Isobaric labeling-based quantitative proteomics of FACS-purified immune cells and epithelial cells from the intestine of Crohn's disease patients reveals proteome changes of potential importance in disease pathogenesis.

Crohn's disease (CD) is a chronic condition characterized by recurrent flares of inflammation in the gastrointestinal tract. Disease etiology is poorly understood and is characterized by dysregulated immune activation that progressively destroys intestinal tissue. Key cellular compartments in disease pathogenesis are the intestinal epithelial layer and its underlying lamina propria. While the epithelium contains predominantly epithelial cells, the lamina propria is enriched in immune cells. Deciphering proteome changes in different cell populations is important to understand CD pathogenesis. Here, using isobaric labeling-based quantitative proteomics, we perform an exploratory study to analyze in-depth proteome changes in epithelial cells, immune cells and stromal cells in CD patients compared to controls using cells purified by FACS. Our study revealed increased proteins associated with neutrophil degranulation and mitochondrial metabolism in immune cells of CD intestinal mucosa. We also found upregulation of proteins involved in glycosylation and secretory pathways in epithelial cells of CD patients, while proteins involved in mitochondrial metabolism were reduced. The distinct alterations in protein levels in immune- versus epithelial cells underscores the utility of proteome analysis of defined cell types. Moreover, our workflow allowing concomitant assessment of cell-type specific changes on an individual basis enables deeper insight into disease pathogenesis.

The developmentally regulated fetal enterocyte gene, ZP4, mediates anti-inflammation by the symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis.

Initial colonizing bacteria play a critical role in completing the development of the immune system in the gastrointestinal tract of infants. Yet, the interaction of colonizing bacterial organisms with the developing human intestine favors inflammation over immune homeostasis. This characteristic of bacterial-intestinal interaction partially contributes to the pathogenesis of necrotizing enterocolitis (NEC), a devastating premature infant intestinal inflammatory disease. However, paradoxically some unique pioneer bacteria (initial colonizing species) have been shown to have a beneficial effect on the homeostasis of the immature intestine and the prevention of inflammation. We have reported that one such pioneer bacterium, Bacteroides fragilis (B. fragilis), and its surface component polysaccharide A (PSA) inhibit IL-1ß-induced inflammation in a human primary fetal small intestinal cell line (H4 cells). In this study, using transcription profiling of H4 cellular RNA after pretreatment with or without PSA before an inflammatory stimulation of IL-1ß, we have begun to further determine the cellular mechanism for anti-inflammation. We show that a developmentally regulated gene, zona pellucida protein 4 (ZP4), is uniquely elevated after IL-1ß stimulation and reduced with PSA exposure. ZP4 was known as a sperm receptor-mediating species-specific binding protein in the initial life of mammals. However, its intestinal epithelial function is unclear. We found that ZP4 is a developmentally regulated gene involved with immune function and regulated by both Toll-like receptor 2 and 4. Knockdown of ZP4-affected PSA inhibited IL-8 mRNA expression in response to IL-1ß. This represents an initial study of ZP4 innate immune function in immature enterocytes. This study may lead to new opportunity for efficient treatment of NEC.NEW & NOTEWORTHY This study extends previous observations to define the cellular mechanisms of polysaccharide A-induced anti-inflammation in immature enterocytes using transcription profiling of enterocyte genes after preexposure to polysaccharide A before an inflammatory stimulus with IL-1ß.

Alternatives to animal testing in basic and preclinical research of atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of increasing prevalence, especially in industrialized countries. Roughly 25% of the children and 1%-3% of adults are affected. Although significant progress has been made in the understanding of the pathogenesis of AD, many aspects remain poorly understood. Moreover, there is a pressing need for improved therapeutic options. Studies to elucidate the pathophysiological pathways of AD and to identify novel therapeutic targets over the last few decades have been conducted almost exclusively in animal models. However, in vitro approaches such as 3D skin disease models have recently emerged due to an increasing awareness of distinct interspecies-related differences that hamper the effective translation of results from animal models to humans. In addition, there is growing political and social pressure to develop alternatives to animal models according to the 3Rs principle (reduction, refinement and replacement of animal models).

Fecal Supernatants from Patients with Crohn's Disease Induce Inflammatory Alterations in M2 Macrophages and Fibroblasts.

