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Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy and one of the most common childhood cancers. Immunosuppressed patients, including HIV-infected patients, are more prone to KSHV-associated disease. KSHV encodes a viral protein kinase (vPK) that is expressed from ORF36. KSHV vPK contributes to the optimal production of infectious viral progeny and upregulation of protein synthesis. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used a bottom-up proteomics approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Subsequently, we validated this interaction using a co-immunoprecipitation assay. We report that both the ubiquitin-like and the catalytic domains of USP9X are important for association with vPK. To uncover the biological relevance of the USP9X/vPK interaction, we investigated whether the knockdown of USP9X would modulate viral reactivation. Our data suggest that depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Understanding how USP9X influences the reactivation of KSHV will provide insights into how cellular deubiquitinases regulate viral kinase activity and how viruses co-opt cellular deubiquitinases to propagate infection. Hence, characterizing the roles of USP9X and vPK during KSHV infection constitutes a first step toward identifying a potentially critical interaction that could be targeted by future therapeutics. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi sarcoma (KS), the plasmablastic form of multicentric Castleman's disease, and primary effusion lymphoma. In sub-Saharan Africa, KS is the most common HIV-related malignancy. KSHV encodes a viral protein kinase (vPK) that aids viral replication. To elucidate the interactions of vPK with cellular proteins in KSHV-infected cells, we used an affinity purification approach and identified host protein ubiquitin-specific peptidase 9X-linked (USP9X) as a potential interactor of vPK. Depletion of USP9X inhibits both viral reactivation and the production of infectious virions. Overall, our data suggest a proviral role for USP9X.
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Herpesvirus Humano 8 , Sarcoma de Kaposi , Ubiquitina Tiolesterasa , Niño , Humanos , Enzimas Desubicuitinizantes , Herpesvirus Humano 8/fisiología , Infecciones por VIH/complicaciones , Linfoma de Efusión Primaria , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/virología , Ubiquitina Tiolesterasa/genética , Proteínas Virales/genéticaRESUMEN
Answer questions and earn CME/CNE The human body harbors enormous numbers of microbiota that influence cancer susceptibility, in part through their prodigious metabolic capacity and their profound influence on immune cell function. Microbial pathogens drive tumorigenesis in 15% to 20% of cancer cases. Even larger numbers of malignancies are associated with an altered composition of commensal microbiota (dysbiosis) based on microbiome studies using metagenomic sequencing. Although association studies cannot distinguish whether changes in microbiota are causes or effects of cancer, a causative role is supported by rigorously controlled preclinical studies using gnotobiotic mouse models colonized with one or more specific bacteria. These studies demonstrate that microbiota can alter cancer susceptibility and progression by diverse mechanisms, such as modulating inflammation, inducing DNA damage, and producing metabolites involved in oncogenesis or tumor suppression. Evidence is emerging that microbiota can be manipulated for improving cancer treatment. By incorporating probiotics as adjuvants for checkpoint immunotherapy or by designing small molecules that target microbial enzymes, microbiota can be harnessed to improve cancer care. CA Cancer J Clin 2017;67:326-344. © 2017 American Cancer Society.
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Microbiota , Neoplasias/microbiología , Neoplasias/terapia , Animales , Carcinogénesis , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Disbiosis , Humanos , Metagenómica , Medicina de PrecisiónRESUMEN
Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.
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Microbioma Gastrointestinal/efectos de los fármacos , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/farmacología , Bacterias/efectos de los fármacos , Modelos Animales de Enfermedad , Disbiosis/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Femenino , Glucuronidasa/metabolismo , Humanos , Irinotecán/farmacología , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológicoRESUMEN
The intestinal microbiome encodes vast metabolic potential, and multidisciplinary approaches are enabling a mechanistic understanding of how bacterial enzymes impact the metabolism of diverse pharmaceutical compounds, including chemotherapeutics. Microbiota alter the activity of many drugs and chemotherapeutics via direct and indirect mechanisms; some of these alterations result in changes to the drug's bioactivity and bioavailability, causing toxic gastrointestinal side effects. Gastrointestinal toxicity is one of the leading complications of systemic chemotherapy, with symptoms including nausea, vomiting, diarrhea, and constipation. Patients undergo dose reductions or drug holidays to manage these adverse events, which can significantly harm prognosis, and can result in mortality. Selective and precise targeting of the gut microbiota may alleviate these toxicities. Understanding the composition and function of the microbiota may serve as a biomarker for prognosis, and predict treatment efficacy and potential adverse effects, thereby facilitating personalized medicine strategies for cancer patients.
