Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.

Body weight (BW) loss and reduced body mass index (BMI) are the most common peripheral alterations in Huntington disease (HD) and have been found in HD mutation carriers and HD animal models before manifestation of neurological symptoms. This suggests that, at least in the early disease stage, these changes could be due to abnormal tissue growth rather than tissue atrophy. Also, BW and BMI are reported to be more affected in males than females in HD animal models and patients. Here, we confirmed sex-dependent growth alterations in the BACHD rat model for HD and investigated the associated contributing factors. We assessed body weight longitudinally and musculoskeletal and brain growth as well as growth-related hormones transversally in male and female rats. Our results showed growth abnormalities along with decreased plasma testosterone and insulin-like growth factor 1 (IGF-1) levels only in males. Moreover, we demonstrated correlations between growth parameters, IGF-1 and testosterone. Our analyses further revealed an aberrant transcription of testosterone biosynthesis-related genes in the testes of BACHD rats with undisturbed luteinizing hormone (LH)/cAMP/PKA signalling, which plays a key role in regulating the transcription process of some of these genes. In line with these results in BACHD rats, analyses in the R6/2 mouse model of HD showed sex-specific BW reduction in males, reduced circulating levels of testosterone and dysregulation of testosterone biosynthesis-related genes. Our findings support the view that mutant huntingtin may induce abnormal growth in males via dysregulation of gene transcription in the testis, which in turn can affect testosterone biosynthesis.

Background Cellular hallmarks of Huntington Disease include aggregate formation in neurons and neuronal loss. Encouraging findings on neuroprotection in AD and PD have led us to explore the effects of a novel α2-adrenoceptor (α2A-AR) antagonist, Beditin, in HD cell models. α2-ARs have been implicated in the reduction of oxidative stress, neuroprotection and reduction of excitotoxicity. Aim We sought to assess the neuroprotective properties of α2-AR inhibition with beditin in cell models of HD. Methods α2A-AR levels were assessed in cell models of HD and brain homogenates of R6/2 mice. In STHdhQ111/Q111 cells, effects of beditin were assessed by LDH and TUNEL cytotoxicity assays. Levels of mHTT and autophagy markers were analyzed by western blot and HTT levels by TR-FRET. Aggregation was analyzed by fluorescence microscopy and filter trap in an HEK293T eGFP-exon1-fragment overexpression model. Results/Outcome α2A-AR levels were found to be increased in STHdh Q111/Q111 cells. Beditin did not only decrease cytotoxicity in this cell line, but it also showed effects on the levels of HTT fragments. It further reduced aggregation in an overexpression cell model. mTOR independent induction of autophagy was observed, possibly contributing to the mHTT reducing effects. Conclusion In line with findings in AD and PD, we could confirm the neuroprotective effects of α2-AR inhibition and showed a reduction of aggregates in an in vitro model. The study suggests α2-ARs as a therapeutic target in HD and pharmacological manipulation of the receptor by beditin can be used to further characterize the molecular mechanisms underlying the observed effects.

Intrastriatal administration of mesenchymal stem cells (MSCs) has shown beneficial effects in rodent models of Huntington disease (HD). However, the invasive nature of surgical procedure and its potential to trigger the host immune response may limit its clinical use. Hence, we sought to evaluate the non-invasive intranasal administration (INA) of MSC delivery as an effective alternative route in HD. GFP-expressing MSCs derived from bone marrow were intranasally administered to 4-week-old R6/2 HD transgenic mice. MSCs were detected in the olfactory bulb, midbrain and striatum five days post-delivery. Compared to phosphate-buffered saline (PBS)-treated littermates, MSC-treated R6/2 mice showed an increased survival rate and attenuated circadian activity disruption assessed by locomotor activity. MSCs increased the protein expression of DARPP-32 and tyrosine hydroxylase (TH) and downregulated gene expression of inflammatory modulators in the brain 7.5 weeks after INA. While vehicle treated R6/2 mice displayed decreased Iba1 expression and altered microglial morphology in comparison to the wild type littermates, MSCs restored both, Iba1 level and the thickness of microglial processes in the striatum of R6/2 mice. Our results demonstrate significantly ameliorated phenotypes of R6/2 mice after MSCs administration via INA, suggesting this method as an effective delivering route of cells to the brain for HD therapy.

One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds - the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.

