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Background: The gut is an environment in which the immune system closely interacts with a vast number of foreign antigens, both inert such as food and alive, from the viral, bacterial, fungal and protozoal microbiota. Within this environment, germinal centres, which are microanatomical structures where B cells affinity-mature, are chronically present and active., Main Body: The functional mechanism by which gut-associated germinal centres contribute to gut homeostasis is not well understood. Additionally, the role of T cells in class switching to immunoglobulin A and the importance of B cell affinity maturation in homeostasis remains elusive. Here, we provide a brief overview of the dynamics of gut-associated germinal centres, T cell dependency in Immunoglobulin A class switching, and the current state of research regarding the role of B cell selection in germinal centres in the gut under steady-state conditions in gnotobiotic mouse models and complex microbiota, as well as in response to immunization and microbial colonization. Furthermore, we briefly link those processes to immune system maturation and relevant diseases., Conclusion: B cell response at mucosal surfaces consists of a delicate interplay of many dynamic factors, including the microbiota and continuous B cell influx. The rapid turnover within gut-associated germinal centres and potential influences of an early-life window of immune system imprinting complicate B cell dynamics in the gut., (© 2024. The Author(s).)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Animal welfare can be viewed as the result of integrating repeated affective evaluations of success in coping with environmental challenges, i.e., subjective challenge adequacy.  The present work summarizes why established physiological and behavioral welfare parameters are inadequate to assess challenge adequacy. Behavioral tests based on the mood-congruent judgment effect and physiologic parameters based on components of the somatotropic axis are proposed as an alternative. Here, the judgment bias measures an animal's subjective confidence to cope successfully with a challenge, which is in turn modulated by the animal's previous experience. The somatotropic axis incorporates the insulin-like growth factor (IGF) and its binding proteins (IGFBP), which are involved in the regulation of metabolism and growth. First results indicate that in particular IGF-1 and IGFBP-3 react with higher latency and higher inertness to short-term stressful events than established physiological stress parameters. Before these indicators can be utilized for overall welfare assessment, further validation studies are necessary that provide more insights into how repeatable the measurements are under different conditions and which other factors may confound the measures.

Objectives: Several clinical disease activity indices (DAIs) have been developed to noninvasively assess mucosal healing in pediatric Crohn's disease (CD). However, their clinical application can be complex. Therefore, we present a new way to identify the most informative biomarkers for mucosal inflammation from current markers in use and, based on this, how to obtain an easy-to-use DAI for clinical practice. A further aim of our proof-of-concept study is to demonstrate how the performance of such a new DAI can be compared to that of existing DAIs., Methods: The data of two independent study cohorts, with 167 visits from 109 children and adolescents with CD, were evaluated retrospectively. A variable selection based on a Bayesian ordinal regression model was applied to select clinical or standard laboratory parameters as predictors, using an endoscopic outcome. The predictive performance of the resulting model was compared to that of existing pediatric DAIs., Results: With our proof-of-concept dataset, the resulting model included C-reactive protein (CRP) and fecal calprotectin (FC) as predictors. In general, our model performed better than the existing DAIs. To show how our Bayesian approach can be applied in practice, we developed a web application for predicting disease activity for a new CD patient or visit., Conclusions: Our work serves as a proof-of-concept, showing that the statistical methods used here can identify biomarkers relevant for the prediction of a clinical outcome. In our case, a small number of biomarkers is sufficient, which, together with the web interface, facilitates the clinical application. However, the retrospective nature of our study, the rather small amount of data, and the lack of an external validation cohort do not allow us to consider our results as the establishment of a novel DAI for pediatric CD. This needs to be done with the help of a prospective study with more data and an external validation cohort in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Wirthgen, Weber, Kubickova-Weber, Schiller, Schiller, Radke and Däbritz.)

Background: Complications of Crohn's disease (CD) often impair patients' quality of life. It is necessary to predict and prevent these complications (surgery, stricturing [B2]/penetrating [B3] disease behavior, perianal disease, growth retardation and hospitalization). Our study investigated previously suggested and additional predictors by analyzing data of the CEDATA-GPGE registry., Methods: Pediatric patients (< 18 years) diagnosed with CD with follow up data in the registry were included in the study. Potential risk factors for the selected complications were evaluated by performing Kaplan-Meier survival curves and cox regression models., Results: For the complication surgery, the potential risk factors older age, B3 disease, severe perianal disease and initial therapy with corticosteroids at the time of diagnosis were identified. Older age, initial therapy with corticosteroids, low weight-for-age, anemia and emesis predict B2 disease. Low weight-for-age and severe perianal disease were risk factors for B3 disease. Low weight-for-age, growth retardation, older age, nutritional therapy, and extraintestinal manifestations (EIM) of the skin were identified as risk factors for growth retardation during the disease course. High disease activity and treatment with biologicals were predictors for hospitalization. As risk factors for perianal disease, the factors male sex, corticosteroids, B3 disease, a positive family history and EIM of liver and skin were identified., Conclusion: We confirmed previously suggested predictors of CD course and identified new ones in one of the largest registries of pediatric CD patients. This may help to better stratify patients' according to their individual risk profile and choose appropriate treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Klamt, de Laffolie, Wirthgen, Stricker, Däbritz and the CEDATA-GPGE study group.)

