RESUMO
Food fortification is an effective strategy to address vitamin A (VitA) deficiency, which is the leading cause of childhood blindness and drastically increases mortality from severe infections. However, VitA food fortification remains challenging due to significant degradation during storage and cooking. We utilized an FDA-approved, thermostable, and pH-responsive basic methacrylate copolymer (BMC) to encapsulate and stabilize VitA in microparticles (MPs). Encapsulation of VitA in VitA-BMC MPs greatly improved stability during simulated cooking conditions and long-term storage. VitA absorption was nine times greater from cooked MPs than from cooked free VitA in rats. In a randomized controlled cross-over study in healthy premenopausal women, VitA was readily released from MPs after consumption and had a similar absorption profile to free VitA. This VitA encapsulation technology will enable global food fortification strategies toward eliminating VitA deficiency.
Assuntos
Deficiência de Vitamina A , Vitamina A , Feminino , Ratos , Animais , Alimentos Fortificados , Estudos Cross-Over , Culinária , MicronutrientesRESUMO
Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K2(SL)6K2 (abbreviated as K2), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K2 hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K2 hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K2. This imine interaction was also observed to improve K2 + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K2, R-Man loaded in K2, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K2 can be used to normalize the release of O-Man to that of R-Man. Further, since K2 itself is an adjuvant, a combination of O-Man and K2 could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy.
Assuntos
Nanofibras , Humanos , Nanofibras/química , Mananas , Preparações de Ação Retardada , Hidrogéis/química , Peptídeos/químicaRESUMO
Vaccination in the developing world is hampered by limited patient access, which prevents individuals from receiving the multiple injections necessary for protective immunity. Here, we developed an injectable microparticle formulation of the inactivated polio vaccine (IPV) that releases multiple pulses of stable antigen over time. To accomplish this, we established an IPV stabilization strategy using cationic polymers for pH modulation to enhance traditional small-molecule-based stabilization methods. We investigated the mechanism of this strategy and showed that it was broadly applicable to all three antigens in IPV. Our lead formulations released two bursts of IPV 1 month apart, mimicking a typical vaccination schedule in the developing world. One injection of the controlled-release formulations elicited a similar or better neutralizing response in rats, considered the correlate of protection in humans, than multiple injections of liquid vaccine. This single-administration vaccine strategy has the potential to improve vaccine coverage in the developing world.
Assuntos
Esquemas de Imunização , Vacina Antipólio de Vírus Inativado/administração & dosagem , Vacinação/métodos , Animais , Modelos Animais de Doenças , Feminino , Humanos , Injeções/métodos , Microesferas , Poliomielite/prevenção & controle , Ratos , Ratos WistarRESUMO
Influenza kills 30,000 to 40,000 people each year in the United States and causes 10 times as many hospitalizations. A common complication of influenza is bacterial superinfection, which exacerbates morbidity and mortality from the viral illness. Recently, methicillin-resistant Staphylococcus aureus (MRSA) has emerged as the dominant pathogen found in bacterial superinfection, with Streptococcus pneumoniae a close second. However, clinicians have few tools to treat bacterial superinfection. Current therapy for influenza/bacterial superinfection consists of treating the underlying influenza infection and adding various antibiotics, which are increasingly rendered ineffective by rising bacterial multidrug resistance. Several groups have recently proposed the use of the antiviral cytokine interferon lambda (IFN-λ) as a therapeutic for influenza, as administration of pegylated IFN-λ improves lung function and survival during influenza by reducing the overabundance of neutrophils in the lung. However, our data suggest that therapeutic IFN-λ impairs bacterial clearance during influenza superinfection. Specifically, mice treated with an adenoviral vector to overexpress IFN-λ during influenza infection exhibited increased bacterial burdens upon superinfection with either MRSA or S. pneumoniae Surprisingly, adhesion molecule expression, antimicrobial peptide production, and reactive oxygen species activity were not altered by IFN-λ treatment. However, neutrophil uptake of MRSA and S. pneumoniae was significantly reduced upon IFN-λ treatment during influenza superinfection in vivo Together, these data support the theory that IFN-λ decreases neutrophil motility and function in the influenza-infected lung, which increases the bacterial burden during superinfection. Thus, we believe that caution should be exercised in the possible future use of IFN-λ as therapy for influenza.