An original immuno-regulatory strategy against inflammatory bowel diseases based on the use of 28 kDa glutathione S-transferase (P28GST), a unique schistosome protein, was recently proposed. Improvement of intestinal inflammation occurs through restoration of the immunological balance between pro-inflammatory T-helper 1 (Th1) responses and both T-helper 2 (Th2) and regulatory responses. However, detailed mechanisms explaining how P28GST prevents colitis and promotes gut homeostasis remain unknown. Considering the complex interplay between the adaptive and innate immune system and the intestinal microbiota, we raised the question of the possible role of the microbial ecosystem in the anti-inflammatory effects mediated by the helminth-derived P28GST protein. We first analyzed, by 16S rRNA sequencing, the bacterial profiles of mice fecal microbiota at several time points of the P28GST-immunomodulation period prior to trinitrobenzene sulfonic acid (TNBS)-colitis. The influence of gut microbiota in the P28GST-mediated anti-inflammatory effects was then assessed by fecal microbiota transplantation experiments from P28GST-immunized mice to either conventional or microbiota depleted naïve recipient mice. Finally, the experimental data were supplemented by the temporal fecal microbiota compositions of P28GST-treated Crohn's disease patients from a pilot clinical study (NCT02281916). The P28GST administration slightly modulated the diversity and composition of mouse fecal microbiota while it significantly reduced experimental colitis in mice. Fecal microbiota transplantation experiments failed to restore the P28GST-induced anti-inflammatory effects. In Crohn's disease patients, P28GST also induced slight changes in their overall fecal bacterial composition. Collectively, these results provide key elements in both the anti-inflammatory mechanisms and the safe therapeutic use of immunomodulation with such promising helminth-derived molecules.

Proteolytic machineries execute vital cellular functions and their disturbances are implicated in diverse medical conditions, including neurodegenerative diseases. Interestingly, calpains, a class of Ca2+-dependent regulatory proteases, can modulate the degradational system of autophagy by cleaving proteins involved in this pathway. Moreover, both machineries are common players in many molecular pathomechanisms and have been targeted individually or together, as a therapeutic strategy in experimental setups. In this review, we briefly introduce calpains and autophagy, with their roles in health and disease, and focus on their direct pathologically relevant interplay in neurodegeneration and beyond. The modulation of calpain activity may comprise a promising treatment approach to attenuate the deregulation of these two essential mechanisms.

Lactic acid bacteria (LAB) are representative members of multiple ecosystems on earth, displaying dynamic interactions within animal and plant kingdoms in respect with other microbes. This highly heterogeneous phylogenetic group has coevolved with plants, invertebrates, and vertebrates, establishing either mutualism, symbiosis, commensalism, or even parasitism-like behavior with their hosts. Depending on their location and environment conditions, LAB can be dominant or sometimes in minority within ecosystems. Whatever their origins and relative abundance in specific anatomic sites, LAB exhibit multifaceted ecological and functional properties. While some resident LAB permanently inhabit distinct animal mucosal cavities, others are provided by food and may transiently occupy the gastrointestinal tract. It is admitted that the overall gut microbiome has a deep impact on health and diseases. Here, we examined the presence and the physiological role of LAB in the healthy human and several animal microbiome. Moreover, we also highlighted some dysbiotic states and related consequences for health, considering both the resident and the so-called "transionts" microorganisms. Whether LAB-related health effects act collectively or follow a strain-specificity dogma is also addressed. Besides the highly suggested contribution of LAB to interplay with immune, metabolic, and even brain-axis regulation, the possible involvement of LAB in xenobiotic detoxification processes and metal equilibrium is also tackled. Recent technological developments such as functional metagenomics, metabolomics, high-content screening and design in vitro and in vivo experimental models now open new horizons for LAB as markers applied for disease diagnosis, susceptibility, and follow-up. Moreover, identification of general and more specific molecular mechanisms based on antioxidant, antimicrobial, anti-inflammatory, and detoxifying properties of LAB currently extends their selection and promising use, either as probiotics, in traditional and functional foods, for dedicated treatments and mostly for maintenance of normobiosis and homeostasis.

