Establishment of a Mucin Secreting Cell Line Cx-03 from an Uterine Carcino Sarcoma.

PURPOSE: The identification of novel cell lines which combine the most important properties of mucosal membranes in terms of drug absorption, transmembrane transport and mucus secretion can help to establish improved and meaningful test systems for pharmacological and infectiological studies. METHODS: We have established a novel mucus secreting tumor cell line (Cx-03) derived from a female patient who underwent radical hysterectomy after diagnosis of a large malignant carcino sarcoma (Muellerian mixed tumor). Via xenotransplantation in SCID beige mice, recultivation and subcloning a stable cell line was established from primary tumor cells. RESULTS: Human origin and novelty of the cell line was determined by karyotype analysis and STR fingerprint. During growth cells produce considerable amounts of a PAS positive viscoelastic mucus. Immunostaining revealed expression of mucins and the mucin modifier CLCA1. We demonstrate in initial electrophysiological experiments that confluent, polarized monolayers of Cx-03 are formed (on PCF-filter supports) that exhibit stable electrical resistance (> 600 Ω cm2). Confluent Cx-03 monolayers express barrier-forming tight junction proteins claudin-1 and -4 which co-localize with zonula occludens protein-1 (ZO-1) at cell-cell contacts. CONCLUSIONS: Mucus secretion is a rare property among mammalian cell lines. In combination with its ability to form polarized monolayers Cx-03 might contribute as a novel cell based model for drug absorption, transport and barrier studies.

Arcobacter butzleri isolates exhibit pathogenic potential in intestinal epithelial cell models.

AIMS: The pathogenic potential of Arcobacter butzleri isolates on human (HT-29/B6) and porcine epithelial (IPEC-J2) cells was investigated by in vitro assays. METHODS AND RESULTS: Five of six A. butzleri isolates were able to adhere and invade HT-29/B6 cells while only four isolates adhered and two invaded IPEC-J2 cells. Two non- or poorly invasive A. butzleri isolates were highly cytotoxic to differentiated HT-29/B6 cells but none to IPEC-J2 cells as determined by WST-assays. Epithelial integrity of cell monolayers, monitored by measurement of the transepithelial electrical resistance (TER), was decreased by all A. butzleri isolates in HT-29/B6 and IPEC-J2 cells to 30-15% and 90-50% respectively. CONCLUSION: The A. butzleri strain-specific pathomechanisms observed with the human colon cell line HT-29/B6, like adhesion, invasion and cytotoxicity might all contribute to epithelial barrier dysfunction, which could explain a leak-flux type of diarrhoea in humans. In contrast, porcine cells seem to be less susceptible to A. butzleri. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacter butzleri has enteric pathogenic potential, characterized by defined interactions with human epithelial cells and strain-specific pathomechanisms.

Two-point phase correlations of a one-dimensional bosonic Josephson junction.

We realize a one-dimensional Josephson junction using quantum degenerate Bose gases in a tunable double well potential on an atom chip. Matter wave interferometry gives direct access to the relative phase field, which reflects the interplay of thermally driven fluctuations and phase locking due to tunneling. The thermal equilibrium state is characterized by probing the full statistical distribution function of the two-point phase correlation. Comparison to a stochastic model allows us to measure the coupling strength and temperature and hence a full characterization of the system.

Resveratrol Prevents Campylobacter jejuni-Induced Leaky gut by Restoring Occludin and Claudin-5 in the Paracellular Leak Pathway.

