RESUMEN
IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.
Asunto(s)
Interleucina-12 , Neoplasias , Humanos , Ratones , Animales , Interleucina-12/metabolismo , Neoplasias/tratamiento farmacológico , Citocinas , Transducción de Señal , Índice Terapéutico , Microambiente TumoralRESUMEN
Throughout the course of infection, many pathogens encounter bactericidal conditions that threaten the viability of the bacteria and impede the establishment of infection. Bile is one of the most innately bactericidal compounds present in humans, functioning to reduce the bacterial burden in the gastrointestinal tract while also aiding in digestion. It is becoming increasingly apparent that pathogens successfully resist the bactericidal conditions of bile, including bacteria that do not normally cause gastrointestinal infections. This review highlights the ability of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter (ESKAPE), and other enteric pathogens to resist bile and how these interactions can impact the sensitivity of bacteria to various antimicrobial agents. Given that pathogen exposure to bile is an essential component to gastrointestinal transit that cannot be avoided, understanding how bile resistance mechanisms align with antimicrobial resistance is vital to our ability to develop new, successful therapeutics in an age of widespread and increasing antimicrobial resistance.
Asunto(s)
Antibacterianos/metabolismo , Bacterias/patogenicidad , Bilis/metabolismo , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Humanos , Intestino Delgado/microbiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , VirulenciaRESUMEN
Intestinal helminth infections elicit Th2-type immunity, which influences host immune responses to additional threats, such as allergens, metabolic disease, and other pathogens. Th2 immunity involves a shift of the CD4+ T-cell population from type-0 to type-2 (Th2) with increased abundance of interleukin (IL)-4 and IL-13. This study sought to investigate if existing gut-restricted intestinal helminth infections impact bacterial-induced acute airway neutrophil recruitment. C57BL/6 mice were divided into four groups: uninfected; helminth-Heligmosomoides polygyrus infected; Pseudomonas aeruginosa infected; and coinfected. Mice infected with H. polygyrus were incubated for 2 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage, blood, and lung samples were analyzed. Interestingly, infection with gut-restricted helminths resulted in immunological and structural changes in the lung. These changes include increased lung CD4+ T cells, increased Th2 cytokine expression, and airway goblet cell hyperplasia. Furthermore, coinfected mice exhibited significantly more airspace neutrophil infiltration at 6 hours following P. aeruginosa infection and exhibited an improved rate of survival compared with bacterial infected alone. These results suggest that chronic helminth infection of the intestines can influence and enhance acute airway neutrophil responses to P. aeruginosa infection.
Asunto(s)
Helmintiasis/patología , Parasitosis Intestinales/patología , Pulmón/microbiología , Nematospiroides dubius/aislamiento & purificación , Neutrófilos/inmunología , Pseudomonas aeruginosa/metabolismo , Animales , Helmintiasis/inmunología , Helmintiasis/microbiología , Mediadores de Inflamación/metabolismo , Parasitosis Intestinales/inmunología , Parasitosis Intestinales/microbiología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Nematospiroides dubius/patogenicidad , Células Th2/inmunologíaRESUMEN
Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography, a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.
Asunto(s)
Antígenos de Plaqueta Humana/metabolismo , Fibrosis Quística/inmunología , Neutrófilos/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Mucosa Respiratoria/inmunología , Migración Transendotelial y Transepitelial , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Comunicación Celular , Línea Celular , Quimiotaxis , Técnicas de Cocultivo , Citosol/metabolismo , Humanos , Leucotrieno B4/metabolismo , Ratones , Neutrófilos/microbiología , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/patología , Tomografía de Coherencia ÓpticaRESUMEN
Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.
