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The review focused mainly on the pathogenesis of hepatogenous diabetes (HD) in liver cirrhosis (LC). This review reveals parallels between the mechanisms of metabolic dysfunction observed in LC and type II diabetes (T2DM), suggesting a shared pathway leading to HD. It underscores the role of insulin in HD pathogenesis, highlighting key factors such as insulin signaling, glucose metabolism, insulin resistance (IR), and the influence of adipocytes. Furthermore, the impact of adipose tissue accumulation, fatty acid metabolism, and pro-inflammatory cytokines like Tumor necrosis factor-α (TNF-α) on IR are discussed in the context of HD. Altered signaling pathways, disruptions in the endocrine system, liver inflammation, changes in muscle mass and composition, and modifications to the gut microbiota collectively contribute to the complex interplay linking cirrhosis and HD. This study highlights how important it is to identify and treat this complex condition in cirrhotic patients by thoroughly analyzing the link between cirrhosis, IR, and HD. It also emphasizes the vitality of targeted interventions. Cellular and molecular investigations into IR have revealed potential therapeutic targets for managing and preventing HD.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Cirrosis Hepática , Humanos , Cirrosis Hepática/metabolismo , Resistencia a la Insulina/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Animales , Transducción de Señal , Insulina/metabolismoRESUMEN
Ulcerative colitis (UC) is a chronic problem of the intestine and relapsing in nature. Biochanin A is a nature-derived isoflavonoid and has numerous bioactivities. However, its role against UC and intestinal inflammation remains obscure. We aimed to comprehensively explore the pharmacological effect of biochanin A in alleviating colitis and to evaluate the potential mechanisms. Initially, we explored the anti-inflammatory action of biochanin A (15, 30, and 60 µM) by employing lipopolysaccharide (LPS)-activated RAW 264.7 cells. In RAW 264.7 cells under LPS stimulation, biochanin A inhibited the elevation of reactive oxygen species (ROS) (p < 0.0001), interleukin (IL)-1ß (p < 0.0001), IL-18 (p < 0.01), and tumor necrosis factor (TNF)-α (p < 0.01) release, nitrite production (p < 0.0001), and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. Next, we studied the effectiveness of biochanin A (20 and 40 mg/kg) in mouse colitis induced with dextran sulfate sodium (DSS) by assessing colon length, disease activity index (DAI) scoring, and performing colonoscopy and histological analysis. The pro-inflammatory cytokines were estimated using ELISA. Western blot studies were performed to assess underlying mechanisms. In mice, biochanin A treatment alleviated DAI score (p < 0.0001), restored colon length (p < 0.05) and morphology, and re-established colon histopathology. Biochanin A affects the phosphorylation of proteins associated with NF-κB (p65) and mitogen-activated protein kinase (MAPK) axis and regulates colonic inflammation by reducing the expression of inflammatory cytokines and myeloperoxidase (MPO) activity. Altogether, our findings support the idea that the anticolitis potential of biochanin A is allied with anti-inflammatory activity by inhibiting the MAPK/NF-κB (p65) axis. Hence, biochanin A may be an alternative option to alleviate the risk of colitis.
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Colitis Ulcerosa , Genisteína , Factor de Transcripción ReIA , Animales , Genisteína/farmacología , Ratones , Células RAW 264.7 , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Factor de Transcripción ReIA/metabolismo , Masculino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Sulfato de Dextran/toxicidadRESUMEN
Chronic kidney disease (CKD) adversely affects the heart. The underlying mechanism and the interplay between the kidney and the heart are still obscure. We examined the cardiac effect using the unilateral ureteral obstruction (UUO)-induced CKD pre-clinical model in mice. Echocardiography, histopathology of the heart, myocardial mRNA expression of ANP and BNP, the extent of fibrotic (TGF-ß, α-SMA, and collagen I) and epigenetic (histone deacetylases, namely HDAC3, HDAC4, and HDAC6) proteins, and myocardial inflammatory response were assessed. Six weeks of post-UUO surgery, we observed a compromised left-ventricular wall thickness and signs of cardiac hypertrophy, accumulation of fibrosis associated, and inflammatory proteins in the heart. In addition, we observed a perturbation of epigenetic proteins, especially HDAC3, HDAC4, and HDAC6, in the heart. Pharmacological inhibition of HDAC6 using ricolinostat (RIC) lessened cardiac damage and improved left-ventricular wall thickness. The RIC treatment substantially restored the serum cardiac injury markers, namely creatine kinase-MB and lactate dehydrogenase (LDH) activities, ANP and BNP mRNA expression, and heart histological changes. The extent of myocardial fibrotic proteins, phospho-NF-κB (p65), and pro-inflammatory cytokines (TNF-α, IL-18, and IL-1ß) were significantly decreased in the RIC treatment group. Further findings revealed the CKD-induced infiltration of CD3, CD8a, CD11c, and F4/80 positive inflammatory cells in the heart. Treatment with RIC substantially reduced the myocardial infiltration of these inflammatory cells. From these findings, we believe that CKD-induced myocardial HDAC6 perturbation has a deteriorative effect on the heart, and inhibition of HDAC6 can be a promising approach to alleviate CKD-induced myocardial remodeling.