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Antineoplásicos/efectos adversos , Enfermedades Gastrointestinales/inducido químicamente , Microbioma Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/efectos de los fármacos , Antineoplásicos/administración & dosificación , Enfermedades Gastrointestinales/microbiología , Enfermedades Gastrointestinales/fisiopatología , Microbioma Gastrointestinal/fisiología , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/fisiopatología , Humanos , Medicina de Precisión/métodos , Medicina de Precisión/tendencias , Probióticos/administración & dosificaciónRESUMEN
Recent advances suggest that in vivo reprogramming of endogenous cell populations provides a viable alternative for neuron replacement. Astrocytes and oligodendrocyte precursor cells can be induced to transdifferentiate into neurons in the CNS, but, in these instances, reprogramming requires either transgenic mice or retroviral-mediated gene expression. We developed a microRNA (miRNA)-GFP construct that in vitro significantly reduced the expression of polypyrimidine tract-binding protein, and, subsequently, we packaged this construct in a novel oligodendrocyte preferring adeno-associated virus vector. Ten days after rat striatal transduction, the vast majority of the GFP-positive cells were oligodendrocytes, but 6 weeks to 6 months later, the majority of GFP-positive cells exhibited neuronal morphology and co-localized with the neuronal marker NeuN. Patch-clamp studies on striatal slices established that the GFP-positive cells exhibited electrophysiological properties indicative of mature neurons, such as spontaneous action potentials and spontaneous inhibitory postsynaptic currents. Also, 3 months after striatal vector administration, GFP-positive terminals in the ipsilateral globus pallidus or substantia nigra retrogradely transported fluorescent beads back to GFP-positive striatal cell bodies, indicating the presence of functional presynaptic terminals. Thus, this viral vector approach provides a potential means to harness resident oligodendrocytes as an endogenous source for in vivo neuronal replacement.
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Transdiferenciación Celular/genética , Reprogramación Celular/genética , Cuerpo Estriado/citología , Vectores Genéticos/genética , Neuronas/citología , Oligodendroglía/citología , Animales , Línea Celular , Dependovirus/genética , Humanos , Neuronas/metabolismo , Oligodendroglía/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , RatasRESUMEN
UNLABELLED: Understanding the entry and trafficking mechanism(s) of recombinant adeno-associated virus (rAAV) into host cells can lead to evolution in capsid and vector design and delivery methods, resulting in enhanced transduction and therapeutic gene expression. Variability of findings regarding the early entry pathway of rAAV supports the possibility that rAAV, like other viruses, can utilize more than one infectious entry pathway. We tested whether inhibition of macropinocytosis impacted rAAV transduction of HeLa cells compared to hepatocellular carcinoma cell lines. We found that macropinocytosis inhibitor cytochalasin D blocked rAAV transduction of HeLa cells (>2-fold) but enhanced (10-fold) transduction in HepG2 and Huh7 lines. Similar results were obtained with another macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA). The augmented transduction was due to neither viral binding nor promoter activity, affected multiple rAAV serotypes (rAAV2, rAAV2-R585E, and rAAV8), and influenced single-stranded and self-complementary virions to comparable extents. Follow-up studies using CDC42 inhibitor ML141 and p21-activated kinase 1 (PAK1) siRNA knockdown also resulted in enhanced HepG2 transduction. Microscopy revealed that macropinocytosis inhibition correlated with expedited nuclear entry of the rAAV virions into HepG2 cells. Enhancement of hepatocellular rAAV transduction extended to the mouse liver in vivo (4-fold enhancement) but inversely blocked heart tissue transduction (13-fold). This evidence of host cell-specific rAAV entry pathways confers a potent means for controlling and enhancing vector delivery and could help unify the divergent accounts of rAAV cellular entry mechanisms. IMPORTANCE: There is a recognized need for improved rAAV vector targeting strategies that result in delivery of fewer total particles, averting untoward toxicity and/or an immune response against the vector. A critical step in rAAV transduction is entry and early trafficking through the host cellular machinery, the mechanisms of which are under continued study. However, should the early entry and trafficking mechanisms of rAAV differ across virus serotype or be dependent on host cell environment, this could expand our ability to target particular cells and tissue for selective transduction. Thus, the observation that inhibiting macropinocytosis leads to cell-specific enhancement or inhibition of rAAV transduction that extends to the organismic level exposes a new means of modulating vector targeting.
