Synbiotic supplementation with prebiotic green banana resistant starch and probiotic Bacillus coagulans spores ameliorates gut inflammation in mouse model of inflammatory bowel diseases.

PURPOSE: The research goal is to develop dietary strategies to help address the growing incidence of inflammatory bowel diseases (IBD). This study has investigated the effectiveness of green banana resistant starch (GBRS) and probiotic Bacillus coagulans MTCC5856 spores for the amelioration of dextran-sulfate sodium (DSS)-induced colitis in mice. METHODS: Eight-week-old C57BL/6 mice were fed standard rodent chow diet supplemented with either B. coagulans, GBRS or its synbiotic combination. After 7 days supplementation, colitis was induced by adding 2% DSS in drinking water for 7 days while continuing the supplemented diets. Animal health was monitored and after 14 days all animals were sacrificed to measure the biochemical and histochemical changes associated with each supplement type. RESULTS: The disease activity index and histological damage score for DSS-control mice (6.1, 17.1, respectively) were significantly higher (p < 0.0001) than the healthy mice. Synbiotic supplementation alleviated these markers (- 67%, - 94% respectively) more adequately than B. coagulans (- 52%, - 58% respectively) or GBRS (- 57%, - 26%, respectively) alone. Compared to DSS-control synbiotic supplementation significantly (p < 0.0001) maintained expressions of tight junction proteins. Moreover, synbiotic effects accounted for ~ 40% suppression of IL-1ß and ~ 29% increase in IL-10 levels in serum while also reducing C-reactive protein (- 37%) compared to that of the DSS-control. While, B. coagulans alone could not induce additional levels of short-chain fatty acid (SCFA) production beyond the caecum, the synbiotic combination with GBRS resulted in substantial increased SCFA levels across the whole length of the colon. CONCLUSION: The synbiotic supplementation with B. coagulans and GBRS ameliorated the overall inflammatory status of the experimental IBD model via synergistic functioning. This supports researching its application in mitigating inflammation in human IBD.

Does NLRP3 Inflammasome and Aryl Hydrocarbon Receptor Play an Interlinked Role in Bowel Inflammation and Colitis-Associated Colorectal Cancer?

Inflammation is a hallmark in many forms of cancer; with colitis-associated colorectal cancer (CAC) being a progressive intestinal inflammation due to inflammatory bowel disease (IBD). While this is an exemplification of the negatives of inflammation, it is just as crucial to have some degree of the inflammatory process to maintain a healthy immune system. A pivotal component in the maintenance of such intestinal homeostasis is the innate immunity component, inflammasomes. Inflammasomes are large, cytosolic protein complexes formed following stimulation of microbial and stress signals that lead to the expression of pro-inflammatory cytokines. The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome has been extensively studied in part due to its strong association with colitis and CAC. The aryl hydrocarbon receptor (AhR) has recently been acknowledged for its connection to the immune system aside from its role as an environmental sensor. AhR has been described to play a role in the inhibition of the NLRP3 inflammasome activation pathway. This review will summarise the signalling pathways of both the NLRP3 inflammasome and AhR; as well as new-found links between these two signalling pathways in intestinal immunity and some potential therapeutic agents that have been found to take advantage of this link in the treatment of colitis and CAC.

NLRP3 inflammasome in colitis and colitis-associated colorectal cancer.

A low level of inflammation is an integral part of the balance between the immune system and the microbiota in the high antigen environment of the gastrointestinal tract and maintains homeostasis. A failure of this balance can lead to chronic intestinal inflammation and increase the chances to develop colorectal cancer significantly. The underlying mechanisms that link inflammation and carcinogenesis are not clear but the molecular platforms of the inflammasomes have been implicated. Inflammasomes are molecule complexes that are assembled in response to microbial components or cellular danger signals and facilitate the production of bioactive pro-inflammatory cytokines. One inflammasome in particular, NLRP3, has been analysed extensively in its contribution to colitis and has been shown to be associated with the development of colitis-associated colorectal cancer. This review will summarise the role of NLRP3 in intestinal inflammation, discuss some of the triggers of inflammation in the gastrointestinal tract such as diet and introduce some opportunities to use this inflammasome as therapeutic target for the treatment of colitis and colitis-associated colorectal cancer.

