas/genética , RNA Mensageiro/análiseRESUMO
Signal transducer and activator of transcription (Stat)4 and Stat6 are transcription factors that provide type 1 and type 2 response, respectively. Here, we explored the role of Stat4 and Stat6 in innate immunity during septic peritonitis. Stat4-/- and Stat6-/- mice were resistant to the lethality compared with wild-type (WT) mice. At the mechanistic level, bacterial levels in Stat6-/- mice were much lower than in WT mice, which was associated with increased peritoneal levels of interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, macrophage-derived chemokine (MDC), and C10, known to enhance bacterial clearance. In Stat4-/- mice, hepatic inflammation and injury during sepsis were significantly ameliorated without affecting local responses. This event was associated with increased hepatic levels of IL-10 and IL-13, while decreasing those of macrophage inflammatory protein (MIP)-2 and KC. Sepsis-induced renal injury was also abrogated in Stat4-/- mice, which was accompanied by decreased renal levels of MIP-2 and KC without altering IL-10 and IL-13 levels. Thus, Stat6-/- and Stat4-/- mice appeared to be resistant to septic peritonitis by enhancing local bacterial clearance and modulating systemic organ damage, respectively, via balancing cytokine responses. These results clearly highlight an important role of local type 1 and systemic type 2 cytokine response in protective immunity during sepsis, which can be regulated by Stat proteins.
Assuntos
Proteínas de Ligação a DNA/imunologia , Peritonite/imunologia , Sepse/imunologia , Transdução de Sinais/imunologia , Transativadores/imunologia , Animais , Quimiocina CCL22 , Quimiocinas/metabolismo , Quimiocinas CC/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Imunidade Inata , Interleucina-12/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Insuficiência de Múltiplos Órgãos/imunologia , Peritônio/microbiologia , Peritonite/sangue , Peritonite/microbiologia , Fator de Transcrição STAT4 , Fator de Transcrição STAT6 , Sepse/sangue , Sepse/microbiologia , Transativadores/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune demyelinating disease of the central nervous system that serves as an animal model for multiple sclerosis. Antigen-specific tolerance regimens, including oral tolerance, have been used prophylactically to prevent development of acute EAE as well as a number of other autoimmune diseases. Two mechanisms have been proposed to explain the immunologic basis for disease inhibition: bystander immune suppression and clonal anergy/deletion. This report demonstrates a novel mechanism for monocyte chemotactic protein (MCP)-1 as a regulatory factor of oral tolerance. Oral administration of proteolipid protein peptide (PLP139-151) increased MCP-1 expression in the intestinal mucosa, Peyer's patch, and mesenteric lymph nodes. Increase in MCP-1 expression resulted in downregulation of mucosal interleukin (IL)-12 expression with concomitant increase in mucosal IL-4 expression. Functionally, MCP-1 upregulation was shown to regulate oral tolerance induction by the ability of antibodies to MCP-1 to inhibit tolerance induction. The anti-MCP-1 abrogation of oral tolerance induction also resulted in restoration of mucosal IL-12 expression as well as peripheral antigen-specific T helper cell 1 responses. These results demonstrate a novel and important role for MCP-1 in the regulation or oral tolerance for the prevention and treatment of autoimmune disease.
Assuntos
Autoantígenos/administração & dosagem , Quimiocina CCL2/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica , Imunidade nas Mucosas/imunologia , Proteína Proteolipídica de Mielina/imunologia , Células Th1/imunologia , Administração Oral , Animais , Regulação para Baixo , Feminino , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-4/imunologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos , Proteína Proteolipídica de Mielina/administração & dosagem , Nódulos Linfáticos Agregados/imunologia , RNA Mensageiro/genética , Regulação para CimaRESUMO
The Jarisch-Herxheimer Reaction (J-HR) is a clinical syndrome occurring soon after the first adequate dose of an antimicrobial drug to treat infectious diseases such as Lyme disease, syphilis, and relapsing fever. Previous attempts to identify factors mediating this reaction, that may cause death, have been unsuccessful. We conducted a prospective trial in Addis Ababa, Ethiopia on 17 patients treated with penicillin for proven louse-borne relapsing fever due to Borrelia recurrentis to evaluate the association of symptoms with plasma levels of tumor necrosis factor (TNF), interleukins 6, and 8 (IL-6 and -8). 14 of the 17 (82%) patients experienced a typical J-HR consisting of rigors, a rise in body temperature (1.06 +/- 0.2 degrees C) peaking at 2 h, leukopenia (7.4 +/- 0.6 x 10(-3) cells/mm3) at 4 h, a slight decrease, and then rise of mean arterial blood pressure. Spirochetes were cleared from blood in 5 +/- 1 h after penicillin. There were no fatalities, but constitutional symptoms were severe during J-HR. Plasma TNF, IL-6, and -8 were raised in several patients on admission, but a seven-, six-, and fourfold elevation of these plasma cytokine concentrations over admission levels was detected, respectively, occurring in transient form coincidental with observed pathophysiological changes of J-HR. Elevated plasma cytokine levels were not detected in the three patients who did not suffer J-HR. We conclude that the severe pathophysiological changes characterizing the J-HR occurring on penicillin treatment of louse-borne relapsing fever are closely associated with transient elevation of plasma TNF, IL-6, and -8 concentrations.
