RESUMO
Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Clostridioides difficile , Enterocolite Pseudomembranosa , Glucosiltransferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Cricetinae , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/enzimologia , Enterocolite Pseudomembranosa/genética , Enterocolite Pseudomembranosa/patologia , Feminino , Deleção de Genes , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Masculino , CamundongosRESUMO
Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1 Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-dependent interferon-ß production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.
Assuntos
Caspases/metabolismo , Suscetibilidade a Doenças/enzimologia , Salmonelose Animal/enzimologia , Adjuvantes Imunológicos/farmacologia , Animais , Caspases Iniciadoras , Morte Celular , Células Cultivadas , Regulação da Expressão Gênica , Inflamassomos/imunologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Salmonelose Animal/genética , Salmonella typhimurium/fisiologia , Transdução de SinaisRESUMO
Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.
Assuntos
Junções Aderentes/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Infecções Estafilocócicas/metabolismo , Vasculite/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Junções Aderentes/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Toxinas Bacterianas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Hemolisinas/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Camundongos Knockout , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologia , Vasculite/genética , Vasculite/microbiologia , Vasculite/patologiaRESUMO
Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Infecções por Salmonella/transmissão , Salmonella typhimurium/patogenicidade , Administração Oral , Animais , Citocinas/imunologia , Transmissão de Doença Infecciosa , Camundongos , Células Mieloides/imunologia , Células Mieloides/patologia , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologiaRESUMO
BACKGROUND & AIMS: Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology. METHODS: We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. RESULTS: H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5(+) stem cell proliferation, and up-regulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. CONCLUSIONS: H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.
Assuntos
Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/microbiologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomarcadores/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Mucosa Gástrica/metabolismo , Genótipo , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Hiperplasia , Cinética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Organoides , Fenótipo , Receptores Acoplados a Proteínas G/genética , Células-Tronco/metabolismo , Células-Tronco/patologia , Técnicas de Cultura de Tecidos , VirulênciaRESUMO
Recent studies of epithelial tissues have revealed the presence of tissue-specific stem cells that are able to establish multiple cell lineages within an organ. The stem cells give rise to progenitors that replicate before differentiating into specific cell lineages. The mechanism by which homeostasis is established between proliferating stem or progenitor cells and terminally differentiated cells is unclear. This study demonstrates that Agr2 expression by mucous neck cells in the stomach promotes the differentiation of multiple cell lineages while also inhibiting the proliferation of stem or progenitor cells. When Agr2 expression is absent, gastric mucous neck cells increased in number as does the number of proliferating cells. Agr2 expression loss also resulted in the decline of terminally differentiated cells, which was supplanted by cells that exhibited nuclear SOX9 labeling. Sox9 expression has been associated with progenitor and stem cells. Similar effects of the Agr2 null on cell proliferation in the intestine were also observed. Agr2 consequently serves to maintain the balance between proliferating and differentiated epithelial cells.
Assuntos
Diferenciação Celular , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Mucoproteínas/biossíntese , Células-Tronco/metabolismo , Estômago/embriologia , Animais , Proliferação de Células , Hiperplasia , Camundongos , Camundongos Mutantes , Mucoproteínas/genética , Proteínas Oncogênicas , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/patologia , Estômago/patologiaRESUMO
Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). Here, we report the identification of STF-083010, a novel small-molecule inhibitor of Ire1. STF-083010 inhibited Ire1 endonuclease activity, without affecting its kinase activity, after endoplasmic reticulum stress both in vitro and in vivo. Treatment with STF-083010 showed significant antimyeloma activity in model human MM xenografts. Similarly, STF-083010 was preferentially toxic to freshly isolated human CD138(+) MM cells compared with other similarly isolated cell populations. The identification of this novel Ire1 inhibitor supports the hypothesis that the Ire1-XBP1 axis is a promising target for anticancer therapy, especially in the context of MM.