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Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 µg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.
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Supervivencia Celular , Modelos Animales de Enfermedad , Matriz Extracelular , Glaucoma , Fármacos Neuroprotectores , Extractos Vegetales , Especies Reactivas de Oxígeno , Células Ganglionares de la Retina , Solanum nigrum , Animales , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Glaucoma/tratamiento farmacológico , Glaucoma/patología , Glaucoma/metabolismo , Ratas , Solanum nigrum/química , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Supervivencia Celular/efectos de los fármacos , Masculino , Conejos , Presión Intraocular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) continues to pose a significant health challenge and is often diagnosed at advanced stages. Metabolic reprogramming is a hallmark of many cancer types, including HCC and it involves alterations in various metabolic or nutrient-sensing pathways within liver cells to facilitate the rapid growth and progression of tumours. However, the role of STAT3-NFκB in metabolic reprogramming is still not clear. APPROACH AND RESULTS: Diethylnitrosamine (DEN) administered animals showed decreased body weight and elevated level of serum enzymes. Also, Transmission electron microscopy (TEM) analysis revealed ultrastructural alterations. Increased phosphorylated signal transducer and activator of transcription-3 (p-STAT3), phosphorylated nuclear factor kappa B (p-NFκß), dynamin related protein 1 (Drp-1) and alpha-fetoprotein (AFP) expression enhance the carcinogenicity as revealed in immunohistochemistry (IHC). The enzyme-linked immunosorbent assay (ELISA) concentration of IL-6 was found to be elevated in time dependent manner both in blood serum and liver tissue. Moreover, immunoblot analysis showed increased level of p-STAT3, p-NFκß and IL-6 stimulated the upregulation of mitophagy proteins such as Drp-1, Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK-1). Meanwhile, downregulation of Poly [ADP-ribose] polymerase 1 (PARP-1) and cleaved caspase 3 suppresses apoptosis and enhanced expression of AFP supports tumorigenesis. The mRNA level of STAT3 and Drp-1 was also found to be significantly increased. Furthermore, we performed high-field 800 MHz Nuclear Magnetic Resonance (NMR) based tissue and serum metabolomics analysis to identify metabolic signatures associated with the progression of liver cancer. The metabolomics findings revealed aberrant metabolic alterations in liver tissue and serum of 75th and 105th days of intervention groups in comparison to control, 15th and 45th days of intervention groups. Tissue metabolomics analysis revealed the accumulation of succinate in the liver tissue samples, whereas, serum metabolomics analysis revealed significantly decreased circulatory levels of ketone bodies (such as 3-hydroxybutyrate, acetate, acetone, etc.) and membrane metabolites suggesting activated ketolysis in advanced stages of liver cancer. CONCLUSION: STAT3-NFκß signaling axis has a significant role in mitochondrial dysfunction and metabolic alterations in the development of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedades Mitocondriales , Transducción de Señal , Animales , alfa-Fetoproteínas/metabolismo , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Interleucina-6/metabolismo , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/metabolismo , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Liver fibrosis is a significant health burden worldwide and has emerged as the leading cause of Hepatocellular carcinoma (HCC) incidence. Mitochondria are the dynamic organelles that regulate the differentiation, survival, and polarization of macrophages. Nuclear-DNA-associated proteins, micro-RNAs, as well as macrophage polarization are essential for maintaining intracellular and extra-cellular homeostasis in the liver parenchyma. Dysregulated mitochondrial coding genes (ETS complexes I, II, III, IV, and V), non-coding RNAs (mitomiRs), and nuclear alteration lead to the production of reactive oxygen species (ROS) and inflammation which are implicated in the transition of liver