Listeria monocytogenes is a facultative intracellular bacterium that causes foodborne disease in humans. L. monocytogenes invade the gut mucosa and then disseminate, causing systemic infections associated with high mortality rates in immunocompromised individuals. It is unknown how L. monocytogenes traffic to the mesenteric lymph nodes, which represent an important bottleneck for systemic spread. In addition, little is known about the gastrointestinal stage of infection due to the general resistance of mice to oral infection with L. monocytogenes. Our laboratory developed a novel foodborne mouse model of listeriosis utilizing a murinized strain of L. monocytogenes to investigate the gastrointestinal stage of infection. First, we found that the majority of L. monocytogenes isolated from the intestinal tissue and MLN were extracellular; however, the minimal fraction of intracellular L. monocytogenes was vital for persistence in the gut and spread to the MLN. The vast majority of cell-associated L. monocytogenes in the MLN were adhered to inflammatory monocytes, but these cells did not support the intracellular growth of L. monocytogenes. A minor proportion of L. monocytogenes were associated with migratory dendritic cells in the intestinal lamina propria and MLN, but like monocytes, these cells did not appear to serve as an intracellular growth niche for L. monocytogenes. Lastly, extracellular L. monocytogenes were observed migrating in mesenteric lymphatic vessels that drain from the intestine to the MLN, suggesting that L. monocytogenes can spread beyond the intestinal mucosa independent of migratory immune cells. Overall, these studies are the first to characterize the interaction of L. monocytogenes with immune cells in the intestine and MLN following foodborne infection and suggest that extracellular, and not cytosolic L. monocytogenes, primarily drive innate immune responses in the gut.

O linfonodo é um órgão linfoide secundário que apresenta uma arquitetura altamente organizada com diferentes compartimentos para tipos celulares específicos. Dentre as células estruturais que compõem este órgão, as células estromais como células fibroblásticas reticulares (FRCs) e células duplo negativas (DNCs) parecem ter papel importante na modulação da resposta imunológica e na tolerância periférica. As FRCs são caracterizadas pela expressão de podoplanina (gp38, PDPN) e localizam-se principalmente na zona de células T, enquanto as DNCs (gp38-) apresentam fenótipo, localização e função pouco descritos. Embora estas células tenham sido muito estudadas em modelos murinos os estudos sobre FRCs e DNCs humanas são escassos e, portanto nosso estudo deve contribuir para a compreensão da biologia e a função dessas células, podendo favorecer o conhecimento sobre a eficiência e as disfunções da resposta imune no linfonodo. Com esse intuito, isolamos e caracterizamos fenotípica e funcionalmente as FRCs e DNCs de linfonodos de pacientes com câncer, diverticulite e doadores de fígado. Nossos resultados mostraram a integridade e a distribuição celular no linfonodo. As células aderentes derivadas dos linfonodos estudados preecheram todos os critérios internacionais de caracterização de estroma, e, portanto, foram consideradas células estromais. Através da expressão de gp38 identificamos duas subpopulações de celulas estromais: FRCs (gp38+ e CD31-) e DNCs (gp38- e CD31-) e verificamos que as frequências destas células variam entre as amostras, sugerindo que a doença pode interferir na composição celular estromal dos linfonodos. As duas populações celulares foram estimuladas com citocinas inflamatórias como IFN-y ou TNF-alfa + IL-1beta por 24 e 48 horas e avaliadas quanto à expressão gênica e proteica. Em condições homeostáticas, genes relacionados com a indução e controle da proliferação foram diferencialmente expressos nas FRCs e DNCs, este dado foi confirmado in vitro, uma vez que as FRCs apresentaram maior potencial proliferativo em relação às DNCs. O estímulo com IFN-y induziu aumento de expressão nas DNCs e FRCs para citocinas, quimiocinas, moléculas de histocompatibilidade e moléculas envolvidas na regulação da resposta imunológica. Em resposta ao estímulo com TNF-alfa +IL-1beta, observamos aumento na expressão de moléculas comuns ao estímulo com IFN-?, entretanto, também observamos