Assuntos
Citotoxinas/farmacologia , Endorribonucleases/antagonistas & inibidores , Mieloma Múltiplo/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ácidos Borônicos/administração & dosagem , Bortezomib , Células Cultivadas , Citotoxinas/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Camundongos , Modelos Biológicos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazinas/administração & dosagem , Especificidade por Substrato/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Sulfonamidas/uso terapêutico , Tiofenos/administração & dosagem , Tiofenos/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QD as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance, and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding nontumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD(2) to integrin α(v)ß(3)-positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD-modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron-based nanoprobes in the clinical setting in the near future.
Assuntos
Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Neoplasias Experimentais/patologia , Pontos Quânticos , Imagem Corporal Total/métodos , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Host-adapted strains of Salmonella are capable of establishing a persistent infection in their host often in the absence of clinical disease. The mouse model of Salmonella infection has primarily been used as a model for the acute systemic disease. Therefore, the sites of long-term S. typhimurium persistence in the mouse are not known nor are the mechanisms of persistent infection clearly understood. Here, we show that S. typhimurium can persist for as long as 1 yr in the mesenteric lymph nodes (MLNs) of 129sv Nramp1(+)(/)(+) (Slc11a1(+)(/)(+)) mice despite the presence of high levels of anti-S. typhimurium antibody. Tissues from 129sv mice colonized for 60 d contain numerous inflammatory foci and lesions with features resembling S. typhi granulomas. Tissues from mice infected for 365 d have very few organized inflammatory lesions, but the bacteria continue to persist within macrophages in the MLN and the animals generally remain disease-free. Finally, chronically infected mice treated with an interferon-gamma neutralizing antibody exhibited symptoms of acute systemic infection, with evidence of high levels of bacterial replication in most tissues and high levels of fecal shedding. Thus, interferon-gamma, which may affect the level of macrophage activation, plays an essential role in the control of the persistent S. typhimurium infection in mice.
Assuntos
Proteínas de Transporte de Cátions/fisiologia , Interferon gama/antagonistas & inibidores , Macrófagos/imunologia , Macrófagos/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Transporte de Cátions/genética , Doença Crônica , Linfonodos/imunologia , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Testes de Neutralização , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/isolamento & purificação , Fatores de TempoRESUMO
In contrast to chronic/long-term stress that suppresses/dysregulates immune function, an acute/short-term fight-or-flight stress response experienced during immune activation can enhance innate and adaptive immunity. Moderate ultraviolet-B (UV) exposure provides a non-invasive system for studying the naturalistic emergence, progression and regression of squamous cell carcinoma (SCC). Because SCC is an immunoresponsive cancer, we hypothesized that short-term stress experienced before UV exposure would enhance protective immunity and increase resistance to SCC. Control and short-term stress groups were treated identically except that the short-term stress group was restrained (2.5h) before each of nine UV-exposure sessions (minimum erythemal dose, 3-times/week) during weeks 4-6 of the 10-week UV exposure protocol. Tumors were measured weekly, and tissue collected at weeks 7, 20, and 32. Chemokine and cytokine gene expression was quantified by real-time PCR, and CD4+ and CD8+ T cells by flow cytometry and immunohistochemistry. Compared to controls, the short-term stress group showed greater cutaneous T-cell attracting chemokine (CTACK)/CCL27, RANTES, IL-12, and IFN-gamma gene expression at weeks 7, 20, and 32, higher skin infiltrating T cell numbers (weeks 7 and 20), lower tumor incidence (weeks 11-20) and fewer tumors (weeks 11-26). These results suggest that activation of short-term stress physiology increased chemokine expression and T cell trafficking and/or function during/following UV exposure, and enhanced Type 1 cytokine-driven cell-mediated immunity that is crucial for resistance to SCC. Therefore, the physiological fight-or-flight stress response and its adjuvant-like immuno-enhancing effects, may provide a novel and important mechanism for enhancing immune system mediated tumor-detection/elimination that merits further investigation.
