Kinome Analysis of Cattle Peripheral Lymph Nodes to Elucidate Differential Response to Salmonella spp.

Salmonella spp., contained within the peripheral lymph nodes (PLNs) of cattle, represents a significant source of contamination of ground beef. Herein is the first report where species-specific kinome peptide arrays designed for bovine biology were used to further the understanding of Salmonella spp. within these PLNs. For the purpose of this research, multiple comparisons of sub-iliac lymph nodes were made to include nodes from feedlot cattle that were infected with Salmonella spp. to those that were non-infected; seasonal differences in feedlot cattle harvested in either August or January; cull dairy cows compared to feedlot cattle; and PLNs from cattle experimentally inoculated with Salmonella spp. versus naturally infected animals. The first comparison of Salmonella-positive and -negative PLNs found that considering the kinotypes for these animals, the major distinguishing difference was not the presence or absence of Salmonella spp. in the PLNs but the concentration. Further, the majority of pathways activated were directly related to immune responses including innate immunity, thus Salmonella spp. within the PLNs activates the immune system in that node. Results from the comparison of feedlot cattle and cull dairy cows suggests that a Salmonella spp.-negative animal, regardless of type, has a more consistent kinome profile than that of a Salmonella spp.-positive animal and that the differences between feedlot and cull dairy cattle are only pronounced when the PLNs are Salmonella spp. positive. PLNs collected in the winter showed a much more consistent kinome profile, regardless of Salmonella status, suggesting that in the winter these cattle are similar, and this is not affected by the presence of Salmonella spp., whereas significant variability among kinotypes was observed for PLNs collected in the summer. The most distinct clustering of kinotypes observed in this study was related to how the animal was infected with Salmonella spp. There were significant differences in the phosphorylation state of the immune response peptides between experimentally and naturally infected animals, suggesting that the immune system is activated in a significantly different manner when comparing these routes of infection. Increasing our understanding of Salmonella spp. within cattle, and specifically within the PLNs, will ultimately help design effective pre-harvest intervention strategies as well as appropriate experimentation to validate those technologies.

Ultrasound-guided placement of an anchor wire or injection of methylene blue to aid in the intraoperative localization and excision of peripheral lymph nodes in dogs and cats

OBJECTIVE To evaluate ultrasound-guided placement of an anchor wire (AW) or injection of methylene blue (MB) to aid in the intraoperative localization of peripheral lymph nodes in dogs and cats. ANIMALS 125 dogs and 10 cats with a total of 171 lymphadenectomies. PROCEDURES Medical records of dogs and cats that underwent peripheral lymphadenectomies with or without (N) the AW or MB localization technique were reviewed. Data retrieved included clinical, surgical, and histologic findings. The proportions of successful lymphadenectomies, lymph node characteristics, and complications among the 3 groups were analyzed. RESULTS 143 (84%) lymph nodes were successfully excised. Lymphadenectomy success was significantly affected by the localization technique, with 94% for group AW, 87% for group MB, and 72% for group N. Lymph node size was smaller in groups AW and MB, compared with group N. Duration of lymphadenectomy was shorter in group AW, compared with groups MB and N, and in group MB, compared with group N. Intra- (7%) and postoperative (10%) complications and final diagnosis did not significantly differ among groups. CONCLUSIONS AND CLINICAL RELEVANCE Both lymph node localization techniques were highly successful and reduced surgery time, compared with unassisted lymphadenectomy. Specifically, these techniques were effective for localization of normal-sized and nonpalpable lymph nodes and were efficient and practical options for peripheral lymphadenectomies, particularly for those that were small or nonpalpable.

Comparison of shear-wave velocities obtained with shear-wave elastography of various peripheral lymph nodes in healthy Beagles

OBJECTIVE To compare shear-wave velocities (SWVs) with shear-wave elastography of various peripheral lymph nodes (LNs). ANIMALS 11 healthy Beagles. PROCEDURES For each dog, bilateral mandibular, medial retropharyngeal, superficial cervical, axillary, superficial inguinal, and popliteal LNs were evaluated with shear-wave elastography in sagittal and transverse scanning planes. Depth of each lymph node was recorded, and intra- and interobserver reliability was determined. RESULTS SWVs for all LNs were significantly higher in the sagittal scanning plane, compared with those in the transverse scanning plane. The SWV of the most superficial LN, the mandibular LN, was significantly higher, compared with that for the other LNs, except for the medial retropharyngeal LN. The SWV of the deepest LN, the medial retropharyngeal LN, was as high as that for the mandibular LN. Intra- and interobserver reliability was excellent. CONCLUSIONS AND CLINICAL RELEVANCE SWVs for normal peripheral LNs of Beagles may serve as a reference to compare with those for other breeds and diseased LNs. Scanning plane, LN depth, and interfering tissues between the LN and the transducer may affect SWV. Shear-wave elastography may not be operator dependent.

