RESUMO
Angiogenesis is a normal physiological process that also contributes to diabetic retinopathy-related complications and facilitates tumor metastasis by promoting the hematogenic dissemination of malignant cells from solid tumors. Here, we investigated the in vitro, ex vivo, and in vivo anti-angiogenic activity of phloridzin docosahexaenoate (PZ-DHA), a novel ω-3 fatty acid ester of a flavonoid precursor. Human umbilical vein endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC) treated with a sub-cytotoxic concentration of PZ-DHA to assess in vitro anti-angiogenic activity showed impaired tubule formation on a Matrigel matrix. Ex vivo angiogenesis was measured using rat thoracic aortas, which exhibited reduced vessel sprouting and tubule formation in the presence of PZ-DHA. Female BALB/c mice bearing VEGF165- and basic fibroblast growth factor-containing Matrigel plugs showed a significant reduction in blood vessel development following PZ-DHA treatment. PZ-DHA inhibited HUVEC and HMVEC proliferation, as well as the migration of HUVECs in gap closure and trans-well cell migration assays. PZ-DHA inhibited upstream and downstream components of the Akt pathway and vascular endothelial growth factor (VEGF165)-induced overexpression of small molecular Rho GTPases in HUVECs, suggesting a decrease in actin cytoskeletal-mediated stress fiber formation and migration. Taken together, these findings reveal the potential of combined food biomolecules in PZ-DHA to inhibit angiogenesis.
Assuntos
Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana , Camundongos Endogâmicos BALB C , Humanos , Animais , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos , Diferenciação Celular/efeitos dos fármacos , Ratos , Ácidos Docosa-Hexaenoicos/farmacologia , Inibidores da Angiogênese/farmacologia , Florizina/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Flavonoides/farmacologia , AngiogêneseRESUMO
BACKGROUND: Inflammation is the body's response to injury or infection and is important for healing and eliminating pathogens; however, prolonged inflammation is damaging and may lead to the development of chronic inflammatory disorders. Recently, there has been interest in exploiting antimicrobial peptides (AMPs) that exhibit immunoregulatory activities to treat inflammatory diseases. METHODS: In this study, we investigated the immunomodulatory effects of lactoferrin-derived lactoferricin AMPs from three different species (bovine, mouse, and human) with subtle differences in their amino acid sequences that alter their antimicrobial action; to our knowledge, no other studies have compared their immunomodulatory effects. Macrophages, key players in the induction and propagation of inflammation, were used to investigate the effects of species-specific lactoferricin peptides on inflammatory processes. RESULTS: Bovine lactoferricin was the only one of the three peptides studied that downregulated lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-6, in both human and mouse macrophages. Lactoferricin regulated inflammation through targeting LPS-activated nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Although the immunoregulatory role of lactoferricin during an inflammatory response in vivo is yet to be elucidated, further investigation with the use of animal models is warranted by the current findings. CONCLUSIONS: The ability of lactoferricin, especially that of bovine origin, to downregulate macrophage-mediated inflammatory responses suggests potential for the development of this peptide as a novel immunotherapeutic agent in the treatment of chronic inflammatory conditions.
Assuntos
Lactoferrina , Macrófagos , Animais , Bovinos , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Lactoferrina/efeitos adversos , Lactoferrina/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Peptídeos/farmacologiaRESUMO
BACKGROUND/AIM: Hepcidin is a cationic acute phase reactant synthesized by the liver. It has bactericidal properties and is a major regulator of iron homeostasis. Cationic antimicrobial peptides represent an innate antimicrobial defense system. We hypothesized that, like other cationic antimicrobial peptides, hepcidin is cytotoxic to cancer cells. MATERIALS AND METHODS: The cytotoxicity of human hepcidin against myeloma cells was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and DNA fragmentation assays. Plasma membrane damage was quantified by propidium iodide (PI) staining. Cell membrane changes were visualized by scanning electron microscopy. RESULTS: Hepcidin impaired myeloma cell survival and induced DNA fragmentation. PI staining and scanning electron microscopy revealed hepcidin-induced disruption of the plasma membrane. CONCLUSION: Human hepcidin is an anti-cancer peptide that induces myeloma cell lysis, and therefore may play a role in innate anticancer immunity. To our knowledge, this is the first biological function ascribed to human hepcidin that is not related to its antimicrobial and iron-regulatory properties.
