Alteration of the fecal microbiota and serum metabolite profiles in dogs with idiopathic inflammatory bowel disease.

Idiopathic inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal (GI) disease in dogs. The combination of an underlying host genetic susceptibility, an intestinal dysbiosis, and dietary/environmental factors are suspected as main contributing factors in the pathogenesis of canine IBD. However, actual mechanisms of the host-microbe interactions remain elusive. The aim of this study was to compare the fecal microbiota and serum metabolite profiles between healthy dogs (n = 10) and dogs with IBD before and after 3 weeks of medical therapy (n = 12). Fecal microbiota and metabolite profiles were characterized by 454-pyrosequencing of 16 S rRNA genes and by an untargeted metabolomics approach, respectively. Significantly lower bacterial diversity and distinct microbial communities were observed in dogs with IBD compared to the healthy control dogs. While Gammaproteobacteria were overrepresented, Erysipelotrichia, Clostridia, and Bacteroidia were underrepresented in dogs with IBD. The functional gene content was predicted from the 16 S rRNA gene data using PICRUSt, and revealed overrepresented bacterial secretion system and transcription factors, and underrepresented amino acid metabolism in dogs with IBD. The serum metabolites 3-hydroxybutyrate, hexuronic acid, ribose, and gluconic acid lactone were significantly more abundant in dogs with IBD. Although a clinical improvement was observed after medical therapy in all dogs with IBD, this was not accompanied by significant changes in the fecal microbiota or in serum metabolite profiles. These results suggest the presence of oxidative stress and a functional alteration of the GI microbiota in dogs with IBD, which persisted even in the face of a clinical response to medical therapy.

Serum metabolomics identifies citrulline as a predictor of adverse outcomes in an equine model of gut-derived sepsis.

Acute laminitis is an inflammatory disease of the equine foot that often occurs secondarily to sepsis or systemic inflammation associated with gastrointestinal disease. It has been suggested that laminitis is similar to multiple organ dysfunction syndrome in humans, although in horses the weight-bearing laminar epithelium of the foot appears to be the tissue most sensitive to insult and the first "organ" to fail. Metabolomics performed on serum samples collected before (Con) and after (Lmn) experimental induction of gastrointestinal-associated sepsis in six horses detected 1,177 metabolites of both mammalian and bacterial origin in equine serum. Network and correlation analyses suggested a dysregulation of fatty acid metabolism in the Lmn group, as well as an accumulation of organic acids such as lactate. Furthermore, concentrations of the amino acid citrulline were decreased in Lmn samples from all study animals, suggesting that citrulline might be useful as a biomarker to identify critically ill animals that are at risk of developing laminitis. We therefore established normal ranges of plasma citrulline concentrations in a separate group of horses (n = 36) and tested the ability of citrulline to predict adverse outcomes (laminitis or death) in critically ill horses (n = 23). Plasma citrulline was significantly lower in critically ill horses that went on to experience adverse outcomes (n = 6). Further study is required to accurately determine a diagnostic cutoff, but the present data are suggestive of the predictive value of citrulline as a biomarker for laminar failure in equine sepsis.

Effects of histidine-rich glycoprotein on cerebral blood vessels.

Delayed cerebral vasospasm is thought to be caused by factors released from a subarachnoid blood clot. Because vasospasm occurs several days after hemorrhage, we hypothesized that clotted blood releases vasoactive factors as it ages. Targeted proteomics identified histidine-rich glycoprotein (HRG) as a potentially vasoactive factor released within the first 72 hours of clot formation. In vitro studies revealed that HRG caused moderate (~30%) dilation of cannulated cerebral arterioles and proliferation of cerebrovascular endothelial cells. We conclude that HRG released from clotted blood, while unlikely to contribute to cerebral vasospasm, might provide important vasodilatory or angiogenic stimuli after hemorrhagic stroke.

Cellular and humoral immunity in chronic equine laminitis.

