Diagnostic utility of the total nucleated cell count for differentiation of septic and sterile peritoneal effusions in dogs.

BACKGROUND: Rapid and accurate diagnosis of septic peritonitis is critical for initiating appropriate medical and surgical management. OBJECTIVES: The aim of this study was to determine the diagnostic utility of the total nucleated cell count (TNCC), absolute neutrophil count, neutrophil percentage, and total protein (TP) to distinguish septic versus non-septic peritoneal effusions in dogs. METHODS: Electronic medical records were retrospectively searched for peritoneal fluid samples from 2008 to 2018 and classified as septic or non-septic based on bacterial culture and/or cytology results. Receiver operator characteristic curves (ROCs) were used to describe the overall diagnostic utility of each test, with optimal cutpoints analyzed to dichotomize continuous variables. Positive and negative likelihood ratios were calculated at these cutpoints. RESULTS: A total of 166 unique samples, including 87 septic and 79 non-septic peritoneal effusions, were included. There were no significant differences in dog sex, age, or days hospitalized between groups. Septic effusions had significantly higher TP, TNCC, absolute neutrophil count, and neutrophil percentage compared with non-septic effusions. The area under the curve of the ROC curves was TNCC (0.80), absolute neutrophil count (0.80), neutrophil percentage (0.64), and TP (0.63). For TNCC and absolute neutrophil count, optimal cutoffs were 17.13 × 103 cells/µL and 19.88 × 103 cells/µL, resulting in positive and negative likelihood ratios of 2.39 and 0.28 and 2.85 and 0.28, respectively. CONCLUSIONS: Total nucleated cell counts and absolute neutrophil counts aid in the differentiation of septic and non-septic peritoneal effusions with similar diagnostic utility but are not sufficiently sensitive or specific to use without concurrent microscopic evaluation.

Performance of a manually operated salad spinner centrifuge for serum separation in the healthy domestic horse (Equus caballus) and southern white rhinoceros (Ceratotherium simum).

BACKGROUND: Field veterinarians and researchers studying wild species, such as the southern white rhinoceros, often work in remote areas with limited access to standard laboratory equipment, hindering the ability to measure serum analytes. OBJECTIVES: The first objective was to produce an inexpensive, manually operated centrifuge that could accept standard laboratory tubes by modifying a consumer-grade salad spinner with low-cost materials. The second objective was to compare biochemistry analysis results obtained from equine and southern white rhinoceros serum separated by traditional laboratory and manual salad spinner centrifugation. METHODS: We optimized the design and serum separation protocol using non-anticoagulated equine blood. Equine and rhinoceros serum samples were separated by manual salad spinner or traditional laboratory centrifugation. Measured analytes included sodium, potassium, chloride, urea nitrogen, creatinine, phosphorous, total calcium, magnesium, glucose, total protein, albumin, globulin, creatinine kinase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, total bilirubin, bicarbonate, sorbitol dehydrogenase, and triglycerides. Results obtained from serum separated by each centrifugation technique were compared by Deming regression and Bland-Altman analyses. RESULTS: A tube adaptor insert modeled after a swing angle rotor and a two-step salad spinner centrifugation yielded serum comparable to traditional laboratory centrifugation. For the majority of analytes, no proportional or constant biases were detected between centrifugation methods. A positive proportional bias in the measurement of ALP in serum separated by manual centrifugation was detected in both equine and rhinoceros samples. CONCLUSIONS: Manual centrifugation with a modified salad spinner yields diagnostic quality serum suitable for the measurement of most standard biochemistry analytes.

Evaluation and establishment of reference intervals using the i-STAT1 blood chemistry analyzer in turkeys.

In veterinary medicine, point-of-care testing techniques have become popular, since they provide immediate results and only small amounts of blood are needed. The handheld i-STAT1 blood analyzer is used by poultry researchers and veterinarians; however, no studies have evaluated the accuracy of this analyzer determined reference intervals in turkey blood. The objectives of this study were to 1) investigate the effect of storage time on turkey blood analytes, 2) compare the results obtained by the i-STAT1 analyzer to those obtained by the GEM Premier 3000, a conventional laboratory analyzer, and 3) establish reference intervals for blood gases and chemistry analytes in growing turkeys using the i-Stat. For the first and second objectives, we used the CG8+ i-STAT1 cartridges to test blood from 30 healthy turkeys in triplicate and once with the conventional analyzer. To establish the reference intervals, we tested a total 330 blood samples from healthy turkeys from 6 independent flocks during a 3-yr period. Blood samples were then divided into brooder (<1 wk) and growing (1-12 wk of age). Friedman's test demonstrated significant time-dependent changes in blood gas analytes, but not for electrolytes. Bland-Altman analysis revealed that there was agreement between the i-STAT1 and the GEM Premier 300 for most of the analytes. However, Passing-Bablok regression analysis identified constant and proportional biases in the measurement of multiple analytes. Tukey's test revealed significant differences in the whole blood analytes between the means of brooding and growing birds. The data presented in the present study provide a basis for measuring and interpreting blood analytes in the brooding and growing stages of the turkey lifecycle, offering a new approach to health monitoring in growing turkeys.