Intestinal macrophages and fibroblasts act as microenvironmental sentinels mediating inflammation and disease progression in Crohn's disease (CD). We aimed to establish the effects of fecal supernatants (FSs) from patients with CD on macrophage and fibroblast phenotype and function. FS were obtained by ultracentrifugation, and the metabolites were analyzed. Monocyte-derived M2 macrophages and fibroblasts were conditioned with FS, and secreted proteins, surface proteins and gene expression were analyzed. M2 macrophage efferocytosis was evaluated. Patients with CD (n = 15) had a skewed fecal metabolite profile compared to healthy subjects (HS, n = 10). FS from CD patients (CD-FS) induced an anti-inflammatory response in M2 macrophages with higher expression of IL-10, IL1RA and CD206 as compared to healthy FS (HS-FS) while the efferocytotic capacity was unaltered. CD-FS did not affect extracellular matrix production from fibroblasts, but increased expression of the pro-inflammatory proteins IL-6 and MCP-1. Conditioned media from M2 macrophages treated with CD-FS modulated gene expression in fibroblasts for TGFß superfamily members and reduced IL-4 expression compared to HS-FS. We show that M2 macrophages and fibroblasts react abnormally to the fecal microenvironment of CD patients, resulting in altered protein expression related to inflammation but not fibrosis. This implies that the gut microbiota and its metabolites have an important role in the generation and/or perpetuation of inflammation in CD.

The potential role of adherence factors in probiotic function in the gastrointestinal tract of adults and pediatrics: a narrative review of experimental and human studies.

Numerous studies point to the important role of probiotic bacteria in gastrointestinal health. Probiotics act through mechanisms affecting enteric pathogens, epithelial barrier function, immune signaling, and conditioning of indigenous microbiota. Once administered, probiotics reach the gastrointestinal tract and interact with the host through bacterial surface molecules, here called adhesion factors, which are either strain- or specie-specific. Probiotic adhesion, through structural adhesion factors, is a mechanism that facilitates persistence within the gastrointestinal tract and triggers the initial host responses. Thus, an understanding of specific probiotic adhesion mechanisms could predict how specific probiotic strains elicit benefits and the potential of adherence factors as a proxy to predict probiotic function. This review summarizes the present understanding of probiotic adherence in the gastrointestinal tract. It highlights the bacterial adhesion structure types, their molecular communication with the host and the consequent impact on intestinal diseases in both adult and pediatric populations. Finally, we discuss knockout/isolation studies as direct evidence for adhesion factors conferring anti-inflammatory and pathogen inhibition properties to a probiotic.What is known: Probiotics can be used to treat clinical conditions.Probiotics improve dysbiosis and symptoms.Clinical trials may not confirm in vitro and animal studies.What is new: Adhesion structures may be important for probiotic function.Need to systematically determine physical characteristics of probiotics before selecting for clinical trials.Probiotics may be genetically engineered to add to clinical efficacy.

Systematic Review of Human and Animal Evidence on the Role of Buckwheat Consumption on Gastrointestinal Health.

BACKGROUND: Buckwheat is a commonly cultivated crop with growing evidence that it is beneficial to gastrointestinal (GI) health. This systematic review summarizes the role of buckwheat in modifying GI health outcomes and microbiomes. METHODS: Four medical databases and Google Scholar were systematically searched. Clinical trials, observational studies, animal in vivo, and in vitro studies with human and animal GI-derived samples were included. RESULTS: There were 32 studies (one randomized controlled trial [RCT], one non-randomized trial, 3 observational, 9 in vitro, and 18 animal in vivo studies) included. In preclinical studies, buckwheat extracts were observed to have cytotoxic potential against human-derived GI cancer cell lines. Animals fed with buckwheat had lower GI mucosal inflammation, higher alpha diversity in the GI microbiome, and higher levels of fecal short-chain fatty acids. Human evidence studies and clinical trials were limited and predominantly of moderate risk of bias. The majority of in vitro studies with GI-derived samples and in vivo studies were reliable without restrictions in study design. CONCLUSION: In vivo and in vitro studies show that buckwheat may have potential GI benefits due to its anti-oxidant and anti-inflammatory potential; however, human evidence remains limited, and its impact on health in humans remains to be elucidated in future trials.

MEFV and NLRP3 Inflammasome Expression Is Attributed to Immature Macrophages and Correlates with Serum Inflammatory Proteins in Crohn´s Disease Patients.

