Chee-Hoon Chang

Clinical and immunologic assessment of sepsis and the systemic inflammatory response syndrome in cats

OBJECTIVE To compare clinical findings and inflammatory mediator production among cats with sepsis, cats with noninfectious systemic inflammatory response syndrome (SIRS), and healthy cats. DESIGN Case-control study. ANIMALS Cats with sepsis (n = 16) or SIRS (19) and 8 healthy control cats. PROCEDURES Clinical variables were recorded for each cat, and plasma tumor necrosis factor (TNF) and interleukin (IL)-1β activities and IL-6 and CXC chemokine ligand (CXCL)-8 concentrations were determined at initial evaluation. RESULTS Clinicopathologic abnormalities associated with sepsis in cats included a high band neutrophil percentage, eosinopenia, hyponatremia, hypochloremia, hypoalbuminemia, hypocalcemia, and hyperbilirubinemia. When the sepsis and SIRS groups were compared, the only significant differences in the CBC and plasma biochemical findings were band neutrophil percentage and albumin concentration. Cats with sepsis had significantly greater plasma TNF activity than did healthy cats and were more likely to have detectable concentrations of IL-6 than were cats with SIRS or healthy cats. Plasma IL-1β activity did not differ among groups, and CXCL-8 was not detectable in most (32/43) cats. Mortality rate was not significantly greater for cats with sepsis (7/16) than for cats with SIRS (5/19). Plasma IL-1β activity and IL-6 and chloride concentrations were the only variables correlated with nonsurvival in the sepsis group. CONCLUSIONS AND CLINICAL RELEVANCE Cats with sepsis may have various clinicopathologic abnormalities but are more likely to have a high band neutrophil percentage and hypoalbuminemia than cats with noninfectious SIRS. Plasma interleukin-1β activity and plasma IL-6 and chloride concentrations may be useful prognostic biomarkers for septic cats.

Age‐associated changes to pathogen‐associated molecular pattern‐induced inflammatory mediator production in dogs

OBJECTIVE To determine whether older dogs will have a more pronounced pro-inflammatory response and blunted anti-inflammatory response to pathogen-associated molecular patterns (PAMPs) compared with younger dogs. DESIGN Prospective. SETTING University teaching hospital. ANIMALS Thirty-eight privately owned sexually altered dogs of various ages. INTERVENTIONS Blood was collected for HCT, WBC count, plasma biochemical analysis, and whole blood culture. Whole blood was stimulated with lipopolysaccharide (LPS) or, lipoteichoic acid or, peptidoglycan or, addition of phosphate-buffered saline. Tumor necrosis factor (TNF), interleukin (IL)-6, and IL-10 production from whole blood were compared among young, middle aged, and geriatric dogs. MEASUREMENTS AND MAIN RESULTS LPS, lipoteichoic acid, and peptidoglycan stimulated significant TNF, IL-6, and IL-10 production from canine whole blood compared with phosphate-buffered saline. Whole blood from geriatric dogs had a blunted IL-10 response to LPS stimulation and middle-aged dogs had increased LPS-induced TNF production compared with the other groups. CONCLUSION PAMPs from gram-positive and gram-negative bacteria stimulate TNF, IL-6, and IL-10 production from canine whole blood. The inflammatory mediator response to PAMPs from gram-negative bacteria alters with age and may be one factor contributing to mortality in geriatric dogs with sepsis.Objective – To determine whether older dogs will have a more pronounced pro-inflammatory response and blunted anti-inflammatory response to pathogen-associated molecular patterns (PAMPs) compared with younger dogs. Design – Prospective. Setting – University teaching hospital. Animals – Thirty-eight privately owned sexually altered dogs of various ages. Interventions – Blood was collected for HCT, WBC count, plasma biochemical analysis, and whole blood culture. Whole blood was stimulated with lipopolysaccharide (LPS) or, lipoteichoic acid or, peptidoglycan or, addition of phosphate-buffered saline. Tumor necrosis factor (TNF), interleukin (IL)-6, and IL-10 production from whole blood were compared among young, middle aged, and geriatric dogs. Measurements and Main Results – LPS, lipoteichoic acid, and peptidoglycan stimulated significant TNF, IL-6, and IL-10 production from canine whole blood compared with phosphate-buffered saline. Whole blood from geriatric dogs had a blunted IL-10 response to LPS stimulation and middle-aged dogs had increased LPS-induced TNF production compared with the other groups. Conclusion – PAMPs from gram-positive and gram-negative bacteria stimulate TNF, IL-6, and IL-10 production from canine whole blood. The inflammatory mediator response to PAMPs from gram-negative bacteria alters with age and may be one factor contributing to mortality in geriatric dogs with sepsis.

