The relationship between small heat shock proteins and redox homeostasis during acute heat stress in chickens.

As heat stress is a major emerging issue in poultry farming, investigations on the molecular mechanisms of the heat-triggered cellular response in chickens are of special importance. In the present study, 32-day-old Ross 308 broiler chickens were subjected to 37 °C environmental temperature combined with 50% relative humidity for 4 or 8 h respectively. Following sampling, redox parameters such as malondialdehyde (MDA), reduced glutathione (GSH), protein carbonyl levels as well as glutathione peroxidase activity were assessed in liver, spleen, and kidney homogenates. The concentrations of small heat shock proteins (sHSP-s) HSP27, αA- and αB-crystallins were also investigated. Among these organs, the liver was found the most susceptible to heat-provoked oxidative stress, indicated by enhanced lipid peroxidation and rapid activation of protective pathways, including the definite increase of glutathione peroxidase activity and the excessive utilization of αA- and αB-crystallin proteins. Heat-associated decline of protein carbonylation and GSH content was observed in the liver in correlation with the increased involvement of αA- and αB-crystallins in cellular defense, resulting supposedly in an overcompensation mechanism. These data highlight the hepatic sensitivity to acute heat shock, potential adaptation mechanisms, and the specific role of sHSP-s in the restoration of physiologic cell function.

Effect of dietary cereal type, crude protein and butyrate supplementation on metabolic parameters of broilers.

This study investigates the metabolic effects of maize- or wheat-based diets with normal (NP) and lowered (LP) dietary crude protein level [the latter supplemented with limiting amino acids and sodium (n-)butyrate at 1.5 g/kg diet] at different phases of broiler fattening. Blood samples of Ross 308 broilers were tested at the age of 1, 3 and 6 weeks. Total protein (TP) concentration increased in wheat-based and decreased in LP groups in week 3, while butyrate reduced albumin/TP ratio in week 1. Uric acid level was elevated by wheat-based diet in week 1 and by wheat-based diet and butyrate in week 3, but decreased in LP groups in weeks 3 and 6. Aspartate aminotransferase activity was increased by wheat-based diet in week 3, and creatine kinase activity was intensified by LP in weeks 3 and 6. Blood glucose level decreased in wheat-based groups in week 3; however, triglyceride concentration was augmented in the same groups in week 3. No change of glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide and insulin concentration was observed. In conclusion, an age-dependent responsiveness of broilers to dietary factors was found, dietary cereal type was a potent modulator of metabolism, and a low crude protein diet supplemented with limiting amino acids might have a beneficial impact on the growth of chickens.

Effects of butyrate on the insulin homeostasis of chickens kept on maize- or wheat-based diets.

The aim of the present study was to investigate the effects of butyrate as a feed supplement on the expression of insulin signalling proteins as potent regulators of metabolism and growth in Ross 308 broiler chickens fed maize- or wheat-based diets. Both diets were supplemented with non-protected butyrate (1.5 and 3.0 g/kg of diet, respectively) or with protected butyrate (0.2 g/kg of diet); the diet of the control groups was prepared without any additives (control). On day 42 of life, systemic blood samples were drawn for analyses of glucose and insulin concentrations, and tissue samples (liver, gastrocnemius muscle and subcutaneous adipose tissue) were taken for Western blotting examinations. The expression of key insulin signalling proteins (IRß, PKCζ and mTOR) was assessed by semiquantitative Western blotting from the tissues mentioned. The type of diet had a remarkable influence on the insulin homeostasis of chickens. The wheat-based diet significantly increased IRß and mTOR expression in the liver as well as mTOR and PKCζ expression in the adipose tissue when compared to animals kept on a maize-based diet. IRß expression in the liver was stimulated by the lower dose of non-protected butyrate as well, suggesting the potential of butyrate as a feed additive to affect insulin sensitivity. Based on the results obtained, the present study shows new aspects of nutritional factors by comparing the special effects of butyrate as a feed additive and those of the cereal type, presumably in association with dietary non-starch polysaccharide- (NSP-) driven enteric shortchain fatty acid release including butyrate, influencing insulin homeostasis in chickens. As the tissues of chickens have physiologically lower insulin sensitivity compared to mammals, diet-associated induction of the insulin signalling pathway can be of special importance in improving growth and metabolic health.