Sialylated milk oligosaccharides and glycoconjugates have several positive effects on the mucosal barrier, the gut microbiome, and an effective immune system. For this reason, they are important biomolecules for mammary gland health and optimal development of offspring. In milk, the major sialic acid, N-acetylneuraminic acid (Neu5Ac), can be attached as monosialyl-residues or as polymers. To investigate the sialylation processes during lactation of German Holstein cows, we analyzed udder tissue in addition to milk at different time points of lactation. The analysis of the milk samples revealed that both the levels of Neu5Ac and its polymer, polysialic acid (polySia), rapidly decreased during the first three days of lactation, and a high interindividual variance was observed. In mature milk, however, the sialylation status remains relatively constant. The results indicate that mammary gland epithelial cells are one source for milk polySia, since immunohistochemistry of udder tissue exhibited strong polySia staining in these cells. Furthermore, both polysialyltransferases, ST8SiaII and ST8SiaIV, are expressed. Based on known functions of monosialyl residues and polySia, we discuss the potential impact of these biomolecules and the consequences of the heterogeneous sialylation status of milk in relation to udder health and offspring health.

Background: Monocyte-derived macrophages or dendritic cells are of increasing interest for cellular therapeutic products to treat inflammation-related diseases and cancer. However, the isolation method and the culture conditions applied influence the functionality of cells. For some approaches, the adhesion-induced differentiation into macrophages must be prevented to maintain functions attributed to circulating monocytes. The effects of the isolation method on the functionality of non-adherent peripheral monocytes have not yet been investigated., Methods: The present study examines the impact of the isolation method on cell viability, growth, metabolism, inflammation-induced cytokine response, migratory capacity, and adherence of non-adherent human peripheral monocytes. The monocytes were isolated by magnetic sorting using either positive or negative selection and cultured in cell-repellent plates., Results: The purity and yield of monocytes were higher after positive selection. However, the adherence and migratory capacity, cytokine response, and metabolic activity were decreased compared to negatively selected monocytes. The impaired functionality presented in combination with cell shrinking, thus, indicates the start of cell viability loss. Negatively selected non-adherent monocytes showed no impairment in functionality, and the viability remained high. In conclusion, this approach is better suited for conducting ex vivo modifications of monocytes prior to the intended experimental setup or therapeutic application.

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented., Competing Interests: The authors declare no conflict of interest.

Background: Animal model studies revealed that the application of 1-methyltryptophan (1-MT), a tryptophan (TRP) analog, surprisingly increased plasma levels of the TRP metabolite, kynurenic acid (KYNA). Under inflammatory conditions, KYNA has been shown to mediate various immunomodulatory effects. Therefore, the present study aims to confirm and clarify the effects of 1-MT on TRP metabolism in mice as well as in humans. Methods: Splenocytes from Balb/C or indoleamine 2,3-dioxygenase knockout ( IDO1 -/- ) mice or whole human blood were stimulated with 1-MT for 6, 24, or 36 h. C57BL/6 mice received 1-MT in drinking water for 5 days. Cell-free supernatants and plasma were analyzed for TRP and its metabolites by tandem mass spectrometry (MS/MS). Results: 1-MT treatment induced an increase in TRP and its metabolite, KYNA in Balb/C, IDO -/- mice, and in human blood. Concurrently, the intermediate metabolite kynurenine (KYN), as well as the KYN/TRP ratio, were reduced after 1-MT treatment. The effects of 1-MT on TRP metabolites were similar after the in vivo application of 1-MT to C57BL/6 mice. Conclusions: The data indicate that 1-MT induced an increase of KYNA ex vivo and in vivo confirming previously described results. Furthermore, the results of IDO -/- mice indicate that this effect seems not to be mediated by IDO1. Due to the proven immunomodulatory properties of KYNA, a shift toward this branch of the kynurenine pathway (KP) may be one potential mode of action by 1-MT and should be considered for further applications., (Copyright © 2020 Wirthgen, Leonard, Scharf and Domanska.)

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO2) are the key enzymes of tryptophan (TRP) metabolism in the kynurenine pathway (KP). Both enzymes function as indicators of immunosuppression and poor survival in cancer patients. Direct or indirect targeting of either of these substances seems thus reasonable to improve therapy options for patients. In this study, glioblastoma multiforme (GBM) as well as head and neck squamous cell carcinomas (HNSCC) were examined because of their different mechanisms of spontaneous and treatment-induced immune escape. Effects on gene expression and protein levels were examined. Accompanying assessment of TRP metabolites from treated GBM cell culture supernatants was conducted. Our results show a heterogeneous and inversely correlated expression profile of TRP-metabolizing genes among GBM and HNSCC cells, with low, but inducible IDO1 expression upon IFNγ treatment. TDO2 expression was higher in GBM cells, while genes encoding kynurenine aminotransferases were mainly confined to HNSCC cells. These data indicate that the KP is active in both entities, with however different enzymes involved in TRP catabolism. Upon treatment with Temozolomide, the standard of care for GBM patients, IDO1 was upregulated. Comparable, although less pronounced effects were seen in HNSCC upon Cetuximab and conventional drugs (i.e., 5-fluorouracil, Gemcitabine). Here, IDO1 and additional genes of the KP ( KYAT1, KYAT2 , and KMO ) were induced. Vice versa, the novel yet experimental cyclin-dependent kinase inhibitor Dinaciclib suppressed KP in both entities. Our comprehensive data imply inhibition of the TRP catabolism by Dinaciclib, while conventional chemotherapeutics tend to activate this pathway. These data point to limitations of conventional therapy and highlight the potential of targeted therapies to interfere with the cells' metabolism more than anticipated., (Copyright © 2020 Riess, Schneider, Kehnscherper, Gesche, Irmscher, Shokraie, Classen, Wirthgen, Domanska, Zimpfer, Strüder, Junghanss and Maletzki.)