expressão de moléculas de citocinas, quimiocinas inflamatórias e moléculas de histocmpatibilidade especificamente relacionados a este sinal em ambas as populações. Em conjunto, nossos dados sugerem que DNCs e FRCs apresentam diferenças no perfil de resposta segundo os estímulos inflamatórios aos quais estão expostas, aumentando a expressão diferencial de moléculas envolvidas na regulação positiva e negativa da resposta imune / The lymph node is a secondary lymphoid organ that has a highly organized architecture with different compartments for specific cell types. Among the structural cells that comprise this organ, stromal fibroblastic reticular cells (FRCs) and double negative cells (DNCs) seems to play an important role in modulating the immune response and peripheral tolerance. FRCs are characterized by podoplanin (gp38, PDPN) expression and are located mainly in the T cell zone, while DNCs (gp38-) present phenotype, location and function not well described. Although these cells have been studied in murine models, studies on human FRCs and DNCs are limited and therefore our study should contribute to the understanding of biology and function of these cells and should promote knowledge of efficiency and disorders in the lymph node immune response. For this purpose, we have isolated and characterized phenotypic and functionally lymph nodes derived FRCs and DNCs from patients with cancer, diverticulitis and liver donors. Our results showed lymph node integrity and its cellular distribution. Adherent cells lymph nodes-derived fullfill the international criteria for stroma characterization, and therefore, they have been considered stromal cells. Using gp38 expression we were able to identify two stromal cells subpopulations: FRCs (gp38 + and CD31-) and DNCs (gp38- and CD31-) and found that this cells frequency varies among samples, suggesting that the disease may interfere with lymph nodes stromal cell composition. These two cells populations were stimulated with inflammatory cytokines such as IFN-y or TNF-alfa + IL-1beta for 24 and 48 hours and evaluated for gene and protein expression. In homeostatic conditions, genes involved in the induction and control of proliferation were differentially expressed by FRCs and DNCs, this data has been confirmed in vitro, since the FRCs showed higher proliferative potential compared to DNCs. IFN-y stimulation induced increase DNCs and FRCs expression for cytokines, chemokines, histocompatibility molecules and molecules involved in regulating the immune response.In response to TNF-alfa + IL-1beta stimulation, we observed common molecules expressed by the IFN-? stimulation, however, we also observed expression of cytokines, chemokines and histocompatibility molecules specifically related to this signal in both cells populations. Together, our data suggest that DNCs and FRCs differ in the response profile according to inflammatory stimuli to which they are exposed, increasing the differential expression of molecules involved in the positive and negative regulation of immune response

Orientador: Maria Jaqueline Mamprim / Resumo: A leishmaniose visceral canina geralmente acomete órgãos do sistema mononuclear fagocitário, entretanto, há evidências de que possa lesionar outros órgãos. Pouco se sabe sobre a patogenia da doença na cavidade torácica. Ainda não há descrições radiológicas das alterações torácicas de cães leishmanióticos. O presente estudo realizou avaliação tomográfica em 35 cadáveres de cães positivos e sintomáticos para a doença. Foi possível confirmar linfadenomegalia esternal e visibilizar os linfonodos mediastinais e traqueobrônquicos. Todos os animais apresentaram opacificações do tipo vidro fosco no parênquima pulmonar. Também se observaram opacificações do tipo linear, nodular e banda parenquimal nos pulmões e, ainda, espessamento e dilatação brônquica. A análise histopatológica foi realizada em 13 pulmões e revelou infiltrado inflamatório em diferentes graus, edema, congestão, espessamento de septo alveolar e brônquios congestos. Tais alterações tomográficas e histopatológicas são semelhantes às descritas em humanos. A tomografia computadorizada se mostra uma ferramenta de grande valia para avaliação da cavidade torácica de cães acometidos pela leishmaniose visceral. O acometimento torácico pela leishmaniose