Asunto(s)
Proteínas Bacterianas/inmunología , Leucotrieno B4/inmunología , Infiltración Neutrófila/inmunología , Infecciones por Pseudomonas/inmunología , Migración Transendotelial y Transepitelial/inmunología , Animales , Western Blotting , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Pseudomonas aeruginosa/patogenicidad , Virulencia/inmunologíaRESUMEN
In the present study, human atherosclerotic carotid arteries were examined following endarterectomy for the presence of the Gram-positive bacterium Propionibacterium acnes and its potential association with biofilm structures within the arterial wall. The P. acnes 16S rRNA gene was detectable in 4 of 15 carotid artery samples, and viable P. acnes was one among 10 different bacterial species recoverable in culture. Fluorescence in situ hybridization analysis of 5 additional atherosclerotic carotid arteries demonstrated biofilm bacteria within all samples, with P. acnes detectable in 4 samples. We also demonstrated that laboratory-grown cultures of P. acnes biofilms were susceptible to induction of a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of iron-bound transferrin or with free iron. The production and release of lipolytic and proteolytic extracellular enzymes by P. acnes were shown to increase in iron-induced dispersed biofilms, and these dispersion-induced P. acnes VP1 biofilms showed increased expression of mRNAs for the triacylglycerol lipases PPA2105 and PPA1796 and the hyaluronate lyase PPA380 compared to that in untreated biofilms. These results demonstrate that P. acnes can infect the carotid arteries of humans with atherosclerosis as a component of multispecies biofilms and that dispersion is inducible for this organism, at least in vitro, with physiologically relevant levels of norepinephrine resulting in the production and release of degradative enzymes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas , Arterias Carótidas/microbiología , Enfermedades de las Arterias Carótidas/microbiología , Norepinefrina/metabolismo , Péptido Hidrolasas/metabolismo , Propionibacterium acnes/aislamiento & purificación , Proteínas Bacterianas/genética , Secuencia de Bases , Humanos , Hierro/metabolismo , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Propionibacterium acnes/enzimología , Propionibacterium acnes/genética , Propionibacterium acnes/fisiologíaRESUMEN
UNLABELLED: Atherosclerosis, a disease condition resulting from the buildup of fatty plaque deposits within arterial walls, is the major underlying cause of ischemia (restriction of the blood), leading to obstruction of peripheral arteries, congestive heart failure, heart attack, and stroke in humans. Emerging research indicates that factors including inflammation and infection may play a key role in the progression of atherosclerosis. In the current work, atherosclerotic carotid artery explants from 15 patients were all shown to test positive for the presence of eubacterial 16S rRNA genes. Density gradient gel electrophoresis of 5 of these samples revealed that each contained 10 or more distinct 16S rRNA gene sequences. Direct microscopic observation of transverse sections from 5 diseased carotid arteries analyzed with a eubacterium-specific peptide nucleic acid probe revealed these to have formed biofilm deposits, with from 1 to 6 deposits per thin section of plaque analyzed. A majority, 93%, of deposits was located proximal to the internal elastic lamina and associated with fibrous tissue. In 6 of the 15 plaques analyzed, 16S rRNA genes from Pseudomonas spp. were detected. Pseudomonas aeruginosa biofilms have been shown in our lab to undergo a dispersion response when challenged with free iron in vitro. Iron is known to be released into the blood by transferrin following interaction with catecholamine hormones, such as norepinephrine. Experiments performed in vitro showed that addition of physiologically relevant levels of norepinephrine induced dispersion of P. aeruginosa biofilms when grown under low iron conditions in the presence but not in the absence of physiological levels of transferrin. IMPORTANCE: The association of bacteria with atherosclerosis has been only superficially studied, with little attention focused on the potential of bacteria to form biofilms within arterial plaques. In the current work, we show that bacteria form biofilm deposits within carotid arterial plaques, and we demonstrate that one species we have identified in plaques can be stimulated in vitro to undergo a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of transferrin. Biofilm dispersion is characterized by the release of bacterial enzymes into the surroundings of biofilm microcolonies, allowing bacteria to escape the biofilm matrix. We believe these enzymes may have the potential to damage surrounding tissues and facilitate plaque rupture if norepinephrine is able to stimulate biofilm dispersion in vivo. This research, therefore, suggests a potential mechanistic link between hormonal state and the potential for heart attack and stroke.