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BACKGROUND: The clustering of immune repertoire data is challenging due to the computational cost associated with a very large number of pairwise sequence comparisons. To overcome this limitation, we developed Anchor Clustering, an unsupervised clustering method designed to identify similar sequences from millions of antigen receptor gene sequences. First, a Point Packing algorithm is used to identify a set of maximally spaced anchor sequences. Then, the genetic distance of the remaining sequences to all anchor sequences is calculated and transformed into distance vectors. Finally, distance vectors are clustered using unsupervised clustering. This process is repeated iteratively until the resulting clusters are small enough so that pairwise distance comparisons can be performed. RESULTS: Our results demonstrate that Anchor Clustering is faster than existing pairwise comparison clustering methods while providing similar clustering quality. With its flexible, memory-saving strategy, Anchor Clustering is capable of clustering millions of antigen receptor gene sequences in just a few minutes. CONCLUSIONS: This method enables the meta-analysis of immune-repertoire data from different studies and could contribute to a more comprehensive understanding of the immune repertoire data space.
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Algoritmos , Receptores de Antígenos , Análisis por ConglomeradosRESUMEN
BACKGROUND: Canine epitheliotropic cutaneous T-cell lymphoma (eCTCL) is thought to represent a disease homologue to human mycosis fungoides (MF). In human MF, neoplastic cells are phenotypically consistent with resident effector memory T cells, a population that remains for an extended period within tissue without circulating. Dogs with eCTCL often present with lesions in multiple locations, raising the question of whether the neoplasm is of the same T-cell subpopulation or not. OBJECTIVES: To characterize the antigen receptor gene rearrangements of lymphocytes from skin and blood of dogs with eCTCL to determine if neoplastic clones are identical. ANIMALS: Fourteen dogs with eCTCL. MATERIALS AND METHODS: Histological and immunohistochemical examination, and PCR for antigen receptor rearrangement (PARR) for T-cell receptor gamma (TRG) performed on multiple cutaneous biopsy samples and blood. RESULTS: All skin biopsies contained cluster of differentiation (CD)3-positive neoplastic lymphocytes. Within individual dogs, all skin biopsies revealed identical TRG clonality profiles, suggesting that the same neoplastic clone was present in all sites. In the blood, a matching clone was found in six of 14 dogs, a unique clone was observed in nine of 14 dogs, and no clone was detected in two of 14 dogs. CONCLUSIONS: These findings show that canine eCTCL lesions in multiple locations harbour the same neoplastic clone, neoplastic lymphocytes do not remain fixed to the skin and instead can circulate via blood, differing clones can be identified in skin versus blood, and circulating neoplastic cells can be detected without lymphocytosis.
Contexte - On pense que le lymphome T cutané épithéliotrope canin (eCTCL) représente une maladie homologue au mycosis fongoïde (MF) humain. Dans le MF humain, les cellules néoplasiques sont phénotypiquement compatibles avec les cellules T mémoire effectrices résidentes, une population qui reste pendant une période prolongée dans les tissus sans circuler. Les chiens atteints d'eCTCL présentent souvent des lésions à plusieurs endroits, ce qui soulève la question de savoir si le néoplasme appartient ou non à la même sous-population de lymphocytes T. Objectifs - Caractériser les réarrangements du gène du récepteur antigénique des lymphocytes de la peau et du sang des chiens atteints d'eCTCL afin de déterminer si les clones néoplasiques sont identiques. Animaux - Quatorze chiens avec eCTCL. Matériels et méthodes - Examen histologique et immunohistochimique, et PCR pour le réarrangement des récepteurs antigéniques (PARR) pour le récepteur gamma des lymphocytes T (TRG) effectués sur plusieurs échantillons de biopsie cutanée et de sang. Résultats - Toutes les biopsies cutanées contenaient des amas de lymphocytes néoplasiques positifs à la différenciation (CD)3. Chez les chiens individuels, toutes les biopsies cutanées ont révélé des profils de clonalité TRG identiques, suggérant que le même clone néoplasique était présent dans tous les sites. Dans le sang, un clone correspondant a été trouvé chez six des 14 chiens, un clone unique a été observé chez neuf des 14 chiens et aucun clone n'a été détecté chez deux des 14 chiens. Conclusions - Ces résultats montrent que les lésions eCTCL canines à plusieurs endroits abritent le même clone néoplasique, les lymphocytes néoplasiques ne restent pas fixés à la peau et peuvent plutôt circuler par le sang, différents clones peuvent être identifiés dans la peau par rapport au sang, et les cellules néoplasiques circulantes peuvent être détecté sans lymphocytose.