The complex nature of inflammatory bowel disease (IBD) often results in treatment failure for many patients. With some patients cycling through multiple therapies before achieving a sustained period of remission, the ability to predict a patient's response to therapeutics could decrease the time from active disease to clinical remission and mucosal healing. The prospect of such individualized treatment of IBD would be aided by accurate biomarkers, both fecal and serological, which have to date shown value as indicators of IBD activity. Here we review the utility of generic biomarkers for inflammation or mucosal healing, such as calprotectin, C-reactive protein (CRP), and fecal hemoglobin (fHb) as predictors of response to treatment of IBD. We further provide a deeper insight into the utility of monitoring the thiopurine treatment by thiopurine metabolites or alternative hematologic parameters. In light of multiple recent publications of biomarkers and biological therapy, our focus in this review is predicting response to thiopurine treatment only, that is, Azathioprine and 6-Mercaptopurine., (Copyright © 2020 Cornish, Wirthgen and Däbritz.)

Immunooncology is still a growing area in cancer therapy. Drugs within this therapeutic approach do not directly target/attack the tumor but interfere with immune checkpoints and target or reprogram key metabolic pathways critical for anti-cancer immune defense. Indolamine 2,3-dioxygenase 1 (IDO1) and the tryptophan (TRP)-kynurenine pathway were identified as critical mechanisms in cancer immune escape and their inhibition as an approach with promising therapeutic potential. Particularly, a multitude of IDO1 inhibiting tryptophan analogs are widely applied in several clinical trials. However, this therapy results in a variety of implications for the patient's physiology. This is not only due to the inhibition of an enzyme important in almost every organ and tissue in the body but also because of the general nature of the inhibitor as an analog of a proteinogenic amino acid as well as the initiation of cellular detoxification known to affect inflammatory pathways. In this review we provide a deeper insight into the physiological consequences of an IDO1 inhibiting therapy based on TRP related molecules. We discuss potential side and off-target effects that contribute to the interpretation of unexpected positive as well as negative results of ongoing or discontinued clinical studies while we also highlight the potential of these inhibitors independent of the IDO1 signaling pathway.

An enhanced indoleamine 2,3-dioxygenase 1 (IDO1) activity is associated with an increased mortality risk in sepsis patients. Thus, the preventive inhibition of IDO1 activity may be a promising strategy to attenuate the severity of septic shock. 1-methyltryptophan (1-MT) is currently in the interest of research due to its potential inhibitory effects on IDO1 and immunomodulatory properties. The present study aims to investigate the protective and immunomodulatory effects of 1-methyltryptophan against endotoxin-induced shock in a porcine in vivo model. Effects of 1-MT were determined on lipopolysaccharide (LPS)-induced tryptophan (TRP) degradation, immune response and sickness behaviour. 1-MT increased TRP and its metabolite kynurenic acid (KYNA) in plasma and tissues, suppressed the LPS-induced maturation of neutrophils and increased inactivity of the animals. 1-MT did not inhibit the LPS-induced degradation of TRP to kynurenine (KYN)-a marker for IDO1 activity-although the increase in KYNA indicates that degradation to one branch of the KYN pathway is facilitated. In conclusion, our findings provide no evidence for IDO1 inhibition but reveal the side effects of 1-MT that may result from the proven interference of KYNA and 1-MT with aryl hydrocarbon receptor signalling. These effects should be considered for therapeutic applications of 1-MT.

Insulin-like growth factors (IGFs) play a critical role in fetal growth, and components of the IGF system have been associated with fetal growth restriction in women. In human pregnancy, the proteolytic cleavage of insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-4, releases free IGF for respective action at the tissue level. The aim of the present study was to determine IGFBP-2, IGFBP-3, and IGFBP-4 concentrations by Western ligand blotting during pregnancy until day 100 in cows and to compare these concentrations with those of non-pregnant cows and cows undergoing embryonic/fetal mortality. Therefore, two study trials (I and II) and an in vitro study were conducted. In study I, 43 cows were not pregnant, 34 cows were pregnant, and 4 cows were undergoing fm. In study II, 500 cows were examined, and 7 cases of pregnancy loss between days 24-27 and 34-37 after artificial insemination (AI, late embryonic mortality; em) and 8 cases of pregnancy loss between days 34-37 and 54-57 after AI (late embryonic mortality and early fetal mortality; em/fm) were defined from the analyses of 30 pregnant and 20 non-pregnant cows randomly selected for insulin-like growth factor 1 and IGFBP analyses. In vitro serum from pregnant ( n  = 3) and non-pregnant ( n  = 3) cows spiked after incubation with recombinant human (rh) IGFBP-4 for 24 h, and IGFBP-4 levels were analyzed before and after incubation to detect proteolytic degradation. The IGFBP-2, -3, and -4 concentrations did not decline during early pregnancy in cows, while IGFBP-4 concentrations were comparable between pregnant and non-pregnant cows, irrespective of low proteolytic activity, which was also demonstrated in cows. Interestingly, cows with em or fm showed distinct IGFBP patterns. The IGFBP-2 and -3 concentrations were higher ( P  < 0.05) in cows with fm compared to pregnant. The IGFBP-4 levels were significantly higher in cows developing fm. Thus, distinct differences in the circulating IGFBP concentrations could be associated with late embryonic and early fetal losses in cattle.