A human origin strain Lactobacillus acidophilus DDS-1 exhibits superior in vitro probiotic efficacy in comparison to plant or dairy origin probiotics.

Background: The health benefits of probiotics are well established and known to be strain-specific. However, the role of probiotics obtained from different origins and their efficacy largely remains unexplored. The aim of this study is to investigate the in vitro efficacy of probiotics from different origins. Methods: Probiotic strains utilized in this study include Lactobacillus acidophilus DDS-1 (human origin), Bifidobacterium animalis ssp. lactis UABla-12 (human origin), L. plantarum UALp-05 (plant origin) and Streptococcus thermophilus UASt-09 (dairy origin). Screening assays such as in vitro digestion simulation, adhesion, cell viability and cytokine release were used to evaluate the probiotic potential. Results: All strains showed good resistance in the digestion simulation process, especially DDS-1 and UALp-05, which survived up to a range of 107 to 108 CFU/mL from an initial concentration of 109 CFU/mL. Two human colonic mucus-secreting cells, HT-29 and LS174T, were used to assess the adhesion capacity, cytotoxicity/viability, and cytokine quantification. All strains exhibited good adhesion capacity. No significant cellular cytotoxicity or loss in cell viability was observed. DDS-1 and UALp-05 significantly upregulated anti-inflammatory IL-10 and downregulated pro-inflammatory TNF-α cytokine production. All the strains were able to downregulate IL-8 cytokine levels. Conclusion: Of the 4 strains tested, DDS-1 demonstrated superior survival rates, good adhesion capacity and strong immunomodulatory effect under different experimental conditions.

Idebenone Protects against Spontaneous Chronic Murine Colitis by Alleviating Endoplasmic Reticulum Stress and Inflammatory Response.

Endoplasmic reticulum (ER) stress in intestinal secretory goblet cells has been linked to the development of ulcerative colitis (UC). Emerging evidence suggests that the short chain quinone drug idebenone displays anti-inflammatory activity in addition to its potent antioxidant and mitochondrial electron donor properties. This study evaluated the impact of idebenone in Winnie mice, that are characterized by spontaneous chronic intestinal inflammation and ER stress caused by a missense mutation in the mucin MUC2 gene. Idebenone (200 mg/kg) was orally administered daily to 5-6 weeks old Winnie mice over a period of 21 days. Idebenone treatment substantially improved body weight gain, disease activity index (DAI), colon length and histopathology score. Immunohistochemistry revealed increased expression of MUC2 protein in goblet cells, consistent with increased MUC2 mRNA levels. Furthermore, idebenone significantly reduced the expression of the ER stress markers C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6) and X-box binding protein-1 (XBP-1) at both mRNA and protein levels. Idebenone also effectively reduced pro-inflammatory cytokine levels in colonic explants. Taken together, these results indicate that idebenone could represent a potential therapeutic approach against human UC by its strong anti-inflammatory activity and its ability to reduce markers of ER stress.

Modulating the Microbiome and Immune Responses Using Whole Plant Fibre in Synbiotic Combination with Fibre-Digesting Probiotic Attenuates Chronic Colonic Inflammation in Spontaneous Colitic Mice Model of IBD.

A probiotic and prebiotic food ingredient combination was tested for synergistic functioning in modulation of the colonic microbiome and remediation of the gastrointestinal immune and inflammatory responses in a spontaneous colitic mouse model. Bacillus coagulans MTCC5856 spores with capability to metabolise complex plant polysaccharides were supplemented with complex whole-plant prebiotic sugarcane fibre (PSCF). The combined and individual efficacies were tested for their influence on the outcomes of chronic inflammation in Muc2 mutant colitic Winnie mice. The mice were fed normal chow diet supplemented with either ingredient or a combination for 21 days. Synbiotic combined supplementation ameliorated clinical symptoms and histological colonic damage scores more effectively than either B. coagulans or PSCF alone. PSCF and B. coagulans alone also induced considerable immunomodulatory effects. Synbiotic supplementation however was the most efficacious in modulating the overall immune profile compared to the unsupplemented Winnie-control. The augmented synbiotic effect could potentially be due to a combination of increased levels of fermentation products, direct immune-modulating abilities of the components, their capability to reduce colonic epithelial damage and/or modulation of the microbiota. The beneficial effects of the supplementation with a complex plant fibre and a fibre-degrading probiotic parallel the effects seen in human microbiota with high plant fibre diets.

Synbiotic Supplementation Containing Whole Plant Sugar Cane Fibre and Probiotic Spores Potentiates Protective Synergistic Effects in Mouse Model of IBD.

Inflammatory bowel diseases (IBD) are a chronic inflammatory disorders with increasing global incidence. Synbiotic, which is a two-point approach carrying probiotic and prebiotic components in mitigating inflammation in IBD, is thought to be a pragmatic approach owing to the synergistic outcomes. In this study, the impacts of dietary supplementation with probiotic Bacillus coagulans MTCC5856 spores (B. coagulans) and prebiotic whole plant sugar cane fibre (PSCF) was assessed using a murine model of IBD. Eight-week-old C57BL/6 mice were fed a normal chow diet supplemented with either B. coagulans, PSCF or its synbiotic combination. After seven days of supplementation, colitis was induced with dextran sulfate sodium (DSS) in drinking water for seven days during the continuation of the supplemented diets. Synbiotic supplementation ameliorated disease activity index and histological score (-72%, 7.38, respectively), more effectively than either B. coagulans (-47%, 10.1) and PSCF (-53%, 13.0) alone. Synbiotic supplementation also significantly (p < 0.0001) prevented the expression of tight junction proteins and modulated the altered serum IL-1ß (-40%), IL-10 (+26%), and C-reactive protein (CRP) (-39%) levels. Synbiotic supplementations also raised the short-chain fatty acids (SCFA) profile more extensively compared to the unsupplemented DSS-control. The synbiotic health outcome effect of the probiotic and prebiotic combinations may be associated with a synergistic direct immune-regulating efficacy of the components, their ability to protect epithelial integrity, stimulation of probiotic spores by the prebiotic fibre, and/or with stimulation of greater levels of fermentation of fibres releasing SCFAs that mediate the reduction in colonic inflammation. Our model findings suggest synbiotic supplementation should be tested in clinical trials.

Lactobacillus acidophilus DDS-1 Modulates Intestinal-Specific Microbiota, Short-Chain Fatty Acid and Immunological Profiles in Aging Mice.

Distribution of the microbiota varies according to the location in the gastrointestinal (GI) tract. Thus, dysbiosis during aging may not be limited to faecal microbiota and extend to the other parts of the GI tract, especially the cecum and colon. Lactobacillus acidophilus DDS-1, a probiotic strain, has been shown to modulate faecal microbiota and its associated metabolic phenotype in aging mice. In the present study, we investigated the effect of L. acidophilus DDS-1 supplementation on caecal- and mucosal-associated microbiota, short-chain fatty acids (SCFAs) and immunological profiles in young and aging C57BL/6J mice. Besides differences in the young and aging control groups, we observed microbial shifts in caecal and mucosal samples, leading to an alteration in SCFA levels and immune response. DDS-1 treatment increased the abundances of beneficial bacteria such as Akkermansia spp. and Lactobacillus spp. more effectively in caecal samples than in mucosal samples. DDS-1 also enhanced the levels of butyrate, while downregulating the production of inflammatory cytokines (IL-6, IL-1ß, IL-1α, MCP-1, MIP-1α, MIP-1ß, IL-12 and IFN-γ) in serum and colonic explants. Our findings suggest distinct patterns of intestinal microbiota, improvements in SCFA and immunological profiles with DDS-1 supplementation in aging mice.

MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice.

