Probiotaceuticals: Back to the future?

Probiotic research has undergone some exciting and unanticipated changes in direction since the 2010 commentary by GSH, which speculated on probiotics being ultimately utilized as "factories" capable of releasing pharmaceutical-grade metabolites with therapeutic potential for a wide range of primarily gastrointestinal disorders. Indeed, the unrelenting search for new alternatives to antibiotics has further stimulated the development of "next-generation" probiotics. Postbiotics, defined as inanimate microorganisms and/or their components that confer a health benefit on the host, remain at the forefront of current probiotic research, with increasing numbers of probiotic species, strains, and substrains now being identified and further exploited as pharmabiotics; probiotics with a proven pharmacologic role in health and disease that have been subjected to clinical trial prior to approval by regulatory bodies. However, perhaps the most unanticipated probiotic development over the past 15 y has been the emergence of psychobiotics with the potential to improve aspects of mental health, such as depression and anxiety, through the release of bioactive metabolites. Moreover, the recent identification of pharmacobiotics, probiotics capable of facilitating the effectiveness of conventional pharmaceutical drugs, is opening new avenues for probiotic applications to combat a range of diseases, including cancers of the digestive system. Although in its infancy, recent reports of oncobiotics with antineoplastic properties are further expanding the potential for certain next-generation probiotics to impact current cancer treatment regimens and possibly even contribute to cancer prevention. Looking to the next 15 y of probiotic development, one could perhaps predict the ultimate development of regulatory-approved xenopostbiotic formulations comprising metabolites with the capacity to improve digestive health, decrease the severity of intestinal disease, and increase the effectiveness of conventional pharmaceuticals, whereas simultaneously improving cognitive functioning and mental welfare. Although speculative, these xenopostbiotic formulations could prove especially effective for the adjunctive treatment of serious chronic diseases such as cancer.

Deferiprone-Gallium-Protoporphyrin Chitogel Decreases Pseudomonas aeruginosa Biofilm Infection without Impairing Wound Healing.

Pseudomonas aeruginosa is one of the most common pathogens encountered in clinical wound infections. Clinical studies have shown that P. aeruginosa infection results in a larger wound area, inhibiting healing, and a high prevalence of antimicrobial resistance. Hydroxypyridinone-derived iron chelator Deferiprone (Def) and heme analogue Gallium-Protoporphyrin (GaPP) in a chitosan-dextran hydrogel (Chitogel) have previously been demonstrated to be effective against PAO1 and clinical isolates of P. aeruginosa in vitro. Moreover, this combination of these two agents has been shown to improve sinus surgery outcomes by quickly reducing bleeding and preventing adhesions. In this study, the efficacy of Def-GaPP Chitogel was investigated in a P. aeruginosa biofilm-infected wound murine model over 6 days. Two concentrations of Def-GaPP Chitogel were investigated: Def-GaPP high dose (10 mM Def + 500 µg/mL GaPP) and Def-GaPP low dose (5 mM Def + 200 µg/mL GaPP). The high-dose Def-GaPP treatment reduced bacterial burden in vivo from day 2, without delaying wound closure. Additionally, Def-GaPP treatment decreased wound inflammation, as demonstrated by reduced neutrophil infiltration and increased anti-inflammatory M2 macrophage presence within the wound bed to drive wound healing progression. Def-GaPP Chitogel treatment shows promising potential in reducing P. aeruginosa cutaneous infection with positive effects observed in the progression of wound healing.

Emu Oil and zinc monoglycerolate independently reduce disease severity in a rat model of ulcerative colitis.

Ulcerative colitis is characterized by colonic inflammation. Previously, Emu Oil protected the intestine against experimentally-induced inflammatory intestinal disorders. Zinc monoglycerolate (ZMG) polymer, formed by heating zinc oxide with glycerol, demonstrated anti-inflammatory and wound healing properties. We aimed to determine whether ZMG, alone or in combination with Emu Oil, could reduce acute colitis severity in rats. Male Sprague Dawley rats (n = 8/group) were orally-administered either vehicle, ZMG, Emu Oil (EO) or ZMG combined with EO (ZMG/EO) daily. Rats were provided ad libitum access to drinking water (Groups 1-4) or dextran sulphate sodium (DSS; 2%w/v; Groups 5-8) throughout the trial (days 0-5) before euthanasia on day 6. Disease activity index, crypt depth, degranulated mast cells (DMCs) and myeloperoxidase (MPO) activity were assessed. p < 0.05 was considered significant. DSS increased disease severity (days 3-6) compared to normal controls (p < 0.05). Importantly, in DSS-administered rats, ZMG/EO (day 3) and ZMG (day 6) reduced disease activity index compared to controls (p < 0.05). Following DSS consumption, distal colonic crypts lengthened (p < 0.01), occurring to a greater extent with EO compared to ZMG and ZMG/EO (p < 0.001). DSS increased colonic DMC numbers compared to normal controls (p < 0.001); an effect decreased only by EO (p < 0.05). Colonic MPO activity increased following DSS consumption (p < 0.05); notably, ZMG, EO and ZMG/EO treatments decreased MPO activity compared to DSS controls (p < 0.001). EO, ZMG and ZMG/EO did not impact any parameter in normal animals. Emu Oil and ZMG independently decreased selected indicators of colitic disease severity in rats; however, the combination did not reveal any additional benefit.