Assuntos
Antineoplásicos/farmacologia , Hepcidinas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Metabolismo Energético/efeitos dos fármacos , Humanos , Camundongos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/ultraestruturaRESUMO
Culprits of cancer development, metastasis, and drug resistance, cancer stem cells (CSCs) are characterized by specific markers, active developmental signaling pathways, metabolic plasticity, increased motility, invasiveness, and epithelial-mesenchymal transition. In breast cancer, these cells are often more prominent in aggressive disease, are amplified in drug-resistant tumors, and contribute to recurrence. For breast cancer, two distinct CSC populations exist and are typically defined by CD44+/CD24- cell surface marker expression or increased aldehyde dehydrogenase (ALDH) activity. These CSC populations share many of the same properties but also exhibit signaling pathways that are more active in CD44+/CD24- or ALDH+ populations. Understanding these CSC populations and their shared or specific signaling pathways may lead to the development of novel therapeutic strategies that will improve breast cancer patient outcomes. Herein, we review the current evidence and assess published patient tumor datasets of sorted breast CSC populations for evidence of heightened prostaglandin E2 (PGE2) signaling and activity in these breast CSC populations. PGE2 is a biologically active lipid mediator and in cancer PGE2 promotes tumor progression and poor patient prognosis. Overall, the data suggests that PGE2 signaling is important in propagating breast CSCs by enhancing inherent tumor-initiating capacities. Development of anti-PGE2 signaling therapeutics may be beneficial in inhibiting tumor growth and limiting breast CSC populations.
RESUMO
Phytochemicals are the basis for many anticancer drugs currently in clinical use, as well as a potential source of future cancer treatments. Some phytochemicals have been found to modify the expression of checkpoint inhibitors of the immune response, as well as kill cancer cells. Cancer cells, in turn, may evade detection by the immune system by expressing molecules such as programmed death ligand 1 (PD-L1) that interacts with programmed cell death 1 (PD-1) on T cells to inhibit T cell activation and effector function. Phytochemicals have direct effects on cancer cells and/or T cells that may impact PD-L1/PD1 interactions, although this may vary depending on the phytochemical in question. Flow cytometric analysis of cancer cells stained with anti-PD-L1 antibodies following treatment with a given phytochemical enables the detection of any alteration in PD-L1 expression. The effect of the phytochemical on T cell function can be assessed using proliferation assays (e.g., tritiated thymidine incorporation, flow cytometric analysis of Oregon Green 488-stained cells) and enzyme-linked immunosorbent assay of interleukin-2 content in culture supernatants. Additional study is needed to better understand the impact of phytochemicals on cancer immunotherapy.
Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Neoplasias/imunologia , Compostos Fitoquímicos/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Adesão Celular , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoterapia , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Neoplasias/tratamento farmacológico , Compostos Fitoquímicos/uso terapêutico , Linfócitos T/metabolismoRESUMO
Breast cancer is a leading cause of cancer-related death in women; however, chemotherapy of breast cancer is often hindered by dose-limiting toxicities, demonstrating the need for less toxic approaches to treatment. Since the rapid growth and metabolism of breast cancer cells results in an increased requirement for iron, withdrawal of bioavailable iron using highly selective iron chelators has been suggested to represent a new approach to breast cancer treatment. Here we show that the recently developed iron-binding polymer DIBI inhibited the growth of five different breast cancer cell lines (SK-BR3, MDA-MB-468, MDA-MB-231, MCF-7, and T47D). In cultures of MDA-MB-468 breast cancer cells, which were most sensitive to DIBI-mediated growth inhibition, iron withdrawal was associated with increased expression of transferrin receptor 1 and ferritin H mRNA but decreased expression of ferroportin mRNA. MDA-MB-468 cells that were exposed to DIBI experienced double-strand DNA breaks during the S phase of the cell cycle. DNA damage was not mediated by reactive oxygen species (ROS) since DIBI-treated MDA-MB-468 cells exhibited a reduction in intracellular ROS. DIBI-treated MDA-MB-468 cells also showed increased sensitivity to growth inhibition by the chemotherapeutic drugs cisplatin, doxorubicin, and 4-hydroperoxy cyclophosphamide (active metabolite of cyclophosphamide). Combination treatment of MDA-MB-468 cells with DIBI and cisplatin caused greater DNA damage than either treatment alone, which was also associated with an increase in apoptotic cell death. Taken together, these findings suggest that DIBI-mediated iron withdrawal may enhance the effect of chemotherapeutic agents used in breast cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Dano ao DNA , Quelantes de Ferro/farmacologia , Polímeros/farmacologia , Piridinas/farmacologia , Piridonas/farmacologia , Fase S/efeitos dos fármacos , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Quelantes de Ferro/química , Polímeros/química , Piridinas/química , Piridonas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Antibody-mediated crosslinking of Thy-1 [also known as cluster of differentiation (CD)90], results in a T cell receptor (TcR)like signal; however, the impact of Thy1 stimulation in comparison to TcR stimulation on T cell activation and effector function has yet to be fully elucidated. In the present study, the outcome of Thy1 and TcRinduced stimulation of T cells was investigated in mice, using fragment crystalizable (Fc) receptorbound antibodies and costimulatory signals provided by syngeneic lipopolysaccharidematured bone marrowderived dendritic cells. Compared with TcR signaling, Thy1 signaling initiated a less robust proliferative response in T cells, as determined by tritiatedthymidine incorporation. In addition, enzymelinked immunosorbent assays revealed that interleukin2 production was reduced, and the expression of CD25 and cyclin D3 was weaker in Thy1stimulated cells, as determined by western blotting; however, the expression of cyclindependent kinase 6 was similar to that in TcRinduced T cells. Furthermore, western blotting demonstrated that the phosphorylation of ζ-chainassociated protein kinase 70 and extracellular signalregulated kinase 1/2 was delayed following Thy1 stimulation. DNA fragmentation assays revealed that cytotoxic effector function was also slower to develop in Thy1stimulated T cells, required more time to be effective and was largely Ca2+independent; these findings suggested that Fas ligand rather than granuleassociated perforin was involved in T cell effector function. In conclusion, the present results suggested that Thy1 signaling may contribute to the regulation of T cell homeostasis and the development of nonspecific T cellmediated cytotoxicity. However, further studies are required to elucidate the exact physiological roles of TcRlike signals that result from Thy1 crosslinking and to investigate the molecular mechanisms that are involved.