Comparison of shear-wave velocities obtained with shear-wave elastography of various peripheral lymph nodes in healthy Beagles

OBJECTIVE To compare shear-wave velocities (SWVs) with shear-wave elastography of various peripheral lymph nodes (LNs). ANIMALS 11 healthy Beagles. PROCEDURES For each dog, bilateral mandibular, medial retropharyngeal, superficial cervical, axillary, superficial inguinal, and popliteal LNs were evaluated with shear-wave elastography in sagittal and transverse scanning planes. Depth of each lymph node was recorded, and intra- and interobserver reliability was determined. RESULTS SWVs for all LNs were significantly higher in the sagittal scanning plane, compared with those in the transverse scanning plane. The SWV of the most superficial LN, the mandibular LN, was significantly higher, compared with that for the other LNs, except for the medial retropharyngeal LN. The SWV of the deepest LN, the medial retropharyngeal LN, was as high as that for the mandibular LN. Intra- and interobserver reliability was excellent. CONCLUSIONS AND CLINICAL RELEVANCE SWVs for normal peripheral LNs of Beagles may serve as a reference to compare with those for other breeds and diseased LNs. Scanning plane, LN depth, and interfering tissues between the LN and the transducer may affect SWV. Shear-wave elastography may not be operator dependent.

Evaluation of a pre-harvest bacteriophage therapy for control of Salmonella within bovine peripheral lymph nodes

A series of proof of concept studies were developed to determine if a commercial bacteriophage (phage) cocktail could be utilized for the mitigation of Salmonella in bovine peripheral lymph nodes (LN). The first objective sought to determine if exogenous phage could be isolated from the LN following administration. If successful, the second objective sought to determine if once in the LN, could the phage effectively reduce Salmonella . Salmonella Montevideo was inoculated intradermally in multiple sites and administrations, later followed by delivery of the phage cocktail subcutaneously in two injections around each of the right and left prescapular and subiliac LN. At the conclusion of each study, animals were euthanized and the popliteal and above LN examined. The first study was successful, in that transmission electron microscopy revealed the presence of phage in the LN of the treated cattle, that were identical to the strains in the cocktail. Concentrations of phage were increased ( P < 0.01) in the pre-scapular and subiliac LN in the phage-treated versus control cattle. Subsequent studies modified the protocols to increase Salmonella and phage concentrations within the LN. Overall concentrations of Salmonella were increased in the LN compared to the first study and phage treatment decreased ( P < 0.01) Salmonella in the some of the LN. Phage concentrations were numerically ( P = 0.12), but not statistically, increased in the treated cattle. The final study was modified, hypothesizing that a 48h post-mortem period prior to LN removal would facilitate phage/ Salmonella interaction, however, there were no differences ( P > 0.10) in Salmonella concentrations among treatments. Results demonstrated that Salmonella- specific phages administered to live cattle can translocate to the LN, however once in the LN they had limited to no effect on Salmonella within these nodes.

IMMU-32. IDENTIFICATION OF TUMOR-ANTIGEN SPECIFIC T CELLS AND THE EFFICACY OF IMMUNOTHERAPY VACCINES FOR GLIOBLASTOMA ANTIGENS DETERMINED USING CANCER IMMUNOGENOMICS APPROACH