Assuntos
Antivirais/uso terapêutico , Influenza Humana/complicações , Influenza Humana/tratamento farmacológico , Interferons/uso terapêutico , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Superinfecção/tratamento farmacológico , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/patologia , Superinfecção/etiologia , Estados UnidosRESUMO
We previously showed that the mitochondrial fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) is expressed in alveolar type II pneumocytes and that LCAD-/- mice have altered breathing mechanics and surfactant defects. Here, we hypothesized that LCAD-/- mice would be susceptible to influenza infection. Indeed, LCAD-/- mice demonstrated increased mortality following infection with 2009 pandemic influenza (A/CA/07/09). However, the mortality was not due to increased lung injury, as inflammatory cell counts, viral titers, and histology scores all showed non-significant trends toward milder injury in LCAD-/- mice. To confirm this, LCAD-/- were infected with a second, mouse-adapted H1N1 virus (A/PR/8/34), to which they responded with significantly less lung injury. While both strains become increasingly hypoglycemic over the first week post-infection, LCAD-/- mice lose body weight more rapidly than wild-type mice. Surprisingly, while acutely fasted LCAD-/- mice develop hepatic steatosis, influenza-infected LCAD-/- mice do not. They do, however, become more hypothermic than wild-type mice and demonstrate increased blood lactate values. We conclude that LCAD-/- mice succumb to influenza from bioenergetic starvation, likely due to increased reliance upon glucose for energy.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/genética , Técnicas de Silenciamento de Genes , Vírus da Influenza A Subtipo H1N1/fisiologia , Pulmão/patologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Animais , Peso Corporal , Feminino , Hipotermia/etiologia , Hipotermia/genética , Hipotermia/patologia , Hipotermia/virologia , Pulmão/virologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/virologiaRESUMO
Seasonal and pandemic influenza is a cause of morbidity and mortality worldwide. Most people infected with influenza virus display mild-to-moderate disease phenotypes and recover within a few weeks. Influenza is known to cause persistent alveolitis in animal models; however, little is known about the molecular pathways involved in this phenotype. We challenged C57BL/6 mice with influenza A/PR/8/34 and examined lung pathologic processes and inflammation, as well as transcriptomic and epigenetic changes at 21 to 60 days after infection. Influenza induced persistent parenchymal lung inflammation, alveolar epithelial metaplasia, and epithelial endoplasmic reticulum stress that were evident after the clearance of virus and resolution of morbidity. Influenza infection induced robust changes in the lung transcriptome, including a significant impact on inflammatory and extracellular matrix protein expression. Despite the robust changes in lung gene expression, preceding influenza (21 days) did not exacerbate secondary Staphylococcus aureus infection. Finally, we examined the impact of influenza on miRNA expression in the lung and found an increase in miR-155. miR-155 knockout mice recovered from influenza infection faster than controls and had decreased lung inflammation and endoplasmic reticulum stress. These data illuminate the dynamic molecular changes in the lung in the weeks after influenza infection and characterize the repair process, identifying a novel role for miR-155.
Assuntos
Epigênese Genética , Pulmão/metabolismo , Pulmão/virologia , Infecções por Orthomyxoviridae/genética , Transcriptoma/genética , Cicatrização/genética , Animais , Progressão da Doença , Estresse do Retículo Endoplasmático/genética , Epitélio/patologia , Perfilação da Expressão Gênica , Inflamação/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/etiologia , Pneumonia/microbiologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
IL-13 and IL-17A, produced mainly by Th2 and Th17 cells, respectively, have an influential role in asthma pathogenesis. We examined the role of IL-13 and IL-17A in mediating airway hyperresponsiveness (AHR), lung inflammation, and mucus metaplasia in a dual Th2/Th17 model of asthma. IL-13 and/or IL-17A were neutralized using mAbs. Th2/Th17 adoptive transfer induced a mixed asthma phenotype characterized by elevated eosinophilia and neutrophilia, tissue inflammation, mucus metaplasia, and AHR that were partially reversible with steroid treatment. Pulmonary inflammation and quasi-static lung compliance were largely unaffected by neutralization of IL-13 and/or IL-17A. However, neutralization of IL-13 alone or in combination with IL-17A significantly attenuated AHR and mucus metaplasia. Further, STAT6 activation was attenuated following IL-13 and IL-13/IL-17A Ab treatment. We next assessed the role of STAT6 in Th2/Th17-mediated allergic airway disease using STAT6(-/-) mice. STAT6(-/-) mice adoptively transferred with Th2/Th17 cells had decreased AHR compared with controls. These data suggest that IL-13 drives AHR and mucus metaplasia in a STAT6-dependent manner, without directly contributing to airway or tissue inflammation. IL-17A independently contributes to AHR, but it only partially mediates inflammation and mucus metaplasia in a mixed Th2/Th17 model of steroid-resistant asthma.
