Person: Fasano, Alessio
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Fasano
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Alessio
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Fasano, Alessio
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Publication Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family(Frontiers Media S.A., 2018) Scheffler, Lucas; Crane, Alyce; Heyne, Henrike; Tönjes, Anke; Schleinitz, Dorit; Ihling, Christian H.; Stumvoll, Michael; Freire, Rachel; Fiorentino, Maria; Fasano, Alessio; Kovacs, Peter; Heiker, John T.Background: There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity. Methods: Serum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. Results: As zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry, and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family. Conclusion: Our study suggests that the zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.Publication Human Fetal-Derived Enterospheres Provide Insights on Intestinal Development and a Novel Model to Study Necrotizing Enterocolitis (NEC)(Elsevier, 2018) Senger, Stefania; Ingano, Laura; Freire, Rachel; Anselmo, Antony; Zhu, Weishu; Sadreyev, Ruslan; Walker, William Allan; Fasano, AlessioBackground & Aims Untreated necrotizing enterocolitis (NEC) can lead to massive inflammation resulting in intestinal necrosis with a high mortality rate in preterm infants. Limited access to human samples and relevant experimental models have hampered progress in NEC pathogenesis. Earlier evidence has suggested that bacterial colonization of an immature and developing intestine can lead to an abnormally high inflammatory response to bacterial bioproducts. The aim of our study was to use human fetal organoids to gain insights into NEC pathogenesis. Methods: RNA sequencing analysis was performed to compare patterns of gene expression in human fetal-derived enterospheres (FEnS) and adult-derived enterospheres (AEnS). Differentially expressed genes were analyzed using computational techniques for dimensional reduction, clustering, and gene set enrichment. Unsupervised cluster analysis, Gene Ontology, and gene pathway analysis were used to predict differences between gene expression of samples. Cell monolayers derived from FEnS and AEnS were evaluated for epithelium function and responsiveness to lipopolysaccharide and commensal bacteria. Results: Based on gene expression patterns, FEnS clustered according to their developmental age in 2 distinct groups: early and late FEnS, with the latter more closely resembling AEnS. Genes involved in maturation, gut barrier function, and innate immunity were responsible for these differences. FEnS-derived monolayers exposed to either lipopolysaccharide or commensal Escherichia coli showed that late FEnS activated gene expression of key inflammatory cytokines, whereas early FEnS monolayers did not, owing to decreased expression of nuclear factor-κB–associated machinery. Conclusions: Our results provide insights into processes underlying human intestinal development and support the use of FEnS as a relevant human preclinical model for NEC. Accession number of repository for expression data: GSE101531.Publication Compositional and Functional Differences in the Human Gut Microbiome Correlate with Clinical Outcome following Infection with Wild-Type Salmonella enterica Serovar Typhi(American Society for Microbiology, 2018) Zhang, Yan; Brady, Arthur; Jones, Cheron; Song, Yang; Darton, Thomas C.; Jones, Claire; Blohmke, Christoph J.; Pollard, Andrew J.; Magder, Laurence S.; Fasano, Alessio; Sztein, Marcelo B.; Fraser, Claire M.ABSTRACT Insights into disease susceptibility as well as the efficacy of vaccines against typhoid and other enteric pathogens may be informed by better understanding the relationship between the effector immune response and the gut microbiota. In the present study, we characterized the composition (16S rRNA gene profiling) and function (RNA sequencing [RNA-seq]) of the gut microbiota following immunization and subsequent exposure to wild-type Salmonella enterica serovar Typhi in a human challenge model to further investigate the central hypothesis that clinical outcomes may be linked to the gut microbiota. Metatranscriptome analysis of longitudinal stool samples collected from study subjects revealed two