Introducción- se cree que el linfoma epiteliotrópico cutáneo de células T canino (eCTCL) representa una enfermedad homóloga a la micosis fungoide (MF) humana. En la MF humana, las células neoplásicas son fenotípicamente consistentes con las células T de memoria efectoras residentes, una población que permanece durante un período prolongado dentro del tejido sin circular. Los perros con eCTCL a menudo presentan lesiones en múltiples ubicaciones, lo que plantea la cuestión de si la neoplasia es de la misma subpoblación de células T o no. Objetivos- caracterizar los reordenamientos del gen del receptor de antígeno de los linfocitos de la piel y la sangre de perros con eCTCL para determinar si los clones neoplásicos son idénticos. Animales- catorce perros con eCTCL. Materiales y métodos - Examen histológico e inmunohistoquímico, y PCR para el reordenamiento del receptor de antígeno (PARR) para el receptor de células T gamma (TRG) realizado en múltiples muestras de biopsia cutánea y sangre. Resultados- todas las biopsias de piel contenían linfocitos neoplásicos positivos para grupos de diferenciación (CD)3. Dentro de perros individuales, todas las biopsias de piel revelaron perfiles de clonalidad de TRG idénticos, lo que sugiere que el mismo clon neoplásico estaba presente en todos los sitios. En la sangre, se encontró un clon compatible en seis de 14 perros, se observó un clon único en nueve de 14 perros y no se detectó ningún clon en dos de 14 perros. Conclusiones- estos hallazgos muestran que las lesiones de eCTCL canino en múltiples ubicaciones albergan el mismo clon neoplásico, los linfocitos neoplásicos no permanecen fijados a la piel y, en cambio, pueden circular a través de la sangre, se pueden identificar diferentes clones en la piel versus la sangre y las células neoplásicas circulantes pueden ser identificadas sin presencia de linfocitosis.
Contexto - Acredita-se que o linfoma epiteliotrópico cutâneo de células T canino (eCTCL) representa uma doença análoga à micose fungoide (MF) humana. Na MF humana, as células neoplásicas são fenotipicamente consistentes com células T efetoras de memória residentes, uma população que permanece por um período extenso no tecido sem entrar na circulação. Os cães com eCTCL frequentemente apresentam lesões em múltiplos locais, levantando a questão de se a neoplasia é da mesma subpopulação de células T ou não. Objetivos - Caracterizar os rearranjos dos genes receptores de antígenos dos linfócitos da pele e do sangue de cães com eCTCL para determinar se os clones neoplásicos são idênticos. Animais - Quatorze cães com eCTCL. Materiais e métodos - Exame histológico e imunohistoquímico, e PCR para rearranjo de receptor de antígeno (PARR) para o receptor Gama de células T (TRG) realizado em múltiplas amostras de biópsia cutânea e sangue. Resultados - Todas as biópsias cutâneas continham clusters de diferenciação linfócitos T (CD)3- positivos. Entre os indivíduos, todas as biópsias cutâneas revelaram perfis de clonalidade de TGR idênticos em seis dos 14 cães, sugerindo que a mesma célula neoplásica estava presente em todos os locais. No sangue, um clone correspondente foi encontrado em seis dos 14 cães, um clone único foi observado em nove dos 14 cães e nenhum clone foi detectado em dois dos 14 cães. Conclusões - Estes achados demonstraram que as lesões de eCTCL em múltiplos locais possuem o mesmo clone neoplásico, linfócitos neoplásicos não permanecem fixos na pele e podem circular por via sistêmica , diversos tipos de clones podem ser identificados na pele versus sangue, e as células neoplásicas circulantes podem ser detectadas sem linfocitose.
