Evaluation of C-reactive protein and its kinetics as a prognostic indicator in canine leptospirosis.

OBJECTIVE To evaluate C-reactive protein at presentation and during hospitalisation in dogs with acute kidney injury resulting from leptospirosis to compare C-reactive protein at presentation in dogs with acute kidney injury of different aetiology and to study its correlation with markers of inflammation, azotaemia and survival. MATERIALS AND METHODS Prospective observational study of 41 dogs with acute kidney injury secondary to leptospirosis and 15 control dogs with acute kidney injury of different aetiology. C-reactive protein was measured at presentation in both groups and daily for 7 days in a subgroup of 28 dogs with leptospirosis. The associations of C-reactive protein with neutrophil count, albumin, urea, creatinine and survival were analysed. RESULTS C-reactive protein was increased at presentation in all dogs with leptospirosis but was not significantly different from dogs with acute kidney injury of different cause. It was associated with markers of inflammation (neutrophil count, albumin) but not with azotaemia (creatinine, urea). It decreased gradually from presentation to day 4, with significantly lower concentrations in survivors than non-survivors. Initial C-reactive protein was only weakly associated with outcome, but its average concentration from presentation to day 2 was more strongly associated. Absolute and relative changes in C-reactive protein during hospitalisation and creatinine at presentation were not associated with survival. CLINICAL SIGNIFICANCE Serial assessment of C-reactive protein may improve outcome prediction in dogs with leptospirosis compared with a single measurement at presentation or with markers of renal function

Plasma procalcitonin kinetics in healthy dogs and dogs undergoing tibial plateau leveling osteotomy.

BACKGROUND Procalcitonin (PCT) is a well-established biomarker for bacterial infection in human patients. OBJECTIVES We aimed to analyze the kinetics of plasma PCT (pPCT) in healthy dogs and dogs with canine cranial cruciate ligament (CCL) rupture undergoing tibial plateau leveling osteotomy (TPLO). METHODS This prospective, longitudinal study included 15 healthy dogs and 25 dogs undergoing TPLO. Hematology, pPCT, and C-reactive protein (CRP) were assessed on 3 consecutive days in healthy dogs and 1 day preoperatively and days 1, 2, 10, and 56 postoperatively. Inter- and intraindividual variability of pPCT were assessed in healthy dogs. Median pPCT concentrations of dogs with CCL rupture preoperatively were compared with healthy controls, and median pPCT concentrations, as well as percentage change post anesthesia, arthroscopy, and TPLO, were compared with baseline. For the correlation analysis, the Spearman rank correlation test was used. RESULTS Inter- and intraindividual variabilities of pPCT in healthy dogs were 36% and 15%, respectively. Median baseline pPCT concentrations were not significantly different between healthy dogs (118.9 pg/mL; IQR: 75.3-157.3 pg/mL) and dogs undergoing TPLO (95.9 pg/mL; IQR: 63.8-117.0 pg/mL). Plasma PCT concentrations were significantly lower immediately post- than preoperatively (P < 0.001). CRP, WBC, and neutrophil concentrations increased significantly on post-OP day 2 and had normalized by day 10. CONCLUSIONS These results indicate that CCL rupture, as well as anesthesia, arthroscopy, and TPLO combined, are not associated with increased pPCT concentrations in dogs with uncomplicated recovery. Considering the high intraindividual variability, individual serial measurements rather than a population-based reference interval should be considered

Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation

Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNALeu(CUN)) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNALeu(CUN) and tRNALeu(UUR) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNALeu(CUN) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation

Safety Profile of a Virus-Like Particle-Based Vaccine Targeting Self-Protein Interleukin-5 in Horses

