Tilivalline- and Tilimycin-Independent Effects of Klebsiella oxytoca on Tight Junction-Mediated Intestinal Barrier Impairment

Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8

Solid-state structure and anti­microbial and cytotoxicity studies of a cucurbit[6]uril-like Cu6L4 constructed from 3,5-bis­[(1H-tetra­zol-5-yl)meth­yl]-4H-1,2,4-triazol-4-amine

3,5-Bis[(1H-tetra­zol-5-yl)meth­yl]-4H-1,2,4-triazol-4-amine (H2L) associates under deprotonation with CuSO4 in aqueous medium to form a new waisted barrel-shaped M6L4 cluster, namely hexa­aqua­tetra­kis­{μ4-3,5-bis­[(1H-tetra­zol-5-yl)meth­yl]-4H-1,2,4- triazol-4-amine}-μ4-sulfato-hexa­copper(II) sulfate hydrate, [Cu6(SO4) (C6H6N12)4(H2O)6]SO4·nH2O (n = ∼23) (1). Cluster 1 resembles concave cucurbit[6]uril and has one disordered sulfate anion trapped inside the cage, which additionally stabilizes the Cu6 unit. The CuII ions have either a square-pyramidal or a distorted octa­hedral geometry. The equatorial positions are filled by N atoms from the L2− ligand, while the axial positions are occupied by coordinated water mol­ecules and O atoms of the sulfate counter-ion. In the solid state, the Cu6 clusters are connected through a large number of hydrogen bonds formed by uncoordinated water mol­ecules and an additional sulfate anion. The compound shows good anti­microbial activity against E. coli tested with the Kirby Bauer approach. In addition, the cell viability towards HeLa and L-929 cells was studied

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10-/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1β, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of β- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea

Interleukin-7 Links T Lymphocyte and Intestinal Epithelial Cell Homeostasis

Interleukin-7 (IL-7) is a major survival factor for mature T cells. Therefore, the degree of IL-7 availability determines the size of the peripheral T cell pool and regulates T cell homeostasis. Here we provide evidence that IL-7 also regulates the homeostasis of intestinal epithelial cells (IEC), colon function and the composition of the commensal microflora. In the colon of T cell-deficient, lymphopenic mice, IL-7-producing IEC accumulate. IEC hyperplasia can be blocked by IL-7-consuming T cells or the inactivation of the IL-7/IL-7R signaling pathway. However, the blockade of the IL-7/IL-7R signaling pathway renders T cell-deficient mice more sensitive to chemically-induced IEC damage and subsequent colitis. In summary, our data demonstrate that IL-7 promotes IEC hyperplasia under lymphopenic conditions. Under non-lymphopenic conditions, however, T cells consume IL-7 thereby limiting IEC expansion and survival. Hence, the degree of IL-7 availability regulates both, T cell and IEC homeostasis

Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice

Assessing eruption column height in ancient flood basalt eruptions

A buoyant plume model is used to explore the ability of flood basalt eruptions to inject climate-relevant gases into the stratosphere. An example from the 1986 Izu-Oshima basaltic fissure eruption validates the model's ability to reproduce the observed maximum plume heights of 12–16 km above sea level, sustained above fire-fountains. The model predicts maximum plume heights of 13–17 km for source widths of between 4–16 m when 32% (by mass) of the erupted magma is fragmented and involved in the buoyant plume (effective volatile content of 6 wt%). Assuming that the Miocene-age Roza eruption (part of the Columbia River Basalt Group) sustained fire-fountains of similar height to Izu-Oshima (1.6 km above the vent), we show that the Roza eruption could have sustained buoyant ash and gas plumes that extended into the stratosphere at ∼45°N. Assuming 5 km long active fissure segments and 9000 Mt of SO2 released during explosive phases over a 10–15 year duration, the ∼180km of known Roza fissure length could have supported ∼36 explosive events/phases, each with a duration of 3–4 days. Each 5 km fissure segment could have emitted 62 Mt of SO2 per day into the stratosphere while actively fountaining, the equivalent of about three 1991 Mount Pinatubo eruptions per day. Each fissure segment could have had one to several vents, which subsequently produced lava without significant fountaining for a longer period within the decades-long eruption. Sensitivity of plume rise height to ancient atmospheric conditions is explored. Although eruptions in the Deccan Traps (∼66Ma) may have generated buoyant plumes that rose to altitudes in excess of 18 km, they may not have reached the stratosphere because the tropopause was substantially higher in the late Cretaceous. Our results indicate that some flood basalt eruptions, such as Roza, were capable of repeatedly injecting large masses of SO2 into the stratosphere. Thus sustained flood basalt eruptions could have influenced climate on time scales of decades to centuries but the location (i.e., latitude) of the province and relevant paleoclimate is important and must be considered

Anti-Diarrheal Mechanism of the Traditional Remedy Uzara via Reduction of Active Chloride Secretion

BACKGROUND AND PURPOSE: The root extract of the African Uzara plant is used in traditional medicine as anti-diarrheal drug. It is known to act via inhibition of intestinal motility, but malabsorptive or antisecretory mechanisms are unknown yet. EXPERIMENTAL APPROACH: HT-29/B6 cells and human colonic biopsies were studied in Ussing experiments in vitro. Uzara was tested on basal as well as on forskolin- or cholera toxin-induced Cl(-) secretion by measuring short-circuit current (I(SC)) and tracer fluxes of (22)Na(+) and (36)Cl(-). Para- and transcellular resistances were determined by two-path impedance spectroscopy. Enzymatic activity of the Na(+)/K(+)-ATPase and intracellular cAMP levels (ELISA) were measured. KEY RESULTS: In HT-29/B6 cells, Uzara inhibited forskolin- as well as cholera toxin-induced I(SC) within 60 minutes indicating reduced active chloride secretion. Similar results were obtained in human colonic biopsies pre-stimulated with forskolin. In HT-29/B6, the effect of Uzara on the forskolin-induced I(SC) was time- and dose-dependent. Analyses of the cellular mechanisms of this Uzara effect revealed inhibition of the Na(+)/K(+)-ATPase, a decrease in forskolin-induced cAMP production and a decrease in paracellular resistance. Tracer flux experiments indicate that the dominant effect is the inhibition of the Na(+)/K(+)-ATPase. CONCLUSION AND IMPLICATIONS: Uzara exerts anti-diarrheal effects via inhibition of active chloride secretion. This inhibition is mainly due to an inhibition of the Na(+)/K(+)-ATPase and to a lesser extent to a decrease in intracellular cAMP responses and paracellular resistance. The results imply that Uzara is suitable for treating acute secretory diarrhea

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival