Intestinal Immune Dysregulation Driven by Dysbiosis Promotes Barrier Disruption and Bacterial Translocation in Rats With Cirrhosis.

In cirrhosis, intestinal dysbiosis, intestinal barrier impairment, and systemic immune system abnormalities lead to gut bacterial translocation (GBT) and bacterial infection. However, intestinal immune system dysfunction and its contribution to barrier damage are poorly understood. This study correlates immune system dysregulation in the intestines of rats at different stages of CCl4 -induced cirrhosis with barrier function and pathogenic microbiota. The following variables were addressed in the small intestine: intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) activation status and cytokine production (flow cytometry), cytokine mRNA and protein expression (quantitative real-time PCR and immunofluorescence), microbiota composition of ileum content (16S recombinant DNA massive sequencing), permeability (fecal albumin loss), and epithelial junctions (immunohistochemistry and immunofluorescence). The intestinal mucosa in rats with cirrhosis showed a proinflammatory pattern of immune dysregulation in IELs and LPLs, which featured the expansion of activated lymphocytes, switch to a T helper 1 (Th1) regulatory pattern, and Th17 reduction. In rats with cirrhosis with ascites, this state was associated with epithelial junction protein disruption, fecal albumin loss, and GBT. Direct correlations (P < 0.01) were observed between elevated interferon gamma (IFNγ)-expressing T cytotoxic LPLs and fecal albumin and between inflammatory taxa abundance and IFNγ-producing immune cells in the ileum. Bowel decontamination led to redistributed microbiota composition, reduced proinflammatory activation of mucosal immune cells, normalized fecal albumin levels, and diminished GBT; but there were no modifications in Th17 depletion. Conclusion: The intestinal mucosa of rats with cirrhosis acquires a proinflammatory profile of immune dysregulation that parallels the severity of cirrhosis; this impaired intestinal immune response is driven by gut dysbiosis and leads to disrupted barrier function, promoting GBT.

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

BACKGROUND & AIMS: In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. METHODS: Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. RESULTS: Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. CONCLUSIONS: In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis.

Serum microRNAs are key predictors of long-term heart failure and cardiovascular death after myocardial infarction.

BACKGROUND: Patients with acute myocardial infarction (MI) are at high risk of upcoming events, in particular heart failure (HF), but reliable stratification methods are lacking. Our goal was to evaluate the potential role of circulating miRNAs as prognostic biomarkers in patients presenting with MI. METHODS AND RESULTS: We conducted a prospective study among 311 consecutive patients hospitalized with MI (65% ST-segment elevation MI & median age of 55 years) with long-term follow-up. An initial screening was conducted to select candidate miRNAs, with subsequent study of 14 candidate miRNAs. The primary outcome was the composite of hospital admission for HF or cardiovascular death. During a mean follow-up of 2.1 years miR-21-5p, miR-23a-3p, miR27b-3p, miR-122-5p, miR210-3p, and miR-221-3p reliably predicted the primary outcome. Multivariate Cox regression analyses highlighted that miR-210-3p [hazard ratio (HR) 2.65 per 1 SD increase, P < 0.001], miR-23a-3p (HR 2.11 per 1 SD increase, P < 0.001), and miR-221-3p (HR 2.03 per 1 SD increase, P < 0.001) were able to accurately predict the primary outcome, as well as cardiovascular death, HF hospitalizations, and long-term New York Heart Association (NYHA) functional class. These three miRNAs clearly improved the performance of multivariate clinical models: ΔC-statistic = 0.10 [95% confidence interval (CI), 0.03-0.17], continuous net reclassification index = 34.8% (95%CI, 5.8-57.4%), and integrated discrimination improvement (P < 0.001). CONCLUSIONS: This is the largest study evaluating the prognostic value of circulating miRNAs for HF-related events among patients with MI. We show that several miRNAs predict HF hospitalizations, cardiovascular mortality, and poor long-term NYHA status and improve current risk prediction methods.

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity.

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.

Low dose of extracellular vesicles identified that promote recovery after ischemic stroke.

BACKGROUND: Mesenchymal stem cell-derived extracellular vesicles (EVs) are one of the most promising therapeutics in protective and/or regenerative therapy in animal models of stroke using a dose of 100 µg. However, whether EVs dose is related to outcomes is not known. This study aimed to identify the optimal effective dose of EVs from adipose tissue-derived mesenchymal stem cells that promote functional recovery in subcortical stroke. MATERIALS AND METHODS: For this purpose, various doses of EVs were tested in an in vitro oxygen-glucose deprivation (OGD) model of oligodendrocytes and neuronal ischemia. At least 50 µg of EVs were necessary to induce proliferation and differentiation of oligodendrocyte and neurons in OGD conditions. For in vivo study, rats were subjected to subcortical stroke and various doses (50 µg, 100 µg, or 200 µg) of EVs were intravenously administered after 24 h. RESULTS: All the animals in the EV groups showed significant improvement in functional tests, with an increase in tract connectivity and brain repair-associated markers, and a decrease in cell death and in astrocyte-marker expression. Cell proliferation was increased in the groups receiving 50 µg and 100 µg doses. Only the 50-µg dose was associated with significant increases in brain-derived neurotrophic factor expression. CONCLUSION: In conclusion, 50 µg of EVs appears to be the minimal effective dose to enhance protection, brain repair, and recovery in subcortical ischemic stroke.

Mobilization of neural stem cells and generation of new neurons in 6-OHDA-lesioned rats by intracerebroventricular infusion of liver growth factor.

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson's disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine-lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases.