Pathological consequences, metabolism and toxic effects of trichothecene T-2 toxin in poultry.

Contamination of feed with mycotoxins has become a severe issue worldwide. Among the most prevalent trichothecene mycotoxins, T-2 toxin is of particular importance for livestock production, including poultry posing a significant threat to animal health and productivity. This review article aims to comprehensively analyze the pathological consequences, metabolism, and toxic effects of T-2 toxin in poultry. Trichothecene mycotoxins, primarily produced by Fusarium species, are notorious for their potent toxicity. T-2 toxin exhibits a broad spectrum of negative effects on poultry species, leading to substantial economic losses as well as concerns about animal welfare and food safety in modern agriculture. T-2 toxin exposure easily results in negative pathological consequences in the gastrointestinal tract, as well as in parenchymal tissues like the liver (as the key organ for its metabolism), kidneys, or reproductive organs. In addition, it also intensely damages immune system-related tissues such as the spleen, the bursa of Fabricius, or the thymus causing immunosuppression and increasing the susceptibility of the animals to infectious diseases, as well as making immunization programs less effective. The toxin also damages cellular processes on the transcriptional and translational levels and induces apoptosis through the activation of numerous cellular signaling cascades. Furthermore, according to recent studies, besides the direct effects on the abovementioned processes, T-2 toxin induces the production of reactive molecules and free radicals resulting in oxidative distress and concomitantly occurring cellular damage. In conclusion, this review article provides a complex and detailed overview of the metabolism, pathological consequences, mechanism of action as well as the immunomodulatory and oxidative stress-related effects of T-2 toxin. Understanding these effects in poultry is crucial for developing strategies to mitigate the impact of the T-2 toxin on avian health and food safety in the future.

Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

Cecropin A: investigation of a host defense peptide with multifaceted immunomodulatory activity in a chicken hepatic cell culture.

Introduction: Host defense peptides (HDPs) are increasingly referred to as promising candidates for the reduction of the use of conventional antibiotics, thereby combating antibiotic resistance. As HDPs have been described to exert various immunomodulatory effects, cecropin A (CecA) appears to be a potent agent to influence the host inflammatory response. Methods: In the present study, a chicken primary hepatocyte-non-parenchymal cell co-culture was used to investigate the putative immunomodulatory effects of CecA alone and in inflammatory conditions evoked by polyinosinic-polycytidylic acid (Poly I:C). To examine the viability of the cells, the extracellular lactate dehydrogenase (LDH) activity was determined by colorimetric assay. Inflammatory markers interleukin (IL)-8 and transforming growth factor-ß1 (TGF-ß1) were investigated using the ELISA method, whereas concentrations of IL-6, IL-10, and interferon-γ (IFN-γ) were assayed by Luminex xMAP technology. Extracellular H2O2 and malondialdehyde levels were measured by fluorometric and colorimetric methods, respectively. Results: Results of the lower concentrations suggested the safe application of CecA; however, it might contribute to hepatic cell membrane damage at its higher concentrations. We also found that the peptide alleviated the inflammatory response, reflected by the decreased production of the pro-inflammatory IL-6, IL-8, and IFN-γ. In addition, CecA diminished the levels of anti-inflammatory IL-10 and TGF-ß1. The oxidative markers measured remained unchanged in most cases of CecA exposure. Discussion: CecA displayed a multifaceted immunomodulatory but not purely anti-inflammatory activity on the hepatic cells, and might be suggested to maintain the hepatic inflammatory homeostasis in Poly I:C-triggered immune response. To conclude, our study suggests that CecA might be a promising molecule for the development of new immunomodulatory antibiotic-substitutive agents in poultry medicine; however, there is still a lot to clarify regarding its cellular effects.

Investigation of the effects of T-2 toxin in chicken-derived three-dimensional hepatic cell cultures.

