In vitro evaluation of defined oligosaccharide fractions in an equine model of inflammation

Background: Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented in vivo. Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidate the effects of selected oligosaccharide fractions on the lipopolysaccharide (LPS) induced inflammatory response in equine peripheral blood mononuclear cells (PBMCs). We investigated three different products containing either galacto-oligosaccharides (GOS) alone, a combination of GOS with fructo-oligosaccharides (FOS), and a triple combination of GOS and FOS with acidic oligosaccharides (AOS), at different concentrations. These products have been used in an identical composition in various previously published in vivo experiments. As the selected oligosaccharide fractions were derived from natural products, the fractions contained defined amounts of mono- and disaccharides and minor amounts of endotoxin, which was taken into account in the design of the study and the analysis of data. Acquired data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses.Results: Exposing cultured PBMCs to either GOS or GOS/FOS fractions resulted in a substantial dose-dependent increase of tumour necrosis factor-α (TNF-α) production in LPS challenged PBMCs. In contrast, incubation with GOS/FOS/AOS resulted in a dose-dependent reduction of both TNF-α and interleukin-10 production following LPS challenge. In addition, incubation with GOS/FOS/AOS significantly increased the apparent PBMC viability, indicating a protective or mitogenic effect. Furthermore, mono- and disaccharide control fractions significantly stimulated the inflammatory response in LPS challenged PBMCs as well, though to a lesser extent than GOS and GOS/FOS fractions.Conclusions: We found distinct immunomodulating effects of the investigated standardised oligosaccharide fractions, which either stimulated or suppressed the LPS induced inflammatory response in PBMCs. Both scenarios require additional investigation, to elucidate underlying modulatory mechanisms, and to translate this knowledge into the clinical application of oligosaccharide supplements in foals and other neonates

Immunomodulation in foals : opportunities and challenges

This thesis aims to explore options and targets for immunomodulation in horses, particularly in foals, which are prone to bacterial infections during the first months of life. The thesis particularly focuses on the possibilities to modulate Toll-like receptors (TLRs), which are a crucial link between innate and adaptive immune responses, regulating both tolerance to commensal bacterial flora as well as defensive immune responses to potentially harmful pathogens. Several methods to modulate inflammatory responses in equine immune cells were investigated ex vivo, inducing inflammation by means of a standardised lipopolysaccharide (LPS) challenge, hence activating TLR-4. Concurrent administration of the synthetic TLR-2 ligand Pam3-Cys-Ser-Lys4 (PCSK), which previously has been shown to mitigate intestinal inflammatory processes in rodent species, did not cause significant changes in the ex vivo induced inflammatory response, neither in immune cells of adult horses nor in cells obtained from neonatal foals, indicating the need to test potential immune-modulators in the target animal species. Aside from this, we performed several studies using specific carbohydrate fractions in comparable ex vivo models of inflammation. Previously performed studies in humans and rodent species have indicated that non-digestible carbohydrates, particularly natural oligosaccharides from different sources, seemed to modulate TLR signalling in the intestinal tract by acting as prebiotic. In addition, there is increasing evidence for a direct impact of oligosaccharides on TLR signalling. The in vitro studies in this thesis support the latter hypothesis. For instance, we demonstrated that the carbohydrate fraction of equine colostrum dose-dependently suppressed LPS-induced inflammatory responses in equine peripheral blood mononuclear cells (PBMCs). In addition, we tested several commercially produced oligosaccharide products, derived from cow’s milk and botanicals. We found distinct effects of several specific oligosaccharide fractions, either enhancing or suppressing the ex vivo induced inflammation. Both scenarios can be useful for the future development and optimisation of oligosaccharide supplements, depending on the clinical context in which they are applied. In the final part of the thesis we describe an in vivo pilot study concerning oral supplementation of galacto-oligosaccharides in foals. During the period of investigation, no significant effects of the treatment were observed on the apparent clinical and blood parameters for health and immune status. However, in PBMCs derived from these foals after four weeks of treatment, a standardised LPS challenge resulted in significantly lower relative mRNA expression levels of pro-inflammatory cytokines in the treated foals compared with the control group. In addition to these in vivo experiments, the first results of the characterisation of the oligosaccharides in equine colostrum are presented in the thesis. These preliminary results reveal distinct oligosaccharide patterns in horse colostrum and identify several unique oligosaccharides, specific for the horse. In conclusion, our data provide the first evidence for efficacy of specific immunomodulatory oligosaccharides in the horse and hence serve as starting point for the development of methods to improve immunocompetence in young foals, in particular by modulating TLR signallin

