A study linking toll-like receptors and irinotecan-induced gastrointestinal mucositis.

Gastrointestinal mucositis (GIM) has become increasingly recognised as a major toxicity of cancer treatment. The efficacy and safe use of irinotecan (a topoisomerase I inhibitor chemotherapeutic drug) is compromised because of GIM. Severe GIM often necessitates dose reduction or treatment discontinuation thus compromising patient survival. In rat studies, irinotecan has been shown to cause apoptosis, histological damage, inflammation and activation of signalling pathways. These signalling pathways include nuclear factor kappa B (NF-κB), tumour necrosis factor (TNF)/stress and toll-like receptors (TLR). The stimulation of TLRs can lead to early or late NF-κB activation, which up-regulates many genes involved in the development of GIM, including pro-inflammatory cytokines. Despite extensive research in this area, there is currently no clinically therapeutic intervention to prevent GIM development following irinotecan administration. Therefore, research focusing on designing therapeutic strategies targeted to specific pathological pathways is greatly needed. In the light of newly emerging roles in inflammatory diseases for TLR2 and TLR4, these receptors have gained significant attention in the development of GIM. TLRs play a role in NF-κB regulation, pro-inflammatory cytokine activation, intestinal inflammation, and regulation of proliferation and apoptosis. Given that these cellular events are key characteristics of GIM, it is suggested that they may be central mediators of the injury process. Recently, it was reported that TLR2 and TLR9 are involved in doxorubicin-induced GIM, and TLR4 is involved in 5-fluorouracil- and methotrexate-induced GIM, providing further evidence as targets for intervention. As such, the overarching objective of this PhD project was to investigate the involvement of TLR2, TLR4 and pro-inflammatory cytokines in irinotecan-induced GIM and the effect of their inhibition, by the antidepressant drug amitriptyline (AMI), on the development of GIM. The first study of this research project investigated the involvement of TLR2, TLR4 and pro-inflammatory cytokines in irinotecan-induced diarrhoea. TLR2, TLR4, interlukin-1 beta (IL-1β), TNF and interleukin-6 (IL-6) mRNA and protein expression was investigated in the colon and jejunum of Dark Agouti rats treated with irinotecan (200 mg/kg intraperitoneally). The expression of each marker (at 72 and 96 h) was compared between rats that developed diarrhoea and rats that did not develop diarrhoea following treatment. These two time points have shown to present maximum damage severity and diarrhoea occurrence, respectively, following irinotecan administration in our rat model. Results showed that mRNA expression of TLR2, TLR4, IL-1β and TNF increased significantly in the colon of rats that developed diarrhoea at 96 h compared to rats that did not develop diarrhoea. TLR2, TLR4 and IL-1β protein expression significantly increased in the apical region of the colonic crypts of the same group compared to the control. This indicated a strong relationship between these inflammatory mediators and severity of mucosal injury. The second study of this project investigated the effect of AMI, a TLR2 and TLR4 inhibitor, in irinotecan-induced GIM. Clinical markers, histological changes, gene expression, and inflammation were compared between Wistar rats treated with irinotecan (125 mg/kg intraperitoneally, administered at 0 h), and combination of irinotecan and AMI (20 mg/kg intraperitoneally; administered at -24, -16 h and 0 h). Rats were then killed at 6, 48 and 96 h post treatment. Results showed that AMI reduced early-onset diarrhoea and colonic epithelial apoptosis in the colon at 6 h post treatment. However, rats were not protected against weight loss, histological damage, distress symptoms, inflammation and late-onset diarrhoea. PCR array analysis showed a significant decrease in caspase-4, IL-1β and IL-1 receptor 2 and increase in interferon γ receptor 1 (INFγ) mRNA expression in rats treated with irinotecan and AMI compared to rats treated with irinotecan alone. AMI was not able to protect rats against GIM but had protective effect from early-onset diarrhoea and anti-apoptotic effects. The study limitation was the increase in toxic symptoms after treatment with both drugs compared to irinotecan alone. This could be related to the ability of AMI to alter irinotecan metabolism through inhibiting the detoxification of the active metabolite SN-38 thus increasing the cytotoxicity of irinotecan. This could explain partially the histological damage, distress symptoms, inflammation and late diarrhoea occurrence after treatment with both drugs. The third study investigated the ability of AMI to inhibit apoptosis induced by SN-38, the active metabolite of irinotecan, in a rat intestinal epithelial cell line, IEC-6. Cells were treated for 24 h with SN-38 (IC₅₀ of apoptosis = 8.7 μM) alone or in combination with AMI (1 μM). At this dose, AMI inhibits TLR2 and TLR4 and is within the therapeutic range. Apoptosis and mRNA expression of TLR2, TLR4 and pro-inflammatory cytokines were investigated. Results showed that treatment with SN-38 was associated with increased mRNA expression of TLR2, TLR4 and pro-inflammatory cytokines compared to AMI treated cells. It also showed that treatment with AMI attenuated apoptosis when administered with SN-38. Treatment with AMI and SN-38 was associated with significant reduction in mRNA expression of TLR2, TLR4 and IL-1β compared to SN-38 treated cells. However, TNF expression increased after treatment with both drugs compared to SN-38 treated cells, suggesting and that an alternative pathway to TLRs is activated which leads to TNF upregulation in IEC-6 cells. In conclusion, this thesis provides an overview of the involvement of TLR2 and TLR4 in irinotecan-induced GIM and the effect of AMI on intestinal injury. Despite the use of AMI for neuropathic and cancer pain, drug interactions should be considered for chemotherapeutic treatment safety and efficacy. Furthermore, the role of TLR2 and TLR4 should be investigated by other specific inhibitors to avoid the “off target” effects of AMI. Findings may then be translated for effective prevention of GIM occurrence clinically.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201

