saline-lavage-for-sampling-of-the-canine-nasal-immune-microenvironment

Saline Lavage for Sampling of the Canine Nasal Immune Microenvironment

Evaluating the local immune microenvironment of the canine nasal cavity can be important for investigating normal tissue health and disease conditions, ...

Colorado State University

Densitometric quantitation using serum protein electrophoresis (SPE) is used to monitor monoclonal proteins (M-proteins) in human patients but has not been validated in the dog. Serum globulin concentrations, species-specific radial immunodiffusion (RID), and ELISAs are currently used in veterinary medicine.

Validation and method comparison of the use of densitometry to quantify monoclonal proteins in canine sera.

A 12-y-old spayed female Schipperke dog with a previous diagnosis of inflammatory bowel disease was presented with a 2-mo history of severe colitis. The patient's condition progressed to hepatopathy, pneumonia, and dermatitis following management with prednisolone and dexamethasone sodium phosphate. Colonic biopsies identified severe necrosuppurative colitis with free and intracellular parasitic zoites. Postmortem examination confirmed extensive chronic-active ulcerative colitis, severe acute necrotizing hepatitis and splenitis, interstitial pneumonia, ulcerative dermatitis, myelitis (bone marrow), and mild meningoencephalitis with variable numbers of intracellular and extracellular protozoal zoites. PCR on samples of fresh colon was positive for . Immunohistochemistry identified  tachyzoites in sections of colon, and a single tissue cyst in sections of brain. Administration of immunosuppressive drugs may have allowed systemic dissemination of  from the intestinal tract.

Disseminated  infection in a dog with severe colitis.

Current recommendations for monitoring disease progression and response to treatment in humans with multiple myeloma include evaluation of serum paraprotein (M-protein) concentration. Densitometry, species-specific radial immunodiffusion (RID) and ELISA methods can be used to quantify M-proteins.

Retrospective evaluation of the use of the International Myeloma Working Group response criteria in dogs with secretory multiple myeloma.

Routine electrophoresis [agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE)] and species-specific immunofixation (IF) can be used alone or in combination to detect immunoglobulin paraprotein (M-protein) and diagnose secretory myeloma-related disorders (sMRD).

Diagnostic performance of routine electrophoresis and immunofixation for the detection of immunoglobulin paraproteins (M-Proteins) in dogs with multiple myeloma and related disorders: Part 1 - Current performance.

The diagnostic performance of routine electrophoresis (agarose gel electrophoresis [AGE] and capillary zone electrophoresis [CZE]) and species-specific immunofixation (IF) for the detection of immunoglobulin paraproteins (M-proteins) and diagnosis of secretory myeloma-related disorders (sMRD) can be improved. Available canine IF targets were IgG-FC, IgA, IgM, light chain (LC), IgG4, and free LC (fLC) antibodies.

Diagnostic performance of routine electrophoresis and immunofixation for the detection of immunoglobulin paraproteins (M-Proteins) in dogs with multiple myeloma and related disorders: Part 2-Toward improved diagnostic performance.

Bullous amyloidosis is a rare disease in humans that has not been described in a veterinary species in the peer-reviewed literature. The human disease is characterised by haemorrhagic vesicles and bullae on the skin and mucosae, which form due to amyloid deposition.

Bullous amyloidosis in a horse: first description in veterinary medicine.

Densitometric quantification of myeloma paraproteins (M-proteins) is used to monitor secretory myeloma related disorders in humans and dogs. The previous work in dogs used agarose gel electrophoresis (AGE) but did not establish if other methods of serum protein electrophoresis, such as capillary zone electrophoresis (CZE), were comparable.

Method comparison for serum protein electrophoretic M-protein quantification: Agarose gel electrophoresis and capillary zone electrophoresis in canine and feline sera.

Three dogs under 12 months old were diagnosed with atypical multiple myeloma (MM), having an aggressive multifocal anaplastic round cell sarcoma in bone marrow, viscera, and/or peripheral blood, which were confirmed by cytology and immunohistochemistry to be of plasma cell origin. The intramedullary sarcomas caused myelophthisis, osteolysis, and hypercalcemia. Complete or free light chain monoclonal gammopathy in the serum and/or urine was demonstrated by protein electrophoresis and immunofixation. The polymerase chain reaction for antigen receptor rearrangement assay performed on 2 cases identified a clonally rearranged immunoglobulin gene. Neoplastic cells lacked expression of CD45, CD3, CD18, CD21, CD34, and MHCII by flow cytometry. Immunohistochemistry revealed MUM1 immunoreactivity of the neoplastic cells. Combining all data, the diagnosis was MM. An aggressive form of MM in young dogs should be a differential diagnosis for patients with an immunoglobulin-productive, B cell-clonal, CD45-negative, MUM1-positive discrete cell neoplasm arising from the bone marrow.

