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Supplementary Figure from B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy.Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3–specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed.In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3–specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3–specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3–specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

Canine cutaneous histiocytomas originate from Langerhans cells. Multiple histiocytomas are referred to as cutaneous Langerhans cell histiocytosis. Feline pulmonary Langerhans cell histiocytosis causes respiratory failure owing to extensive lung infiltration. Localized and disseminated histiocytic sarcomas usually arise from interstitial dendritic cells. Primary sites include spleen, lung, skin, brain (meninges), lymph node, bone marrow, and synovial tissues of limbs. An initially indolent form of localized histiocytic sarcomas, progressive histiocytosis, originates in the skin of cats. Hemophagocytic histiocytic sarcomas originates in splenic red pulp macrophages. Canine reactive histiocytoses (systemic histiocytosis and cutaneous histiocytosis) are complex inflammatory diseases with underlying immune dysregulation.

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.To characterize the antigen receptor gene rearrangements of lymphocytes from skin and blood of dogs with eCTCL to determine if neoplastic clones are identical.Fourteen dogs with eCTCL.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.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.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.Hintergrund - Das epitheliotrope kutane T-Zell Lymphom des Hundes (eCTCL) dürfte eine Krankheit widerspiegeln, die der Mycosis fungoides (MF) des Menschen entspricht. Bei der MF des Menschen sind die neoplastischen Zellen phänotypisch gleich wie die lokalen Effektor Memory T Zellen, eine Zellpopulation, die über einen ausgedehnten Zeitraum ohne zu zirkulieren im Gewebe bleibt. Hunde mit eCTCL werden oft mit Hautveränderungen in verschiedenen Körperregionen vorgestellt, was die Frage aufwirft, ob es sich bei dieser Neoplasie um dieselbe -Zell Unterpopulation handelt oder nicht. Ziele - Eine Charakterisierung des Antigenrezeptorgen Arrangements der Lymphozyten der Haut und aus dem Blut von Hunden mit eCTCL, um festzustellen, ob die neoplastischen Klone identisch sind. Tiere - Vierzehn Hunde mit eCTCL. Materialien und Methoden - Es wurde eine histologische und immunhistochemische Untersuchung sowie ein PCR für das Antigen Rezeptor Rearrangement (PARR) des T-Zell Rezeptor Gamma (TRG) an verschiedenen Hautbiopsieproben und im Blut durchgeführt. Ergebnisse - Alle Hautbiopsien beinhalteten Differenzierungskluster (CD)-3-positive neoplastische Lymphozyten. Innerhalb der individuellen Hunde zeigten alle Hautbiopsien identische TRG Klonalitätsprofile, was auf das Vorhandensein desselben neoplastischen Klons an allen Hautstellen hinweist. Im Blut wurde ein übereinstimmender Klon bei sechs von 14 Hunden gefunden, ein neuer Klon wurde bei neun von 14 Hunden gefunden und bei zwei von 14 Hunden wurde kein Klon gefunden. Schlussfolgerungen - Diese Ergebnisse zeigen, dass die Hautveränderungen beim eCTCL des Hundes an multiplen Stellen denselben neoplastischen Klon beherbergen, dass die neoplastischen Lymphozyten nicht in der Haut fixiert bleiben, sondern über das Blut zirkulieren können, dass unterschiedliche Klone in der Haut versus im Blut identifiziert werden können, und dass zirkulierende neoplastische Zellen auch ohne Lymphozyten gefunden werden können.背景 - 犬の上皮向性皮膚T細胞リンパ腫(eCTCL)は、ヒトの菌状息肉症(MF)と相同性のある疾患であると考えられている。ヒトのMFでは、腫瘍細胞は、循環せずに組織内に長期間留まる集団である常在エフェクターメモリーT細胞と表現型的に一致している。eCTCLの犬は、しばしば複数の場所に病変を呈するため、腫瘍が同じT細胞亜集団のものかどうかという疑問が生じる。 目的 - 本研究の目的は、eCTCLを発症した犬の皮膚および血液中のリンパ球の抗原受容体遺伝子の再配列を特徴づけ、腫瘍性クローンが同一であるかどうかを判断することであった。 被検動物 - eCTCLを発症した犬14頭。 材料と方法 - 複数の皮膚生検材料および血液について、組織学的および免疫組織化学的検査、T細胞受容体ガンマ(TRG)に対する抗原受容体再配列(PARR)のPCRを実施した。 結果 - すべての皮膚生検で、分化クラスタ(CD)3陽性の腫瘍性リンパ球が検出された。個々の犬では、すべての皮膚生検で同一のTRGクローナリティプロファイルが得られ、同じ腫瘍クローンがすべての部位に存在することが示唆された。血液中では、14頭中6頭で一致したクローンが見つかり、14頭中9頭で固有のクローンが観察され、14頭中2頭ではクローンが検出されなかった。 結論-これらの知見は、犬のeCTCL病変が複数の部位に同一の腫瘍性クローンを保有していること、腫瘍性リンパ球は皮膚に固定されず血液を介して循環すること、皮膚と血液で異なるクローンを同定できること、循環腫瘍細胞はリンパ球増殖なしに検出できることを示すものであった。.背景-犬趋上皮性皮肤 T 细胞淋巴瘤(eCTCL)被认为是人类蕈样肉芽肿(MF)的同源疾病。在人类MF中，肿瘤细胞在表型上与驻留效应记忆T细胞一致，该细胞群在组织内保持较长时间而不循环。患有eCTCL的犬通常存在多个部位的病变，提出了肿瘤是否为相同T细胞亚群的问题。 目的-描述eCTCL患犬的皮肤和血液淋巴细胞的抗原受体基因重排特征，以确定肿瘤克隆是否相同。 动物-14只eCTCL患犬。 材料和方法-对多个皮肤活检样本和血液进行组织学和免疫组织化学检查，以及T细胞受体γ (TRG) 抗原受体重排 (PARR) 的PCR。 结果-所有皮肤活检均含有分化簇 (CD)3 阳性肿瘤淋巴细胞。在个体犬中，所有皮肤活检均显示相同的TRG克隆特征，表明所有部位均存在相同的肿瘤克隆。在血液中，在6/14只犬中发现了匹配的克隆，在9/14只犬中观察到了独特的克隆，在2/14只犬中未检测到克隆。 结论-这些发现表明，多个位置的犬eCTCL 病变中含有相同的肿瘤克隆，肿瘤淋巴细胞不会保持固定在皮肤上，而是可以通过血液循环，在皮肤与血液中发现不同克隆，并且可以检测循环肿瘤细胞，而不会出现血液淋巴细胞增多。.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.

This review is on global wildland fire management research needs from the standpoint of “integrated fire management”. It seeks to apply a characterisation of fires to frame research needs, and also recognise some differences in research needs between “normal wildfires” and “extreme wildfire events” and draw some distinctions between the needs for developing and developed countries. In the past, the dominant approach to fires in developed countries has been to suppress them including prohibition of fire use. In developing countries, the approach has tended to be similar. However, fires are a landscape problem in both developed and developing contexts, not resulting from insufficient or inadequate means of suppression, but from fuel continuity and accumulation. The impacts of fires are becoming higher profile, due to sizes and intensity in part but also from land use and demographic changes and their interactions, which see more people, more assets and ecological and economic values affected and publicised. Not fully appreciating the ecological role, impact, social, cultural and economic context in which fires are occurring, and the contributing factors and underlying causes of the fire problem, has seen planning, policy, development and other influences, alter landscapes and made populations increasingly vulnerable. Key to successfully integrating ecology, society and fire management technologies is effective analysis of the situation. This requires strengthened focus, including through use of social science and related disciplines. Damaging fires suggest that suppression alone is not sufficient to deal with wildfire, with the damage and loss that results in some cases being catastrophic. The implications of the limits of suppression need to be a component for planning risk reduction, readiness, response and of research. Similarly, comprehensive analyses and figures for damage and losses, including suppression costs, would have implications for wildland fire planning and investment. Existing research findings need to be applied and further research undertaken as necessary. Application to wildland fire management globally of existing research, and further research as needed, is required to analyse, select appropriate strategies, and apply management, monitor implementation and enable continuous improvement to reduce vulnerability and underpin resilience. This approach is termed “integrated fire management” and is particularly valuable for developing countries. For them, there may be the potential to reduce or avoid the wildfire damage and loss trends experienced in developed countries, through development and application of what has already been researched and operationalised in other contexts.

Epizootic bovine abortion (EBA) is an arthropod-borne bacterial disease that causes significant economic loss for cattle producers in the western United States. The etiologic agent

A combination of cellular and humoral immunological assaults directed against follicular matrical cells is suspected to cause alopecia areata (AA) in humans. The specific aims of this study were to determine whether cytotoxic T lymphocytes (CTL) were increased in the blood and skin lesions of canine AA. Additionally, we wished to determine if circulating antifollicular antibodies were present in the dog. Finally, we aimed to investigate whether bulbar inflammation was associated with decreased matrical cell proliferation and keratinocyte apoptosis. Canine AA lesions were infiltrated by intrabulbar CTL, perifollicular helper T cells and dendritic cells. A higher percentage of CTL was present in canine AA peripheral blood compared with that of normal dogs. Tissue-fixed and circulating antifollicular IgG antibodies were detected. Reduced matrical proliferation and increased keratinocyte apoptosis was observed in inflamed hair bulbs. This study demonstrates the existence of antifollicular cell-mediated and humoral immunological responses in canine AA. Resume— L'association d'agressions immunologiques cellulaires et humorales dirigees contre les cellules matricielles folliculaires est suspectee d'etre a l'origine de l'Alopecie Areata chez l'homme. Le but de cette etude etait de montrer si le nombre de lymphocytes T cytotoxiques est augmente dans le sang et dans les lesions cutanees lors d'Alopecie Areata chez le chien. En plus, nous souhaitions montrer la presence d'anticorps circulants antifolliculaire chez le chien. Enfin, nous voulions voir si l'inflammation du bulbe pileux etait associee a une diminution de la proliferation des cellules matricielles et une apoptose des keratinocytes. Lors d'Alopecie Areata chez le chien, existe une infiltration lesionnelle par des lymphocytes T cytotoxiques intrabulbaires, des lymphocytes T auxiliaires perifolliculaires et des cellules dentritiques. Le pourcentage de lymphocytes T cytotoxiques etait plus important dans le sang peripherique provenant de sujets a Alopecie Areata que dans le sang d'animaux sains. Par ailleurs, des anticorps antifolliculaire (IgG) circulants et fixes dans les tissus sont detectes. Une reduction de la proliferation des cellules matricielles et une augmentation de l'apoptose des keratinocytes sont observees dans les bulbes pileux atteints. Cette etude demontre l'existence de reponses immunologiques cellulaires et humorales contre le follicule pileux dans l'Alopecie Areata du chien. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunite contre le follicule pileux dans l'Alopecie Areata du chien). Veterinary Dermatology 1996; 7: 67–79.] Resumen Se cree que la alopecia areata (AA) en la especie humana resulta de la combinacion de la accion inmunologica celular y humoral contra celulas de la matriz folicular. Los objetivos especificos de este estudio fueron determinar si los linfocitos T citotoxicos (CTL) se encontraban aumentados en sangre y en las lesiones cutaneas de animales con AA. Ademas, descamos investigar la posible presencia de anticuerpos antifoliculares circulantes en el perro. Finalmente, nos propusimos investigar si la inflamacion bulbar se encontraba asociada a una disminucion en la proliferacion de celulas de la matriz y a la apoptosis de queratinocitos. Las lesiones de la AA canina mostraban infiltracion por CTL intrabulbares, celulas T colaboradoras y celulas dendriticas. Se encontro un porcentaje mayor de CTL en la sangre periferica de perros con AA respecto a perros normales. Se detectaron anticuerpos IgG antifoliculares circulantes y en tejido fijado. Se observo una disminucion en la proliferacion matrical y un aumento en queratinocitos apoptoticos en los bulbos foliculares inflamados. Este estudio demuestra la existencia de respuestas inmunitarias antifoliculares de tipo celular y humoral en la AA canina. [Olivry, T., Moore, P.F., Naydan, D.K., Puget, B.J., Affolter, V.K., Kline, A.E. Antifollicular cell-mediated and humoral immunity in canine alopecia areata (Immunidad antifolicular en la alopecia areata canina). Veterinary Dermatology 1996; 7: 67–79.] Zusammenfassung— Eine Kombinaton von zellularen und humoralen immunologischen Angriffen, die gegen die follikularen Matrixzellen gerichtet sind, wird als Ursache bei der Alopecia areata (AA) des Menschen vermutet. Die besondere Absicht dieser Studie war, festzustellen, ob die zytotoxischen T-Lymphozyten (CTL) im Blut und in den Hautveranderungen bei kaniner AA erhoht sind. Zusatzlich wollten wir feststellen, ob beim Hund zirkulierende antifollikulare Antikorper vorkommen. Schlieslich beabsichtigten wir, zu untersuchen, ob die bulbare Entzundung mit verminderter Matrixzellproliferation und Keratinozytenapoptose einhergeht. Die Veranderungen bei kaniner AA wurden von intrabulbaren CTL, perifollikularen T-Helferzellen und dendritischen Zellen infiltriert. Verglichen mit dem peripheren Blut gesunder Hunde kam bei kaniner AA eine hoherer Prozentsatz an CTL vor. Es wurden gewebsfixierte und zirkulierende antifollikulare IgG-Antikorper festgestellt. In entzundeten Haarbulbi wurde eine reduzierte Matrixproliferation und eine erhohte Keratinozytenapoptose beobachtet. Diese Studie zeigt die Existenz von antifollikularen zellvermittelten und humoralen immunologischen Reaktionen bei kaniner AA. [Olivry, T., Moore, P. F., Naydan, D. K., Puget, B. J., Affolter, V. K., Kline, A. E. Antifollicular immunity in canine alopecia areata (Antifollikulare Immunitat bei der Alopecia areata des Hundes). Veterinary Dermatology 1996; 7: 67–79.]

A 5‐year‐old, spayed female French Bulldog presented with multiple papules on the skin of the scapular area. Histopathological examination of punch biopsy samples revealed dense infiltration of small lymphoid cells in the superficial dermis and in areas surrounding hair follicles. Immunohistochemical analysis indicated that these cells were positive for CD3, CD4, and TCRαβ, but negative for CD1c, CD8α, CD8β, CD11c, CD20, CD45RA, CD90, MHC‐II, and TCRγδ. In addition, CD45 was highly expressed, and the proliferation fraction was very low. Molecular clonality of T‐cell receptor G chains yielded a clonal result. The skin lesions were surgically excised because they had progressed to the lateral front leg. Postoperative clinical course was favorable, and recurrence was not observed until the dog died in a traffic accident, approximately 1 year later.