fibrosis into HCC. Recent findings indicated the protecting effect of E74-like factor 3/peroxisome proliferator-activated receptor-γ (Elf-3/PPAR-γ). HDAR-y inhibits the deacetylation of PPAR-y and maintains the PPAR-y pathway. Elf-3 plays a tumor suppressive role through epithelial-mesenchymal transition-related gene and zinc finger E-box binding homeobox 2 (ZEB-2) domain. Additionally, the development of HCC includes the PI3K/Akt/mTOR and transforming Growth Factor ß (TGF-ß) pathway that promotes the Epithelial-mesenchymal transition (EMT) through Smad/Snail/Slug signaling cascade. In contrast, the TLR2/NOX2/autophagy axis promotes M2 polarization in HCC. Thus, a thorough understanding of the mitochondrial and nuclear reciprocal relationship related to macrophage polarization could provide new research opportunities concerning diseases with a significant impact on liver parenchyma towards developing liver fibrosis or liver cancer. Moreover, this knowledge can be used to develop new therapeutic strategies to treat liver diseases.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores Activados del Proliferador del Peroxisoma/farmacología , Línea Celular Tumoral , Transducción de Señal , Cirrosis Hepática/patología , Mitocondrias/metabolismo , Macrófagos/metabolismo , Transición Epitelial-MesenquimalRESUMEN
Neurological disorders are possibly the most prevalent and have been identified to occur among individuals with autism beyond chance. These disorders encompass a diverse range of consequences with neurological causes and have been regarded as a major threat to public mental health. There is no tried-and-true approach for completely protecting the nervous system. Therefore, plant-derived compounds have developed significantly nowadays. Coumestrol (CML) is a potent isoflavone phytoestrogen with a protective effect against neurological dysfunction and has been discovered to be structurally and functionally similar to estrogen. In recent years, more research has been undertaken on phytoestrogens. This research demonstrates the biological complexity of phytoestrogens, which consist of multiple chemical families and function in various ways. This review aimed to explore recent findings on the most significant pharmacological advantages of CML by emphasising neurological benefits. Numerous CML extraction strategies and their pharmacological effects on various neurological disorders, including PD, AD, HD, anxiety, and cognitive impairments, were also documented.
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The gastrointestinal (GI) epithelium plays a major role in nutrient absorption, barrier formation, and innate immunity. The development of organoid-based methodology has significantly impacted the study of the GI epithelium, particularly in the fields of mucosal biology, immunity, and host-microbe interactions. Various effects on the GI epithelium, such as genetics and nutrition, impact patients and alter disease states. Thus, incorporating these effects into organoid-based models will facilitate a better understanding of disease progression and offer opportunities to evaluate therapeutic candidates. One condition that has a significant effect on the GI epithelium is malnutrition, and studying the mechanistic impacts of malnutrition would enhance our understanding of several pathologies. Therefore, the goal of this study was to begin to develop methodology to generate viable malnourished organoids with accessible techniques and resources that can be used for a wide array of mechanistic studies. By selectively limiting distinct macronutrient components of organoid media, we were able to successfully culture and evaluate malnourished organoids. Genetic and protein-based analyses were used to validate the approach and confirm the presence of known biomarkers of malnutrition. Additionally, as proof-of-concept, we utilized malnourished organoid-derived monolayers to evaluate the effect of malnourishment on barrier formation and the ability of the bacterial pathogen Shigella flexneri to infect the GI epithelium. This work serves as the basis for new and exciting techniques to alter the nutritional state of organoids and investigate the related impacts on the GI epithelium.
