Lactotrehalose, an Analog of Trehalose, Increases Energy Metabolism Without Promoting Clostridioides difficile Infection in Mice.

BACKGROUND & AIMS: Trehalose is a disaccharide that might be used in the treatment of cardiometabolic diseases. However, trehalose consumption promotes the expansion of Clostridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase. Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lactotrehalose) has the same metabolic effects of trehalose without expanding C difficile. METHODS: We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells, and glucose tra2nsport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose, and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for toxins A (ToxA) or B (ToxB) by enzyme-linked immunosorbent assay. Other mice were given trehalose or lactotrehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, RNA sequencing, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S ribosomal RNA sequencing and principal component analyses. RESULTS: Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight-to-body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched-chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue after CD027 infection. CONCLUSIONS: Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for the treatment of diabetes and nonalcoholic fatty liver disease.

Helicobacter suis affects the health and function of porcine gastric parietal cells.

The stomach of pigs at slaughter age is often colonized by Helicobacter (H.) suis, which is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. It is associated with chronic gastritis, gastric ulceration and other gastric pathological changes in both hosts. Parietal cells are highly specialized, terminally differentiated epithelial cells responsible for gastric acid secretion and regulation. Dysfunction of these cells is closely associated with gastric pathology and disease. Here we describe a method for isolation and culture of viable and responsive parietal cells from slaughterhouse pigs. In addition, we investigated the interactions between H. suis and gastric parietal cells both in H. suis-infected six-month-old slaughter pigs, as well as in our in vitro parietal cell model. A close interaction of H. suis and parietal cells was observed in the fundic region of stomachs from H. suis positive pigs. The bacterium was shown to be able to directly interfere with cultured porcine parietal cells, causing a significant impairment of cell viability. Transcriptional levels of Atp4a, essential for gastric acid secretion, showed a trend towards an up-regulation in H. suis positive pigs compared to H. suis-negative pigs. In addition, sonic hedgehog, an important factor involved in gastric epithelial differentiation, gastric mucosal repair, and stomach homeostasis, was also significantly up-regulated in H. suis positive pigs. In conclusion, this study describes a successful approach for the isolation and culture of porcine gastric parietal cells. The results indicate that H. suis affects the viability and function of this cell type.

Helicobacter pullorum cytolethal distending toxin targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial cells.

Helicobacter pullorum, a bacterium initially isolated from poultry, has been associated with human digestive disorders. However, the factor responsible for its cytopathogenic effects on epithelial cells has not been formally identified. The cytopathogenic alterations induced by several human and avian H. pullorum strains were investigated on human intestinal epithelial cell lines. Moreover, the effects of the cytolethal distending toxin (CDT) were evaluated first by using a wild-type strain and its corresponding cdtB isogenic mutant and second by delivering the active CdtB subunit of the CDT directly into the cells. All of the H. pullorum strains induced cellular distending phenotype, actin cytoskeleton remodeling, and G2/M cell cycle arrest. These effects were dependent on the CDT, as they were (1) not observed in response to a cdtB isogenic mutant strain and (2) present in cells expressing CdtB. CdtB also induced an atypical delocalization of vinculin from focal adhesions to the perinuclear region, formation of cortical actin-rich large lamellipodia with an upregulation of cortactin, and decreased cellular adherence. In conclusion, the CDT of H. pullorum is responsible for major cytopathogenic effects in vitro, confirming its role as a main virulence factor of this emerging human pathogen.

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection.

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens.

Digital dermatitis in cattle is associated with an excessive innate immune response triggered by the keratinocytes.