Feline indolent cutaneous T-cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL.To report the clinical outcome of three cats with ICL treated with hypofractionated electron-beam radiotherapy (RT).Three privately owned cats with ICL.Medical records and client surveys were reviewed. A diagnosis of probable ICL was based on history, clinical presentation and histopathological findings, and confirmed using CD3 immunohistochemical analysis and PCR for antigen receptor gene rearrangement (PARR). All cats were treated with hypofractionated RT (four fractions of 8 Gy).All cats presented with skin lesions characterised by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Before hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T-cell receptor gamma gene rearrangement. After RT, two cats showed histological improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histological resolution of lesions with only minimal residual lymphocytes. One cat was determined to have a complete clinical response while the other showed partial responses. No acute adverse effects of radiation were observed; chronic effects included leukotrichia, partial alopecia and mild fibrosis. All clients reported improvement in quality of life for their cats.Clinical and histological improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.Le lymphome à cellule T cutané indolent félin (ICL) est une néoplasie rare. Il n'y a pas de consensus sur les recommandations de traitement pour l'ICL.Décrire l'évolution clinique de trois chats avec ICT traités avec radiothérapie (RT) hypo-fractionnée par faisceaux.Trois chats de propriétaires avec ICL. MATÉRIELS ET MÉTHODES: Les données médicales et les suivis de clients ont été revus. Un diagnostic de probable ICL basé sur les commémoratifs, la présentation clinique et les données histopathologiques et confirmé par analyse immunohistochimique et PCR pour PARR (antigen receptor gene rearrangement). Tous les chats ont été traités par RT hypo-fractionnée (quatre fractions de 8 Gy). RÉSULTATS: Tous les chats présentaient des lésions cutanées caractérisées par de l'érythème et de l'alopécie, réfractaires aux traitements antérieurs avec des corticoïdes systémiques. Avant le traitement par RT hypofractionnée, les lésions étaient histologiquement décrites comme une infiltration dermique diffuse avec CD3+. Une analyse moléculaire de clonalité a révélé un réarrangement clonal du gène gamma du récepteur cellulaire T. Après RT, deux chats ont montré une amélioration histologique définie par une diminution de l'infiltration de lymphocytes avec un infiltrat cellulaire présent uniquement dans le derme le plus profond; un chat avait une résolution quasi complète des lésions avec seulement des lymphocytes résiduels minimes. Un chat avait une rémission clinique complète alors que les autres présentaient des réponses partielles. Aucun effet indésirable aigu des radiations n'a été observé; les effets chroniques incluaient une leucotrichie, une alopécie partielle et une fibrose modérée. Tous les clients ont rapporté une amélioration de la qualité de vie de leur chat.L'amélioration clinique et histologique de ces chats suggère que la RT hypofractionnée peut être utile dans les modalités de traitement des chats avec ICL.INTRODUCCIÓN: el linfoma de células T cutáneo indolente felino (ICL) es una enfermedad neoplásica poco común. Actualmente no existe consenso sobre las recomendaciones de tratamiento para ICL. OBJETIVO: describir el resultado clínico de tres gatos con ICL tratados con radioterapia de haz de electrones (RT) hipofraccionada. ANIMALES: tres gatos de propietarios particulares con ICL. MATERIALES Y MÉTODOS: se revisaron los historiales médicos y las encuestas a los clientes. El diagnóstico de ICL probable se basó en la historia, la presentación clínica y los hallazgos histopatológicos, y se confirmó mediante análisis inmunohistoquímico de CD3 y PCR para el reordenamiento del gen del receptor de antígeno (PARR). Todos los gatos fueron tratados con RT hipofraccionada (cuatro fracciones de 8 Gy). RESULTADOS: Todos los gatos presentaron lesiones cutáneas caracterizadas por eritema y alopecia refractarias al tratamiento previo con glucocorticoides sistémicos. Antes del tratamiento con RT hipofraccionada, histológicamente se describía que las lesiones presentaban infiltración difusa de la dermis con linfocitos T CD3 +. El análisis de clonalidad molecular reveló un reordenamiento del gen gamma del receptor de células T clonal. Después de la RT, dos gatos mostraron una mejoría histológica definida por una menor infiltración de linfocitos, con infiltrado celular presente solo en la dermis más profunda; un gato tuvo una resolución histológica casi completa de las lesiones con solo un mínimo de linfocitos residuales. Se determinó que un gato tuvo una respuesta clínica completa mientras que el otro mostró respuesta parcial. No se observaron efectos adversos agudos de la radiación; los efectos crónicos incluyeron leucotriquia, alopecia parcial y fibrosis leve. Todos los clientes informaron una mejora en la calidad de vida de sus gatos. CONCLUSIONES E IMPORTANCIA CLÍNICA: la mejora clínica e histológica en estos gatos sugiere que la RT hipofraccionada puede ser una modalidad de tratamiento útil para los gatos con ICL.Das feline indolente kutane T-Zell Lymphom (ICL) ist eine seltene neoplastische Erkrankung. Es gibt zurzeit keinen Konsens in Bezug auf Behandlungsempfehlungen für ICL. ZIEL: Ein Fallbericht über das klinische Ergebnis dreier Katzen mit ICL, die mit hypofraktionierter Elektronenstrahl-Radiotherapie (RT) behandelt worden waren.Drei Katzen mit ICL, die in Privatbesitz waren.Die Patientenkarteien und Fragebögen von KlientInnen wurden durchgesehen. Die Diagnose einer möglichen ICL basierte auf der Anamnese, der klinischen Präsentation und den Histopathologie Befunden, und wurde mittels CD3 immunhistochemischer Analyse und PCR für Antigen Rezeptor Gen Rearrangement (PARR) bestätigt. Alle Katzen wurden mit hypofraktionierter RT (vier Fraktionen von 8 Gy) behandelt.Alle Katzen wurden mit Hautveränderungen vorgestellt, die durch Erythem und Alopezie charakterisiert waren, die nach einer vorangegangenen Behandlung mit systemischen Glukokortikoiden wieder auftraten. Vor der Behandlung mit hypofraktionierter RT wurden die Veränderungen histologisch als diffuse Infiltration der Dermis mit CD3+ Zellen beschrieben. Die molekulare Klonalitätsanalyse zeigte ein klonales T-Zell Rezeptor Gamma Gen Rearrangement. Nach der RT zeigten zwei Katzen histologisch eine Verbesserung, die durch eine Abnahme der Lymphozyteninfiltration definiert wurde, wobei das zelluläre Infiltrat nur in der tieferen Dermis auftrat; eine Katze zeigte histologisch ein fast vollständiges Verschwinden der Veränderungen mit nur mehr minimalen Rest Lymphozyten. Bei einer Katze wurde festgehalten, dass sie eine völlige klinische Heilung zeigte, während die anderen nur eine partielle Verbesserung zeigten. Es wurden keine akuten Nebenwirkungen auf die Bestrahlung beobachtet; chronische Auswirkungen bestanden in Form einer Leukotrichie, teilweisen Alopezie und einer milden Fibrose. Alle BesitzerInnen berichteten von einer verbesserten Lebensqualität ihrer Katzen.Die klinische und histologische Verbesserung bei diesen Katzen weist darauf hin, dass die hypofraktionierte RT eine nützliche Behandlungsmodalität für Katzen mit ICL sein kann.背景: 猫の低悪性度皮膚T細胞リンパ腫 (ICL) は、まれな腫瘍性疾患である。現在、ICLの推奨治療法に関するコンセンサスはない。 目的: 本研究の目的は、低分割電子ビーム放射線療法 (RT) で治療されたICLの3頭の猫の臨床転帰を報告することであった。 被験動物: ICLを使用した3頭のオーナー所有猫。 材料と方法: 医療記録および顧客調査をレビューした。可能性のあるICLの診断は、病歴、臨床症状、および組織病理学的所見に基づいており、CD3免疫組織化学的解析および抗原受容体遺伝子再構成 (PARR) のPCR法を使用して確認された。すべての猫は低分割RT(8 Gyの4分割) で治療された。 結果: すべての猫は、以前の全身性糖質コルチコイドによる治療に抵抗性を示し、紅斑および脱毛症を特徴とする皮膚病変を呈した。低分割放射線治療の前に、病変は、CD3背景: 猫惰性皮肤T细胞淋巴瘤(ICL)是一种不常见的肿瘤性疾病。目前对ICL的治疗建议尚未达成共识。 目的: 报告3只ICL猫接受大分割电子束放疗(RT)治疗的临床结果。 动物: 3只ICL私家猫。 材料和方法: 审查病历和客户调查。可能的ICL诊断是基于病史、临床表现和组织病理学结果, 并使用CD3免疫组织化学分析和抗原受体基因重排(PARR)的PCR进行证实。所有猫均采用大分割放疗(4次8 Gy) 。 结果: 所有猫均表现为皮肤病变, 特征为红斑和脱毛, 既往全身性糖皮质激素治疗难以控制。在大分割放疗治疗前, 病变在组织学上被描述为CD3+T细胞真皮弥漫性浸润。分子克隆性分析发现克隆性T细胞受体γ基因重排。RT后, 2只猫显示组织学改善, 定义为淋巴细胞浸润减少, 细胞浸润仅存在于较深的真皮中; 一只猫的病变几乎完全组织学消退, 仅有极少量残留淋巴细胞。一只猫确定为临床完全缓解, 而另一只猫显示部分缓解。未观察到放疗的急性不良反应; 慢性效应包括白毛症、部分脱毛和轻度纤维化。所有客户均报告其猫的生活质量改善。 结论和临床重要性: 这些猫的临床和组织学改善表明, 大分割RT可成为ICL猫的有用治疗方式。.O linfoma cutâneo indolente de células T em felinos (LCI) é uma doença neoplásica incomum. Não há consenso atual sobre as recomendações de tratamento para o LCI.Relatar o desfecho de três casos de LCI em gatos tratados com radioterapia hipofracionada por feixe de elétrons (RT).Três gatos de propriedade privada apresentando LCI MATERIAIS E MÉTODOS: Foram revisados os prontuários e os questionários preenchidos pelos clientes. O diagnóstico de um provável LCI foi baseado no histórico, apresentação clínica e achados histopatológicos, e confirmado utilizando análise imunohistoquímica de CD3 e PCR para rearranjo do gene receptor do antígeno (antigen receptor gene rearrangement - PARR). Todos os gatos foram tratados com RT hipofracionada (quatro frações de 8 Gy).Todos os gatos apresentaram lesões cutâneas caracterizadas por eritema e alopecia refratárias aos tratamentos anteriores com glicocorticoides sistêmicos. Antes do tratamento com RT hipofracionada, as lesões histopatológicas descritas foram presença de infiltração difusa de células T CD3+ na derme. A análise molecular de clonalidade demonstrou rearranjo do gene gama de receptores de células T. Após a RT, dois gatos demonstraram melhora histológica caracterizada pela redução da infiltração de linfócitos e presença de infiltrado celular apenas na derme profunda; um gato apresentou quase resolução histológica completa restando apenas quantidade mínima de linfócitos residuais. Um gato apresentou resposta clínica completa, enquanto os outros apenas parcial. Não foram observadas reações adversas agudas à radiação; os efeitos crônicos observados foram leucotriquia, alopecia parcial e fibrose. Todos os clientes relataram melhora na qualidade de vida dos seus animais. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: A melhora clínica e histopatológica nesses gatos sugere que a RT hipofracionada uma modalidade terapêutica válida para gatos com LCI.