The insulin-like growth factors IGF-1 and IGF-2 play important roles in the growth, development, and metabolism of teleost fish. We isolated cDNA sequences of igf1, and igf2 genes from maraena whitefish. We quantified the mRNA and protein expressions of IGFs in different tissues of marketable juvenile maraena whitefish. Moreover, we analyzed the gene expression profiles during maraena whitefish development from unfertilized egg to fingerling and examined the effect of incubation temperature on igf1, and igf2 gene expression during embryonic and early larval development. Transcripts encoding IGF-1 or IGF-2 were detected in all tested tissues, with the greatest abundance in the liver. We measured higher igf2 than igf1 copy numbers in all tissues and at all developmental stages examined, even at advanced juvenile stages. Using the Western blot technique, we demonstrated that several isoforms of IGF-1 are expressed in the liver and gills but not in muscle tissue, indicating tissue-specific protein expression of IGF-1. We observed an accelerated embryonic development with increasing temperature, resulting in shortened hatching periods. Out of the three tested temperatures, we observed the highest hatching rate, larval hatching size, and larval growth at 6 °C. At 9 °C, hatching rate, larval hatching size and larval growth were reduced compared to the values we observed at 4 °C and 6 °C, since incubation temperature might have exceeded the optimum. To our knowledge, our data show for the first time that both igf1 and igf2 expression were upregulated due to elevated incubation temperature within embryonic development of fish. Further, we found significantly higher igf expression for the best-developing larvae (6 °C group) at specific life stages of maraena whitefish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Functional genome analysis usually is performed on the level of genotype-phenotype interaction. However, phenotypes also depend on the relations between genomes and environment. In our experimental system, we observed differential response to environmental factors defined by different conditions of husbandry in a semi-barrier unit or in a SPF (specific pathogen free) barrier unit, which resulted in partial reversal of phenotypes previously observed under semi-barrier conditions. To provide an update of basic phenotypes in unselected and randomly mated controls (DUC) and long-term selected DUhTP (Dummerstorf high treadmill performance) mice in the SPF facility, we compared growth parameters, reproductive performance, the accretion of muscle and fat mass, physical activity, and running performance as well as food intake in all experimental groups. For selected parameters, the comparative analysis spans more than 30 generations. In DUC mice, under SPF conditions a more than threefold (P < 0.0001) higher subcutaneous fat mass, higher muscle mass by about 25% (P < 0.0001), but lower epididymal fat mass in DUhTP mice by about 20% (P < 0.0001) were observed. In SPF husbandry, body weight increased to a stronger extent in adult DUC mice (≈ 20%; P < 0.0001) than in DUhTP mice (≈ 8%; P = 0.001). The concentrations of IGF-1 and IGFBPs in the serum as well as the liver weights were similar in all experimental groups, indicating growth effects independent of the somatotropic axis. Under SPF conditions the litter size at birth increased in DUC mice (P < 0.001) but not in DUhTP mice. The differential effect of husbandry on body weights at day 21 and concentrations of triglycerides in the serum of our model were due to the different diets used in the semi-barrier and in the SPF facility. Our results demonstrate differential trait response to environmental factors resulting in partial phenotype conversion in our experimental system. The existence of conditional phenotypes as a result of genotype-environment interactions points to the importance of environmental factors in functional genome analysis.

In previous work using market-weight pigs, we had demonstrated that insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs) are regulated during shipment characterized by changing conditions of stress due to loading or unloading, transportation, lairage, and slaughter. In addition, we found in a previous study that IGFBP-2 concentrations were lower in pigs transported for longer periods of time. Therefore, we performed a more detailed study on the effects of transport duration and season on the plasma concentrations of IGFs and IGFBPs in adult pigs. For the study, exsanguination blood was collected from 240 market-weight barrows that were transported for 6, 12, or 18 h in January or July. IGF-I and -II were detected using commercial ELISAs whereas IGFBPs were quantified by quantitative Western ligand blotting. In addition, established markers of stress and metabolism were studied in the animals. The results show that plasma concentrations of IGFBP-3 were significantly reduced after 18 h of transport compared to shorter transport durations (6 and 12 h; p  < 0.05). The concentrations of IGF-I in plasma were higher ( p  < 0.001) in pigs transported 12 h compared to shorter or longer durations. Season influenced plasma concentrations of IGFBP-3 and IGF-II ( p  < 0.05 and p  < 0.01, respectively). Neither transport duration nor differential environmental conditions of winter or summer had an effect on glucocorticoids, albumin, triglycerides, or glucose concentrations ( p  > 0.05). However, low-density lipoprotein concentrations decreased after 18 h compared to 6 h of transport ( p  < 0.05), whereas high-density lipoprotein concentrations were higher ( p  < 0.05) in pigs transported for 12 or 18 h compared to those transported for only 6 h. Our findings indicate differential regulation of IGF-compounds in response to longer transport duration or seasonal changes and support current evidence of IGFs and IGFBPs as innovative animal-based indicators of psycho-social or metabolic stress in pigs.

