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A 9-year-old female spayed Boston Terrier presented for diagnostic investigation of lethargy, poor appetite, weight loss, and a marked leukocytosis. Significant muscle wasting and a palpable abdominal mass were present on physical examination. Abdominal imaging revealed the mass to be of small intestinal origin; consequently, an intestinal resection and anastomosis were performed without complication. The histopathologic diagnosis was a gastrointestinal stromal tumor, verified by immunohistochemical positivity to CD117 (KIT). Two weeks after discharge, the leukocytosis had resolved. Though the exact molecular mediator of the severe leukocytosis was undetermined, resolution following tumor removal suggests a paraneoplastic cause. To the authors' knowledge, this is the first reported case of probable paraneoplastic leukocytosis secondary to a gastrointestinal stromal tumor in the dog. Gastrointestinal tract imaging should be performed when this uncommon hematologic abnormality is present. Published version

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45(+) immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Hepatorenal fibrocystic disease (HRFCD) is a genetically inherited disorder related to primary cilia dysfunction in which patients display varying levels of fibrosis, bile duct expansion, and inflammation. In mouse models of HRFCD, the phenotype is greatly impacted by the genetic background in which the mutation is placed. Macrophages are a common factor associated with progression of HRFCD and are also strongly influenced by the genetic background. These data led us to hypothesize that macrophage subtypes that change in relation to the genetic background are responsible for the variable phenotypic outcomes in HRFCD. To test this hypothesis, we utilized a mouse model of HRFCD (Ift88(Orpk) mice) on the C57BL/6 and BALB/c inbred backgrounds that have well-documented differences in macrophage subtypes. Our analyses of infiltrating macrophage subtypes confirm that genetic strain influences the subtype of infiltrating macrophage present during normal postnatal liver development and in Ift88(Orpk) livers (Ly6c(lo) in C57BL/6 vs Ly6c(hi) in BALB/c). Each infiltrating macrophage subtype was similarly associated with a unique phenotypic outcome as analysis of liver tissue shows that C57BL/6 Ift88(Orpk) mice have increased bile duct expansion, but reduced levels of fibrosis compared to BALB/c Ift88(Orpk) livers. RNA sequencing data suggest that the ability to infiltrate macrophage subtypes to influence the phenotypic outcome may be due to unique ligand-receptor signaling between infiltrating macrophages and cilia dysfunctional biliary epithelium. To evaluate whether specific macrophage subtypes cause the observed phenotypic divergence, we analyzed the liver phenotype in BALB/c Ift88(Orpk) mice on a CCR2-/- background. Unexpectedly, the loss of Ly6c(hi) macrophages, which were strongly enriched in BALB/c Ift88(Orpk) mice, did not significantly alter liver fibrosis. These data indicate that macrophage subtypes may correlate with HRFCD phenotypic outcome, but do not directly cause the pathology.

Kidney resident macrophages (KRMs) are involved in maintaining renal homeostasis and in controlling the pathological outcome of acute kidney injury and cystic kidney disease in mice. In adult mice, KRMs maintain their population through self-renewal with little or no input from the peripheral blood. Despite recent data suggesting that a transcriptionally similar population of KRM-like cells is present across species, the idea that they are self-renewing and minimally dependent on peripheral blood input in other species has yet to be proven due to the lack of an appropriate model and cross-species expression markers. In this study, we used our recently identified cross-species KRM cell surface markers and parabiosis surgery in inbred Lewis rats to determine if rat KRMs are maintained independent of peripheral blood input, similar to their mouse counterparts. Flow cytometry analysis indicated that parabiosis surgery in the rat results in the establishment of chimerism of T/B cells, neutrophils, and monocyte-derived infiltrating macrophages in the blood, spleen, and kidney 3 wk after parabiosis surgery. Analysis of KRMs using the cell surface markers CD81 and C1q indicated that these cells have minimal chimerism and, therefore, receive little input from the peripheral blood. These data indicate that KRM properties are conserved in at least two different species. NEW & NOTEWORTHY In this report, we performed parabiosis surgery on inbred Lewis rats and showed that rat kidney resident macrophages (KRMs), identified using our novel cross-species markers, are minimally dependent on peripheral blood input. Thus, for the first time, to our knowledge, we confirm that a hallmark of mouse KRMs is also present in KRMs isolated from another species.