Background: Insect bite hypersensitivity (IBH) is an eosinophilic allergic dermatitis of horses caused by type I/IVb reactions against mainly Culicoides bites. The vaccination of IBH-affected horses with equine IL-5 coupled to the Cucumber mosaic virus-like particle (eIL-5-CuMVTT) induces IL-5-specific auto-antibodies, resulting in a significant reduction in eosinophil levels in blood and clinical signs. Objective: the preclinical and clinical safety of the eIL-5-CuMVTT vaccine. Methods: The B cell responses were assessed by longitudinal measurement of IL-5- and CuMVTT-specific IgG in the serum and plasma of vaccinated and unvaccinated horses. Further, peripheral blood mononuclear cells (PBMCs) from the same horses were re-stimulated in vitro for the proliferation and IFN-γ production of specific T cells. In addition, we evaluated longitudinal kidney and liver parameters and the general blood status. An endogenous protein challenge was performed in murine IL-5-vaccinated mice. Results: The vaccine was well tolerated as assessed by serum and cellular biomarkers and also induced reversible and neutralizing antibody titers in horses and mice. Endogenous IL-5 stimulation was unable to re-induce anti-IL-5 production. The CD4+ T cells of vaccinated horses produced significantly more IFN-γ and showed a stronger proliferation following stimulation with CuMVTT as compared to the unvaccinated controls. Re-stimulation using E. coli-derived proteins induced low levels of IFNγ+CD4+ cells in vaccinated horses; however, no IFN-γ and proliferation were induced following the HEK-eIL-5 re-stimulation. Conclusions: Vaccination using eIL-5-CuMVTT induces a strong B-cell as well as CuMVTT-specific T cell response without the induction of IL-5-specific T cell responses. Hence, B-cell unresponsiveness against self-IL-5 can be bypassed by inducing CuMVTT carrier-specific T cells, making the vaccine a safe therapeutic option for IBH-affected horses

Association of markers of endothelial activation and dysfunction with occurrence and outcome of pulmonary hemorrhage in dogs with leptospirosis.

BACKGROUND Endothelial dysfunction might contribute to the development of leptospiral pulmonary hemorrhage syndrome (LPHS). HYPOTHESIS Serum concentrations of markers of endothelial activation and dysfunction are higher in dogs with leptospirosis and correlate with the occurrence of LPHS and a higher case fatality rate. ANIMALS Clinically healthy dogs (n = 31; 10/31 dogs confirmed healthy based on no detected abnormalities on blood work), dogs with leptospirosis with LPHS (n = 17) and without LPHS (n = 15), dogs with acute kidney injury not due to leptospirosis (AKI-nL, n = 34). METHODS Observational study. Serum concentrations of soluble intercellular adhesion molecule 1 (sICAM-1), vascular endothelial growth factor (VEGF), and angiopoietin-2 (Ang-2) at admission were compared between groups. Correlations with outcome and the accuracy to predict LPHS were examined. RESULTS Soluble intercellular adhesion molecule (sICAM-1), VEGF, and Ang-2 concentrations were higher in dogs with AKI-nL (sICAM-1 34.7 ng/mL, interquartile range [IQR] = 24.4-75.5; VEGF 43.1 pg/mL, IQR = 12.3-79.2; Ang-2 8.5 ng/mL, IQR = 6.2-12.3), leptospirosis without LPHS (sICAM-1 45.1 ng/mL, IQR = 30.6-59.0; VEGF 32.4 pg/mL, IQR = 12.5-62.6; Ang-2 9.6 ng/mL, IQR = 6.9-19.3), and LPHS (sICAM-1 69.7 ng/mL, IQR = 42.1-89.1; VEGF 51.8 pg/mL, IQR = 26.3-96.7; Ang-2 8.0 ng/mL, IQR = 5.6-12.2) compared to controls (P < .001). In dogs with leptospirosis, VEGF and sICAM-1 were higher in nonsurvivors (sICAM-1 89.4 ng/mL, IQR = 76.5-101.0; VEGF 117.0 pg/mL, IQR = 90.3-232.4) than survivors (P = .004) and sICAM-1 predicted the development of LPHS. CONCLUSIONS Soluble intercellular adhesion molecule 1, VEGF, and Ang-2 do not discriminate leptospirosis from AKI-nL. In dogs with leptospirosis, sICAM-1 and VEGF predict outcome and sICAM-1 might identify dogs at risk for LPHS

Reduced incidence of insect-bite hypersensitivity in Icelandic horses is associated with a down-regulation of interleukin-4 by interleukin-10 and transforming growth factor-beta1

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a >/=50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH

Analysis of blood degradation products and ferritin in the cerebrospinal fluid of dogs with acute thoracolumbar intervertebral disk extrusion, a prospective pilot study