Efficacy of Various Feed Additives on Performance, Nutrient Digestibility, Bone Quality, Blood Constituents, and Phosphorus Absorption and Utilization of Broiler Chickens Fed Low Phosphorus Diet

The present trial was designed to assess the effect of phytase, multi-strain probiotic, Saccharomyces cerevisiae, and fumaric acid on performance, nutrient digestibility, bone physical parameters and mineralization, blood constituents, bone and gut histomorphology, and duodenal phosphorus transporter genes of broiler chickens fed a decreased non-phytate phosphorus (nPP) diet for 5 weeks. A total of 480 broiler chickens were allotted to six dietary groups and eight replicates each: (1) positive control diet with recommended levels of nPP (PC; 0.48, 0.44, and 0.41% in the three feeding phases); (2) negative control diet with a decreased dietary nPP (NC; 0.28, 0.24, and 0.21% in the three feeding phases); (3) NC + 600 FTU/kg phytase (PHY); (4) NC + 0.05% multi-strain probiotic (PRO); (5) NC + 0.2% Saccharomyces cerevisiae (SC); and (6) NC + 0.2% fumaric acid. Growth performance data were recorded weekly, and blood sampling was performed at days 21 and 35 of age. Bone quality traits, gut and tibia histology, nutrient digestibility, and intestinal gene expression analyses were conducted at the end of the trial (35 days of age). Final body weight and total gain at day 35 of age of the broiler chickens fed with the PHY, PRO, and SC diets were greater (p < 0.01) than in NC, where broilers fed with the PRO and PHY diets had higher values and were similar to that of PC. There was a non-significant variation in the cumulative feed intake among the treatment groups. The PHY and PRO groups had better FCR than the PC group (p < 0.05), and FA and SC had an FCR equivalent to that of PC. The PHY and PRO broilers had greater dressing % than the NC group (p < 0.05) and even better than PC. The PHY, PRO, SC, and FA broilers had higher relative weights of spleen and bursa of Fabricius (p < 0.01) than NC. In comparison to NC, the PHY, PRO, and SC groups improved (p < 0.05) CP, CF, Ca, and P digestibility. Greater tibia breaking strength of the low nPP-supplemented groups was shown to be associated with higher tibia ash, Ca, and P concentrations (p < 0.01) and increased (p < 0.001) tibia cortical area thickness. At days 21 and 35 of age, the dietary supplements to low nPP diets reduced (p < 0.05) serum total cholesterol, triglyceride, triiodothyronine, thyroxine, glucose, and alkaline phosphatase levels, while serum Ca and P concentrations were improved (p < 0.05) compared to NC. All supplements led to enhancement (p < 0.01) in villi height and width and villi absorptive surface area when compared with NC and were even comparable to that of PC. The mRNA expression of NaP-IIb was up-regulated (p < 0.001) in the duodenum of PRO and FA broilers at day 35 of age compared with NC, and their expression levels were similar to that of PC, indicating greater P availability. It is concluded that dietary supplementation of PHY, PRO, SC, and FA to a low nPP diet was advantageous and mitigated the negative impacts of P reduction on the growth performance, health, nutrient digestibility, and bone quality of broilers