Key Points Ly6chi infiltrating macrophage numbers are increased in injured, conditional Ift88 mice, compared with controls. Loss of Ly6chi infiltrating macrophages slows injury-accelerated cystic disease. Ly6chi infiltrating macrophages drive cystic disease in non-Pkd1–deficient cystic models.

Although renal macrophages have been shown to contribute to cystic kidney disease in PKD animal models, it remains unclear if there is a specific macrophage subpopulation involved. Here we analyze changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We demonstrate that CD206+ resident macrophages are minimal in a normal adult kidney but accumulate in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and show this significantly reduces cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increases in kidneys and in the urine from ADPKD patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation and demonstrate that the CD206+ resident macrophages in urine may serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients.

Innate and adaptive immune cells modulate Autosomal Dominant Polycystic Kidney Disease (ADPKD) severity, a common kidney disease with inadequate treatment options. ADPKD shares parallels with cancer where immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have shown that, in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of PD-1 on CD8+ T cells and PD-L1 on macrophages and epithelial cells in Pkd1RC/RC mice versus wildtypes, paralleling disease severity. PD-L1 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti-PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti-PD-1 plus anti-CTLA-4, blocking two immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers. Together, our data suggests that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.

Background Autosomal dominant polycystic kidney disease is caused by genetic mutations in PKD1 or PKD2. Macrophages and their associated inflammatory cytokines promote cyst progression; however, transcription factors within macrophages that control cytokine production and cystic disease are unknown. Methods In these studies, we used conditional Pkd1 mice to test the hypothesis that macrophage-localized interferon regulatory factor-5 (IRF5), a transcription factor associated with production of cyst-promoting cytokines (TNFα, IL-6), is required for accelerated cyst progression in a unilateral nephrectomy (1K) model. Analyses of quantitative real-time PCR (qRT-PCR) and flow-cytometry data 3 weeks post nephrectomy, a time point before the onset of severe cystogenesis, indicate an accumulation of inflammatory infiltrating and resident macrophages in 1K Pkd1 mice compared with controls. qRT-PCR data from FACS cells at this time demonstrate that macrophages from 1K Pkd1 mice have increased expression of Irf5 compared with controls. To determine the importance of macrophage-localized Irf5 in cyst progression, we injected scrambled or IRF5 antisense oligonucleotide (ASO) in 1K Pkd1 mice and analyzed the effect on macrophage numbers, cytokine production, and renal cystogenesis 6 weeks post nephrectomy. Results Analyses of qRT-PCR and IRF5 ASO treatmentsignificantly reduced macrophage numbers, Irf5 expression in resident—but not infiltrating—macrophages, and the severity of cystic disease. In addition, IRF5 ASO treatment in 1K Pkd1 mice reduced Il6 expression in resident macrophages, which was correlated with reduced STAT3 phosphorylation and downstream p-STAT3 target gene expression. Conclusions These data suggest that Irf5 promotes inflammatory cytokine production in resident macrophages resulting in accelerated cystogenesis.

Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to β oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.

BACKGROUND: Mutations affecting cilia proteins have an established role in renal cyst formation. In mice, the rate of cystogenesis is influenced by the age at which cilia dysfunction occurs and whether the kidney has been injured. Disruption of cilia function before postnatal day 12–14 results in rapid cyst formation; however, cyst formation is slower when cilia dysfunction is induced after postnatal day 14. Rapid cyst formation can also be induced in conditional adult cilia mutant mice by introducing renal injury. Previous studies indicate that macrophages are involved in cyst formation, however the specific role and type of macrophages responsible has not been clarified. METHODS: We analyzed resident macrophage number and subtypes during postnatal renal maturation and after renal injury in control and conditional Ift88 cilia mutant mice. We also used a pharmacological inhibitor of resident macrophage proliferation and accumulation to determine the importance of these cells during rapid cyst formation. RESULTS: Our data show that renal resident macrophages undergo a phenotypic switch from R2b (CD11c(lo)) to R2a (CD11c(hi)) during postnatal renal maturation. The timing of this switch correlates with the period in which cyst formation transitions from rapid to slow following induction of cilia dysfunction. Renal injury induces the reaccumulation of juvenile-like R2b resident macrophages in cilia mutant mice and restores rapid cystogenesis. Loss of primary cilia in injured conditional Ift88 mice results in enhanced epithelial production of membrane-bound CSF1, a cytokine that promotes resident macrophage proliferation. Inhibiting CSF1/CSF1-receptor signaling with a CSF1R kinase inhibitor reduces resident macrophage proliferation, R2b resident macrophage accumulation, and renal cyst formation in two mouse models of cystic disease. CONCLUSIONS: These data uncover an important pathogenic role for resident macrophages during rapid cyst progression.