Emu Oil Attenuates Disease Severity and Results in Fewer Large Colonic Tumors in a Mouse Model of Colitis-Associated Colorectal Cancer.

Ulcerative colitis patients have an increased risk of developing colorectal cancer (CRC). The aim of the current study was to determine whether Emu Oil (EO) could reduce the severity of colitis, thereby inhibiting colitis-associated CRC (CA-CRC) development. Female C57BL/6 mice (n = 8/group) were injected (i.p.) with saline or azoxymethane (AOM) (7.4 mg/kg). Mice underwent three dextran sulfate sodium (DSS)/water cycles. Mice were orally-administered either water (160 µL) or EO (80 µL or 160 µL) thrice weekly and euthanized after 12 weeks. AOM/DSS decreased bodyweight compared with normal controls (max. 20%; p < 0.05). In AOM/DSS mice, EO (160 µL) increased bodyweight compared with untreated and 80 µL EO-treated mice (max. 10%; p < 0.05). Both volumes of EO reduced disease activity index (DAI) scores on day 49, 56-63 (max. 40%; p < 0.05), compared with AOM/DSS controls. Histological damage was increased in the distal colon of AOM/DSS mice, and reduced by EO (160 µL; p < 0.05). Mucin-secreting goblet cells were increased by AOM/DSS compared to normal, with no effect observed following EO treatment (p > 0.05). Large tumor numbers were decreased in EO-treated mice (160 µL; 2 ± 0.6) compared with AOM/DSS controls (5 ± 0.7; p < 0.05). EO did not impact overall tumor number (p > 0.05). Other analyses remained unchanged across groups (p > 0.05). EO demonstrates promise as an adjunct to conventional treatment options for colitis management.

Emu Oil and Saireito in combination reduce tumour development and clinical indicators of disease in a mouse model of colitis-associated colorectal cancer.

BACKGROUND: Emu Oil (EO) previously demonstrated therapeutic potential in a mouse model of colitis-associated CRC (CA-CRC). Saireito, a traditional Japanese medicine, has not been investigated in CA-CRC. AIM: To determine whether EO and Saireito could be therapeutic in an azoxymethane (AOM)/dextran sulphate sodium (DSS) model of CA-CRC. METHODS: Female C57BL/6 mice were assigned to groups (n = 10/group); 1) saline control, 2) saline+Saireito, 3) saline+EO, 4) saline+EO/Saireito, 5) AOM/DSS control, 6) AOM/DSS+Saireito, 7) AOM/DSS+EO and 8) AOM/DSS+EO/Saireito. Mice were intraperitoneally injected with saline or AOM (7.4 mg/kg) on day 0 and underwent three DSS/water cycles (2%w/v DSS for 7 days, 14 days water). Mice were orally-gavaged with either water (80 µL), Saireito (80 µL), EO (80 µL) or EO/Saireito (160 µL; 80 µL EO + 80 µL Saireito) thrice weekly. Daily bodyweight and disease activity index (DAI) were recorded and colonoscopies performed on days 20, 41 and 62. Mice were euthanized on day 63. p < 0.05 was considered statistically significant. RESULTS: AOM/DSS induced significant bodyweight loss throughout the trial (max -36%), which was attenuated by Saireito (max +7%), EO (max +5%) and EO/Saireito (max +14%; p < 0.05). AOM/DSS increased DAI compared to saline controls (p < 0.05), which was reduced by Saireito, EO and EO/Saireito (p < 0.05). All treatments reduced colonoscopically-assessed colitis severity (days 20 and 41; p < 0.05). EO/Saireito further decreased colitis severity compared to Saireito and EO alone (day 20; p < 0.05). Finally, EO and EO/Saireito resulted in fewer colonic tumours compared to AOM/DSS controls (p < 0.05). CONCLUSION: Combined EO and Saireito reduced disease and tumour development in AOM/DSS mice, suggesting therapeutic potential in CA-CRC.