Background Chimeric antigen receptor (CAR) T cell therapy has transformed therapy for hematological malignancies but has not yet been established as standard of care for any solid tumors. One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant, immunocompetent animal models. In this study, we sought to establish CAR T cells for naturally occurring canine sarcomas in client owned animals as a model for human CAR T cell therapy. Methods Archived FFPE, freshly isolated canine solid tumor samples as well as tumor lines were tested for B7H3 expression by immunohistochemistry (IHC) and flow cytometry analysis. We designed CARs using the scFv from the human B7H3-specific antibody MGA271 and confirmed the cross-reactivity to canine B7H3 (construct information see figure 1A). A truncated EGFR (tEGFR) was included in the construct to allow for IHC and flow cytometry testing for the presence of CAR T cells. Killing efficiency was evaluated using 3D tumor spheroid killing assays to monitor dynamics. Safety of the CAR products following lymphodepletion was confirmed in two healthy dogs (figure 1B). Results Canine solid tumors were confirmed to be B7H3 positive in almost all cases. Using the GALV-pseudotyped retrovirus system, transduction was efficient with up to 70% CAR+ cells. Post-transduction expansion was over 100 folds. B7H3 CAR transduced canine T cells were able to eliminate B7H3+ canine tumor spheroids effectively (figure 2). Safety of the CAR T cells (dose: 1 × 109/m2) were confirmed in both healthy animals following cyclophosphamide lymphodepletion. After week 6, cetuximab was given to the subjects to deplete EGFR+ cells. Subject 2 experienced fever after CAR T cell administration. Both dogs showed elevated serum ALP and ALT levels and returned to normal (figure 3). No other treatment-related adverse events were observed. Information of the CAR T cell products can be found in table 1. Conclusions We demonstrated that, similar to human cancers, B7H3 is a target in canine solid tumors. We successfully generated canine B7H3 specific CAR T cell products that are highly efficient at killing canine 3D tumor spheroids using a production protocol that closely models human CAR T cell production procedure and confirmed the safety in vivo. We plan to test and optimize various approaches to enhance CAR T cell efficacy for solid tumor treatment both in vitro and in canine sarcoma patients. Ethics Approval The study was approved by Fred Hutchinson Cancer Research Center‘s Institutional Animal Care and Use Committee (IACUC), approval number PROTO201900860

Cutaneous histiocytomas (CH) are derived from epidermal Langerhans cells. Single CH are generally associated with a good prognosis in dogs because most undergo spontaneous remission. However, aggressive behaviour and lymph node metastasis have been reported in a small number of dogs with single CH.To describe the clinical presentation, treatment and disease progression of an aggressive CH located in the ear canal of a dog.An 8-year-old intact male Rottweiler dog.A unilateral ear canal mass was identified as a CH on routine haematoxylin and eosin stained samples. The diagnosis was confirmed by the demonstration of markers associated with Langerhans cells (Iba-1, E-cadherin and CD18) and the absence of markers associated with B cells (CD79a, CD20, Pax5), T cells (CD3), plasma cells (Mum-1) and macrophages (CD11d, CD204).A total ear canal ablation was performed, but tumour cells extended throughout the horizontal canal and to the deep surgical margin. Due to the locally invasive nature of the mass and incomplete excision, adjunctive chemotherapy with CCNU was pursued. No measurable local disease was appreciable at the time of the last treatment. At 250 days post-surgery the dog was euthanized owing to the development of multiple abdominal masses. No evidence of local tumour recurrence was noted.Although single CH are typically associated with benign behaviour, the mass in this dog demonstrated locally invasive behaviour. Cutaneous histiocytomas in the ear canals of dogs may represent a particularly aggressive variant of the condition.Les histiocytomes cutanés (CH) sont issus des cellules de Langerhans épidermiques. Un CH unique est généralement de bon pronostic chez le chien, la majorité rétrocédant spontanément. Cependant, une évolution agressive et des métastases ganglionnaires ont été décrites dans un faible nombre de cas de CH unique.Décrire la présentation clinique, le traitement et l’évolution d'un CH agressif localisé dans le conduit auriculaire d'un chien.Un Rottweiler mâle entier de 8 ans. MATÉRIELS ET MÉTHODES: Une masse unilatérale du conduit auriculaire a été identifié comme un CH par examen histopathologique coloré à l'hémalun-éosine. Le diagnostic a été confirmé par la disposition de marqueurs associés à des cellules de Langerhans (Iba-1, E-cadhérine et CD18) et l'absence de marqueurs associés aux cellules B (CD79a, CD20, Pax5), cellules T (CD3), cellules plasmatiques (Mum-1) et macrophages (CD11d, CD204). RÉSULTATS: Une ablation totale du conduit auriculaire a été réalisée mais les cellules tumorales s’étendaient au delà de la paroi horizontale et aux marges chirurgicales profondes. En raison de la nature localement invasive et de l'exérèse incomplète, une chimiothérapie d'appoint avec CCNU a été instaurée. Aucune lésion mesurable n’était observée lors du dernier traitement. A 250 jours après la chirurgie le chien a été euthanasié en raison du développement de masses abdominales multiples. Aucune preuve de récidive tumorale locale n'a été notée.Bien qu'un CH unique soit typiquement associé à une évolution bénigne, la masse chez ce chien a montré un comportement invasif local. Les histiocytomes cutanés du conduit auriculaire du chien pourraient représenter une variante particulièrement agressive.INTRODUCCIÓN: los histiocitomas cutáneos (CH) se derivan de las células epidérmicas de Langerhans. Los CH únicos generalmente se asocian con un buen pronóstico en los perros porque la mayoría experimenta una remisión espontánea. Sin embargo, se ha reportado un comportamiento agresivo y metástasis en ganglios linfáticos en un pequeño número de perros con CH único. OBJETIVO: describir la presentación clínica, el tratamiento y la progresión de la enfermedad de un CH agresivo ubicado en el canal auditivo de un perro. ANIMAL: un perro Rottweiler macho entero de 8 años. MÉTODOS Y MATERIALES: se identificó una masa unilateral del canal auditivo como CH en muestras teñidas con hematoxilina y eosina de rutina. El diagnóstico fue confirmado por la demostración de marcadores asociados con las células de Langerhans (Iba-1, E-cadherina y CD18) y la ausencia de marcadores asociados con las células B (CD79a, CD20, Pax5), células T (CD3), células plasmáticas (MUM - 1) y macrófagos (CD11d, CD204). RESULTADOS: se realizó una ablación total del canal auditivo, pero las células tumorales se extendieron por todo el canal horizontal y hasta el margen quirúrgico profundo. Debido a la naturaleza localmente invasiva de la masa y la escisión incompleta, se utilizó quimioterapia adyuvante con CCNU. Ninguna enfermedad local visible fue apreciable en el momento del último tratamiento. A los 250 días después de la cirugía, el perro fue sacrificado debido al desarrollo de múltiples masas abdominales. No se observó evidencia de recurrencia tumoral local. CONCLUSIÓN E IMPORTANCIA CLÍNICA: aunque el CH solitario se asocia típicamente con un comportamiento benigno, la masa en este perro demostró un comportamiento localmente invasivo. Los histiocitomas cutáneos en los canales auditivos de los perros pueden representar una variante particularmente agresiva de la afección.Kutane Histiozytome (CH) entspringen den epidermalen Langerhans Zellen. Einzelne CH werden generell mit einer guten Prognose bei Hunden gesehen, weil die meisten eine spontane Remission durchlaufen. Es wurde jedoch auch aggressives Wachstum und Lymphknotenmetastasierung in einer geringen Anzahl von Hunden mit einzelnen CH beschrieben. ZIEL: Eine Beschreibung der klinischen Präsentation, der Behandlung und des Krankheitsfortschritts bei einem aggressiven CH, welcher im Ohrkanal eines Hundes wuchs.Es handelt sich um einen 8-jährigen intakten Rottweilerrüden.Es wurde eine unilaterale Masse im Ohr mittels routinemäßiger Hämatoxylin und Eosin Färbung als CH beschrieben. Die Diagnose wurde durch den Nachweis von Langerhans Zell Markern (Iba-1, E-Cadherin und CD 18), sowie einer Abwesenheit von B-Zell Markern (CD79a, CD20, Pax5), T Zellen (CD3), Plasmazellen (Mum-1) und Makrophagen (CD11d, CD204) bestätigt.Es wurde eine Totale Ohrkanalablation durchgeführt, aber die Tumorzellen erstreckten sich entlang des gesamten Horizontalkanals und in die Tiefe der chirurgischen Exzisionslinie. Aufgrund der lokal invasiven Natur der Masse und der unvollständigen Entfernung wurde eine zusätzliche Chemotherapie mit CCNU durchgeführt. Zum Zeitpunkt der letzten Behandlung konnte lokal keine krankhafte Veränderung mehr festgestellt werden. Zweihundertfünfzig Tage nach der Operation wurde der Hund aufgrund der Entwicklung multipler abdominaler Massen eingeschläfert. Es wurde zu diesem Zeitpunkt kein lokales Wiederwachsen des Tumors festgestellt.背景: 皮膚組織球腫(CH)は表皮ランゲルハンス細胞に由来する。孤立性CHは、ほとんどが自然寛解を経験するため、一般的に犬の良好な予後と関連している。しかしながら、孤立性CHに罹患した少数の犬で悪性挙動およびリンパ節転移が報告されている。 目的: 本研究における目的は、ある犬の外耳道に発生した悪性挙動CHの臨床症状、治療、および疾患の進行を説明することであった。 供試動物: 8歳、雄のロットワイラー。 材料と方法: 通常のヘマトキシリンおよびエオジン染色サンプルにより片側外耳道腫瘍をCHと特定した。診断は、ランゲルハンス細胞(Iba-1、E-カドヘリン、CD18)関連マーカーの実証およびB細胞(CD79a、CD20、Pax5)、T細胞(CD3)、形質細胞( Mum-1)およびマクロファージ(CD11d、CD204)関連マーカーの欠如によって確認した。 結果: 全耳道切除術を実施したが、腫瘍細胞は水平耳道全体および深部手術縁まで広がっていた。腫瘤の局所侵襲性および不完全切除のため、CCNUによる補助化学療法を実施した。最終治療点では、測定可能な局所疾患は認められなかった。犬は手術後250日で、複数の腹部腫瘤が発生したため、安楽死させられた。局所腫瘍再発の証拠は認められなかった。 結論と臨床的重要性: 通常、孤立性CHは良性挙動に関連付けられているが、供試犬の腫瘍は局所的に侵襲的な挙動を示した。犬の外耳道の皮膚組織球腫は、この疾患において特に攻撃的な変異型である可能性がある。.背景: 皮肤组织细胞瘤 (CH) 来源于表皮郎格罕细胞。因为犬的单个CH大多数能自愈,所以通常预后良好。然而,在少数单个 CH患犬的病例报告中,发现了侵袭行为和淋巴结转移。 目的: 描述位于犬耳道的侵袭性CH的临床表现、治疗和发病过程。 动物: 8 岁、未去势雄性罗威纳犬。 方法和材料: 在常规苏木精和伊红染色样本中,单侧耳道肿块被确定为 CH。通过证明郎格罕细胞标记物(Iba-1、E-cadherin 和 CD18)以及缺乏 B 细胞 (CD79a、CD20、Pax5)、T 细胞 (CD3)、浆细胞 (Mum-1) 和巨噬细胞 (CD11d、CD204) 标记物来确诊。 结果: 进行全耳道切除,但肿瘤细胞延伸至整个水平耳道和深部手术边缘。由于肿块局部侵袭性强,切除不彻底,给予CCNU 作为辅助化疗。末次治疗时未检测到局部疾病。术后 250 天,由于出现多个腹部肿块,对犬实施安乐死。未观察到局部肿瘤复发证据。 结论和临床重要性: 尽管单个CH的生物学行为通常为良性,但该犬的肿块表现出局部侵袭行为。犬耳道中的皮肤组织细胞瘤可能代表了该疾病一种特殊的具有侵袭性的变体。.Os histiocitomas cutâneos (HC) são derivados de células de Langerhan epidérmicas. O HC único está geralmente associado a um bom prognóstico em cães, porque a maioria sofre remissão espontânea. No entanto, comportamento agressivo e metástase para linfonodos foram relatados em um pequeno número de cães com HC único.Descrever a apresentação clínica, o tratamento e a progressão da doença de um HC agressivo localizado no conduto auditivo de um cão.Um cão Rottweiler macho intacto de 8 anos de idade. MÉTODOS E MATERIAIS: Uma massa unilateral no conduto auditivo foi classificada como HC nas amostras coradas com hematoxilina e eosina. O diagnóstico foi confirmado pela demonstração de marcadores associados às células de Langerhans (Iba - 1, E - caderina e CD18) e pela ausência de marcadores associados às células B (CD79a, CD20, Pax5), células T (CD3), plasmócitos (Mum-1) e macrófagos (CD11d, CD204).Realizou-se ablação total do canal auditivo, mas as células tumorais se estenderam por todo o conduto horizontal e até a margem cirúrgica profunda. Devido à natureza localmente invasiva da massa e excisão incompleta, foi realizada quimioterapia adjuvante com CCNU. Nenhuma doença local mensurável foi observada no momento do último tratamento. Aos 250 dias após a cirurgia, o cão foi eutanasiado devido ao desenvolvimento de várias massas abdominais. Nenhuma evidência de recorrência local do tumor foi observada. CONCLUSÃO E IMPORTÂNCIA CLÍNICA: Embora um único HC esteja tipicamente associado a um comportamento benigno, a massa neste cão demonstrou um comportamento localmente invasivo. Histiocitomas cutâneos nos condutos auditivos de cães podem representar uma variante particularmente agressiva da doença.