Despite being one of the most common contaminants of poultry feed, the molecular effects of T-2 toxin on the liver of the exposed animals are still not fully elucidated. To gain more accurate understanding, the effects of T-2 toxin were investigated in the present study in chicken-derived three-dimensional (3D) primary hepatic cell cultures. 3D spheroids were treated with three concentrations (100, 500, 1000 nM) of T-2 toxin for 24 h. Cellular metabolic activity declined in all treated groups as reflected by the Cell Counting Kit-8 assay, while extracellular lactate dehydrogenase activity was increased after 500 nM T-2 toxin exposure. The levels of oxidative stress markers malondialdehyde and protein carbonyl were reduced by the toxin, suggesting effective antioxidant compensatory mechanisms of the liver. Concerning the pro-inflammatory cytokines, IL-6 concentration was decreased, while IL-8 concentration was increased by 100 nM T-2 toxin exposure, indicating the multifaceted immunomodulatory action of the toxin. Further, the metabolic profile of hepatic spheroids was also modulated, confirming the altered lipid and amino acid metabolism of toxin-exposed liver cells. Based on these results, T-2 toxin affected cell viability, hepatocellular metabolism and inflammatory response, likely carried out its toxic effects by affecting the oxidative homeostasis of the cells.

Immunomodulatory properties of chicken cathelicidin-2 investigated on an ileal explant culture.

As the threat posed by antimicrobial resistance grows more crucial, the development of compounds that can replace antibiotics becomes increasingly vital. Chicken cathelicidin-2 (Cath-2) belongs to the group of Host Defense Peptides (HDPs), which could provide a feasible solution for the treatment of gastrointestinal infections in poultry. It is a small peptide produced by the heterophil granulocytes of chickens as part of the innate immune response, and its immunomodulatory activity has already been demonstrated in several cell types. In this study, the effects of Cath-2 on the intestinal immune response were examined using ileal explant cultures isolated from chicken. Regarding our results, Cath-2 displayed a potent anti-inflammatory effect as it alleviated the LTA-caused elevation of interleukin (IL)-6 and IL-2 concentrations, and that of the IFN-γ/IL-10 ratio, furthermore, it increased the concentration of IL-10, alleviating the LTA-evoked decreased level of the anti-inflammatory cytokine. Moreover, when applied alone, it elevated the concentrations of IL-6, CXCLi2, and IL-2, providing evidence of its complex immunomodulatory mechanisms. In summary, Cath-2 was able to modulate the immune response of the intestinal wall not only by reducing pro-inflammatory cytokine release, but also through immune stimulation, demonstrating that it has the ability to improve innate immunity via a complex mechanism that may make it a suitable candidate for the control of intestinal infections.

Epigenetic effects of dietary butyrate on hepatic histone acetylation and enzymes of biotransformation in chicken.

The aim of the study was to investigate the in vivo epigenetic influences of dietary butyrate supplementation on the acetylation state of core histones and the activity of drug-metabolising microsomal cytochrome P450 (CYP) enzymes in the liver of broiler chickens in the starter period. One-day-old Ross 308 broilers were fed a starter diet without or with sodium butyrate (1.5 g/kg feed) for 21 days. After slaughtering, nucleus and microsome fractions were isolated from the exsanguinated liver by multi-step differential centrifugation. Histone acetylation level was detected from hepatocyte nuclei by Western blotting, while microsomal CYP activity was examined by specific enzyme assays. Hyperacetylation of hepatic histone H2A at lysine 5 was observed after butyrate supplementation, providing modifications in the epigenetic regulation of cell function. No significant changes could be found in the acetylation state of the other core histones at the acetylation sites examined. Furthermore, butyrate did not cause any changes in the drugmetabolising activity of hepatic microsomal CYP2H and CYP3A37 enzymes, which are mainly involved in the biotransformation of most xenobiotics in chicken. These data indicate that supplementation of the diet with butyrate probably does not have any pharmacokinetic interactions with simultaneously applied xenobiotics.

Feline Uroepithelial Cell Culture as a Novel Model of Idiopathic Cystitis: Investigations on the Effects of Norepinephrine on Inflammatory Response, Oxidative Stress, and Barrier Function.

