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Featured Research

Research published by VCCIS' members
ACVIM consensus statement on the diagnosis of immune-mediated hemolytic anemia in dogs and cats
Oliver A Garden 1, Linda Kidd 2, Angela M Mexas 3, Yu-Mei Chang 4, Unity Jeffery 5, Shauna L Blois 6, Jonathan E Fogle 7, Amy L MacNeill 8, George Lubas 9, Adam Birkenheuer 7, Simona Buoncompagni 10, Julien R S Dandrieux 11, Antonio Di Loria 12, Claire L Fellman 13, Barbara Glanemann 4, Robert Goggs 14, Jennifer L Granick 15, Dana N LeVine 16, Claire R Sharp 17, Saralyn Smith-Carr 18, James W Swann 19, Balazs Szladovits 4, February 2019

Immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune-mediated erythrocyte destruction, and adverse consequences of long-term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence-based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.

Subcategories:  American College of Veterinary Internal Medicine Consensus Statements on IMHA
ACVIM consensus statement on the treatment of immune-mediated hemolytic anemia in dogs
James W Swann 1, Oliver A Garden 2, Claire L Fellman 3, Barbara Glanemann 4, Robert Goggs 5, Dana N LeVine 6, Andrew J Mackin 7, Nathaniel T Whitley, March 2019

Immune-mediated hemolytic anemia (IMHA) causes severe anemia in dogs and is associated with considerable morbidity and mortality. Treatment with various immunosuppressive and antithrombotic drugs has been described anecdotally and in previous studies, but little consensus exists among veterinarians as to the optimal regimen to employ and maintain after diagnosis of the disease. To address this inconsistency and provide evidence-based guidelines for treatment of IMHA in dogs, we identified and extracted data from studies published in the veterinary literature. We developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria, explanation of treatment regimens, and validity of statistical methods. In combination with our clinical experience and comparable guidelines for humans afflicted with autoimmune hemolytic anemia, we used the conclusions of this process to make a set of clinical recommendations regarding treatment of IMHA in dogs, which we refined subsequently by conducting several iterations of Delphi review. Additionally, we considered emerging treatments for IMHA in dogs and highlighted areas deserving of future research. Comments were solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted for publication. The resulting document is intended to provide clinical guidelines for management of IMHA in dogs. These guidelines should be implemented pragmatically, with consideration of animal, owner, and veterinary factors that may vary among cases.

Subcategories:  American College of Veterinary Internal Medicine Consensus Statements on IMHA
Allergy, inflammation, hepatopathy and coagulation biomarkers in dogs with suspected anaphylaxis due to insect envenomation
Kate Turner*, Corrin Boyd, Gabriele Rossi, Claire R. Sharp, Melissa A. Claus, Abbie Francis, Lisa Smar, August 2022

To compare concentrations of biomarkers of; allergy [mast cell tryptase (MCT) and histamine], inflammation [interleukin (IL)-6,-10, and−18, CXCL8, CCL2, keratinocyte chemoattractant (KC), C-reactive protein (CRP)], endothelial glycocalyx shedding (hyaluronan), coagulation [prothrombin time, activated partial thromboplastin time, fibrinogen concentration, and von Willebrand Factor antigen, protein C (PC) and antithrombin (AT) activity], and hepatopathy [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin] between dogs with anaphylaxis after suspected insect exposure, dogs with critical illness, and healthy dogs.

Subcategories:  General
Biomarkers of endothelial activation and inflammation in dogs with organ dysfunction secondary to sepsis
Sarah Gaudette, Lisa Smart, Andrew P. Woodward, Claire R. Sharp, Dez Hughes, Simon R. Bailey, Julien R. S. Dandrieux, Leilani Santos, Manuel Boller, July 2023

Alteration in endothelial function during sepsis is thought to play a key role in the progression of organ failure. We herein compared plasma concentrations of endothelial activation biomarkers vascular endothelial growth factor (VEGF), hyaluronan (HA), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF), as well as inflammatory mediator concentrations (IL-6, IL-8, IL-10, C-reactive protein and monocyte chemoattractant protein-1) in dogs with sepsis to healthy dogs.