Background The CAG repeat expansion in the HTT gene is a dynamic mutation varying in length in tissues and between generations. The mechanisms behind the instability of CAG expansions are polymerase slippage and DNA repair failure. BRCA1 and BRCA2 are well characterized DNA repair genes and mutations increase the risk of developing breast and ovarian cancer for carriers. Aim Therefore, in our study we analysed CAG lengths in various polyglutamine containing genes to investigate the potential effect of mutations in the DNA repair genes BRCA1 and BRCA2 on CAG length. Methods In a cohort of patients from Department of Obstetrics and Gynecology in Tuebingen carrying BRCA1 or BRCA2 mutations, CAG lengths in 6 different polyglutamine disease genes (HD, SCA1, SCA2, SCA3, SCA6 and SCA7) were determined by fragment length analysis. CAG lengths were classified as non-pathological, intermediate and pathological for each disorder. The findings were compared to data on the general population and to a control group from the Department of Obstetrics and Gynecology in Tuebingen. Results We have found no difference in the overall CAG repeat length in patients carrying BRCA1 or BRCA2 mutations. However, the frequency of patients carrying intermediate CAG lengths was higher in patients carrying BRCA1 and BRCA2 mutations in comparison to the general population. Conclusion The frequency of intermediate alleles was in our cohort higher than reported for the general population. They further link BRCA mutations to defects in DNA repair possibly contributing to the expansion of the CAG repeats with a mechanism common to different polyglutamine disorders. Further studies are needed in order to evaluate the correlation of specific mutations with CAG size and CAG size with disease prognosis of BRCA1 or BRCA2 mutation carriers.

Background In Huntington disease (HD), the negative impacts of metabolic dysfunctions heavily affects the quality of life of patients. As energy metabolism is centrally regulated by the hypothalamus via a number of proteins, we hypothesize that mutant huntingtin (mHTT) disrupts selective proteins resulting in metabolic disturbances. Aims To investigate disruptions of energy metabolism caused by mHTT in the hypothalamus. Methods A viral-mediated microRNA that could downregulate both wild-type and mutant HTT was administered to the hypothalamus of BACHD rats using bilateral stereotaxic injections. To compare the effects of mHTT downregulation at the time of intervention, BACHD rats were treated at 1 and 6 months of age, which correspond to the early disease and symptomatic stages respectively. Wild-type littermates treated with vector formulation buffer and BACHD rats treated with an empty vector were included as control groups. Brain tissues were harvested 5 months post-treatment for protein analyses. Results Our findings showed that, protein expressions of neuropeptide Y receptor 5 (NPY5R), orexin receptor 1 (OX1R) and leptin receptor were significantly altered in at least one of the disease stages in the hypothalamus of the empty vector-treated BACHD rats when compared to the wild-type littermates. Downregulation of mHTT in the hypothalamus restored the protein expressions of NPY5R, OX1R and leptin receptor in the hypothalamus to respective levels as in the wild-type littermates. Conclusions The physiological functions of the hypothalamus are disrupted by mHTT in HD. Nevertheless, mHTT suppression in the hypothalamus can be an effective approach for restoring the perturbed neuroendocrine axes regulating energy metabolism.

Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells' tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.

Abstract Introduction: The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a fundamental role in many cellular processes like growth, survival, proliferation, differentiation and motility. In cancers several mutations have been identified that lead to constitutive activation of PI3K. PQR530 is a novel, ATP site directed inhibitor of all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 that is currently in pre-clinical development. PQR530 potently binds to its targets, inhibits cell proliferation and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR530 inhibits PI3K signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been found for PQR530 during GLP toxicological testing in rats and dogs. Increase in insulin and blood glucose, a treatable class effect of PI3K inhibitors, has been observed after PQR530 administration to mice. Investigation of mutagenicity and hERG binding resulted in a clean profile. PQR530 exhibited dose-proportional pharmacokinetics (PK) in male C57BL/6J mice. A maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (7.8 μg/ml and 112.6 μg/ml, respectively) indicating that efficacious concentrations were reached in both tissues. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. PQR530 potently inhibited PI3K signaling in vivo for several hours after administration of a single oral dose of 50 mg/kg. Tumor growth was significantly decreased in SUDHL-6 lymphoma, RIVA lymphoma and OVCAR-3 ovarian cancer mouse xenografts using daily, oral administration. Conclusion: PQR530 is a potent, ATP competitive pan-PI3K and mTORC1/2 inhibitor. The physico-chemical properties of PQR530 result in good oral bioavailability and excellent brain penetration. PQR530 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Rageot D, et al., Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor, abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Petra Hillmann, Denise Rageot, Florent Beaufils, Anna Melone, Alexander Sele, Robert A. Ettlin, Jürgen Mestan, Vladimir Cmiljanovic, Marc Lang, Elisabeth Singer, Carolin Walter, Hoa HP Nguyen, Paul Hebeisen, Matthias P. Wymann, Doriano Fabbro. Pharmacological characterization of the selective, orally bioavailable, potent dual PI3K/mTORC1/2 inhibitor PQR530 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2017-159