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Enfermedades de los Perros , Linfoma Cutáneo de Células T , Micosis Fungoide , Neoplasias Cutáneas , Perros , Animales , Humanos , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/veterinaria , Micosis Fungoide/patología , Micosis Fungoide/veterinaria , Linfoma Cutáneo de Células T/veterinaria , Linfoma Cutáneo de Células T/patología , Piel/patología , Biopsia/veterinaria , Enfermedades de los Perros/patologíaRESUMEN
BACKGROUND: The domestic cat (Felis catus) is an important companion animal and is used as a large animal model for human disease. However, the comprehensive study of adaptive immunity in this species is hampered by the lack of data on lymphocyte antigen receptor genes and usage. The objectives of this study were to annotate the feline T cell receptor (TR) loci and to characterize the expressed repertoire in lymphoid organs of normal cats using high-throughput sequencing. RESULTS: The Felis catus TRG locus contains 30 genes: 12 TRGV, 12 TRGJ and 6 TRGC, the TRB locus contains 48 genes: 33 TRBV, 2 TRBD, 11 TRBJ, 2 TRBC, the TRD locus contains 19 genes: 11 TRDV, 2 TRDD, 5 TRDJ, 1 TRDC, and the TRA locus contains 127 genes: 62 TRAV, 64 TRAJ, 1 TRAC. Functional feline V genes form monophyletic clades with their orthologs, and clustering of multimember subgroups frequently occurs in V genes located at the 5' end of TR loci. Recombination signal (RS) sequences of the heptamer and nonamer of functional V and J genes are highly conserved. Analysis of the TRG expressed repertoire showed preferential intra-cassette over inter-cassette rearrangements and dominant usage of the TRGV2-1 and TRGJ1-2 genes. The usage of TRBV genes showed minor bias but TRBJ genes of the second J-C-cluster were more commonly rearranged than TRBJ genes of the first cluster. The TRA/TRD V genes almost exclusively rearranged to J genes within their locus. The TRAV/TRAJ gene usage was relatively balanced while the TRD repertoire was dominated by TRDJ3. CONCLUSIONS: This is the first description of all TR loci in the cat. The genomic organization of feline TR loci was similar to that of previously described jawed vertebrates (gnathostomata) and is compatible with the birth-and-death model of evolution. The large-scale characterization of feline TR genes provides comprehensive baseline data on immune repertoires in healthy cats and will facilitate the development of improved reagents for the diagnosis of lymphoproliferative diseases in cats. In addition, these data might benefit studies using cats as a large animal model for human disease.
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Gatos/genética , Sitios Genéticos/genética , Tejido Linfoide/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Inmunidad Adaptativa/genética , Secuencia de Aminoácidos , Animales , Gatos/inmunología , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Filogenia , Receptores de Antígenos de Linfocitos T/clasificación , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Evolution of indolent to aggressive lymphoma has been described in dogs but is difficult to distinguish from the de novo development of a second, clonally distinct lymphoma. Differentiation of these scenarios can be aided by next generation sequencing (NGS)-based assessment of clonality of lymphocyte antigen receptor genes. CASE PRESENTATION: An 8-year-old male intact Mastiff presented with generalized lymphadenomegaly was diagnosed with nodal T zone lymphoma (TZL) based on cytology, histopathology, immunohistochemistry and flow cytometry. Thirteen months later, the dog re-presented with progressive lymphadenomegaly, and based on cytology and flow cytometry, a large B cell lymphoma (LBCL) was diagnosed. Sequencing-based clonality testing confirmed the de novo development of a LBCL and the persistence of a TZL. CONCLUSIONS: The occurrence of two distinct lymphoid neoplasms should be considered if patient features and tumor cytomorphology or immunophenotype differ among sequential samples. Sequencing-based clonality testing may provide conclusive evidence of two concurrent and distinct clonal lymphocyte populations, termed most appropriately "composite lymphoma".
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Enfermedades de los Perros/patología , Linfoma de Células B Grandes Difuso/veterinaria , Linfoma de Células T/veterinaria , Animales , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/uso terapéutico , Antineoplásicos Hormonales/administración & dosificación , Antineoplásicos Hormonales/uso terapéutico , Clorambucilo/administración & dosificación , Clorambucilo/uso terapéutico , Perros , Linfoma de Células B Grandes Difuso/complicaciones , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células T/complicaciones , Linfoma de Células T/patología , Masculino , Prednisona/administración & dosificación , Prednisona/uso terapéuticoRESUMEN
Canine nonepitheliotropic cutaneous T-cell lymphomas (NECTCL) are poorly characterized. In humans, a number of distinct subtypes of NECTCL have been recognized, including subcutaneous panniculitis-like T-cell lymphoma (SPTCL). Five dogs with subcutaneous T-cell lymphomas histologically similar to SPTCL in humans are herein described. The mean age was 8.5 years (5.5 to 12 years). No breed or sex predilection was identified in this small cohort. Two dogs presented with an acute onset of multiple skin masses and 3 dogs had solitary masses with subsequent development of multiple smaller masses within 0.5 to 2 months post-diagnosis without treatment. Locations, when specified, included shoulder, neck, and ventral abdomen. Two dogs were euthanized following diagnosis and one dog treated with chemotherapy (CCNU) survived 7 months post-diagnosis. Histologically, all cases were characterized by proliferations of either small to intermediate or large sized, CD3-positive T cells that infiltrated the subcutis in a lace-like pattern and frequently rimmed adipocytes. No epitheliotropism was observed, neoplastic cells were often karyorrhectic, and there were regions of extensive necrosis. Heavy infiltrates of histiocytes with prominent phagocytosis masked the lymphoid neoplastic cell population in some sections. A clonal T-cell receptor gamma gene rearrangement was found in 4 of the 5 cases. While SPTCLs typically have a less aggressive clinical course in humans, their biological behavior in dogs remains to be determined. In summary, SPTCL may represent a distinct entity in dogs and needs to be accurately diagnosed to better determine clinical behavior.