Assuntos
Asma/imunologia , Interleucina-13/imunologia , Interleucina-17/imunologia , Hipersensibilidade Respiratória/imunologia , Transferência Adotiva , Animais , Asma/patologia , Modelos Animais de Doenças , Resistência a Medicamentos , Immunoblotting , Metaplasia/imunologia , Metaplasia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Muco/imunologia , Reação em Cadeia da Polimerase , Hipersensibilidade Respiratória/patologia , Células Th17/imunologia , Células Th2/imunologia , TranscriptomaRESUMO
The retinal pigment epithelium located between the neurosensory retina and the choroidal vasculature is critical for the function and maintenance of both the photoreceptors and underlying capillary endothelium. While the trophic role of retinal pigment epithelium on choroidal endothelial cells is well recognized, the existence of a reciprocal regulatory function of endothelial cells on retinal pigment epithelium cells remained to be fully characterized. Using a physiological long-term co-culture system, we determined the effect of retinal pigment epithelium-endothelial cell heterotypic interactions on cell survival, behaviour and matrix deposition. Human retinal pigment epithelium and endothelial cells were cultured on opposite sides of polyester transwells for up to 4 weeks in low serum conditions. Cell viability was quantified using a trypan blue assay. Cellular morphology was evaluated by H&E staining, S.E.M. and immunohistochemistry. Retinal pigment epithelium phagocytic function was examined using a fluorescent bead assay. Gene expression analysis was performed on both retinal pigment epithelium and endothelial cells by quantitative PCR. Quantification of extracellular matrix deposition was performed on decellularized transwells stained for collagen IV, fibronectin and fibrillin. Our results showed that presence of endothelial cells significantly improves retinal pigment epithelium maturation and function as indicated by the induction of visual cycle-associated genes, accumulation of a Bruch's membrane-like matrix and increase in retinal pigment epithelium phagocytic activity. Co-culture conditions led to increased expression of anti-angiogenic growth factors and receptors in both retinal pigment epithelium and endothelial cells compared to monoculture. Tube-formation assays confirmed that co-culture with retinal pigment epithelium significantly decreased the angiogenic phenotype of endothelial cells. These findings provide evidence of critical interdependent interactions between retinal pigment epithelium and endothelial cell involved in the maintenance of retinal homeostasis.
Assuntos
Comunicação Celular , Técnicas de Cocultura/métodos , Células Endoteliais da Veia Umbilical Humana/citologia , Epitélio Pigmentado da Retina/citologia , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fagocitose , Epitélio Pigmentado da Retina/metabolismoRESUMO
RATIONALE: Infection with Pneumocystis, an opportunistic fungal pathogen, can result in fulminant pneumonia in the clinical setting of patients with immunosuppression. In murine models, Pneumocystis has previously been shown to induce a CD4+ T cell-dependent eosinophilic response in the lung capable of providing protection. OBJECTIVES: We sought to explore the role of Pneumocystis in generating asthma-like lung pathology, given the natural eosinophilic response to infection. METHODS: Pneumocystis infection or antigen treatment was used to induce asthma-like pathology in wild-type mice. The roles of CD4+ T cells and eosinophils were examined using antibody depletion and knockout mice, respectively. The presence of anti-Pneumocystis antibodies in human serum samples was detected by ELISA and Western blotting. MEASUREMENTS AND MAIN RESULTS: Pneumocystis infection generates a strong type II response in the lung that requires CD4+ T cells. Pneumocystis infection was capable of priming a Th2 response similar to that of a commonly studied airway allergen, the house dust mite. Pneumocystis antigen treatment was also capable of inducing allergic inflammation in the lung, resulting in anti-Pneumocystis IgE production, goblet cell hyperplasia, and increased airway resistance. In the human population, patients with severe asthma had increased levels of anti-Pneumocystis IgG and IgE compared with healthy control subjects. Patients with severe asthma with elevated anti-Pneumocystis IgG levels had worsened symptom scores and lung parameters such as decreased forced expiratory volume and increased residual volume compared with patients with severe asthma who had low anti-Pneumocystis IgG. CONCLUSIONS: The present study demonstrates for the first time, to our knowledge, that Pneumocystis is an airway allergen capable of inducing asthma-like lung pathology.