Abstract Background Hemorrhage in the spinal canal leads to further damage of the spinal cord influencing outcome in dogs with intervertebral disk (IVD) extrusion. The aim of the study was to evaluate blood degradation products and ferritin in the cerebrospinal fluid (CSF) of dogs with thoracolumbar IVD extrusion, and their association to clinical parameters and MRI findings. Results In the CSF of dogs with IVD extrusion, both net oxyhemoglobin absorption (NOA) and net bilirubin absorption (NBA) were significantly higher compared to the control groups of dogs with steroid responsive meningitis arteritis (SRMA) and idiopathic epilepsy (IE) (P < 0.001), but NOA compared to the idiopathic epilepsy group contaminated artificially with blood (IEc) was not (P = 0.890). Ferritin concentration was significantly higher in dogs with IVD extrusion compared to dogs with IE (P = 0.034), but not to dogs with SRMA (P = 0.526). There was no association between NOA, NBA or ferritin concentration and severity or duration of clinical signs. In dogs with a higher ferritin concentration the outcome was better (P = 0.018). In dogs with evidence of hemorrhage on MRI, NOA and NBA were significantly higher (P = 0.016, P = 0.009), but not ferritin (P = 0.0628). Conclusion and clinical importance Quantification of blood degradation products and ferritin in the CSF of dogs to assess subarachnoidal hemorrhage is feasible; however, larger case numbers are needed to evaluate the relevance of NBA and ferritin as prognostic indicators

Regulatory T Cells in Early Life: Comparative Study of CD4⁺CD25^high T Cells from Foals and Adult Horses

<div><p>The immune system of mammals is subject to continuous development during the postnatal phase of life. Studies following the longitudinal development of the immune system in healthy children are limited both by ethical considerations and sample volumes. Horses represent a particular valuable large animal model for T regulatory (Treg) cells and allergy research. We have recently characterised Treg cells from horses, demonstrated their regulatory capability and showed both their expansion and induction <i>in vitro</i>. Insect bite hypersensitivity (IBH) is a common allergy in horses resembling atopic dermatitis and studies have shown that first exposure to allergens in adult life results in an increased incidence of IBH. The aim of the present study was to characterize circulating CD4⁺CD25^highFoxP3⁺cells in foals, evaluate their suppressive capability and their in vitro induction compared to adult horses. 19 foals (age range, 1–5 months), their adult mothers and six one-year-old horses (yearlings) were included in the study. The proportion of FoxP3⁺ cells within the circulating CD4⁺CD25^high population was significantly higher in foals (47%) compared to their mothers (18%) and to yearlings (26%). Treg cells from foals also displayed a higher suppressive capability. Furthermore, CD4⁺CD25^high cells in foals could be induced <i>in vitro</i> from CD4⁺CD25⁻ cells in a significantly higher proportion compared to mares. These cells also displayed a significantly enhanced suppressive capability. In summary these findings support the notion that exposure of horses to allergens during maturation of the immune system assists the establishment of induced (i)Treg driven tolerance.</p></div

Induction of CD4⁺CD25^high cells from CD4⁺CD25⁻ cells from foals and mares.

<p>1x10⁷ CD4⁺CD25⁻ lymphocytes sorted from freshly isolated PBMC of foals (<b>F</b>, n = 3, age 1–2 months) and mares (<b>M</b>, n = 3, 5–17 years) were cultured with the cocktail. 4 days later, the cells were harvested, stained for CD25 and resorted for induced CD4^<b>+</b>CD25^high (_<b>I</b><b>CD4</b>^<b>+</b><b>CD25</b>^<b>high</b>) cells. <b>A)</b> Proportion of _ICD4⁺CD25^high cells from foals and mares were measured using flow cytometry. 5x10⁵ CD4^<b>+</b>CD25^<b>−</b> cells sorted from freshly isolated PBMC were labelled with CFSE and cultured with 5x10⁵ irradiated allogeneic PBMC alone <b>(CD4</b>^<b>+</b><b>CD25</b>^<b>−</b>) or in the presence of _ICD4^<b>+</b>CD25^high<b>(CD4</b>^<b>+</b><b>CD25</b>^<b>−</b><b>+</b>_<b>I</b><b>CD4</b>^<b>+</b><b>CD25</b>^<b>high</b>) cells in a ratio of 1: 0.25. After 4 days, the cells were harvested and stained for FoxP3 and IL-10. <b>B) P</b>ercentage of proliferation was measured by flow cytometry. Results are shown as percentage inhibition of proliferation of <b>CD4</b>^<b>+</b><b>CD25</b>^<b>−</b> cells by _ICD4^<b>+</b>CD25^high cells following stimulation with irradiated allogeneic PBMC. <b>C)</b> Percentage of single positive <b>FoxP3</b>^<b>+</b>IL-10⁻, single positive <b>IL-10</b>^<b>+</b>FoxP3⁻ and double positive <b>IL-10</b>^<b>+</b><b>FoxP3</b>^<b>+</b> was measured by flow cytometry. Each data point represents 1 horse horizontal bars within values denote medians. An asterisk with line indicates statistical significance difference. Comparisons were performed using non-parametric Mann-Witney U test. *p ≤0.05 was considered significant.</p