Background Resident macrophages regulate homeostatic and disease processes in multiple tissues, including the kidney. Despite having well defined markers to identify these cells in mice, technical limitations have prevented identification of a similar cell type across species. The inability to identify resident macrophage populations across species hinders the translation of data obtained from animal model to human patients. Methods As an entry point to determine novel markers that could identify resident macrophages across species, we performed single-cell RNA sequencing (scRNAseq) analysis of all T and B cell–negative CD45 + innate immune cells in mouse, rat, pig, and human kidney tissue. Results We identified genes with enriched expression in mouse renal resident macrophages that were also present in candidate resident macrophage populations across species. Using the scRNAseq data, we defined a novel set of possible cell surface markers (Cd74 and Cd81) for these candidate kidney resident macrophages. We confirmed, using parabiosis and flow cytometry, that these proteins are indeed enriched in mouse resident macrophages. Flow cytometry data also indicated the existence of a defined population of innate immune cells in rat and human kidney tissue that coexpress CD74 and CD81, suggesting the presence of renal resident macrophages in multiple species. Conclusions Based on transcriptional signatures, our data indicate that there is a conserved population of innate immune cells across multiple species that have been defined as resident macrophages in the mouse. Further, we identified potential cell surface markers to allow for future identification and characterization of this candidate resident macrophage population in mouse, rat, and pig translational studies.

A large, firm, multi-cystic mammary gland mass grew slowly over 4 y in a 12-y-old, female Finn–Shetland cross sheep. A diagnosis of epithelial malignancy was suspected following fine-needle aspiration cytology at 30 mo after initial observation. The sheep was euthanized when the flock was downsized 18 mo later. A field postmortem examination revealed a large mammary mass, but an absence of metastases to internal organs. Imprint cytology of the mammary tissue supported a benign proliferative process. Histologically, mammary tissue was obliterated by cystic, tubular, and papillary adenomatous arrangements of mammary epithelium, with an anaplastic component, consistent with mammary carcinoma arising in an adenoma. IHC showed strong nuclear positivity to the antibody against progesterone receptor and minimal positivity to the antibody against estrogen receptor alpha expression. Intrinsic subtyping for basal or luminal epithelial origin was attempted through adaptation of companion animal IHC classification panels; high- and low-molecular-weight cytokeratins (CK5, CK8, CK18) failed to stain, but p63 expression for basal epithelium was positive.

Author(s): Toyoda, Izumi; Vernau, William; Sturges, Beverly K; Vernau, Karen M; Rossmeisl, John; Zimmerman, Kurt; Crowe, Chelsea M; Woolard, Kevin; Giuffrida, Michelle; Higgins, Robert J; Dickinson, Peter J | Abstract: BackgroundHistiocytic sarcoma affecting the central nervous system (CNS HS) in dogs may present as primary or disseminated disease, often characterized by inflammation. Prognosis is poor, and imaging differentiation from other CNS tumors can be problematic.ObjectiveTo characterize the clinicopathological inflammatory features, breed predisposition, and survival in dogs with CNS HS.AnimalsOne hundred two dogs with HS, 62 dogs with meningioma.MethodsRetrospective case series. Records were reviewed for results of cerebrospinal fluid (CSF) analysis, CBC, treatment, and outcome data.ResultsPredisposition for CNS HS was seen in Bernese Mountain Dogs, Golden Retrievers, Rottweilers, Corgis, and Shetland Sheepdogs (P ≤ .001). Corgis and Shetland Sheepdogs had predominantly primary tumors; Rottweilers had exclusively disseminated tumors. Marked CSF inflammation was characteristic of primary rather than disseminated HS, and neoplastic cells were detected in CSF of 52% of affected dogs. Increased neutrophil to lymphocyte ratios were seen in all groups relative to controls (P l.008) but not among tumor subtypes. Definitive versus palliative treatment resulted in improved survival times (P l .001), but overall prognosis was poor.Conclusions and clinical importanceClinicopathological differences between primary and disseminated HS suggest that tumor biological behavior and origin may be different. Corgis and Shetland Sheepdogs are predisposed to primary CNS HS, characterized by inflammatory CSF. High total nucleated cell count and the presence of neoplastic cells support the use of CSF analysis as a valuable diagnostic test. Prognosis for CNS HS is poor, but further evaluation of inflammatory mechanisms may provide novel therapeutic opportunities.