visceral canina deve ser considerado em diagnósticos diferenciais quando se observam opacificações do tipo vidro fosco e lineares e linfadenomegalia em exames tomográficos do tórax de cães em áreas endêmicas. / Abstract: Canine visceral leishmaniasis usually affects organs of the mononuclear phagocytic system, however, there is evidence that it can damage other organs. Little is known about the pathogenesis of the disease in the chest cavity. There are still no radiological descriptions of the thoracic changes in leishmaniotic dogs. In this study computed tomography in 35 cadavers of dogs positive and symptomatic for the disease was performed. It was possible to confirm sternal lymphadenomegaly and to visualize the mediastinal and tracheobronchial lymph nodes. All animals presented ground-glass opacifications in the lung parenchyma. Linear, nodular and parenchymal band opacifications were also observed in the lungs, as well as bronchial thickening and dilation. Histopathological analysis was performed in 13 lungs and revealed inflammatory infiltrate in different degrees, edema, congestion, thickening of the alveolar septum and congested bronchi. Such tomographic and histopathologic changes are similar to those described in humans. Computed tomography is a valuable tool for assessing the chest cavity of dogs affected by visceral leishmaniasis. Thoracic involvement by canine visceral leishmaniasis should be considered as differential diagnoses when ground-glass and linear opacifications and lymphadenomegaly are observed in tomographic examinations of the chest of dogs in endemic areas. / Doutor

Le virus du syndrome dysgénésique et respiratoire porcin (SDRPv) est un pathogène à l’origine de problèmes respiratoires et de reproduction. La réponse immunitaire face au SDRPv est caractérisée par une virémie persistante et un retard dans la mise en place des anticorps neutralisants. Les macrophages alvéolaires (AM) sont la cible principale du virus mais plusieurs études in vitro suggèrent une infection des cellules dendritiques (DC). Dans ce manuscrit, de nouvelles cibles cellulaires du virus ont été découvertes dans le poumon et dans les ganglions trachéo-bronchiques. Les cellules du parenchyme pulmonaire AM-like sont infectés in vivo par le SDRPv de Type 1 alors que les cDC1, cDC2 et moDC du poumon ne sont pas infectées. La souche Lena Type 1.3 se distingue des autres souches par sa capacité à induire une polarisation vers une réponse Th1 in vitro. Une expérience de transfert de lymphocytes T mémoires in vivo suggèrent une réponse cellulaire accrue et délétère lors de l’infection par la souche Lena. Trois populations de macrophages ont été identifiées dans les ganglions et nommées : macrophages périfolliculaires (PFMacro), macrophages des cordons lymphatiques (cordMacro) et macrophages efférents (effMacro). Les effMacro et les PFMacro sont infectés par les souches européennes. Quant à la réponse humorale, 5 stades de différenciation des LB dans les ganglions ont été identifiés et une étude de l’impact de l’infection sur la différenciation des lymphocytes B est en cours. / Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded RNA virus that causes reproductive failure and respiratory problems in swine. Immune response against PRRSV is characterized by a persistent viremia and a delay in neutralizing antibodies’ production. Main targets of PRRSV are Alveolar Macrophages (AM) but in vitro studies suggest that PRRSV could replicate in dendritic cells (DC). In this manuscript, new cellular targets of PRRSV in the lung and the tracheo-bronchial lymph nodes have been depicted. This work revealed that pulmonary parenchymal AM-like are susceptible to PRRSV in vivo. Moreover, study of DC infection in the lung reveals that cDC1, cDC2 and moDC are not infected by Type 1 PRRSV. In tracheo-bronchial lymph nodes, 3 populations of pig macrophages (Macro) have been identified and called perifollicular macrophages (PFMacro), lymphatic cord macrophages (cordMacro) and efferent macrophages (effMacro). In vivo infection revealed that PFMacro and effMacro are susceptible to Type 1 PRRSV infection. In order to understand the delay in humoral response establishment, 5 populations of B cells have been identified in the lymph nodes. The interaction of SDRPv with these populations is under study.