Feline idiopathic cystitis (FIC) is one of the most common urinary tract disorders in domestic cats. As stress is suggested to play a key role in the pathogenesis of FIC, the effects of norepinephrine (NE) as a stress mediator were investigated on a novel feline primary uroepithelial cell culture, serving as an in vitro model of the disease. The uroepithelial cells gained from the mucosa of the bladder of a euthanized cat were cultured for 6 days and were acutely exposed to NE (10, 100, and 1000 µM) for 1 h. NE increased the metabolic activity of the cultured cells and elevated the extracellular concentrations of the pro-inflammatory mediators interleukin-6 (IL-6) and stromal cell derived factor 1 (SDF-1), confirming that NE can trigger an inflammatory response in the uroepithelium. Cellular protein carbonyl levels were increased by NE exposure, while malondialdehyde and glucose regulated protein 78 concentrations remained unchanged, indicating that NE may provoke the oxidative damage of proteins without inducing lipid peroxidation or endoplasmic reticulum stress. Further, it can be strongly suggested that an acute NE challenge might diminish the barrier function of uroepithelial cells, as reflected by the decreased glycosaminoglycan concentration, claudin-4 protein expression, and reduced TER values of the NE-treated cell cultures. Based on these results, short-term NE exposure mimicking acute stress can provoke an inflammatory response and decrease the barrier integrity of cultured feline uroepithelial cells. Hence, it is highly expected that stress-associated NE release may play an important mediatory role in the pathogenesis of FIC.

Molecular effects of intermittent stress on primary feline uroepithelial cell culture as an in vitro model of feline idiopathic cystitis.

Introduction: The most common cause of feline lower urinary tract disease (FLUDT) is feline idiopathic cystitis (FIC), which is a complex multifactorial disease with symptoms including stranguria, dysuria, hematuria, and pain during urination. The development of these symptoms is often triggered by stress, and in case of chronic stress, these symptoms will many times return. One of the most important stress hormones in the pathogenesis of FIC is norepinephrine (NE), as persistently elevated level of this hormone can be measured in the blood of cats with FIC. However, it is not well understood if recurrently elevated level of NE has any direct effect on urinary bladder, therefore the aim of this study was to investigate the molecular effects of intermittent NE exposure on feline primary uroepithelial cell culture. Methods: Primary uroepithelial cells were gained from the mucosa of the bladder of a euthanized cat, and were cultured for 6 days, then they were exposed to 10, 100, and 1,000 µM NE treatment for 3 × 1 h, including a 1 h long regeneration period between exposures. Results: NE was able to trigger pro-inflammatory response and oxidative stress in the uroepithelial cells by increasing the level of stromal cell derived factor 1 (SDF-1) and H2O2 in cell culture media. In addition, NE increased the permeability of the uroepithelium, since decreased glycosaminoglycan (GAG) concentration, tight junction protein claudin-4 content, and TER values were measured after the NE treatments. Discussion: Based on these results it can be concluded that recurrent stress mimicked by 3×1 h NE treatment has a direct molecular effect on the uroepithelial cells, which leads to inflammatory response, oxidative stress and decreased barrier function of the uroepithelium. Therefore, intermittent release of NE may have an important role in the pathogenesis of FIC and the results of this study may contribute to a better understanding of the development of this illness.

Modulation of the immune response by the host defense peptide IDR-1002 in chicken hepatic cell culture.

IDR-1002, a synthetic host defense peptide (HDP), appears to be a potential candidate for the treatment of bacterial infections and the consequent inflammatory response due to its potent immunomodulatory activity. This is of relevance to the emerging issue of antimicrobial resistance in the farming sector. In this study, the effects of IDR-1002 were investigated on a chicken hepatocyte‒non-parenchymal cell co-culture, and the results revealed that IDR-1002 had complex effects on the regulation of the hepatic innate immunity. IDR-1002 increased the levels of both RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and Macrophage colony stimulating factor (M-CSF), suggesting the peptide plays a role in the modulation of macrophage differentiation, also reflected by the reduced concentrations of interleukin (IL)-6 and IL-10. The pro-inflammatory cytokine release triggered by the bacterial cell wall component lipoteichoic acid (LTA) was ameliorated by the concomitantly applied IDR-1002 based on the levels of IL-6, chicken chemotactic and angiogenic factor (CXCLi2) and interferon (IFN)-γ. Moreover, the production of nuclear factor erythroid 2-related factor 2 (Nrf2), an essential transcription factor in the antioxidant defense pathway, was increased after IDR-1002 exposure, while protein carbonyl (PC) levels were also elevated. These findings suggest that IDR-1002 affects the interplay of the cellular immune response and redox homeostasis, thus the peptide represents a promising tool in the treatment of bacterially induced inflammation in chickens.

