RESUMO
Severe sepsis and septic shock are important causes of death in intensive care units. Although Gram-negative infections were predominant in the 1960s, Gram-positive infections have increased in the past two decades and now account for about half of the cases of severe sepsis. In this study, we examined the effect of a Limulus anti-LPS factor (LALF)-derived peptide on lung and liver Th1/Th2 cytokine mRNA levels during a Gram-positive sepsis. We also examined the morphopathological changes observed in these organs during the disease. Mice challenged with a high dose of Staphylococcus haemolyticus showed severe damage in lung. In contrast, the liver of challenged mice showed an accumulation of bacterial particles in the sinusoids, associated with a severe inflammatory response due to high levels of tissue mRNA proinflammatory cytokines. Treatment with the peptide LALF(32-51) ameliorated the sepsis-induced effects in the lung and liver and increased the survival of mice in a dose- and time-dependent manner. Pretreatment with the peptide LALF(32-51) differentially regulates TNF-alpha, IFN-gamma, IL-12p40, IL-2 and IL-10 mRNA levels in lung and liver of peptide-treated mice, and limits the systemic inflammatory response. These findings support for the first time the effectiveness of an LALF-derived peptide in the treatment of a Gram-positive sepsis. Modulation of the Th1/Th2 pattern in tissues relevant for sepsis correlates with an improved outcome of the disease as denoted by increased survival.
Assuntos
Anti-Infecciosos/farmacologia , Hormônios de Invertebrado/química , Fragmentos de Peptídeos/farmacologia , Sepse/prevenção & controle , Infecções Estafilocócicas/prevenção & controle , Células Th1/imunologia , Células Th2/imunologia , Animais , Anti-Infecciosos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Citocinas/sangue , Citocinas/genética , Regulação da Expressão Gênica , Fígado/patologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , RNA Mensageiro/sangue , Sepse/imunologia , Sepse/mortalidade , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/mortalidade , Staphylococcus haemolyticus , Taxa de Sobrevida , Fatores de TempoRESUMO
Sepsis in experimental animals and humans has been associated with perturbed immune response. A major event contributing to the decrease in immune functions in septic disorders seems to be the inadequate balance of cytokines mediating the interactions between the innate and adaptive immune systems. We previously observed that a cyclic peptide derived from the Limulus anti-LPS factor (LALF), which partially protect mice from endotoxic shock lethality, has the ability to modulate cytokine secretion in vitro. We herein examined the effects of the LALF(31-52) peptide in an experimental model of Gram-negative peritoneal sepsis and analyzed the cytokine gene expression in the spleen and liver of peptide-treated mice. The prophylactic administration of LALF(31-52) abrogated the systemic TNF-alpha response, reduced organ damage and increased the survival of infected mice. Histological examination of spleen and liver in peptide-treated mice showed prevention of tissue damage induced by the high dose of Pseudomonas aeruginosa. This treatment modulates the cytokine gene expression in these tissues, stimulating IL-2, IL-12 and IL-13 mRNA synthesis, while IL-4 and IL-10 mRNA expression was not modified. This cytokine profile induced by the LALF-derived peptide seems to be favorable for host resistance against Gram-negative bacteria acute infection. In addition, peptide treatment was effective after the initiation of the systemic inflammatory response, promoting a significant increase in mice survival. These results further demonstrate the immunomodulatory potential of LALF(31-52) and are relevant for the design of prophylactic and therapeutic strategies for acute bacteria infection and sepsis, especially for preventing or ameliorating host immunity defects in these disorders.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Citocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios de Invertebrado/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Fragmentos de Peptídeos/uso terapêutico , Doença Aguda , Animais , Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Interleucina-12/biossíntese , Interleucina-13/biossíntese , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors. This process is thought to be of great importance in systemic inflammatory reactions such as septic shock. A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP. We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99. Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding. In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction. Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect. The mutant Lys95 was the most active in blocking LPS binding to LBP. These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis.
Assuntos
Proteínas de Fase Aguda , Alanina/metabolismo , Substituição de Aminoácidos/genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Biblioteca de Peptídeos , Alanina/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Cromatografia Líquida de Alta Pressão , Lipopolissacarídeos/antagonistas & inibidores , Mutagênese , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologiaRESUMO
We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2'-O-deoxycytidine 3A. We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-[n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl]-2'-deoxycytidine compounds, and confirmed their presence by 1H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.