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Dependovirus/fisiología , Transducción Genética , Internalización del Virus , Línea Celular , Dependovirus/genética , Vectores Genéticos , HumanosRESUMEN
The metabolic differences between B-NHL and primary human B cells are poorly understood. Among human B-cell non-Hodgkin lymphomas (B-NHL), primary effusion lymphoma (PEL) is a unique subset that is linked to infection with Kaposi's sarcoma-associated herpesvirus (KSHV). We report that the metabolic profiles of primary B cells are significantly different from that of PEL. Compared with primary B cells, both aerobic glycolysis and fatty acid synthesis (FAS) are up-regulated in PEL and other types of nonviral B-NHL. We found that aerobic glycolysis and FAS occur in a PI3K-dependent manner and appear to be interdependent. PEL overexpress the fatty acid synthesizing enzyme, FASN, and both PEL and other B-NHL were much more sensitive to the FAS inhibitor, C75, than primary B cells. Our findings suggest that FASN may be a unique candidate for molecular targeted therapy against PEL and other B-NHL.
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Linfocitos B/metabolismo , Ácidos Grasos/biosíntesis , Glucólisis/fisiología , Linfoma de Células B/metabolismo , Linfoma de Efusión Primaria/metabolismo , Redes y Vías Metabólicas/fisiología , Transducción de Señal/fisiología , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Biología Computacional , Ácido Graso Sintasas/antagonistas & inhibidores , Humanos , Immunoblotting , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Undernutrition in children commonly disrupts the structure and function of the small intestinal microbial community, leading to enteropathies, compromised metabolic health, and impaired growth and development. The mechanisms by which diet and microbes mediate the balance between commensal and pathogenic intestinal flora remain elusive. In a murine model of undernutrition, we investigated the direct interactions Giardia lamblia, a prevalent small intestinal pathogen, on indigenous microbiota and specifically on Lactobacillus strains known for their mucosal and growth homeostatic properties. Our research reveals that Giardia colonization shifts the balance of lactic acid bacteria, causing a relative decrease in Lactobacillus spp . and an increase in Bifidobacterium spp . This alteration corresponds with a decrease in multiple indicators of mucosal and nutritional homeostasis. Additionally, protein-deficient conditions coupled with Giardia infection exacerbate the rise of primary bile acids and susceptibility to bile acid-induced intestinal barrier damage. In epithelial cell monolayers, Lactobacillus spp . mitigated bile acid-induced permeability, showing strain-dependent protective effects. In vivo, L. plantarum, either alone or within a Lactobacillus spp consortium, facilitated growth in protein-deficient mice, an effect attenuated by Giardia , despite not inhibiting Lactobacillus colonization. These results highlight Giardia's potential role as a disruptor of probiotic functional activity, underscoring the imperative for further research into the complex interactions between parasites and bacteria under conditions of nutritional deficiency.
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BACKGROUND: Understanding the cause vs consequence relationship of gut inflammation and microbial dysbiosis in inflammatory bowel diseases (IBD) requires a reproducible mouse model of human-microbiota-driven experimental colitis. RESULTS: Our study demonstrated that human fecal microbiota transplant (FMT) transfer efficiency is an underappreciated source of experimental variability in human microbiota-associated (HMA) mice. Pooled human IBD patient fecal microbiota engrafted germ-free (GF) mice with low amplicon sequence variant (ASV)-level transfer efficiency, resulting in high recipient-to-recipient variation of microbiota composition and colitis severity in HMA Il-10-/- mice. In contrast, mouse-to-mouse transfer of mouse-adapted human IBD patient microbiota transferred with high efficiency and low compositional variability resulting in highly consistent and reproducible colitis phenotypes in recipient Il-10-/- mice. Engraftment of human-to-mouse FMT stochastically varied with individual transplantation events more than mouse-adapted FMT. Human-to-mouse FMT caused a population bottleneck with reassembly of microbiota composition that was host inflammatory environment specific. Mouse-adaptation in the inflamed Il-10-/- host reassembled a more aggressive microbiota that induced more severe colitis in serial transplant to Il-10-/- mice than the distinct microbiota reassembled in non-inflamed WT hosts. CONCLUSIONS: Our findings support a model of IBD pathogenesis in which host inflammation promotes aggressive resident bacteria, which further drives a feed-forward process of dysbiosis exacerbated by gut inflammation. This model implies that effective management of IBD requires treating both the dysregulated host immune response and aggressive inflammation-driven microbiota. We propose that our mouse-adapted human microbiota model is an optimized, reproducible, and rigorous system to study human microbiome-driven disease phenotypes, which may be generalized to mouse models of other human microbiota-modulated diseases, including metabolic syndrome/obesity, diabetes, autoimmune diseases, and cancer. Video Abstract.