Interruption of the programmed death 1 (PD-1) / programmed death ligand 1 (PD-L1) pathway is an established and effective therapeutic strategy in human oncology and holds promise for veterinary oncology. We report the generation and characterization of monoclonal antibodies specific for canine PD-1 and PD-L1. Antibodies were initially assessed for their capacity to block the binding of recombinant canine PD-1 to recombinant canine PD-L1 and then ranked based on efficiency of binding as judged by flow cytometry. Selected antibodies were capable of detecting PD-1 and PD-L1 on canine tissues by flow cytometry and Western blot. Anti-PD-L1 worked for immunocytochemistry and anti-PD-1 worked for immunohistochemistry on formalin-fixed paraffin embedded canine tissues, suggesting the usage of this antibody with archived tissues. Additionally, anti-PD-L1 (JC071) revealed significantly increased PD-L1 expression on canine monocytes after stimulation with peptidoglycan or lipopolysaccharide. Together, these antibodies display specificity for the natural canine ligand using a variety of potential diagnostic applications. Importantly, multiple PD-L1-specific antibodies amplified IFN-γ production in a canine peripheral blood mononuclear cells (PBMC) concanavlin A (Con A) stimulation assay, demonstrating functional activity.

Small cell intestinal lymphoma has not been well characterized in dogs. The objective of this study was to describe clinical characteristics and outcome in dogs with small cell intestinal lymphoma. We hypothesized that affected dogs would have prolonged survival compared with high-grade gastrointestinal (GI) lymphoma. Pathology records were searched for dogs with histologically confirmed small cell GI lymphoma. Seventeen dogs with confirmed small cell intestinal lymphoma were identified, and clinical and outcome data were retrospectively collected. Histopathology was reviewed by a board-certified pathologist, and tissue sections were subjected to immunophenotyping and molecular clonality assessment. All dogs had small cell, T-cell, lymphoma confirmed within various regions of small intestine, with 1 dog also having disease in abdominal lymph nodes. All dogs had clinical signs attributable to GI disease; diarrhoea (n = 13) was most common. Ultrasonographic abnormalities were present in 8 of 13 dogs with abnormal wall layering (n = 7) and hyperechoic mucosal striations (n = 7) representing the most common findings. In total, 14 dogs received some form of treatment. The median survival time (MST) for all dogs was 279 days and the MST for the 14 dogs that received any treatment was 628 days. Dogs with anaemia and weight loss at presentation had significantly shorter survival times and dogs that received a combination of steroids and an alkylating agent had significantly longer survival times. Small cell, T-cell, intestinal lymphoma is a distinct disease process in dogs, and those undergoing treatment may experience prolonged survival.

