Removal of nitrates from high-salinity wastewaters from desulphurization process with denitrifying bacteria encapsulated in Lentikats Biocatalyst

Successful elimination of high concentrations of N–NO-x (up to 250 mg/L) from high salinity wastewaters (up to 35 g/L Cl- + 17 g/L SO4 2-) originating from desulphurization process within coal power stations was achieved using pure cultures of denitrifying bacteria encapsulated in porous polyvinyl alcohol lenses (so called Lentikats Biocatalyst, LB). Laboratory batch tests revealed inhibitory influence of the raw wastewater on the denitrification activity, which was partially mitigated by the addition of P–PO4 3-. In following continuous tests, the denitrification activities reached the range 150–450 mg N/h/kg LB, i.e., values suitable for industrial scale applications. The higher activities were achieved under a lower salinity, higher N–NOx- influent concentrations and a prolonged retention time. The effluent N–NOx- concentrations were below the determination limit of 5 mg/L. After a period of 3 months, a significant decrease of denitrification activity of Lentikats Biocatalyst was observed. Addition of nutrients into the wastewater enabled fast regeneration of the initial activity. The overall results proved the applicability of Lentikats Biocatalysts for the removal of nitrates from high-salinity desulphurization water and other industrial wastewaters of similar character

Pepsin digest of wheat gliadin fraction increases production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB signaling pathway and an NLRP3 inflammasome activation.

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1β and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1β and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1β production in celiac PBMC show that PDWGF-induced de novo pro-IL-1β synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1β. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1β release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3(-/-) and ASC(-/-) knockout mice. Moreover, treatment of human PBMC as well as MyD88(-/-) and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)(-/-) BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1β in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD

TLR signaling is required for pro-IL-1β synthesis in response to PDWGF.

<p>WT BMDC and MyD88−/−, TRIF−/−, and IL-1R−/− KO BMDC were treated with PDWGF alone or in combination with ATP and (<b>A</b>) IL-1β production was evaluated after 24 h. (<b>B</b>) Cell lysates were evaluated for <i>de novo</i> synthesis of pro-IL-1β (<b>C</b>) WT BMDC and TLR2−/−, TLR4−/−, and TLR2/4−/− KO BMDC were treated with PDWGF alone or in combination with ATP, and IL-1β production was evaluated after 24 h. (<b>D</b>) Cell lysates were evaluated for <i>de novo</i> synthesis of pro-IL-1β. Data in (A) and (C) are expressed as mean ± SD from 5 independent experiments. *P<0.05, **P<0.01 vs. WT BMDC. Blots in (B) and (D) are representative from 3 independent experiments. β-actin was used as a loading control. (<b>E</b>) Celiac PBMC were treated with PDWGF alone or in combination with anti-TLR4 or anti-TLR2 Ab. IL-1β secretion was evaluated after 24 h. LPS was used as a positive control. Mean ± SD, 8 independent experiments. **P<0.01, ***P<0.001 vs. cells without anti-TLR Ab.</p

Genotype frequencies for <i>NALP1</i> (rs12150220) and <i>NALP3</i> (rs10754558).

<p>OR- odds ratio, CI- confidence interval.</p

PDWGF stimulates BMDC to IL-1β production through NLRP3 and ASC.

<p>(<b>A</b>) BMDC from WT, NLRP3−/− and ASC−/− mice were exposed to PDWGF (100 µg/ml) alone for 24 h; or first PDWGF was added for 21.5 h, the subsequently ATP (2 mM) was added for additional 2.5 h. IL-1β was measured in culture supernatants. (<b>B</b>) Flow-cytometric evaluation of PDWGF-induced maturation assessed by CD40, CD80, and CD86 expression on BMDC from WT and NLRP3−/− mice. WT and NLRP3−/− BMDC were cultured with 100 µg/ml of PDWGF (green), as well as 0.1 µg/ml of LPS (red) or 100 µg/ml of OVA (grey-filled) as positive and negative controls, respectively. Isotype controls are represented in black overlays. (<b>C</b>) Cells were preincubated with caspase-1 inhibitor Z-YVAD-fmk for 30 min, and then exposed to PDWGF in combination with ATP. Production of IL-1β was measured in culture supernatants. Results are expressed as mean ± SD from 4 independent experiments. The levels of significance for KO BMDC vs. WT BMDC are indicated as follows: *P<0.05, **P<0.01, and ***P<0.001.</p

PDWGF-induced IL-1β production from celiac patient PBMC is modulated by K+ efflux, but is independent of the P2X7 receptor; as shown by (A) ELISA, mean ± SD, n = 10, ***P<0.001 vs. PDWGF-treated cells; and by (B) Western blot.

<p>Representative blots from 5 independent experiments are shown. (<b>C</b>) Inhibition of ROS modulate PDWGF-induced IL-1β secretion, mean ± SD, n = 10; as well as (D) pro-IL-1β production from PBMC of CD patients. Representative blots from 3 independent experiments are shown. β-actin was used as a loading control. ***P<0.001 vs. PDWGF-treated cells.</p

Reduction and alkylation (R/A) of PDWGF led to abrogated IL-1β production.

<p>PDWGF as well as α-amylase inhibitor (non-treated or R/A treated) were added to celiac PBMC. IL-1β secretion was evaluated after 24 h. Results are shown as the percentage of the cytokine production from 5 CD patients. The data were normalized to the result from PDWGF-treated cells which was set as 100%. Mean ± SD, 5 independent experiments. ***P<0.001 vs. non-treated PDWGF.</p

The role of caspase-1 in PDWGF-treated PBMC.

<p>(<b>A</b>) Caspase-1 inhibitor Z-YVAD-fmk reduced PDWGF-induced IL-1β, but not IL-1α or TNF-α production. Mean ± SD, n = 10 independent experiments, ***P<0.001 compared to PDWGF-treated cells. (<b>B</b>) Western blot analysis for the expression of pro-IL-1β and mature IL-1β and (<b>C</b>) for caspase-1 and caspase-1 p10 in cell lysates (CL) and cell culture supernatants (CS) from PBMC. Representative blots from 5 independent experiments are shown. β-actin was used as a loading control. (<b>D</b>) The fold increase (FI) (densitometry analysis) of the quantity of caspase-1 p10 normalized to non-activated cells. Mean ± SD, n = 5 independent experiments, *P<0.05 compared to non-activated cells. (<b>E, F</b>) Direct activation of caspase-1 in PDWGF-treated PBMC, assessed by flow cytometry using a cell-permeable fluorescent probe. Results are shown as (<b>E</b>) a representative histogram from 2 CD patients and 1 HD; and (<b>F</b>) as the percentage of the MFI from 12 CD patients and 10 HD. The data were normalized to the result from untreated cells, which was set as 100%. Mean ± SD, *P<0.05 compared to untreated cells. CD, celiac disease patients; HD, healthy donors.</p

PDWGF digest induces IL-1β, IL-18, and IL-1α release in monocytes and PBMC from CD patients.

<p>IL-1β (A), IL-18 (B) and IL-1α (C) levels were quantified in cell supernatants by ELISA. Data are given as mean ± SD from 39 patients and 15 healthy donors (HD). *P<0.05, **P<0.01 (CD vs. HD). (<b>D</b>). PDWGF-induced activation of PBMC is not due to LPS contamination. Results are shown as the percentage of the cytokine production from 4 CD patients. The data were normalized to the result from untreated cells which was set as 100%. Mean ± SD, n = 4 independent experiments, ***P<0.001 compared to untreated cells.</p