Assuntos
Interleucina-6/sangue , Interleucina-8/sangue , Febre Recorrente/sangue , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Humanos , Cinética , Masculino , Febre Recorrente/fisiopatologiaRESUMO
Polymorphonuclear leukocytes (PMNs) characterize the pathology of T cell-mediated autoimmune diseases and delayed-type hypersensitivity reactions (DTHRs) in the skin, joints, and gut, but are absent in T cell-mediated autoimmune diseases of the brain or pancreas. All of these reactions are mediated by interferon gamma-producing type 1 T cells and produce a similar pattern of cytokines. Thus, the cells and mediators responsible for the PMN recruitment into skin, joints, or gut during DTHRs remain unknown. Analyzing hapten-induced DTHRs of the skin, we found that mast cells determine the T cell-dependent PMN recruitment through two mediators, tumor necrosis factor (TNF) and the CXC chemokine macrophage inflammatory protein 2 (MIP-2), the functional analogue of human interleukin 8. Extractable MIP-2 protein was abundant during DTHRs in and around mast cells of wild-type (WT) mice but absent in mast cell-deficient WBB6F(1)-Kit(W)/Kit(W-)(v) (Kit(W)/Kit(W)(-v)) mice. T cell-dependent PMN recruitment was reduced >60% by anti-MIP-2 antibodies and >80% in mast cell-deficient Kit(W)/Kit(W)(-v) mice. Mast cells from WT mice efficiently restored DTHRs and MIP-2-dependent PMN recruitment in Kit(W)/Kit(W)-(v) mice, whereas mast cells from TNF(-/)- mice did not. Thus, mast cell-derived TNF and MIP-2 ultimately determine the pattern of infiltrating cells during T cell-mediated DTHRs.
Assuntos
Quimiotaxia de Leucócito , Hipersensibilidade Tardia/imunologia , Leucócitos/imunologia , Mastócitos/imunologia , Monocinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Quimiocina CXCL2 , Fatores Quimiotáticos/metabolismo , Dermatite de Contato/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neutrófilos/imunologia , Cloreto de Picrila , Proteínas Proto-Oncogênicas c-kit/genética , Pele/imunologia , Linfócitos T/imunologiaRESUMO
We investigated the role of interleukin 8 (IL-8) in mediating angiogenesis in human bronchogenic carcinoma. Increased quantities of IL-8 were detected in tumor tissue as compared with normal lung tissue. Immunohistochemical staining of tumors revealed primary localization of IL-8 to individual tumor cells and demonstrated the capacity of tumor to elaborate IL-8. Functional studies that used tissue homogenates of tumors demonstrated the induction of both in vitro endothelial cell chemotaxis and in vivo corneal neovascularization. It is important to note that the addition of neutralizing antisera to IL-8 to these assays resulted in the marked and specific attenuation of these responses. Our observations definitively establish IL-8 as a primary mediator of angiogenesis in bronchogenic carcinoma and offer a potential target for immunotherapies against solid malignancies.
Assuntos
Carcinoma Broncogênico/irrigação sanguínea , Interleucina-8/fisiologia , Neoplasias Pulmonares/irrigação sanguínea , Neovascularização Patológica/etiologia , Animais , Quimiotaxia , Endotélio Vascular/citologia , Feminino , Humanos , Imuno-Histoquímica , Interleucina-8/antagonistas & inibidores , Pulmão/química , Ratos , Ratos Endogâmicos F344RESUMO
Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1(-/-) mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1(-/-) mice, as was expression of IL-4, IL-5, and interferon gamma in splenocytes. In contrast, MCP-1(-/-) mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.