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Enfermedades de los Perros/patología , Linfoma de Células T/veterinaria , Paniculitis/veterinaria , Animales , Enfermedades de los Perros/clasificación , Perros , Femenino , Linfoma de Células T/clasificación , Linfoma de Células T/patología , Masculino , Paniculitis/clasificación , Paniculitis/patología , Piel/patologíaRESUMEN
Leukemia is broadly divided into acute and chronic lymphocytic and myeloid types based on the proportion of blasts, morphology of cells, and expression of specific antigens on neoplastic cells. Classifying leukemia in horses can be challenging if blasts predominate and since few antibodies to identify cell types are available. The objective of this study was to describe in detail the clinical and pathologic features of acute leukemia in horses. Twelve horses ranging from 0.2 to 25.9 years of age were diagnosed with acute leukemia. Six cases were classified as acute lymphocytic leukemia (ALL) based on predominance of blasts, lack of granulocytic or monocytic differentiation, and detection of CD3, CD20, and/or CD79a antigens by immunohistochemistry. Six other cases were classified as acute myeloid leukemia (AML) with myelomonocytic ( n = 4), basophilic ( n = 1), and eosinophilic ( n = 1) differentiation based on > 20% bone marrow blasts and partial leukocytic differentiation. Reactivity with antibodies to Iba-1/AIF-1, CD172a, and CD163 was determined for all cases of AML. Eleven horses had thrombocytopenia, 10 had neutropenia, 8 had anemia, all had blasts on blood films, and none had leukocytosis. Ten horses had increased serum acute phase proteins. Bone marrow cellularity ranged from 30% to 100%, and the proportion of blasts ranged from 80% to 100% and 30% to 60% in ALL and AML, respectively. Horses were severely ill at diagnosis and euthanized within days or weeks. Unique features of acute leukemia in horses compared to other species were variable lymphocyte antigen expression (ALL) and frequent inflammation (ALL and AML).
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Enfermedades de los Caballos/patología , Leucemia/patología , Animales , Progresión de la Enfermedad , Enfermedades de los Caballos/clasificación , Caballos , Leucemia/clasificación , Leucemia-Linfoma Linfoblástico de Células Precursoras , Trombocitopenia/patologíaRESUMEN
CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity.
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Anticuerpos Monoclonales/química , Antígenos CD1/química , Antígenos CD1/genética , Sitios Genéticos , Proteínas Recombinantes de Fusión , Alelos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Antígenos CD1/metabolismo , Secuencia de Bases , Biología Computacional , Perros , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Unión Proteica , Isoformas de Proteínas , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Introduction: The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. Methods: Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Results: Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. Discussion: Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.
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In drug development, assessing the toxicity of candidate compounds is crucial for successfully transitioning from preclinical research to early-stage clinical trials. Drug safety is typically assessed using animal models with a manual histopathological examination of tissue sections to characterize the dose-response relationship of the compound - a time-intensive process prone to inter-observer variability and predominantly involving tedious review of cases without abnormalities. Artificial intelligence (AI) methods in pathology hold promise to accelerate this assessment and enhance reproducibility and objectivity. Here, we introduce TRACE, a model designed for toxicologic liver histopathology assessment capable of tackling a range of diagnostic tasks across multiple scales, including situations where labeled data is limited. TRACE was trained on 15 million histopathology images extracted from 46,734 digitized tissue sections from 157 preclinical studies conducted on Rattus norvegicus. We show that TRACE can perform various downstream toxicology tasks spanning histopathological response assessment, lesion severity scoring, morphological retrieval, and automatic dose-response characterization. In an independent reader study, TRACE was evaluated alongside ten board-certified veterinary pathologists and achieved higher concordance with the consensus opinion than the average of the pathologists. Our study represents a substantial leap over existing computational models in toxicology by offering the first framework for accelerating and automating toxicological pathology assessment, promoting significant progress with faster, more consistent, and reliable diagnostic processes.