Impacts of Dietary Lysine and Crude Protein on Performance, Hepatic and Renal Functions, Biochemical Parameters, and Histomorphology of Small Intestine, Liver, and Kidney in Broiler Chickens

The present study aimed to investigate the effects of increasing dietary lysine (Lys) levels with an adequate dietary crude protein (CP) content, as well as the effects of a reduction in dietary CP content with the recommended amino acid (AAs) level, on the performance, blood biochemical parameters, and histomorphology of the duodenum, liver, and kidney in broiler chickens. A total of 500 broiler chickens were randomly distributed into five dietary treatment groups, following a completely randomized design, where, at the beginning, the control group (C) was fed a diet containing the standard CP and Lys levels: 23% CP with 1.44% Lys during the starter period; 21.5% CP with 1.29% Lys during the growing period; and 19.5% CP with 1.16% Lys during the finishing period. The Lys content was increased by 10% above the recommended control basal requirements in the second group (Gr1) and by 20% in the third group (Gr2), while using the same recommended CP percentage as the C group. The fourth group (Gr3) had a 1% lower CP content and the fifth group had a 2% lower CP content than the C group, with the same recommended AA level as the C group. Increasing the Lys content in the Gr1 group improved the broilers’ weight gains (p p > 0.05) the live weight gain, feed intake, or feed conversion ratio (FCR) of the broilers compared with those fed with the C diet. Blood total bilirubin, direct and indirect bilirubin, triglycerides, cholesterol, low-density lipoprotein (LDL), and very LDL were not different among the experimental groups. However, blood aspartate aminotransferase levels were increased (p p p p < 0.05), while the low-CP diets resulted in shorter villi length and width, along with degenerated areas and lymphocytic infiltration. Low dietary CP content induced hepatocyte disorganization and moderate degeneration, along with vacuolated hepatic cells, excessive connective tissue, and lymphocytic infiltration. The cortical regions of the kidney exhibited obvious alterations in the Gr3 and Gr4 groups and large interstitial spaces were found between tubules. Renal tubules in the Gr3 and Gr4 groups were smaller in size and some of these tubules were atrophied. In conclusion, reducing dietary CP levels to 1% or 2% lower than the recommended level did not negatively affect growth performance, inducing minimal influence on the blood metabolic indicators of health status, and resulting in moderate alterations to the histomorphology of the duodenum, liver, and kidney. Furthermore, increasing the Lys content by 10% above the recommended level improved the growth performance, health status, and histomorphology of the duodenum, liver, and kidney in broiler chickens

Impacts of Dietary Lysine and Crude Protein on Performance, Hepatic and Renal Functions, Biochemical Parameters, and Histomorphology of Small Intestine, Liver, and Kidney in Broiler Chickens

