Effects of Dietary Fibre from the Traditional Indonesian Food, Green Cincau (Premna oblongifolia Merr.) on Preneoplastic Lesions and Short Chain Fatty Acid Production in an Azoxymethane Rat Model of Colon Cancer.

Green cincau (Premna oblongifolia Merr.) is a traditional food of Indonesia and provides a natural source of dietary fibre and antioxidants. This study evaluated the ability of green cincau, and other dietary fibres with or without the addition of anti-oxidant, epigallocatechin-3-gallate (EGCG), to prevent colorectal cancer in a 12 week azoxymethane (AOM) rat model. While all dietary treatments stimulated short chain fatty acid production (SCFA) in the digesta and faeces, no one treatment was able to significantly protect against aberrant crypt formation (ACF), when compared to the control diet. However, feeding green cincau leaves or extracts did not result in an increase in ACF compared to the control diet. Unexpectedly, when the dietary fibre source was pectin, 0.1% EGCG increased proliferative activity and liver lipid peroxidation when compared to the control diet containing cellulose. Examination of faecal microbial communities identified the presence of short chain acid producing bacteria, but a distinct community profile was not observed from any individual diet group. Overall, this research implies that combining dietary fibre with an antioxidant does not automatically equate to a beneficial response. Further work is required to investigate the health-promoting properties of green cincau.

Protein kinase cα regulates the expression of complement receptor Ig in human monocyte-derived macrophages.

The complement receptor Ig (CRIg) is selectively expressed by macrophages. This receptor not only promotes the rapid phagocytosis of bacteria by macrophages but also has anti-inflammatory and immunosuppressive functions. Previous findings have suggested that protein kinase C (PKC) may be involved in the regulation of CRIg expression in human macrophages. We have now examined the role of PKCα in CRIg expression in human monocyte-derived macrophages (MDM). Macrophages nucleofected with plasmid containing short hairpin RNA against PKCα showed markedly reduced expression of PKCα, but normal PKCζ expression, by Western blotting analysis, and vice versa. PKCα-deficient MDM showed increased expression of CRIg mRNA and protein (both the long and short form), an increase in phagocytosis of complement-opsonized Candida albicans, and decreased production of TNF-α and IL-6. TNF-α caused a marked decrease in CRIg expression, and addition of anti-TNF mAb to the TNF-α-producing MDMs increased CRIg expression. PKCα-deficient macrophages also showed significantly less bacterial LPS-induced downregulation of CRIg. In contrast, cells deficient in PKCα showed decreased expression of CR type 3 (CR3) and decreased production of TNF-α and IL-6 in response to LPS. MDM developed under conditions that increased expression of CRIg over CR3 showed significantly reduced production of TNF-α in response to opsonized C. albicans. The findings indicate that PKCα promotes the downregulation of CRIg and upregulation of CR3 expression and TNF-α and IL-6 production, a mechanism that may promote inflammation.

Dipeptidyl peptidase 9 substrates and their discovery: current progress and the application of mass spectrometry-based approaches.

The enzyme members of the dipeptidyl peptidase 4 (DPP4) gene family have the very unusual capacity to cleave the post-proline bond to release dipeptides from the N-terminus of peptide/protein substrates. DPP4 and related enzymes are current and potential therapeutic targets in the treatment of type II diabetes, inflammatory conditions and cancer. Despite this, the precise biological function of individual dipeptidyl peptidases (DPPs), other than DPP4, and knowledge of their in vivo substrates remains largely unknown. For many years, identification of physiological DPP substrates has been difficult due to limitations in the available tools. Now, with advances in mass spectrometry based approaches, we can discover DPP substrates on a system wide-scale. Application of these approaches has helped reveal some of the in vivo natural substrates of DPP8 and DPP9 and their unique biological roles. In this review, we provide a general overview of some tools and approaches available for protease substrate discovery and their applicability to the DPPs with a specific focus on DPP9 substrates. This review provides comment upon potential approaches for future substrate elucidation.

Are the Traditional Medical Uses of Muricidae Molluscs Substantiated by Their Pharmacological Properties and Bioactive Compounds?