Assuntos
Quimiocina CCL2/fisiologia , Citocinas/biossíntese , Inflamação/imunologia , Monócitos/fisiologia , Animais , Antígenos de Helmintos/imunologia , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Dermatite de Contato/imunologia , Modelos Animais de Doenças , Feminino , Marcação de Genes , Granuloma do Sistema Respiratório/imunologia , Granuloma do Sistema Respiratório/parasitologia , Hipersensibilidade Tardia/imunologia , Interferon gama/biossíntese , Interleucina-4/biossíntese , Interleucina-5/biossíntese , Masculino , Camundongos , Monócitos/imunologia , Mycobacterium bovis/imunologia , Fenótipo , Esquistossomose mansoni/imunologia , Tuberculose/imunologiaRESUMO
The success of solid tumor growth and metastasis is dependent upon angiogenesis. Neovascularization within the tumor is regulated, in part, by a dual and opposing system of angiogenic and angiostatic factors. We now report that IP-10, a recently described angiostatic factor, as a potent angiostatic factor that regulates non-small cell lung cancer (NSCLC)-derived angiogenesis, tumor growth, and spontaneous metastasis. We initially found significantly elevated levels of IP-10 in freshly isolated human NSCLC samples of squamous cell carcinoma (SCCA). In contrast, levels of IP-10 were equivalent in either normal lung tissue or adenocarcinoma specimens. The neoplastic cells in specimens of SCCA were the predominant cells that appeared to express IP-10 by immunolocalization. Neutralization of IP-10 in SCCA tumor specimens resulted in enhanced tumor-derived angiogenic activity. Using a model of human NSCLC tumorigenesis in SCID mice, we found that NSCLC tumor growth was inversely correlated with levels of plasma or tumor-associated IP-10. IP-10 in vitro functioned as neither an autocrine growth factor nor as an inhibitor of proliferation of the NSCLC cell lines. Reconstitution of intratumor IP-10 for a period of 8 wk resulted in a significant inhibition of tumor growth, tumor-associated angiogenic activity and neovascularization, and spontaneous lung metastases, whereas, neutralization of IP-10 for 10 wk augmented tumor growth. These findings support the notion that tumor-derived IP-10 is an important endogenous angiostatic factor in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Quimiocinas CXC , Citocinas/fisiologia , Neoplasias Pulmonares/patologia , Neovascularização Patológica , Animais , Quimiocina CXCL10 , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos SCID , Metástase Neoplásica/patologia , CoelhosRESUMO
Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.
Assuntos
Eosinófilos/imunologia , Hipersensibilidade/imunologia , Receptores de Quimiocinas/deficiência , Células Th2/imunologia , Administração por Inalação , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Baratas/imunologia , Citocinas/genética , Citocinas/metabolismo , Relação Dose-Resposta Imunológica , Eosinófilos/citologia , Granuloma/imunologia , Granuloma/patologia , Hipersensibilidade/genética , Hipersensibilidade/patologia , Imunidade Celular/genética , Imunidade Celular/imunologia , Injeções Subcutâneas , Interleucina-5/sangue , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Óvulo/imunologia , RNA Mensageiro/metabolismo , Receptores CCR8 , Receptores de Quimiocinas/genética , Schistosoma mansoni/imunologia , Células Th1/imunologiaRESUMO
Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective viral RNAs featuring large deletions during replication. These defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity-of-infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early-passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated next-generation sequencing libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and P2 virions and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than P2 virions and that specific populations of D-RNAs are differentially expressed between intracellular and extracellular compartments. Importantly, these trends were likewise observed in a murine model of CHIKV AF15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that is conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which were confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.IMPORTANCE Our understanding of viral defective RNAs (D-RNAs), or truncated viral genomes, comes largely from passaging studies in tissue culture under artificial conditions and/or packaged viral RNAs. Here, we show that specific populations of alphavirus D-RNAs arise de novo and that they are not packaged into virions, thus imposing a transmission bottleneck and impeding their prior detection. This raises important questions about the roles of D-RNAs, both in nature and in tissue culture, during viral infection and whether their influence is constrained by packaging requirements. Further, during the course of these studies, we found a novel type of alphavirus D-RNA that is enriched intracellularly; dubbed subgenomic D-RNAs (sgD-RNAs), they are defined by deletion boundaries between the capsid-E3 region and the E1-3' untranslated region (UTR) and are common to chikungunya, Mayaro, Sindbis, and Aura viruses. These sgD-RNAs are enriched intracellularly and do not appear to be selectively packaged, and additionally, they may exist as subgenome-derived transcripts.