The present study aimed to investigate the effects of increasing dietary lysine (Lys) levels with an adequate dietary crude protein (CP) content, as well as the effects of a reduction in dietary CP content with the recommended amino acid (AAs) level, on the performance, blood biochemical parameters, and histomorphology of the duodenum, liver, and kidney in broiler chickens. A total of 500 broiler chickens were randomly distributed into five dietary treatment groups, following a completely randomized design, where, at the beginning, the control group (C) was fed a diet containing the standard CP and Lys levels: 23% CP with 1.44% Lys during the starter period; 21.5% CP with 1.29% Lys during the growing period; and 19.5% CP with 1.16% Lys during the finishing period. The Lys content was increased by 10% above the recommended control basal requirements in the second group (Gr1) and by 20% in the third group (Gr2), while using the same recommended CP percentage as the C group. The fourth group (Gr3) had a 1% lower CP content and the fifth group had a 2% lower CP content than the C group, with the same recommended AA level as the C group. Increasing the Lys content in the Gr1 group improved the broilers&rsquo; weight gains (p &lt; 0.05) during the starter, growing, and finishing periods. Decreasing dietary CP with the standard AA levels (Gr3 and Gr4) did not significantly affect (p &gt; 0.05) the live weight gain, feed intake, or feed conversion ratio (FCR) of the broilers compared with those fed with the C diet. Blood total bilirubin, direct and indirect bilirubin, triglycerides, cholesterol, low-density lipoprotein (LDL), and very LDL were not different among the experimental groups. However, blood aspartate aminotransferase levels were increased (p &lt; 0.05) in the Gr1 and Gr3 groups compared with the other treatment groups. All dietary treatments decreased the serum creatinine levels (p &lt; 0.05) compared with the C group. The Gr2 broilers had greater serum total protein and globulin (p &lt; 0.05) than those receiving the other treatments. Increasing dietary Lys levels resulted in a significant improvement in duodenum villus height and width (p &lt; 0.05), while the low-CP diets resulted in shorter villi length and width, along with degenerated areas and lymphocytic infiltration. Low dietary CP content induced hepatocyte disorganization and moderate degeneration, along with vacuolated hepatic cells, excessive connective tissue, and lymphocytic infiltration. The cortical regions of the kidney exhibited obvious alterations in the Gr3 and Gr4 groups and large interstitial spaces were found between tubules. Renal tubules in the Gr3 and Gr4 groups were smaller in size and some of these tubules were atrophied. In conclusion, reducing dietary CP levels to 1% or 2% lower than the recommended level did not negatively affect growth performance, inducing minimal influence on the blood metabolic indicators of health status, and resulting in moderate alterations to the histomorphology of the duodenum, liver, and kidney. Furthermore, increasing the Lys content by 10% above the recommended level improved the growth performance, health status, and histomorphology of the duodenum, liver, and kidney in broiler chickens

Immune response, hematological traits, biochemical blood parameters, and histological status of laying hens influenced by dietary chitosan-oligosaccharides

ABSTRACT: This experiment aimed to examine the effect of chitosan-oligosaccharides (COS) supplementation in laying hens' diets affected their immune response, hematological characteristics, blood biochemical parameters, and histological status. At the age of 34 wk, 200 laying hens and 20 cocks of the Mandarah chicken strain were allotted into four groups, each consisting of 50 hens and five cocks. The first group acted as a control group, fed on a basal diet. The second, third, and fourth experimental groups each received 0.1, 0.2, and 0.5 g/kg of COS in addition to a base diet. Birds received COS at various dosages had significantly (P ˂ 0.05) increased serum concentration of immunoglobulins, avian influenza, and Newcastle disease antibodies compared with the control birds. Moreover, adding COS at level 0.2 g/kg diet insignificantly enhanced immune response than the rest of the treatment groups. Also, treated birds with COS at different levels had insignificantly improved hematological parameters such as red blood cells, white blood cells, hemoglobin and hematocrit compared to the control group. Birds fed COS at all levels had significantly decreased serum cholesterol, triglycerides, Ca++ and alanine aminotransferase concentrations compared with control birds. In addition, compared to the control group, chitosan-treated birds showed enhanced histological examination of the small intestine, isthmus, and testis, notably in birds given COS at 0.1 g/kg diet compared to other treated birds. Cocks fed COS at all levels improved testicular tissues and increased the number and diameter of seminiferous tubules compared with control birds Morphological examination of the ileum showed increased villi number, height, and crypt depth. It is possible to conclude that laying hens' physiological performance and general health can be effectively improved by using chitosan at 0.1 or 2 g/kg diet levels enhanced immune response