In human medicine, narrow-band ultraviolet B (NB-UVB) phototherapy has been used to treat various T-cell-mediated skin diseases. However, the effect of NB-UVB on inflamed canine skin remains uncertain.To investigate the effect of NB-UVB phototherapy on the skin of dogs with hapten-induced contact dermatitis.Seven healthy beagles without skin problems.Dogs were irradiated with varying doses of NB-UVB to determine the minimal erythema dose (MED). After determining the MEDs of six dogs (excluding one of the seven whose skin did not show a visible reaction), we investigated the effect of NB-UVB on their inflamed skin by topically applying 2,4-dinitrochlorobenzene (DNCB), which causes type 1 helper T cell (Th1)- and cytotoxic T-cell (Tc)1-induced skin inflammation. We then irradiated the skin with NB-UVB. We analysed the treated skin samples via histopathological and immunohistochemical methods, and TdT-mediated dUTP nick-end labelling (TUNEL) to demonstrate apoptotic cells. We also analysed the cytokine gene transcription via real-time quantitative reverse transcription PCR.The NB-UVB MEDs caused mild inflammatory changes yet no severe epidermal exfoliations in the irradiated skin. In DNCB-treated skin irradiated by the NB-UVB MEDs, TUNEL-positive dermal apoptotic cells were increased significantly compared with those of DNCB-treated, nonirradiated skin. INF-γ and TNF-α transcription levels in DNCB-treated, irradiated skin were significantly lower than those in the DNCB-treated, nonirradiated skin.Phototherapy using NB-UVB MEDs attenuated cutaneous Th1 and Tc1 cytokine responses with minimal skin damage in a canine model of hapten-induced contact dermatitis.En médecine humaine, la photothérapie ultraviolette B (NB-UVB) à bande étroite a été utilisée pour traiter diverses maladies de la peau à médiation par les lymphocytes T. Cependant, l'effet de NB-UVB sur la peau canine enflammée reste incertain.Étudier l'effet de la photothérapie NB-UVB sur la peau de chiens atteints de dermatite de contact induite par l'haptène.Sept beagles en bonne santé sans problèmes de peau. MÉTHODES ET MATÉRIEL: Les chiens ont été irradiés avec des doses variables de NB-UVB pour déterminer la dose minimale d'érythème (DEM). Après avoir déterminé les MED de six chiens (à l'exclusion de l'un des sept dont la peau n'a pas montré de réaction visible), nous avons étudié l'effet du NB-UVB sur leur peau enflammée en appliquant localement du 2,4-dinitrochlorobenzène (DNCB), qui provoque un inflammation de type 1 de la peau induite par les lymphocytes T auxiliaires (Th1) et les lymphocytes T cytotoxiques (Tc)1. Nous avons ensuite irradié la peau avec du NB-UVB. Nous avons analysé les échantillons de peau traités via des méthodes histopathologiques et immunohistochimiques, et le marquage dUTP nick-end (TUNEL) médié par TdT pour démontrer les cellules apoptotiques. Nous avons également analysé la transcription du gène des cytokines par PCR de transcription inverse quantitative en temps réel. RÉSULTATS: Les MED NB-UVB ont provoqué de légers changements inflammatoires, mais aucune exfoliation épidermique grave de la peau irradiée. Dans la peau traitée au DNCB irradiée par les MED NB-UVB, les cellules apoptotiques dermiques positives pour TUNEL ont augmenté de manière significative par rapport à celles de la peau non irradiée traitée au DNCB. Les niveaux de transcription d'INF-γ et de TNF-α dans la peau irradiée traitée au DNCB étaient significativement inférieurs à ceux de la peau non irradiée traitée au DNCB.La photothérapie utilisant des MED NB-UVB a atténué les réponses des cytokines cutanées Th1 et Tc1 avec des lésions cutanées minimales dans un modèle canin de dermatite de contact induite par l'haptène.INTRODUCCIÓN: en medicina humana, la fototerapia ultravioleta B de banda estrecha (NB-UVB) se ha utilizado para tratar diversas enfermedades de la piel mediadas por células T. Sin embargo, el efecto de NB-UVB sobre la piel canina inflamada sigue siendo incierto. OBJETIVOS: investigar el efecto de la fototerapia NB-UVB en la piel de perros con dermatitis de contacto inducida por hapteno. ANIMALES: Siete perros Beagle sanos sin problemas en la piel. MÉTODOS Y MATERIALES: los perros se irradiaron con dosis variables de NB-UVB para determinar la dosis mínima de eritema (MED). Después de determinar las MEDs de seis perros (excluyendo uno de los siete cuya piel no mostró una reacción visible), investigamos el efecto de NB-UVB en su piel inflamada aplicando tópicamente 2,4-dinitroclorobenceno (DNCB), que causa inflamación de la piel por tipo 1 linfocito T colaborador (Th1) y linfocito T citotóxico (Tc) 1. Luego irradiamos la piel con NB-UVB. Analizamos las muestras de piel tratadas a través de métodos histopatológicos e inmunohistoquímicos, y marcaje de extremos rotos (Nick-end labelling) dUTP mediado por TdT (TUNEL) para demostrar células apoptóticas. También analizamos la transcripción de genes de citoquinas mediante PCR cuantitativa de transcripción inversa en tiempo real. RESULTADOS: las NB-UVB MEDs causaron cambios inflamatorios leves pero no exfoliaciones epidérmicas graves en la piel irradiada. En la piel tratada con DNCB irradiada por las MEDs de NB-UVB, las células apoptóticas dérmicas positivas a TUNEL aumentaron significativamente en comparación con las de la piel no irradiada tratada con DNCB. Los niveles de transcripción de INF-γ y TNF-α en la piel irradiada tratada con DNCB fueron significativamente más bajos que los de la piel no irradiada tratada con DNCB. CONCLUSIÓN Y RELEVANCIA CLÍNICA: la fototerapia con NB-UVB MEDs atenuó las respuestas de las citoquinas cutáneas Th1 y Tc1 con un daño cutáneo mínimo en un modelo canino de dermatitis de contacto inducida por hapteno.In der Humanmedizin wird die Schmalband Ultraviolet B (NB-UVB) Phototherapie verwendet, um verschiedene T-Zell-mediierte Hauterkrankungen zu behandeln. Die Auswirkung von NB-UVB auf entzündete Hundehaut bleibt jedoch noch unklar.Eine Untersuchung der Auswirkung von NB-UVB Phototherapie auf die Haut von Hunden mit Hapten-induzierter Kontaktdermatitis.Sieben gesunde Beagles ohne Hautprobleme.Die Hunde wurden mit unterschiedlichen Dosen an NB-UVB bestrahlt, um die minimale Erythem Dosis (MED) zu bestimmen. Nach Bestimmung der MEDs bei sechs Hunden (einer der sieben Hunde wurde ausgenommen, da seine Haut keine sichtbare Reaktion zeigte), untersuchten wir die Auswirkung von NB-UVB auf ihre entzündete Haut durch topische Verabreichung von 2,3-Dinitrochlorobenzen (DNCB), welches eine Typ 1 Helfer T Zell (Th1)- und zytotoxische T-Zell (Tc)1-induzierte Hautentzündung verursacht. Danach wurde die Haut mit NB-UVB bestrahlt. Wir analysierten die behandelten Hautproben mittels histopathologischer und immunhistochemischer Methoden und TdT-mediierter dUTP Nick-Ende Färbung (TUNEL), um apoptotische Zellen zu demonstrieren. Wir analysierten auch die Zytokin Gentranskription mittels Real-Time Quantitativer Reverser Transkriptions PCR.Die NB-UVB MEDs verursachten in der bestrahlten Haut milde entzündliche Veränderungen, jedoch keine hochgradigen epidermalen Exfoliationen. Bei der DNCB-behandelten Haut, die mit NB-UVB MEDS bestrahlt worden war, nahm die Anzahl der TUNEL-positiven dermalen apoptotischen Zellen im Vergleich zu den mit DNCB-behandelter, nicht bestrahlter Haut signifikant zu. INF-γ und TNF-α Transkriptionswerte bei DNCB-behandelter, bestrahlter Haut waren signifikant niedriger als jene in der DNCB-behandelten, nicht bestrahlten Haut.Phototherapie mittels NB-UVB MEDS verminderte die kutane Th1 und Tc1 Zytokin Antwort bei minimalen Hautveränderungen in einem Hundemodell von Hapten-induzierter Kontaktdermatitis.背景: ヒトでは、ナローバンド紫外線B(NB-UVB) 光線療法は、様々なT細胞介在性皮膚疾患の治療に用いられている。しかし、炎症を起こした犬の皮膚に対するNB-UVBの効果はまだ不明である。 目的: 本研究の目的は、ハプテン誘発性接触皮膚炎のイヌの皮膚に対するNB-UVB光線療法の効果を調査することであった。 被検動物: 皮膚に問題のない健常ビーグル7頭。 材料と方法: 犬に様々な線量のNB-UVBを照射し、最小紅斑量 (MED) を決定した。6頭の犬 (皮膚が目に見える反応を示さなかった7頭のうち1頭を除く) のMEDを決定した後、1型ヘルパーT細胞 (Th1) および細胞障害性T細胞 (Tc)1誘発性皮膚炎症を引き起こす2,4-ジニトロクロロベンゼン (DNCB) を局所的に塗布することで、炎症を起こした皮膚に対するNB-UVBの効果を調査した。次に、この皮膚にNB-UVBを照射した。照射した皮膚サンプルは、病理組織学的および免疫組織化学的手法により解析し、TdT-mediated dUTP nick-end labelling (TUNEL)によりアポトーシス細胞を検出した。また、リアルタイム定量逆転写PCRでサイトカイン遺伝子の転写を解析した。 結果: NB-UVB MEDは、照射された皮膚に軽度の炎症性変化を引き起こしたが、重度の表皮剥離はなかった。NB-UVB MEDを照射したDNCB処理皮膚では, TUNEL陽性の真皮アポトーシス細胞がDNCB処理した非照射皮膚に比べて有意に増加した。DNCB処理した照射皮膚のINF-γおよびTNF-α転写レベルは、DNCB処理した非照射皮膚の転写レベルに比べて有意に低かった。 結論と臨床的妥当性: NB-UVB MEDを用いた光線療法は、ハプテン誘発性接触皮膚炎イヌモデルにおいて、皮膚の損傷を最小限に抑えながら、皮膚のTh1およびTc1サイトカイン反応を弱めた。.背景: 在人类医学中, 窄谱中波紫外线(NB-UVB)光疗已被用于治疗各种T细胞介导的皮肤病。然而, NB-UVB对犬发炎皮肤的影响仍不确定。 目的: 半抗原诱导出犬接触性皮炎,研究NB-UVB光疗对皮肤的影响。 动物: 7只健康比格犬, 无皮肤问题。 方法和材料: 用不同剂量的NB-UVB照射犬, 测定最小红斑剂量(MED)。在确定了6只犬的MED后 (不包括1只皮肤无可见反应的犬) , 我们研究NB-UVB对发炎皮肤的影响,通过局部涂抹2,4-二硝基氯苯(DNCB),引起1型辅助性T细胞(Th1)和细胞毒性T细胞(Tc)1诱导的皮肤炎症。然后我们用NB-UVB照射皮肤。我们通过组织病理学和免疫组织化学方法分析了治疗的皮肤样本, 并通过TdT介导的dUTP缺口末端标记(TUNEL)证明凋亡细胞。我们还通过实时定量逆转录PCR分析了细胞因子基因转录。 结果: NB-UVB MED在照射皮肤中引起轻度炎症变化, 但无重度表皮剥脱。DNCB处理、经NB-UVB MEDs照射皮肤, TUNEL阳性真皮凋亡细胞较DNCB处理、未照射皮肤显著增加。DNCB处理、照射皮肤的INF-γ和TNF-α转录水平显著低于DNCB处理、未照射皮肤。 结论和临床相关性: 在半抗原诱导的接触性皮炎犬模型中, 使用NB-UVB MED的光疗减弱了皮肤Th1和Tc1细胞因子反应, 减轻了皮肤损伤。.Na medicina humana, a fototerapia ultravioleta B de banda curta (NB-UVB) tem sido usada para tratar várias doenças cutâneas mediadas por células T. No entanto, o efeito de NB-UVB na pele canina inflamada permanece incerto.Investigar o efeito da fototerapia NB-UVB na pele de cães com dermatite de contato induzida por hapteno.Sete beagles saudáveis sem diagnóstico de dermatopatia. MÉTODOS E MATERIAIS: Os cães foram irradiados com doses variadas de NB-UVB para determinar a dose eritema mínima (MED). Depois de determinar as MEDs de seis cães (excluindo um dos sete cuja pele não mostrou uma reação visível), investigamos o efeito de NB-UVB em sua pele inflamada aplicando topicamente 2,4-dinitroclorobenzeno (DNCB), que causa inflamação cutânea mediada por células T-helper 1 (Th1) e por células T citotóxicas (Tc) 1. Em seguida, irradiamos a pele com NB-UVB. Analisamos as amostras de pele tratadas por meio de métodos histopatológicos e imuno-histoquímicos e marcação com dUTP nick-end (TUNEL) mediada por TdT para demonstrar células apoptóticas. Também analisamos a transcrição do gene da citocina via PCR de transcrição reversa quantitativa em tempo real.As MEDs de NB-UVB causaram leves alterações inflamatórias, mas não geraram esfoliação epidérmica grave na pele irradiada. Na pele tratada com DNCB irradiada pelas MEDs de NB-UVB, as células apoptóticas dérmicas positivas para TUNEL aumentaram significativamente em comparação com aquelas da pele não irradiada tratada com DNCB. Os níveis de transcrição de INF-γ e TNF-α em pele irradiada tratada com DNCB foram significativamente mais baixos do que na pele não irradiada tratada com DNCB. CONCLUSÃO E RELEVÂNCIA CLÍNICA: A fototerapia com MEDs de NB-UVB atenuou as respostas cutâneas de citocinas Th1 e Tc1 com injúria cutânea mínima em um modelo canino de dermatite de contato induzida por hapteno.

