In Vivo and In Vitro Evidence of the Involvement of CXCL1, a Keratinocyte-Derived Chemokine, in Equine Laminitis

Background: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans.
Objectives: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis.
Animals: Twenty adult horses.
Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression.
Results: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [ P = .0001]) and DTP groups (21-fold [ P = .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases ( P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H₂O₂ (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA ( P = .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes.
Conclusion and Clinical Importance: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.     

Analysis of synovial fluid from clinically normal alpacas and llamas

OBJECTIVE: To establish reference range values for synovial fluid from clinically normal New World camelids. ANIMALS: 15 llamas and 15 alpacas. PROCEDURE: Llamas and alpacas were anesthetized with an IM injection of a xylazine hydrochloride, butorphanol tartrate, and ketamine hydrochloride combination. Synovial fluid (1 to 2 ml) was obtained by aseptic arthrocentesis from the radiocarpal and tarsocrural joints. Synovial fluid evaluation included determination of total nucleated cell count (NCC), absolute number and percentage of polymorphonuclear (PMN) and mononuclear leukocytes, total protein, and specific gravity. RESULTS: Synovial fluid evaluation revealed a total NCC of 100 to 1,400 cells/microl (mean +/- SD, 394.8+/-356.2 cells/microl; 95% confidence interval [CI], 295.2 to 494.6 cells/microl). Mononuclear leukocytes were the predominant cell type with lymphocytes, composing 50 to 90% (mean, 75.6+/-172%; 95% CI, 70.8 to 80.4%) of the mononuclear leukocytes. Approximately 0 to 12% (mean, 1.3+/-2.9%; 95% CI, 0.49 to 2.11%) of the cells were PMN leukocytes. Total protein concentrations ranged from 2.0 to 3.8 g/dl (mean, 2.54+/-0.29 g/dl; 95% CI, 2.46 to 2.62 g/dl); the specific gravity ranged between 1.010 and 1.026 (mean, 1.017+/-0.003; 95% CI, 1.016 to 1.018). CONCLUSION AND CLINICAL RELEVANCE: In llamas and alpacas, significant differences do not exist between species or between limbs (left vs right) or joints (radiocarpal vs tarsocrural) for synovial fluid values. Total NCC and absolute number and percentage of PMN and mononuclear leukocyte are similar to those of other ruminants and horses. However, synovial fluid total protein concentrations in New World camelids are high, compared with other domestic species.     

Blackspot bruise dependent changes in enzyme activity and gene expression in Lemhi russet potato

Blackspot bruise-induced changes in enzyme activity and gene expression were examined in the bruise susceptible cultivar Lemhi. The activities of several enzymes involved with the synthesis and metabolism of phenolic intermediates were examined over the time course of blackspot bruise development in tubers. A large, transient increase (200-fold) in phenylalanine ammonia lyase (PAL) activity was observed, while other enzymes examined (chorismate mutase, tyrosine ammonia-lyase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, and polyphenol oxidase) showed either modest or no activation. Maximal PAL activity was observed 48 hours after bruise induction, well after the discoloration reactions were complete. The change in PAL activity was associated with a transient increase in messenger RNA coding for PAL. In addition, messenger RNAs encoding two stress-induced genes, ubiquitin and the 70 kD heat shock protein, were transiendy induced by bruising. Physical impact also induced a marked decrease, followed by recovery, in the messenger RNA level for patatin, a primary storage protein in the tuber. These results suggest that the stress resulting in blackspot bruising elicits a wound response similar to those observed in other injuries to plant tissues.     

Distribution of insulin receptor and insulin‐like growth factor‐1 receptor in the digital laminae of mixed‐breed ponies: An immunohistochemical study

Reasons for performing study: Hyperinsulinaemia has been implicated in the pathogenesis of laminitis; however, laminar cell types responding to insulin remain poorly characterised. Objectives: To identify laminar cell types expressing insulin receptor (IRc) and/or insulin‐like growth factor‐1 receptor (IGF‐1R); and to evaluate the effect of dietary nonstructural carbohydrate (NSC) on their expression. Methods: Mixed‐breed ponies (n = 22) received a conditioning hay chop diet (NSC ～6%); following acclimation, ponies were stratified into lean (n = 11, body condition score [BCS]≤4) or obese (n = 11, BCS ≥7) groups and each group further stratified to remain on the low NSC diet (n = 5 each for obese and lean) or receive a high NSC diet (total diet ～42% NSC; n = 6 each for obese and lean) for 7 days. Laminar samples were collected at the end of the feeding protocol and stained immunohistochemically for IRc and IGF‐1R. The number of IRc(+) cells was quantified; distribution of IGF‐1R was qualitatively described. Laminar IRc content was assessed via immunoblotting. Results: The number of IRc(+) cells was greater in the laminae of high NSC ponies than low NSC ponies (P = 0.001); there was a positive correlation between the change in serum insulin concentration and number of IRc(+) cells (r²= 0.74; P<0.0001). No epithelial IRc(+) cells were observed; IRc(+) cells were absent from the deep dermis. Analysis of serial sections identified IRc(+) cells as endothelial cells. The distribution of IGF‐1R was more extensive than that of IRc, with signal in vascular elements, epithelial cells and fibroblasts. Conclusions: Increased dietary NSC results in increased laminar endothelial IRc expression. Laminar keratinocytes do not express IRc, suggesting that insulin signalling in laminar epithelial cells must be mediated through other receptors (such as IGF‐1R). Potential relevance: Manipulation of signalling downstream of IRc and IGF‐1R may aid in treatment and prevention of laminitis associated with hyperinsulinaemia.