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Modelos Animales de Enfermedad , Disbiosis , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Interleucina-10 , Animales , Humanos , Ratones , Enfermedades Inflamatorias del Intestino/microbiología , Disbiosis/microbiología , Interleucina-10/genética , Colitis/microbiología , Heces/microbiología , Colon/microbiología , Ratones Noqueados , Ratones Endogámicos C57BL , Femenino , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Inflamación , MasculinoRESUMEN
Women are at significantly greater risk of metabolic dysfunction after menopause, which subsequently leads to numerous chronic illnesses. The gut microbiome is associated with obesity and metabolic dysfunction, but its interaction with female sex hormone status and the resulting impact on host metabolism remains unclear. Herein, we characterized inflammatory and metabolic phenotypes as well as the gut microbiome associated with ovariectomy and high-fat diet feeding, compared to gonadal intact and low-fat diet controls. We then performed fecal microbiota transplantation (FMT) using gnotobiotic mice to identify the impact of ovariectomy-associated gut microbiome on inflammatory and metabolic outcomes. We demonstrated that ovariectomy led to greater gastrointestinal permeability and inflammation of the gut and metabolic organs, and that a high-fat diet exacerbated these phenotypes. Ovariectomy also led to alteration of the gut microbiome, including greater fecal ß-glucuronidase activity. However, differential changes in the gut microbiome only occurred when fed a low-fat diet, not the high-fat diet. Gnotobiotic mice that received the gut microbiome from ovariectomized mice fed the low-fat diet had greater weight gain and hepatic gene expression related to metabolic dysfunction and inflammation than those that received intact sham control-associated microbiome. These results indicate that the gut microbiome responds to alterations in female sex hormone status and contributes to metabolic dysfunction. Identifying and developing gut microbiome-targeted modulators to regulate sex hormones may be useful therapeutically in remediating menopause-related diseases.
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Microbioma Gastrointestinal , Humanos , Femenino , Ratones , Animales , Microbioma Gastrointestinal/fisiología , Obesidad/metabolismo , Hígado/metabolismo , Dieta Alta en Grasa/efectos adversos , Inflamación/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Ratones Endogámicos C57BLRESUMEN
Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial ß-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation. We demonstrate that a range of FDA-approved drugs prevent this reactivation by intercepting the catalytic cycle of the enzymes in a conserved fashion. Finally, we find that inhibiting GUS in conventional mice reduces free serotonin and increases its inactive glucuronide in the serum and intestines. Our results illuminate the indispensability of gut microbial enzymes in sustaining endobiotic homeostasis and indicate that therapeutic disruptions of this metabolism promote interindividual response variabilities.
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Microbioma Gastrointestinal , Glucuronidasa , Homeostasis , Animales , Microbioma Gastrointestinal/efectos de los fármacos , Ratones , Glucuronidasa/metabolismo , Ratones Endogámicos C57BL , Serotonina/metabolismo , Glucurónidos/metabolismo , Humanos , Intestinos/microbiología , Masculino , Vida Libre de GérmenesRESUMEN
The world is witnessing a global increase in the urban population, particularly in developing Asian and African countries. Concomitantly, the global burden of non-communicable diseases (NCDs) is rising, markedly associated with the changing landscape of lifestyle and environment during urbanization. Accumulating studies have revealed the role of the gut microbiome in regulating the immune and metabolic homeostasis of the host, which potentially bridges external factors to the host (patho-)physiology. In this review, we discuss the rising incidences of NCDs during urbanization and their links to the compositional and functional dysbiosis of the gut microbiome. In particular, we elucidate the effects of urbanization-associated factors (hygiene/pollution, urbanized diet, lifestyles, the use of antibiotics, and early life exposure) on the gut microbiome underlying the pathogenesis of NCDs. We also discuss the potential and feasibility of microbiome-inspired and microbiome-targeted approaches as novel avenues to counteract NCDs, including fecal microbiota transplantation, diet modulation, probiotics, postbiotics, synbiotics, celobiotics, and precision antibiotics.