Beneficial cross-protection of allergen-specific immunotherapy on airway eosinophilia using unrelated or a partial repertoire of allergen(s) implicated in experimental feline asthma

The study hypothesis was that in experimentally asthmatic cats rush immunotherapy (RIT) using allergens not completely matched with sensitizing allergen(s) would at least partially attenuate the asthmatic phenotype and modulate the aberrant immune response. In phase I, cats sensitized to Bermuda grass allergen (BGA), house dust mite allergen (HDMA) or placebo received BGA RIT. In phase II, cats dually sensitized to BGA and HDMA received RIT using BGA, HDMA or placebo. Efficacy of RIT was assessed using percentage bronchoalveolar lavage fluid (BALF) eosinophils. Additionally, a variety of immunologic assays were performed. Eosinophilic airway inflammation significantly decreased over time in asthmatic cats given RIT using sensitizing allergen or unrelated allergen (P<0.001). In dually sensitized cats, single allergen RIT but not placebo reduced airway eosinophilia (P=0.038). Differences in allergen-specific lymphocyte proliferation, in the number of IL-10 producing cells and in the percentage T regulatory cells were detected between asthmatic cats getting RIT and controls. Cross-protection manifested by reduced airway eosinophilia was noted in cats treated with RIT allergens which did not completely match allergen used in asthma induction. However, the mechanism of immunologic tolerance may differ when improperly matched allergens to the sensitizing allergens are used in RIT.

Upregulation of Proinflammatory Cytokine Production in Response to Bacterial Pathogen-Associated Molecular Patterns in Dogs with Diabetes Mellitus Undergoing Insulin Therapy

Background: Metabolic alterations associated with diabetes mellitus alter innate immunity. Dogs often develop infectious or inflammatory complications related to diabetes mellitus, yet little is known about the effects of diabetes mellitus on the immune system in this species. Methods: Prospective evaluation in dogs with poorly regulated spontaneous type 1 diabetes mellitus (T1DM). In vitro leukocyte cytokine response to lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PG) was compared between dogs with T1DM and healthy dogs. Additionally, the effect of acute in vitro glucose exposure on leukocyte tumor necrosis factor (TNF) production from healthy dogs was measured. Results: Leukocytes from dogs with T1DM had significantly greater TNF production after LTA and PG stimulation compared with leukocytes from healthy dogs. Leukocyte interleukin (IL)-6 production was greater after stimulation with LPS, LTA, PG, and phosphate-buffered saline in the T1DM group. No such difference was noted when evaluating IL-10 production between groups regardless of stimulant. Dogs with T1DM had significantly greater IL-6 to IL-10 production ratios than healthy dogs. Acute exposure to dextrose did not augment cytokine production from healthy canine leukocytes. Conclusions: Dogs with T1DM have altered innate immunity characterized by upregulation of proinflammatory cytokine production without a concurrent change in anti-inflammatory cytokine production. This may be one explanation for the common infectious and inflammatory complications associated with T1DM in dogs.

The impact of oral versus inhaled glucocorticoids on allergen specific IgE testing in experimentally asthmatic cats