Campylobacter jejuni is a bacterial human pathogen causing gastroenteritis and sequelae like irritable bowel syndrome. Epidemiologists count the human campylobacteriosis by C. jejuni as the most common foodborne zoonosis and bacterial diarrheal disease worldwide. Based on bioinformatics predictions for potential protective compounds in campylobacteriosis, the question was raised whether the plant-based polyphenol resveratrol is sufficient to attenuate intestinal epithelial damage induced by C. jejuni. We investigated this by performing experimental infection studies in an epithelial cell culture and the secondary abiotic IL-10-/- mouse model. In C. jejuni-infected human colonic HT-29/B6 cell monolayers, transepithelial electrical resistance (TER) was decreased and the paracellular marker flux of fluorescein (332 Da) increased. Concomitantly, the tight junction (TJ) proteins occludin and claudin-5 were re-distributed off the tight junction domain. This was accompanied by an increased induction of epithelial apoptosis, both changes contributing to compromised barrier function and the opening of the leak pathway induced by C. jejuni. In parallel, the recovery experiments with the application of resveratrol revealed a functional improvement of the disturbed epithelial barrier in both models in vitro and in vivo. During treatment with resveratrol, TJ localization of occludin and claudin-5 was fully restored in the paracellular domain of HT-29/B6 cells. Moreover, resveratrol decreased the rate of epithelial apoptosis. These resveratrol-induced molecular and cellular effects would therefore be expected to improve epithelial barrier function, thereby minimizing the so-called leaky gut phenomenon. In conclusion, the induction of the leak pathway by C. jejuni and the restoration of barrier function by resveratrol demonstrates its effectiveness as a potential preventive or therapeutic method of mitigating the leaky gut associated with campylobacteriosis.

Serial femtosecond and serial synchrotron crystallography can yield data of equivalent quality: A systematic comparison.

For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins, reasonable data statistics can be obtained with approximately 5000 room-temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that the quality of diffraction data obtained from these samples is linked to the properties of the crystals rather than to the radiation source. Therefore, for other systems with similar properties, time-resolved experiments can be conducted at the radiation source that best matches the desired time resolution.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

This corrects the article DOI: 10.1038/mi.2017.66.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

Campylobacter jejuni is the most prevalent cause of foodborne bacterial enteritis worldwide. Patients present with diarrhea and immune responses lead to complications like arthritis and irritable bowel syndrome. Although studies exist in animal and cell models, we aimed at a functional and structural characterization of intestinal dysfunction and the involved regulatory mechanisms in human colon. First, in patients' colonic biopsies, sodium malabsorption was identified as an important diarrheal mechanism resulting from hampered epithelial ion transport via impaired epithelial sodium channel (ENaC) ß- and γ-subunit. In addition, barrier dysfunction from disrupted epithelial tight junction proteins (claudin-1, -3, -4, -5, and -8), epithelial apoptosis, and appearance of lesions was detected, which cause leak-flux diarrhea and can perpetuate immune responses. Importantly, these effects in human biopsies either represent direct action of Campylobacter jejuni (ENaC impairment) or are caused by proinflammatory signaling (barrier dysfunction). This was revealed by regulator analysis from RNA-sequencing (cytometric bead array-checked) and confirmed in cell models, which identified interferon-γ, TNFα, IL-13, and IL-1ß. Finally, bioinformatics' predictions yielded additional information on protective influences like vitamin D, which was confirmed in cell models. Thus, these are candidates for intervention strategies against C. jejuni infection and post-infectious sequelae, which result from the permissive barrier defect along the leaky gut.

Laboratory evaluation of two bi-level toothbrush products for subgingival access and gingival margin cleaning.

Recent technological advances have enabled the production of split or feathered endings of individual toothbrush filaments. The purpose of this laboratory study was to compare a bi-level manual toothbrush containing standard end-rounded bristles with the same shaped toothbrush containing feathered filaments, 3 mm deep, on the outer raised bi-level rows to achieve subgingival access (SA) and remove artificial plaque at the gingival margin, or gingival margin cleaning (GMC). Simulated gingivae were prepared with a 0.2 mm space between the gingivae and tooth surface. The toothbrushes were aligned with the papillae at the gingival margin, and were tested using wet brushing conditions for 60 seconds at two strokes per second with a 15 mm stroke. Four toothbrushes of each type were evaluated four times, for a total of 16 assays per design. The SA maximum depth was recorded on pressure-sensitive paper under the simulated gingivae, and the length of gingival margin cleaning was recorded as the GMC efficacy. Readings were performed with 3x magnification by one investigator. In these laboratory assays, the bi-level toothbrush with feathered outer-row bristles significantly (p < 0.001) increased the ability of the bristles to achieve SA by 35.7% and GMC efficacy by 54.5% compared to an identical bi-level toothbrush with end-rounded bristles.

TcpC protein from E. coli Nissle improves epithelial barrier function involving PKCζ and ERK1/2 signaling in HT-29/B6 cells.