Hepatorenal fibrocystic disease (HRFCD) is characterized by cysts in the kidney and liver with associated fibrosis and is the result of defects in proteins required for cilia function or assembly. Previous reports indicate that macrophages, mainly M2-like macrophages, contribute to HRFCD, although the origin of these cells (yolk sac-derived resident macrophages vs. bone marrow-derived infiltrating macrophages) and their contribution to the observed phenotypes are unknown. We utilize a congenital model of cilia dysfunction (IFT88Orpk) to study the importance of macrophages in HRFCD. Our data show a rapid expansion of the bile duct region and development of fibrosis between 2 and 4 wk of age. Immunofluorescence microscopy analysis reveals an accumulation of F4/80+ macrophages in regions exhibiting biliary hyperplasia in IFT88Orpk mice. Flow cytometry data show that cilia dysfunction leads to an accumulation of infiltrating macrophages (CD11bhi, F4/80lo) and a reduction of resident macrophage (CD11blo, F4/80hi) number. A majority of the infiltrating macrophages are Ly6chi profibrogenic macrophages. Along with the accumulation of immune cells, expression of proinflammatory and profibrotic transcripts, including TGF-β, TNF-α, IL-1β, and chemokine (C-C) motif ligand 2, is increased. Quantitative RT-PCR analysis of flow-sorted cells shows enhanced expression of CCL2 in cholangiocytes and enhanced expression of VEGF-A and IL-6 in Ly6chi macrophages. Genetic inhibition of Ly6chi macrophage accumulation in IFT88Orpk FVB CCR2−/− mice reduced biliary fibrosis but did not affect epithelial expansion. Collectively, these studies suggest that biliary epithelium with defects in primary cilia preferentially recruits Ly6chi infiltrating macrophages, which promote fibrotic progression in HRFCD pathogenesis. NEW & NOTEWORTHY These studies are the first to address the contribution of the infiltrating and resident macrophage niche during progression of hepatorenal fibrocystic disease (HRFCD). We show that the number of infiltrating macrophages is significantly upregulated in HRFCD mouse models. Finally, we show that prevention of Ly6chi infiltrating macrophage accumulation significantly reduces biliary fibrosis, but not biliary hyperplasia, suggesting that this population may be responsible for the fibrotic progression of the disease in HRFCD patients.

Background Stereotactic brain biopsy (SBB) is a technique that allows for definitive diagnosis of brain lesions. Little information is available regarding the diagnostic utility of SBB in dogs with intracranial diseases. Objective To investigate the diagnostic accuracy (DA) of SBB in dogs with brain tumors. Animals Thirty-one client-owned dogs that underwent SBB followed by surgical resection or necropsy examinations. Methods Retrospective observational study. Two pathologists blinded to SBB and reference standard diagnoses reviewed histologic specimens and typed and graded tumors according to World Health Organization and revised canine glioma classification criteria. Agreement between tumor type and grade from SBB were compared to reference standards and assessed using kappa statistics. Patient and technical factors associated with agreement also were examined. Results Stereotactic brain biopsy specimens were obtained from 24 dogs with gliomas and 7 with meningiomas. Tumor type agreement between SBB and the reference standard was observed in 30/31 cases (kappa = 0.95). Diagnostic concordance was perfect for meningiomas. Grade agreement among gliomas was observed in 18/23 cases (kappa = 0.47). Stereotactic brain biopsy underrepresented the reference standard glioma grade in cases with disagreement. The DA of SBB was 81%, with agreement noted in 56/69 biopsy samples. Smaller tumors and fewer SBB specimens obtained were significantly associated with diagnostic discordance. Conclusions and Clinical Importance The DA of SBB readily allows for the diagnosis of common brain tumors in dogs. Although glioma grade discordance was frequent, diagnoses obtained from SBB are sufficient to currently inform therapeutic decisions. Multiple SBB specimens should be collected to maximize DA. National Institutes of Health/National Cancer Institute [P01CA207206, R01CA213423] National Institutes of Health/National Cancer Institute, Grant/Award Number: P01CA207206 and R01CA213423