Marine molluscs from the family Muricidae hold great potential for development as a source of therapeutically useful compounds. Traditionally known for the production of the ancient dye Tyrian purple, these molluscs also form the basis of some rare traditional medicines that have been used for thousands of years. Whilst these traditional and alternative medicines have not been chemically analysed or tested for efficacy in controlled clinical trials, a significant amount of independent research has documented the biological activity of extracts and compounds from these snails. In particular, Muricidae produce a suite of brominated indoles with anti-inflammatory, anti-cancer and steroidogenic activity, as well as choline esters with muscle-relaxing and pain relieving properties. These compounds could explain some of the traditional uses in wound healing, stomach pain and menstrual problems. However, the principle source of bioactive compounds is from the hypobranchial gland, whilst the shell and operculum are the main source used in most traditional remedies. Thus further research is required to understand this discrepancy and to optimise a quality controlled natural medicine from Muricidae.

Identifying natural substrates for dipeptidyl peptidases 8 and 9 using terminal amine isotopic labeling of substrates (TAILS) reveals in vivo roles in cellular homeostasis and energy metabolism.

Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.

Light-dark cycles may influence in situ soil bacterial networks and diurnally-sensitive taxa.

Soil bacterial taxa have important functional roles in ecosystems (e.g. nutrient cycling, soil formation, plant health). Many factors influence their assembly and regulation, with land cover types (e.g. open woodlands, grasslands), land use types (e.g. nature reserves, urban green space) and plant-soil feedbacks being well-studied factors. However, changes in soil bacterial communities in situ over light-dark cycles have received little attention, despite many plants and some bacteria having endogenous circadian rhythms that could influence soil bacterial communities. We sampled surface soils in situ across 24-h light-dark cycles (at 00:00, 06:00, 12:00, 18:00) at two land cover types (remnant vegetation vs. cleared, grassy areas) and applied 16S rRNA amplicon sequencing to investigate changes in bacterial communities. We show that land cover type strongly affected soil bacterial diversity, with soils under native vegetation expressing 15.4%-16.4% lower alpha diversity but 4.9%-10.6% greater heterogeneity than soils under cleared vegetation. In addition, we report time-dependent and site-specific changes in bacterial network complexity and between 598-922 ASVs showing significant changes in relative abundance across times. Native site node degree (bacterial interactions) at the phylum level was 16.0% higher in the early morning than in the afternoon/evening. Our results demonstrate for the first time that light-dark cycles have subtle yet important effects on soil bacterial communities in situ and that land cover influences these dynamics. We provide a new view of soil microbial ecology and suggest that future studies should consider the time of day when sampling soil bacteria.

Proteotransciptomics of the Most Popular Host Sea Anemone Entacmaea quadricolor Reveals Not All Toxin Genes Expressed by Tentacles Are Recruited into Its Venom Arsenal.

While the unique symbiotic relationship between anemonefishes and sea anemones is iconic, it is still not fully understood how anemonefishes can withstand and thrive within the venomous environment of their host sea anemone. In this study, we used a proteotranscriptomics approach to elucidate the proteinaceous toxin repertoire from the most common host sea anemone, Entacmaea quadricolor. Although 1251 different toxin or toxin-like RNA transcripts were expressed in E. quadricolor tentacles (0.05% of gene clusters, 1.8% of expression) and 5375 proteins were detected in milked venom, only 4% of proteins detected in venom were putative toxins (230), and they only represent on average 14% of the normalised protein expression in the milked venom samples. Thus, most proteins in milked venom do not appear to have a toxin function. This work raises the perils of defining a dominant venom phenotype based on transcriptomics data alone in sea anemones, as we found that the dominant venom phenotype differs between the transcriptome and proteome abundance data. E. quadricolor venom contains a mixture of toxin-like proteins of unknown and known function. A newly identified toxin protein family, Z3, rich in conserved cysteines of unknown function, was the most abundant at the RNA transcript and protein levels. The venom was also rich in toxins from the Protease S1, Kunitz-type and PLA2 toxin protein families and contains toxins from eight venom categories. Exploring the intricate venom toxin components in other host sea anemones will be crucial for improving our understanding of how anemonefish adapt to the venomous environment.

The macroecology of butyrate-producing bacteria via metagenomic assessment of butyrate production capacity.