MCC950 a potent, highly specific small molecule inhibitor of canonical and noncanonical activation of NLRP3 inflammasome has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colonic inflammation has not yet been reported. In the present study we investigated the effect of MCC950 in a spontaneous chronic colitis mouse model Winnie, which mimics human ulcerative colitis. Oral administration of 40 mg/kg MCC950 commencing at Winnie week seven for three weeks significantly improved body weight gain, colon length, colon weight to body weight ratio, disease activity index and histopathological scores. MCC950 significantly suppressed release of proinflammatory cytokines IL-1ß, IL-18, IL1-α, IFNγ, TNF-α, IL6, IL17, chemokine MIP1a and Nitric Oxide in colonic explants. Moreover, MCC950 resulted in a significant decrease of IL-1ß release and activation of caspase-1 in colonic explants and macrophage cells isolated from Winnie. Complete inhibition with MCC950 in Winnie colonic explants shows, for the first time, the contribution of inflammatory effects resulting exclusively from canonical and noncanonical NLRP3 inflammasome activation in colitis. Taken together, our results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides avenue for a potential novel therapeutic agent for human inflammatory bowel diseases.

NLRP3 Inhibitors as Potential Therapeutic Agents for Treatment of Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a group of intestinal disorders characterised by chronic relapsing inflammation of the small intestine and colon. IBD manifests as either ulcerative colitis or Crohn's disease and increases the risk of developing colorectal cancer. METHODS: Here, we have reviewed current treatment regimen for IBD utilising anti-inflammatory drugs, immune system suppressors and antibiotics or a combination of these. However, these therapeutics lead to a number of adverse effects, remission or significant non-responsiveness creating an urgent need to develop potent drugs with novel mechanisms of action for IBD. RESULTS: The inflammasome is a multiprotein complex that assembles to a key innate immune system signalling platform and is involved in the pathogenesis of inflammatory and autoimmune diseases. A number of investigations show that the NLRP3 inflammasome recognizes microbial and cell stress components and serve as a platform for caspase-1 activation and pro-inflammatory cytokine IL-1ß, IL18 maturation. Although the exact aetiology of IBD is unknown, uncontrolled NLRP3 Inflammasome activation has shown to play a major role in the chronic intestinal inflammation and mature IL-1ß and IL18 are consistently associated with increased colitis and colitis associated colorectal cancer development. CONCLUSION: In this review, we discuss the experimental NLRP3 inhibitors that have been investigated in IBD experimental models. The potential mechanism of action of these inhibitors such as inhibiting NF-κB activation and decreasing mitochondrial reactive oxygen species are discussed in detail. We further expand the controversial role of NLRP3 in IBD and future issues that might arise from the long term use of NLRP3 inhibitors in IBD therapy.

A rapid amplification/detection assay for analysis of Mycobacterium tuberculosis using an isothermal and silicon bio-photonic sensor complex.

Global tuberculosis (TB) control is hampered by cost and slow or insensitive diagnostic methods to be used for TB diagnosis in clinic. Thus, TB still remains a major global health problem. The failure to rapidly and accurately diagnose of TB has posed significant challenges with consequent secondary resistance and ongoing transmission. We developed a rapid Mycobacterium tuberculosis (MTB) amplification/detection method, called MTB isothermal solid-phase amplification/detection (MTB-ISAD), that couples isothermal solid-phase amplification and a silicon biophotonics-based detection sensor to allow the simultaneous amplification and detection of MTB in a label-free and real-time manner. We validated the clinical utility of the MTB-ISAD assay by detecting MTB nucleic acid in sputum samples from 42 patients. We showed the ability of the MTB-ISAD assay to detect MTB in 42 clinical specimens, confirming that the MTB-ISAD assay is fast (<20 min), highly sensitive, accurate (>90%, 38/42), and cost-effective because it is a label-free method and does not involve thermal cycling. The MTB-ISAD assay has improved time-efficiency, affordability, and sensitivity compared with many existing methods. Therefore, it is potentially adaptable for better diagnosis across various clinical applications.

Mach-Zehnder interferometer (MZI) point-of-care system for rapid multiplexed detection of microRNAs in human urine specimens.

MicroRNAs have been identified as promising biomarkers for human diseases. The development of a point-of-care (POC) test for the disease-associated miRNAs would be especially beneficial, since miRNAs are unexpectedly well preserved in various human specimens, including urine. Here, we present the Mach-Zehnder interferometer-miRNA detection system capable of detecting multiple miRNAs in clinical urine samples rapidly and simultaneously in a label-free and real-time manner. Through measurement of the light phase change, the MZI sensor provides an optical platform for fast profiling of small molecules with improved accuracy. We demonstrate that this system could specifically detect target miRNAs (miR-21, and let-7a), and even identify the single nucleotide polymorphism of the let-7 family of miRNAs from synthetic and cell line samples. The clinical applicability of this system is confirmed by simultaneously detecting two types of miRNAs in urine samples of bladder cancer patients in a single reaction, with a detection time of 15 min. The POC system can be expanded to detect a number of miRNAs of different species and should be useful for a variety of clinical applications requiring at or near the site of patient care.