Abstract Introduction: The mammalian target of rapamycin (mTOR) signaling pathway is an integrating factor in cell physiology that influences many processes like growth, metabolism and proliferation. mTOR signaling is constitutively activated in many cancers. Rapamycin is an allosteric inhibitor of mTOR that targets a subset of mTOR functions via inhibition of the mTORC1 complex. An ATP site-directed mTORC1/2 inhibitor that fully blocks all mTOR functions is desirable as cancer therapeutic. PQR620 is a novel, ATP site directed inhibitor of mTOR that is currently in pre-clinical development. PQR620 potently binds to its target (Kd = 6 nM) and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR620 inhibits mTOR signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been observed in mice (MTD = 150 mg/kg). A 14 day GLP toxicological study in rats showed very good tolerability (MTD = 30 mg/kg). Only minor toxicities such as dose-related changes in body weight and blood count were observed. PQR620 was administered to male C57BL/6J mice for a pharmacokinetic (PK) and pharmacodynamics (PD) evaluation. After oral application PQR620 exhibited dose-proportional PK, a maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (4.8 μg/ml and 7.7 μg/ml, respectively). In muscle, Cmax (7.6 μg/ml) was reached after 2 hours. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC0-tz (area under the curve)) was 20.5 μg*h/ml in plasma, while it was approximately 30% higher in both, brain and thigh muscle (30.6 and 32.3 μg*h/ml, respectively). PQR620 potently inhibited mTOR signaling in vivo after administration of a single oral dose of 50 mg/kg. Importantly, no effect on plasma insulin levels was observed. In an OVCAR-3, ovarian carcinoma mouse xenograft, PQR620 effectively attenuated tumor growth using daily, oral dosing. Conclusion: PQR620 potently inhibits mTORC1/2 in vitro and in vivo. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Beaufils F, Rageot D, et al., Structure-Activity Relationship Studies, Synthesis and Biological Evaluation of PQR620, a Highly Potent and Selective mTORC1/2 Inhibitor, AACR annual meeting 2016 Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Alexander Sele, Marc Lang, Juergen Mestan, Robert A. Ettlin, Petra Hillmann, Vladimir Cmiljanovic, Carolin Walter, Elisabeth Singer, Hoa HP Nguyen, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393A.

Background The pathogenesis of HD is not entirely understood, but it is clear that the expression of the disease-causing mutant huntingtin protein leads to the disruption of a variety of cellular functions, which ultimately results in cell death. A hallmark of HD is the accumulation of intracellular inclusions of mHTT, the mHTT aggregates. Although the toxicity of mHTT aggregates is still debated, various studies have demonstrated that induction of autophagy, the major pathway for the degradation of mHTT aggregates, leads to increased cell viability. ATP- competitive mTOR inhibitors have been shown to be more efficient in induction of autophagy and reduction of protein aggregates than allosteric mTOR inhibitors, like Rapamycin. Aim The ability of catalytic mTOR inhibitors to cross the blood-brain barrier, however, is limited and mTOR inhibition in brain is insufficient. Therefore two novel mTOR inhibitors were evaluated for their efficacy in mTOR inhibition in an HD context, by assessing their potential for inducing autophagy and reducing HTT levels. Methods Expression levels of autophagic markers and well-studied mTOR targets were analysed by western blot analysis in STHdhQ7/Q7 and STHdhQ111/Q111 cells and brain samples from mice treated with both compounds. Cytotoxic effects were monitored in STHdh cells. For studying HTT levels HEK293T cells were transiently transfected with HTT- Exon 1- eGFP constructs with either 19 or 51 Q and analysed by fluorescent imaging and filter trap assay. Results Treatment with both compounds led to decreased cytotoxicity in STHdh cells. The phosphorylation level of mTOR targets 4EBP1 and ribosomal protein S6 were found to be reduced, while LC3B – II levels increased in vitro and in vivo, indicating inhibition of mTOR activity and induction of autophagy. Furthermore reduction of aggregates was demonstrated upon treatment with both substances. Conclusion Originally derived from cancer treatment, these second generation mTOR inhibitors show brain penetrance and effectiveness in cell models of HD, which suggests them as potentially relevant in treatment of neurodegenerative disorders.