Emu oil and grape seed extract reduce tumour burden and disease parameters in murine colitis-associated colorectal cancer.

Ulcerative colitis is an incurable condition whereby patients are at an increased risk of developing colorectal cancer (CRC). We aimed to investigate the combination of Emu oil (EO) and grape seed extract (GSE) in an azoxymethane (AOM)/dextran sulphate sodium (DSS) model of colitis-associated CRC (CA-CRC). C57BL/6 mice (n = 10/group) were injected i.p. with saline or AOM (7.4 mg/kg) and underwent three DSS/water cycles. Mice were orally-gavaged thrice weekly with water (80 µl), EO (80 µl), GSE (80 µl; 400 mg/kg) or combined EO/GSE (160 µl). Mice were euthanized on day 63. AOM/DSS induced significant bodyweight loss (max -21%) and increased disease activity index (DAI) (max +83%) throughout the trial (P < 0.05). EO (max -53%), GSE (max -51%) and EO/GSE (max -71%) reduced DAI scores in AOM/DSS mice in all DSS cycles (P < 0.05). EO/GSE-treatment in AOM/DSS mice resulted in further DAI reduction compared with EO (max -62%) and GSE (max -71%) alone (P < 0.05). AOM/DSS mice presented with severe colonoscopically-assessed colitis at all time-points, which was reduced by EO, GSE and EO/GSE (P < 0.05). EO, GSE and EO/GSE reduced the number of colonic tumours compared with AOM/DSS controls (P < 0.05). Myeloperoxidase (acute inflammation) and fluorescein isothiocyanate-dextran levels (intestinal permeability) were increased in AOM/DSS controls (P < 0.05). EO (-58%) and EO/GSE (-77%) reduced fluorescein isothiocyanate-dextran compared with AOM/DSS controls (P < 0.05), with no effect on myeloperoxidase. Histologically-assessed severity scores were increased in the distal colon of AOM/DSS mice compared with saline (P < 0.05), with no effect observed following treatment. The combination of EO and GSE improved clinical indicators and reduced colonic tumours in AOM/DSS treated mice, suggesting potential in CA-CRC management.

Orally administered emu oil attenuates disease in a mouse model of Crohn's-like colitis.

Crohn's disease is a severe, incurable inflammatory bowel disease. Orally administered emu oil has demonstrated anti-inflammatory properties in previous models of gastrointestinal disease. We aimed to determine whether orally administered emu oil could attenuate disease in a mouse model of Crohn's-like colitis. Female ARC(s) mice (CD-1 equivalent, n = 10/group) were intra-rectally administered water (120 µL) or trinitrobenzene sulfonic acid (TNBS; 3 mg in 50% ethanol; 120 µL bolus) on day 0. Mice were orally administered water (80 µL) or emu oil (80 µL or 160 µL) daily for five days and euthanized on day six. Bodyweight and disease activity were recorded daily. Colonoscopy, burrowing activity, facial grimace, histological parameters (damage severity, small intestinal villus height/crypt depth and colonic crypt depth), myeloperoxidase activity and intestinal permeability were assessed. P < 0.05 was considered statistically significant. TNBS decreased bodyweight (days 1, 2, 4; P < 0.05) and increased disease activity (days 1-6; P < 0.01), compared to normal controls. Emu oil (80 µL) attenuated disease activity on days 5-6 (P < 0.05), although bodyweight loss was not significantly impacted (P > 0.05). Facial grimace and colonoscopy scores were significantly increased in TNBS-control mice; effects attenuated by both volumes of emu oil (P < 0.001). TNBS increased histological damage severity compared to normal controls (P < 0.05); an effect attenuated by 80 µL emu oil (proximal and distal colon; P < 0.05) and 160 µL emu oil (distal colon; P < 0.01). In the ileum, villus height and crypt depth were unaffected by TNBS or emu oil treatment compared to normal (P > 0.05). TNBS-induced distal colonic crypt lengthening was unaffected following emu oil administration (P > 0.05). Remaining parameters, including burrowing, myeloperoxidase activity and intestinal permeability, were unchanged across all treatment groups (P > 0.05). In normal mice, emu oil treatment did not significantly impact any parameter compared to normal controls. In conclusion, emu oil reduced overall disease severity and facial grimace scores in TNBS mice. These results suggest therapeutic potential for orally administered emu oil in the management of Crohn's disease.