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BACKGROUND: Cutaneous T-cell lymphoma (CTCL) in dogs is a heterogeneous disease complex, which consists of nonepitheliotropic (NE) and epitheliotropic forms. These lymphomas are readily recognized by the presence of dominant populations of cytologically atypical lymphocytes. OBJECTIVE: The objective of this study was to introduce the key features of inflamed NE-CTCL, which is easily confused with reactive, inflammatory histiocytic disease. ANIMALS: Twenty-four dogs (mean age 7.5 years) presented with inflamed NE-CTCL. Lesions presented as nodules, plaques or masses. An initial diagnosis of cutaneous reactive histiocytosis (11 dogs) or histiocytic neoplasia (three dogs) was made by primary pathologists. METHODS: Lesions were assessed by histology and immunohistochemistry to detect canine leukocyte antigens. Lesional genomic DNA was extracted and gene rearrangement analysis of the T-cell receptor γ locus was assessed. RESULTS: The cutaneous lesions consisted of pleocellular infiltration of the dermis with variable extension into the subcutis. The lesions often surrounded vessels and adnexae. Epitheliotropism was minimal or lacking. Small lymphocytes, plasma cells and intermediate to large, cytologically atypical lymphocytes were scattered between prominent histiocytic infiltrates. Atypical lymphocytes often had marked variation in the intensity of CD3 expression. Molecular clonality analysis of the T-cell receptor γ locus revealed clonal expansion of T cells in 22 of 23 dogs tested. CONCLUSION: The recognition of inflamed NE-CTCL and its differentiation from cutaneous reactive histiocytosis depends on careful assessment of lymphocyte morphology and immunostaining patterns. Confirmation of the diagnosis is best accomplished by T-cell antigen receptor gene rearrangement analysis.
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Enfermedades de los Perros/diagnóstico , Linfoma Cutáneo de Células T/veterinaria , Animales , Enfermedades de los Perros/clasificación , Enfermedades de los Perros/patología , Perros , Femenino , Regulación Neoplásica de la Expresión Génica , Linfoma Cutáneo de Células T/clasificación , Linfoma Cutáneo de Células T/diagnóstico , Linfoma Cutáneo de Células T/patología , Masculino , Mutación , Organofosfatos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
The dog is valued as a companion animal and increasingly recognized as a model for human disorders. Given the importance of T cells in health and disease, comprehensive knowledge of canine T cells can contribute to our understanding of pathogenesis mechanisms and inform the development of new treatment strategies. However, the diversity of canine T cells is still poorly understood mainly due to the lack of species-reactive antibodies for use in flow cytometry. The aim of this study was to generate a detailed atlas of peripheral blood TCRαß+ T cells of healthy dogs using single-cell RNA-sequencing (scRNAseq) combined with immune repertoire sequencing. A total of 22 TCRαß+ T cell clusters were identified, which were classified into three major groups: CD4-dominant (11 clusters), CD8A-dominant (8 clusters), and CD4/CD8A-mixed (3 clusters). Based on differential gene expression, distinct differentiation states (naïve, effector, memory, exhausted) and lineages (e.g. CD4 T helper and regulatory T cells) could be distinguished. Importantly, several T cell populations were identified, which have not been described in dogs before. Of particular note, our data provide first evidence for the existence of canine mucosa-associated invariant T cell (MAIT)-like cells, representing one of three newly identified FCER1G+ innate-like CD8A+ T cell populations in the peripheral blood of healthy dogs. In conclusion, using scRNAseq combined with immune repertoire sequencing we were able to resolve canine TCRαß+ T cell populations at unprecedented resolution. The peripheral blood TCRαß+ T cell atlas of healthy dogs generated here represents an important reference data set for future studies and is of relevance for identifying new targets for T cell-specific therapies.
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Células T Invariantes Asociadas a Mucosa , Receptores de Antígenos de Linfocitos T alfa-beta , Perros , Animales , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transcriptoma , Linfocitos T Reguladores , Diferenciación CelularRESUMEN
While some companion animals have been shown to be susceptible to SARS-CoV-2, their role in the COVID-19 pandemic has remained poorly investigated. Equids are susceptible to SARS-CoV-2 based on the similarity of the human ACE-2 receptor and reports of infection. Clinical disease and prevalence factors associated with SARS-CoV-2 infection in equids have not yet been investigated. The aim of this study was to determine the seroprevalence of SARS-CoV-2 and selected prevalence factors in 1186 equids presented for various conditions to a Veterinary Medical Teaching Hospital over a two-year period. Blood samples were tested for SARS-CoV-2 antibodies using an ELISA targeting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Further, selected prevalence factors (season, age, breed, sex, presenting complaint) were retrieved from the medical records. No information was available on whether the horses had come into contact with COVID-19-positive individuals. Among the study animals, 42/1186 (3.5%) horses had detectable SARS-CoV-2 antibodies. Amongst the prevalence factors investigated, only seasonality (spring) was associated with a greater frequency of seropositivity to SARS-CoV-2. Horses with medical and surgical complaints were more likely to test seropositive to SARS-CoV-2 compared to horses presented for routine health care procedures, suggesting more frequent and/or longer interactions with individuals with COVID-19. While horses can become infected with SARS-CoV-2 via the occasional spillover from COVID-19 individuals, clinical disease expression remains subclinical, making horses an unlikely contributor to the spread of SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Antivirales , California , COVID-19/epidemiología , COVID-19/veterinaria , Caballos/virología , Hospitales de Enseñanza , Pandemias , Estudios Seroepidemiológicos , Hospitales VeterinariosRESUMEN