Assuntos
Cálcio/metabolismo , Citotoxicidade Imunológica , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos Thy-1/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Replicação do DNA , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteína Ligante Fas/metabolismo , Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Fosforilação , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Since iron uptake is essential for cell growth, rapidly dividing cancer cells are sensitive to iron depletion. To explore the effect of iron withdrawal on cancer cell growth, mouse and human mammary carcinoma cells (4T1 and MDA-MB-468, respectively) and mouse and human fibrosarcoma cells (L929 and HT1080, respectively) were cultured in the absence or presence of DIBI, a novel iron-chelating polymer containing hydroxypyridinone iron-ligand functionality. Cell growth was measured by a colorimetric assay for cell metabolic activity. DIBI-treated 4T1, MDA-MB-468, L929 and HT1080 cells, as well as their normal counterparts, showed a dose- and time-dependent reduction in growth that was selective for human cancer cells and mouse fibrosarcoma cells. The inhibitory effect of DIBI on fibrosarcoma and mammary carcinoma cell growth was reversed by addition of exogenous iron in the form of iron (III) citrate, confirming the iron selectivity of DIBI and that its inhibitory activity was iron-related. Fibrosarcoma and mammary carcinoma cell growth inhibition by DIBI was associated with S-phase cell cycle arrest and low to moderate levels of cell death by apoptosis. Consistent with apoptosis induction following DIBI-mediated iron withdrawal, fibrosarcoma and mammary carcinoma cells exhibited mitochondrial membrane permeabilization. A comparison of DIBI to other iron chelators showed that DIBI was superior to deferiprone and similar to or better than deferoxamine for inhibition of fibrosarcoma and mammary carcinoma cell growth. These findings suggest that iron withdrawal from the tumor microenvironment with a selective and potent iron chelator such as DIBI may prevent or inhibit tumor progression.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Fibrossarcoma/tratamento farmacológico , Quelantes de Ferro/farmacologia , Deficiências de Ferro , Neoplasias Mamárias Animais/tratamento farmacológico , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Feminino , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Humanos , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Células Tumorais CultivadasRESUMO
[6]-Gingerol, [8]-gingerol, and [10]-gingerol are pungent components of fresh ginger, extracts of which inhibit various components of the inflammatory response. Because little is known regarding the effect of gingerols with different unbranched alkyl side chain lengths on the activation and effector function of T lymphocytes, we compared the effects of [6]-gingerol, [8]-gingerol, and [10]-gingerol on murine T lymphocyte proliferation, expression of CD25 and CD69 activation markers, cytokine synthesis, and interleukin (IL)-2 receptor signaling. All three gingerols inhibited DNA synthesis by T lymphocytes, as well as interferon-γ synthesis. In contrast, only [8]-gingerol and [10]-gingerol inhibited CD25 and CD69 expression, and IL-2 synthesis. None of the gingerols affected IL-4 synthesis. Exogenous IL-2 enhanced T lymphocyte proliferation in the presence of [6]-gingerol but did not significantly increase T lymphocyte proliferation in the presence of [8]-gingerol or [10]-gingerol. In line with this finding, [8]-gingerol and [10]-gingerol impaired IL-2-induced proliferation of CTLL-2 cells, but constitutive CD25 expression was unaffected, indicating inhibition of IL-2 receptor signaling. In general, [10]-gingerol and [8]-gingerol were more potent inhibitors of T lymphocytes than [6]-gingerol. Suppression of T lymphocyte responses by gingerols suggests that these phytochemicals may be beneficial in chronic inflammatory conditions associated with excessive or inappropriate T lymphocyte activation.
Assuntos
Catecóis/farmacologia , Álcoois Graxos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Zingiber officinale/química , Interferon gama/imunologia , Interleucina-2/imunologia , Interleucina-2/metabolismo , Interleucina-4/imunologia , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
Gad-1 and Gad-2 are antimicrobial peptide (AMP) sequences encoded by paralogous genes. They are rich in histidine, which suggests that their activity might be pH-dependent. We examined their structure-function relationships with a view to learning how to improve AMP therapeutic ratios. Activity assays with Gram-negative bacteria and cancer cell lines demonstrate that Gad-2 is substantially more active at slightly acidic pH than it is at neutral pH. By contrast, the activity of Gad-1 at lower pH is similar to its activity at pH7. Circular dichroism spectra indicate that the greater functional plasticity of Gad-2 correlates with a greater structural plasticity; Gad-2's percent helicity varies dramatically with altered pH and lipid environment. Interestingly, Gad-2's highest levels of helicity do not correspond to the conditions where it is most active. High resolution solution NMR structures were determined in SDS micelles at pH5, conditions that induce an intermediate