Luteolin: A Phytochemical to Mitigate S. Typhimurium Flagellin-Induced Inflammation in a Chicken In Vitro Hepatic Model.

The use of natural feed supplements is an alternative tool to diminish the damage caused by certain bacteria, improving animal health and productivity. The present research aimed to investigate the proinflammatory effect of flagellin released from the bacterial flagellum of Salmonella enterica serovar Typhimurium and to attenuate the induced inflammation with luteolin as a plant-derived flavonoid on a chicken primary hepatocyte-non-parenchymal cell co-culture. Cells were cultured in a medium supplemented with 250 ng/mL flagellin and 4 or 16 µg/mL luteolin for 24 h. Cellular metabolic activity, lactate dehydrogenase (LDH) activity, interleukin-6, 8, 10 (IL-6, IL-8, IL-10), interferon-α, γ (IFN-α, IFN-γ), hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were determined. Flagellin significantly increased the concentration of the proinflammatory cytokine IL-8 and the ratio of IFN-γ/IL-10, while it decreased the level of IL-10, indicating that the model served adequate to study inflammation in vitro. Luteolin treatment at 4 µg/mL did not prove to be cytotoxic, as reflected by metabolic activity and extracellular LDH activity, and significantly reduced the flagellin-triggered IL-8 release of the cultured cells. Further, it had a diminishing effect on the concentration of IFN-α, H2O2 and MDA and restored the level of IL-10 and the ratio of IFN-γ/IL-10 when applied in combination with flagellin. These results suggest that luteolin at lower concentrations may protect hepatic cells from an excessive inflammatory response and act as an antioxidant to attenuate oxidative damage.

Detrimental consequences of tebuconazole on redox homeostasis and fatty acid profile of honeybee brain.

Excessive use of azole fungicides in agriculture poses a potential threat to honeybees and other pollinator insects; however, the detailed effects of these molecules remain largely unclear. Hence, in the present study it was aimed to investigate the acute sublethal effects of tebuconazole on the redox homeostasis and fatty acid composition in the brain of honeybees. Our findings demonstrate that tebuconazole decreased total antioxidant capacity, the ratio of reduced to oxidized glutathione and disturbed the function of key antioxidant defense enzymes along with the induction of lipid peroxidation indicated by increased malondialdehyde levels, while it also altered the fatty acid profile of the brain. The present study highlights the negative impact of tebuconazole on honeybees and contributes to the understanding of potential consequences related to azole exposure on pollinator insects' health, such as the occurrence of colony collapse disorder.

Altered Intestinal Production of Volatile Fatty Acids in Dogs Triggered by Lactulose and Psyllium Treatment.

The intestinal microbiome of dogs can be influenced by a number of factors such as non-starch polysaccharides as well as some non-digestible oligo- and disaccharides. These molecules are only decomposed by intestinal anaerobic microbial fermentation, resulting in the formation of volatile fatty acids (VFAs), which play a central role in maintaining the balance of the intestinal flora and affecting the health status of the host organism. In the present study, the effects of lactulose and psyllium husk (Plantago ovata) were investigated regarding their influence on concentrations of various VFAs produced by the canine intestinal microbiome. Thirty dogs were kept on a standard diet for 15 days, during which time half of the animals received oral lactulose once a day, while the other group was given a psyllium-supplemented diet (in 0.67 and in 0.2 g/kg body weight concentrations, respectively). On days 0, 5, 10 and 15 of the experiment, feces were sampled from the rectum, and the concentration of each VFA was determined by GC-MS (gas chromatography−mass spectrometry). Lactulose administration caused a significant increase in the total VFA concentration of the feces on days 10 and 15 of the experiment (p = 0.035 and p < 0.001, respectively); however, in the case of psyllium supplementation, the concentration of VFAs showed a significant elevation only on day 15 (p = 0.003). Concentrations of acetate and propionate increased significantly on days 5, 10 and 15 after lactulose treatment (p = 0.044, p = 0.048 and p < 0.001, respectively). Following psyllium administration, intestinal acetate, propionate and n-butyrate production were stimulated on day 15, as indicated by the fecal VFA levels (p = 0.002, p = 0.035 and p = 0.02, respectively). It can be concluded that both lactulose and psyllium are suitable for enhancing the synthesis of VFAs in the intestines of dogs. Increased acetate and propionate concentrations were observed following the administration of both supplements; however, elevated n-butyrate production was found only after psyllium treatment, suggesting that the applied prebiotics may exert slightly different effects in the hindgut of dogs. These findings can be also of great importance regarding the treatment and management of patients suffering from intestinal disorders as well as hepatic encephalopathy due to portosystemic shunt.