Total protein concentration as a predictor of neoplastic peritoneal and pleural effusions of dogs.

BACKGROUND: The diagnosis of neoplastic cavitary effusions requires the identification of neoplastic cells in effusions, yet the cytologic appearance of neoplastic effusions can be highly variable due to the varied mechanisms of formation. Additional parameters might aid in the interpretation of equivocal cytologic results. OBJECTIVES: Our goal was to evaluate whether total protein concentrations can be used to support the diagnosis of neoplasia in the peritoneal and pleural effusions of dogs with lower cellularities (≤5000 nucleated cells/µL). METHODS: Pleural and peritoneal fluid analyses from dogs presented to the University of Illinois Veterinary Teaching Hospital between 2014 and 2019 were evaluated retrospectively. Effusions were categorized as neoplastic or non-neoplastic based on histology or cytology. Non-neoplastic effusions were subcategorized according to mechanism: decreased oncotic pressure, increased hydrostatic pressure, increased vascular permeability, leakage of urine, and leakage of lymph. The TP and blood albumin to fluid TP ratio (Albblood :TPfluid ) were compared among groups. RESULTS: Twenty-seven neoplastic and 65 non-neoplastic cases were evaluated. TP was higher in the neoplastic group (P = .001) than in the non-neoplastic group. Neoplastic effusions had a lower Albblood :TPfluid than non-neoplastic (P = .001), and effusions with Albblood :TPfluid of ≤0.6 were 5.6 times more likely to be neoplastic (95% CI 1.69-17.36; P = .003). CONCLUSIONS: Fluid TP concentrations were significantly greater in neoplastic than non-neoplastic effusions; however, given the considerable overlap between groups, the diagnostic utility of this difference is low. A neoplastic etiology might be more likely in cases with an Albblood :TPfluid ≤0.6.

Macrophage uptake of oxidized and acetylated low-density lipoproteins and generation of reactive oxygen species are regulated by linear stiffness of the growth surface.

Macrophages are key players in the development of atherosclerosis: they scavenge lipid, transform into foam cells, and produce proinflammatory mediators. At the same time, the arterial wall undergoes profound changes in its mechanical properties. We recently showed that macrophage morphology and proinflammatory potential are regulated by the linear stiffness of the growth surface. Here we asked whether linear stiffness also regulates lipid uptake by macrophages. We cultured murine bone marrow-derived macrophages (BMMs) on polyacrylamide gels modeling stiffness of healthy (1kPa) and diseased (10-150kPa) blood vessels. In unprimed BMMs, increased linear stiffness increased uptake of oxidized (oxLDL) and acetylated (acLDL) low density lipoproteins and generation of reactive oxygen species, but did not alter phagocytosis of bacteria or silica particles. Macrophages adapted to stiff growth surfaces had increased mRNA and protein expression of two key lipoprotein receptors: CD36 and scavenger receptor b1. Regulation of the lipoprotein receptor, lectin-like receptor for ox-LDL, was more complex: mRNA expression decreased but surface protein expression increased with increased stiffness. Focal adhesion kinase was required for maximal uptake of oxLDL, but not of acLDL. Uptake of oxLDL and acLDL was independent of rho-associated coiled coil kinase. Through pharmacologic inhibition and genetic deletion, we found that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, plays an inhibitory role in the uptake of acLDL, but not oxLDL. Together, these results implicate mechanical signaling in the uptake of acLDL and oxLDL, opening up the possibility of new pharmacologic targets to modulate lipid uptake by macrophages in vivo.

Comparison of portable and conventional laboratory analyzers for biochemical tests in chickens.