Chronic equine laminitis causes persistent pain and lameness in affected animals and often necessitates euthanasia when pain management strategies become ineffective. Published studies as well as anecdotal reports suggest that this chronic inflammatory disease is associated with systemic alterations in immune responsiveness, perhaps involving an autoimmune component. We investigated this broad hypothesis by measuring a variety of immune indicators in healthy control horses (CON) and horses with chronic laminitis (LMN). We found that white blood cells from LMN horses produced more IFNγ than did cells from CON horses when stimulated in vitro with polyinosinic-polycytidylic acid [poly(I:C)], possibly due to an elevated number of circulating monocytes. No differences between groups were observed in plasma concentrations of IgG, IgA, IgM, IgE, or rheumatoid factor. Laminar tissue from LMN horses expressed elevated levels of keratinocyte damage-related genes as well as inflammatory cytokines and chemokines, which corresponded with a modest amount of neutrophil infiltration as shown by histological staining of fixed tissue and accumulation of neutrophil elastase protein. Taken together, our results do not support the hypothesis of an autoimmune component in chronic laminitis, although the strong induction of neutrophil chemokines and the presence of tissue neutrophils suggests that this cell type is likely involved in perpetuating the inflammation and tissue damage associated with this disease.

Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis.

BACKGROUND: The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis. RESULTS: Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks. CONCLUSIONS: Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis.

Plasma proteomics shows an elevation of the anti-inflammatory protein APOA-IV in chronic equine laminitis.

BACKGROUND: Equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry. In acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. Horses that survive the acute phase transition into a chronic form of laminitis, which is often termed "founder". Some evidence suggests that chronic laminar inflammation might be associated with alterations in the endocrine and immune systems. We investigated this broad hypothesis by using DIGE to assess global differences in the plasma proteome between horses with chronic laminitis and controls. RESULTS: We identified 16 differentially expressed proteins; the majority of these were involved in the interrelated coagulation, clotting, and kininogen cascades. Clinical testing of functional coagulation parameters in foundered horses revealed a slight delay in prothrombin (PT) clotting time, although most other indices were within normal ranges. Upregulation of the intestinal apolipoprotein APOA-IV in horses with chronic laminitis was confirmed by western blot. CONCLUSIONS: Our results support the hypothesis that localized laminar inflammation may be linked to systemic alterations in immune regulation, particularly in the gastrointestinal system. Gastrointestinal inflammation has been implicated in the development of acute laminitis but has not previously been associated with chronic laminitis.

Differential remodeling responses of cerebral and skeletal muscle arterioles in a novel organ culture system.

Evidence suggests that maladaptive changes in the cerebral microcirculation may contribute to ischemia in numerous diseases. We sought, therefore, to develop an ex vivo organ culture system to study early changes in cerebral arteriolar structure and function, and to compare associated findings to those for non-cerebral arterioles. Pilot studies revealed that rabbit cerebral arterioles maintained contractility longer when cultured in media containing rabbit-specific plasma rather than fetal bovine serum. Cerebral and skeletal muscle arterioles were cultured in a pressure myograph for 5 days; maximum dilatory and contractile responses were measured at 0, 1, 3, and 5 days. Passive properties were preserved in cerebral arterioles over the entire culture period, although skeletal muscle arterioles underwent constrictive remodeling. Cerebral arterioles also maintained a myogenic capability over the entire culture period, albeit at progressively larger diameters, whereas the skeletal muscle arterioles did so only over 3 days. Culture in rabbit serum, which contains numerous growth factors and clotting factors, did not induce or increase inward remodeling in cerebral or skeletal arterioles. These results suggest inherent, organ-specific differences in arteriolar remodeling, and that extensive results in the literature on non-cerebral arterioles should not be extrapolated to predict responses in the cerebral microcirculation.

Perivascular tethering modulates the geometry and biomechanics of cerebral arterioles.

Recent studies have renewed interest in the effects of perivascular tethering on vascular mechanics, particularly growth and remodeling. We quantified effects of axial and circumferential tethering on rabbit pial arterioles from the ventral occipital lobe of the brain. The homeostatic axial pre-stretch, which is influenced by perivascular tethering, was measured in situ to be 1.24±0.04. Using a cannulated microvessel preparation, wall mechanics were then quantified in vitro for isolated arterioles at low (1.10) or normal (1.24) values of axial stretch and for tethered arterioles having perivascular support. Axial stretch did not significantly affect changes in circumferential stretch or stress upon pressurization, but circumferential tethering caused arteriolar geometry to change from a circular cross-section at normal pressure to an elliptical one at pressures above and below normal. Calculations suggested that the observed levels of ellipticity could cause a modest decrease in volumetric blood flow, but also a pronounced variation in shear stress around the circumference of the arteriole. An elliptical cross-section could thus increase vascular resistance or promote luminal remodeling at pressures different from normal. This characterization of effects of perivascular tethering on pial arterioles should prove useful in future studies of roles of perturbed cerebral blood flow on the propensity of the cerebral microcirculation to remodel.