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Microbioma Gastrointestinal , Desnutrición , Humanos , Estado Nutricional , Epitelio , OrganoidesRESUMEN
Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide. Cytochrome P450 2E1 (CYP2E1) is an enzyme, primarily involved in the metabolism of xenobiotics and procarcinogens. The present study was designed to investigate the potential role of CYP2E1 triggered endoplasmic reticulum stress in the progression of HCC through inhibition of apoptosis. In vitro CYP2E1 promotes HepG2 cell migration, reduced chromatin condensation, enhanced intracellular ROS accumulation and induce cell cycle progression. Conversely this effect was averted by CYP2E1 siRNA, selective inhibitor Diallyl sulphide (DAS) and antioxidants (vitamin C and E). In vivo Diethylnitrosamine (DEN) induced HCC rats showed decreased body weight and increased relative liver weight. Moreover, macro trabecular-massive HCC (MTM-HCC) histological subtyping showed pathological features like well-differentiated tumors, micro-trabecular and pseudo glandular patterns, megakaryocytes and cholestasis. Masson's trichrome staining revealed an intensive accumulation of collagen fibers in the extracellular matrix (ECM). Increased CYP2E1, VEGF and PCNA enhance the carcinogenicity as revealed in immunohistochemistry results. Immunoblot analysis showed reduced expression of copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) in cytosolic as well as mitochondrial fraction of rat liver tissue respectively. Also, increased level of CYP2E1 stimulated the upregulation of unfolded proteins response (UPR) and ER stress-related proteins such as Glucose regulatory protein 78 (GRP78), activating transcription factor 6 (ATF6) and CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP). Meanwhile, CYP2E1 stimulated ER-stress reduces BCL2 and downregulates the cleaved caspase 3 thus suppresses apoptosis. in. Furthermore, immunofluorescence revealed increased expression level of α-SMA in the HCC rat liver tissue. The level of CYP2E1 mRNA was significantly increased. Altogether, these findings indicate that CYP2E1 has a dynamic role in the pathogenesis of HCC and might be a budding agent in liver carcinogenesis therapy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Ratas , Factor de Transcripción Activador 6 , Apoptosis , Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Neoplasias Hepáticas/metabolismo , Factores de Transcripción , Factor de Transcripción CHOP , HumanosRESUMEN
There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.
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Disentería Bacilar , Vacunas contra la Shigella , Shigella , Humanos , Niño , Animales , Ratones , Preescolar , Shigella flexneri , Vacunas Conjugadas , Disentería Bacilar/prevención & control , Lipopolisacáridos , Antígenos O , Anticuerpos Antibacterianos , Inmunoglobulina GRESUMEN
Hepatocellular carcinoma (HCC) is among the deadliest cancer in human malignancies. It is the most common and severe type of primary liver cancer. However, the molecular mechanisms underlying HCC pathogenesis remain poorly understood. Long non-coding RNAs (lncRNAs), a new kind of RNA and epigenetic factors, play a crucial role in tumorigenesis and the progression of HCC. LncRNAs are capable of promoting the autophagy, proliferation, and migration of tumor cells by targeting and modulating the expression of downstream genes in signaling pathways related to cancer; these transcripts modify the activity and expression of various tumor suppressors and oncogenes. LncRNAs could act as biomarkers for treatment approaches such as immunotherapy, chemotherapy, and surgery to effectively treat HCC patients. Improved knowledge regarding the aetiology of HCC may result from an advanced understanding of lncRNAs. Enhanced oxidative stress in the mitochondrial and Endoplasmic reticulum leads to the activation of unfolded protein response pathway that plays a crucial role in the pathophysiology of hepatocellular carcinoma. The mutual regulation between LncRNAs and Endoplasmic reticulum (ER) stress in cancer and simultaneous activation of the unfolded protein response (UPR) pathway determines the fate of tumor cells in HCC. Mitochondria-associated lncRNAs work as essential components of several gene regulatory networks; abnormal regulation of mitochondria-associated lncRNAs may lead to oncogenesis, which provides further insight into the understanding of tumorigenesis and therapeutic strategies.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , ARN Largo no Codificante , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Regulación Neoplásica de la Expresión Génica , Carcinogénesis/genética , Transformación Celular NeoplásicaRESUMEN
There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.
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Vacunas contra el Cólera , Cólera , Vibrio cholerae O1 , Administración Oral , Anticuerpos Antibacterianos , Preescolar , Cólera/prevención & control , Humanos , Inmunoglobulina A , Inmunoglobulina G , Inmunoglobulina M , Células B de Memoria , Vacunas ConjugadasRESUMEN
The molecular complexity of host-pathogen interactions remains poorly understood in many infectious diseases, particularly in humans due to the limited availability of reliable and specific experimental models. To bridge the gap between classical two-dimensional culture systems, which often involve transformed cell lines that may not have all the physiologic properties of primary cells, and in vivo animal studies, researchers have developed the organoid model system. Organoids are complex three-dimensional structures that are generated in vitro from primary cells and can recapitulate key in vivo properties of an organ such as structural organization, multicellularity, and function. In this review, we discuss how organoids have been deployed in exploring Salmonella infection in mice and humans. In addition, we summarize the recent advancements that hold promise to elevate our understanding of the interactions and crosstalk between multiple cell types and the microbiota with Salmonella. These models have the potential for improving clinical outcomes and future prophylactic and therapeutic intervention strategies.