Solid phase nucleic acid extraction technique in a microfluidic chip using a novel non-chaotropic agent: dimethyl adipimidate.

Here, we present a silicon microfluidic system for the purification and extraction of nucleic acids from human body fluid samples utilizing a dimethyl adipimidate (DMA)-based solid-phase extraction method. We propose DMA, which has been used as an amino-reactive cross-linking agent within cells and proteins, as a non-chaotropic reagent for the capture of nucleic acids to overcome the limitations of existing chaotropic and non-chaotropic techniques such as low binding efficiency, PCR inhibition and so on. DMA contains bi-functional imidoesters that form reversible cross-linking structures with DNA therefore providing a high surface-area to volume ratio for capturing DNA without structurally modifying microfluidic channels. In this work, we have first demonstrated highly efficient capture and purification of genomic DNA (T24 cell line) with DMA using a label-free silicon microring resonator sensor device. In addition, we observed the improvement of the DNA amplification efficiency by using the proposed technique for both the genetic (HRAS) and epigenetic (RARß) analysis of DNA biomarkers. Particularly, we confirmed that the DMA-based solid-phase extraction technique can be applied for the extraction of genomic DNA with higher purity (p < 0.001) using human body fluids (blood and urine) in silicon microfluidic devices compared to other chaotropic methods. Therefore, the proposed technique would be able to harmonize with a micro-total analysis system platform for the analysis of genetic and epigenetic DNA biomarkers related to human diseases in the field of point-of-care (POC) diagnostic applications.

Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers.

Here, we first present an isothermal solid-phase amplification/detection (ISAD) technique for the detection of single-point mutations that can be performed without labelling in real-time by utilizing both silicon microring-based solid-phase amplification and isothermal recombinase polymerase amplification (RPA). The ISAD technique was performed on a silicon microring device with a plastic chamber containing 10 µL of the reaction mixture, and characterized with an assay for the detection of the HRAS (Harvey RAS) gene single-point mutation. For the solid-phase amplification, the primer of the gene was directly attached to the surface of the device via an amine modification reaction. The amplified DNA was detected, without a label, by measuring the optical wavelength shift of the silicon microring resonator during the reaction. We demonstrated that the sensitivity of the ISAD technique was 100-times higher than that of RPA and conventional PCR methods. Moreover, this technique can be used to distinguish a single-point mutation of the HRAS gene via target amplification. This novel DNA amplification/detection technique will be useful for the detection of sequence alterations such as mutations and single-nucleotide polymorphisms as DNA biomarkers in human diseases.

Label-free, PCR-free chip-based detection of telomerase activity in bladder cancer cells.

Bladder cancer is one of the most common cancers in Worldwide. The determination of urinary telomerase activity is a promising tool for the diagnosis of bladder carcinoma owing to the high rate of expression of telomerase in cancer cells. Typical assay for telomerase activity is the telomeric repeat amplification protocol (TRAP) based on polymerase chain reaction (PCR) amplification. However, TRAP assay is susceptible to PCR-derived artifacts and requires time-consuming procedure, expensive equipments and reagents. To develop a new method for telomerase activity assay that is fast, simple, and cost-effective, we have examined a silicon-based microring resonator biosensor to detect label-free, PCR-free telomerase activity using telomerase extracted from two bladder cancer cell lines, J-82 and HT-1376, in a buffer solution and spiked urine. With telomerase primer immobilized microring resonator sensor system, we successfully demonstrate the detection of telomerse extracted from as little as 10 cells/µL and 100 cells/µL in buffer and urine, respectively. Especially, the results represented here is the first demonstration of the detection of telomerase activity in human urine on the chip-based system. From the results, we expect that the silicon photonic microring resonator system can provide a powerful tool for cost-effective and sensitive telomerase activity detection in urinary bladder cancer.