Glucocorticoids (GCs) are palliative for allergic asthma, but allergen-specific immunotherapy (ASIT), which relies on identification of allergens, represents a potentially curative treatment. The purpose of this study was to determine if oral or inhaled GCs would interfere with identification of sensitizing allergens. The hypothesis was that oral but not inhaled GCs would interfere with accurate allergen-specific IgE identification determined by skin and serum testing in experimentally asthmatic cats. Asthma was induced in 18 cats using Bermuda grass allergen (BGA). Cats (n=6/group) were randomized to receive oral GCs (10mg prednisolone q 24 h), inhaled GCs (600 μg budesonide q 24 h) or placebo (q 24 h PO) for one month. Intradermal skin testing (IDST) and serum BGA-specific IgE were measured prior to, during and after treatment. A paired t test was used to compare groups pre- and post-treatment (P<0.05 significant). IDST reactivity was eliminated in 4/6, 3/6, and 1/6 cats receiving oral GCs, inhaled GCs, and placebo respectively. Two weeks after stopping treatment, IDST was again positive in all cats. Serum IgE reactivity to BGA was not significantly diminished by any treatment. In conclusion, a two-week withdrawal from GCs is adequate for IDST, but may not be necessary for serum IgE testing.

Flow cytometric determination of allergen-specific T lymphocyte proliferation from whole blood in experimentally asthmatic cats

The ability to quantify feline lymphocyte proliferation, especially to specific antigen or allergen, would be valuable in experimental models and naturally developing disease where activated lymphocytes drive immune responses. Traditional proliferation assays may pose radioactivity hazards, lack the ability to distinguish viable from non-viable cells, and cannot discriminate individual populations of proliferating lymphocytes (e.g., the CD4+ T cell class). We hypothesized that in an experimental model of feline allergic asthma a four-color flow cytometric assay capable of simultaneously detecting division, viability and cell surface markers (pan T cell marker CD5 or CD4) would allow characterization of lymphocytes stimulated ex vivo using the sensitizing allergen, Bermuda grass (BGA). Peripheral blood mononuclear cells were harvested from eight experimentally asthmatic cats to validate and optimize use of a cell proliferation dye or bromodeoxyuridine (BrdU) with BGA-specific stimulation in a lymphocyte proliferation flow cytometric assay. Only the latter reagent was suitable in the cat. After a 3 day incubation, antibodies with different fluorochromes were used to identify BrdU, viable cells, CD5 and CD4 for subsequent flow cytometric analysis. In asthmatic cats, the group mean ± SEM of proliferating CD5+ lymphocytes was 2.3 ± 0.5%. The group mean ± SEM of proliferating CD4+ lymphocytes was 1.2 ± 0.3%. Flow cytometry is a sensitive method for detecting simultaneous proliferation and viability of very minor populations of allergen-specific lymphocytes in experimentally asthmatic cats.

Oral glucocorticoids diminish the efficacy of allergen-specific immunotherapy in experimental feline asthma.

Allergen-specific rush immunotherapy (RIT) shows promise in treating asthma; however, pet cats will likely require at least initial concurrent glucocorticoids (GCs) to control serious clinical signs. How the immunosuppressive effects of GCs would impact RIT in cats is unknown. The hypothesis of this study was that oral, but not inhaled GCs will diminish the efficacy of RIT in experimental feline asthma. Cats (n=6/group) were sensitized using Bermuda grass allergen (BGA) and randomized to receive BGA-specific RIT for 9 months with an oral GC (prednisolone 10mg daily), inhaled GC (fluticasone 220 μg twice daily), or placebo administered for the first 6 months. Bronchoalveolar lavage fluid (BALF) percent eosinophils and other immunological assays were performed. Eosinophilic airway inflammation was suppressed in all groups at month 6 of RIT (group mean ± SD, 5 ± 2%, 13 ± 4%, and 7 ± 2% for oral GC, inhaled GC, and placebo, respectively; P=0.291). BALF percent eosinophils significantly increased over time only in oral GC/RIT cats between months 6 and 9 (P=0.031). Placebo/RIT cats had significant decreases over time in BGA-specific serum IgE (P=0.031). Concentration of interleukin (IL)-5 in BALF significantly increased over time in inhaled GC/RIT cats (P=0.031). No significant differences were found between groups at month 6 or over time in each group for BGA-specific lymphocyte blastogenesis, percent blood T regulatory cells, or number of IL-10-producing cells. Given the significant increase of airway eosinophilia over time in RIT cats initially treated with an oral GC, inhaled GCs might be better for dampening eosinophilic inflammation until RIT normalizes the dysregulated immune system.