Subcategories:  General
CD4+CD25+ regulatory T cells are infected and activated during acute FIV infection
Angela M Mexas 1, Jonathan E Fogle, Wayne A Tompkins, Mary B Tompkins, August 2008

HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Treg cells). Treg cells have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Treg cells become infected and activated during the acute infection with FIV leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU1 and blood and lymph node cells were collected at weekly intervals following inoculation. Real-time RT-PCR was used to determine plasma viremia and the relative expression of FIV, FoxP3, TGF-beta, and GAPDH mRNA copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of surface TGF-beta and intracellular FoxP3 in CD4+CD25+ and CD4+CD25- T cells at each time-point. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays. Our results showed that peak viremia occurred at 2 weeks post infection and correlated with maximal infectivity in CD4+CD25+ T cell populations. FIV-gag-mRNA levels were higher in CD4+CD25+ T cells than CD4+CD25- T cells throughout the acute phase of infection. Induction of FoxP3 and TGF-beta indicated activation of Treg cells during the acute stage infection, which was confirmed by Treg cell suppression of IL-2 production by CD4+ Th cells in an ELISPOT assay. Our findings support the hypothesis that early activation of Treg immunosuppressor function may limit an effective anti-FIV response, contributing to the establishment of chronic infection and the immunodeficiency caused by this virus.

Subcategories:  Regulatory T Cells and Myeloid-Derived Suppressor Cells in Dogs and Cats
Cell-Free DNA and DNase Activity in Dogs with Immune-Mediated Hemolytic Anemia
U. Jeffery, L. Ruterbories, R. Hanel, D.N. LeVine, August 2017

Immune-mediated hemolytic anemia (IMHA) in dogs has a high risk of thrombosis and is associated with marked neutrophilia and necrosis. Cell death and release of neutrophil extracellular traps contribute to increased serum concentrations of cell-free DNA, and in human autoimmune disease reduced DNase activity further increases cell-free DNA. Free DNA in blood has prothrombotic properties and could contribute to hypercoagulability in IMHA.

Subcategories:  Neutrophil Extracellular Traps (NETs), Microparticles, and Thrombosis in IMHA
Characterisation of the Immunophenotype of Dogs with Primary Immune-Mediated Haemolytic Anaemia
James W. Swann,1,2 Kelly Woods,2 Ying Wu,2 Barbara Glanemann,1 and Oliver A. Garden1,2,*, December 2016

Immune-mediated haemolytic anaemia (IMHA) is reported to be the most common autoimmune disease of dogs, resulting in significant morbidity and mortality in affected animals. Haemolysis is caused by the action of autoantibodies, but the immunological changes that result in their production have not been elucidated.

Subcategories:  Regulatory T Cells and Myeloid-Derived Suppressor Cells in Dogs and Cats
Defining sepsis in small animals
Stefano Cortellini, Amy E. DeClue, Massimo Giunti, Robert Goggs, Kate Hopper, Julie M Menard, Rodrigo C Rabelo, Elizabeth A Rozanski, Claire R. Sharp, Deborah C. Silverstein, Virginia Sinnott-Stutzman, Giacomo Stanzani, January 2024

To discuss the definitions of sepsis in human and veterinary medicine.

Subcategories:  General
Demographic Characteristics, Survival and Prognostic Factors for Mortality in Cats with Primary Immune-Mediated Hemolytic Anemia
J.W. Swann, B. Szladovits, B. Glanemann, December 2015

  Immune-mediated hemolytic anemia (IMHA) is uncommon in cats, but may result in severe disease. Demographic predispositions for development of the disease and prognostic factors for mortality have not been investigated previously.

Subcategories:  Clinical Immunology Research
Development and implementation of a novel immune thrombocytopenia bleeding score for dogs
Kelly M. Makielski, Marjory B. Brooks, Chong Wang, Jonah N. Cullen, Annette M. O'Connor, Dana N. LeVine, April 2018

A method of quantifying clinical bleeding in dogs with immune thrombocytopenia (ITP) is needed because ITP patients have variable bleeding tendencies that inconsistently correlate with platelet count. A scoring system will facilitate patient comparisons and allow stratification based on bleeding severity in clinical trials.