BACKGROUND: Digital Dermatitis (DD) is a common disease of dairy cows, the pathogenesis of which is still not clear. This study examined some host responses associated with the typical lesions, in an attempt to further elucidate the pathogenesis of the disease. Twenty four samples representing the 5 different clinical stages of DD (M0-M4) were collected from slaughtered cattle for histopathological and immunological analyses. RESULTS: Significant increases in total epidermal thickness were found in M2, M3, and M4 when compared with M0 and M1. M3 samples, when compared with M0 and M1, were characterized by a significant increase in the thickness of the keratin layer. Counts of both eosinophils and neutrophils were at a maximum in the M2 stage and decreased in the M3 and M4 stage. A significant increase in IL8 expression was observed in the M2-M3 stages of the disease and immunohistochemical staining showed the source as keratinocytes, suggesting an important role for keratinocyte-derived IL8 in the pathogenesis of DD. CONCLUSION: Results of the present study point to a strong stimulation of the innate immune response at the level of the keratinocytes throughout most of the clinical stages, and a delayed response of the adaptive immune reaction.

Current and future methods of probiotic therapy for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a complex intestinal disease that primarily affects premature neonates. Given its significant mortality and morbidity, there is an urgent need to develop improved prophylactic measures against the disease. One potential preventative strategy for NEC is the use of probiotics. Although there has been significant interest for decades in probiotics in neonatal care, no clear guidelines exist regarding which probiotic to use or for which patients, and no FDA-approved products exist on the market for NEC. In addition, there is lack of agreement regarding the benefits of probiotics in neonates, as well as some concerns about the safety and efficacy of available products. We discuss currently available probiotics as well as next-generation probiotics and novel delivery strategies which may offer an avenue to capitalize on the benefits of probiotics, while minimizing the risks. Thus, probiotics may still prove to be an effective prevention strategy for NEC, although further product development and research is needed to support use in the preterm population.

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip.

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease.

Development of a novel definitive scoring system for an enteral feed-only model of necrotizing enterocolitis in piglets.

Introduction: Necrotizing enterocolitis (NEC) is a complex inflammatory disorder of the human intestine that most often occurs in premature newborns. Animal models of NEC typically use mice or rats; however, pigs have emerged as a viable alternative given their similar size, intestinal development, and physiology compared to humans. While most piglet NEC models initially administer total parenteral nutrition prior to enteral feeds, here we describe an enteral-feed only piglet model of NEC that recapitulates the microbiome abnormalities present in neonates that develop NEC and introduce a novel multifactorial definitive NEC (D-NEC) scoring system to assess disease severity. Methods: Premature piglets were delivered via Caesarean section. Piglets in the colostrum-fed group received bovine colostrum feeds only throughout the experiment. Piglets in the formula-fed group received colostrum for the first 24 h of life, followed by Neocate Junior to induce intestinal injury. The presence of at least 3 of the following 4 criteria were required to diagnose D-NEC: (1) gross injury score ≥4 of 6; (2) histologic injury score ≥3 of 5; (3) a newly developed clinical sickness score ≥5 of 8 within the last 12 h of life; and (4) bacterial translocation to ≥2 internal organs. Quantitative reverse transcription polymerase chain reaction was performed to confirm intestinal inflammation in the small intestine and colon. 16S rRNA sequencing was performed to evaluate the intestinal microbiome. Results: Compared to the colostrum-fed group, the formula-fed group had lower survival, higher clinical sickness scores, and more severe gross and histologic intestinal injury. There was significantly increased bacterial translocation, D-NEC, and expression of IL-1α and IL-10 in the colon of formula-fed compared to colostrum-fed piglets. Intestinal microbiome analysis of piglets with D-NEC demonstrated lower microbial diversity and increased Gammaproteobacteria and Enterobacteriaceae. Conclusions: We have developed a clinical sickness score and a new multifactorial D-NEC scoring system to accurately evaluate an enteral feed-only piglet model of NEC. Piglets with D-NEC had microbiome changes consistent with those seen in preterm infants with NEC. This model can be used to test future novel therapies to treat and prevent this devastating disease.

