Coen Govers

Optimized triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

Scope Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for β-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays. Methods and results Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination. Conclusion The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

The effects of Lactobacillus plantarum on small intestinal barrier function and mucosal gene transcription; a randomized double-blind placebo controlled trial

The aim of this study was to investigate the effects of three Lactobacillus plantarum strains on in-vivo small intestinal barrier function and gut mucosal gene transcription in human subjects. The strains were selected for their differential effects on TLR signalling and tight junction protein rearrangement, which may lead to beneficial effects in a stressed human gut mucosa. Ten healthy volunteers participated in four different intervention periods: 7-day oral intake of either L. plantarum WCFS1, CIP104448, TIFN101 or placebo, proceeded by a 4 weeks wash-out period. Lactulose-rhamnose ratio (an indicator of small intestinal permeability) increased after intake of indomethacin, which was given as an artificial stressor of the gut mucosal barrier (mean ratio 0.06 ± 0.04 to 0.10 ± 0.06, p = 0.001), but was not significantly affected by the bacterial interventions. However, analysis in small intestinal biopsies, obtained by gastroduodenoscopy, demonstrated that particularly L. plantarum TIFN101 modulated gene transcription pathways related to cell-cell adhesion with high turnover of genes involved in tight- and adhesion junction protein synthesis and degradation (e.g. actinin alpha-4, metalloproteinase-2). These effects were less pronounced for L. plantarum WCFS1 and CIP104448. In conclusion, L. plantarum TIFN101 induced the most pronounced probiotic properties with specific gene transcriptional effects on repair processes in the compromised intestine of healthy subjects.

Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells

T cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100(280-288)/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer(®), when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells.

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies.

T cell receptor fused to CD3ζ: Transmembrane domain of CD3ζ prevents TCR mis-pairing, whereas complete CD3ζ directs functional TCR expression

textabstractTCR gene therapy represents a feasible and promising treatment for patients with cancer and virus infections. Currently, this treatment rationale is hampered by diluted surface expression of the TCR transgene and generation of potentially self reactive T-cells, both a direct consequence of mis-pairing with endogenous TCR chains. As we reported previously (Gene Ther 16:1369, 2000; J Immunol 180:7736, 2008), TCR mis-pairing can be successfully addressed by a TCR:CD3ζ fusion protein (i.e., TCR:ζ). Here, we set out to minimize the content of CD3ζ in TCR:ζ, specific for MAGEA1/ HLA-A1, without compromising TCR pairing and function. Domain-exchange and 3D-modeling strategies defined a set of minimal TCR:ζ variants, which, together with a murinized and cysteine-modified TCR (TCR:mu+cys), were tested for functional TCR expression and TCR pairing. Our data with Jurkat T cells show that the CD3ζ transmembrane domain is important for cell-surface expression, whereas the CD3ζ intracellular domain is crucial for T-cell activation. Notably, inability of TCR:ζ to mis-pair was not observed for TCR:mu+cys, which depended exclusively on the transmembrane domain of CD3ζ and could not be recapitulated by a limited number of structurally defined CD3ζ transmembrane amino acids. The extracellular CD3ζ domain was dispensable for TCR:ζs ability to prevent TCR mis-pairing, bind pMHC and mediate NFAT activation. In primary human T cells, however, minimal TCR:ζ without CD3ζs extracellular domain but not TCR:ζ nor TCR:mu+cys revealed compromised cell surface expression and T cell function. Taken together, our study demonstrates that CD3ζs transmembrane domain dictates TCR:ζs inability to TCR mis-pair, but only TCR coupled to complete CD3ζ and not its minimal variants were functionally expressed in primary T cells.

The Effect of Tomatine on Gene Expression and Cell Monolayer Integrity in Caco-2

More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1β, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations <20 µg/mL of tomatine, either undigested or in vitro digested, do not compromise the viability of Caco-2 cells and stimulate cytokine expression. This effect of tomatine, in vitro digested tomatine or in vitro digested tomatine with tomato matrix differs slightly, probably due to variations of bioactivity or bioavailability of the tomatine. The results lead to the hypothesis that tomatine acts as hormetic compound that can induce beneficial or risk toxic effects whether used in low or high dose.

Consumption of β-glucans to spice up T cell treatment of tumors: a review

ABSTRACT Introduction: Adoptive T-cell treatments of solid cancers have evolved into a robust therapy with objective response rates surpassing those of standardized treatments. Unfortunately, only a limited fraction of patients shows durable responses, which is considered to be due to a T cell-suppressive tumor microenvironment (TME). Here we argue that naturally occurring β-glucans can enable reversion of such T cell suppression by engaging innate immune cells and enhancing numbers and function of lymphocyte effectors. Areas covered: This review summarizes timely reports with respect to absorption, trafficking and immune stimulatory effects of β-glucans, particularly in relation to innate immune cells. Furthermore, we list effects toward well-being and immune functions in healthy subjects as well as cancer patients treated with orally administered β-glucans, extended with effects of β-glucan treatments in mouse cancer models. Expert opinion: Beta-glucans, when present in food and following uptake in the proximal gut, stimulate immune cells present in gut-associated lymphoid tissue and initiate highly conserved pro-inflammatory pathways. When tested in mouse cancer models, β-glucans result in better control of tumor growth and shift the TME toward a T cell-sensitive environment. Along these lines, we advocate that intake of β-glucans provides an accessible and immune-potentiating adjuvant when combined with adoptive T-cell treatments of cancer.

Water-Soluble Polysaccharide Extracts from the Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) with HMGCR Inhibitory Activity

Water extracts from Pleurotus ostreatus containing no statins showed 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR) inhibitory activity (in vitro) that might be due to specific water-soluble polysaccharides (WSPs); when isolated and deproteinized, increasing concentrations of the WSP extract induced higher inhibition. The WSP extract contained mainly β-glucans, mannogalactans, and glycogen (e.g., α-glucans), although derivatives or fragments with lower molecular weights (between 14 and 3.5 kDa) were present and were able to induce the inhibitory activity. The extract contained more β-(1→3)-glucans than β-(11→3),(11→6)-glucans, and they partially survived digestion and managed to pass through Caco2 cell monolayers to the lower compartment after in vitro digestion and transport experiments. The WSP might also modulate Caco2 membrane integrity.