Intestinal stem cells promote crypt fission during postnatal growth of the small intestine.

OBJECTIVE: Wnt-ß-catenin signalling is essential for intestinal stem cells. Our aim was to investigate the relationship between intestinal stem cells and crypt fission which peaks during infancy. DESIGN: Duodenal biopsies were obtained during endoscopy to assess the severity of reflux oesophagitis of 15 infants, children and teenagers, which would not affect the duodenum. Samples of small intestine were also obtained from rats 7-72 days of life. Crypt fission was assessed using microdissection of 100 whole crypts and recording the percentage of bifid crypts. Intestinal LGR5+ stem cells were identified by in situ hybridisation. Rats were treated with Dickkopf to block Wnt-ß-catenin signalling. RESULTS: Crypt fission peaked during infancy before declining after 3-4 years in humans and after 21 days of life in rats. Occasional mitotic figures were seen in bifid crypts during early fission. Stem cells were elevated for a greater period during infancy and childhood in humans. Clustering of Paneth cells was present around the stem cells at the crypt base. Dickkopf reduced the number of stem cells and crypt fission to 45% and 29%, respectively, of control values, showing dependence of both crypt fission and Lgr5+ stem cells on Wnt signalling. However, Dickkopf did not decrease mitotic count per crypt, indicating a difference in signalling between stem cells and their progeny in the transit amplifying zone. CONCLUSION: Crypt fission peaks during infancy and is dependent on intestinal stem cells. This is relatively hidden by 'a cloak of invisibility' due to the low proliferation of stem cells.

Affective state determination in a mouse model of colitis-associated colorectal cancer.

Behavioural indicators of affective state, including burrowing, clinical scores and the Mouse Grimace Score have not yet been validated in mouse models of chronic gastrointestinal disease. Additionally, a comparison of these methods has not been characterised. This study aimed to determine which behavioural assessment was the optimal indicator of disease, evidenced by correlation with clinically-assessed measures, in an azoxymethane (AOM)/dextran sulphate sodium (DSS) mouse model of colitis-associated colorectal cancer. C57BL/6 mice were allocated to four groups (n = 10/group); 1) saline control, 2) saline+buprenorphine, 3) AOM+DSS+water, 4) AOM+DSS+buprenorphine. Mice were gavaged thrice weekly with water or buprenorphine (0.5mg/kg; 80µL) for 9 weeks. Disease activity index (DAI) was measured daily; burrowing and grimace analyses occurred on days -1, 5, 19, 26, 40, 47 and 61. Colonoscopies were performed on days 20, 41 and 62. All animals were euthanized on day 63. Burrowing activity and retrospective grimace analyses were unaffected (P>0.05), whilst DAI was significantly increased (P<0.05) in mice with colitis-associated colorectal cancer compared to normal controls. In addition, DAI was positively correlated with colonoscopically-assessed severity and tumour number (P<0.05). We conclude that traditional measures of DAI or clinical scoring provide the most reliable assessment of wellbeing in mice with colitis-associated colorectal cancer.

Chemotherapy-induced mucositis development in a murine model of colitis-associated colorectal cancer.

Objectives: Ulcerative colitis is an incurable inflammatory bowel disease that increases the risk of colorectal cancer (CRC). 5-Fluorouracil (5-FU) is the predominant chemotherapy for CRC patients; however, undesirable side-effects, including mucositis, are common. This study utilised 5-FU-treatment in a model of colitis-associated CRC to develop a pre-clinical setting of intestinal mucositis coincident with manifestation of CRC.Materials/methods: On day 0, female C57BL/6 mice (n = 10/group); (1) saline control, (2) AOM/DSS control, or (3) AOM/DSS + 5-FU were injected with saline or AOM (i.p; 7.4 mg/kg). Groups 2 and 3 underwent cycles of seven days 2%w/v DSS followed by 14 days plain water. After three cycles, 5-FU was administered weekly (i.p; 75 mg/kg) to group 3 for five weeks. Clinical indicators were measured daily and colonoscopy performed at four time-points. Mice were euthanized at 13 weeks (day 91). Intestinal sections were collected for histological and biochemical analyses. p < .05 was considered significant.Results: AOM/DSS resulted in bodyweight loss, increased disease activity index, colitis-severity and tumour number compared to saline controls (p < .05). 5-FU-treatment in AOM/DSS mice decreased bodyweight and disease activity index at selected time-points compared to AOM/DSS controls (p < .05). 5-FU did not impact colitis-severity or overall tumour burden; although, resulted in fewer small tumours compared to AOM/DSS controls (<2mm; p < .05). AOM/DSS increased histological severity scores in intestinal sections (p < .05), however, 5-FU-treatment did not further increase histologically-assessed disease severity (p > .05).Conclusion: Weekly 5-FU administration at a dose of 75 mg/kg was insufficient to reduce overall tumour burden or induce intestinal mucositis in the AOM/DSS mouse model.