Protective effects of chicoric acid on polyinosinic-polycytidylic acid exposed chicken hepatic cell culture mimicking viral damage and inflammation.

Virus induced damage triggered by excessive inflammation and free radical production is a major threat in the poultry industry, leading to low productivity even in vaccinated flocks. The purpose of the study was to induce inflammation with the viral double-stranded RNA analog polyinosinic-polycytidylic acid (poly I:C) on chicken primary hepatocyte - non-parenchymal cell co-cultures to investigate the immunomodulatory and cell protectant effects of chicoric acid (CA) in comparison to N-acetylcysteine (NAC). Poly I:C significantly elevated the activity of the cell damage marker, lactate dehydrogenase (LDH) and the concentration of inflammatory cytokines (IL-6, IL-8, IFN-α, IFN-γ and M-CSF) in the culture medium and decreased cellular metabolic activity. CA significantly reduced the elevated LDH and cytokine levels in a dose-dependent manner, moreover, the higher (100 µg/mL) concentration of CA even elevated the level of metabolic activity. In contrast, 10 µg/mL NAC treatment decreased the level of each inflammatory cytokine but did not rectify cell damage or metabolic depression. The results indicate, that CA, present in common plants of the Asteraceae family, proves to be a beneficial hepatoprotective, and along with NAC, an immunomodulatory supplement in vitro under a stimulus mimicking viral infection.

Modulation of Hepatic Insulin and Glucagon Signaling by Nutritional Factors in Broiler Chicken.

Influencing the endocrine metabolic regulation of chickens by nutritional factors might provide novel possibilities for improving animal health and productivity. This study was designed to evaluate the impact of dietary cereal type (wheat-based (WB) vs. maize-based (MB) diets), crude protein level (normal (NP) vs. lowered (LP)), and sodium (n-)butyrate (1.5 g/kg diet) supplementation (vs. no butyrate) on the responsiveness of hepatic glucagon receptor (GCGR), insulin receptor beta (IRß) and mammalian target of rapamycin (mTOR) in the phase of intensive growth of chickens. Liver samples of Ross 308 broiler chickens (Gallus gallus domesticus) were collected on day 21 for quantitative real-time polymerase chain reaction and Western blot analyses. Hepatic GCGR and mTOR gene expressions were up-regulated by WB and LP diet. GCGR and IRß protein level decreased in groups with butyrate supplementation; however, the quantity of IRß and mTOR protein increased in WB groups. Based on these data, the applied dietary strategies may be useful tools to modulate hepatic insulin and glucagon signaling of chickens in the period of intensive growth. The obtained results might contribute to the better understanding of glycemic control of birds and increase the opportunity of ameliorating insulin sensitivity, hence, improving the production parameters and the welfare of broilers.

Immunomodulatory effects of chicken cathelicidin-2 on a primary hepatic cell co-culture model.