Assuntos
Carcinoma de Células Escamosas/imunologia , Imunidade Celular/imunologia , Estresse Psicológico/imunologia , Animais , Comportamento Animal/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Contagem de Células , Quimiocinas/biossíntese , Citocinas/biossíntese , Feminino , Expressão Gênica , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Estresse Psicológico/psicologia , Sobrevida , Raios UltravioletaRESUMO
Skeletal malformations in captive-bred, adult Xenopus spp., have not previously been reported. Here we describe 10 sexually mature, genetically modified laboratory frogs (6 Xenopus laevis and 4 Xenopus tropicalis) with axial skeletal abnormalities. The young adult frogs were described by veterinary staff as presenting with "hunchbacks," but were otherwise considered to be in good health. All affected frogs were genetically engineered using various techniques: transcription activator-like effector nucleases (TALEN) editing using thyroid hormone receptor α TALEN mRNA, restriction enzyme-mediated integration methods involving insertion of the inducible transgene pCAR/TRDN, or via I-SceI meganuclease transgenesis using either pDRTREdpTR-HS4 or pDPCrtTA-TREG-HS4 plasmid sequences. Radiographic findings (6 frogs) and gross necropsy (10 frogs) revealed vertebral column malformations and sacroiliac deformities that resulted in moderate to severe kyphosis and kyphoscoliosis. These findings were confirmed and additional skeletal abnormalities were identified using computed tomography to create a 3D reconstruction of 4 frogs. Additional findings visible on the 3D reconstructions included incomplete vertebral segmentation, malformed transverse processes, and a short and/or curved urostyle. Histopathologic findings included misshapen intervertebral joints with nonconforming articular surfaces, narrowed joint cavities, flattened or irregularly-formed articular cartilage, irregular maturation lines and nonpolarized chondrocytes, excess fibrocartilage, and evidence of irregular bone resorption and growth. While the specific etiology of the vertebral skeletal abnormalities remains unclear, possibilities include: 1) egg/oocyte physical manipulation (dejellying, microinjection, fertilization, etc.), 2) induction and expression of the transgenes, 3) inactivation (knockout) of existing genes by insertional mutagenesis, or 4) a combination of the above. Furthermore, the possibility of undetected changes in the macro or microenvironment, or a feature of the genetic background of the affected frogs cannot be ruled out.
Assuntos
Técnicas de Transferência de Genes , Animais , Animais Geneticamente Modificados , Humanos , RNA Mensageiro , Transgenes , Xenopus/genética , Xenopus laevis/genéticaRESUMO
Rationale: Localized blood-brain barrier (BBB) opening can be achieved with minimal to no tissue damage by applying pulsed focused ultrasound alongside a low microbubble (MB) dose. However, relatively little is known regarding how varying treatment parameters affect the degree of neuroinflammation following BBB opening. The goal of this study was to evaluate the activation of an inflammatory response following BBB opening as a function of applied acoustic pressure using two different microbubble doses. Methods: Mice were treated with 650 kHz ultrasound using varying acoustic peak negative pressures (PNPs) using two different MB doses, and activation of an inflammatory response, in terms of microglial and astrocyte activation, was assessed one hour following BBB opening using immunohistochemical staining. Harmonic and subharmonic acoustic emissions (AEs) were monitored for all treatments with a passive cavitation detector, and contrast-enhanced magnetic resonance imaging (CE-MRI) was performed following BBB opening to quantify the degree of opening. Hematoxylin and eosin-stained slides were assessed for the presence of microhemorrhage and edema. Results: For each MB dose, BBB opening was achieved with minimal activation of microglia and astrocytes using a PNP of 0.15 MPa. Higher PNPs were associated with increased activation, with greater increases associated with the use of the higher MB dose. Additionally, glial activation was still observed in the absence of histopathological findings. We found that CE-MRI was most strongly correlated with the degree of activation. While acoustic emissions were not predictive of microglial or astrocyte activation, subharmonic AEs were strongly associated with marked and severe histopathological findings. Conclusions: Our study demonstrated that there were mild histologic changes and activation of the acute inflammatory response using PNPs ranging from 0.15 MPa to 0.20 MPa, independent of MB dose. However, when higher PNPs of 0.25 MPa or above were applied, the same applied PNP resulted in more severe and widespread histological findings and activation of the acute inflammatory response when using the higher MB dose. The potential activation of the inflammatory response following ultrasound-mediated BBB opening should be considered when treating patients to maximize therapeutic benefit.