Summary A 14-year-old domestic longhaired cat presented with a 2-year history of nasal discharge and a recent onset of inappetence and submandibular lymphadenopathy. The cat was humanely destroyed after developing severe respiratory distress. Necropsy examination revealed thickened nasal turbinates and soft palate, and friable red–tan material within the frontal sinus, nasal cavity and nasopharynx. The lungs contained multifocal irregular friable tan nodules. Multiple lymph nodes were enlarged, friable and red–tan in colour. Histopathology revealed a mature type extramedullary plasmacytoma (EMP) within the frontal sinus, nasal cavity, soft palate, larynx, trachea, lungs and multiple lymph nodes. The lymph nodes and larynx also contained marked granulomatous inflammation with extensive intrahistiocytic (and lesser amounts of extracellular) lambda light chain amyloid, confirmed by electron microscopy and immunohistochemistry. Neoplastic cells expressed CD79a and MUM1. This is the first report of an infiltrative EMP of the feline respiratory tract with lymph node metastasis and predominantly intrahistiocytic amyloid.

An 11-year-old, 443-kg Haflinger mare was presented to the North Carolina State University Veterinary Teaching Hospital with a 2-week history of lethargy and a 3-day duration of anorexia, pyrexia, tachycardia, and ventral edema. Severe pitting edema, peripheral lymphadenopathy, and a caudal abdominal mass were noted on physical examination. An extreme leukocytosis (154.3 × 103 /μL) and microscopic hematologic findings suggestive of myelomonocytic leukemia were observed. Serum protein electrophoresis revealed a monoclonal gammopathy and urine protein electrophoresis revealed a monoclonal light chain proteinuria. Necropsy and histopathology confirmed widespread neoplastic infiltration in many organs with a heterogenous population of cells; there was no apparent evidence of bone marrow involvement. Immunohistochemistry confirmed presence of a majority of B cells with a limited antigen expression, admixed with a lower number of T cells. Molecular clonality analysis of IgH2, IgH3, and kappa-deleting element (KDE, B cell) on whole blood and KDE on infiltrated tissues revealed clonal rearrangements, and the KDE intron clones that amplified in blood and in infiltrated tissue were identical. In contrast, the clonality analysis of T-cell receptor γ revealed no clonality on blood cells and infiltrated tissues. In conjunction with the histopathologic changes, the lesion was interpreted to be composed of neoplastic B cells with a reactive T-cell population. Polymerase chain reaction testing for equine herpes virus 5 was negative. The final diagnosis was diffuse large B-cell lymphoma with a marked hematogenous component.

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.

Placentitis has been reported as the most important cause of equine abortions, stillbirths, and perinatal deaths in horses. Most cases are caused by bacteria and less commonly by fungal elements. The aim of this report is to describe the clinical presentation of a fungal placentitis caused by Aspergillus terrerus. A 5-year-old thoroughbred maiden mare at the 217th day of gestation presented with some classic signs of placentitis (premature udder development and milk dripping). All ultrasonographic findings were consistent with a live fetus and a severe placentitis. On vaginal examination, purulent discharge was found coming from the external cervical os. Samples sent for culture yielded very small numbers of mixed growth including Enterococcus faecalis (by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer), Streptococcus viridans, and Aspergillus terreus, and polymerase chain reaction was positive for Aspergillus terreus and Pseudomonas. The mare was placed on broad-spectrum antimicrobials, nonsteroidal anti-inflammatories, and hormonal and antifungal treatment. The fetus kept on developing and growing despite the placentitis for 14 days until the demise of the fetus in utero occurred. Aspergillus terreus was isolated from the chorionic surface but not from the fetus. Fungal placentitis is not very commonly found in mares. The extent of the placental lesions and the severity of the placentitis contributed to the death of the fetus. This is one of the few case reports available describing fungal placentitis. Aspergillus terreus has not been previously reported as a cause of placentitis.

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.

OBJECTIVES To describe a small subset of canine solitary cutaneous histiocytoma in which lymph node metastasis has been documented. METHODS Cases of dogs with solitary cutaneous histiocytoma lesions and regional lymph node metastasis diagnosed via histopathology were found through a retrospective search of the databases of IDEXX Laboratories and the University of California, Davis Veterinary Medical Teaching Hospital Clinical Diagnostic Laboratories. Information on signalment, history and clinical follow-up was obtained from the submittal form and/or via a questionnaire to the submitting veterinarian. Slides were available for review in seven cases and when possible immunohistochemistry was reviewed or performed by a single pathologist. RESULTS Eight cases met the inclusion criteria. The neoplasms had the typical appearance of histiocytomas. All tested samples were immunoreactive for CD18 and lacked immunoreactivity for other lymphocyte markers and CD11d. Immunoreactivity for E-cadherin varied among the neoplasms tested. Outcome was known for five dogs and at the time of manuscript preparation three of those dogs were alive 1682 days, 570 days and 318 days post-diagnosis. Of the other two dogs with known outcome, one was euthanased shortly after diagnosis and another was hit by a car. Of the dogs that were eventually lost to follow-up, one was reported to be disease-free 1003 days after diagnosis. CLINICAL SIGNIFICANCE Metastatic histiocytoma is rarely reported and distinction from aggressive disease processes such as histiocytic sarcoma may be difficult. Based upon a small number of cases with known outcomes, some dogs with solitary metastatic histiocytoma may experience favourable outcomes.

Feline primary cutaneous lymphomas (FPCLs) account for 0.2% to 3% of all lymphomas in cats and are more frequently dermal nonepitheliotropic small T-cell tumors. Emergence of FPCL seems unrelated to feline leukemia virus (FeLV) serological positivity or to skin inflammation. A total of 17 cutaneous lymphomas with a history of vaccine injection at the site of tumor development were selected from 47 FPCLs. Clinical presentation, histology, immunophenotype, FeLV p27 and gp70 expression, and clonality were assessed. A majority of male (12/17), domestic short-haired (13/17) cats with a mean age of 11.3 years was reported. Postinjection time of development ranged from 15 days to approximately 9 years in 5 cats. At diagnosis, 11 of 17 cats had no evidence of internal disease. Lymphomas developed in interscapular (8/17), thoracic (8/17), and flank (1/17) cutaneous regions; lacked epitheliotropism; and were characterized by necrosis (16/17), angiocentricity (13/17), angioinvasion (9/17), angiodestruction (8/17), and peripheral inflammation composed of lymphoid aggregates (14/17). FeLV gp70 and/or p27 proteins were expressed in 10 of 17 tumors. By means of World Health Organization classification, immunophenotype, and clonality, the lesions were categorized as large B-cell lymphoma (11/17), anaplastic large T-cell lymphoma (3/17), natural killer cell–like (1/17) lymphoma, or peripheral T-cell lymphoma (1/17). Lineage remained uncertain in 1 case. Cutaneous lymphomas at injection sites (CLIS) shared some clinical and pathological features with feline injection site sarcomas and with lymphomas developing in the setting of subacute to chronic inflammation reported in human beings. Persistent inflammation induced by the injection and by reactivation of FeLV expression may have contributed to emergence of CLIS.