RESUMO
Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Erros Inatos do Metabolismo Lipídico/metabolismo , Pneumopatias/etiologia , Surfactantes Pulmonares/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Adulto , Animais , Brônquios/metabolismo , Linhagem Celular Tumoral , Coenzima A/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Ácidos Graxos/metabolismo , Feminino , Homozigoto , Humanos , Lactente , Recém-Nascido , Pulmão/metabolismo , Pneumopatias/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxigênio/metabolismo , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Polimorfismo Genético , Alvéolos Pulmonares/metabolismoRESUMO
Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection.
Assuntos
Infecções por Escherichia coli/microbiologia , Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/microbiologia , Pneumonia Bacteriana/microbiologia , Pneumonia/microbiologia , Receptor de Interferon alfa e beta/fisiologia , Infecções Estafilocócicas/microbiologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/virologia , Suscetibilidade a Doenças , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/virologia , Vírus da Influenza A/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/imunologia , Pneumonia/virologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/virologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/virologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidadeRESUMO
Pneumonia is a leading cause of death worldwide. Staphylococcal aureus can be a cause of severe pneumonia alone or a common pathogen in secondary pneumonia following influenza. Recently, we reported that preceding influenza attenuated the Type 17 pathway, increasing the lung's susceptibility to secondary infection. IL-1ß is known to regulate host defense, including playing a role in Th17 polarization. We examined whether IL-1ß signaling is required for S. aureus host defense and whether influenza infection impacted S. aureus-induced IL-1ß production and subsequent Type 17 pathway activation. Mice were challenged with S. aureus (USA 300), with or without preceding Influenza A/PR/8/34 H1N1 infection. IL-1R1(-/-) mice had significantly higher S. aureus burden, increased mortality, and decreased Type 17 pathway activation following S. aureus challenge. Coinfected mice had significantly decreased IL-1ß production versus S. aureus infection alone at early time points following bacterial challenge. Preceding influenza did not attenuate S. aureus-induced inflammasome activation, but there was early suppression of NF-κB activation, suggesting an inhibition of NF-κB-dependent transcription of pro-IL-1ß. Furthermore, overexpression of IL-1ß in influenza and S. aureus-coinfected mice rescued the induction of IL-17 and IL-22 by S. aureus and improved bacterial clearance. Finally, exogenous IL-1ß did not significantly rescue S. aureus host defense during coinfection in IL-17RA(-/-) mice or in mice in which IL-17 and IL-22 activity were blocked. These data reveal a novel mechanism by which Influenza A inhibits S. aureus-induced IL-1ß production, resulting in attenuation of Type 17 immunity and increased susceptibility to bacterial infection.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Interleucina-1beta/metabolismo , Infecções por Orthomyxoviridae/imunologia , Pneumonia Estafilocócica/imunologia , Staphylococcus aureus/imunologia , Animais , Carga Bacteriana/genética , Carga Bacteriana/imunologia , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/virologia , Ativação Enzimática/imunologia , Inflamassomos/imunologia , Interleucina-17/biossíntese , Interleucina-1beta/biossíntese , Interleucinas/biossíntese , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Receptores Tipo I de Interleucina-1/deficiência , Receptores Tipo I de Interleucina-1/genética , Transdução de Sinais , Interleucina 22RESUMO
Influenza A represents a significant cause of morbidity and mortality worldwide. Bacterial complications of influenza A confer the greatest risk to patients. TH17 pathway inhibition has been implicated as a mechanism by which influenza A alters bacterial host defense. Here we show that preceding influenza causes persistent Staphylococcus aureus infection and suppression of TH17 pathway activation in mice. Influenza does not inhibit S. aureus binding and uptake by phagocytic cells but instead attenuates S. aureus induced TH17 related antimicrobial peptides necessary for bacterial clearance in the lung. Importantly, exogenous lipocalin 2 rescued viral exacerbation of S. aureus infection and decreased free iron levels in the bronchoalveolar lavage from mice coinfected with S. aureus and influenza. These findings indicate a novel mechanism by which influenza A inhibits TH17 immunity and increases susceptibility to secondary bacterial pneumonia. Identification of new mechanisms in the pathogenesis of bacterial pneumonia could lead to future therapeutic targets.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/microbiologia , Pneumonia Estafilocócica/microbiologia , Staphylococcus aureus/imunologia , Análise de Variância , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Coinfecção/microbiologia , Coinfecção/virologia , Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A/patogenicidade , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Estafilocócica/imunologia , Pneumonia Estafilocócica/virologia , Staphylococcus aureus/patogenicidade , Células Th17RESUMO
Influenza infection is widespread in the United States and the world. Despite low mortality rates due to infection, morbidity is common and little is known about the molecular events involved in recovery. Influenza infection results in persistent distal lung remodeling, and the mechanism(s) involved are poorly understood. Recently IL-22 has been found to mediate epithelial repair. We propose that IL-22 is critical for recovery of normal lung function and architecture after influenza infection. Wild-type and IL-22(-/-) mice were infected with influenza A PR8/34 H1N1 and were followed up for up to 21 days post infection. IL-22 receptor was localized to the airway epithelium in naive mice but was expressed at the sites of parenchymal lung remodeling induced by influenza infection. IL-22(-/-) mice displayed exacerbated lung injury compared with wild-type mice, which correlated with decreased lung function 21 days post infection. Epithelial metaplasia was observed in wild-type mice but was not evident in IL-22(-/-) animals that were characterized with an increased fibrotic phenotype. Gene expression analysis revealed aberrant expression of epithelial genes involved in repair processes, among changes in several other biological processes. These data indicate that IL-22 is required for normal lung repair after influenza infection. IL-22 represents a novel pathway involved in interstitial lung disease.
