Proteomics, the study of the structure and function of proteins, has been used to investigate candidate biomarkers and study the pathophysiology of various diseases. Biomarkers may be useful in diagnosing, monitoring and assessing treatment responses. This review provides an overview of proteomic techniques, describes the workflow and pipeline for biomarker discovery, and evaluates the current literature on proteomic studies in veterinary gastroenterology in dogs and cats. Current studies in this field are advancing our understanding of disease pathophysiology in conditions such as chronic enteropathy (CE). For biomarker development, proteomic research in veterinary gastroenterology is still in the early discovery phase. Substantial work on verification and validation is required before proteomic biomarkers can be reliably translated into clinical practice in companion animals.

BACKGROUND:Bilirubin encephalopathy is a poorly recognized complication in dogs with immune-mediated hemolytic-anemia (IMHA). HYPOTHESIS/OBJECTIVES:Assess serial trends of hyperbilirubinemia and the association between bilirubin concentrations and neurologic signs in dogs with IMHA. ANIMALS:Eighty one dogs with non-associative IMHA and hyperbilirubinemia. METHODS:Multicenter retrospective cohort study. The signalment, clinical signs, clinicopathological data, treatment, and outcome were evaluated. Bilirubin concentrations were recorded at the baseline, peak, initial decrease, and normalization. Univariable logistic regression was used to determine the association between neurologic signs and hyperbilirubinemia. RESULTS:The median bilirubin concentrations at the baseline, peak, and initial decrease were 2.5 (IQR, 1.4-4.8), 3.7 (IQR, 1.8-24.2), and 1.1 mg/dL (IQR, 0.5-4.3; 43, 64, and 19 μmol/L), respectively. Twenty percent (16/81) of dogs developed neurologic signs. Neurologic signs included stupor, non-ambulatory tetraparesis, and generalized seizures. A significant association was found between the presence of neurologic signs and the baseline, peak, and fold-change of bilirubin concentration (P < .001). The odds of having neurologic signs were 12.2 (95% CI, 3.1-48.2) for dogs with baseline bilirubin concentrations ≥3.3 mg/dL (≥57.5 μmol/L), and 93.3 (95% CI, 11.0-795.5) for dogs with peak bilirubin concentrations ≥13.9 mg/dL (≥239.5 μmol/L). CONCLUSIONS AND CLINICAL IMPORTANCE:Although the causation of the neurologic signs cannot be attributed solely to bilirubin based on our study, these findings emphasize the importance of monitoring serum bilirubin concentrations and the development of neurologic signs in dogs with IMHA. The results reflect findings in our study population and may not be directly applicable to all dogs with IMHA.

The overall goal of this work was to assess the ability of Natural Killer cells to kill cultures of patient-derived glioblastoma cells. Herein we report impressive levels of NK-92 mediated killing of various patient-derived glioblastoma cultures observed at ET (effector: target) ratios of 5:1 and 1:1. This enabled direct comparison of the degree of glioblastoma cell loss across a broader range of glioblastoma cultures. Importantly, even at high ET ratios of 5:1, there are always subpopulations of glioblastoma cells that prove very challenging to kill that evade the NK-92 cells. Of value in this study has been the application of ECIS (Electric Cell–Substrate Impedance Sensing) biosensor technology to monitor the glioblastoma cells in real-time, enabling temporal assessment of the NK-92 cells. ECIS has been powerful in revealing that at higher ET ratios, the glioblastoma cells are acutely sensitive to the NK-92 cells, and the observed glioblastoma cell death is supported by the high-content imaging data. Moreover, long-term ECIS experiments reveal that the surviving glioblastoma cells were then able to grow and reseed the culture, which was evident 300–500 h after the addition of the NK-92 cells. This was observed for multiple glioblastoma lines. In addition, our imaging provides evidence that some NK-92 cells appear to be compromised early, which would be consistent with potent evasive mechanisms by the glioblastoma tumour cells. This research strongly highlights the potential for NK-92 cells to kill glioblastoma tumour cells and provides a basis to identify the mechanism utilised by the surviving glioblastoma cells that we now need to target to achieve maximal cytolysis of the resistant glioblastoma cells. It is survival of the highly resistant glioblastoma clones that results in tumour relapse.