Assuntos
Alphavirus/genética , Vírus Chikungunya/genética , Vírus Defeituosos/genética , RNA Viral/genética , Recombinação Genética , Alphavirus/classificação , Animais , Linhagem Celular , Febre de Chikungunya , Chlorocebus aethiops , Culicidae , Variação Genética , Camundongos , Células VeroRESUMO
Movement of neutrophils from the bloodstream to inflamed tissue depends on the activation of both the neutrophil and the endothelial cell. Endothelial cells lining the postcapillary venule respond to proinflammatory mediators by expressing adhesion molecules and synthesizing a variety of neutrophil-activating factors. Endothelial cell production of a 77-amino acid variant of interleukin-8 (IL-8) was found to be a requirement for the invasion of neutrophils through a vessel wall model. IL-8 secreted by cytokine- or lipopolysaccharide-stimulated endothelial cells induced the rapid shedding of neutrophil lectin adhesion molecule-1, the up-regulation of leukocyte beta 2 integrins, and the attachment and transmigration of the neutrophils. Thus, endogenous endothelial IL-8 regulates transvenular traffic during acute inflammatory responses.
Assuntos
Endotélio Vascular/fisiologia , Interleucina-8/fisiologia , Neutrófilos/fisiologia , Antígenos CD/metabolismo , Northern Blotting , Antígenos CD18 , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Células Cultivadas , Quimiotaxia de Leucócito , Selectina E , Expressão Gênica , Humanos , Técnicas In Vitro , Integrinas/metabolismo , Molécula 1 de Adesão Intercelular , Interleucina-1/farmacologia , Interleucina-8/genética , Lipopolissacarídeos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Human endothelial cells produced a neutrophil chemotactic factor (NCF) upon stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or lipopolysaccharide (LPS). The expression of endothelial cell-derived NCF messenger RNA and biological activity was both time- and concentration-dependent. Maximal NCF mRNA expression occurred at 10 and at 2 nanograms per milliliter for TNF and IL-1 beta, respectively; mRNA expression was first observed 1 hour after stimulation and was maintained for at least 24 hours. In situ hybridization analysis showed that NCF mRNA peaked in treated cells by 24 hours, whereas unstimulated cells were negative. These studies demonstrated that endothelial cells may participate in neutrophil-mediated inflammation by synthesizing a chemotactic factor in response to specific monokines and LPS.
Assuntos
Fatores Quimiotáticos/genética , Endotélio Vascular/fisiologia , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Northern Blotting , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Interleucina-8 , Sondas de Oligonucleotídeos , Fatores de TempoRESUMO
Angiogenic factors produced by monocytes-macrophages are involved in the pathogenesis of chronic inflammatory disorders characterized by persistent angiogenesis. The possibility was tested that interleukin-8 (IL-8), which is a cytokine that is chemotactic for lymphocytes and neutrophils, is also angiogenic. Human recombinant IL-8 was potently angiogenic when implanted in the rat cornea and induced proliferation and chemotaxis of human umbilical vein endothelial cells. Angiogenic activity present in the conditioned media of inflamed human rheumatoid synovial tissue macrophages or lipopolysaccharide-stimulated blood monocytes was equally blocked by antibodies to either IL-8 or tumor necrosis factor-alpha. An IL-8 antisense oligonucleotide specifically blocked the production of monocyte-induced angiogenic activity. These data suggest a function for macrophage-derived IL-8 in angiogenesis-dependent disorders such as rheumatoid arthritis, tumor growth, and wound repair.