level of helicity in the peptides. Gad-1 is more helical than Gad-2, with both peptides exhibiting the greatest helical tendencies in their central region and lowest helicity in their N-termini. The high resolution structures suggest that maximum activity relies on the appropriate balance between an N-terminal region with mixed hydrophobic/hydrophilic structure features and an amphipathic central and C-terminal region. Taken together with previous studies, our results suggest that to improve the therapeutic ratio of AMPs, consideration should be given to including sequential histidine-pairs, keeping the overall charge of the peptide modest, and retaining a degree of structural plasticity and imperfect amphipathicity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Peixes/química , Gadus morhua/metabolismo , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Peixes/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/farmacologia , Relação Estrutura-AtividadeRESUMO
Piperine, a piperidine alkaloid present in black pepper, inhibits the growth of cancer cells, although the mechanism of action is not well understood. In this study, we show that piperine (75-150 µM) inhibited the growth of several colon cancer cell lines but had little effect on the growth of normal fibroblasts and epithelial cells. Piperine inhibited HT-29 colon carcinoma cell proliferation by causing G1 phase cell cycle arrest that was associated with decreased expression of cyclins D1 and D3 and their activating partner cyclin-dependent kinases 4 and 6, as well as reduced phosphorylation of the retinoblastoma protein and up-regulation of p21/WAF1 and p27/KIP1 expression. In addition, piperine caused hydroxyl radical production and apoptosis that was partially dependent on the production of reactive oxygen species. Piperine-treated HT-29 cells showed loss of mitochondrial membrane integrity and cleavage of poly (ADP-ribose) polymerase-1, as well as caspase activation and reduced apoptosis in the presence of the pan-caspase inhibitor zVAD-FMK. Increased expression of the endoplasmic reticulum stress-associated proteins inositol-requiring 1α protein, C/EBP homologous protein, and binding immunoglobulin protein, and activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, as well as decreased phosphorylation of Akt and reduced survivin expression were also observed in piperine-treated HT-29 cells. Furthermore, piperine inhibited colony formation by HT-29 cells, as well as the growth of HT-29 spheroids. Cell cycle arrest and endoplasmic reticulum stress-associated apoptosis following piperine treatment of HT-29 cells provides the first evidence that piperine may be useful in the treatment of colon cancer.
Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Benzodioxóis/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Fase G1/efeitos dos fármacos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Neoplasias do Colo/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HT29 , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Piper nigrum/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Survivina , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Epidemiological studies show that fruit- and vegetable-rich diets are associated with a reduced risk of developing certain forms of cancer, including breast cancer. In this study we demonstrate that a subcytotoxic concentration of apigenin, which is a flavone found at high concentrations in parsley, onions, grapefruit, oranges, and chamomile tea, inhibited DNA synthesis in a panel of human breast cancer cell lines (MDA-MB-231, MBA-MB-468, MCF-7, SK-BR-3). Decreased proliferation of MDA-MB-468 cells in the presence of apigenin was associated with G2/M phase cell cycle arrest and the production of reactive oxygen species. Apigenin-treated MDA-MB-468 cells also showed reduced phosphorylation of Akt (protein kinase B), which is an essential effector serine/threonine kinase in the phosphatidylinositide 3-kinase pathway that promotes tumor growth and progression. However, exposure to the antioxidant reduced glutathione failed to reverse apigenin-mediated inhibition of Akt phosphorylation and cell proliferation, indicating that these effects were not due to oxidative stress. Taken together, these findings suggest that low-dose apigenin has the potential to slow or prevent breast cancer progression.
Assuntos
Apigenina/farmacologia , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ciclo Celular/efeitos dos fármacos , Humanos , Células MCF-7 , FosforilaçãoRESUMO
Preterm infants are especially susceptible to late-onset sepsis that is often due to Gram-positive bacterial infections resulting in substantial morbidity and mortality. Herein, we will describe neonatal innate immunity to Staphylococcus spp. comparing differences between preterm and full-term newborns with adults. Newborn innate immunity is distinct demonstrating diminished skin integrity, impaired Th1-polarizing responses, low complement levels, and diminished expression of plasma antimicrobial proteins and peptides, especially in preterm newborns. Characterization of distinct aspects of the neonatal immune response is defining novel approaches to enhance host defense to prevent and/or treat staphylococcal infection in this vulnerable population.