stable patterns of gene expression for the human gut microbiota, dominated by transcripts from either Methanobrevibacter or a diverse representation of genera in the Firmicutes phylum. Immunization with one of two live oral attenuated vaccines against S. Typhi had minimal effects on the composition or function of the gut microbiota. It was observed that subjects harboring the methanogen-dominated transcriptome community at baseline displayed a lower risk of developing symptoms of typhoid following challenge with wild-type S. Typhi. Furthermore, genes encoding antioxidant proteins, metal homeostasis and transport proteins, and heat shock proteins were expressed at a higher level at baseline or after challenge with S. Typhi in subjects who did not develop symptoms of typhoid. These data suggest that functional differences relating to redox potential and ion homeostasis in the gut microbiota may impact clinical outcomes following exposure to wild-type S. Typhi.Publication Celiac Disease Histopathology Recapitulates Hedgehog Downregulation, Consistent with Wound Healing Processes Activation(Public Library of Science, 2015) Senger, Stefania; Sapone, Anna; Fiorentino, Maria; Mazzarella, Giuseppe; Lauwers, Gregory Y.; Fasano, AlessioBackground: In celiac disease (CD), intestinal epithelium damage occurs secondary to an immune insult and is characterized by blunting of the villi and crypt hyperplasia. Similarities between Hedgehog (Hh)/BMP4 downregulation, as reported in a mouse model, and CD histopathology, suggest mechanistic involvement of Hh/BMP4/WNT pathways in proliferation and differentiation of immature epithelial cells in the context of human intestinal homeostasis and regeneration after damage. Herein we examined the nature of intestinal crypt hyperplasia and involvement of Hh/BMP4 in CD histopathology. Methods and Findings: Immunohistochemistry, qPCR and in situ hybridization were used to study a cohort of 24 healthy controls (HC) and 24 patients with diagnosed acute celiac disease (A-CD) intestinal biopsies. In A-CD we observed an increase in cells positive for Leucin-rich repeat-containing G protein-coupled receptor 5 (LGR5), an epithelial stem cell specific marker and expansion of WNT responding compartment. Further, we observed alteration in number and distribution of mesenchymal cells, predicted to be part of the intestinal stem cells niche. At the molecular level we found downregulation of indian hedgehog (IHH) and other components of the Hh pathway, but we did not observe a concurrent downregulation of BMP4. However, we observed upregulation of BMPs antagonists, gremlin 1 and gremlin 2. Conclusions: Our data suggest that acute CD histopathology partially recapitulates the phenotype reported in Hh knockdown models. Specifically, Hh/BMP4 paradigm appears to be decoupled in CD, as the expansion of the immature cell population does not occur consequent to downregulation of BMP4. Instead, we provide evidence that upregulation of BMP antagonists play a key role in intestinal crypt hyperplasia. This study sheds light on the molecular mechanisms underlying CD histopathology and the limitations in the use of mouse models for celiac disease.Publication Celiac Disease Genomic, Environmental, Microbiome, and Metabolomic (CDGEMM) Study Design: Approach to the Future of Personalized Prevention of Celiac Disease(MDPI, 2015) Leonard, Maureen; Camhi, Stephanie; Huedo-Medina, Tania B.; Fasano, AlessioIn the past it was believed that genetic predisposition and exposure to gluten were necessary and sufficient to develop celiac disease (CD). Recent studies however suggest that loss of gluten tolerance can occur at any time in life as a consequence of other environmental stimuli. Many environmental factors known to influence the composition of the intestinal microbiota are also suggested to play a role in the development of CD. These include birthing delivery mode, infant feeding, and antibiotic use. To date no large-scale longitudinal studies have defined if and how gut microbiota composition and metabolomic profiles may influence the loss of gluten tolerance and subsequent onset of CD in genetically-susceptible individuals. Here we describe a prospective, multicenter, longitudinal study of infants at risk for CD which will employ a blend of basic and applied studies to yield fundamental