Background The STHdh cell lines are immortalised striatal precursor cells from wild type and Hdh Q111 knock-in mice and are commonly used to study the molecular aspects of HD. Morphological differences between the wild type and mutant cell lines exist, but are rarely described or clearly considered in published reports. Aims The aim was to characterise cell size and proliferation differences in the STHdh cells, to investigate the importance of these phenotypes for HD and their possible confounding nature. Methods Cell size, cell proliferation, mTOR-related cell signalling and chromosome content were assessed in wild type STHdh Q7/7 and HD mutant STHdh Q111/111 cell lines as well as in primary cultures of mouse embryonic fibroblasts (MEF) established from the same mouse model (MEFHdh Q7/7 and MEFHdh Q111/111 cells). Cell viability and cell death were measured in STHdh cells using standard fluorometric assays and flow cytometry. Results STHdh Q111/111 as well as MEFHdh Q111/111 cells were smaller, showed higher proliferation rates and higher levels of phosphorylated mTOR pathway components compared to their wild type counterpart. Both STHdh cell lines displayed chromosome multiplications already at early passages, although the phenotype was more severe in STHdh Q7/7 cells. No marked chromosome abnormalities were found in the MEF cells. Results from fluorometric cell viability assays indicated that STHdh Q111/111 cells had reduced cell viability and increased cell mortality. This was not supported by the results from flow cytometry, the readouts of which are likely to be unaffected by cell size and proliferation. Conclusions Differences in cell size and proliferation are characteristics of STHdh Q111/111 cells and might be caused by altered mTOR-related signalling. These phenotypes appear to be a general feature of HD, as they are also found in the second cell model. The different degree of genomic instability in wild type and mutant STHdh cells puts the usefulness of the wild type cells as controls in question. Furthermore, cell size and proliferation phenotypes are likely to confound test results and lead to inaccurate conclusions. Thus, our observations suggest that careful experimental design and well-considered data analysis are crucial when using this cell model.

A weakening of the gut mucous barrier permits an increase in the access of intestinal luminal contents to the epithelial cells, which will trigger the inflammatory response. In inflammatory bowel diseases, there is an inappropriate and ongoing activation of the immune system, possibly because the intestinal mucus is less protective against the endogenous microflora. General strategies aimed at improving the protection of the intestinal epithelium are still missing. We generated a transgenic mouse that secreted a molecule consisting of 12 consecutive copies of a mucin domain into its intestinal mucus, which is believed to modify the mucus layer by establishing reversible interactions. We showed that the mucus gel was more robust and that mucin O-glycosylation was altered. Notably, the gut epithelium of transgenic mice housed a greater abundance of beneficial Lactobacillus spp. These modifications were associated with a reduced susceptibility of transgenic mice to chemically induced colitis. Furthermore, transgenic mice cleared faster Citrobacter rodentium bacteria which were orally given and mice were more protected against bacterial translocation induced by gavage with adherent-invasive Escherichia coli. Our data show that delivering the mucin CYS domain into the gut lumen strengthens the intestinal mucus blanket which is impaired in inflammatory bowel diseases.

BACKGROUND:Inflammatory bowel diseases (IBD) patients are abnormally colonized by adherent-invasive Escherichia coli (AIEC). NOD2 gene mutations impair intracellular bacterial clearance. We evaluated the impact of antibiotic treatment on AIEC colonization in wildtype (WT) and NOD2 knockout mice (NOD2KO) and the consequences on intestinal inflammation.METHODS:After 3 days of antibiotic treatment, mice were infected for 2 days with 10⁹ CFU AIEC and sacrificed 1, 5, and 60 days later. In parallel, mice were challenged with AIEC subsequent to a dextran sodium sulfate (DSS) treatment and sacrificed 9 days later. Ileum, colon, and mesenteric tissues were sampled for AIEC quantification and evaluation of inflammation.RESULTS:Without antibiotic treatment, AIEC was not able to colonize WT and NOD2KO mice. Compared with nontreated animals, antibiotic treatment led to a significant increase in ileal and colonic colonization of AIEC in WT and/or NOD2KO mice. Persistent AIEC colonization was observed until day 5 only in NOD2KO mice, disappearing at day 60. Mesenteric translocation of AIEC was observed only in NOD2KO mice. No inflammation was observed in WT and NOD2KO mice treated with antibiotics and infected with AIEC. During DSS-induced colitis, colonization and persistence of AIEC was observed in the colon. Moreover, a dramatic increase in clinical, histological, and molecular parameters of colitis was observed in mice infected with AIEC but not with a commensal E. coli strain.CONCLUSIONS:Antibiotic treatment was necessary for AIEC colonization of the gut and mesenteric tissues and persistence of AIEC was dependent on NOD2. AIEC exacerbated a preexisting DSS-induced colitis in WT mice.