Cathelicidin-2 is an antimicrobial peptide (AMP) produced as part of the innate immune system of chickens and might be a new candidate to combat infection and inflammation within the gut-liver axis. Studying the hepatic immune response is of high importance as the liver is primarily exposed to gut-derived pathogen-associated molecular patterns. The aim of the present study was to assess the effects of chicken cathelicidin-2 alone or combined with lipoteichoic acid (LTA) or phorbol myristate acetate (PMA) on cell viability, immune response and redox homeostasis in a primary hepatocyte-non-parenchymal cell co-culture of chicken origin. Both concentrations of cathelicidin-2 decreased the cellular metabolic activity and increased the extracellular lactate dehydrogenase (LDH) activity reflecting reduced membrane integrity. Neither LTA nor PMA affected these parameters, and when combined with LTA, cathelicidin-2 could not influence the LDH activity. Cathelicidin-2 had an increasing effect on the concentration of the proinflammatory CXCLi2 and interferon- (IFN-)γ, and on that of the anti-inflammatory IL-10. Meanwhile, macrophage colony stimulating factor (M-CSF), playing a complex role in inflammation, was diminished by the AMP. LTA elevated IFN-γ and decreased M-CSF levels, while PMA only increased the concentration of M-CSF. Both concentrations of cathelicidin-2 increased the H2O2 release of the cells, but the concentration of malondialdehyde as a lipid peroxidation marker was not affected. Our findings give evidence that cathelicidin-2 can also possess anti-inflammatory effects, reflected by the alleviation of the LTA-triggered IFN-γ elevation, and by reducing the M-CSF production induced by PMA. Based on the present results, cathelicidin-2 plays a substantial role in modulating the hepatic immune response with a multifaceted mode of action. It was found to have dose-dependent effects on metabolic activity, membrane integrity, and reactive oxygen species production, indicating that using it in excessively high concentrations can contribute to cell damage. In conclusion, cathelicidin-2 seems to be a promising candidate for future immunomodulating drug development with an attempt to reduce the application of antibiotics.

Dietary Protected Butyrate Supplementation of Broilers Modulates Intestinal Tight Junction Proteins and Stimulates Endogenous Production of Short Chain Fatty Acids in the Caecum.

Short chain fatty acid (SCFA) butyrate has various beneficial effects on the gut microbiota as well as on the overall health status and metabolism of the host organism. The modulatory role of butyrate on gut barrier integrity reflected by tight junction protein expression has been already described in mammalian species. However, there is limited information available regarding chickens. Therefore, the main aim of this study was to monitor the effects of protected butyrate on claudin barrier protein and monocarboxylate transporter 1 abundance in different gastrointestinal segments of chickens as well as the growth performance of broiler chickens. The effect of protected butyrate on the caecal microbiota was monitored by quantifying the concentrations of total eubacteria and key enzymes of butyrate production. Furthermore, intestinal SCFA concentrations were also measured. Based on the data obtained, protected butyrate increased the overall performance as well as the barrier integrity of various gut segments. Protected butyrate also positively affected the SCFA concentration and composition. These findings provide valuable insight into the complex effects of protected butyrate on broiler gut health, highlighting the beneficial effects in improving intestinal barrier integrity and performance parameters.

Interspecies Comparisons of the Effects of Potential Antiviral 3-Amidinophenylalanine Derivatives on Cytochrome P450 1A2 Isoenzyme.

In vitro models of animals vulnerable to SARS-CoV-2 infection can support the characterization of effective antiviral drugs, such as synthetic inhibitors of the transmembrane protease serine 2 (TMPRSS2). Changes in cytochrome P450 (CYP) 1A2 activities in the presence of the potential TMPRSS2/matriptase inhibitors (MI) were measured using fluorometric and luminescent assays. Furthermore, the cytotoxicity of these inhibitors was evaluated using the MTS method. In addition, 60 min-long microsomal stability assays were performed using an UPLC-MS/MS procedure to elucidate depletion rates of the inhibitors. CYP1A2 was influenced significantly by MI-463 and MI-1900 in rat microsomes, by MI-432 and MI-482 in beagle microsomes, and by MI-432, MI-463, MI-482, and MI-1900 in cynomolgus monkey microsomes. The IC50 values in monkey microsomes were 1.30 ± 0.14 µM, 2.4 ± 1.4 µM, 0.21 ± 0.09 µM, and 1.1 ± 0.8 µM for inhibitors MI-432, MI-463, MI-482, and MI-1900, respectively. The depletion rates of the parent compounds were lower than 50%, independently of the investigated animal species. The host cell factor TMPRSS2 is of key importance for the cross-species spread of SARS-CoV-2. Studies of the in vitro biotransformation of TMPRSS2 inhibitors provide additional information for the development of new antiviral drugs.