Butyrate-producing bacteria are found in many outdoor ecosystems and host organisms, including humans, and are vital to ecosystem functionality and human health. These bacteria ferment organic matter, producing the short-chain fatty acid butyrate. However, the macroecological influences on their biogeographical distribution remain poorly resolved. Here we aimed to characterise their global distribution together with key explanatory climatic, geographical and physicochemical variables. We developed new normalised butyrate production capacity (BPC) indices derived from global metagenomic (n = 13,078) and Australia-wide soil 16S rRNA (n = 1331) data, using Geographic Information System (GIS) and modelling techniques to detail their ecological and biogeographical associations. The highest median BPC scores were found in anoxic and fermentative environments, including the human (BPC = 2.99) and non-human animal gut (BPC = 2.91), and in some plant-soil systems (BPC = 2.33). Within plant-soil systems, roots (BPC = 2.50) and rhizospheres (BPC = 2.34) had the highest median BPC scores. Among soil samples, geographical and climatic variables had the strongest overall effects on BPC scores (variable importance score range = 0.30-0.03), with human population density also making a notable contribution (variable importance score = 0.20). Higher BPC scores were in soils from seasonally productive sandy rangelands, temperate rural residential areas and sites with moderate-to-high soil iron concentrations. Abundances of butyrate-producing bacteria in outdoor soils followed complex ecological patterns influenced by geography, climate, soil chemistry and hydrological fluctuations. These new macroecological insights further our understanding of the ecological patterns of outdoor butyrate-producing bacteria, with implications for emerging microbially focused ecological and human health policies.

Childcare centre soil microbiomes are influenced by substrate type and surrounding vegetation condition.

Urban development has profoundly reduced human exposure to biodiverse environments, which is linked to a rise in human disease. The 'biodiversity hypothesis' proposes that contact with diverse microbial communities (microbiota) benefits human health, as exposure to microbial diversity promotes immune training and regulates immune function. Soils and sandpits in urban childcare centres may provide exposure to diverse microbiota that support immunoregulation at a critical developmental stage in a child's life. However, the influence of outdoor substrate (i.e., sand vs. soil) and surrounding vegetation on these environmental microbiota in urban childcare centres remains poorly understood. Here, we used 16S rRNA amplicon sequencing to examine the variation in bacterial communities in sandpits and soils across 22 childcare centres in Adelaide, Australia, plus the impact of plant species richness and habitat condition on these bacterial communities. We show that sandpits had distinct bacterial communities and lower alpha diversity than soils. In addition, we found that plant species richness in the centres' yards and habitat condition surrounding the centres influenced the bacterial communities in soils but not sandpits. These results demonstrate that the diversity and composition of childcare centre sandpit and soil bacterial communities are shaped by substrate type, and that the soils are also shaped by the vegetation within and surrounding the centres. Accordingly, there is potential to modulate the exposure of children to health-associated bacterial communities by managing substrates and vegetation in and around childcare centres.

6-bromoisatin found in muricid mollusc extracts inhibits colon cancer cell proliferation and induces apoptosis, preventing early stage tumor formation in a colorectal cancer rodent model.

Muricid molluscs are a natural source of brominated isatin with anticancer activity. The aim of this study was to examine the safety and efficacy of synthetic 6-bromoisatin for reducing the risk of early stage colorectal tumor formation. The purity of 6-bromoisatin was confirmed by 1H NMR spectroscopy, then tested for in vitro and in vivo anticancer activity. A mouse model for colorectal cancer was utilized whereby colonic apoptosis and cell proliferation was measured 6 h after azoxymethane treatment by hematoxylin and immunohistochemical staining. Liver enzymes and other biochemistry parameters were measured in plasma and haematological assessment of the blood was conducted to assess potential toxic side-effects. 6-Bromoisatin inhibited proliferation of HT29 cells at IC50 223 µM (0.05 mg/mL) and induced apoptosis without increasing caspase 3/7 activity. In vivo 6-bromoisatin (0.05 mg/g) was found to significantly enhance the apoptotic index (p≤0.001) and reduced cell proliferation (p≤0.01) in the distal colon. There were no significant effects on mouse body weight, liver enzymes, biochemical factors or blood cells. However, 6-bromoisatin caused a decrease in the plasma level of potassium, suggesting a diuretic effect. In conclusion this study supports 6-bromoisatin in Muricidae extracts as a promising lead for prevention of colorectal cancer.

Purified brominated indole derivatives from Dicathais orbita induce apoptosis and cell cycle arrest in colorectal cancer cell lines.