Use of the Rat Grimace Scale to Evaluate Visceral Pain in a Model of Chemotherapy-Induced Mucositis.

The rat grimace scale (RGS) is a measure of spontaneous pain that evaluates pain response. The ability to characterize pain through a non-invasive method has considerable utility for numerous animal models of disease, including mucositis, a painful, self-limiting side-effect of chemotherapy treatment. Preclinical studies investigating novel therapeutics for mucositis often focus on pathological outcomes and disease severity. These investigations fail to measure pain, in spite of reduction of pain being a key clinical therapeutic goal. This study assessed the utility of the RGS for pain assessment in a rat model of mucositis, and whether changes in disease activity index (DAI) and open field test (OFT) reflected the grimace responses recorded. Sixty tumor-bearing female Dark Agouti rats were injected with either saline or 5-Fluourouracil alone, or with co-administration of opioid analgesics. Whilst differences in DAI were observed between treatment groups, no difference in RGS scores or OFT were demonstrated. Significant increases in grimace scores were observed across time. However, whilst a statistically significant change may have been noted, the biological relevance is questionable in terms of practical usage, since an observer is only able to score whole numbers. Development of effective pain assessment methods in animal models is required to improve welfare, satisfy regulatory requirements, and increase translational validity of the model to human patients.

Prebiotics Fructo-, Galacto-, and Mannan-Oligosaccharide Do Not Protect against 5-Fluorouracil-Induced Intestinal Mucositis in Rats.

BACKGROUND: Prebiotics selectively stimulate the growth of beneficial bacteria within the gastrointestinal tract, and have been investigated in human and animal studies for their capacity to improve intestinal health. OBJECTIVE: We investigated the prebiotics fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), and mannan-oligosaccharide (MOS) for their potential to alleviate intestinal damage in rats. METHODS: Female Dark Agouti rats (6-8 wk old, 110-150 g) were allocated to 1 of the following treatment groups (n = 8/group): saline/water, saline/FOS, saline/GOS, saline/MOS, 5-fluorouracil (5FU)/water, 5FU/FOS, 5FU/GOS, and 5FU/MOS. Rats were pretreated with either 5% GOS, MOS, or FOS or vehicle (water) from day -12 to day 0. On day 0, rats received a single intraperitoneal injection of saline or 5FU. Metabolic data were recorded daily and all rats were killed on day 3. Histopathology was quantified in hematoxylin and eosin-stained sections. Intestinal sucrase and myeloperoxidase activity were quantified by biochemical assay. Fecal SCFAs-acetic, propionic, and butyric acid-were also measured. Statistical analysis was by repeated-measures, 2-factor ANOVA or Kruskal-Wallis and Mann-Whitney U test; P < 0.05 was considered statistically significant. RESULTS: Body weight was significantly decreased in all treatment groups after 5FU injection, with no change in body weight observed in any prebiotic treatment group. Total food intake was lower by ≥7% in the GOS treatment group pre-5FU than in all other groups (P < 0.05). Ileal villus height was 18% higher in GOS-treated rats pre-5FU than in respective water controls (P < 0.05). Jejunal and ileal villus height and crypt depth were significantly decreased in all treatment groups after 5FU injection, with no prebiotic effect observed. SCFAs were differentially increased in prebiotic treatment groups compared with water-only controls (P < 0.05). CONCLUSIONS: FOS, GOS, and MOS have differential effects in modifying small intestinal pathology and SCFA profiles in rats with healthy and damaged small intestinal mucosa.

Emu Oil reduces disease severity in a mouse model of chronic ulcerative colitis.