Superior performance of biofilm versus planktonic Limosilactobacillus reuteri in protection of the intestines and brain in a piglet model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related death in premature infants. Its etiology is multifactorial, with intestinal dysbiosis playing a major role. Probiotics are a logical preventative therapy for NEC, however their benefits have been inconsistent. We previously developed a novel probiotic delivery system in which planktonic (free-living) Limosilactobacillus reuteri (Lr) is incubated with biocompatible dextranomer microspheres (DM) loaded with maltose (Lr-DM-maltose) to induce biofilm formation. Here we have investigated the effects of Lr-DM-maltose in an enteral feed-only piglet model of NEC. We found a significant decrease in the incidence of Definitive NEC (D-NEC), death associated with D-NEC, and activated microglia in the brains of piglets treated with Lr-DM-maltose compared to non-treated piglets. Microbiome analyses using 16S rRNA sequencing of colonic contents revealed a significantly different microbial community composition between piglets treated with Lr-DM-maltose compared to non-treated piglets, with an increase in Lactobacillaceae and a decrease in Clostridiaceae in Lr-DM-maltose-treated piglets. Furthermore, there was a significant decrease in the incidence of D-NEC between piglets treated with Lr-DM-maltose compared to planktonic Lr. These findings validate our previous results in rodents, and support future clinical trials of Lr in its biofilm state for the prevention of NEC in premature neonates.

T cell counts and IL-6 concentration in blood of North African COVID-19 patients are two independent prognostic factors for severe disease and death.

The immune system plays a crucial role in the response against severe acute respiratory syndrome coronavirus 2 with significant differences among patients. The study investigated the relationships between lymphocyte subsets, cytokines, and disease outcomes in patients with coronavirus disease 2019 (COVID-19). The measurements of peripheral blood lymphocytes subsets and cytokine levels were performed by flow cytometry for 57 COVID-19 patients. Patients were categorized into two groups according to the severity of the disease (nonsevere vs. severe). Total lymphocytes, T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells were decreased in COVID-19 patients and statistical differences were found among different severity of illness and survival states (P ˂ 0.01). The levels of IL-6 and IL-10 were significantly higher in severe and death groups and negatively correlated with lymphocyte subsets counts. The percentages of Th17 in the peripheral blood of patients were higher than those of healthy controls whereas the percentages of Th2 were lower. For the severe cases, the area under receiver operating characteristic (ROC) curve of IL-6 was the largest among all the immune parameters (0.964; 95% confidence interval: 0.927-1.000, P < 0.0001). In addition, the preoperative IL-6 concentration of 77.38 pg/ml was the optimal cutoff value (sensitivity: 84.6%, specificity: 100%). Using multivariate logistic regression analysis and ROC curves, IL-6 > 106.44 pg/ml and CD8+ T cell counts <150 cells/µl were found to be associated with mortality. Measuring the immune parameters and defining a risk threshold can segregate patients who develop a severe disease from those with a mild pathology. The identification of these parameters may help clinicians to predict the outcome of the patients with high risk of unfavorable progress of the disease.

A Mouse Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is an acute inflammatory disease that unforeseeably develops in very low birth weight premature infants. NEC is characterized by impairment of the intestinal barrier resulting in intestinal necrosis and multisystem organ failure. Animal models of NEC have contributed significantly to a better understanding of the underlying molecular mechanisms of the disease and facilitated the exploration of potential new therapeutic strategies. Here, we provide a detailed protocol that recapitulates some of the main histological and transcriptional features of human NEC in newborn mice.

Indole-3-Carbinol-Dependent Aryl Hydrocarbon Receptor Signaling Attenuates the Inflammatory Response in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) causes significant morbidity and mortality in premature infants; therefore, the identification of therapeutic and preventative strategies against NEC remains a high priority. The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) is well known to contribute to the regulation of intestinal microbial communities and amelioration of intestinal inflammation. However, the role of AhR signaling in NEC is unclear. Experimental NEC was induced in 4-d-old wild-type mice or mice lacking AhR expression in the intestinal epithelial cells or AhR expression in CD11c+ cells (AhRΔCD11c) by subjecting animals to twice daily hypoxic stress and gavage feeding with formula supplemented with LPS and enteric bacteria. During NEC, compared with wild-type mice treated with vehicle, littermates treated with an AhR proligand, indole-3-carbinol, had reduced expression of Il1b and Marco, a scavenger receptor that mediates dendritic cell activation and the recognition and clearance of bacterial pathogens by macrophages. Furthermore, indole-3-carbinol treatment led to the downregulation of genes involved in cytokine and chemokine, as revealed by pathway enrichment analysis. AhR expression in the intestinal epithelial cells and their cre-negative mouse littermates were similarly susceptible to experimental NEC, whereas AhRΔCD11c mice with NEC exhibited heightened inflammatory responses compared with their cre-negative mouse littermates. In seeking to determine the mechanisms involved in this increased inflammatory response, we identified the Tim-4- monocyte-dependent subset of macrophages as increased in AhRΔCD11c mice compared with their cre-negative littermates. Taken together, these findings demonstrate the potential for AhR ligands as a novel immunotherapeutic approach to the management of this devastating disease.

