A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni.

The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.

Investigation of Cellular Function and DNA Integrity during 2D in vitro Culture of Human Salivary Gland Epithelial Cells.

In vitro culture of human salivary gland epithelial cells (SGEC) is still a challenge. A high quantity and quality of cells are needed for the cultivation of 3D matrices. Furthermore, it is known that DNA damage is supposed to be an important factor involved in carcinogenesis. This study investigates cellular function and DNA integrity of human SGEC during 3 passage steps in 2 groups (group 1: n = 10; group 2: n = 9). Cellular function was analyzed by immunofluorescence, transmission electron microscopy (TEM), and quantitative real-time polymerase chain reaction (qPCR). DNA integrity was tested via the comet assay. Immunohistochemistry and qPCR results showed stable α-amylase and pan-cytokeratin levels; TEM revealed functional cells; and no significant DNA damage could be detected in the comet assay during 3 culture steps. The study shows that not only at cellular but also at DNA level human SGEC can be safely quantified over 3 passages for preclinical tissue engineering without loss of differentiation and function.

Obstructed defecation-an enteric neuropathy? An exploratory study of patient samples.

PURPOSE: Although various strategies exist for chronic constipation therapy, the pathogenesis of chronic constipation is still not completely understood. The aim of this exploratory experimental study is to elucidate alterations of the autonomous enteric nervous system at the molecular level in patients with obstructed defecation, who represent one of the most predominant groups of constipated patients. METHODS: Full-thickness rectal wall samples of patients with obstructed defecation were analyzed and compared with controls. Differential gene expression analyses by RNA-Seq transcriptome profiling were performed and gene expression profiles were assigned to gene ontology pathways by application of different biological libraries. RESULTS: Analysis of the transcriptome showed that genes associated with the enteric nervous system functions were significantly downregulated in patients with obstructed defecation. These affected functions included developmental processes and synaptic transmission. CONCLUSIONS: Our results therefore indicate that obstructed defecation may represent an enteric neuropathy, comparable to Hirschsprung disease and slow-transit constipation.

Novel Focus of Sin Nombre Virus in Peromyscus eremicus Mice, Death Valley National Park, California, USA.

The deer mouse (Peromyscus maniculatus) is the primary reservoir for Sin Nombre virus (SNV) in the western United States. Rodent surveillance for hantavirus in Death Valley National Park, California, USA, revealed cactus mice (P. eremicus) as a possible focal reservoir for SNV in this location. We identified SNV antibodies in 40% of cactus mice sampled.

Regulators of gene expression in Enteric Neural Crest Cells are putative Hirschsprung disease genes.

The enteric nervous system (ENS) is required for peristalsis of the gut and is derived from Enteric Neural Crest Cells (ENCCs). During ENS development, the RET receptor tyrosine kinase plays a critical role in the proliferation and survival of ENCCs, their migration along the developing gut, and differentiation into enteric neurons. Mutations in RET and its ligand GDNF cause Hirschsprung disease (HSCR), a complex genetic disorder in which ENCCs fail to colonize variable lengths of the distal bowel. To identify key regulators of ENCCs and the pathways underlying RET signaling, gene expression profiles of untreated and GDNF-treated ENCCs from E14.5 mouse embryos were generated. ENCCs express genes that are involved in both early and late neuronal development, whereas GDNF treatment induced neuronal maturation. Predicted regulators of gene expression in ENCCs include the known HSCR genes Ret and Sox10, as well as Bdnf, App and Mapk10. The regulatory overlap and functional interactions between these genes were used to construct a regulatory network that is underlying ENS development and connects to known HSCR genes. In addition, the adenosine receptor A2a (Adora2a) and neuropeptide Y receptor Y2 (Npy2r) were identified as possible regulators of terminal neuronal differentiation in GDNF-treated ENCCs. The human orthologue of Npy2r maps to the HSCR susceptibility locus 4q31.3-q32.3, suggesting a role for NPY2R both in ENS development and in HSCR.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Public Health Response to Aedes aegypti and Ae. albopictus Mosquitoes Invading California, USA.