Inflammasomes are intracellular protein complexes whose activation results in proinflammatory cytokines. Inflammasomes are implicated in Crohn´s disease (CD) pathogenesis, yet the contribution of inflammasomes in intestinal epithelial cells (IECs) versus lamina propria (LP) macrophages is poorly understood. Whether inflammasome expression in intestinal tissue reflects the serum inflammatory protein profile of patients is also not known. We aimed to determine the intestinal cell types where inflammasome expression is increased in CD and if they correlate with the serum protein profile. RT-PCR and NanoString nCounter technology were used to characterize inflammasome gene expression in CD patients and controls. The mucosa, LP and IEC cell fractions and FACS-sorted cells were analyzed. Proximity extension assay with a 92-protein panel was used to determine the serum inflammatory protein profile. Compositional analysis was used to correlate ileum inflammasome gene expression with intestinal mononuclear phagocyte populations. We show that NLRP3 and MEFV inflammasome sensors and downstream effector expression including IL-1ß are increased in inflamed mucosa of IBD patients and correlate with disease activity. Inflammasome gene expression increased with the abundance of immature intestinal macrophages, and increased IL-1ß released by CD LP cells correlated with immature macrophage frequency. Inflammasome gene expression was also increased in circulating monocytes, the precursors of immature intestinal macrophages. Finally, the serum inflammatory profile of CD patients correlates with ileal expression of genes related to NLRP3 and MEFV inflammasomes. Overall, we show that MEFV and NLRP3 inflammasome expression in CD intestine is attributed to the accumulation of immature macrophages and correlates with serum inflammatory proteins.

TREM-1+ Macrophages Define a Pathogenic Cell Subset in the Intestine of Crohn's Disease Patients.

BACKGROUND AND AIMS: Uncontrolled activation of intestinal mononuclear phagocytes [MNPs] drives chronic inflammation in inflammatory bowel disease [IBD]. Triggering receptor expressed on myeloid cells 1 [TREM-1] has been implicated in the pathogenesis of IBD. However, the role of TREM-1+ cell subsets in driving IBD pathology and the link with clinical parameters are not understood. We investigated TREM-1 expression in human intestinal MNP subsets and examined blocking TREM-1 as a potential IBD therapy. METHODS: TREM-1 gene expression was analysed in intestinal mucosa, enriched epithelial and lamina propria [LP] layers, and purified cells from controls and IBD patients. TREM-1 protein on immune cells was assessed by flow cytometry and immunofluorescence microscopy. Blood monocyte activation was examined by large-scale gene expression using a TREM-1 agonist or LP conditioned media [LP-CM] from patients in the presence or absence of TREM-1 and tumour necrosis factor [TNF] antagonist antibodies. RESULTS: TREM-1 gene expression increases in intestinal mucosa from IBD patients and correlates with disease score. TREM-1+ cells, which are mainly immature macrophages and CD11b+ granulocytes, increase among LP cells from Crohn's disease patients and their frequency correlates with inflammatory molecules in LP-CM. LP-CM from Crohn's disease patients induces an inflammatory transcriptome in blood monocytes, including increased IL-6 expression, which is reduced by simultaneous blocking of TREM-1 and TNF. CONCLUSIONS: High intestinal TREM-1 expression, reflecting a high frequency of TREM-1+ immature macrophages and TREM-1+CD11b+ granulocytes, is linked to the deleterious inflammatory microenvironment in IBD patients. Therefore, blocking the TREM-1 pathway, especially simultaneously with anti-TNF therapy, has potential as a new IBD therapy.

A survey on patients medication reconciliation process in an oncological hospital.

OBJECTIVES: The purpose of this study was to assess the impact of medication reconciliation in the clinical practice from a hospital pharmacist point of view. METHODS: A survey of the medication taken by cancer patients was performed on admission and on discharge in an oncological hospital, and then the subjects were followed up until discharge for 8 weeks. The pharmacist entered the data collected into a computer based tool which, by using Screening Tool of Older Persons' Potentially Inappropriate Prescriptions (STOPP criteria) and Micromedex™ interactions database, automatically produces a report indicating the possible inconsistencies. The report is to check all potentially inappropriate prescriptions (PIPs) correlated to the drugs assumption by the patient. The appropriateness of the medication was scored using a Medication Appropriateness Index (MAI index) which was used to reconcile the medication list accordingly. RESULTS: Patients reconciled at admission were 98, while patients reconciled at discharge were 90, 8 patients dropped out due to death. After the intervention of the hospital pharmacist, the average value of MAI index showed a significant reduction (3,391 to 2,552 p=0.039) and the median number of drugs prescribed per patient was decreased (7 vs 6; p=0.8058). CONCLUSION: Our study demonstrated that the forms used in the reconciliation process, in particular the record card, is a promising method to increase the quality of the information related to drug use in clinical decisions. We think that medication reconciliation softwares should be widely used by health care professionals involved in the recording of drug history or prescription process.