Assuntos
Epitélio/patologia , Epitélio/virologia , Interleucinas/metabolismo , Pulmão/patologia , Pulmão/virologia , Infecções por Orthomyxoviridae/patologia , Cicatrização , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Colágeno/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Epitélio/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interleucinas/deficiência , Pulmão/fisiopatologia , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/fisiopatologia , Infecções por Orthomyxoviridae/virologia , Receptores de Interleucina/metabolismo , Testes de Função Respiratória , Transdução de Sinais/genética , Interleucina 22RESUMO
AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.
Assuntos
Diferenciação Celular , Ácidos Graxos , Miócitos Cardíacos , Miofibrilas , Fosforilação Oxidativa , Proteína 2 de Ligação ao Retinoblastoma , Humanos , Células Cultivadas , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Histonas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/enzimologia , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/genética , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Miofibrilas/enzimologia , Oxirredução , Regiões Promotoras Genéticas , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genéticaRESUMO
Surgical intervention is the most common first-line treatment for severe traumatic brain injuries (TBIs) associated with high intracranial pressure, while the complexity of these surgical procedures often results in complications. Surgeons often struggle to comprehensively evaluate the TBI status, making it difficult to select the optimal intervention strategy. Here, we introduce a fluorescence imaging-based technology that uses high-quality silver indium selenide-based quantum dots (QDs) for integrated TBI diagnosis and surgical guidance. These engineered, poly(ethylene glycol)-capped QDs emit in the near-infrared region, are resistant to phagocytosis, and importantly, are ultrastable after the epitaxial growth of an aluminum-doped zinc sulfide shell in the aqueous phase that renders the QDs resistant to long-term light irradiation and complex physiological environments. We found that intravenous injection of QDs enabled both the precise diagnosis of TBI in a mouse model and, more importantly, the comprehensive evaluation of the TBI status before, during, and after an operation to distinguish intracranial from superficial hemorrhages, provide real-time monitoring of the secondary hemorrhage, and guide the decision making on the evacuation of intracranial hematomas. This QD-based diagnostic and monitoring system could ultimately complement existing clinical tools for treating TBI, which may help surgeons improve patient outcomes and avoid unnecessary procedures.
Assuntos
Lesões Encefálicas Traumáticas , Pontos Quânticos , Pontos Quânticos/química , Lesões Encefálicas Traumáticas/diagnóstico por imagem , Animais , Camundongos , Imagem Óptica , Água/química , Fluorescência , Índio/química , Masculino , Polietilenoglicóis/químicaRESUMO
Toll-like receptors (TLRs) recognize pathogen- and damage-associated molecular patterns and, in turn, trigger the release of cytokines and other immunostimulatory molecules. As a result, TLR agonists are increasingly being investigated as vaccine adjuvants, though many of these agonists are small molecules that quickly diffuse away from the vaccination site, limiting their co-localization with antigens and, thus, their effect. Here, the small-molecule TLR7 agonist 1V209 is conjugated to a positively-charged multidomain peptide (MDP) hydrogel, K 2 , which was previously shown to act as an adjuvant promoting humoral immunity. Mixing the 1V209-conjugated K 2 50:50 with the unfunctionalized K 2 produces hydrogels that retain the shear-thinning and self-healing physical properties of the original MDP, while improving the solubility of 1V209 more than 200-fold compared to the unconjugated molecule. When co-delivered with ovalbumin as a model antigen, 1V209-functionalized K 2 produces antigen-specific IgG titers that were statistically similar to alum, the gold standard adjuvant, and a significantly lower ratio of Th2-associated IgG1 to Th1-associated IgG2a than alum, suggesting a more balanced Th1 and Th2 response. Together, these results suggest that K 2 MDP hydrogels functionalized with 1V209 are a promising adjuvant for vaccines against infectious diseases, especially those benefiting from a combined Th1 and Th2 immune response. Table of Contents: Activation of toll-like receptors (TLRs) stimulates a signaling cascade to induce an immune response. A TLR7 agonist was conjugated to an injectable peptide hydrogel, which was then used to deliver a model vaccine antigen. This platform produced antibody titers similar to the gold standard adjuvant alum and demonstrated an improved balance between Th1- and Th2-mediated immunity over alum.