The probiotic Escherichia coli Nissle 1917 (EcN) is widely used to maintain remission in ulcerative colitis. This is thought to be mediated by various immunomodulatory and barrier-stabilizing effects in the intestine. In this study, the mechanisms of barrier modulation by EcN were studied in the human epithelial HT-29/B6 cell culture model.EcN supernatant increased transepithelial resistance (TER) and reduced permeability to mannitol because of sealing of the paracellular passage pathway as revealed by two-path impedance spectroscopy. This increase in TER was attributed to the TcpC protein of EcN. TcpC induced protein kinase C-ζ (PKCζ) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which in turn resulted in upregulation of the barrier-forming tight junction protein claudin-14. By specific silencing of protein expression by small interfering RNA (siRNA), the sealing function of claudin-14 was confirmed. In conclusion, the TcpC protein of EcN affects innate immunity by improving intestinal barrier function through upregulation of claudin-14 via PKCζ and ERK1/2 signaling.

Interferometry with non-classical motional states of a Bose-Einstein condensate.

The Ramsey interferometer is a prime example of precise control at the quantum level. It is usually implemented using internal states of atoms, molecules or ions, for which powerful manipulation procedures are now available. Whether it is possible to control external degrees of freedom of more complex, interacting many-body systems at this level remained an open question. Here we demonstrate a two-pulse Ramsey-type interferometer for non-classical motional states of a Bose-Einstein condensate in an anharmonic trap. The control sequences used to manipulate the condensate wavefunction are obtained from optimal control theory and are directly optimized to maximize the interferometric contrast. They permit a fast manipulation of the atomic ensemble compared to the intrinsic decay processes and many-body dephasing effects. This allows us to reach an interferometric contrast of 92% in the experimental implementation.

Integrated Mach-Zehnder interferometer for Bose-Einstein condensates.

Particle-wave duality enables the construction of interferometers for matter waves, which complement optical interferometers in precision measurement devices. This requires the development of atom-optics analogues to beam splitters, phase shifters and recombiners. Integrating these elements into a single device has been a long-standing goal. Here we demonstrate a full Mach-Zehnder sequence with trapped Bose-Einstein condensates confined on an atom chip. Particle interactions in our Bose-Einstein condensate matter waves lead to a nonlinearity, absent in photon optics. We exploit it to generate a non-classical state having reduced number fluctuations inside the interferometer. Making use of spatially separated wave packets, a controlled phase shift is applied and read out by a non-adiabatic matter-wave recombiner. We demonstrate coherence times a factor of three beyond what is expected for coherent states, highlighting the potential of entanglement as a resource for metrology. Our results pave the way for integrated quantum-enhanced matter-wave sensors.

Short-term and medium-term clinical outcomes of Campylobacter concisus infection.

There are only sparse data on the short-term and medium-term clinical impacts of Campylobacter concisus infection. A clinical study was performed during a 2-year period to determine the clinical manifestations in C. concisus-positive adult patients. A case patient was defined as an adult patient (≥18 years) with a C. concisus-positive stool sample during the study period. Clinical data were obtained with use of a questionnaire supplemented with the patients' medical records, if any. The short-term and medium-term clinical manifestations in these patients were compared with those of patients with Campylobacter jejuni/Campylobacter coli infection. One hundred and seventy-four C. concisus patients and 196 C. jejuni/C. coli patients participated in the study. Patients with pre-existing inflammatory bowel disease or microscopic colitis or enteric co-infection were excluded from review of the clinical manifestations. Comparison of the short-term clinical manifestations in 139 C. concisus patients with those in 187 C. jejuni/C. coli patients showed a significantly lower prevalence of fever, chills, mucus and blood in stools, and weight loss. However, 80% of C. concisus patients, but only 32% of C. jejuni/C. coli patients, had diarrhoea for >2 weeks. After a 6-month follow-up period, 12% of C. concisus patients were diagnosed with microscopic colitis, whereas no C. jejuni/C. coli patients were diagnosed with non-infective colitis. Irritable bowel symptoms were common in both groups at follow-up. C. concisus infection seems to cause a milder course of acute gastroenteritis than C. jejuni/C. coli infection, but is associated with more prolonged diarrhoea.