Intracellular Delivery of an Antibody Targeting Gasdermin-B Reduces HER2 Breast Cancer Aggressiveness.

PURPOSE: Gasdermin B (GSDMB) overexpression/amplification occurs in about 60% of HER2 breast cancers, where it promotes cell migration, resistance to anti-HER2 therapies, and poor clinical outcome. Thus, we tackle GSDMB cytoplasmic overexpression as a new therapeutic target in HER2 breast cancers. EXPERIMENTAL DESIGN: We have developed a new targeted nanomedicine based on hyaluronic acid-biocompatible nanocapsules, which allow the intracellular delivery of a specific anti-GSDMB antibody into HER2 breast cancer cells both in vitro and in vivo. RESULTS: Using different models of HER2 breast cancer cells, we show that anti-GSDMB antibody loaded to nanocapsules has significant and specific effects on GSDMB-overexpressing cancer cells' behavior in ways such as (i) lowering the in vitro cell migration induced by GSDMB; (ii) enhancing the sensitivity to trastuzumab; (iii) reducing tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts; and (iv) diminishing lung metastasis in MDA-MB-231-HER2 cells in vivo. Moreover, at a mechanistic level, we have shown that AbGB increases GSDMB binding to sulfatides and consequently decreases migratory cell behavior and may upregulate the potential intrinsic procell death activity of GSDMB. CONCLUSIONS: Our findings portray the first evidence of the effectiveness and specificity of an antibody-based nanomedicine that targets an intracellular oncoprotein. We have proved that intracellular-delivered anti-GSDMB reduces diverse protumor GSDMB functions (migration, metastasis, and resistance to therapy) in an efficient and specific way, thus providing a new targeted therapeutic strategy in aggressive HER2 cancers with poor prognosis.

Neuroprotective activity of peripherally administered liver growth factor in a rat model of Parkinson's disease.

Liver growth factor (LGF) is a hepatic mitogen purified some years ago that promotes proliferation of different cell types and the regeneration of damaged tissues, including brain tissue. Considering the possibility that LGF could be used as a therapeutic agent in Parkinson's disease, we analyzed its potential neuroregenerative and/or neuroprotective activity when peripherally administered to unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. For these studies, rats subjected to nigrostriatal lesions were treated intraperitoneally twice a week with LGF (5 microg/rat) for 3 weeks. Animals were sacrificed 4 weeks after the last LGF treatment. The results show that LGF stimulates sprouting of tyrosine hydroxylase-positive terminals and increases tyrosine hydroxylase and dopamine transporter expression, as well as dopamine levels in the denervated striatum of 6-OHDA-lesioned rats. In this structure, LGF activates microglia and raises tumor necrosis factor-alpha protein levels, which have been reported to have a role in neuroregeneration and neuroprotection. Besides, LGF stimulates the phosphorylation of MAPK/ERK1/2 and CREB, and regulates the expression of proteins which are critical for cell survival such as Bcl2 and Akt. Because LGF partially protects dopamine neurons from 6-OHDA neurotoxicity in the substantia nigra, and reduces motor deficits in these animals, we propose LGF as a novel factor that may be useful in the treatment of Parkinson's disease.

Liver growth factor promotes the survival of grafted neural stem cells in a rat model of Parkinson's disease.

Neural stem cells (NSCs) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nerve tissue. Several studies have focused on the ability of the neurotrophic factors coadministration to improve the efficiency of grafted NSCs. Liver growth factor (LGF) is an hepatic mitogen that promotes regeneration of damaged tissues, including brain tissue. It has neurogenic activity and has partially restored the nigrostriatal dopaminergic system in an experimental model of Parkinson's disease. Present results demonstrate that in the dopamine- depleted striatum of 6-hydroxydopamine-lesioned rats, grafted NSCs retained their ability to differentiate into neurons, astrocytes, and oligodendrocytes. NSCs also differentiated into microglia/macrophages and endothelial cells. Thus, 23 ± 5.6% of them were inmunoreactive for isolectin IB4, and a small population integrated into blood vessels, showing an endothelial-like morphology. Intrastriatal infusion of LGF promoted the viability of the implants, and favored their differentiation to an endothelial-like phenotype. Moreover, LGF infusion raised the expression of the anti-apoptotic protein Bcl-2 by 3.9 ± 0.9 fold without affecting the levels of the pro-apoptotic protein Bax. Since LGF-treated rats also showed a significant reduction in apomorphine-induced rotational behavior, our results suggest that administration of this factor might be a convenient treatment for Parkinson's disease cell replacement therapies based on NSCs transplantation.

Applications of human umbilical cord blood cells in central nervous system regeneration.

In recent decades, there has been considerable amount of information about embryonic stem cells (ES). The dilemma facing scientists interested in the development and use of human stem cells in replacement therapies is the source of these cells, i.e. the human embryo. There are many ethical and moral problems related to the use of these cells. Hematopoietic stem cells from umbilical cord blood have been proposed as an alternative source of embryonic stem cells. After exposure to different agents, these cells are able to express antigens of diverse cellular lineages, including the neural type. The In vitro manipulation of human umbilical cord blood (hUCB) cells has shown their stem capacity and plasticity. These cells are easily accessible, In vitro amplifiable, well tolerated by the host, and with more primitive molecular characteristics that give them great flexibility. Overall, these properties open a promising future for the use of hUCB in regenerative therapies for the Central Nervous System (CNS). This review will focus on the available literature concerning umbilical cord blood cells as a therapeutic tool for the treatment of neurodegenerative diseases.