Subcategories:  Clinical Immunology Research
Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia
Unity Jeffery 1, Kayoko Kimura 2, Robert Gray 3, Paul Lueth 2, Bryan Bellaire 2, Dana LeVine 4, October 2015

Neutrophil extracellular traps (NETs) are webs of DNA and protein with both anti-microbial and pro-thrombotic properties which have not been previously reported in dogs. To confirm dog neutrophils can form NETs, neutrophils were isolated from healthy dogs, and stimulated in vitro with 2μM, 8μM, 31μM, and 125μM platelet activating factor (PAF) or 0.03μM, 0.1μM, 0.4μM, 1.6μM and 6.4μM phorbol-12-myristate-13-acetate (PMA). Extracellular DNA was measured using the cell impermeable dye Sytox Green every hour for 4h. At 4h, extracellular DNA was significantly greater than non-stimulated cells at concentrations ≥31μM and ≥0.1μM for PAF and PMA, respectively. Cells stimulated with 31.25μM PAF reached maximal fluorescence by 1h, whereas maximal fluorescence was not achieved until 2h for cells stimulated with 0.1μM PMA. Immunofluorescent imaging using DAPI and anti-elastase antibody confirmed that extracellular DNA is released as NETs. As NETs have been implicated in thrombosis, nucleosomes, a marker correlated with NET formation, were measured in the serum of dogs with the thrombotic disorder primary immune-mediated hemolytic anemia (IMHA) (n=7) and healthy controls (n=20) using a commercially available ELISA. NETs were significantly higher in IMHA cases than controls (median 0.12 and 0.90, respectively, p=0.01), but there were large positive interferences associated with hemolysis and icterus. In summary, the study is the first to describe NET generation by canine neutrophils and provides preliminary evidence that a marker associated with NETs is elevated in IMHA. However, this apparent elevation must be interpreted with caution due to the effect of interference, emphasizing the need for a more specific and robust assay for NETs in clinical samples.

Subcategories:  Neutrophil Extracellular Traps (NETs), Microparticles, and Thrombosis in IMHA
Endothelial alterations in a canine model of immune thrombocytopenia
Dana N. LeVine, Rachel E. Cianciolo, Keith E. Linder, Petra Bizikova, Adam J. Birkenheuer, Marjory B. Brooks, Abdelghaffar K. Salous, Shila K. Nordone, Dwight A. Bellinger, Henry Marr, Sam L. Jones, Thomas H. Fischer, Yu Deng,Marshall Mazepa & Nigel S. Key, December 2015

Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.

Subcategories:  Clinical Immunology Research
In vivo depletion of CD4+CD25+ regulatory T cells in cats
S Rochelle Smithberg 1, Jonathan E Fogle, Angela M Mexas, Stacie K Reckling, Susan M Lankford, Mary B Tompkins, Gregg A Dean, October 2007

To establish a characterized model of regulatory T cell (Treg) depletion in the cat we assessed the kinetics of depletion and rebound in peripheral and central lymphoid compartments after treatment with anti-CD25 antibody as determined by cell surface markers and FOXP3 mRNA expression. An 82% decrease in circulating CD4+CD25+ Tregs was observed by day 11 after treatment. CD4+CD25+ cells were also reduced in the thymus (69%), secondary lymphoid tissues (66%), and gut (67%). Although CD4+CD25+ cells rebound by day 35 post-treatment, FOXP3 levels remain depressed suggesting anti-CD25 antibody treatment has a sustainable diminutive effect on the Treg population. To determine whether CD25+ Treg depletion strategies also deplete activated CD25+ effector cells, cats were immunized with feline immunodeficiency virus (FIV) p24-GST recombinant protein, allowing them to develop a measurable memory response, prior to depletion with anti-CD25 antibody. Anti-FIV p24-GST effector cell activity in peripheral blood after depletion was sustained as determined by antigen-specific T cell proliferation and humoral responses against FIV p24-GST with an ELISA for antigen-specific feline IgG. Furthermore, development of an anti-mouse response in Treg-depleted cats was similar to control levels indicating the retained capacity to respond to a novel antigen. We conclude that despite alterations in CD25+ cell levels during depletion, the feline immune system remains functional. We demonstrate here a model for the study of disease pathogenesis in the context of reduced numbers of immunosuppressive CD4+CD25+ Tregs throughout the feline immune system.