Murine astrovirus tropism for goblet cells and enterocytes facilitates an IFN-λ response in vivo and in enteroid cultures.

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air-liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

Interleukin-22 signaling attenuates necrotizing enterocolitis by promoting epithelial cell regeneration.

Necrotizing enterocolitis (NEC) is a deadly intestinal inflammatory disorder that primarily affects premature infants and lacks adequate therapeutics. Interleukin (IL)-22 plays a critical role in gut barrier maintenance, promoting epithelial regeneration, and controlling intestinal inflammation in adult animal models. However, the importance of IL-22 signaling in neonates during NEC remains unknown. We investigated the role of IL-22 in the neonatal intestine under homeostatic and inflammatory conditions by using a mouse model of NEC. Our data reveal that Il22 expression in neonatal murine intestine is negligible until weaning, and both human and murine neonates lack IL-22 production during NEC. Mice deficient in IL-22 or lacking the IL-22 receptor in the intestine display a similar susceptibility to NEC, consistent with the lack of endogenous IL-22 during development. Strikingly, treatment with recombinant IL-22 during NEC substantially reduces inflammation and enhances epithelial regeneration. These findings may provide a new therapeutic strategy to attenuate NEC.

Global hypermethylation of intestinal epithelial cells is a hallmark feature of neonatal surgical necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) remains one of the overall leading causes of death in premature infants, and the pathogenesis is unpredictable and not well characterized. The aim of our study was to determine the molecular phenotype of NEC via transcriptomic and epithelial cell-specific epigenomic analysis, with a specific focus on DNA methylation. METHODS: Using laser capture microdissection, epithelial cell-specific methylation signatures were characterized by whole-genome bisulfite sequencing of ileal and colonic samples at the time of surgery for NEC and after NEC had healed at reanastomosis (n = 40). RNA sequencing was also performed to determine the transcriptomic profile of these samples, and a comparison was made to the methylome data. RESULTS: We found that surgical NEC has a considerable impact on the epigenome by broadly increasing DNA methylation levels, although these effects are less pronounced in genomic regions associated with the regulation of gene expression. Furthermore, NEC-related DNA methylation signatures were influenced by tissue of origin, with significant differences being noted between colon and ileum. We also identified numerous transcriptional changes in NEC and clear associations between gene expression and DNA methylation. CONCLUSIONS: We have defined the intestinal epigenomic and transcriptomic signatures during surgical NEC, which will advance our understanding of disease pathogenesis and may enable the development of novel precision medicine approaches for NEC prediction, diagnosis and phenotyping.

Impact of Toll-Like Receptor 4 Signaling in Necrotizing Enterocolitis: The State of the Science.

Necrotizing enterocolitis (NEC) remains a leading cause of preterm infant mortality. NEC is multifactorial and believed a consequence of intestinal immaturity, microbial dysbiosis, and an exuberant inflammatory response. Over the past decade, exaggerated Toll-like receptor 4 (TLR4) activity in the immature intestine of preterm neonates emerged as an inciting event preceding NEC. Increased TLR4 signaling in epithelial cells results in the initiation of an uncontrolled immune response and destruction of the mucosal barrier. This article discusses the state of the science of the molecular mechanisms involved in TLR4-mediated inflammation during NEC and the development of new therapeutic strategies to prevent NEC.

Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway.