Aedes aegypti and Ae. albopictus mosquitoes, primary vectors of dengue and chikungunya viruses, were recently detected in California, USA. The threat of potential local transmission of these viruses increases as more infected travelers arrive from affected areas. Public health response has included enhanced human and mosquito surveillance, education, and intensive mosquito control.

Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling.

Conjugates of methylated cyclodextrin derivatives and hydroxyethyl starch (HES): Synthesis, cytotoxicity and inclusion of anaesthetic actives.

The mono-6-deoxy-6-azides of 2,6-di-O-methyl-ß-cyclodextrin (DIMEB) and randomly methylated-ß-cyclodextrin (RAMEB) were conjugated to propargylated hydroxyethyl starch (HES) by Cu(+)-catalysed [2 + 3] cycloaddition. The resulting water soluble polymers showed lower critical solution temperatures (LCST) at 52.5 °C (DIMEB-HES) and 84.5 °C (RAMEB-HES), respectively. LCST phase separations could be completely avoided by the introduction of a small amount of carboxylate groups at the HES backbone. The methylated CDs conjugated to the HES backbone exhibited significantly lower cytotoxicities than the corresponding monomeric CD derivatives. Since the binding potentials of these CD conjugates were very high, they are promising candidates for new oral dosage forms of anaesthetic actives.

Enhancing pre-clinical research with simplified intestinal cell line models.

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings.

Locked Out: Phoenixin-14 Does Not Cross a Stem-Cell-Derived Blood-Brain Barrier Model.

Phoenixin-14 is a recently discovered peptide regulating appetite. Interestingly, it is expressed in the gastrointestinal tract; however, its supposed receptor, GPR173, is predominantly found in hypothalamic areas. To date, it is unknown how peripherally secreted phoenixin-14 is able to reach its centrally located receptor. To investigate whether phoenixin is able to pass the blood-brain barrier, we used an in vitro mono-culture blood-brain barrier (BBB) model consisting of brain capillary-like endothelial cells derived from human induced-pluripotent stem cells (hiPSC-BCECs). The passage of 1 nMol and 10 nMol of phoenixin-14 via the mono-culture was measured after 30, 60, 90, 120, 150, 180, 210, and 240 min using a commercial ELISA kit. The permeability coefficients (PC) of 1 nMol and 10 nMol phoenixin-14 were 0.021 ± 0.003 and 0.044 ± 0.013 µm/min, respectively. In comparison with the PC of solutes known to cross the BBB in vivo, those of phoenixin-14 in both concentrations are very low. Here, we show that phoenixin-14 alone is not able to cross the BBB, suggesting that the effects of peripherally secreted phoenixin-14 depend on a co-transport mechanism at the BBB in vivo. The mechanisms responsible for phoenixin-14's orexigenic property along the gut-brain axis warrant further research.

Biomimetic Connection of Transcutaneous Implants with Skin.

Bacterial infection is a crucial complication in implant restoration, in particular in permanent skin-penetrating implants. Therein, the resulting gap between transcutaneous implant and skin represents a permanent infection risk, limiting the field of application and the duration of application. To overcome this limitation, a tight physiological connection is required to achieve a biological and mechanical welding for a long-term stable closure including self-healing probabilities. This study describes a new approach, wherein the implant is connected covalently to a highly porous electrospun fleece featuring physiological dermal integration potential. The integrative potential of the scaffold is shown in vitro and confirmed in vivo, further demonstrating tissue integration by neovascularization, extracellular matrix formation, and prevention of encapsulation. To achieve a covalent connection between fleece and implant surface, self-initiated photografting and photopolymerization of hydroxyethylmethacrylate is combined with a new crosslinker (methacrylic acid coordinated titanium-oxo clusters) on proton-abstractable implant surfaces. For implant modification, the attached fleece is directed perpendicular from the implant surface into the surrounding dermal tissue. First in vitro skin implantations demonstrate the implants' dermal integration capability as well as wound closure potential on top of the fleece by epithelialization, establishing a bacteria-proof and self-healing connection of skin and transcutaneous implant.

Overcoming the blood-brain barrier? - prediction of blood-brain permeability of hydrophobically modified polyethylenimine polyplexes for siRNA delivery into the brain with in vitro and in vivo models.