Assuntos
DNA/análise , DNA/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/síntese química , Desoxicitidina/metabolismo , Desoxicitidina/química , Escherichia coli , Ligação de Hidrogênio , Immunoblotting , Espectroscopia de Ressonância Magnética , Plasmídeos/metabolismo , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
We have previously reported that IFNalpha-chronic treatment for 41 days induced a partial phenotype reversion on HeLa cells along with a down-regulation of HPV18 mRNA levels. However, tumorigenicity of these cells in nude mice was unchanged. Interestingly, after 1 year of IFNalpha-chronic exposition, HeLa cells failed to induce s.c. tumors when injected into nude mice. In such experimental conditions both HPV18 DNA integration pattern and viral DNA copy number present in HeLa cells remained intact in the nontumorigenic phenotype cells. As result of the treatment with IFNalpha, HeLa cells rendered more resistant to lysis mediated by activated natural killer cells in vitro. Furthermore, IFNalpha-chronic treatment was able to induce VEGF and decrease bFGF mRNA expression, suggesting a potential effect on the angiogenic behavior of these tumoral cells. Thus, long-term treatment of HeLa cells with IFNalpha can accomplish a reversion of the malignant phenotype by a sequential multistep mechanism, in which the antiangiogenic effect of IFNalpha could be one of the contributing events.
Assuntos
Interferon-alfa/farmacologia , Neovascularização Patológica , Animais , Northern Blotting , Southern Blotting , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Fatores de Crescimento Endotelial/biossíntese , Feminino , Fator 2 de Crescimento de Fibroblastos/biossíntese , Dosagem de Genes , Células HeLa , Humanos , Interferon alfa-2 , Células Matadoras Naturais/metabolismo , Linfocinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Papillomaviridae/química , Papillomaviridae/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Previous studies have shown that cyclic peptides corresponding to residues 35 to 52 of the Limulus antilipopolysaccharide (anti-LPS) factor (LALF) bind and neutralize LPS-mediated in vitro and in vivo activities. Therapeutic approaches based on agents which bind and neutralize LPS activities are particularly attractive because these substances directly block the primary stimulus for the entire proinflammatory cytokine cascade. Here we describe new activities of the LALF(31-52) peptide, other than its LPS binding ability. Surprisingly, supernatants from human mononuclear cells stimulated with the LALF peptide are able to induce in vitro antiviral effects on the Hep-2 cell line mediated by gamma interferon (IFN-gamma) and IFN-alpha. Analysis of the effect of LALF(31-52) on tumor necrosis factor (TNF) and nitric oxide (NO) production by LPS-stimulated peritoneal macrophages revealed that a pretreatment with the peptide decreased LPS-induced TNF production but did not affect NO generation. This indicates that the LALF peptide modifies the LPS-induced response. In a model in mice with peritoneal fulminating sepsis, LALF(31-52) protected the mice when administered prophylactically, and this effect is related to reduced systemic TNF-alpha levels. This study demonstrates, for the first time, the anti-inflammatory properties of the LALF-derived peptide. These properties widen the spectrum of the therapeutic potential for this LALF-derived peptide and the molecules derived from it. These agents may be useful in the prophylaxis and therapy of viral and bacterial infectious diseases, as well as for septic shock.
Assuntos
Anti-Infecciosos/imunologia , Caranguejos Ferradura/imunologia , Hormônios de Invertebrado/imunologia , Lipopolissacarídeos/imunologia , Peptídeos/imunologia , Animais , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos , Proteínas de Artrópodes , Humanos , Hormônios de Invertebrado/química , CamundongosRESUMO
p27(Kip1) is one of the key regulatory proteins in cell cycle through inhibition of pRB phosphorylation by suppression of the activity of several cyclin/Cdk complexes. The expression of p27(Kip1) has been shown to be controlled by a posttranslational mechanism, although vitamin D(3) and neuronal differentiation can also induce its mRNA. Recently, the p27(Kip1) promoter was isolated and sequenced from a human leukocyte genomic library. In this report, we demonstrate that IFNalpha 2b, activates the human p27(Kip1) promoter-driven luciferase reporter gene in transient expression assays in H82 cells. This induction might involve two IRF 1-like binding sites present in the p27(Kip1) promoter. To our knowledge this is the first report on the direct activation of the human p27(Kip1) promoter by IFNalpha 2b.