Background/Objectives: Brain cancers offer poor prognoses to patients accompanied by symptoms that drastically impact the patient and their family. Brain tumours recruit local non-transformed cells to provide trophic support and immunosuppression within the tumour microenvironment, supporting tumour progression. Given the localisation and supportive role of pericytes at the brain vasculature, we explored the potential for brain pericytes to contribute to the brain cancer microenvironment. Methods: To investigate this, primary brain pericytes were treated with factors commonly upregulated in brain cancers. Immunofluorescent labelling identified changes to brain pericyte cell signalling, cytometric bead array measured inflammatory secretion, and flow cytometry investigated brain pericyte phagocytosis. Results: The TGFβ superfamily cytokines TGFβ and GDF-15 activated SMAD2/3 and inhibited C/EBP-δ, revealing a potential mechanism behind the pleiotropic action of TGFβ on brain pericytes. IL-17 induced secretion of IL-6 without activating NFκB, STAT1, SMAD2/3, or C/EBP-δ signalling pathways. IL-27 and IFNγ induced STAT1 signalling and significantly reduced brain pericyte phagocytosis. The remaining brain cancer-derived factors did not induce a measured response, indicating that these factors may act on other cell types or require co-stimulation with other factors to produce significant effects. Conclusions: We identify several brain cancer-secreted factors which alter relevant brain pericyte functions. This reveals mechanisms through which brain tumours may regulate brain pericyte activity and these data start to uncover the supportive role these cells may play in brain cancers.

Background iPSC-derived cells are increasingly used to model complex diseases in vitro because they can be patient derived and can differentiate into any cell in the adult human body. Recent studies have demonstrated the generation of brain pericytes using a neural crest-based differentiation protocol. However, the inflammatory response of these iPSC-derived brain pericytes has not been investigated. We aimed to investigate the response of iPSC-derived brain pericytes to common inflammatory stimuli, thereby assessing the suitability of these cells to study inflammatory disease. Methods Brain pericytes were differentiated from iPSCs for 42 days. The expression of brain pericyte markers was assessed by RT-qPCR and immunofluorescent staining at days 0, 15, 21, and 42 of differentiation to validate the brain pericyte-like phenotype. Nuclear localisation of NFκB and STAT1 was assessed by immunofluorescence following IL-1β- and TNF-treatment in day 21 and day 42 iPSC-derived pericytes, and primary human pericytes. Cytometric bead array assessed the concentration of secreted inflammatory factors in the cell medium and phagocytosis was investigated using fluorescent carboxylated beads and flow cytometry. Results At day 42 of differentiation, but not at day 21, cells expressed brain pericyte markers. Generally, iPSC-derived pericytes lacked consistent responses to inflammatory treatment compared to primary human pericytes. Day 21 and 42 iPSC-derived pericytes exhibited a NFκB response to IL-1β treatment comparable to primary human pericytes. Day 21 iPSC-derived pericytes exhibited a STAT1 response with IL-1β treatment which was absent in day 42 cells, but present in a subset of primary human pericytes. TNF treatment presented similar NFκB responses between day 21 and 42 iPSC-derived and primary human pericytes, but a STAT1 response was again present in a subset of primary human pericytes which was absent in both day 21 and day 42 iPSC-derived pericytes. Numerous differences were observed in the secretion of cytokines and chemokines following treatment of iPSC-derived and primary human pericytes with IL-1β and TNF. iPSC-derived pericytes exhibited greater rates of phagocytosis than primary human pericytes. Conclusions With the increase in iPSC-derived cells in research, labs should undertake validation of lineage specificity when adapting an iPSC-derived differentiation protocol. In our hands, the inflammatory response of iPSC-derived pericytes was different to that of primary human pericytes, raising concern regarding the use of iPSC-derived pericytes to study neuroinflammatory disease. ![Figure][1] Graphical Abstract Brain pericytes can be generated from iPSCs. The work presented here shows the generation of phenotypically distinct pericytes from the original protocol, demonstrating the significant variability present within some iPSC differentiation protocols. Furthermore, functional differences are demonstrated between iPSC-derived brain pericytes and primary brain pericytes, revealing limitations in the use of iPSC-derived brain pericytes to model brain pericyte biology. What is already known about this topic? Brain pericyte-like cells can be generated from induced pluripotent stem cells, however their responses to inflammatory stimuli has not been assessed. What does this study add? iPSC-derived brain pericytes exhibit different inflammatory responses compared to primary brain pericytes, showing that some iPSC-derived cell models are not appropriate for modelling all aspects of a cell’s biology. Furthermore, the iPSC-derived pericytes generated here were markedly different to those generated from the original article. It is therefore important for each lab to optimise the generation of iPSC-derived cell in their own hands to account for potential inter-lab variability. ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