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The thymus generates cells of the T cell lineage that seed the lymphatic and blood systems. Transcription factor regulatory networks control the lineage programming and maturation of thymic precursor cells. Whether extrathymic antigenic events, such as the microbial colonization of the mucosal tract also shape the thymic T cell repertoire is unclear. We show here that intestinal microbes influence the thymic homeostasis of PLZF-expressing cells in early life. Impaired thymic development of PLZF+ innate lymphocytes in germ-free (GF) neonatal mice is restored by colonization with a human commensal, Bacteroides fragilis, but not with a polysaccharide A (PSA) deficient isogenic strain. Plasmacytoid dendritic cells influenced by microbes migrate from the colon to the thymus in early life to regulate PLZF+ cell homeostasis. Importantly, perturbations in thymic PLZF+ cells brought about by alterations in early gut microbiota persist into adulthood and are associated with increased susceptibility to experimental colitis. Our studies identify a pathway of communication between intestinal microbes and thymic lymphocytes in the neonatal period that can modulate host susceptibility to immune-mediated diseases later in life.
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Microbioma Gastrointestinal , Linfocitos/inmunología , Timo/crecimiento & desarrollo , Animales , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bacteroides fragilis/fisiología , Diferenciación Celular , Colitis/genética , Colitis/inmunología , Colitis/microbiología , Colon/microbiología , Humanos , Linfocitos/citología , Ratones , Ratones Endogámicos C57BL , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc/inmunología , Timo/citología , Timo/inmunologíaRESUMEN
SALMONELLA ENTERICA: serovar Typhi is the etiologic agent of typhoid fever, a major public health problem in the developing world. Moving toward and adhering to the intestinal epithelium represents key initial steps of infection by S. Typhi. We examined the role of the S. Typhi yrbE gene, which encodes an inner membrane phospholipid transporter, in these interactions with epithelial cells. Disruption of yrbE resulted in elevated expression of flagellin and a hypermotile phenotype. It also significantly reduced the ability of S. Typhi to adhere to the HeLa epithelial cell line and to polarized primary epithelial cells derived from human ileal organoids. Interestingly, the yrbE-deficient strain of S. Typhi induced higher production of interleukin-8 from the primary human ileal epithelial cell monolayers compared to the wild-type bacteria. Deletion of the flagellin gene (fliC) in the yrbE-deficient S. Typhi inhibited motility and attenuated interleukin-8 production, but it did not correct the defect in adhesion. We also disrupted yrbE in S. Typhimurium. In contrast to the results in S. Typhi, the deficiency of yrbE in S. Typhimurium had no significant effect on flagellin expression, motility or adhesion to HeLa cells. Correspondingly, the lack of yrbE also had no effect on association with the intestine or the severity of intestinal inflammation in the mouse model of S. Typhimurium infection. Thus, our results point to an important and serovar-specific role played by yrbE in the early stages of intestinal infection by S. Typhi.
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Adhesión Bacteriana , Flagelina/genética , Proteínas de Transporte de Membrana/fisiología , Infecciones por Salmonella/microbiología , Salmonella typhi/fisiología , Animales , Proteínas Bacterianas/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Perros , Células Epiteliales/microbiología , Flagelina/metabolismo , Regulación Bacteriana de la Expresión Génica , Células HeLa , Interacciones Microbiota-Huesped , Humanos , Inflamación/microbiología , Interleucina-8/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Movimiento , Fosfolípidos/metabolismo , Salmonella typhimurium/fisiología , Índice de Severidad de la EnfermedadRESUMEN
The intestinal microbiota has several effects on host physiology. Previous work from our laboratory demonstrated that the microbiota influences systemic iron homeostasis in mouse colitis models by altering inflammation-induced expression of the iron-regulating hormone hepcidin. In the present study, we examined the impact of the gut commensal bacterium Bacteroides fragilis on the expression of the iron exporter ferroportin, the target of hepcidin action, in macrophages, the cell type that plays a pivotal role in iron recycling. Mouse bone marrow-derived macrophages were exposed to B. fragilis and were analyzed by quantitative real-time polymerase chain reaction and Western blotting. We found that B. fragilis down-regulated ferroportin transcription independently of bacterial viability. Medium conditioned by the bacteria also reduced ferroportin expression, indicating the involvement of soluble factors, possibly Toll-like receptor ligands. Consistent with this idea, several of these ligands were able to down-regulate ferroportin. The B. fragilis-induced decrease in ferroportin was functionally important since it produced a significant increase in intracellular iron concentrations that prevented the effects of the iron chelator deferoxamine on Salmonella-induced IL-6 and IL-1ß production. Our results thus reveal that B. fragilis can influence macrophage iron handling and inflammatory responses by modulating ferroportin expression.