RESUMO
Although zinc deficiency (secondary to malnutrition) has long been considered an important contributor to morbidity and mortality of infectious disease (e.g. diarrhea disorders), epidemiologic data (including randomized controlled trials with supplemental zinc) for such a role in lower respiratory tract infection are somewhat ambiguous. In the current study, we provide the first preclinical evidence demonstrating that although diet-induced acute zinc deficiency (Zn-D: ~50% decrease) did not worsen infection induced by either influenza A (H1N1) or methicillin-resistant staph aureus (MRSA), Zn-D mice were sensitive to the injurious effects of superinfection of H1N1 with MRSA. Although the mechanism underlying the sensitivity of ZnD mice to combined H1N1/MRSA infection is unclear, it was noteworthy that this combination exacerbated lung injury as shown by lung epithelial injury markers (increased BAL protein) and decreased genes related to epithelial integrity in Zn-D mice (surfactant protein C and secretoglobins family 1A member 1). As bacterial pneumonia accounts for 25%-50% of morbidity and mortality from influenza A infection, zinc deficiency may be an important pathology component of respiratory tract infections.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Desnutrição , Staphylococcus aureus Resistente à Meticilina , Pneumonia Bacteriana , Animais , Camundongos , Pneumonia Bacteriana/complicações , Staphylococcus aureus , ZincoRESUMO
Maintaining safe and potent pharmaceutical drug levels is often challenging. Multidomain peptides (MDPs) assemble into supramolecular hydrogels with a well-defined, highly porous nanostructure that makes them attractive for drug delivery, yet their ability to extend release is typically limited by rapid drug diffusion. To overcome this challenge, we developed self-assembling boronate ester release (SABER) MDPs capable of engaging in dynamic covalent bonding with payloads containing boronic acids (BAs). As examples, we demonstrate that SABER hydrogels can prolong the release of five BA-containing small-molecule drugs as well as BA-modified insulin and antibodies. Pharmacokinetic studies revealed that SABER hydrogels extended the therapeutic effect of ganfeborole from days to weeks, preventing Mycobacterium tuberculosis growth better than repeated oral administration in an infection model. Similarly, SABER hydrogels extended insulin activity, maintaining normoglycemia for six days in diabetic mice after a single injection. These results suggest that SABER hydrogels present broad potential for clinical translation.
RESUMO
Porous scaffolds have the ability to minimize transport barriers for both two- (2D) and three-dimensional tissue engineering. However, current porous scaffolds may be non-ideal for 2D tissues such as epithelium due to inherent fabrication-based characteristics. While 2D tissues require porosity to support molecular transport, pores must be small enough to prevent cell migration into the scaffold in order to avoid non-epithelial tissue architecture and compromised function. Though electrospun meshes are the most popular porous scaffolds used today, their heterogeneous pore size and intense topography may be poorly-suited for epithelium. Porous scaffolds produced using other methods have similar unavoidable limitations, frequently involving insufficient pore resolution and control, which make them incompatible with 2D tissues. In addition, many of these techniques require an entirely new round of process development in order to change material or pore size. Herein we describe "pore casting," a fabrication method that produces flat scaffolds with deterministic pore shape, size, and location that can be easily altered to accommodate new materials or pore dimensions. As proof-of-concept, pore-cast poly(ε-caprolactone) (PCL) scaffolds were fabricated and compared to electrospun PCL in vitro using canine kidney epithelium, human colon epithelium, and human umbilical vein endothelium. All cell types demonstrated improved morphology and function on pore-cast scaffolds, likely due to reduced topography and universally small pore size. These results suggest that pore casting is an attractive option for creating 2D tissue engineering scaffolds, especially when the application may benefit from well-controlled pore size or architecture.