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Microbioma Gastrointestinal , Microbiota , Enfermedades no Transmisibles , Probióticos , Humanos , Microbioma Gastrointestinal/fisiología , Urbanización , Enfermedades no Transmisibles/terapia , Enfermedades no Transmisibles/tratamiento farmacológico , Trasplante de Microbiota Fecal , Antibacterianos/uso terapéutico , Disbiosis/tratamiento farmacológico , PrebióticosRESUMEN
Primary effusion lymphoma (PEL) constitutes a subset of non-Hodgkin lymphoma whose incidence is highly increased in the context of HIV infection. Kaposi sarcoma-associated herpesvirus is the causative agent of PEL. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in cell proliferation and survival, and this pathway is dysregulated in many different cancers, including PEL, which display activated PI3K, Akt, and mammalian target of rapamycin (mTOR) kinases. PELs rely heavily on PI3K/Akt/mTOR signaling, are dependent on autocrine and paracrine growth factors, and also have a poor prognosis with reported median survival times of less than 6 months. We compared different compounds that inhibit the PI3K/Akt/mTOR pathway in PEL. Although compounds that modulated activity of only a single pathway member inhibited PEL proliferation, the use of a novel compound, NVP-BEZ235, that dually inhibits both PI3K and mTOR kinases was significantly more efficacious in culture and in a PEL xenograft tumor model. NVP-BEZ235 was effective at low nanomolar concentrations and has oral bioavailability. We also report a novel mechanism for NVP-BEZ235 involving the suppression of multiple autocrine and paracrine growth factors required for lymphoma survival. Our data have broad applicability for the treatment of cytokine-dependent tumors with PI3K/mTOR dual inhibitors.
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Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Linfoma de Efusión Primaria/tratamiento farmacológico , Linfoma de Efusión Primaria/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinolinas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Comunicación Autocrina/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Comunicación Paracrina/efectos de los fármacos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Rosiglitazona , Serina-Treonina Quinasas TOR , Tiazolidinedionas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Success of live biotherapeutics depends upon sustained and durable engraftment of beneficial microbes with robust functional output. In this issue of Cell Host & Microbe, Button et al. (2022) report that a human milk oligosaccharide-Bifidobacterium synbiotic delivers by supporting functional engraftment in healthy adults without antibiotic administration.
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Simbióticos , Adulto , Bifidobacterium , Humanos , Leche Humana , OligosacáridosRESUMEN
The hydrolysis of xenobiotic glucuronides by gut bacterial glucuronidases reactivates previously detoxified compounds resulting in severe gut toxicity for the host. Selective bacterial ß-glucuronidase inhibitors can mitigate this toxicity but their impact on wider host metabolic processes has not been studied. To investigate this the inhibitor 4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine (UNC10201652, Inh 9) was administered to mice to selectively inhibit a narrow range of bacterial ß-glucuronidases in the gut. The metabolomic profiles of the intestinal contents, biofluids, and several tissues involved in the enterohepatic circulation were measured and compared to control animals. No biochemical perturbations were observed in the plasma, liver or gall bladder. In contrast, the metabolite profiles of urine, colon contents, feces and gut wall were altered compared to the controls. Changes were largely restricted to compounds derived from gut microbial metabolism. This work establishes that inhibitors targeted towards bacterial ß-glucuronidases modulate the functionality of the intestinal microbiota without adversely impacting the host metabolic system.
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Microbioma Gastrointestinal , Glucuronidasa , Ratones , Animales , Glucuronidasa/metabolismo , Microbioma Gastrointestinal/fisiología , Xenobióticos , Bacterias/metabolismo , MorfolinasRESUMEN
The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement. We used a human HER2/neu-specific IgE to arm human MCs through the high affinity IgE receptor (FcεRI). The ability of MCs to bind to and induce apoptosis of HER2/neu-positive cancer cells in vitro and in vivo was assessed. The interactions between MCs and cancer cells were investigated in real time using confocal microscopy. The mechanism of action using cytotoxic MCs was examined using gene array profiling. Genetically manipulating autologous MC to assess the effects of MC-specific mediators have on apoptosis of tumor cells was developed using siRNA. We found that HER2/neu tumor-specific IgE-sensitized MCs bound, penetrated, and killed HER2/neu-positive tumor masses in vitro. Tunneling nanotubes formed between MCs and tumor cells are described that parallel tumor cell apoptosis. In solid tumor, human breast cancer (BC) xenograft mouse models, infusion of HER2/neu IgE-sensitized human MCs co-localized to BC cells, decreased tumor burden, and prolonged overall survival without indications of toxicity. Gene microarray of tumor cells suggests a dependence on TNF and TGFß signaling pathways leading to apoptosis. Knocking down MC-released tryptase did not affect apoptosis of cancer cells. These studies suggest MCs can be polarized from Type I hypersensitivity-mediating cells to cytotoxic cells that selectively target tumor cells and specifically triggered to release anti-tumor mediators. A strategy to investigate which MC mediators are responsible for the observed tumor killing is described so that rational decisions can be made in the future when selecting which mediators to target for deletion or those that could further polarize them to cytotoxic MC by adding other known anti-tumor agents. Using autologous human MC may provide further options for cancer therapeutics that offers a unique anti-cancer mechanism of action using tumor targeted IgE's.