Tryptophan metabolites are known to participate in the regulation of many cells of the immune system and are involved in various immune-mediated diseases and disorders. Kynurenic acid (KYNA) is a product of one branch of the kynurenine pathway of tryptophan metabolism. The influence of KYNA on important neurophysiological and neuropathological processes has been comprehensively documented. In recent years, the link of KYNA to the immune system, inflammation, and cancer has become more apparent. Given this connection, the anti-inflammatory and immunosuppressive functions of KYNA are of particular interest. These characteristics might allow KYNA to act as a "double-edged sword." The metabolite contributes to both the resolution of inflammation and the establishment of an immunosuppressive environment, which, for instance, allows for tumor immune escape. Our review provides a comprehensive update of the significant biological functions of KYNA and focuses on its immunomodulatory properties by signaling via G-protein-coupled receptor 35 (GPR35)- and aryl hydrocarbon receptor-mediated pathways. Furthermore, we discuss the role of KYNA-GPR35 interaction and microbiota associated KYNA metabolism for gut homeostasis.

Cytokines are required for normal growth and development of the mammary gland and TGF-β prominently represents an established effector of apoptosis, e.g., during involution of the mammary gland. By the control of intracellular signaling pathways, including JAK/STAT, MAPK, PI-3K, and NF-κB, cytokines efficiently regulate cell proliferation and inflammation in the breast. Therefore, cytokines are discussed also in a context of malignant mammary growth. As a group of tissue hormones produced by somatic cells or by cells from the immune system, cytokines are defined by their immunomodulatory potential. Over the past 40 years, multiple cytokines were identified in colostrum and milk. Importantly, cytokines derived from mammary secretions after birth are required for maturation of the immune system in the developing gastrointestinal tract from the suckling. Moreover, recent studies have further assessed the particular interactions between probiotic bacterial strains and cytokines. In light of the increasing prevalence of inflammatory diseases of the gastrointestinal system, the effects of probiotic microorganisms during milk fermentation may have immunotherapeutic potential in the future., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The acceptance of animal products is increasingly associated with standardized animal welfare, which relates to appropriate animal husbandry from birth to slaughter. In particular, shipment to the slaughterhouse is considered as a critical process exposing the animals to a number of, in part severe, stressors. New biomarkers may be useful for the assessment of animal welfare. The IGF-system has been assessed in a commercial pig transport in conjunction with established markers of stress response. Furthermore, the effect of repeated restraint as an experimental model for repeated acute stress was investigated. During shipment from farm to slaughterhouse, plasma concentrations of IGFBP-3 and IGFBP-2 were significantly reduced (p < 0.01). After shipment, the plasma concentrations of IGFBP-5, glucocorticoids and IL-2 increased but decreased after lairage (p < 0.05) whereas IGF-1 decreased after shipment (p < 0.01). Repeated acute stress increased concentrations of IGFBP-3 and IGF-1 in exsanguination blood (p < 0.05). Differential IGF- signatures can indicate altered endocrine or metabolic control and thus contain complex animal-related information. The somatotropic axis may be of particular interest when established biomarkers such as cortisol, glucose, or lactate cannot be used for the assessment of animal stress or welfare.

Circulating insulin-like growth factor 1 (IGF-1) may be directly associated with colorectal cancer risk, and IGF binding protein 3 (IGFBP-3) is one of the most abundantly expressed binding proteins in various cancers. Calcium intakes, primarily from food, have been directly associated with circulating IGF-1, but whether supplemental calcium affects IGF-1 and IGFBP-3 is unknown. We tested the effects of 1.0 and 2.0 g of supplemental elemental calcium daily on circulating IGF-1 and IGFBP-3 concentrations in colorectal adenoma patients in a randomized, double-blinded, placebo-controlled clinical trial (n = 193). IGF-1 and IGFBP-3 were quantified using enzyme-linked immunoassay and quantitative Western ligand blot, respectively. We also assessed cross-sectional associations of these biomarkers with participants' baseline characteristics. We found no appreciable effect of calcium relative to placebo on circulating IGF-1, IGFBP-3, or the IGF-1:IGFBP-3 molar ratio. Mean IGF-1 concentrations were 11.1% higher in those with greater milk intakes (P = 0.05). Mean IGF-1 and IGFBP-3 concentrations were, respectively, 18.0% (P = 0.003) and 16.5% (P = 0.01) higher in men and were monotonically lower with increasing age (both P = 0.01). IGFBP-3 was 17.7% higher among those with higher relative to no alcohol consumption (P = 0.04). While these results support previous findings that IGF-1 concentrations are higher with greater milk intakes, and IGF-1 and IGFBP-3 concentrations differ according to sex and age, they provide no evidence to suggest that supplemental calcium appreciably affects circulating IGF-1, IGFBP-3, or the IGF-1:IGFBP-3 molar ratio in sporadic colorectal adenoma patients., (© 2017 Wiley Periodicals, Inc.)