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Bacteroides fragilis/inmunología , Proteínas de Transporte de Catión/inmunología , Regulación hacia Abajo/inmunología , Homeostasis/inmunología , Hierro/inmunología , Macrófagos/inmunología , Animales , Macrófagos/microbiología , RatonesRESUMEN
Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Vesículas Citoplasmáticas/patología , Células Epiteliales/microbiología , Mucosa Intestinal/microbiología , Infecciones por Salmonella/patología , Salmonella typhimurium/patogenicidad , Sumoilación , Proteínas de Unión al GTP rab/metabolismo , Células Cultivadas , Vesículas Citoplasmáticas/microbiología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/crecimiento & desarrollo , Proteínas de Unión al GTP rab/química , Proteínas de Unión a GTP rab7RESUMEN
Post-translational modification pathways such as SUMOylation are integral to all cellular processes and tissue homeostasis. We investigated the possible involvement of SUMOylation in the epithelial signalling in Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD). Initially in a murine model of IBD, induced by dextran-sulfate-sodium (DSS mice), we observed inflammation accompanied by a lowering of global SUMOylation of colonic epithelium. The observed SUMOylation alteration was due to a decrease in the sole SUMO E2 enzyme (Ubc9). Mass-spectrometric analysis revealed the existence of a distinct SUMOylome (SUMO-conjugated proteome) in DSS mice with alteration of key cellular regulators, including master kinase Akt1. Knocking-down of Ubc9 in epithelial cells resulted in dramatic activation of inflammatory gene expression, a phenomenon that acted via reduction in Akt1 and its SUMOylated form. Importantly, a strong decrease in Ubc9 and Akt1 was also seen in endoscopic biopsy samples (N = 66) of human CD and UC patients. Furthermore, patients with maximum disease indices were always accompanied by severely lowered Ubc9 or SUMOylated-Akt1. Mucosal tissues with severely compromised Ubc9 function displayed higher levels of pro-inflammatory cytokines and compromised wound-healing markers. Thus, our results reveal an important and previously undescribed role for the SUMOylation pathway involving Ubc9 and Akt1 in modulation of epithelial inflammatory signalling in IBD.
Asunto(s)
Regulación hacia Abajo , Enfermedades Inflamatorias del Intestino/fisiopatología , Mucosa Intestinal/enzimología , Sumoilación , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Adolescente , Animales , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Persona de Mediana Edad , Transducción de Señal/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Adulto JovenRESUMEN
Post translational modification pathways regulate fundamental processes of cells and thus govern vital functions. Among these, particularly the modification with Small Ubiquitin-like Modifiers (SUMO) is being recognized as a pathway crucial for cell homeostasis and health. Understandably, bacterial pathogens intervene with the SUMO pathway of the host for ensuring successful infection. Among the bacterial pathogens known to target host sumoylation varied points of intervention are utilized. Majority of them including Salmonella Typhimurium, Shigella flexneri and Listeria monocytogenes target the E2 conjugating enzyme Ubc9. While others, such as Xanthomonase compestris, target the desumoylation machineries mimicking cysteine protease activity. Still others such as Ehrlichia chaffeensis and Anaplasma phagocytophilum utilize host SUMO-machinery for sumoylating their own effectors. Together such changes lead to modulation of host proteome and transcriptome thereby leading to major alterations in signal transduction that favor invasion and bacterial multiplication. Such interplay between bacterial pathogens and host sumoylation has added a new dimension to host-pathogen biology and its understanding could be vital for developing potential therapeutic intervention strategies.