BACKGROUND: Necrotizing meningoencephalitis (NME) in the pug dogs is a fatal neuroinflammatory disease associated with rapid progression and poor response to conventional immunosuppressive therapy. Diagnosis is typically made after severe neurological abnormalities have manifested. HYPOTHESIS/OBJECTIVE: Pug dogs at genetic risk for NME might manifest neurological abnormalities before developing pathognomonic clinical signs of NME. ANIMALS: Thirty-six pug dogs less than 4 years of age asymptomatic for NME. METHODS: Prospective observational cohort study with germline genome-wide genotyping. Neurological examinations were performed 4 weeks apart to document reproducible findings of central nervous system disease. Magnetic resonance imaging, cerebrospinal fluid analysis, and testing for infectious diseases were performed in all pugs with reproducible abnormalities detected on neurological examination. RESULTS: The overall risk allele frequency in this cohort was 40%; 5 (14%) dogs were high risk, 19 (53%) dogs were medium risk, and 12 (33%) dogs were low genetic risk for NME. Reproducible abnormalities detected on neurological examination were identified in 8/24 (33%) genetically at-risk dogs and 0/12 (0%) low risk dogs. Clinical abnormalities included multifocal spinal pain in 8/8, reduced menace response in 5/8, and lateralizing postural reaction deficits in 5/8 pugs. There was a strong association between genotype risk and the presence of this clinical phenotype (P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest the presence of a novel early clinical phenotype of NME in apparently asymptomatic genetically at-risk pugs which might be used to plan early diagnostic and therapeutic clinical trials.
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Enfermedades de los Perros , Meningoencefalitis , Animales , Perros , Enfermedades de los Perros/líquido cefalorraquídeo , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/genética , Frecuencia de los Genes , Meningoencefalitis/líquido cefalorraquídeo , Meningoencefalitis/genética , Meningoencefalitis/veterinaria , Fenotipo , Estudios ProspectivosRESUMEN
BACKGROUND: T cell clonality assays in veterinary medicine currently target only the T cell receptor gamma (TRG) locus. Existing assays have suboptimal sensitivity because of insufficient primer coverage of all possible rearrangements. OBJECTIVE: Develop higher sensitivity clonality assays targeting the TRG, delta (TRD), and beta (TRB) loci in cats. ANIMALS: Cats with histopathologically confirmed lymphoma (n = 89), non-lymphoma (n = 35), and possible hepatic small cell lymphoma (n = 31). METHODS: Molecular clonality assay development utilizing our recently reported topology and expressed repertoire data of the T cell receptor loci in cats. Determination of clonality status of lymphoma, non lymphoma, and possible hepatic small cell lymphoma samples, and calculation of assay sensitivity and specificity. RESULTS: The new multiplex TRG assay yielded the highest sensitivity (95.5%). All assays yielded 100% specificity except for the new multiplex TRG assay (97.3%). The combination of the new TRG and TRB assays yielded sensitivity of 98.9% and specificity of 97.0%. The new TRG assay detected clonality in 17/31 possible small cell lymphoma livers, whereas an existing TRG assay detected clonality in 6/31 livers. CONCLUSIONS AND CLINICAL IMPORTANCE: The assessment of multiple T cell loci compensates for the potential shortcomings of individual assays. Using a combination of molecular clonality assays will increase the overall sensitivity for the diagnosis of T-cell lymphoma in cats, especially intestinal, and hepatic small cell lymphoma. Hepatic small cell lymphomas detected by the new TRG assay utilized rarely expressed V and J genes not recognized by previous assays, likely indicating unique biology of hepatic small cell lymphoma in cats.
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Enfermedades de los Gatos , Linfoma de Células T , Animales , Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/genética , Gatos , Reordenamiento Génico , Linfoma de Células T/diagnóstico , Linfoma de Células T/genética , Linfoma de Células T/veterinaria , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfocitos TRESUMEN
Lymphoma is the most common hematopoietic tumour in dogs and is remarkably similar to the human disease. Tumour biomarker discovery is providing new tools for diagnostics and predicting therapeutic response and clinical outcome. MicroRNAs are small non-coding RNAs that participate in post-transcriptional gene regulation and their aberrant expression can impact genes involved in cancer. The aim of this study was to characterize microRNA expression in lymph nodes and plasma from dogs with multicentric B or T cell lymphoma compared to healthy control dogs. We further compared expression between lymph nodes and corresponding plasma samples and assessed changes in expression at relapse compared to time of diagnosis. Lastly, we investigated microRNAs for association with clinical outcome in patients treated with CHOP chemotherapy. A customized PCR array was utilized to profile 38 canine target microRNAs. Quantification was performed using real time RT-qPCR and relative expression was determined by the delta-delta Ct method. In lymph nodes, there were 16 microRNAs with significantly altered expression for B cell lymphoma and 9 for T cell lymphoma. In plasma, there were 15 microRNAs altered for B cell lymphoma and 3 for T cell lymphoma. The majority of microRNAs did not have correlated expression between lymph node and plasma and only 8 microRNAs were significantly different between diagnosis and relapse. For B cell lymphoma, 8 microRNAs had differential expression in the non-remission group compared to dogs that completed CHOP in complete remission. Four of these microRNAs were also altered in patients that died prior to one-year. Kaplan-Meier survival curves for high versus low microRNA expression revealed that 10 microRNAs were correlated with progression-free survival and 3 with overall survival. This study highlights microRNAs of interest for canine multicentric lymphoma. Future goals include development of microRNA panels that may be useful as biomarkers with the intent to provide improved outcome prediction to veterinary cancer patients.