Assuntos
Barreira Hematoencefálica/efeitos da radiação , Sistemas de Liberação de Medicamentos/métodos , Inflamação/metabolismo , Microbolhas , Terapia por Ultrassom/métodos , Animais , Astrócitos/metabolismo , Química Encefálica/efeitos da radiação , Feminino , Camundongos , Microglia/metabolismo , Ondas UltrassônicasRESUMO
BACKGROUND: Neuromodulation by transcranial focused ultrasound (FUS) offers the potential to non-invasively treat specific brain regions, with treatment location verified by magnetic resonance acoustic radiation force imaging (MR-ARFI). OBJECTIVE: To investigate the safety of these methods prior to widespread clinical use, we report histologic findings in two large animal models following FUS neuromodulation and MR-ARFI. METHODS: Two rhesus macaques and thirteen Dorset sheep were studied. FUS neuromodulation was targeted to the primary visual cortex in rhesus macaques and to subcortical locations, verified by MR-ARFI, in eleven sheep. Both rhesus macaques and five sheep received a single FUS session, whereas six sheep received repeated sessions three to six days apart. The remaining two control sheep did not receive ultrasound but otherwise underwent the same anesthetic and MRI procedures as the eleven experimental sheep. Hematoxylin and eosin-stained sections of brain tissue (harvested zero to eleven days following FUS) were evaluated for tissue damage at FUS and control locations as well as tissue within the path of the FUS beam. TUNEL staining was used to evaluate for the presence of apoptosis in sheep receiving high dose FUS. RESULTS: No FUS-related pre-mortem histologic findings were observed in the rhesus macaques or in any of the examined sheep. Extravascular red blood cells (RBCs) were present within the meninges of all sheep, regardless of treatment group. Similarly, small aggregates of perivascular RBCs were rarely noted in non-target regions of neural parenchyma of FUS-treated (8/11) and untreated (2/2) sheep. However, no concurrent histologic abnormalities were observed, consistent with RBC extravasation occurring as post-mortem artifact following brain extraction. Sheep within the high dose FUS group were TUNEL-negative at the targeted site of FUS. CONCLUSIONS: The absence of FUS-related histologic findings suggests that the neuromodulation and MR-ARFI protocols evaluated do not cause tissue damage.
Assuntos
Encéfalo/diagnóstico por imagem , Técnicas de Imagem por Elasticidade/métodos , Imageamento por Ressonância Magnética/métodos , Estimulação Elétrica Nervosa Transcutânea/métodos , Ultrassonografia Doppler Transcraniana/métodos , Animais , Encéfalo/fisiologia , Macaca mulatta , Espectroscopia de Ressonância Magnética/métodos , Masculino , OvinosRESUMO
PURPOSE: To evaluate the feasibility of magnetic resonance (MR) imaging-guided percutaneous cryoablation of normal canine prostates and to identify MR imaging features that accurately predict the area of tissue damage at a microscopic level. MATERIALS AND METHODS: Six adult male mixed-breed dogs were anesthetized, intubated, and placed in a 0.5-T open MR imaging system. A receive-only endorectal coil was placed, and prostate location and depth were determined on T1-weighted fast spin-echo (FSE) MR imaging. After placement of cryoprobes and temperature sensors, three freezing protocols were used to ablate prostate tissue. Ice ball formation was monitored with T1-weighted FSE imaging. Tissue necrosis area was assessed with contrast-enhanced weighted MR imaging and compared with histopathologic findings. RESULTS: A total of 12 cryolesions (mean size, 1.2 cm) were bilaterally created in six prostates. Ice ball formation was oval and signal-free on T1-weighted FSE sequences in all cases. Postprocedural contrast-enhanced MR imaging typically showed a nonenhancing area of low signal intensity centrally located within the frozen area, surrounded by a bright enhancing rim in all cases. On histopathologic examination, two distinct zones were identified within cryolesions. Centrally, a necrotic zone with complete cellular destruction and hemorrhage was found. Between this necrotic zone and normal glandular tissue, a zone of fragmented and intact glands, interstitial edema, and rare acute inflammatory cells was seen. Correlation between nonenhancement on contrast-enhanced weighted MR images and tissue necrosis on pathologic examination was consistent within all six dogs. CONCLUSIONS: MR imaging-guided cryoablation of the prostate is technically feasible. The nonenhancing area on postablation contrast-enhanced weighted MR imaging accurately predicts the area of cryoablation-induced tissue necrosis on pathologic analysis.