IGFs and IGF-binding proteins (IGFBPs) are abundantly present in milk and in dairy products. Compared to the IGFs, the IGFBP have received less attention in milk, although truncated IGFBPs and IGFBP-glycosylation have been described in milk. Thereby, complex control of local IGF-effects can be assumed on the levels of IGFBPs, proteases, and protease inhibitors. The present review collects the current knowledge both on presence and regulation of IGFs and IGFBPs in milk particularly from dairy animal species. As a rule higher levels of IGF-I, IGF-II, and IGFBPs are measured around parturition if compared to later time-points of lactation. In all farm animal species included in this review, it is found that the relative abundancies of IGFBPs in milk and serum are similar, with IGFBP-3 and -2 characterized by higher concentrations if compared to IGFBP-4 or -5. The concentrations of IGFs and IGFBPs in milk or dairy products can be altered by hormones, dairy processing, or fermentation. Because milk can be used for non-invasive biomarker research, quality management, and health monitoring, we discuss novel directions of IGF-analysis and potential on-site biomarker research in milk., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The pre-weaning period is critical for calf health and growth, and intensive milk feeding programs may assist postnatal development by improving body growth and organ maturation. The aim of the present work was to study the effects of ad libitum milk replacer (MR) feeding on the growth, metabolic adaptation, health, and immune status of newborn calves. Twenty-eight newborn Holstein and Holstein x Charolais crossbred calves were fed ad libitum (ADLIB) or in restricted amounts (6 liters per day; RES) during the first five weeks of life. The MR intake in the ADLIB treatment was gradually reduced at weeks 6 and 7, and all calves then received 6 liters of MR per day until day 60. Blood samples were collected to measure the plasma concentrations of metabolites, insulin, insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP), immunoglobulins, and acute phase proteins. The expression of mRNA associated with both the somatotropic axis and gluconeogenic enzymes was measured in the liver on day 60. Intensive feeding improved MR intake and growth in ADLIB without influencing concentrate intake. Carcass weight, perirenal fat, and muscle mass were greater in ADLIB. Plasma concentrations of glucose, triglycerides, insulin, and IGF-I were greater, whereas plasma concentrations of β-hydroxybutyrate, total protein, albumin, urea, IGFBP-2 and -4, and fibrinogen were lower at distinct time points in ADLIB. The hepatic mRNA expression of cytosolic phosphoenolpyruvate carboxykinase was greater in ADLIB. Most metabolic and endocrine differences occurred during the MR feeding period, but a slightly greater concentrate intake was associated with increased plasma IGF-I and insulin at the end of the study. The immune and health status of the calves were not affected by MR feeding. However, increased plasma fibrinogen in the RES group suggested differences in the acute phase response., Competing Interests: The authors have declared that no competing interests exist.

Dummerstorf marathon mice (DUhTP) are characterized by increased accretion of peripheral body fat with fast mobilization in response to mild physical activity if running wheels were included in their home cages. The obese phenotype coincides with elevated hepatic lipogenesis if compared to unselected controls. We now asked, if microRNA (miRNA) species present in the liver may contribute to the obese phenotype of DUhTP mice and if miRNAs respond to mild physical activity in our mouse model. Total RNA was extracted from livers of sedentary or physically active marathon mice and controls and analyzed by array hybridization or real-time PCR using locked nucleic acid probes. Pathway analysis of altered miRNA concentrations identified fatty acid biosynthesis as the most important target for the effects of miRNAs in the liver. A miRNA signature consisting of miR-21, 27, 33, 122, and 143 was present at higher abundance (p < 0.01) in the liver of sedentary or active DUhTP mice indicating involvement of miRNAs with hepatic lipogenesis. Furthermore, in protein lysates from the liver of DUhTP mice, significantly reduced concentrations of total and phosphorylated AKT and lower levels of phosphorylated AMPK were found (p < 0.05). Our results indicate active involvement of miRNAs in the control of hepatic energy metabolism and discuss effects on signal transduction as a potentially direct effect of miR-143 in the liver of DUhTP mice.

Increased activity of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is associated with immunological and neurological disorders, and inhibition of its enzyme activity could be a therapeutic approach for treatment of these disorders. The aim of the present study was to establish a large animal model to study the accumulation of the potential IDO inhibitor 1-methyltryptophan (1-MT) in blood and different organs of domestic pigs (Sus scrofa domestica). Because 1-MT has not been previously evaluated in pigs, the pharmacokinetics of a single subcutaneous 1-MT application was investigated. Based on this kinetic study, a profile for repeated 1-MT applications over a period of five days was simulated and tested. The results show that a single administration of 1-MT increases its concentrations in blood, with the maximum concentration being obtained at 12 h. Repeated daily injections of 1‑MT generated increasing plasma concentrations followed by a steady-state after two days. Twelve hours after the final application, accumulation of 1-MT was observed in the brain and other organs, with a substantial variability among various tissues. The concentrations of 1-MT measured in plasma and tissues were similar to, or even higher, than those of tryptophan. Our data indicate that repeated subcutaneous injections of 1-MT provide a suitable model for accumulation of 1-MT in plasma and tissues of domestic pigs. These findings provide a basis for further research on the immunoregulatory functions of IDO in a large animal model.

The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P<0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P<0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P<0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Impaired growth is often associated with an extension of lifespan. However, the negative correlation between somatic growth and life expectancy is only true within, but not between, species. This can be observed because smaller species have, as a rule, a shorter lifespan than larger species. In insects and worms, reduced reproductive development and increased fat storage are associated with prolonged lifespan. However, in mammals the relationship between the dynamics of reproductive development, fat metabolism, growth rate, and lifespan are less clear. To address this point, female transgenic mice that were overexpressing similar levels of either intact (D-mice) or mutant insulin-like growth factor-binding protein-2 (IGFBP-2) lacking the Arg-Gly-Asp (RGD) motif (E- mice) were investigated. Both lines of transgenic mice exhibited a similar degree of growth impairment (-9% and -10%) in comparison with wild-type controls (C-mice). While in D-mice, sexual maturation was found to be delayed and life expectancy was significantly increased in comparison with C-mice, these parameters were unaltered in E-mice in spite of their reduced growth rate. These observations indicate that the RGD-domain has a major influence on the pleiotropic effects of IGFBP-2 and suggest that somatic growth and time of sexual maturity or somatic growth and life expectancy are less closely related than thought previously., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