The blood-brain barrier (BBB) is a highly selective biological barrier that represents a major bottleneck in the treatment of all types of central nervous system (CNS) disorders. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach, e.g., for brain tumors, by downregulating brain tumor-related genes and inhibiting tumor growth via RNA interference. In an effort to develop efficient siRNA nanocarriers for crossing the BBB, we utilized polyethyleneimine (PEI) polymers hydrophobically modified with either stearic-acid (SA) or dodecylacrylamide (DAA) subunits and evaluated their suitability for delivering siRNA across the BBB in in vitro and in vivo BBB models depending on their structure. Physicochemical characteristics of siRNA-polymer complexes (polyplexes (PXs)), e.g., particle size and surface charge, were measured by dynamic light scattering and laser Doppler anemometry, whereas siRNA condensation ability of polymers and polyplex stability was evaluated by spectrophotometric methods. The composition of the biomolecule corona that absorbs on polyplexes upon encountering physiological fluids was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Cellular internalization abilities of PXs into brain endothelial cells (hCMEC/D3) was confirmed, and a BBB permeation assay using a human induced pluripotent stem cell (hiPSC)-derived BBB model revealed similar abilities to cross the BBB for all formulations under physiological conditions. However, biodistribution studies of radiolabeled PXs in mice were inconsistent with in vitro results as the detected amount of radiolabeled siRNA in the brain delivered with PEI PXs was higher compared to PEI-SA PXs. Taken together, PEI PXs were shown to be a suitable nanocarrier to deliver small amounts of siRNA across the BBB into the brain but more sophisticated human BBB models that better represent physiological conditions and biodistribution are required to provide highly predictive in vitro data for human CNS drug development in the future.

Correction: A forty-year review of Rocky Mountain spotted fever cases in California shows clinical and epidemiologic changes.

[This corrects the article DOI: 10.1371/journal.pntd.0010738.].

A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection.

Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.

Human isogenic cells of the neurovascular unit exert transcriptomic cell type-specific effects on a blood-brain barrier in vitro model of late-onset Alzheimer disease.

BACKGROUND: The function of the blood-brain barrier (BBB) is impaired in late-onset Alzheimer disease (LOAD), but the associated molecular mechanisms, particularly with respect to the high-risk APOE4/4 genotype, are not well understood. For this purpose, we developed a multicellular isogenic model of the neurovascular unit (NVU) based on human induced pluripotent stem cells. METHODS: The human NVU was modeled in vitro using isogenic co-cultures of astrocytes, brain capillary endothelial-like cells (BCECs), microglia-like cells, neural stem cells (NSCs), and pericytes. Physiological and pathophysiological properties were investigated as well as the influence of each single cell type on the characteristics and function of BCECs. The barriers established by BCECs were analyzed for specific gene transcription using high-throughput quantitative PCR. RESULTS: Co-cultures were found to tighten the barrier of BCECs and alter its transcriptomic profile under both healthy and disease conditions. In vitro differentiation of brain cell types that constitute the NVU was not affected by the LOAD background. The supportive effect of NSCs on the barrier established by BCECs was diminished under LOAD conditions. Transcriptomes of LOAD BCECs were modulated by different brain cell types. NSCs were found to have the strongest effect on BCEC gene regulation and maintenance of the BBB. Co-cultures showed cell type-specific functional contributions to BBB integrity under healthy and LOAD conditions. CONCLUSIONS: Cell type-dependent transcriptional effects on LOAD BCECs were identified. Our study suggests that different brain cell types of the NVU have unique roles in maintaining barrier integrity that vary under healthy and LOAD conditions. .

Challenges in Permeability Assessment for Oral Drug Product Development.