Assuntos
Criocirurgia/métodos , Imageamento por Ressonância Magnética/métodos , Próstata/patologia , Próstata/cirurgia , Prostatectomia/métodos , Cirurgia Assistida por Computador/métodos , Animais , Cães , Estudos de Viabilidade , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Transmission is an essential stage of a pathogen's life cycle and remains poorly understood. We describe here a model in which persistently infected 129X1/SvJ mice provide a natural model of Salmonella enterica serovar Typhimurium transmission. In this model only a subset of the infected mice, termed supershedders, shed high levels (>10(8) CFU/g) of Salmonella serovar Typhimurium in their feces and, as a result, rapidly transmit infection. While most Salmonella serovar Typhimurium-infected mice show signs of intestinal inflammation, only supershedder mice develop colitis. Development of the supershedder phenotype depends on the virulence determinants Salmonella pathogenicity islands 1 and 2, and it is characterized by mucosal invasion and, importantly, high luminal abundance of Salmonella serovar Typhimurium within the colon. Immunosuppression of infected mice does not induce the supershedder phenotype, demonstrating that the immune response is not the main determinant of Salmonella serovar Typhimurium levels within the colon. In contrast, treatment of mice with antibiotics that alter the health-associated indigenous intestinal microbiota rapidly induces the supershedder phenotype in infected mice and predisposes uninfected mice to the supershedder phenotype for several days. These results demonstrate that the intestinal microbiota plays a critical role in controlling Salmonella serovar Typhimurium infection, disease, and transmissibility. This novel model should facilitate the study of host, pathogen, and intestinal microbiota factors that contribute to infectious disease transmission.
Assuntos
Intestinos/microbiologia , Infecções por Salmonella/microbiologia , Infecções por Salmonella/transmissão , Salmonella typhimurium/fisiologia , Salmonella typhimurium/patogenicidade , Fatores de Virulência/metabolismo , Animais , Antibacterianos/farmacologia , Fezes/microbiologia , Feminino , Hospedeiro Imunocomprometido , Camundongos , Fatores de TempoRESUMO
Phantoms are widely used during the development of new imaging systems and algorithms. For development and optimization of new imaging systems such as tomosynthesis, where conventional image quality metrics may not be applicable, a realistic phantom that can be used across imaging systems is desirable. A novel anthropomorphic lung phantom was developed by plastination of an actual pig lung. The plastinated phantom is characterized and compared with reference to in vivo images of the same tissue prior to plastination using high resolution 3D CT. The phantom is stable over time and preserves the anatomical features and relative locations of the in vivo sample. The volumes for different tissue types in the phantom are comparable to the in vivo counterparts, and CT numbers for different tissue types fall within a clinically useful range. Based on the measured CT numbers, the phantom cardiac tissue experienced a 92% decrease in bulk density and the phantom pulmonary tissue experienced a 78% decrease in bulk density compared to their in vivo counterparts. By-products in the phantom from the room temperature vulcanizing silicone and plastination process are also identified. A second generation phantom, which eliminates most of the by-products, is presented. Such anthropomorphic phantoms can be used to evaluate a wide range of novel imaging systems.