The family of steroid hormones is quite attractive for the approach of phenotype monitoring in farm animals. Therefore, we developed a new protocol for the quantitative analysis of natural steroids in follicular fluid from dairy cows. The corresponding steroid profile, which consists of progesterone, corticosterone, hydrocortisone, testosterone, and androstenedione covering three distinct steroid classes, was determined by LC/MS. Quantification is achieved by use of steroid standards diluted in steroid-free follicular fluid as calibrators. Thus, the new protocol does not require deuterated standards. In order to correct for conditional performance of the analytical system we have used dexamethasone as an internal standard. The method was validated according to EMA guidelines. Within- and between-day variations were below 20% for most parameters assessed. All steroids assessed had lower limits of quantification in the range of 2.1 to 4.4ng/ml. We have established a simple and sensitive analytical system in order to step towards a broader and cost-efficient phenotyping analysis in follicular fluid from dairy cows., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recent studies suggest that insulin-like growth factor-binding protein-2 (IGFBP-2) affects both growth and metabolism. Whereas negative growth effects are primarily due to negative interference with IGF-I, the mechanisms for metabolic interference of IGFBP-2 are less clear. As we demonstrate, overexpression of IGFBP-2 in transgenic mice is correlated with a decelerated clearance of blood glucose after oral administration. IGFBP-2 carries an integrin-binding domain (RGD motif), which has been shown to also mediate IGF-independent effects. We thus asked if higher serum levels of IGFBP-2 without an intact RGD motif would also partially block blood glucose clearance after oral glucose application. In fact, transgenic mice overexpressing mutated IGFBP-2 with higher levels of IGFBP-2 carrying an RGE motif instead of an RGD were not characterized by decelerated glucose clearance. Impaired glucose tolerance was correlated with lower levels of GLUT4 present in plasma membranes isolated from muscle tissues after glucose challenge. At the same time, activation of TBC1D1 was depressed in mice overexpressing wild-type but not mutated IGFBP-2. Although we do not have reason to assume altered activation of IGF-I receptor or PDK1/Akt activation in both models, we have identified increased levels of integrin-linked kinase and focal adhesion kinase dependent on the presence of the RGD motif. From our results we conclude that impaired glucose clearance in female IGFBP-2 transgenic mice is dependent on the presence of the RGD motif and that translocation of GLUT4 in the muscle may be regulated by IGFBP-2 via RGD-dependent mechanisms., (Copyright © 2015 the American Physiological Society.)

In humans, mutations in IGF1 or IGF1R cause intrauterine and postnatal growth restriction; however, data on mutations in IGF2, encoding insulin-like growth factor (IGF) II, are lacking. We report an IGF2 variant (c.191C→A, p.Ser64Ter) with evidence of pathogenicity in a multigenerational family with four members who have growth restriction. The phenotype affects only family members who have inherited the variant through paternal transmission, a finding that is consistent with the maternal imprinting status of IGF2. The severe growth restriction in affected family members suggests that IGF-II affects postnatal growth in addition to prenatal growth. Furthermore, the dysmorphic features of affected family members are consistent with a role of deficient IGF-II levels in the cause of the Silver-Russell syndrome. (Funded by Bundesministerium für Bildung und Forschung and the European Union.).

The degradation of tryptophan (TRP) along the kynurenine pathway plays a crucial role as a neuro- and immunomodulatory mechanism in response to inflammatory stimuli, such as lipopolysaccharides (LPS). In endotoxemia or sepsis, an enhanced activation of the rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO) is associated with a higher mortality risk. It is assumed that IDO induced immunosuppressive effects provoke the development of a protracted compensatory hypoinflammatory phase up to a complete paralysis of the immune system, which is characterized by an endotoxin tolerance. However, the role of IDO activation in the development of life-threatening immunoparalysis is still poorly understood. Recent reports described the impact of inflammatory IDO activation and aryl hydrocarbon receptor- (AhR-) mediated pathways on the development of LPS tolerance and immune escape of cancer cells. These immunosuppressive mechanisms offer new insights for a better understanding of the development of cellular dysfunctions in immunoparalysis. This review provides a comprehensive update of significant biological functions of TRP metabolites along the kynurenine pathway and the complex regulation of LPS-induced IDO activation. In addition, the review focuses on the role of IDO-AhR-mediated immunosuppressive pathways in endotoxin tolerance and carcinogenesis revealing the significance of enhanced IDO activity for the establishment of life-threatening immunoparalysis in sepsis.

This study was conducted to determine if the main components of the somatotropic axis change during the early phase of pregnancy in the maternal blood system and whether differences exist on day 18 after pregnancy recognition by the maternal organism. Blood samples of pregnant heifers (Holstein Friesian; n = 10 after embryo transfer) were obtained on the day of ovulation (day 0), as well as on days 7, 14, 16 and 18 and during pregnant, non-pregnant and negative control cycles. The concentrations of progesterone (P4), oestrogen, growth hormone (GH), insulin-like growth factor-1 and -2 (IGF1, -2) and IGF-binding protein-2, -3 and -4 (IGFBP2, -3, -4) were measured. The mRNA expressions of growth hormone receptor 1A, IGF1, IGF2, IGFBP2, IGFBP3 and IGFBP4 were detected using RT-qPCR in liver biopsy specimens (day 18). In all groups, total serum IGF1 decreased from day 0 to 16. Notably, IGFBP4 maternal blood concentrations were lower during pregnancy than during non-pregnant cycles and synchronized control cycles. It can be speculated that the lower IGFBP4 in maternal blood may result in an increase of free IGF1 for local action. Further studies regarding IGFBP4 concentration and healthy early pregnancy are warranted.

