A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility.

Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.

PARK7/DJ-1 deficiency impairs microglial activation in response to LPS-induced inflammation.

BACKGROUND: Specific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases, including Parkinson's disease (PD). However, the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here, according to the multiple-hit hypothesis, which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors, we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency, a genetic cause of PD, during lipopolysaccharide (LPS)-induced inflammation. METHODS: Using a combination of single-cell RNA-sequencing, bulk RNA-sequencing, multicolor flow cytometry and immunofluorescence analyses, we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-h intraperitoneal injection with LPS. For translational perspectives, we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs). RESULTS: By excluding the contribution of other immune brain resident and peripheral cells, we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype, specially related to type II interferon and DNA damage response signaling, when compared with wildtype microglia, in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level, with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice, both at 6 and 24 h after acute inflammation, as also observed in BMDMs. CONCLUSIONS: Taken together, our results show that, under inflammatory conditions, PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses, which may play a causative role in PD onset and progression.

Allergic airway inflammation delays glioblastoma progression and reinvigorates systemic and local immunity in mice.

BACKGROUND: Numerous patient-based studies have highlighted the protective role of immunoglobulin E-mediated allergic diseases on glioblastoma (GBM) susceptibility and prognosis. However, the mechanisms behind this observation remain elusive. Our objective was to establish a preclinical model able to recapitulate this phenomenon and investigate the role of immunity underlying such protection. METHODS: An immunocompetent mouse model of allergic airway inflammation (AAI) was initiated before intracranial implantation of mouse GBM cells (GL261). RAG1-KO mice served to assess tumor growth in a model deficient for adaptive immunity. Tumor development was monitored by MRI. Microglia were isolated for functional analyses and RNA-sequencing. Peripheral as well as tumor-associated immune cells were characterized by flow cytometry. The impact of allergy-related microglial genes on patient survival was analyzed by Cox regression using publicly available datasets. RESULTS: We found that allergy establishment in mice delayed tumor engraftment in the brain and reduced tumor growth resulting in increased mouse survival. AAI induced a transcriptional reprogramming of microglia towards a pro-inflammatory-like state, uncovering a microglia gene signature, which correlated with limited local immunosuppression in glioma patients. AAI increased effector memory T-cells in the circulation as well as tumor-infiltrating CD4+ T-cells. The survival benefit conferred by AAI was lost in mice devoid of adaptive immunity. CONCLUSION: Our results demonstrate that AAI limits both tumor take and progression in mice, providing a preclinical model to study the impact of allergy on GBM susceptibility and prognosis, respectively. We identify a potentiation of local and adaptive systemic immunity, suggesting a reciprocal crosstalk that orchestrates allergy-induced immune protection against GBM.

Localized cutaneous calcinosis associated with leptospirosis in a 4-month-old beagle puppy.

A 4-month-old male beagle dog was presented for a 15-day history of firm cutaneous nodules. Histopathological examination of skin biopsies revealed calcinosis cutis. However, re-evaluation 40 d later confirmed spontaneous resolution of the lesions without specific treatment. Two weeks before development of the skin lesions, this dog had been hospitalized and treated for acute renal and hepatic disease attributed to leptospirosis, with both PCR and serology positive for Leptospira australis. Calcinosis cutis secondary to a systemic disease (leptospirosis, blastomycosis) has been rarely reported. In this case, the suspected pathogenesis included organic stress (cortisol hypersecretion) and abnormal calcium/phosphorus metabolism during acute renal failure. To our knowledge, this is the third published case of cutis calcinosis associated with leptospirosis in dogs. Key clinical message: Previous leptospirosis should be considered in a dog with calcinosis cutis. The cutaneous lesions appeared after acute leptospirosis and regressed spontaneously.

Calcinose cutanée localisée associée à une leptospirose chez un chiot Beagle de 4 mois. Un Beagle mâle de 4 mois a été présenté en consultation à la suite de l'apparition de nodules cutanés fermes 15 jours auparavant. L'examen histopathologique de biopsies cutanées a révélé une calcinose cutanée. Le contrôle à 40 jours a confirmé une résolution spontanée des lésions sans traitement spécifique. Deux semaines avant le développement des lésions cutanées, ce chien avait été hospitalisé et traité pour une maladie rénale et hépatique, attribuée à une leptospirose. Les examens PCR et sérologique étaient positifs pour Leptospira australis. La calcinose cutanée secondaire à une maladie systémique (leptospirose, blastomycose) est rarement rapportée et le mécanisme étiopathogénique suspecté comprenait un stress organique (hypersécrétion de cortisol) et un déséquilibre du métabolisme phosphocalcique lors de l'épisode d'insuffisance rénale aiguë. À notre connaissance, il s'agit du troisième cas publié de calcinose cutanée associée à la leptospirose chez le chien.Message clinique clé:Une potentielle leptospirose antérieure doit être mentionnée chez un chien atteint de calcinose cutanée. Les lésions cutanées semblent apparaître de manière décalée et régresser spontanément.(Traduit par Claude Muller).

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

Transcriptional variations in the wider peritumoral tissue environment of pancreatic cancer.

Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples. Pancreatic ductal adenocarcinomas (PDAC) and cystic tumours were most different in these non-tumorous tissues surrounding them, whereas the actual tumours exhibited rather similar transcript patterns. The environment of cystic tumours was transcriptionally nearly identical to normal pancreas tissue. In contrast, the tissue around PDAC behaved a lot like the tumour, indicating some kind of field defect, while showing far less molecular resemblance to both chronic pancreatitis and healthy tissue. This suggests that the major pathogenic difference between cystic and ductal tumours may be due to their cellular environment rather than the few variations between the tumours. Lack of correlation between DNA methylation and transcript levels makes it unlikely that the observed field defect in the peritumoral tissue of PDAC is controlled to a large extent by such epigenetic regulation. Functionally, a strikingly large number of autophagy-related transcripts was changed in both PDAC and its peritumoral tissue, but not in other pancreatic tumours. A transcription signature of 15 autophagy-related genes was established that permits a prognosis of survival with high accuracy and indicates the role of autophagy in tumour biology.

Preliminary Report of Percutaneous Cholecystostomy as Diagnosis and Treatment of Biliary Tract Trauma.

BACKGROUND: Biliary leak following severe blunt liver injuries is a complex problem becoming more frequent with improvements in non-operative management. Standard treatment requires main bile duct drainage usually performed by endoscopic sphincterotomy and stent placement. We report our experience with cholecystostomy as a first minimally invasive diagnostic and therapeutic approach. METHODS: We performed a retrospective analysis of consecutive patients with post-traumatic biliary leak between 2006 and 2015. In the first period (2006-2010), biliary fistula was managed using perihepatic drainage and endoscopic, percutaneous or surgical main bile duct drainage. After 2010, cholecystostomy as an initial minimally invasive approach was performed. RESULTS: Of 341 patients with blunt liver injury, 18 had a post-traumatic biliary leak. Ten patients received standard treatment and eight patients underwent cholecystostomy. The cholecystostomy (62.5%) and the standard treatment (80%) groups presented similar success rates as the first biliary drainage procedure (p = 0.41). Cholecystostomy presented no severe complications and resulted, when successful, in a bile flow rate inversion between the perihepatic drains and the gallbladder drain within a median [IQR] 4 days [1-7]. The median time for bile leak resolution was 26 days in the cholecystostomy group and 39 days in the standard treatment group (p = 0.09). No significant difference was found considering median duration of hospital stay (54 and 74 days, respectively, p = 0.37) or resuscitation stay (17.5 and 19.5 days, p = 0.59). CONCLUSION: Cholecystostomy in non-operative management of biliary fistula after blunt liver injury could be an effective, simple and safe first-line procedure in the diagnostic and therapeutic approach of post-traumatic biliary tract injuries.

RNA sequencing and transcriptome arrays analyses show opposing results for alternative splicing in patient derived samples.

BACKGROUND: RNA sequencing (RNA-seq) and microarrays are two transcriptomics techniques aimed at the quantification of transcribed genes and their isoforms. Here we compare the latest Affymetrix HTA 2.0 microarray with Illumina 2000 RNA-seq for the analysis of patient samples - normal lung epithelium tissue and squamous cell carcinoma lung tumours. Protein coding mRNAs and long non-coding RNAs (lncRNAs) were included in the study. RESULTS: Both platforms performed equally well for protein-coding RNAs, however the stochastic variability was higher for the sequencing data than for microarrays. This reduced the number of differentially expressed genes and genes with predictive potential for RNA-seq compared to microarray data. Analysis of this variability revealed a lack of reads for short and low abundant genes; lncRNAs, being shorter and less abundant RNAs, were found especially susceptible to this issue. A major difference between the two platforms was uncovered by analysis of alternatively spliced genes. Investigation of differential exon abundance showed insufficient reads for many exons and exon junctions in RNA-seq while the detection on the array platform was more stable. Nevertheless, we identified 207 genes which undergo alternative splicing and were consistently detected by both techniques. CONCLUSIONS: Despite the fact that the results of gene expression analysis were highly consistent between Human Transcriptome Arrays and RNA-seq platforms, the analysis of alternative splicing produced discordant results. We concluded that modern microarrays can still outperform sequencing for standard analysis of gene expression in terms of reproducibility and cost.

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A.

BACKGROUND: Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM. METHODS: Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells. RESULTS: We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion. CONCLUSIONS: This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors.

Cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine cortex.

The neuronal coding of stimulus-to-action sequences is believed to involve the release of dopamine (DA) and norepinephrine (NE). The electrochemical similarity of these monoamines, however, confounds real-time measurements of their release. Here we report cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) that use the specificity of G protein-coupled receptors (GPCRs) to discriminate nanomolar concentrations of DA and NE. CNiFERs were implanted into the frontal cortex of mice to measure the timing of neurotransmitter release during classical conditioning with the use of two-photon microscopy. The onset of DA release correlated with that of licking and shifted from the time of the reward toward that of the cue upon conditioning. In contrast, concurrent release of NE did not correlate with licking or the cue. This generation of CNiFERs provides unique tools to assess the release of monoamines. The molecular design of these CNiFERs may be generalized to realize CNiFERs for any molecule that activates a GPCR.