Comparison of direct and indirect bronchoprovocation testing using ventilator-acquired pulmonary mechanics in healthy cats and cats with experimental allergic asthma

Airway hyperresponsiveness (AHR) is a key feature of asthma and can be measured using bronchoprovocation. Direct (methacholine, MCh) or indirect (adenosine-5-monophosphate, AMP; or mannitol) bronchoprovocants are used in human patients, the latter inducing AHR only with pre-existing airway inflammation. The present study compared the responses to direct (MCh) and indirect (mannitol, AMP) bronchoprovocation in healthy and asthmatic cats (n=6/group). The order of bronchoprovocant was randomized using a published table of random numbers and there was a 1-month washout before crossover to the next treatment. Pulmonary mechanics were measured in anesthetized and mechanically ventilated cats using a critical care ventilator. Saline at baseline and increasing doses of each bronchoprovocant were aerosolized for 30 s, followed by 4 min of data collection between doses. The endpoint for each bronchoprovocant was reached when airway resistance exceeded 200% of baseline values (EC200Raw). There was a significant difference (P<0.001) in the airway response of asthmatic vs. healthy cats over the range of MCh concentrations, despite there being no significant difference in the EC200Raw between the groups. Response to MCh was significantly greater (P<0.05) in asthmatic than in healthy cats at MCh concentrations as low as 0.0625 mg/mL. For AMP, a small subset of asthmatics (n=2/6) responded at low concentrations; four asthmatic cats and all healthy cats failed to respond even to the highest concentrations of AMP. One asthmatic cat but no healthy cats responded to mannitol. In conclusion, MCh discriminated asthmatic from healthy cats but neither AMP nor mannitol was an effective bronchoprovocant in this model.

Noninvasive Recognition and Biomarkers of Early Allergic Asthma in Cats Using Multivariate Statistical Analysis of NMR Spectra of Exhaled Breath Condensate

Asthma is prevalent in children and cats, and needs means of noninvasive diagnosis. We sought to distinguish noninvasively the differences in 53 cats before and soon after induction of allergic asthma, using NMR spectra of exhaled breath condensate (EBC). Statistical pattern recognition was improved considerably by preprocessing the spectra with probabilistic quotient normalization and glog transformation. Classification of the 106 preprocessed spectra by principal component analysis and partial least squares with discriminant analysis (PLS-DA) appears to be impaired by variances unrelated to eosinophilic asthma. By filtering out confounding variances, orthogonal signal correction (OSC) PLS-DA greatly improved the separation of the healthy and early asthmatic states, attaining 94% specificity and 94% sensitivity in predictions. OSC enhancement of multi-level PLS-DA boosted the specificity of the prediction to 100%. OSC-PLS-DA of the normalized spectra suggest the most promising biomarkers of allergic asthma in cats to include increased acetone, metabolite(s) with overlapped NMR peaks near 5.8 ppm, and a hydroxyphenyl-containing metabolite, as well as decreased phthalate. Acetone is elevated in the EBC of 74% of the cats with early asthma. The noninvasive detection of early experimental asthma, biomarkers in EBC, and metabolic perturbation invite further investigation of the diagnostic potential in humans.

Hyperascorbaemia in dogs admitted to a teaching hospital intensive care unit

OBJECTIVE To determine whether or not dogs develop a deficiency of ascorbic acid during hospitalisation in an intensive care unit. METHODS Blood samples were collected daily for up to three days from dogs hospitalised in an intensive care unit for 36 to 72 hours (n = 16) or ê72 hours (n = 20) and from healthy dogs (n = 13). Plasma total ascorbic acid concentrations were measured using a colorimetric method involving a reaction between ascorbic acid, 2,6 dichlorophenol-indophenol, thiourea and dinitrophenyl hydrazine. Additionally, clinical data were recorded for each patient. RESULTS Dogs hospitalised for ê72 hours had significantly greater plasma ascorbic acid concentrations on day 3 compared to days 1 and 2. There was no difference in plasma ascorbic acid concentrations between days 1 and 2 for dogs hospitalised for 36 to 72 hours. Plasma ascorbic acid concentrations were significantly greater for each day of sampling for the hospitalised dogs compared to the control dogs. CLINICAL SIGNIFICANCE Plasma ascorbic acid concentrations appear to increase during hospitalisation, and supplementation may not be indicated in dogs hospitalised in an intensive care unit.