A 5-year-old male castrated ferret was presented for evaluation of chronic diarrhea, unresponsive to medical therapy. Initial examination and minimum database findings failed to determine an underlying etiology for the ferret's gastrointestinal disorder. Biopsies obtained by exploratory surgery confirmed a diagnosis of epitheliotropic gastrointestinal T-cell lymphoma. Several treatment strategies were discussed, and ultimately palliative care with prednisolone was elected. Over the course of treatment, the ferret developed concurrent pancreatic beta-cell adenomas (insulinoma) and a left adrenocortical carcinoma. At the time of necropsy, nearly 15 months after the exploratory surgery, the lymphoma appeared to be in regression despite the use of only palliative therapy.

While dogs are increasingly being utilized as large-animal models of disease, important features of age-related immunosenescence in the dog have yet to be evaluated due to the lack of defined naive vs. memory T lymphocyte phenotypes. We therefore performed multi-color flow cytometry on peripheral blood mononuclear cells from young and aged beagles, and determined the differential cytokine production by proposed memory subsets. CD4+ and CD8+ T lymphocytes in aged dogs displayed increased cytokine production, and decreased proliferative capacity. Antibodies targeting CD45RA and CD62L, but less so CD28 or CD44, defined canine cells that consistently exhibited properties of naive-, central memory-, effector memory-, and terminal effector-like CD4+ and CD8+ T lymphocyte subsets. Older dogs demonstrated decreased frequencies of naive-like CD4+ and CD8+ T lymphocytes, and an increased frequency of terminal effector-like CD8+ T lymphocytes. Overall findings revealed that aged dogs displayed features of immunosenescence similar to those reported in other species.

Our laboratory has serially reported on the virologic and immunopathologic features of a cohort of experimental feline immunodeficiency virus (FIV)-infected cats for more than eight years. At 8.09 years post infection (PI), one of these animals entered the terminal stage of infection, characterized by undulating hyperthermia, progressive anorexia, weight loss, and pancytopenia; the animal was not responsive to therapeutic interventions, necessitating euthanasia six weeks later (8.20 years PI). Subsequent analyses indicated that neoplastic lymphocytes infiltrated multiple cervical lymph nodes and a band-like region of the mucosal lamina propria within a segment of the intestine. Immunohistochemistry and T cell clonality testing determined that the nodal and intestinal lesions were independently arising from CD3 T cell lymphomas. In-situ RNA hybridization studies indicated that diffuse neoplastic lymphocytes from the cervical lymph node contained abundant viral nucleic acid, while viral nucleic acid was not detectable in lymphocytes from the intestinal lymphoma lesion. The proviral long terminal repeat (LTR) was amplified and sequenced from multiple anatomic sites, and a common clone containing a single nucleotide polymorphism was determined to be defective in response to phorbol myristate acetate (PMA)-mediated promoter activation in a reporter gene assay. This assay revealed a previously unidentified PMA response element within the FIV U3 region 3’ to the TATA box. The possible implications of these results on FIV-lymphoma pathogenesis are discussed.

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.

Histiocytic proliferative disorders are commonly observed in dogs and less often cats. Histiocytic disorders occur in most of the dendritic cell (DC) lineages. Canine cutaneous histiocytoma originates from Langerhans cells (LCs) indicated by expression of CD1a, CD11c/CD18, and E-cadherin. When histiocytomas occur as multiple lesions in skin with optional metastasis to lymph nodes and internal organs, the disease resembles cutaneous Langerhans cell histiocytosis of humans. Langerhans cell disorders do not occur in feline skin. Feline pulmonary LCH has been recognized as a cause of respiratory failure due to diffuse pulmonary infiltration by histiocytes, which express CD18 and E-cadherin and contain Birbeck’s granules. In dogs and cats, histiocytic sarcomas (HS) arise from interstitial DCs that occur in most tissues of the body. Histiocytic sarcomas begin as localized lesions, which rapidly disseminate to many organs. Primary sites include spleen, lung, skin, brain (meninges), lymph node, bone marrow, and synovial tissues of limbs. An indolent form of localized HS, progressive histiocytosis, originates in the skin of cats. Hemophagocytic HS originates in splenic red pulp and bone marrow macrophages in dogs and cats. In dogs, histiocytes in hemophagocytic HS express CD11d/CD18, which is a leuko-integrin highly expressed by macrophages in splenic red pulp and bone marrow. Canine reactive histiocytic diseases, systemic histiocytosis (SH) and cutaneous histiocytosis, are complex inflammatory diseases with underlying immune dysregulation. The lesions are dominated by activated interstitial DCs and lymphocytes, which invade vessel walls and extend as vasocentric infiltrates in skin, lymph nodes, and internal organs (SH).

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

Very little is known about the occurrence of immune system cells in the canine uterus. The aim of this study was to generate information about lymphocyte subsets that are present in the healthy canine uterus and that are recruited under inflammatory conditions caused by pyometra. Using immunohistochemistry and flow cytometry, a significant influx of γδ T lymphocytes was found in pyometra samples mainly due to recruitment of γδ(+)/CD8(-) T lymphocytes. The relative expression of genes encoding selected cytokines/chemokines was evaluated in samples from healthy and pyometra-affected uteri. Expression of pro-inflammatory cytokines (including IL-1β, TNF-α, IL-8, IL-17 and IFN-γ) and chemokines (including CXCL10, CCL4 and CCL5) was upregulated in pyometra samples confirming the presence of inflammation. In contrast, the expression of the homeostatic chemokine CCL25 and of the anti-inflammatory cytokine IL-10 was downregulated and unchanged, respectively.

Background Leukemia/lymphoma cell lines have been critical in the investigation of the pathogenesis and therapy of hematological malignancies. While human LL cell lines have generally been found to recapitulate the primary tumors from which they were derived, appropriate characterization including cytogenetic and transcriptional assessment is crucial for assessing their clinical predictive value. Results In the following study, five canine LL cell lines, CLBL-1, Ema, TL-1 (Nody-1), UL-1, and 3132, were characterized using extensive immunophenotyping, karyotypic analysis, oligonucleotide array comparative genomic hybridization (oaCGH), and gene expression profiling. Genome-wide DNA copy number data from the cell lines were also directly compared with 299 primary canine round cell tumors to determine whether the cell lines represent primary tumors, and, if so, what subtype each most closely resembled. Conclusions Based on integrated analyses, CLBL-1 was classified as B-cell lymphoma, Ema and TL-1 as T-cell lymphoma, and UL-1 as T-cell acute lymphoblastic leukemia. 3132, originally classified as a B-cell lymphoma, was reclassified as a histiocytic sarcoma based on characteristic cytogenomic properties. In combination, these data begin to elucidate the clinical predictive value of these cell lines which will enhance the appropriate selection of in vitro models for future studies of canine hematological malignancies. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0836-z) contains supplementary material, which is available to authorized users.

Primary and secondary nervous system involvement occurs in 4% and 5%–12%, respectively, of all canine non-Hodgkin lymphomas. The recent new classification of canine malignant lymphomas, based on the human World Health Organization classification, has been endorsed with international acceptance. This histological and immunocytochemical classification provides a unique opportunity to study the histologic anatomic distribution patterns in the central and peripheral nervous system of these defined lymphoma subtypes. In this study, we studied a cohort of 37 dogs with lymphoma, which at necropsy had either primary (n = 1, 2.7%) or secondary (n = 36; 97.3%) neural involvement. These T- (n = 16; 43.2%) or B-cell (n = 21; 56.8%) lymphomas were further classified into 12 lymphoma subtypes, with predominant subtypes including peripheral T-cell lymphoma (PTCL) or diffuse large B-cell lymphoma (DLBCL), respectively. This systematic study identified 6 different anatomically based histologically defined patterns of lymphoma infiltration in the nervous system of dogs. Different and distinct combinations of anatomical patterns correlated with specific lymphoma subtypes. Lymphoma infiltration within the meningeal, perivascular, and periventricular compartments were characteristic of DLBCL, whereas peripheral nerve involvement was a frequent feature of PTCL. Similarly cell counts above 64 cells/μL in cerebrospinal samples correlated best with marked meningeal and periventricular lymphoma infiltration histologically. Prospective studies are needed in order to confirm the hypothesis that these combinations of histological neuroanatomic patterns reflect targeting of receptors specific for the lymphoma subtypes at these various sites.

The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines.

Background Splenic fibrohistiocytic nodules (SFHN) are commonly diagnosed. It is suspected that these represent a heterogeneous group of malignant and nonmalignant diseases, separation of which could improve the ability of clinicians to prognosticate for dogs. Hypothesis/Objectives Immunohistochemistry will differentiate histologic diagnoses within the group of SFHN; survival after splenectomy is associated with those histologic types. Animals Thirty-two dogs with SFHN treated by or under direction from veterinary oncologists. Methods Retrospective case record analysis from dogs followed from splenectomy until death. Clinical, histopathologic, and immunohistochemistry data analyzed for an association with survival time. Results Thirty-two dogs had SFHN; grade 1 (2 dogs), grade 2 (9 dogs), and grade 3 (lymphoid percentage