Atypical multiple myeloma in 3 young dogs.

Canine acute leukaemia is a heterogeneous neoplasm with multiple phenotypes. Criteria to subtype acute leukaemia by flow cytometry have not been validated. The goal of this study was to develop a panel of antibodies and objective antigen expression criteria for the assignment of lymphoid or myeloid lineage by flow cytometry. We isolated mRNA from the blood of 45 CD34+ acute leukaemia cases and measured expression of 43 genes that represent lymphoid and myeloid lineages using NanoString technology. We determined differentially expressed genes between major groups identified by unsupervised hierarchical clustering. We then evaluated the expression of antigens by flow cytometry to determine if cases could be assigned to a lineage. Two groups were identified by gene expression. Group 1/LYMPH overexpressed lymphoid-associated genes (ex. DNTT) and had a higher percentage of CD5 + CD3- cells by flow cytometry. Group 2/MYELO overexpressed myeloid-associated genes (ex. ANPEP/CD13) and had a higher percentage of class II major histocompatibility complex (MHCII)- CD14+ and/or CD18 + CD4- cells. We proposed that >12.5% CD5 + CD3- cells in the blood was indicative of lymphoid lineage, and > 3.0% CD14 + MHCII- cells or > 18% CD18 + MHCII-CD4- cells was indicative of myeloid lineage. 15/15 cases that met the proposed criteria for acute lymphocytic leukaemia were in LYMPH group and 12/15 cases that met the proposed criteria for acute myeloid leukaemia were in MYELO group. The majority of CD34+ cases that did not meet either immunophenotyping lineage criterion (12/13) clustered within the LYMPH group. In conclusion, currently available antibodies can be useful for determining canine acute leukaemia subtypes.

Using digital RNA counting to establish flow cytometry diagnostic criteria for subtypes of CD34+ canine acute leukaemia.

Hyperglobulinemia is reported in 26% of canine chronic B-cell lymphocytic leukemia (B-CLL) cases. However, few cases have been characterized by protein electrophoresis and immunofixation (IF), and the incidence of a monoclonal protein (M-protein) is unknown using these techniques.

Detection and Characterization of Paraproteinemia in Canine Chronic B-cell Lymphocytic Leukemia Using Routine and Free Light Chain Immunofixation.

Translationally relevant animal models are essential for the successful translation of basic science findings into clinical medicine. While rodent models are widely accessible, there are numerous limitations that prevent the extrapolation of findings to human medicine. One approach to overcome these limitations is to use animal models that are genetically diverse and naturally develop disease. For example, pet dogs spontaneously develop diseases that recapitulate the natural progression seen in humans and live in similar environments alongside humans. Thus, dogs represent a useful animal model for many areas of research. Despite the value of the canine model, species specific reagent limitations have hampered in depth characterization of canine immune cells, which constrains the conclusions that can be drawn from canine immunotherapy studies. To address this need, we used single-cell RNA sequencing to characterize the heterogeneity of circulating leukocytes in healthy dogs (n = 7) and osteosarcoma (OS) affected dogs (n = 10). We present a cellular atlas of leukocytes in healthy dogs, then employ the dataset to investigate the impact of primary OS tumors on the transcriptome of circulating leukocytes. We identified 36 unique cell populations amongst dog circulating leukocytes, with a remarkable amount of heterogeneity in CD4 T cell subtypes. In our comparison of healthy dogs and dogs with OS, we identified relative increases in the abundances of polymorphonuclear (PMN-) and monocytic (M-) myeloid-derived suppressor cells (MDSCs), as well as aberrations in gene expression within myeloid cells. Overall, this study provides a detailed atlas of canine leukocytes and investigates how the presence of osteosarcoma alters the transcriptional profiles of circulating immune cells.