Asunto(s)
Enfermedades de los Perros/diagnóstico , Linfoma de Células B/diagnóstico , Linfoma de Células T/diagnóstico , MicroARNs/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ciclofosfamida/uso terapéutico , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/genética , Enfermedades de los Perros/mortalidad , Perros , Doxorrubicina/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunofenotipificación , Estimación de Kaplan-Meier , Ganglios Linfáticos/metabolismo , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/genética , Linfoma de Células B/mortalidad , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/genética , Linfoma de Células T/mortalidad , Masculino , MicroARNs/sangre , Recurrencia Local de Neoplasia , Prednisona/uso terapéutico , Supervivencia sin Progresión , Resultado del Tratamiento , Vincristina/uso terapéuticoRESUMEN
Several molecular clonality assays have been developed to assess canine B cell proliferations. These assays were based on different sequence data, utilized different assay designs and employed different testing strategies. This has resulted in a complex body of literature and complicates evidence-based selection of primer sets. In addition, further refinement of primer sets is difficult because it is unknown how well current primer sets cover the expressed sequence repertoire. The objectives of this study were 1) to provide an overview of published IGH clonality assays that highlights key differences in assay design and testing strategy and 2) to propose a novel method for optimizing primer sets that leverages large-scale sequencing data. A review of previously published assays highlighted confounding factors that hamper a direct comparison of performance metrics between studies. These findings illustrate the need for a multi-institutional effort to harmonize veterinary clonality testing. A novel in silico analysis of primer sequences using a large dataset of expressed sequences identified shortfalls of existing primer sets and was used to guide primer optimization. Three optimized primer sets were tested and yielded qualitative sensitivity values between 80-90%. The qualitative sensitivity ranged from 1% to over 50% and was dependent on the size of the neoplastic clone and the sample DNA used. These findings illustrate that inclusion of high-throughput sequencing data for primer design can be a useful tool to guide primer design and optimization. This strategy could be applied to other antigen receptor loci or species to further improve veterinary clonality assays.
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Linfocitos B/citología , Células Clonales , Cartilla de ADN , Perros/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Animales , Perros/genéticaRESUMEN
The ability to mount adaptive immune responses to a diverse array of pathogens is essential to maintaining the health of an individual. The outcome of adaptive immune responses is influenced by the pool of available lymphocyte antigen receptors. Understanding the composition and dynamics of immune repertoires is hence of relevance to characterizing physiologic immunological processes as well as understanding disease pathogenesis. The dog is increasingly recognized as a model for human disease. The objective of this study was to utilize NGS for comprehensive and unbiased analysis of the IGH repertoire in healthy dogs. First, the IGH locus was searched in silico for previously unidentified genes. Second, IGH transcripts from major lymphoid organs were amplified using a 5'RACE approach without V/J primer bias. Third, amplicons were sequenced on an Illumina MiSeq platform, and data were analyzed using the ARResT/Interrogate platform. Data analysis included V/J usage, V-J pairing biases, isotype frequency, CDR3 diversity, convergent recombination, and public repertoires. The results of this study provide a comprehensive IGH repertoire analysis for healthy dogs. These data will allow further improvement of V/J gene-specific primer sets and will serve as baseline for future studies investigating immune repertoires in health and disease.