BACKGROUND Glioblastoma multiforme (GBM) remains a disease with debilitating survival outcomes. Owing to the heterogeneous nature and low mutation burden, identifying multiple antigens inherent to GBM that may serve as targets for immune-based therapies is attractive. Our aim is to develop a personalized immunotherapy approach using cancer immunogenomics for prospectively identifying neoantigens and uniquely expressed tumor proteins and then selectively expanding T cells against these truly tumor-specific antigens and dendritic cell vaccines to boost the T cell responses. METHODS RNAseq and WES was performed for murine KR158-luc GBM tumor. Using a cancer immunogenomics approach that we developed, called the O pen R eading Frame A ntigen N etwork (O.R.A.N.), we identified the immunogenic neoantigens and tumor-associated antigens (TAAs) including cancer testis and developmental antigens, that are aberrantly over-expressed in KR158-luc tumor. All predicted genes were subjected to a gene enrichment strategy and an mRNA library was generated containing predominantly only the target genes but had some background non-specific genes (validated by RNAseq). KR158-luc tumor bearing animals were then treated with dendritic cells loaded with the tumor antigen specific mRNA library. Tumor volume and thus progress was determined using in vivo luciferase imaging technique. Additionally, tetramers specific to several of the predicted antigens were manufactured and the frequency of antigen specific T cells was determined using flow cytometry. RESULTS The dendritic cell vaccines were effective in delaying the progression of KR158-luc tumors and we identified T cells targeting several of our predicted antigens in the tumor bearing animals. The antigen specific T cells were detected in the tumor infiltrating lymphocytes as well as in the peripheral lymph organs. CONCLUSION We developed a dendritic cell-based vaccination approach targeting all neoantigens and TAAs identified as being tumor-specific and validated our developed immunogenomics pipeline by identifying antigen-specific T cells in the tumor bearing animals against novel GBM antigens.

Posttranslational modifications by ADAM10 shape myeloid antigen-presenting cell homeostasis in the splenic marginal zone

The spleen contains phenotypically and functionally distinct conventional dendritic cell (cDC) subpopulations, termed cDC1 and cDC2, which each can be divided into several smaller and less well-characterized subsets. Despite advances in understanding the complexity of cDC ontogeny by transcriptional programming, the significance of posttranslational modifications in controlling tissue-specific cDC subset immunobiology remains elusive. Here, we identified the cell-surface–expressed A-disintegrin-and-metalloproteinase 10 (ADAM10) as an essential regulator of cDC1 and cDC2 homeostasis in the splenic marginal zone (MZ). Mice with a CD11c-specific deletion of ADAM10 (ADAM10ΔCD11c) exhibited a complete loss of splenic ESAMhi cDC2A because ADAM10 regulated the commitment, differentiation, and survival of these cells. The major pathways controlled by ADAM10 in ESAMhi cDC2A are Notch, signaling pathways involved in cell proliferation and survival (e.g., mTOR, PI3K/AKT, and EIF2 signaling), and EBI2-mediated localization within the MZ. In addition, we discovered that ADAM10 is a molecular switch regulating cDC2 subset heterogeneity in the spleen, as the disappearance of ESAMhi cDC2A in ADAM10ΔCD11c mice was compensated for by the emergence of a Clec12a+ cDC2B subset closely resembling cDC2 generally found in peripheral lymph nodes. Moreover, in ADAM10ΔCD11c mice, terminal differentiation of cDC1 was abrogated, resulting in severely reduced splenic Langerin+ cDC1 numbers. Next to the disturbed splenic cDC compartment, ADAM10 deficiency on CD11c+ cells led to an increase in marginal metallophilic macrophage (MMM) numbers. In conclusion, our data identify ADAM10 as a molecular hub on both cDC and MMM regulating their transcriptional programming, turnover, homeostasis, and ability to shape the anatomical niche of the MZ.

Clinical and Radiological Features of Urachal Carcinoma and Infection

PurposeTo explore the clinical and radiological differences between urachal carcinoma and urachal infection.MethodsClinical and imaging information for 13 cases of urachal carcinoma and 14 cases of urachal infection confirmed by pathology were retrospectively analyzed. The size, location, shape, margin, lesion composition, calcification, T1 and T2 signal intensity, peripheral lymph nodes, degree of enhancement, adjacent bladder wall, and apparent diffusion coefficient (ADC) value were examined in both groups, and distinguish features were determined. The student t-test or Mann-Whitney U test was used for quantitative data, and Fisher’s exact test was used for qualitative data. Kappa coefficient consistency test was used to evaluate the interobserver agreement.ResultsSex, hematuria, abdominal pain, calcification, and thickening of adjacent bladder wall can distinguish between urachal carcinoma and urachal infection (p < 0.05). There were no statistical differences in age (p = 0.076), size (p = 0.797), location (p = 0.440), shape (p = 0.449), margin (p = 0.449), lesion composition (p = 0.459), T1 signal intensity (p = 0.196), T2 signal intensity (p = 0.555), peripheral lymph nodes (p = 0.236), degree of enhancements (p = 0.184) and ADC value (p = 0.780) between two groups.ConclusionThe following clinical and imaging features help distinguish urachal carcinoma from urachal infection: sex, hematuria, abdominal pain, calcification, and thickening of the adjacent bladder wall.