Subcategories:  Regulatory T Cells and Myeloid-Derived Suppressor Cells in Dogs and Cats
Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review
Robert Goggs 1, Unity Jeffery 2, Dana N LeVine 3, Ronald H L Li 4, July 2019

Immunothrombosis is a potentially beneficial physiological process that aids innate immunity and host defense against pathogen invasion. However, this process can also be damaging when it occurs to excess or in critical blood vessels. Formation of extracellular traps by leukocytes, particularly neutrophils, is central to our understanding of immunothrombosis. In addition to degranulation and phagocytosis, extracellular traps are the third mechanism by which neutrophils combat potential pathogens. These traps consist of extracellular DNA decorated with bactericidal cellular proteins, including elastase, myeloperoxidase, and cathepsins. Neutrophils can release these structures as part of a controlled cell-death process or via a process termed vital NETosis that enables the cells to extrude DNA but remain viable. There is accumulating evidence that NETosis occurs in companion animals, including dogs, horses, and cats, and that it actively contributes to pathogenesis. Numerous studies have been published detailing various methods for identification and quantification of extracellular trap formation, including cell-free DNA, measurements of histones and proteins such as high-mobility group box-1, and techniques involving microscopy and flow cytometry. Here, we outline the present understanding of these phenomena and the mechanisms of extracellular trap formation. We critically review the data regarding measurement of NETosis in companion animals, summarize the existing literature on NETosis in veterinary species, and speculate on what therapeutic options these insights might present to clinicians in the future.

Subcategories:  Neutrophil Extracellular Traps (NETs), Microparticles, and Thrombosis in IMHA
Phenotypic and transcriptomic characterization of canine myeloid-derived suppressor cells
Michelle R Goulart 1 2, Sabina I Hlavaty 3, Yu-Mei Chang 1, Gerry Polton 4, Anneliese Stell 1, James Perry 3, Ying Wu 1, Eshita Sharma 5, John Broxholme 5, Avery C Lee 6, Balazs Szladovits 1, Mark Turmaine 7, John Gribben 2, Dong Xia 1, Oliver A Garden 8, March 2019

Myeloid-derived suppressor cells (MDSCs) are key players in immune evasion, tumor progression and metastasis. MDSCs accumulate under various pathological states and fall into two functionally and phenotypically distinct subsets that have been identified in humans and mice: polymorphonuclear (PMN)-MDSCs and monocytic (M)-MDSCs. As dogs are an excellent model for human tumor development and progression, we set out to identify PMN-MDSCs and M-MDSCs in clinical canine oncology patients. Canine hypodense MHC class II-CD5-CD21-CD11b+ cells can be subdivided into polymorphonuclear (CADO48A+CD14-) and monocytic (CADO48A-CD14+) MDSC subsets. The transcriptomic signatures of PMN-MDSCs and M-MDSCs are distinct, and moreover reveal a statistically significant similarity between canine and previously published human PMN-MDSC gene expression patterns. As in humans, peripheral blood frequencies of canine PMN-MDSCs and M-MDSCs are significantly higher in dogs with cancer compared to healthy control dogs (PMN-MDSCs: p < 0.001; M-MDSCs: p < 0.01). By leveraging the power of evolution, we also identified additional conserved genes in PMN-MDSCs of multiple species that may play a role in MDSC function. Our findings therefore validate the dog as a model for studying MDSCs in the context of cancer.

Subcategories:  Regulatory T Cells and Myeloid-Derived Suppressor Cells in Dogs and Cats
Procoagulant microparticles in dogs with immune-mediated hemolytic anemia
L Kidd 1, J Geddings 2, Y Hisada 2, M Sueda 3, T Concannon 3, T Nichols 4, E Merricks 4, N Mackman 2, April 2015

Studies of some human prothrombotic diseases suggest that phosphatidylserine-positive (PS+) and tissue factor-positive (TF+) microparticles (MPs) might play a role in the pathogenesis of thrombosis or serve as biomarkers of thrombotic risk.

Subcategories:  Neutrophil Extracellular Traps (NETs), Microparticles, and Thrombosis in IMHA
Systematic Review of Prognostic Factors for Mortality in Dogs with Immune-mediated Hemolytic Anemia
J.W. Swann, B.J. Skelly, January 2015

Treatment of dogs with primary immune-mediated hemolytic anemia (IMHA) is difficult and frequently unrewarding. Prognostic factors have been evaluated in a number of previous studies, and identification of such factors would be beneficial to enable selection of appropriate therapeutic regimens and supportive care.

Subcategories:  Clinical Immunology Research

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