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BACKGROUND & AIMS: Single-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 human beings. METHODS: A total of 12,590 single epithelial cells from 3 independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and their capacity for response to extrinsic signals along the gut axis across different human beings. RESULTS: Cells were assigned to 25 epithelial lineage clusters. Multiple accepted intestinal stem cell markers do not specifically mark all human intestinal stem cells. Lysozyme expression is not unique to human Paneth cells, and Paneth cells lack expression of expected niche factors. Bestrophin 4 (BEST4)+ cells express Neuropeptide Y (NPY) and show maturational differences between the small intestine and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell junctions, and nutrient absorption genes show unappreciated regional expression differences across lineages. The differential expression of receptors and drug targets across lineages show biological variation and the potential for variegated responses. CONCLUSIONS: Our study identifies novel lineage marker genes, covers regional differences, shows important differences between mouse and human gut epithelium, and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves likely functional differences across anatomic regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.
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Mucosa Intestinal , Transcriptoma , Animales , Colon , Epitelio , Humanos , Mucosa Intestinal/metabolismo , Intestino Delgado , Ratones , Transcriptoma/genéticaRESUMEN
Mycophenolate mofetil (MMF) is an important immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage, however, is limited by severe gastrointestinal toxicity that is observed in approximately 45% of MMF recipients. The active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepatic recirculation by bacterial ß-glucuronidase (GUS) enzymes, which reactivate MPA from mycophenolate glucuronide (MPAG) within the gastrointestinal tract. GUS enzymes demonstrate distinct substrate preferences based on their structural features, and gut microbial GUS enzymes that reactivate MPA have not been identified. Here, we compare the fecal microbiomes of transplant recipients receiving MMF to healthy individuals using shotgun metagenomic sequencing. We find that neither microbial composition nor the presence of specific structural classes of GUS genes are sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We next employed a GUS-specific activity-based chemical probe and targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples. The identification of specific GUS enzymes was improved by using the metagenomics data collected from the fecal samples. We found that the presence of GUS enzymes that bind the flavin mononucleotide (FMN) is significantly correlated with efficient MPA reactivation. Furthermore, structural analysis identified motifs unique to these FMN-binding GUS enzymes that provide molecular support for their ability to process this drug glucuronide. These results indicate that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.
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Microbioma Gastrointestinal , Mononucleótido de Flavina , Microbioma Gastrointestinal/fisiología , Glucurónidos , Humanos , Inmunosupresores , Ácido Micofenólico/uso terapéutico , ProteómicaRESUMEN
Commonly used synthetic dietary emulsifiers, including carboxymethylcellulose (CMC) and polysorbate-80 (P80), promote intestinal inflammation. We compared abilities of CMC vs. P80 to potentiate colitis and impact human microbiota in an inflammatory environment using a novel colitis model of ex-germ-free (GF) IL10-/- mice colonized by pooled fecal transplant from three patients with active inflammatory bowel diseases. After three days, mice received 1% CMC or P80 in drinking water or water alone for four weeks. Inflammation was quantified by serial fecal lipocalin 2 (Lcn-2) and after four weeks by blinded colonic histologic scores and colonic inflammatory cytokine gene expression. Microbiota profiles in cecal contents were determined by shotgun metagenomic sequencing. CMC treatment significantly increased fecal Lcn-2 levels compared to P80 and water treatment by one week and throughout the experiment. Likewise, CMC treatment increased histologic inflammatory scores and colonic inflammatory cytokine gene expression compared with P80 and water controls. The two emulsifiers differentially affected specific intestinal microbiota. CMC did not impact bacterial composition but significantly decreased Caudoviricetes (bacteriophages), while P80 exposure non-significantly increased the abundance of both Actinobacteria and Proteobacteria. Commonly used dietary emulsifiers have different abilities to induce colitis in humanized mice. CMC promotes more aggressive inflammation without changing bacterial composition.