Dicathais orbita is a large Australian marine gastropod known to produce bioactive compounds with anticancer properties. In this research, we used bioassay guided fractionation from the egg mass extract of D. orbita using flash column chromatography and identified fractions containing tyrindoleninone and 6-bromoisatin as the most active against colon cancer cells HT29 and Caco-2. Liquid chromatography coupled with mass spectrometry (LCMS) and 1H NMR were used to characterize the purity and chemical composition of the isolated compounds. An MTT assay was used to determine effects on cell viability. Necrosis and apoptosis induction using caspase/LDH assay and flow cytometry (PI/Annexin-V) and cell cycle analysis were also investigated. Our results show that semi-purified 6-bromoisatin had the highest anti-cancer activity by inhibiting cell viability (IC50 = ~100 µM) and increasing caspase 3/7 activity in both of the cell lines at low concentration. The fraction containing 6-bromoisatin induced 77.6% apoptosis and arrested 25.7% of the cells in G2/M phase of cell cycle in HT29 cells. Tyrindoleninone was less potent but significantly decreased the viability of HT29 cells at IC50 = 390 µM and induced apoptosis at 195 µM by increasing caspase 3/7 activity in these cells. This research will facilitate the development of these molluscan natural products as novel complementary medicines for colorectal cancer.

Circulating Dipeptidyl Peptidase Activity Is a Potential Biomarker for Inflammatory Bowel Disease.

INTRODUCTION: Dipeptidyl peptidase (DPP)-4 is part of a larger family of proteases referred to as DPPs. DPP4 has been suggested as a possible biomarker for inflammatory bowel disease (IBD). Circulating DPP4 (cDPP4) enzyme activity was investigated as a potential biomarker for IBD. In addition, DPP enzyme activity and gene expression were quantified in colonic tissue of patients with IBD and non-IBD. METHODS: In study 1, DPP enzyme activity was quantified in plasma samples from 220 patients with IBD (Crohn's disease [CD] n = 130 and ulcerative colitis [UC] n = 90) and non-IBD controls (n = 26) using a colorimetric assay. In study 2, tissue and plasma samples were collected from 26 patients with IBD and 20 non-IBD controls. Plasma C-reactive protein (CRP) was quantified in all patients. Colonic DPP4, DPP8, DPP9, and fibroblast activation protein (FAP) gene expression was determined by quantitative polymerase chain reaction. cDPP and cFAP enzyme activity was also measured. Sensitivity and specificity were determined by receiver operating characteristic curve analysis. RESULTS: In study 1, total cDPP activity was found to differentiate patients with CD with active disease (n = 18) from those in remission (n = 19; sensitivity 78% and specificity 63%). In study 2, total cDPP and cFAP activity was 28% and 48% lower in patients with elevated CRP (>10 mg/L), respectively, compared with patients with normal CRP. Gene expression of DPP4, FAP, and DPP8 was also significantly higher in colonic biopsies from patients with IBD compared with non-IBD patients (P < 0.05). DISCUSSION: Our findings implicate the DPP enzyme family in intestinal inflammation and suggest future biomarker applications to differentiate the pathophysiological aspects of IBD.

Biochemical and histological changes in the small intestine of mice with dextran sulfate sodium colitis.

The dextran sulfate sodium (DSS) model of colitis has been commonly utilized in mice to assess novel treatments for ulcerative colitis. Recent studies have indicated that morphological and biochemical changes extend to the small intestine (SI). This study aimed to characterize histological and biochemical changes in the SI during DSS colitis in wild-type (WT) and DPIV knock-out (DPIV(-/-) ) mice treated with saline or the DPIV inhibitors, Ile-Pyrr-(2-CN)*TFA or Ile-Thia. Groups (n = 10) of DPIV(-/-) and WT mice were orally gavaged twice daily with saline, Ile-Pyrr-(2-CN)*TFA or Ile-Thia. Mice consumed 2% DSS in drinking water for 6 days to induce colitis. Small intestinal tissue was assessed for histological changes, sucrase, and DPIV activity and neutrophil infiltration. Jejunal villus length was increased in all groups after 6 days DSS consumption (P < 0.05). Jejunal DPIV activity was significantly lower by 35% in WT mice receiving Ile-Pyrr-(2-CN)*TFA compared to saline controls. Jejunal MPO activity was significantly increased in the WT + saline and DPIV(-/-) + saline groups following DSS consumption, compared to WT and DPIV(-/-) controls at day 0. Increased sucrase activity was apparent at day 0 in DPIV(-/-) compared to WT mice (P < 0.05). We conclude that DSS-induced damage is not restricted to the colon, but also extends to the small intestine. Furthermore, reduced or absent DPIV activity resulted in functional adaptations to brush border enzyme activity. DPIV inhibitors are now a recognized therapy for type-II diabetes. The work presented here highlights the need to delineate any long-term effects of DPIV inhibitors on SI function, to further validate their safety and tolerability.

Bioactivity of the Murex Homeopathic Remedy and of Extracts from an Australian Muricid Mollusc against Human Cancer Cells.