Assuntos
Antropometria/métodos , Imagens de Fantasmas , Algoritmos , Animais , Vasos Sanguíneos/patologia , Humanos , Processamento de Imagem Assistida por Computador , Pulmão/patologia , Modelos Anatômicos , Modelos Estatísticos , Miocárdio/patologia , Radiografia Torácica/métodos , Silicones/química , Suínos , Tomografia Computadorizada por Raios X/métodosRESUMO
Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive lean weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness, they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role in sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute to chronic cachexia development, pathology and potential reversal.
Assuntos
Bactérias/classificação , Caquexia/etiologia , Disbiose/etiologia , Toxoplasmose Animal/complicações , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Caquexia/imunologia , Caquexia/veterinária , Linhagem Celular , Citocinas/metabolismo , Disbiose/imunologia , Disbiose/veterinária , Feminino , Microbioma Gastrointestinal , Masculino , Camundongos , Fenótipo , Toxoplasma/patogenicidade , Toxoplasma/fisiologia , Toxoplasmose Animal/imunologia , Toxoplasmose Animal/parasitologiaRESUMO
Clostridium difficile is an opportunistic diarrhoeal pathogen, and C. difficile infection (CDI) represents a major health care concern, causing an estimated 15,000 deaths per year in the United States alone 1 . Several enteric pathogens, including C. difficile, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut 2 . Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota-accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant effect on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12 days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that C. difficile burdens are suppressed through the addition of either a diet containing a complex mixture of MACs or a simplified diet containing inulin as the sole MAC source. We show that switches between these dietary conditions are coincident with changes to microbiota membership, its metabolic output and C. difficile-mediated inflammation. Together, our data demonstrate the outgrowth of MAC-utilizing taxa and the associated end products of MAC metabolism, namely, the short-chain fatty acids acetate, propionate and butyrate, are associated with decreased C. difficile fitness despite increased C. difficile toxin expression in the gut. Our findings, when placed into the context of the known fibre deficiencies of a human Western diet, provide rationale for pursuing MAC-centric dietary strategies as an alternate line of investigation for mitigating CDI.
Assuntos
Antibacterianos/efeitos adversos , Infecções por Clostridium/dietoterapia , Carboidratos da Dieta/administração & dosagem , Disbiose/dietoterapia , Plantas/metabolismo , Animais , Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/induzido quimicamente , Infecções por Clostridium/complicações , Carboidratos da Dieta/farmacologia , Modelos Animais de Doenças , Disbiose/etiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Inulina/administração & dosagem , Inulina/farmacologia , Camundongos , Resultado do TratamentoRESUMO
The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S. Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.
Assuntos
Microbioma Gastrointestinal/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Propionatos/metabolismo , Infecções por Salmonella/etiologia , Salmonella typhimurium/patogenicidade , Animais , Derrame de Bactérias/fisiologia , Bacteroides/fisiologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Ácidos Graxos Voláteis/metabolismo , Transplante de Microbiota Fecal , Fezes/microbiologia , Feminino , Enteropatias/microbiologia , Masculino , Camundongos Endogâmicos C57BLRESUMO
The physiological role of an orphan G protein-coupled receptor, LGR5, was investigated by targeted deletion of this seven-transmembrane protein containing a large N-terminal extracellular domain with leucine-rich repeats. LGR5 null mice exhibited 100% neonatal lethality characterized by gastrointestinal tract dilation with air and an absence of milk in the stomach. Gross and histological examination revealed fusion of the tongue to the floor of oral cavity in the mutant newborns and immunostaining of LGR5 expression in the epithelium of the tongue and in the mandible of the wild-type embryos. The observed ankyloglossia phenotype provides a model for understanding the genetic basis of this craniofacial defect in humans and an opportunity to elucidate the physiological role of the LGR5 signaling system during embryonic development.