Antemortem blood biochemical and blood gas analyses are routinely used in health screening and diagnosis of disease in domestic veterinary species. These testing modalities are not routinely performed in poultry, in part, due to the distance from the diagnostic laboratory. Portable blood analyzers such as the i-STAT and VetScan (VS2) can be used to obtain results on the farm without delay, potentially offering a more practical option for poultry practitioners. We investigated the time effect on blood chemistry values and compared the results obtained using the i-STAT and VS2 with those obtained using conventional laboratory analyzers (GEM Premier 3000 and Cobas c501, respectively). We tested blood from 60 healthy chickens. Each sample was tested in triplicate using each of the portable analyzers and once using conventional analyzers. All samples were analyzed within 60 minutes of collection. The concentrations of some analytes were outside the limit of detection of the portable analyzers (i.e., bile acids). Although statistically significant differences were found for some biochemical analytes over time, the actual mean or median differences were too small to be considered of clinical importance. As observed in mammals, significant time-dependent changes in blood gas analytes were observed in whole blood samples exposed to ambient air. Correlation coefficients between portable and conventional analyzers were moderate to high for most of the analytes. For the most part, there was an agreement between the portable and conventional analyzers. We identified constant and proportional biases in the measurement of multiple analytes by both the i-STAT and VS2. Future studies are warranted to establish analyzer-specific reference intervals for poultry.

LACTIC ACIDOSIS INDUCED BY MANUAL RESTRAINT FOR HEALTH EVALUATION AND COMPARISON OF TWO POINT-OF-CARE ANALYZERS IN HEALTHY LOGGERHEAD SEA TURTLES (CARETTA CARETTA).

Sea turtles are often restrained manually for brief periods during veterinary evaluation and care in rescue, rehabilitation, research, and aquarium settings. Blood gas values and lactate are routinely evaluated during triage of sea turtles, and lactate clearance is of prognostic significance in cold-stunned individuals. Although increases in blood lactate have been associated with muscle exertion, experimental forced submergence, trawl and pound net capture, and general anesthesia, changes in blood lactate associated with short periods of manual restraint have not been evaluated. Venous blood gas and lactate values were tested in 16 juvenile loggerhead sea turtles (Caretta caretta) before and after manual restraint for a 15-min routine veterinary examination. The agreement of blood lactate values between two point-of care analyzers (i-STAT and Lactate Plus) was also compared. Blood pH and bicarbonate (HCO3-) decreased significantly (P < 0.001), and partial pressure of carbon dioxide (pCO2) increased significantly (P < 0.0001) after 15 min. Lactate increased significantly between time points for both analyzers (P < 0.0001). Linear regression analysis showed excellent correlation for lactate measurements obtained on both analyzers (r = 0.998). The mean difference in lactate concentrations between the analyzers was statistically significant, indicating that the methods cannot be used interchangeably (P < 0.0001). Deming regression and Bland-Altman plots identified a slight negative proportional bias for lactate measurement by the Lactate Plus compared with the i-STAT. These results suggest that clinicians should evaluate blood gas values and lactate at the beginning of health evaluations and interpret serial lactate values in sea turtles with caution, because even short periods of manual restraint can induce lactic acidosis and considerably influence these values.

Toll-like receptor signaling in macrophages is regulated by extracellular substrate stiffness and Rho-associated coiled-coil kinase (ROCK1/2).

Macrophages participate in immunity, tissue repair and tissue homeostasis. Activation of Toll-like receptors (TLRs) by conserved exogenous or endogenous structures initiates signaling cascades that result in the release of cytokines such as tumor necrosis factor α (TNFα). Extracellular substrate stiffness is known to regulate functions of non-immune cells through a process called mechanotransduction, yet less is known about how physical cues affect macrophage function or TLR signaling. To investigate this question, we cultured murine primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells on fibronectin-coated polyacrylamide (PA) gels of defined stiffnesses (1, 20 and 150 kPa) that approximate the physical properties of physiologic tissues. BMMs on all gels were smaller and more circular than those on rigid glass. Macrophages on intermediate stiffness 20 kPa PA gels were slightly larger and less circular than those on either 1 or 150 kPa. Secretion of the pro-inflammatory cytokine, TNFα, in response to stimulation of TLR4 and TLR9 was increased in macrophages grown on soft gels versus more rigid gels, particularly for BMMs. Inhibition of the rho-associated coiled-coil kinase 1/2 (ROCK1/2), key mediators in cell contractility and mechanotransduction, enhanced release of TNFα in response to stimulation of TLR4. ROCK1/2 inhibition enhanced phosphorylation of the TLR downstream signaling molecules, p38, ERK1/2 and NFκB. Our data indicate that physical cues from the extracellular environment regulate macrophage morphology and TLR signaling. These findings have important implications in the regulation of macrophage function in diseased tissues and offer a novel pharmacological target for the manipulation of macrophage function in vivo.

Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines.