insights into the role of the gut microbiome as an additional factor that may play a key role in early steps involved in the onset of autoimmune disease.Publication Blood–brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders(BioMed Central, 2016) Fiorentino, Maria; Sapone, Anna; Senger, Stefania; Camhi, Stephanie S.; Kadzielski, Sarah; Buie, Timothy; Kelly, Deanna L.; Cascella, Nicola; Fasano, AlessioBackground: Autism spectrum disorders (ASD) are complex conditions whose pathogenesis may be attributed to gene–environment interactions. There are no definitive mechanisms explaining how environmental triggers can lead to ASD although the involvement of inflammation and immunity has been suggested. Inappropriate antigen trafficking through an impaired intestinal barrier, followed by passage of these antigens or immune-activated complexes through a permissive blood–brain barrier (BBB), can be part of the chain of events leading to these disorders. Our goal was to investigate whether an altered BBB and gut permeability is part of the pathophysiology of ASD. Methods: Postmortem cerebral cortex and cerebellum tissues from ASD, schizophrenia (SCZ), and healthy subjects (HC) and duodenal biopsies from ASD and HC were analyzed for gene and protein expression profiles. Tight junctions and other key molecules associated with the neurovascular unit integrity and function and neuroinflammation were investigated. Results: Claudin (CLDN)-5 and -12 were increased in the ASD cortex and cerebellum. CLDN-3, tricellulin, and MMP-9 were higher in the ASD cortex. IL-8, tPA, and IBA-1 were downregulated in SCZ cortex; IL-1b was increased in the SCZ cerebellum. Differences between SCZ and ASD were observed for most of the genes analyzed in both brain areas. CLDN-5 protein was increased in ASD cortex and cerebellum, while CLDN-12 appeared reduced in both ASD and SCZ cortexes. In the intestine, 75% of the ASD samples analyzed had reduced expression of barrier-forming TJ components (CLDN-1, OCLN, TRIC), whereas 66% had increased pore-forming CLDNs (CLDN-2, -10, -15) compared to controls. Conclusions: In the ASD brain, there is an altered expression of genes associated with BBB integrity coupled with increased neuroinflammation and possibly impaired gut barrier integrity. While these findings seem to be specific for ASD, the possibility of more distinct SCZ subgroups should be explored with additional studies.Publication Biomarkers of Environmental Enteropathy, Inflammation, Stunting, and Impaired Growth in Children in Northeast Brazil(Public Library of Science, 2016) Guerrant, Richard L.; Leite, Alvaro M.; Pinkerton, Relana; Medeiros, Pedro H. Q. S.; Cavalcante, Paloma A.; DeBoer, Mark; Kosek, Margaret; Duggan, Christopher; Gewirtz, Andrew; Kagan, Jonathan; Gauthier, Anna; Swann, Jonathan; Mayneris-Perxachs, Jordi; Bolick, David T.; Maier, Elizabeth A.; Guedes, Marjorie M.; Moore, Sean R.; Petri, William A.; Havt, Alexandre; Lima, Ila F.; Prata, Mara de Moura Gondim; Michaleckyj, Josyf C.; Scharf, Rebecca J.; Sturgeon, Craig; Fasano, Alessio; Lima, Aldo A. M.Critical to the design and assessment of interventions for enteropathy and its developmental consequences in children living in impoverished conditions are non-invasive biomarkers that can detect intestinal damage and predict its effects on growth and development. We therefore assessed fecal, urinary and systemic biomarkers of enteropathy and growth predictors in 375 6–26 month-old children with varying degrees of malnutrition (stunting or wasting) in Northeast Brazil. 301 of these children returned for followup anthropometry after 2-6m. Biomarkers that correlated with stunting included plasma IgA anti-LPS and anti-FliC, zonulin (if >12m old), and intestinal FABP (I-FABP, suggesting prior barrier disruption); and with citrulline, tryptophan and with lower serum amyloid A (SAA) (suggesting impaired defenses). In contrast, subsequent growth was predicted in those with higher fecal MPO or A1AT and also by higher L/M, plasma LPS, I-FABP and SAA (showing intestinal barrier disruption and inflammation). Better growth was predicted in girls with higher plasma citrulline and in boys with higher plasma tryptophan. Interactions were also seen with fecal MPO and neopterin in predicting subsequent growth impairment. Biomarkers clustered into markers