Orientador: Antônio José Maria Cataneo / Resumo: Introdução: No melanoma primário, a aplicabilidade da biópsia de linfonodo sentinela (BLS), seguida ou não de esvazimento linfonodal (EL) é conhecida. Na recidiva locorregional (RL) de melanoma, alguns serviços tendem a indicá-la, buscando estadiamento mais acurado para embasar condutas individualizadas aos pacientes, ainda que as evidências sejam insuficientes. Objetivo: Avaliar o sucesso da BLS no encontro do linfonodo sentinela (LNS) e sua positividade na RL. Comparar a sobrevida entre os pacientes com LNS positivo e negativo. Verificar diferença na sobrevida pós EL. Métodos: Revisão sistemática, através das bases MEDLINE via PUBMED, LILACS, SCOPUS, EMBASE e CENTRAL, buscando estudos experimentais e observacionais sobre BLS na RL de melanoma. Desfechos avaliados: sucesso na BLS pelo encontro do LNS, positividade para melanoma no LNS; sobrevida no subgrupo LNS positivo comparado com o negativo; sobrevida livre de doença no subgrupo LNS positivo comparada com o negativo; sobrevida dos pacientes submetidos ao EL. Para metanálises, utilizaram-se RevMan 5.3 e StatsDirect 3.0.121. Resultados: Foram identificados 1872 estudos, destes, seis estudos observacionais foram incluídos, totalizando 449 pacientes. O LNS foi encontrado em 98% das BLS (IC 95-100%, I2=53,7% - seis estudos). LNS com 32% de positividade para melanoma (IC 19-47%, I2= 84,6% - seis estudos). A chance de sobrevida global em cinco anos foi 2,49 vezes maior no subgrupo com LNS negativo (IC 95% 1,41-4,38, I2=0% - qua... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Background: In primary melanoma, the applicability of sentinel lymph node biopsy (SLB), followed or not by complete lymph node dissection (CLND) is known. In locoregional recurrence (LR) of melanoma, some groups may indicate it for more accurate staging to support individualized management, even with scarce evidence. Objective: To evaluate success in SLB and its positivity in LR. Compare survival between patients with positive and negative sentinel lymph node (SLN). Check for survival modification after CLND. Methods: Systematic review through databases such as MEDLINE via PUBMED, LILACS, SCOPUS, EMBASE and CENTRAL, searching for experimental and observational studies on SLB in melanoma LR. Outcomes assessed: success in SLB by finding the SLN, positivity for melanoma in the SLN; survival in the positive SLN subgroup compared to the negative one; disease-free survival in the positive versus negative SLN subgroup; survival of patients undergoing CLND. For meta-analyzes, RevMan 5.3 and StatsDirect 3.0.121 were used. Results: The total number of patients in six observational studies was 449, over 1872 studies indentified. The SNL was found in 98% of SLB (95-100% CI, I2 = 53.7%, 6 studies). SLB detected 32% positivity for melanoma on SNL (CI 19-47%, I2 = 84.6%, 6 studies). The chance of five year overall survival was 2,49 higher in the negative SNL subgroup (95% CI 1.41-4.38, I2 = 0%, 4 studies). Meta-analyzes were not performed due to lack of objective data for disease-free survi... (Complete abstract click electronic access below) / Doutor

Cell therapies are an emerging form of therapy, with the potential to treat and cure a variety of diseases. As more cell therapies become approved and commercialized, challenges remain in the manufacturing of these often single-batch products due to their complexity and patient-to-patient variability, which limit their cost-effectiveness and reproducibility. In this dissertation, we aim to improve the manufacturing of two different cell therapies, namely, organoid-based cell therapies using hydrogel scaffolds, and adoptive cell therapies using deep learning and microfluidics, to facilitate their widespread clinical use. First, we develop new tools to manufacture organoids, which are widespread in drug-screening technologies, but have been sparingly used for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility. Here, we use alginate microwell scaffolds to form pre-vascularized organoids composed of endothelial cells and mesenchymal stem cells, where the size and structure can be readily tuned by varying the cell source, ratio of cells, or size of the microwells. Furthermore, by uncrosslinking the alginate scaffold, the organoids can be harvested in a gentle manner without damaging their structure or impairing their functionality. Finally, we assess the ability of the pre-vascularized organoids to restore vascular perfusion in a mouse model of hindlimb ischemia. By making use of the dynamic nature of hydrogels, this method can offer high yields of reproducible, self-organized multicellular aggregates for use in cell therapies. Next, we shift our focus to the identification of antigen-specific T cells, which is a critical