Objective: Ulcerative colitis (UC) is characterized by mucosal inflammation and ulceration of the large intestine. Emu Oil (EO) has been reported to protect the intestine against mucositis, NSAID-enteropathy, UC-associated colorectal cancer and acute UC. We aimed to determine whether EO could reduce the severity chronic UC in mice. Methods: Female C57BL/6 mice (n = 10/group) were orally administered (gavage) water (Groups 1-2) or EO (Groups 3: low dose-80 µl and 4: high dose-160 µl), thrice weekly. Group 1 mice consumed plain drinking water throughout the trial. Groups 2-4 mice underwent two cycles [each consisting of seven days dextran sulfate sodium (DSS; 2% w/v) and 14 days water], followed by a third DSS week. All mice were euthanized two days later (day 51). Bodyweight, disease activity index (DAI), burrowing activity, myeloperoxidase activity, crypt depth and histologically assessed damage severity were assessed. p < .05 was considered significant. Results: DSS decreased bodyweight and increased DAI compared to normal controls (p < .05), which was partially attenuated by both EO doses (p < .05). Burrowing activity was impaired in DSS-controls compared to normal controls (days 27 and 40); an effect prevented by both EO doses (p < .05). DSS increased colonic myeloperoxidase activity and crypt depth compared to controls (p < .05), with no significant EO effect. Moreover, DSS increased colonic damage severity compared to normal controls (p < .001). Importantly, both EO doses decreased distal colonic damage severity compared to DSS-controls (p < .001). Conclusions: Emu Oil attenuated clinically- and histologically-assessed disease severity in a mouse model of chronic UC. Emu Oil demonstrates promise as an adjunct to conventional treatment options for UC management.

Combined Nutraceuticals: A Novel Approach to Colitis-Associated Colorectal Cancer?

Ulcerative colitis is an unremitting and lifelong inflammatory bowel disease that is increasing in prevalence worldwide. Patients display various clinical symptoms such as abdominal pain, diarrhea and fatigue. The etiology of ulcerative colitis remains unknown and the current pharmaceutical treatments are variably effective and not curative, highlighting the need for improved therapeutic approaches. Furthermore, patients with ulcerative colitis are at an increased risk of developing colorectal cancer. Some naturally sourced agents, named nutraceuticals, have been identified to possess anti-inflammatory and antioxidant properties. Of particular interest is Emu Oil, grape seed extract and Japanese Kampo medicine. Previously, Emu Oil has protected and repaired intestinal damage in models of gastrointestinal diseases including colitis and colitis-associated colorectal cancer. Additionally, grape seed extract possesses anticancer properties in vitro. Moreover, Kampo medicine, composed of herbal ingredients, is widely used in Japan for the treatment of various medical conditions and has demonstrated efficacy in targeting cancer cells in vitro. Nutraceuticals in combination have not yet been widely investigated in a setting of colitis-associated colorectal cancer. Investigation into the efficacy of Emu Oil combined with other nutraceuticals, including grape seed extract and Kampo medicine, is warranted as they may provide a novel approach to conventional colitis and colorectal cancer management.

Active ß-Catenin Signaling in the Small Intestine of Humans During Infancy.

BACKGROUND: Wnt-ß-catenin signaling is essential for homeostasis of intestinal stem cells in mice and is thought to promote intestinal crypt fission. AIMS: The aim of this study was to investigate Wnt-ß-catenin signaling in intestinal crypts of human infants. METHODS: Duodenal biopsies from nine infants (mean, range 0.9 years, 0.3-2 years) and 11 adults (mean, range 43 years, 34-71 years) were collected endoscopically. Active ß-catenin signaling was assessed by cytoplasmic and nuclear ß-catenin, nuclear c-Myc, and cytoplasmic Axin-2 expression in the base of crypts. Tissues were stained by an immunoperoxidase staining technique and quantified as pixel energy using cumulative signal analysis. Data were expressed as mean ± SD and significance assessed by Student's t test. RESULTS: Crypt fission was significantly higher in infants compared to adults (16 ± 8.6% versus 0.7 ± 0.6%, respectively, p < 0.0001). Expression of cytoplasmic and nuclear ß-catenin was 1.8-fold (p < 0.0001) and 2.9-fold (p < 0.0001) higher in infants, respectively, while cytoplasmic Axin-2 was 3.1-fold (p < 0.0001) increased in infants. c-Myc expression was not significantly different between infants and adults. Expression was absent in Paneth cells but present in the transit amplifying zone of crypts. Crypt base columnar cells, which were intercalated between Paneth cells, expressed c-Myc. CONCLUSIONS: Wnt-ß-catenin signaling was active in crypt base columnar cells (i.e., intestinal stem cells) in human infants. This signaling could promote crypt fission during infancy. Wnt-ß-catenin signaling likely acts in concert with other pathways to promote postnatal growth.