Marine molluscs from the family Muricidae are the source of a homeopathic remedy Murex, which is used to treat a range of conditions, including cancer. The aim of this study was to evaluate the in vitro bioactivity of egg mass extracts of the Australian muricid Dicathais orbita, in comparison to the Murex remedy, against human carcinoma and lymphoma cells. Liquid chromatography coupled with mass spectrometry (LC-MS) was used to characterize the chemical composition of the extracts and homeopathic remedy, focusing on biologically active brominated indoles. The MTS (tetrazolium salt) colorimetric assay was used to determine effects on cell viability, while necrosis and apoptosis induction were investigated using flow cytometry (propidium iodide and Annexin-V staining, resp.). Cells were treated with varying concentrations (1-0.01 mg/mL) of crude and semi-purified extracts or preparations (dilute 1 M and concentrated 4 mg/mL) from the Murex remedy (4 h). The Murex remedy showed little biological activity against the majority of cell lines tested. In contrast, the D. orbita egg extracts significantly decreased cell viability in the majority of carcinoma cell lines. Flow cytometry revealed these extracts induce necrosis in HT29 colorectal cancer cells, whereas apoptosis was induced in Jurkat cells. These findings highlight the biomedical potential of Muricidae extracts in the development of a natural therapy for the treatment of neoplastic tumors and lymphomas.

The potential of outdoor environments to supply beneficial butyrate-producing bacteria to humans.

Butyrate is an important mediator of human health and disease. The mechanisms of action of butyrate are becoming increasingly well-known. Many commensal bacteria that inhabit the human gut can synthesise butyrate, which is then absorbed into the human host. Simultaneously, several immune- and inflammatory-mediated diseases are being linked to insufficient exposure to beneficial microbes from our environment, including butyrate-producing bacteria. However, the role of outdoor environmental exposure to butyrate-producing bacteria remains poorly understood. Here we review the literature on the human exposure pathways to butyrate-producing bacteria, with a particular focus on outdoor environmental sources (e.g. associated with plants, plant-based residues, and soil), and the health implications of exposure to them. Emerging evidence suggests that environmental butyrate-producers may help supplement the human gut microbiota and represent an important component of the Biodiversity and Old Friends hypotheses. Improving our understanding of potential sources, precursors, and exposure pathways of environmental butyrate-producers that influence the gut microbiota and butyrate production offers promise to advance multiple disciplines of health and environmental science. We outline research priorities to address knowledge gaps in the outdoor environment-butyrate-health nexus and build knowledge of the potential pathways to help optimise exposure to human-beneficial butyrate-producing bacteria from the outdoor environment during childhood and adulthood.

Regulation of epithelial apical junctions and barrier function by Galpha13.

The epithelial tight junction forms a barrier to paracellular solute movement. In this study we show that the heterotrimeric G-protein Galpha13 regulates the epithelial tight junction barrier. We generated MDCKII kidney epithelial cell lines in which the expression of an active Galpha13 mutant (Galpha13Q226L) could be induced. We demonstrated that Galpha13Q226L expression increased paracellular permeability and caused the disruption and redistribution of proteins comprising the tight junction and the adherens junction away from sites of cell contact and the appearance of basal stress fibers. The effects on the junctional proteins and the actin cytoskeleton were abrogated by the Rho kinase inhibitor Y27632 but not by the Src kinase inhibitor PP2. The Galpha13 mediated increase in permeability was also Src kinase independent but was partly dependent on Rho kinase signalling. Our data establish a link between Galpha13, Rho kinase signaling and epithelial barrier function and not only demonstrate that Galpha13 regulates epithelial apical junction properties but that it does so via signaling pathways that are distinct from the closely related protein Galpha12.

Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DP8).

Dipeptidyl peptidase (DP) 8 belongs to the dipeptidyl peptidase IV gene family. DP8 has been implicated in immune function and asthma, although its biological function is yet unknown. Structures of the homologs, fibroblast activation protein (FAP) and DPIV, are known but the DP8 structure is yet to be resolved. To help characterise the DP8 substrate pocket, mutants of residues lining the pocket were produced at DP8(D772), DP8(Y315), DP8(H434) and DP8(D435) and assessed by substrate kinetics and size-exclusion chromatography. Mutations of DP8(D772A/E/S/V) affected catalysis but did not confer endopeptidase activity. Mutations of DP8(H434F), DP8(D435F) and DP8(Y315F) reduced catalytic activity. Furthermore, mutations to DP8(D772A/E/S/V), DP8(H434F), DP8(D435F) and DP8(Y315F) affected dimer stabilisation. Homology modelling of DP8 using DPIV and FAP crystal structures suggested that DP8(D772), DP8(H434) and DP8(D435) were located at the edge of the S2 catalytic pocket, contributing to the junction between the alpha-beta hydrolase and beta-propeller domains. This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors.