Assuntos
Proteínas de Ciclo Celular , Interferon-alfa/farmacologia , Proteínas Associadas aos Microtúbulos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Supressoras de Tumor , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p27 , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter/efeitos dos fármacos , Humanos , Fator Regulador 1 de Interferon , Interferon alfa-2 , Luciferases/genética , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
Cervical cancer represents the second most common cancer in women worldwide. About 90% of cervical cancer contain high-risk human papillomavirus (HPV) DNA, most often HPV type 16. Animal models and mostly laboratory mice are excellent for carrying out diverse immunological studies. We transfected a fibroblast cell line, 3T3-A31, with human papillomavirus type 16 genome to develop an in vivo/in vitro malignant transformant model. Isolated clones inoculated to immunocompetent mice displayed a tumorigenic phenotype. Small clusters of metastatic cells were found in the liver of animal 45 days after receiving the inoculum. Integrated viral DNA and expression of E7 viral oncogene from the high-risk HPV-16 were demonstrated both in transfectans and tumor-derived cells. The observed high-grade neovascularization was correlated with the upregulation of vascular endothelial growth factor (VEGF) mRNA on HPV-16 transformed fibroblast cells. These observations emphasize the association between papillomavirus expression and progression to malignancy.
Assuntos
Transformação Celular Neoplásica , Transformação Celular Viral , Fibroblastos/virologia , Neoplasias Experimentais/virologia , Papillomaviridae/patogenicidade , Animais , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Feminino , Fibroblastos/patologia , Humanos , Linfocinas/biossíntese , Linfocinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/genética , Neovascularização Patológica , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , RNA Mensageiro/isolamento & purificação , Transfecção , Neoplasias do Colo do Útero/virologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have characterized the binding epitopes of two monoclonal antibodies (MAbs) reacting with human Interleukin-2 (IL-2), using a phage display peptide library. The first antibody (CB-IL2.1) recognizes the sequence LSFL, amino acid 72 to amino acid 80, numbered in the IL-2. The second antibody (CB-IL2.2) binds the sequence TTFM (amino acids 101 to 104) located at the opposite site of the four-helix bundle of IL-2. Enzyme-linked immunoadsorbent assay (ELISA) and Western blot using different IL-2 protein construct expressed in bacteria and phage display demonstrate the specificities of this antibody. The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitope are accessible from the outside in the native IL-2 molecule.
Assuntos
Anticorpos Monoclonais , Epitopos/química , Interleucina-2/química , Interleucina-2/imunologia , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Sítios de Ligação/genética , Epitopos/genética , Humanos , Hibridomas/imunologia , Interleucina-2/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Conformação ProteicaRESUMO
IFNs arrest the growth of a small cell lung cancer (SCLC) cell line NCI-H82 in the G1 phase but not the growth of the derived cell line NCI-H82R. Progression through the G1 phase is controlled by positive and negative regulatory genes. Oncoviral genes can override this control. In this study, we compared the effects of human IFN alpha 2b on the mRNA levels of the Cdk inhibitor p27Kip1 in NCI-H82, NCI-H82R and HPV 16 E7-transfected NCI-H82 cell lines. Induction of the 2-5 Oligoadenylate synthetase (2-5 OAS) gene was used as a marker of IFN-dependent signal transduction The expression of p27Kip1 mRNA increased at 48 and 72 hr after IFN alpha 2b addition in sensitive cells. In contrast, p27Kip1 mRNA had only slight variations in both the resistant and E7-transfected cells. Interestingly, the E7-transfected NCI-H82 cells became resistant to the IFN alpha 2b anti-proliferative effect. Our results suggest that p27Kip1 could be a key mediator of the IFN alpha 2b-induced growth arrest and that HPV 16 E7 might affect p27Kip1 inducibility, originating IFN alpha 2b-resistant cells.