Assuntos
Quimiotaxia/efeitos dos fármacos , Córnea/efeitos dos fármacos , Endotélio Vascular/fisiologia , Interleucina-8/farmacologia , Macrófagos/fisiologia , Neovascularização Patológica , Oligonucleotídeos Antissenso/farmacologia , Animais , Artrite Reumatoide/fisiopatologia , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Córnea/fisiologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Interleucina-8/genética , Camundongos , Dados de Sequência Molecular , Monócitos/fisiologia , Coelhos , Ratos , Proteínas Recombinantes/farmacologia , Líquido Sinovial/fisiologia , Fator de Necrose Tumoral alfa/genética , Veias UmbilicaisRESUMO
BACKGROUND/INTRODUCTION: Sarcoidosis is a multi-systemic disorder of unknown etiology, characterized by the presence of non-caseating granulomas in target organs. In 90% of cases, there is thoracic involvement. Fifty to seventy percent of pulmonary sarcoidosis patients will experience acute, self-limiting disease. For the subgroup of patients who develop persistent disease, no targeted therapy is currently available. AIM: To investigate the potential of the single nucleotide polymorphism (SNP), Toll-like receptor 3 Leu412Phe (TLR3 L412F; rs3775291), as a causative factor in the development of and in disease persistence in pulmonary sarcoidosis. To investigate the functionality of TLR3 L412F in vitro in primary human lung fibroblasts from pulmonary sarcoidosis patients. DESIGN: SNP-genotyping and cellular assays, respectively, were used to investigate the role of TLR3 L412F in the development of persistent pulmonary sarcoidosis. METHODS: Cohorts of Irish sarcoidosis patients (n = 228), healthy Irish controls (n = 263) and a secondary cohort of American sarcoidosis patients (n = 123) were genotyped for TLR3 L412F. Additionally, the effect of TLR3 L412F in primary lung fibroblasts from pulmonary sarcoidosis patients was quantitated following TLR3 activation in the context of cytokine and type I interferon production, TLR3 expression and apoptotic- and fibroproliferative-responses. RESULTS: We report a significant association between TLR3 L412F and persistent clinical disease in two cohorts of Irish and American Caucasians with pulmonary sarcoidosis. Furthermore, activation of TLR3 in primary lung fibroblasts from 412 F-homozygous pulmonary sarcoidosis patients resulted in reduced IFN-ß and TLR3 expression, reduced apoptosis- and dysregulated fibroproliferative-responses compared with TLR3 wild-type patients. DISCUSSION/CONCLUSION: This study identifies defective TLR3 function as a previously unidentified factor in persistent clinical disease in pulmonary sarcoidosis and reveals TLR3 L412F as a candidate biomarker.
Assuntos
Polimorfismo de Nucleotídeo Único , Sarcoidose Pulmonar/genética , Receptor 3 Toll-Like/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Humanos , Irlanda , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Fenótipo , Adulto JovemRESUMO
Various arachidonic acid (AA) metabolites are known to regulate immune cell function(s) and dictate the progression of both acute and chronic inflammatory reactions. Using a model of Schistosoma mansoni egg-induced hypersensitivity granulomas, we have delineated the in vivo effects of inhibitors of cyclooxygenase (CO) and lipoxygenase (LO) pathways on granuloma development and granuloma macrophage I-region-associated (Ia) antigen expression. In addition, by high performance liquid chromatography (HPLC) we have profiled the metabolism of AA by macrophages that are isolated from granulomatous foci, and have biochemically characterized the in vitro specificity and activity of selected CO and LO inhibitors. The development of hypersensitivity-type pulmonary granulomas in mice was dramatically suppressed by inhibitors with anti-LO activity (nordihydroguairetic acid (NDGA), nafazatrom, and BW755c) in a dose-dependent manner, while indomethacin, which is primarily CO-selective, had no significant effect. Furthermore, NDGA and nafazatrom profoundly arrested the normal progression of preformed granulomatous lesions. The inhibitors of the LO pathway also suppressed the in vivo kinetics of Ia antigen expression by granuloma macrophages. In contrast, indomethacin augmented Ia-antigen expression. The major AA metabolites that were synthesized by the granuloma macrophages were shown to be leukotriene C4 and mono-hydroxyeicosatetraenoic acids. HPLC analysis of AA metabolites from granuloma macrophages that were treated with the various inhibitors confirmed that indomethacin was most CO-selective and NDGA most LO-selective. Nafazatrom and BW755c inhibited AA metabolism by both pathways. Notably, high concentrations of the compounds (5 X 10(-5) M) tended to suppress all products. Our results suggest that LO products may be important in the generation and maintenance of immune granulomatous inflammatory responses.