step in the manufacturing of adoptive cell therapy. Conventional techniques for selecting antigen-specific T cells are time-consuming, making them difficult to adapt for large-scale manufacturing, and are limited to pre-defined antigenic peptide sequences. Here we train a deep learning model to rapidly classify videos of antigen-specific CD8+ T cells by distinguishing the distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and dendritic cells (DCs). The model is able to classify high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalizes well to other types of high and low affinity CD8+ T cells. We also show that the experimental addition of anti-CD40 antibodies amplifies the differences between cognate and non-cognate T cells and DCs, thereby improving the model’s ability to discriminate between them. This workflow can be used to better understand the role of cognate T cell – DC interactions in the pathogenesis of cancer and autoimmune diseases, and can be integrated into a device to simplify and accelerate the selection of antigen-specific T cells for use in adoptive cell therapy. Finally, we sought to develop a device to address two other issues associated with the selection of antigen-specific T cells: low-throughput screening, and the inability to assess a mixed population of T cells against a library of antigens, both of which are necessary to identify rare T cells, and improve clinical outcomes of the corresponding cell therapy. A few specialized assays exist that can assess T cells against multiple antigens, but they are often limited by an increased manufacturing burden. Here, we develop a microfluidic artificial lymph node, which is inspired by the efficient selection of antigen-specific T cells in vivo. In particular, our flow-through design consists of multiple compartments, each containing microcarrier beads coated with DCs presenting a distinct antigen, such that T cells that are flowed sequentially through each compartment can stably arrest to cognate DCs, becoming captured in the appropriate compartment. We test a single-compartment device computationally using agent-based simulations, and experimentally using a mixed population of antigen-specific and wild-type (WT) (non-specific) T cells, and in both cases we observe a preferential accumulation of cognate, antigen-specific T cells. This proof-of-concept single-compartment device can be readily scaled up to systematically test many T cells against multiple antigens. Underlying this work is the development of technologies to enable the large-scale manufacturing of cell therapies. Cell therapies are undergoing a transformation to a new class of therapeutic modality, and there are many emerging questions, especially related to the scale-up and scale-out of production processes. Together, this work aims to engineer technologies to improve cell therapy manufacturing processes, facilitate their clinical translation, and ensure their availability to all patients who would benefit from them.

Objective: Background: Rare disease An epithelial inclusion cyst within a lymph node denotes a heterotopic phenomenon. Nodal epithelial inclusion cysts have been reported in a variety of anatomical locations including pelvic, abdominal, mediastinal, and axillary regions. While nodal melanocytic nevus (also known as nevus cell aggregates) is the most common heterotopic phenomena involving the axillary lymph nodes, the presence of benign epithelial inclusion cysts in axillary lymph nodes is a rare but well-reported finding. Such documentation is in part due to assessment of sentinel lymph nodes in breast cancer becoming standard of care. These epithelial inclusion cysts offer a diagnostic pitfall in evaluation of sentinel lymph node in the setting of breast carcinoma. They also complicate assessment of sentinel lymph node during intraoperative frozen sections analysis. Case Report: We report a case of co-existent of benign squamous-type and glandular-type epithelial inclusions cysts in 2 sentinel lymph nodes in a patient with grade III invasive ductal carcinoma involving the left breast. There have been at least 4 cases reported in literature in which benign epithelial inclusion cysts in sentinel lymph nodes were first mistakenly diagnosed as metastatic carcinoma both during intraoperative frozen section analysis and during review of permanent sections. The missed diagnosis could potentially occur intraoperatively during frozen section sentinel lymph node analysis secondarily due to lack of availability of the primary tumor for comparison and inability to use immunohistochemical stains. Conclusions: Pathologists should be aware of this pitfall especially in frozen section analysis of sentinel lymph node to avoid misdiagnosis and its associated potential grave consequences.