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor-replete or specific coagulation factor-deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X-deficient plasma; residual thrombin generation in factor VII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

Role of tissue factor expression in thrombin generation by canine tumor cells.

OBJECTIVE: To measure thrombin generation by high and low tissue factor (TF)-expressing canine cancer cell lines. SAMPLE: Canine cell lines CMT25 (high TF-expressing mammary gland tumor cell line) and HMPOS (low TF-expressing osteosarcoma cell line). PROCEDURES: Thrombin generation by cancer cells was measured in pooled normal canine plasma by use of calibrated automated thrombography without added trigger reagents. Results were expressed as lag time, time to peak thrombin concentration, peak thrombin concentration, and total thrombin concentration or thrombin generation potential. Corn trypsin inhibitor, hirudin, and annexin V were used to inhibit contact activation, thrombin formation, and phosphatidylserine activity, respectively. Pooled normal human plasma deficient in coagulation factors VII, VIII, IX, X, XI, or XII was used to assess the role of individual coagulation factors on thrombin generation. RESULTS: CMT25 generated significantly more thrombin than did HMPOS (mean ± SD, 3,555 ± 604 nM thrombin•min and 636 ± 440 nM thrombin•min, respectively). Thrombin generation of CMT25 was dependent on factor VII and phosphatidylserine and was independent of contact activation. In contrast, thrombin generation of HMPOS was attributed to contact activation. CONCLUSIONS AND CLINICAL RELEVANCE: High TF-expressing canine mammary cancer cells generated thrombin in a plasma milieu in vitro in a factor VII- and phosphatidylserine-dependent manner. These findings support a role for TF in hypercoagulability detected in dogs with mammary gland tumors and potentially for other tumors that strongly express TF.

TLR9 stability and signaling are regulated by phosphorylation and cell stress.

Innate sensing of pathogens elicits protective immune responses through pattern recognition receptors, including Toll-like receptors. Although signaling by Toll-like receptors is regulated at multiple steps, including localization, trafficking, proteolytic cleavage, and phosphorylation, the significance of post-translational modifications and cellular stress response on Toll-like receptor stability and signaling is still largely unknown. In the present study, we investigated the role of cytoplasmic tyrosine motifs in Toll-like receptor-9 stability, proteolytic cleavage, and signaling. We demonstrated that tyrosine phosphorylation is essential for mouse Toll-like receptor-9 protein stability and signaling. Upon inhibition of tyrosine kinases with piceatannol, Toll-like receptor-9 tyrosine phosphorylation induced by CpG deoxyribonucleic acid was inhibited, which correlated with decreased signaling. Furthermore, inhibition of Src kinases with 1-tert-Butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine also inhibited response to CpG deoxyribonucleic acid. Toll-like receptor-9 protein stability was also sensitive to autophagy, the cellular stress response pathway, and infection by a deoxyribonucleic acid virus. Whereas autophagy induced by rapamycin or low serum levels caused a preferential loss of the mature p80 proteolytic cleavage product, infection with herpes simplex virus-1 and induction of cell stress with tunicamycin caused preferential loss of full-length Toll-like receptor-9, which is localized to the endoplasmic reticulum. Our data reveal new information about the stability and signaling of Toll-like receptor-9 and suggest that immune evasion mechanisms may involve targeted loss of innate sensing receptors.

Short upstream region drives dynamic expression of hypoxia-inducible factor 1alpha during Xenopus development.

Hypoxia-inducible factor 1alpha (HIF-1alpha) plays a central role in regulating oxygen-dependent gene expression and is involved in a range of pathways implicated in cellular survival, proliferation, and development. While the posttranslational regulation of HIF-1alpha is well characterized, the relative importance of its control at the transcriptional level during development remains less clear. Although the mouse and human promoter regions have been analyzed in vitro, to date, there has been no in vivo analysis of any vertebrate HIF-1alpha promoter. To investigate the transcriptional regulation of HIF-1alpha during development of the amphibian Xenopus laevis, we have described the gene's expression pattern and isolated the xHIF-1alpha upstream regulatory regions. We show xHIF-1alpha mRNA to be constitutively expressed at low levels throughout embryogenesis, but with significant up-regulation during gastrula stages, and subsequently, in specific regions of the central nervous system and axial tissues. Our functional analysis using a series of truncated xHIF-1alpha promoter constructs demonstrates that a 173-bp region of the proximal promoter, which is 100% conserved among five allelic variants, is sufficient to drive correct expression in transgenic embryos. Although these results are corroborated by a parallel set of in vitro transfection experiments in a Xenopus cell line, some key differences suggest the importance of using transgenic methods in conjunction with in vitro assays.