Assuntos
Anti-Inflamatórios/uso terapêutico , Granuloma/enzimologia , Lipoxigenase/metabolismo , Pneumopatias/enzimologia , Esquistossomose/enzimologia , Animais , Ácido Araquidônico , Ácidos Araquidônicos/antagonistas & inibidores , Feminino , Granuloma/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Indometacina/uso terapêutico , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Óvulo/fisiologia , Schistosoma mansoni/patogenicidadeRESUMO
Ischemia-reperfusion and hyperoxia-induced pulmonary injury are associated with the presence of activated neutrophils (PMN) and cellular injury. Although the signals orchestrating the directed migration of these PMN during the pathogenesis of these disease states remain to be fully elucidated, it appears they may be dependent upon the production of certain neutrophil activating/chemotactic factors such as C5a, leukotriene B4, platelet-activating factor, and IL-8. The production of the latter chemotaxin by mononuclear phagocytes is especially intriguing as these cells can mediate inflammatory cell migration by either directly generating IL-8, or by inducing its production from surrounding nonimmune cells. In light of these observations, we propose that ischemia-reperfusion and oxidant stress, in vivo, may be simulated by anoxia-hyperoxia induced stress in vitro, and that this stress may act as a stimulus for the production of IL-8. We now show that isolated human blood monocytes respond to such an oxygen stress with augmented production of IL-8. In initial studies, monocytes demonstrated an increase in the production of IL-8 under anoxic preconditioning. Subsequently, monocytes were cultured under one of the following conditions for 24 h: (a) room air/5% CO2; (b) 95% N2/5% CO2 for 6 h, followed by room air/5% CO2 for 18 h; (c) 95% N2/5% CO2 for 6 h, followed by 95% O2/5% CO2 for 18 h; (d) room air/5% CO2 for 6 h, followed by 95% O2/5% CO2 for 18 h; or (e) 95% O2/5% CO2. Supernatants were isolated and analyzed for IL-8 antigen by specific IL-8 ELISA, demonstrating the production of monocyte-derived IL-8: 5.9 +/- 0.9, 11.4 +/- 1.7, 21.1 +/- 2.3, 14.6 +/- 2.4, and 26.3 +/- 4.7, ng/ml by designated conditions a, b, c, d, and e listed above, respectively. This variance in IL-8 production reflects altered rates of transcription as shown by Northern blot analysis and nuclear run-off assay. Furthermore, when monocytes were concomitantly treated with LPS (100 ng/ml) under in vitro hyperoxic conditions, both IL-8 steady-state mRNA and antigenic activity were two- to threefold greater than under room air conditions. The association of anoxic preconditioning and oxygen stress with augmented production of monocyte-derived IL-8 support the potential role for ischemia-reperfusion and hyperoxia-induced IL-8 production in vivo, providing a possible mechanism for PMN migration/activation in disease states characterized by altered tissue oxygenation.
Assuntos
Hipóxia/metabolismo , Interleucina-8/biossíntese , Monócitos/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Humanos , Interleucina-8/genética , Lipopolissacarídeos , Dados de Sequência Molecular , Neutrófilos/fisiologia , Oxigênio/toxicidade , RNA Mensageiro/análise , CoelhosRESUMO
Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this study, we compared lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (F-IPF) and from patients undergoing resectional surgery for lung cancer (F-nl) with respect to their capacity for PGE2 synthesis and their expression and regulation of cyclooxygenase (COX) proteins. Basal COX activity, assessed by quantitating immunoreactive PGE2 synthesized from arachidonic acid, was twofold less (P < 0.05) in F-IPF than F-nl. In F-nl, incubation with the agonists PMA, LPS, or IL-1 increased COX activity and protein expression of the inducible form of COX, COX-2, and these responses were inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate increases in COX-2 protein expression or COX activity in response to these agonists. Under conditions of maximal induction, COX activity in F-IPF was sixfold less than that in F-nl (P < 0.05). Our data indicate that F-IPF have a striking defect in their capacity to synthesize the antiinflammatory and antifibrogenic molecule PGE2, apparently because of a diminished induction of COX-2 protein. This reduction in the endogenous capacity of F-IPF to down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response in IPF. Moreover, we believe that this represents the first description of a defect in COX-2 expression in association with a human disease.