T cells can fight cancer, but an immunosuppressive tumor microenvironment (TME) disallows them from carrying out their function over time. Upregulation of inhibitory checkpoint molecules such as programmed cell death protein 1 (PD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) can lead to such an immunosuppressive TME. Despite their widespread use, immune checkpoint blockade (ICB) antibodies targeting checkpoint molecules remain ineffective in most cancer patients. We do not understand why some patients respond to ICB better than others. To understand the heterogeneity of ICB response, we must understand the heterogeneity of the T cell subsets acted upon by such therapies. Here, we ask how T cell subsets change in the presence and absence of ICB. We track T cell clones through their T cell receptor sequences and link phenotypes with T cell receptor specificities. Through multiplexed single cell TCR sequencing, single cell RNA sequencing, and the use of cell- surface CITE-seq antibodies, coupled with surgical biopsy, we longitudinally tracked the fate of individual T cell clones within tumors at baseline and in response to ICB in an immunogenic mouse tumor model. Furthermore, computational clustering of T cells solely based on their gene expression profiles may ignore upstream regulatory mechanisms that control T cell gene expression. Hence, we employed Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to cluster CD8+ and CD4+ T cell phenotypes. VIPER leverages inference of gene regulatory networks to allow full quantitative characterization of protein activity for transcription factors, co-factors, and signaling molecules by assessing the enrichment of their transcriptional targets cell-by-cell among expressed genes. This gave us a window into the transcriptional states and their inferred protein activity. We next developed a computational analysis toolkit to study TCR clonality incorporating sub-sampling of TCR clonotypes, forward and back tracing of shared clones between timepoints, and in turn, inferred shared clonal evolution. We employed the above workflow to MC38 tumor-infiltrating and tumor-draining lymph node-derived CD8+ and CD4+ T cells. We found that T cell phenotypes are highly dynamic within tumors at baseline, in the absence of ICB, particularly within the window that they are responsive to therapy. In the absence of ICB, effector phenotype of CD8+ T cells diminished, while the exhaustion phenotype was enhanced as tumors progressed. Within the CD4+ population, a heterogenous subset of regulatory CD4+ T cells (Tregs) changed phenotype over time, and CD4+ Th1 like effectors, along with stem like progenitor CD4+ showed distinct dynamism. Next, by analyzing responses to therapy within his context, we found that both anti-PD1 and anti-CTLA4 act through distinct mechanisms on CD8+ and CD4+ T cells. Anti-PD1 acted upon intra-tumoral effector CD8+ T cells to slow their progression to terminally differentiated exhausted cells, i.e., increased their persistence within tumors. Anti-CTLA4 therapy increased recruitment of novel effector CD8+ T cell clones to tumors from lymph nodes while diminishing tumor-infiltrating Tregs. ICB also potentiated CD4+ Th1 like phenotype. These results uncovered a behavior pattern of CD8+ and CD4+ T cells within tumors at baseline tumor progression, and then in the presence of ICB. We believe these findings have added to our understanding of the subtleties of T cell phenotypes in tumors, specifically in response to ICB. This will provide a practical framework for designing and validating novel checkpoint blockade therapies in the future.