Intestinal homeostasis is restored in mice following a period of intestinal growth induced by orally administered Emu Oil.

Previously, we reported that orally administered Emu Oil (EO) increases mucosal thickness in the small intestine and colon in rodent models of chemotherapy-induced mucositis and colitis. However, it remains unclear whether mucosal thickening (crypt and villus lengthening) represents a process of normal or aberrant growth. We sought to determine if villus height (VH) and crypt depth (CD) measurements returned to normal in EO-treated rats following withdrawal of EO therapy. Dark agouti rats ( n = 8/group) were gavaged daily for 10 days with water, olive oil (OO), or EO (0.5 mL or 1 mL). Groups of rats were euthanized on days 10 and 17. Intestinal weights, lengths, VH, and CD were quantified. P < 0.05 was considered significant. On day 10, jejuno-ileum weight was increased by OO (26%) and EO (0.5 mL: 15%; 1 mL: 29%) compared to water controls ( P < 0.01), which was normalized by day 17. On days 10 and 17, jejuno-ileum length was greater in OO- (12%) and EO-treated rats (0.5 mL: 8%; 1 mL: 12%; P < 0.05), relative to water controls. On day 10, OO and EO increased ileal VH (OO: 32%; 0.5 EO: 22%; EO: 35%; P < 0.01) and CD (OO: 17%; 0.5 EO: 13%; EO: 22%) compared to water controls. Importantly, however, after withdrawal of all oils, VH and CD measurements returned to normal control values. Moreover, the VH:CD ratio (potential indicator of dysplasia) remained unchanged in all experimental groups on days 10 and 17. The restoration of normal intestinal architecture following cessation of Emu Oil therapy supports its safety for application in intestinal disorders. Impact statement Uncontrolled inflammation and intestinal proliferation can predispose to the development of colorectal cancer. In previous pre-clinical studies, we demonstrated that oral administration of Emu Oil promotes intestinal repair via stimulation of the mucosa in response to tissue injury and inflammation. Therefore, it was important to determine if Emu Oil administration did not promote the precocious development of colorectal cancer. The current study revealed that Emu Oil returned indicators of intestinal proliferation back to normal values after a period of seven days. These data strongly support the safety of Emu Oil for further studies in the context of bowel inflammation.

From the Bottom-Up: Chemotherapy and Gut-Brain Axis Dysregulation.

The central nervous system and gastrointestinal tract form the primary targets of chemotherapy-induced toxicities. Symptoms associated with damage to these regions have been clinically termed chemotherapy-induced cognitive impairment and mucositis. Whilst extensive literature outlines the complex etiology of each pathology, to date neither chemotherapy-induced side-effect has considered the potential impact of one on the pathogenesis of the other disorder. This is surprising considering the close bidirectional relationship shared between each organ; the gut-brain axis. There are complex multiple pathways linking the gut to the brain and vice versa in both normal physiological function and disease. For instance, psychological and social factors influence motility and digestive function, symptom perception, and behaviors associated with illness and pathological outcomes. On the other hand, visceral pain affects central nociception pathways, mood and behavior. Recent interest highlights the influence of functional gut disorders, such as inflammatory bowel diseases and irritable bowel syndrome in the development of central comorbidities. Gut-brain axis dysfunction and microbiota dysbiosis have served as key portals in understanding the potential mechanisms associated with these functional gut disorders and their effects on cognition. In this review we will present the role gut-brain axis dysregulation plays in the chemotherapy setting, highlighting peripheral-to-central immune signaling mechanisms and their contribution to neuroimmunological changes associated with chemotherapy exposure. Here, we hypothesize that dysregulation of the gut-brain axis plays a major role in the intestinal, psychological and neurological complications following chemotherapy. We pay particular attention to evidence surrounding microbiota dysbiosis, the role of intestinal permeability, damage to nerves of the enteric and peripheral nervous systems and vagal and humoral mediated changes.

A Judgement Bias Test to Assess Affective State and Potential Therapeutics in a Rat Model of Chemotherapy-Induced Mucositis.