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Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Animales , Perros , Secuenciación de Nucleótidos de Alto Rendimiento , Isotipos de Inmunoglobulinas/genética , Receptores de Antígenos/genética , Análisis de Secuencia de ADNRESUMEN
In most validation studies of tissue microarrays (TMAs), a fixed number of cores with a given diameter are analyzed to determine the degree of accuracy by which the TMA represents the whole section. The statistical model described in the present study predicts this property for various combinations of 2 core sizes (0.6 mm and 1.2 mm) and different core numbers. The model was based on artificial TMA core biopsies generated from Ki-67 and active caspase-3 immunostains of 40 canine lymphoma samples. Positivity was scored on a continuous scale, and a large number of cells were analyzed with the help of semiautomated cell counting. Despite considerable differences in range and distribution of Ki-67 and active caspase-3 positivity values, the model predictions showed a high degree of agreement for both markers. Comparison of 0.6 mm and 1.2 mm cores indicated that the use of small cores necessitates inclusion of a larger number of samples but requires counting a markedly smaller number of cells. Suitability of TMAs to determine the immunophenotype of the whole section was assessed using 2 different combinations of core sizes and numbers. Both displayed a high degree of concordance with the whole section (kappa(0.6) = 0.79; kappa(1.2) = 0.91). The present study provides a basis for the use of TMAs in future high-throughput immunohistochemical investigations of selected markers in canine lymphomas. The statistical model presented can be used to determine an optimal TMA design depending on a desired accuracy.
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Enfermedades de los Perros/diagnóstico , Inmunohistoquímica/veterinaria , Linfoma/veterinaria , Análisis de Matrices Tisulares/veterinaria , Animales , Caspasa 3 , Enfermedades de los Perros/inmunología , Perros , Inmunofenotipificación , Antígeno Ki-67 , Linfoma/diagnóstico , Linfoma/inmunología , Proteínas de Neoplasias , Reproducibilidad de los ResultadosRESUMEN
CD1 molecules are glycoproteins present primarily on dendritic cells (DCs), which recognize and present a variety of foreign- and self-lipid antigens to T-cells. Humans have five different CD1 isoforms that survey distinct cellular compartments allowing for recognition of a large repertoire of lipids. The canine CD1 family consists of seven functional CD1 molecules (canine CD1a2, CD1a6, CD1a8, CD1a9, CD1b, CD1c and CD1e) and one presumed non-functional isoform (canine CD1d) due to a disrupted gene structure. The aim of this study was to describe in vitro steady-state localization ptterns of canine CD1 isoforms and their correlation with endocytic organelles. GFP-fused canine CD1 293T cell transfectants were stained with markers for early endocytic compartments (EEA-1) and late endocytic/lysosomal compartments (LAMP-1), respectively, and analyzed by confocal microscopy. Canine CD1a molecules localized to the plasma membrane and partially to the early endocytic compartment, but not to late endosomes or lysosomes. In contrast, canine CD1b was highly associated with late endosomal/lysosomal compartments and showed a predominant intracellular expression pattern. Canine CD1c protein expression localized more promiscuously to both the early endosomal compartments and the late endosomal/lysosomal compartments. The canine CD1e molecule showed a strictly intracellular expression with a partial overlap with late endosomal/lysosomal compartments. Lastly, canine CD1d was expressed abnormally showing only a diminished GFP expression. In conclusion, canine CD1 transfectants show distinct localization patterns that are similar to human CD1 proteins with the exception of the canine CD1d isoform, which most likely is non-functional. These findings imply that canine CD1 localization overall resembles human CD1 trafficking patterns. This knowledge is important for the understanding of lipid antigen-receptor immunity in the dog.
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Antígenos CD1/metabolismo , Perros/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD1/genética , Compartimento Celular/inmunología , Perros/genética , Endosomas/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Isoantígenos/genética , Isoantígenos/metabolismo , Lisosomas/inmunología , Microscopía Confocal , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Wildlife populations of conservation concern are limited in distribution, population size and persistence by various factors, including mortality. The fisher (Pekania pennanti), a North American mid-sized carnivore whose range in the western Pacific United States has retracted considerably in the past century, was proposed for threatened status protection in late 2014 under the United States Endangered Species Act by the United States Fish and Wildlife Service in its West Coast Distinct Population Segment. We investigated mortality in 167 fishers from two genetically and geographically distinct sub-populations in California within this West Coast Distinct Population Segment using a combination of gross necropsy, histology, toxicology and molecular methods. Overall, predation (70%), natural disease (16%), toxicant poisoning (10%) and, less commonly, vehicular strike (2%) and other anthropogenic causes (2%) were causes of mortality observed. We documented both an increase in mortality to (57% increase) and exposure (6%) from pesticides in fishers in just the past three years, highlighting further that toxicants from marijuana cultivation still pose a threat. Additionally, exposure to multiple rodenticides significantly increased the likelihood of mortality from rodenticide poisoning. Poisoning was significantly more common in male than female fishers and was 7 times more likely than disease to kill males. Based on necropsy findings, suspected causes of mortality based on field evidence alone tended to underestimate the frequency of disease-related mortalities. This study is the first comprehensive investigation of mortality causes of fishers and provides essential information to assist in the conservation of this species.