Assuntos
Imunidade Inata , Infecções Estafilocócicas/imunologia , Staphylococcus/imunologia , Humanos , Recém-Nascido , Recém-Nascido Prematuro/imunologia , Recém-Nascido Prematuro/metabolismo , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/terapia , Receptores Toll-Like/metabolismoRESUMO
Staphylococcus epidermidis (SE) causes late onset sepsis and significant morbidity in catheterized preterm newborns. Animal models of SE infection are useful in characterizing disease mechanisms and are an important approach to developing improved diagnostics and therapeutics. Current murine models of neonatal bacterial infection employ intraperitoneal or subcutaneous routes at several days of age, and may, therefore, not accurately reflect distinct features of innate immune responses to bacteremia. In this study we developed, validated, and characterized a murine model of intravenous (IV) infection in neonatal mice <24 hours (h) old to describe the early innate immune response to SE. C57BL/6 mice <24 h old were injected IV with 10(6), 10(7), 10(8) colony-forming units (CFU) of SE 1457, a clinical isolate from a central catheter infection. A prospective injection scoring system was developed and validated, with only high quality injections analyzed. Newborn mice were euthanized between 2 and 48 h post-injection and spleen, liver, and blood collected to assess bacterial viability, gene expression, and cytokine production. High quality IV injections demonstrated inoculum-dependent infection of spleen, liver and blood. Within 2 h of injection, SE induced selective transcription of TLR2 and MyD88 in the liver, and increased systemic production of plasma IL-6 and TNF-α. Despite clearance of bacteremia and solid organ infection within 48 h, inoculum-dependent impairment in weight gain was noted. We conclude that a model of IV SE infection in neonatal mice <24 h old is feasible, demonstrating inoculum-dependent infection of solid organs and a pattern of bacteremia, rapid and selective innate immune activation, and impairment of weight gain typical of infected human neonates. This novel model can now be used to characterize immune ontogeny, evaluate infection biomarkers, and assess preventative and therapeutic modalities.
Assuntos
Imunidade Inata/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/imunologia , Animais , Animais Recém-Nascidos , Bacteriemia/sangue , Bacteriemia/genética , Bacteriemia/imunologia , Modelos Animais de Doenças , Estudos de Viabilidade , Regulação da Expressão Gênica , Humanos , Imunidade Inata/genética , Injeções Intravenosas , Interleucina-6/biossíntese , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Transcrição Gênica , Transcriptoma/genética , Fator de Necrose Tumoral alfa/biossíntese , Aumento de Peso/genética , Aumento de Peso/imunologiaRESUMO
Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4(+) T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca(2+) release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4(+)CD25(+) regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.
Assuntos
Curcumina/farmacologia , Imunossupressores/farmacologia , Subunidade alfa de Receptor de Interleucina-2/antagonistas & inibidores , Interleucina-2/antagonistas & inibidores , Receptores de Interleucina-2/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Interleucina-2/biossíntese , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Janus Quinase 3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Adenosine is an endogenous purine metabolite whose concentration in human blood plasma rises from nanomolar to micromolar concentrations during the inflammatory process. Leukocytes express seven-transmembrane adenosine receptors whose engagement modulates Toll-like receptor-mediated cytokine responses, in part via modulation of intracellular cyclic adenosine monophosphate. Adenosine analogs are used clinically to treat arrhythmias and apnea of prematurity. Herein, we consider the potential of adenosine analogs as innate immune response modifiers to prevent and/or treat infection.
Assuntos
Adenosina/farmacologia , Receptores Toll-Like/metabolismo , 5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , AMP Cíclico/metabolismo , Humanos , Imunidade Inata/imunologia , Recém-Nascido , Receptores Toll-Like/imunologiaRESUMO
BACKGROUND: Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24-48 h) were markedly impaired in TLR2-deficient mice. CONCLUSIONS/SIGNIFICANCE: TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.