Chemotherapy-induced mucositis is an extremely painful condition that occurs in 40-60% of patients undergoing chemotherapy. As mucositis currently has no effective treatment, and due to the self-limiting nature of the condition, the major treatment aims are to manage symptoms and limit pain with significance placed on improving patient quality of life. Rodent models are frequently used in mucositis research. These investigations typically assess pathological outcomes, yet fail to include a measure of affective state; the key therapeutic goal. Assessment of cognitive biases is a novel approach to determining the affective state of animals. Consequently, this study aimed to validate a cognitive bias test through a judgement bias paradigm to measure affective state in a rat model of chemotherapy-induced intestinal mucositis. Rats with intestinal mucositis demonstrated a negative affective state, which was partially ameliorated by analgesic administration, whilst healthy rats showed an optimistic response. This study concluded that the judgement bias test was able to evaluate the emotional state of rats with chemotherapy-induced mucositis. These findings provide a foundation for future refinement to the experimental design associated with the animal model that will expedite successful transitioning of novel therapeutics to clinical practice, and also improve humane endpoint implementation.

Emu Oil Improves Clinical Indicators of Disease in a Mouse Model of Colitis-Associated Colorectal Cancer.

BACKGROUND/AIMS: Ulcerative colitis is a remitting and relapsing inflammatory bowel disorder. Current treatments are limited, and if poorly controlled, colitis may progress to colorectal cancer. Previously, Emu Oil protected the intestine in experimental models of gut damage. We aimed to determine whether Emu Oil could reduce the severity of chronic colitis and prevent the onset of neoplasia in a mouse model of colitis-associated colorectal cancer. METHODS: Female C57BL/6 mice were injected (day 0) with azoxymethane, followed by ad libitum access to three dextran sulfate sodium/water cycles (7 days of dextran sulfate sodium and 14 days of water). Mice (n = 9/group) were orally administered either water or Emu Oil (low dose 80 µL or high dose 160 µL), thrice weekly for 9 weeks. Bodyweight and disease activity index were measured daily. Colitis progression was monitored by colonoscopy on days 20, 41 and 62. At killing, tumor number and size were recorded. RESULTS: Azoxymethane/dextran sulfate sodium induced significant bodyweight loss (maximum 24%) which was attenuated by Emu Oil treatment (low dose days 9, 10, 14: maximum 7%; high dose days 7-15, 30-36: maximum 11%; p < 0.05). Emu Oil reduced disease activity index of azoxymethane/dextran sulfate sodium mice at most time points (maximum 20%; p < 0.05). Additionally, Emu Oil reduced colonoscopically assessed colitis severity (days 20 and 62) compared to disease controls (p < 0.05). Finally, in azoxymethane/dextran sulfate sodium mice, low-dose Emu Oil resulted in fewer small colonic tumors (p < 0.05) compared to controls. CONCLUSIONS: Emu Oil improved clinical indicators and reduced severity of colitis-associated colorectal cancer, suggesting therapeutic potential in colitis management.

Effects of commercially produced almond by-products on chemotherapy-induced mucositis in rats.

AIM: To determine if almond extracts reduce the severity of chemotherapy-induced mucositis as determined through biochemical, histological and behavioural markers. METHODS: Intestinal mucositis is a debilitating condition characterized by inflammation and ulceration of the gastrointestinal mucosa experienced by cancer patients undergoing chemotherapy. Certain bioactive plant products have shown promise in accelerating mucosal repair and alleviating clinical symptoms. This study evaluated almond extracts for their potential to reduce the severity of chemotherapy-induced mucositis in Dark Agouti rats. Female Dark Agouti rats were gavaged (days 3-11) with either PBS, almond hull or almond blanched water extract at two doses, and were injected intraperitoneally with 5-fluorouracil (5-FU-150 mg/kg) or saline on day 9 to induce mucositis. Burrowing behavior, histological parameters and myeloperoxidase activity were assessed. RESULTS: Bodyweight was significantly reduced in rats that received 5-FU compared to saline-treated controls (P < 0.05). Rats administered 5-FU significantly increased jejunal and ileal MPO levels (1048%; P < 0.001 and 409%; P < 0.001), compared to healthy controls. Almond hull extract caused a pro-inflammatory response in rats with mucositis as evidenced by increased myeloperoxidase activity in the jejunum when compared to 5-FU alone (rise 50%, 1088 ± 96 U/g vs 723 ± 135 U/g, P = 0.02). Other extract-related effects on inflammatory activity were minimal. 5-FU significantly increased histological severity score compared to healthy controls confirming the presence of mucositis (median of 9.75 vs 0; P < 0.001). The extracts had no ameliorating effect on histological severity score in the jejunum or ileum. Burrowing behavior was significantly reduced in all chemotherapy-treated groups (P = 0.001). The extracts failed to normalize burrowing activity to baseline levels. CONCLUSION: Almond extracts at these dosages offer little beneficial effect on mucositis severity. Burrowing provides a novel measure of affective state in studies of chemotherapy-induced mucositis.