BackgroundChronic enteropathy (CE) is common in dogs and can occur with multiple etiologies including food-responsive enteropathy (FRE) and idiopathic inflammatory bowel disease (IBD). Hypothesis/ObjectiveTo study the protein profile and pathway differences among dogs with FRE, IBD, and healthy controls using serum proteome analysis. AnimalsNine CE dogs with signs of gastrointestinal disease and histologically confirmed chronic inflammatory enteropathy and 16 healthy controls. MethodsA cross-sectional study with cases recruited from 2 veterinary hospitals between May 2019 and November 2020 was performed. Serum samples were analyzed using mass spectrometry-based proteomic techniques. ResultsProteomic profiles showed marked variation in relative protein abundances. Forty-five proteins were significantly (P <= .01) differentially expressed among the dogs with CE and controls with >= 2-fold change in abundance. The fold change of dogs with IBD normalized to controls was more pronounced for the majority of proteins than that seen in the dogs with FRE normalized to control dogs. Proteins involving reactive oxygen species, cytokine activation, acute phase response signaling, and lipid metabolism were altered in dogs with CE. Conclusions and Clinical ImportanceCytokine alterations, acute phase response signaling, and lipid metabolism are likely involved in pathogenesis of CE. Although there are insufficient current data to justify the use of proteomic biomarkers for assessment of CE in dogs, our study identifies potential candidates.

Abstract Melanoma is an aggressive skin cancer with high propensity for brain metastasis. For advanced melanoma, immune checkpoint blockade (ICB) drugs, like pembrolizumab, have shown remarkable promise for progression-free survival. The ICB drugs have primarily targeted melanoma suppression of cytotoxic T-lymphocytes (CTLs). Earlier studies showed positive responses in up to 40% of cases, which has improved with discovery of new inhibitory ligands and combination therapy. However, not all patients respond, likely because of the capacity of cancer cells to regulate inhibitory and activating ligands, circumventing both immunomodulatory axes currently targeted by immunotherapies. In this study, we aim to characterize additional axes, prioritizing NK cells of the innate immune system. NK cells can mediate cytotoxic killing similar to CTLs but possess the advantages of germline-encoded receptors that recognize a broad range of ligands, thus, requiring relatively little priming and circumventing antigen escape. We have acquired up to ten patient-derived New Zealand Melanoma lines in collaboration with Auckland Cancer Society Research Centre, of which three are from brain metastases. We used NanoString technology to assess melanoma gene expression of immunoregulatory ligands governing NK activation, inhibition or both (via dual-functional ligands dictated by NK-receptors). The results showed expression of several inhibitory ligands but very few activating ligands. The expression trends were largely similar but varied in a select few ligands. Flow cytometry was used to characterize cell-surface expression of the immunoregulatory proteins. These were compared with the classical inhibitory checkpoint molecules evidencing that melanoma cells expressed more NK inhibitory molecules. We hypothesize that melanoma cells both express inhibitory ligands and suppress activation ligands, to hinder NK cell activity. Here we aim to identify novel immune axes leading towards expansion of ICB therapies. This will afford clinicians a personalized approach and access to combination immunotherapies, especially in combating refractory metastatic melanoma, which have poor prognoses.

AbstractBackgroundSerum protein biomarkers are used to diagnose, monitor treatment response, and to differentiate various forms of chronic enteropathies (CE) in humans. The utility of liquid biopsy proteomic approaches has not been examined in cats.Hypothesis/ObjectivesTo explore the serum proteome in cats to identify markers differentiating healthy cats from cats with CE.AnimalsTen cats with CE with signs of gastrointestinal disease of at least 3 weeks duration, and biopsy‐confirmed diagnoses, with or without treatment and 19 healthy cats were included.MethodsCross‐sectional, multicenter, exploratory study with cases recruited from 3 veterinary hospitals between May 2019 and November 2020. Serum samples were analyzed and evaluated using mass spectrometry‐based proteomic techniques.ResultsTwenty‐six proteins were significantly (P < .02, ≥5‐fold change in abundance) differentially expressed between cats with CE and controls. Thrombospondin‐1 (THBS1) was identified with >50‐fold increase in abundance in cats with CE (P < 0.001) compared to healthy cats.Conclusions and Clinical ImportanceDamage to the gut lining released marker proteins of chronic inflammation that were detectable in serum samples of cats. This early‐stage exploratory study strongly supports THBS1 as a candidate biomarker for chronic inflammatory enteropathy in cats.

Glioblastoma is refractory to therapy and presents a significant oncological challenge. Promising immunotherapies have not shown the promise observed in other aggressive cancers. The reasons for this include the highly immuno-suppressive tumour microenvironment controlled by the glioblastoma cells and heterogeneous phenotype of the glioblastoma cells. Here, we wanted to better understand which glioblastoma phenotypes produced the regulatory cytokines, particularly those that are implicated in shaping the immune microenvironment. In this study, we employed nanoString analysis of the glioblastoma transcriptome, and proteomic analysis (proteome profiler arrays and cytokine profiling) of secreted cytokines by different glioblastoma phenotypes. These phenotypes were cultured to reflect a spectrum of glioblastoma cells present in tumours, by culturing an enhanced stem-like phenotype of glioblastoma cells or a more differentiated phenotype following culture with serum. Extensive secretome profiling reveals that there is considerable heterogeneity in secretion patterns between serum-derived and glioblastoma stem-like cells, as well as between individuals. Generally, however, the serum-derived phenotypes appear to be the primary producers of cytokines associated with immune cell recruitment into the tumour microenvironment. Therefore, these glioblastoma cells have considerable importance in shaping the immune landscape in glioblastoma and represent a valuable therapeutic target that should not be ignored.