A single-cell RNA sequencing atlas of circulating leukocytes from healthy and osteosarcoma affected dogs.

Peripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms with poor treatment responses and survival times. Canine PTCL clinically and immunophenotypically resembles the most common human subtype, PTCL-not otherwise specified (PTCL-NOS), leading to interest in this canine disease as a naturally occurring model for human PTCL. Gene expression profiling in human PTCL-NOS has helped characterize this ambiguous diagnosis into distinct subtypes, but similar gene expression profiling in canine PTCL is lacking.

The gene expression profile and cell of origin of canine peripheral T-cell lymphoma.

Acute myeloid leukemia (AML) poses significant challenges in veterinary medicine, with limited treatment options and poor survival rates. While substantial progress has been made in characterizing human AML, translating these advancements to veterinary practice has been hindered by limited molecular understanding and diagnostic tools. The case study presented illustrates the application of whole genome sequencing in diagnosing AML in a dog, showcasing its potential in veterinary oncology. Our approach facilitated comprehensive genomic analysis, identifying mutations in genes that may be associated with AML pathogenesis in dogs, such as KRAS, IKZF1, and RUNX1. However, without supportive evidence of its clinical utility (eg, association with response to treatment or prognosis), the information is limited to exploration. This article reviews the comparative features of canine AML with human AML and discusses strategies to shrink the knowledge gap between human and veterinary medicine with cost-effective next-generation sequencing (NGS) techniques. By utilizing these approaches, the unique and shared molecular features with human AML can be identified, aiding in molecular classification and therapeutic development for both species. Despite the promise of NGS, challenges exist in implementing it into routine veterinary diagnostics. Cost considerations, turnaround times, and the need for robust bioinformatics pipelines and quality control measures must be addressed. Most importantly, analytical and clinical validation processes are essential to ensure the reliability and clinical utility of NGS-based assays. Overall, integrating NGS technologies into veterinary oncology holds great potential for advancing our understanding of AML and improving disease stratification, in hopes of improving clinical outcomes.

Article	Year
Validation and method comparison of the use of densitometry to quantify monoclonal proteins in canine sera. Veterinary clinical pathology\| PubMed ID: 31410878	2019
Disseminated infection in a dog with severe colitis. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc\| PubMed ID: 32954990	2020
Retrospective evaluation of the use of the International Myeloma Working Group response criteria in dogs with secretory multiple myeloma. Journal of veterinary internal medicine\| PubMed ID: 33215766	2021
Diagnostic performance of routine electrophoresis and immunofixation for the detection of immunoglobulin paraproteins (M-Proteins) in dogs with multiple myeloma and related disorders: Part 1... Veterinary clinical pathology\| PubMed ID: 33847384	2021
Diagnostic performance of routine electrophoresis and immunofixation for the detection of immunoglobulin paraproteins (M-Proteins) in dogs with multiple myeloma and related disorders: Part 2... Veterinary clinical pathology\| PubMed ID: 33855710	2021
Bullous amyloidosis in a horse: first description in veterinary medicine. Veterinary dermatology\| PubMed ID: 34121242	2021
Method comparison for serum protein electrophoretic M-protein quantification: Agarose gel electrophoresis and capillary zone electrophoresis in canine and feline sera. Veterinary clinical pathology\| PubMed ID: 34862629	2021
Atypical multiple myeloma in 3 young dogs. Veterinary pathology\| PubMed ID: 35400242	2022
Using digital RNA counting to establish flow cytometry diagnostic criteria for subtypes of CD34+ canine acute leukaemia. Veterinary and comparative oncology\| PubMed ID: 35491468	2022
Detection and Characterization of Paraproteinemia in Canine Chronic B-cell Lymphocytic Leukemia Using Routine and Free Light Chain Immunofixation. Veterinary clinical pathology\| PubMed ID: 35883213	2022
A single-cell RNA sequencing atlas of circulating leukocytes from healthy and osteosarcoma affected dogs. Frontiers in immunology\| PubMed ID: 37275879	2023
The gene expression profile and cell of origin of canine peripheral T-cell lymphoma. BMC cancer\| PubMed ID: 38166662	2024
Advancements in genetic analysis: Insights from a case study and review of next-generation sequencing techniques for veterinary oncology applications. Veterinary clinical pathology\| PubMed ID: 39367609	2024

Adam Harris

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