of 1) functional intestinal barrier disruption and translocation, 2) structural intestinal barrier disruption and inflammation and 3) systemic inflammation. Principle components pathway analyses also showed that L/M with %L, I-FABP and MPO associate with impaired growth, while also (like MPO) associating with a systemic inflammation cluster of kynurenine, LBP, sCD14, SAA and K/T. Systemic evidence of LPS translocation associated with stunting, while markers of barrier disruption or repair (A1AT and Reg1 with low zonulin) associated with fecal MPO and neopterin. We conclude that key noninvasive biomarkers of intestinal barrier disruption, LPS translocation and of intestinal and systemic inflammation can help elucidate how we recognize, understand, and assess effective interventions for enteropathy and its growth and developmental consequences in children in impoverished settings.Publication The Role of Gluten in Celiac Disease and Type 1 Diabetes(MDPI, 2015) Serena, Gloria; Camhi, Stephanie; Sturgeon, Craig; Yan, Shu; Fasano, AlessioCeliac disease (CD) and type 1 diabetes (T1D) are autoimmune conditions in which dietary gluten has been proven or suggested to play a pathogenic role. In CD; gluten is established as the instigator of autoimmunity; the autoimmune process is halted by removing gluten from the diet; which allows for resolution of celiac autoimmune enteropathy and subsequent normalization of serological markers of the disease. However; an analogous causative agent has not yet been identified for T1D. Nevertheless; the role of dietary gluten in development of T1D and the potentially beneficial effect of removing gluten from the diet of patients with T1D are still debated. In this review; we discuss the comorbid occurrence of CD and T1D and explore current evidences for the specific role of gluten in both conditions; specifically focusing on current evidence on the effect of gluten on the immune system and the gut microbiota.Publication HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche(Nature Publishing Group, 2017) Xian, Lingling; Georgess, Dan; Huso, Tait; Cope, Leslie; Belton, Amy; Chang, Yu-Ting; Kuang, Wenyong; Gu, Qihua; Zhang, Xiaoyan; Senger, Stefania; Fasano, Alessio; Huso, David L.; Ewald, Andrew J.; Resar, Linda M. S.High-mobility group A1 (Hmga1) chromatin remodelling proteins are enriched in intestinal stem cells (ISCs), although their function in this setting was unknown. Prior studies showed that Hmga1 drives hyperproliferation, aberrant crypt formation and polyposis in transgenic mice. Here we demonstrate that Hmga1 amplifies Wnt/β-catenin signalling to enhance self-renewal and expand the ISC compartment. Hmga1 upregulates genes encoding both Wnt agonist receptors and downstream Wnt effectors. Hmga1 also helps to ‘build' an ISC niche by expanding the Paneth cell compartment and directly inducing Sox9, which is required for Paneth cell differentiation. In human intestine, HMGA1 and SOX9 are positively correlated, and both become upregulated in colorectal cancer. Our results define a unique role for Hmga1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche. This work also suggests that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis.Publication Indications and Use of the Gluten Contamination Elimination Diet for Patients with Non-Responsive Celiac Disease(MDPI, 2017) Leonard, Maureen; Cureton, Pamela; Fasano, AlessioFor the majority of patients diagnosed with celiac disease, once a gluten-free diet is initiated, symptoms improve within weeks and may completely resolve in months. However, up to 30% of patients may show signs, symptoms or persistent small intestinal damage after one year on a gluten-free diet. These patients require evaluation for other common GI etiologies and assessment of their celiac disease status in order to make a diagnosis and suggest treatment. Here, we propose an approach to evaluating patients with celiac disease with persistent symptoms, persistently elevated serology, and or persistent villous atrophy despite a gluten-free diet. We detail how to diagnose and distinguish between non-responsive and refractory celiac disease. Finally, we introduce the indications for use of the gluten contamination elimination diet and provide information for practitioners to implement the diet when necessary in their practice.