Glioblastoma is a highly aggressive brain malignancy commonly refractory to classical and novel chemo‐, radio‐ and immunotherapies, with median survival times of ~15 months following diagnosis. Poor immunological responses exemplified by the downregulation of T‐cell activity, and upregulation of immunosuppressive cells within the tumor microenvironment have limited the effectiveness of immunotherapy in glioblastoma to date. Here we show that glioblastoma cells express a large repertoire of inhibitory checkpoint ligands known to control effector T cell responses. Furthermore, flow cytometry analysis reveals that glioblastoma cells with an enhanced stem cell‐like phenotype express several investigated ligands at significant levels on their cell surface. This reveals that glioblastoma stem‐like cells express suppressive ligands with the potential of suppressing major T cell checkpoint receptors. With this information, it is now essential that we understand the relevance of this extensive repertoire of immune checkpoint ligands and their functional consequence on immune evasion in glioblastoma. This is necessary to develop effective immunotherapeutics and to be able to match treatment to patient, especially in the light of CheckMate 143.

Glioblastoma is considered the most aggressive and lethal form of brain cancer. Glioblastoma tumours are complex, comprising a spectrum of oncogenically transformed cells displaying distinct phenotypes. These can be generated in culture and are called differentiated-glioblastoma cells and glioblastoma stem cells. These cells are phenotypically and functionally distinct, where the stem-like glioblastoma cells give rise to and perpetuate the tumour. Electric cell-substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method for the analysis of cellular behaviour, based on cellular adhesion. Therefore, we asked the question of whether ECIS was suitable for, and capable of measuring the adhesion of glioblastoma cells. The goal was to identify whether ECIS was capable of measuring glioblastoma cell adhesion, with a particular focus on the glioblastoma stem cells. We reveal that ECIS reliably measures adhesion of the differentiated glioblastoma cells on various array types. We also demonstrate the ability of ECIS to measure the migratory behaviour of differentiated glioblastoma cells onto ECIS electrodes post-ablation. Although the glioblastoma stem cells are adherent, ECIS is substantially less capable at reliably measuring their adhesion, compared with the differentiated counterparts. This means that ECIS has applicability for some glioblastoma cultures but much less utility for weakly adherent stem cell counterparts.

Glioblastoma is a highly aggressive brain malignancy commonly refractory to classical and novel chemo-, radio- and immunotherapies, with median survival times of ~15 months following diagnosis. Poor immunological responses exemplified by the downregulation of T-cell activity, and upregulation of immunosuppressive cells within the tumor microenvironment have limited the effectiveness of immunotherapy in glioblastoma to date. Here we show that glioblastoma cells express a large repertoire of inhibitory checkpoint ligands known to control effector T cell responses. Furthermore, flow cytometry analysis reveals that glioblastoma cells with an enhanced stem cell-like phenotype express several investigated ligands at significant levels on their cell surface. This reveals that glioblastoma stem-like cells express suppressive ligands with the potential of suppressing major T cell checkpoint receptors. With this information, it is now essential that we understand the relevance of this extensive repertoire of immune checkpoint ligands and their functional consequence on immune evasion in glioblastoma. This is necessary to develop effective immunotherapeutics and to be able to match treatment to patient, especially in the light of CheckMate 143.

Case summary A 7-year-old male neutered domestic longhair cat was presented with chronic progressive gynaecomastia, polydipsia, polyphagia, weight loss and poor fur regrowth. Sexualised behavioural changes were not reported and virilisation was not present on physical examination. Pertinent haematology, biochemistry and urinalysis findings at the time of referral included mild hypokalaemia. Left adrenomegaly and mild prostatomegaly were identified on a CT scan. Evaluation of adrenal hormones with a low-dose dexamethasone suppression test, serum progesterone, testosterone, oestradiol, plasma aldosterone, renin, plasma metanephrine and normetanephrine measurement supported a diagnosis of hyperprogesteronism, hyperaldosteronism and hypercortisolism. Adrenalectomy was performed and histopathology was consistent with an adrenocortical tumour. Clinical signs and hormone elevations resolved postoperatively. Relevance and novel information To our knowledge, this is the second report of gynaecomastia secondary to an adrenal tumour in a male neutered cat and the first associated with hyperprogesteronism.