Drug permeation across the intestinal epithelium is a prerequisite for successful oral drug delivery. The increased interest in oral administration of peptides, as well as poorly soluble and poorly permeable compounds such as drugs for targeted protein degradation, have made permeability a key parameter in oral drug product development. This review describes the various in vitro, in silico and in vivo methodologies that are applied to determine drug permeability in the human gastrointestinal tract and identifies how they are applied in the different stages of drug development. The various methods used to predict, estimate or measure permeability values, ranging from in silico and in vitro methods all the way to studies in animals and humans, are discussed with regard to their advantages, limitations and applications. A special focus is put on novel techniques such as computational approaches, gut-on-chip models and human tissue-based models, where significant progress has been made in the last few years. In addition, the impact of permeability estimations on PK predictions in PBPK modeling, the degree to which excipients can affect drug permeability in clinical studies and the requirements for colonic drug absorption are addressed.

KBP interacts with SCG10, linking Goldberg-Shprintzen syndrome to microtubule dynamics and neuronal differentiation.

Goldberg-Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction between KBP and SCG10 in vivo. To investigate the possibility of direct interaction between KBP and microtubules, we undertook co-localization and in vitro binding assays, but found no evidence of direct binding. Thus, our data indicate that KBP is involved in neuronal differentiation and that the central and enteric nervous system defects seen in GOSHS are likely caused by microtubule-related defects.

Variants in RET associated with Hirschsprung's disease affect binding of transcription factors and gene expression.

BACKGROUND & AIMS: Two noncoding variations in RET-the T allele of the single nucleotide polymorphism (SNP) rs2435357 (Enh1:C>T) and the A allele of the SNP rs2506004 (Enh2:C>A)-are associated with Hirschsprung's disease. These SNPs are in strong linkage disequilibrium and located in an enhancer element in intron 1 of the RET gene. The T allele of the Enh1 variant results in reduced expression of RET, compared with the C allele, because the T allele disrupts binding to the transcription factor SOX10. We studied whether the A allele of Enh2 (Enh2-A) also affects RET gene expression. METHODS: We evaluated the function of Enh1 and Enh2 using luciferase reporter assays with constructs that contained each allele, separately or in combination. We performed in silico analysis to identify transcription activators or repressors that bind to Enh2-C. RESULTS: The Enh1-T and the Enh2-A alleles reduced expression of the luciferase reporter gene. In silico analysis identified the sequence of Enh2-C and its surrounding sequence (ACGTG) as a potential binding site for the NXF-ARNT2 and SIM2-ARNT2 transcription factor heterodimers. The affinity of NXF-ARNT2 for Enh2-C was confirmed by electrophoresis mobility shift and supershift assays. Transfection of neuroblastoma cell lines with NXF-ARNT2 or SIM2-ARNT2 increased and decreased expression of RET, respectively. CONCLUSIONS: More than one SNP on an associated haplotype can influence gene expression and ultimately disease phenotype. Binding of the transcription factors NXF, ARNT2, and SIM2 to RET depend on the RET polymorphism of Enh2 and affect RET expression and the development of Hirschsprung's disease.

Ability of newly emerged adult Culex quinquefasciatus (Diptera: Culicidae) mosquitoes to exit belowground stormwater treatment systems via lateral conveyance pipes.

Culex quinquefasciatus Say mosquitoes flourish in belowground stormwater systems in the southern United States. Recent evidence suggests that oviposition-site-seeking females may have difficulties locating, entering, and ovipositing inside permanent water chambers when surface entry through pickholes in manhole covers are sealed. It remains unknown, however, if newly emerged adults are able to detect cues necessary to exit these partly sealed systems via lateral conveyance pipes or if they perish belowground. Fourth instar Cx. quinquefasciatus were placed within proprietary belowground stormwater treatment systems to determine the percentage of newly emerged adults able to escape treatment chambers via a single lateral conveyance pipe. Overall, 56% of deployed mosquitoes were captured in adult exit traps with an 1:1 male:female ratio. The percentage of adults captured varied significantly among chambers, but was not associated with structural site characteristics such as the chamber depth or the length and course of conveyance pipe to the exit trap. Empirical observations suggested that longbodied cellar spiders, Pholcus phalangioides (Fuesslin), ubiquitous in these structures, may have reduced adult trap capture. Findings demonstrate that newly emerged Cx. quinquefasciatus can exit subterranean chambers under potentially difficult structural conditions but suggest that a portion may perish in the absence of surface exit points in manhole shafts.