The urinary bladder is especially subject to infection by virtue of its direct connection to the external urethral opening, and it is natural to anticipate the presence of a well-developed immunological mechanism to respond to this potential threat. The present study describes small, very highly vascular lymph nodes located in the wall of the rabbit bladder, which may be involved in a local response to foreign antigens. The vasculature and structure of these lymph nodes was described using a combination of vascular corrosion casting, ink injection, and light and electron microscopy. The distal abdominal aorta was cannulated, and after clearing the bladder vasculature with buffered saline, one of the following procedures was used: 1) the bladder was perfuse-fixed in preparation for light and electron microscopy; 2) the bladder vasculature was filled with India ink for vessel tracing; or 3) vascular corrosion casts of the vasculature were prepared by infusing resin comprised of a mixture of Mercox, methyl methacrylate monomer, and catalyst. The resulting casts were cleaned with KOH, formic acid, and water in preparation for scanning electron microscopy. Vascular casts and India ink injections revealed the presence of a number of isolated capillary tufts consisting of clusters of one to five 'glomeruli,' closely associated with the major vesicular vessels along the lateral walls of the bladder, and supplied by tertiary branches of these vessels. Light and electron microscopy showed that the capillary tufts represented the blood supply to small, ovoid lymph nodes located near the serosal surface of the bladder wall and usually restricted to the basal half of the bladder. These nodes were encapsulated and exhibited subcapsular sinuses, numerous small blood vessels, a limited number of high endothelial cells, and, occasionally, nerves and a follicular substructure. The nodes contained abundant lymphocytes, stellate stromal cells, macrophages, and eosinophils, but lacked the obvious cortical and medullary organization and germinal centers often seen in larger lymph nodes. Vascular corrosion casts, vascular ink injections, and microscopic examination confirmed the presence of small, highly vascular lymph nodes closely associated with the main vesicular vessels along the lateral walls of the rabbit bladder. A follicular substructure of the nodes appears to correspond with the 'glomerular' capillary arrangement within the nodes as seen with corrosion casts. The rich blood supply may be indicative of the high metabolic demand of lymphatic tissue, and may be altered in response to the level of activity of the node. The close association between the lymphatic tissue and the rich blood supply to the nodes may allow a rapid mobilization of lymphocytes during a local immune response to foreign agents.

Equine herpesvirus type 1 (EHV-1) is a common pathogen of horses that typically causes upper respiratory disease, however is also associated with late-term abortion, neonatal foal death and neurologic disease. Once a horse is infected, the virus concentrates to local lymphoid tissue, where it becomes latent. The virus can recrudesce during times of stress, which can lead to the initiation of devastating outbreaks. Some variants of EHV-1 have been associated with more severe disease outcomes. Appropriate outbreak management focuses on minimizing the movement of potentially exposed horses. This approach lacks a strategy for prevention at the level of latency largely due to a knowledge paucity in regards to carriage rate of latent EHV-1. Biosecurity decisions are also dependent on awaiting currently-available diagnostic testing that often take several days for results. Thus, our work has been focused on understanding the carriage rate of the latent virus in different geographic regions as well as improving diagnostic efficiency, both of which are essential for improving the management of EHV-1 disease. Loop mediated isothermal amplification (LAMP) is a method that amplifies nucleic acid rapidly at a constant temperature and is minimally affected by inhibitors that are often found in clinical samples. This procedure can be followed by multiple detection methods. A new, efficient sequencing method, called nanopore sequencing, has been developed in a handheld device, called MinION, that provides thorough output in a timely manner. When combined with LAMP, it has been referred to as LAMPore. The first objective of our work was to estimate the prevalence of latent EHV-1 and compare the frequency of each variant in the submandibular lymph nodes from horses in Virginia. Our second objective was to perform direct DNA sequencing of EHV-1 using the mobile MinION sequencer in combination with LAMP viral enrichment. Our findings demonstrated a low apparent prevalence of latent EHV-1 DNA in submandibular lymph nodes in this population of horses in Virginia as well as successful detection and identification of EHV-1 in equine nasal swab samples using LAMPore sequencing. / Doctor of Philosophy / Horses can develop disease from a virus called equine herpesvirus type 1 (EHV-1). Symptoms can vary from mild respiratory signs to the inability to rise leading to death or euthanasia. Horses transmit this virus to other nearby horses; however, the virus also becomes dormant once a horse is infected, meaning the virus is not infectious but is present within the animal. When the horse undergoes stress, such as during travel or competition, the virus can become active again, leading to the spread to other horses. This results in outbreaks, many of which are devastating to the equine industry. In order to minimize the risks of this virus spreading and causing disease, management is currently focused on minimizing the movement of horses that may have been exposed to the virus. There is little information regarding the number of horses that harbor the dormant virus and the current methods to detect the infectious virus can take multiple days for results. These limit decision-making during the management of an outbreak. Our work seeks to determine the number of horses in a region that harbor EHV-1 and also to test a new, efficient diagnostic method to identify the virus in samples from horses. Our findings showed a low number of horses in Virginia harbor dormant EHV-1 in the lymph nodes under their mandible, a common site of dormancy. Further, we found that our new method of detection was effective in identifying the virus in samples from nasal secretions from horse.