Immunomodulation in foals : opportunities and challenges

This thesis aims to explore options and targets for immunomodulation in horses, particularly in foals, which are prone to bacterial infections during the first months of life. The thesis particularly focuses on the possibilities to modulate Toll-like receptors (TLRs), which are a crucial link between innate and adaptive immune responses, regulating both tolerance to commensal bacterial flora as well as defensive immune responses to potentially harmful pathogens. Several methods to modulate inflammatory responses in equine immune cells were investigated ex vivo, inducing inflammation by means of a standardised lipopolysaccharide (LPS) challenge, hence activating TLR-4. Concurrent administration of the synthetic TLR-2 ligand Pam3-Cys-Ser-Lys4 (PCSK), which previously has been shown to mitigate intestinal inflammatory processes in rodent species, did not cause significant changes in the ex vivo induced inflammatory response, neither in immune cells of adult horses nor in cells obtained from neonatal foals, indicating the need to test potential immune-modulators in the target animal species. Aside from this, we performed several studies using specific carbohydrate fractions in comparable ex vivo models of inflammation. Previously performed studies in humans and rodent species have indicated that non-digestible carbohydrates, particularly natural oligosaccharides from different sources, seemed to modulate TLR signalling in the intestinal tract by acting as prebiotic. In addition, there is increasing evidence for a direct impact of oligosaccharides on TLR signalling. The in vitro studies in this thesis support the latter hypothesis. For instance, we demonstrated that the carbohydrate fraction of equine colostrum dose-dependently suppressed LPS-induced inflammatory responses in equine peripheral blood mononuclear cells (PBMCs). In addition, we tested several commercially produced oligosaccharide products, derived from cow’s milk and botanicals. We found distinct effects of several specific oligosaccharide fractions, either enhancing or suppressing the ex vivo induced inflammation. Both scenarios can be useful for the future development and optimisation of oligosaccharide supplements, depending on the clinical context in which they are applied. In the final part of the thesis we describe an in vivo pilot study concerning oral supplementation of galacto-oligosaccharides in foals. During the period of investigation, no significant effects of the treatment were observed on the apparent clinical and blood parameters for health and immune status. However, in PBMCs derived from these foals after four weeks of treatment, a standardised LPS challenge resulted in significantly lower relative mRNA expression levels of pro-inflammatory cytokines in the treated foals compared with the control group. In addition to these in vivo experiments, the first results of the characterisation of the oligosaccharides in equine colostrum are presented in the thesis. These preliminary results reveal distinct oligosaccharide patterns in horse colostrum and identify several unique oligosaccharides, specific for the horse. In conclusion, our data provide the first evidence for efficacy of specific immunomodulatory oligosaccharides in the horse and hence serve as starting point for the development of methods to improve immunocompetence in young foals, in particular by modulating TLR signallin

Intestinal barrier function in neonatal foals: options for improvement

Gastrointestinal defence in the new-born is limited in comparison to adults, due to an immature epithelial barrier function and deficits in both innate and adaptive immune responses. Consequently, neonates (including foals) are at increased risk of disturbance to mucosal homeostasis during initial intestinal colonisation that may lead to excessive inflammation and bacterial translocation into the bloodstream, resulting in septicaemia. Bacterial recognition by Pattern Recognition Receptors (PRRs) and their downstream regulation of cytokine release have been shown to be pivotal for gastrointestinal mucosal homeostasis and the development of a functional intestinal barrier. Evidence suggests that selective PRR agonists limit the inflammatory responses and improve epithelial barrier function. Milk, and in particular colostrum, contain a broad array of oligosaccharides which seem to act as PRR agonists. This class of compounds forms a source for new dietary formulas that may orchestrate gut colonisation by the commensal flora in the early phase of life and so reduce the risks of inflammation and pathogen invasion

Comparison of Milk Oligosaccharides Pattern in Colostrum of Different Horse Breeds

Colostrum oligosaccharides are known to exhibit prebiotic and immunomodulatory properties. Oligosaccharide composition is species-specific, and equine colostrum has been reported to contain unique oligosaccharides. Therefore, equine oligosaccharides (EMOS) from colostrum from different horse breeds were analyzed by CE-LIF, CE-MSn, HILIC-MSn, and exoglycosidase degradation. Sixteen EMOS were characterized and quantified, of which half were neutral and half were acidic. EMOS showed about 63% structural overlap with human milk oligosaccharides, known for their bioactivity. Seven EMOS were not reported before in equine oligosaccharides literature: neutral Gal(ß1–4)HexNAc, Gal(ß1–4)Hex-Hex, ß4'-galactosyllactose, and lactose-N-hexaose, as well as acidic 6'-Sialyl-Hex-Ac-HexNAc, sialyllacto-N-tetraose-a, and disialylacto-N-tetraose (isomer not further specified). In all colostrum samples, the average oligosaccharide concentration ranged from 2.12 to 4.63 g/L; with ß 6'and 3'- galactosyllactose, 3'-sialyllactose, and disialyllactose as the most abundant of all oligosaccharides (27–59, 16–37, 1–8, and 1–6%, respectively). Differences in presence and in abundance of specific EMOS were evident not only between the four breeds but also within the breed

Enhanced sensitivity to punctate painful stimuli in female patients with chronic low back pain

<p>Abstract</p> <p>Background</p> <p>Chronic low back pain (CLBP) has been shown to be associated with various pathophysiological changes at several level of the sensorimotor system, pointing to a general hypersensitivity in CLBP patients. The aim of the present study was to investigate signs of generalized mechanical pain hypersensitivity in CLBP patients on the hand and on the painful site of the back.</p> <p>Methods</p> <p>Pinprick stimulation according to a validated standardized quantitative sensory testing protocol was used in 14 female CLBP patients and 14 healthy controls (HC) matched for sex and age. Stimulus response functions to pinprick stimulation on the skin were examined at the affected back and reference sites (hand palmar and hand dorsum). Data from CLBP patients were compared with HC and with reference data from the German Research Network on Neuropathic Pain.</p> <p>Results</p> <p>We found significant differences in the stimulus response functions between CLBP patients and HC. Pain ratings to the pinpricks were increased for low and moderate pinprick stimuli in CLBP patients. Importantly, this kind of specific pinprick hyperalgesia was found not only for the affected body site (back), but also for the remote reference sites (hand dorsum and hand palmar).</p> <p>Conclusions</p> <p>We interpret our results as pointing to changes in the nociceptive processing in CLBP at higher levels of the neuraxis, possibly thalamus and/or attentional control, rather than changes of spinal processing. Alternatively, there might be a higher vulnerability to noxious stimulation in CLBP patients.</p