The Ecological Importance of Toxicity: Sea Anemones Maintain Toxic Defence When Bleached.

Cnidarians are amongst the most venomous animals on the planet. They are also under significant threat due to the impacts of climate change. Corals and anemones undergo climate-induced bleaching during extreme environmental conditions, where a loss of symbiotic photosynthetic algae (zooxanthellae) causes whitening in colour, loss of internal food supply, and reduction in health, which can ultimately lead to death. What has yet to be determined is whether bleaching causes a reduction in the production or quality of venom. In this study, the sea anemone Entacmaea quadricolor was exposed to long-term light-induced bleaching to examine the effect that bleaching has on venom. Venom quality and quantity, as determined through lethality and haemolysis measures and nematocyst production was highly preserved over the five-month imposed bleaching event. Maintenance of venom and nematocyst production, despite a loss of an internal food source provided by endosymbiotic algae, indicates both the ecological importance of maintaining toxicity and a remarkable resilience that anemones have to major environmental stressors.

Development of a 13C Stable Isotope Assay for Dipeptidyl Peptidase-4 Enzyme Activity A New Breath Test for Dipeptidyl Peptidase Activity.

Dipeptidyl peptidase-4 inhibitors (DPP4i) are a class of orally available, small molecule inhibitors for the management of Type-II diabetes. A rapid, real-time, functional breath test for DPP4 enzyme activity could help to define DPP4i efficacy in patients that are refractory to treatment. We aimed to develop a selective, non-invasive, stable-isotope 13C-breath test for DPP4. In vitro experiments were performed using high (Caco-2) and low (HeLa) DPP4 expressing cells. DPP gene expression was determined in cell lines by qRT-PCR. A DPP4 selective 13C-tripeptide was added to cells in the presence and absence of the DPP4 inhibitor Sitagliptin. Gas samples were collected from the cell headspace and 13CO2 content quantified by isotope ratio mass spectrometry (IRMS). DPP4 was highly expressed in Caco-2 cells compared to HeLa cells and using the 13C-tripeptide, we detected a high 13CO2 signal from Caco2 cells. Addition of Sitaglitpin to Caco2 cells significantly inhibited this 13CO2 signal. 13C-assay DPP4 activity correlated positively with the enzyme activity detected using a colorimetric substrate. We have developed a selective, non-invasive, 13C-assay for DPP4 that could have broad translational applications in diabetes and gastrointestinal disease.

Human Dendritic Cells Express the Complement Receptor Immunoglobulin Which Regulates T Cell Responses.

The B7 family-related protein V-set and Ig containing 4 (VSIG4), also known as Z39Ig and Complement Immunoglobulin Receptor (CRIg), is the most recent of the complement receptors to be identified, with substantially distinct properties from the classical complement receptors. The receptor displays both phagocytosis-promoting and anti-inflammatory properties. The receptor has been reported to be exclusively expressed in macrophages. We now present evidence, that CRIg is also expressed in human monocyte-derived dendritic cells (MDDC), including on the cell surface, implicating its role in adaptive immunity. Three CRIg transcripts were detected and by Western blotting analysis both the known Long (L) and Short (S) forms were prominent but we also identified another form running between these two. Cytokines regulated the expression of CRIg on dendritic cells, leading to its up- or down regulation. Furthermore, the steroid dexamethasone markedly upregulated CRIg expression, and in co-culture experiments, the dexamethasone conditioned dendritic cells caused significant inhibition of the phytohemagglutinin-induced and alloantigen-induced T cell proliferation responses. In the alloantigen-induced response the production of IFNγ, TNF-α, IL-13, IL-4, and TGF-ß1, were also significantly reduced in cultures with dexamethasone-treated DCs. Under these conditions dexamethasone conditioned DCs did not increase the percentage of regulatory T cells (Treg). Interestingly, this suppression could be overcome by the addition of an anti-CRIg monoclonal antibody to the cultures. Thus, CRIg expression may be a control point in dendritic cell function through which drugs and inflammatory mediators may exert their tolerogenic- or immunogenic-promoting effects on dendritic cells.