The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, the most sensitive pharmacodynamic marker for the action of GH in humans, shows no response to treatment with recombinant human GH and there is little evidence for the effects of GHAs, except when administered at very high doses or when overexpressed. As an alternative, more suitable model, we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for the analysis of rabbit serum and tested precision, sensitivity, linearity and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with recombinant human GH or the GHA Pegvisomant. For a subset of samples, we used LC-MS/MS to measure IGF-I, and quantitative western ligand blot to analyze IGF-binding proteins (IGFBPs). Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, our results show that the sensitivity, precision (1.7-3.3% coefficient of variation) and linearity (90.4-105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I concentration in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple injections of recombinant human GH (IGF-I: 286±22 versus 434±26 ng/ml; P<0.01) and were highly correlated (P<0.0001). Treatment with the GHA lowered IGF-I levels from the fourth injection onwards (P<0.01). In summary, we demonstrated that the IDS-iSYS IGF-I immunoassay can be used in rabbits. Similar to rodents, rabbits display variations in IGF-I depending on sex, age and genetic background. Unlike in rodents, the IGF-I response to treatment with recombinant human GH or a GHA closely mimics the pharmacodynamics seen in humans, suggesting that rabbits are a suitable new model to test human GH agonists and antagonists., (© 2014. Published by The Company of Biologists Ltd.)

Spinal muscular atrophy with respiratory distress type 1 is a neuromuscular disorder characterized by progressive weakness and atrophy of the diaphragm and skeletal muscles, leading to death in childhood. No effective treatment is available. The neuromuscular degeneration (Nmd(2J)) mouse shares a crucial mutation in the immunoglobulin mu-binding protein 2 gene (Ighmbp2) with spinal muscular atrophy with respiratory distress type 1 patients and also displays some basic features of the human disease. This model serves as a promising tool in understanding the complex mechanisms of the disease and in exploring novel treatment modalities such as insulin-like growth factor 1 (IGF1) which supports myogenic and neurogenic survival and stimulates differentiation during development. Here we investigated the treatment effects with polyethylene glycol-coupled IGF1 and its mechanisms of action in neurons and muscles. Polyethylene glycol-coupled IGF1 was applied subcutaneously every second day from post-natal Day 14 to post-natal Day 42 and the outcome was assessed by morphology, electromyography, and molecular studies. We found reduced IGF1 serum levels in Nmd(2J) mice 2 weeks after birth, which was normalized by polyethylene glycol-coupled IGF1 treatment. Nmd(2J) mice showed marked neurogenic muscle fibre atrophy in the gastrocnemius muscle and polyethylene glycol-coupled IGF1 treatment resulted in muscle fibre hypertrophy and slowed fibre degeneration along with significantly higher numbers of functionally active axonal sprouts. In the diaphragm with predominant myogenic changes a profound protection from muscle fibre degeneration was observed under treatment. No effects of polyethylene glycol-coupled IGF1 were monitored at the level of motor neuron survival. The beneficial effects of polyethylene glycol-coupled IGF1 corresponded to a marked activation of the IGF1 receptor, resulting in enhanced phosphorylation of Akt (protein kinase B) and the ribosomal protein S6 kinase in striated muscles and spinal cord from Nmd(2J) mice. Based on these findings, polyethylene glycol-coupled IGF1 may hold promise as a candidate for future treatment trials in human patients with spinal muscular atrophy with respiratory distress type 1.

IGFBP-2 (1) has been described as a brain tumor oncogene (2) and is widely expressed in cancers from different origins (3-8). IGFBP-2 alone cannot cause malignant transformation, yet progression of brain tumors to higher grade (9) and also has been provided as a protective element in earlier stages of multistage colon carcinogenesis (10). Therefore, it is crucial to understand the factors that determine expression patterns of IGFBP-2 under normal and malignant conditions. The present review provides a comprehensive update of known factors that have an impact on expression of IGFBP-2.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway, and its increased activation is associated with immunologic disorders. Because the specific role of IDO activation is not yet completely clear, the aim of the present study was to establish a pig model of IDO activation for further research. The activation of IDO in pigs was induced experimentally by LPS stimulation in vivo and ex vivo. IDO activation was characterized by measuring Trp, Trp metabolites and IDO protein expression in blood, liver, lung, muscle and different brain areas. The results show that the in vivo LPS administration induced increased plasma concentrations of TNF-α and IL-10, a depletion of Trp and an increase of Kyn, indicating an elevated enzymatic activity of IDO. This was supported by an LPS-induced IDO protein expression in blood, liver and lung. The ex vivo LPS stimulation also resulted in increased TNF-α concentrations and an IDO activation, characterized by an increase of Trp metabolites and IDO protein expression. In conclusion, our data emphasize that the LPS stimulation is a suitable model for IDO activation in the domestic pig, which provides a basis for further research on immunoregulatory IDO functions.