Expulsion of parasitic gastrointestinal nematodes requires diverse effector mechanisms coordinated by a Th2-type response. The evolutionarily conserved JmjC protein; Myc Induced Nuclear Antigen (Mina) has been shown to repress IL4, a key Th2 cytokine, suggesting Mina may negatively regulate nematode expulsion. Here we report that expulsion of the parasitic nematode Trichuris muris was indeed accelerated in Mina deficient mice. Unexpectedly, this was associated not with an elevated Th2- but rather an impaired Th1-type response. Further reciprocal bone marrow chimera and conditional KO experiments demonstrated that retarded parasite expulsion and a normal Th1-type response both required Mina in intestinal epithelial cells (IECs). Transcriptional profiling experiments in IECs revealed anti-microbial α-defensin peptides to be the major target of Mina-dependent retention of worms in infected mice. In vitro exposure to recombinant α-defensin peptides caused cytotoxic damage to whipworms. These results identify a latent IEC-intrinsic anthelmintic pathway actively constrained by Mina and point to α-defensins as important effectors that together with Mina may be attractive therapeutic targets for the control of nematode infection.

Stem Cell-Derived Models of Viral Infections in the Gastrointestinal Tract.

Studies on the intestinal epithelial response to viral infection have previously been limited by the absence of in vitro human intestinal models that recapitulate the multicellular complexity of the gastrointestinal tract. Recent technological advances have led to the development of "mini-intestine" models, which mimic the diverse cellular nature and physiological activity of the small intestine. Utilizing adult or embryonic intestinal tissue, enteroid and organoid systems, respectively, represent an opportunity to effectively model cellular differentiation, proliferation, and interactions that are specific to the specialized environment of the intestine. Enteroid and organoid systems represent a significant advantage over traditional in vitro methods because they model the structure and function of the small intestine while also maintaining the genetic identity of the host. These more physiologic models also allow for novel approaches to investigate the interaction of enteric viruses with the gastrointestinal tract, making them ideal to study the complexities of host-pathogen interactions in this unique cellular environment. This review aims to provide a summary on the use of human enteroid and organoid systems as models to study virus pathogenesis.

A SNP uncoupling Mina expression from the TGFß signaling pathway.

INTRODUCTION: Mina is a JmjC family 2-oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell-type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1-region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. METHODS: Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1-region SNPs perturbs a Mina cis-regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus-spanning 26-kilobase genomic interval. RESULTS: We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c-but not C57Bl/6 allele-abolished both Smad3 binding and transforming growth factor beta (TGFß) responsiveness. CONCLUSIONS: Our results demonstrate the TGFß signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFß signaling pathway.

Effect of an Ostertagia ostertagi infection on the transcriptional stability of housekeeping genes in the bovine abomasum.

Quantitative Real-Time PCR (qRT-PCR) is a widely used tool to study host responses against parasites. A crucial step in the gene quantification process is the normalization of the expression data against stable housekeeping genes (HKGs). However, in recent years, several reports have showed that the transcriptional levels of such HKGs can change dramatically, especially when cellular changes appear in the tissues investigated. The aim of the current study was to assess the variability of 11 putative HKGs in bovine abomasal tissue during an infection with the parasitic nematode Ostertagia ostertagi. Gene transcription levels of selected potential HKGs were measured by qRT-PCR and the expression stabilities evaluated using geNorm, NormFinder, and The Mann-Whitney-U test. The analysis showed that all the putative HKGs considered in this study, including the ones selected by geNorm and NormFinder, were found to be significantly upregulated in infected animals compared to the controls, clearly suggesting that none of these genes can actually be used as a HKG. The greatest alterations in gene transcription levels appeared at 24 dpi, which might be due to the dramatic changes in cell populations occurring in the abomasal tissue at this infection time point. To demonstrate the effect of normalizing target gene transcription levels with unstable HKGs, IL4 transcription levels were assessed using different normalization procedures. Our findings clearly showed that gene expression levels determined using HKGs differed significantly from those determined without normalization. The potential for HKG selection to impact candidate transcript levels is therefore an important consideration for studies of parasite infected tissue.