Machine-learning methods can assist with the medical decision-making processes at the both the clinical and diagnostic levels. In this article, we first review historical milestones and specific applications of computer-based medical decision support tools in both veterinary and human medicine. Next, we take a mechanistic look at 3 archetypal learning algorithms—naive Bayes, decision trees, and neural network—commonly used to power these medical decision support tools. Last, we focus our discussion on the data sets used to train these algorithms and examine methods for validation, data representation, transformation, and feature selection. From this review, the reader should gain some appreciation for how these decision support tools have and can be used in medicine along with insight on their inner workings.

Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80(hi)-expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow–derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII(–) KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.

Intraocular neoplasms in dogs are uncommon in comparison with other anatomic locations [1–4]. Over 75% of these cases are attributed to melanocytic neoplasia [1]. A majority of the remaining cases (~22%) are comprised of lymphoma, metastatic neoplasia, iridociliary epithelial tumors, optic nerve meningiomas, and histiocytic sarcomas in decreasing order of frequency [1, 2]. Rarely, optic nerve astrocytomas have been reported in humans, dogs, and horses [5–7]. In humans, astrocytomas involving the optic nerve are uncommon accounting for only about 1% of neoplasms at this site in comparison with 25% of neoplasms occurring in the brain [5, 8–14]. Most of these neoplasms occur unilaterally, are benign, and arise in children under 10 years of age; those involving the cerebellum have a more favorable prognosis [8, 14]. These younger patients commonly have an underlying familiar disorder such as neurofibromatosis. In older patients in their fourth and fifth decade of life, astrocytomas more commonly involve the cerebrum with a less favorable prognosis and can be associated with familiar disorders such as Li–Fraumeni syndrome [13, 15–17]. Most canine ocular astrocytoma cases are sporadic and not associated with any familial disorder [1, 13, 18–21]. However, Thomas et al. demonstrated a genomic risk factor associated with frequency of chromosome copy number aberrations within canine brain astrocytomas and tumor grade [22]. Similar to humans, canine astrocytomas account for less than 1% of ocular and optic nerve neoplasms and 10–36% of primary intracranial neoplasms [1, 4, 16–18, 20, 21, 23]. There appears to be a breed predisposition for development of intracranial astrocytomas in English Toy Spaniels, Boston Terriers, French Bulldogs, Boxers, and English Bulldog with a peak prevalence at 7– 8 years and 1.5 odds ratio in favor of larger versus smaller breeds [13, 23]. These risk factors have not been shown in association with canine ocular forms of this neoplasm [1, 16, 17]. Gender as a risk factor has not been described for either anatomic location [1, 13, 16–18, 23]. Case reports related to ocular astrocytomas are still rare in veterinary literature. The purpose of this report is to add to this sparse body of information. This report presents the clinical, histological, and immunohistochemical features of an optic nerve astrocytoma in a dog.

Polycystic Kidney Disease (PKD) triggers a robust immune system response including changes in both innate and adaptive immunity. These changes involve immune cells (e.g., macrophages and T cells) as well as cytokines and chemokines (e.g., MCP-1) that regulate the production, differentiation, homing, and various functions of these cells. This review is focused on the role of the immune system and its associated factors in the pathogenesis of PKDs as evidenced by data from cell-based systems, animal models, and PKD patients. It also highlights relevant pre-clinical and clinical studies that point to specific immune system components as promising candidates for the development of prognostic biomarkers and therapeutic strategies to improve PKD outcomes.

Several innate immune response components were recognized as outcome predictors in autosomal dominant polycystic kidney disease (ADPKD) and their causative role in disease pathogenesis was confirmed in animal models. In contrast, the role of adaptive immunity in ADPKD remains relatively unexplored. Therefore, we evaluated T cell populations in kidney and urine of ADPKD patients using flow cytometry and confocal immunofluorescence microscopy approaches. These analyses revealed ADPKD‐associated overall increases in the number of intrarenal CD4 and CD8 T cells that were associated with a loss of polarity in distribution between the cortex and medulla (higher in medulla vs. cortex in controls). Also, the urinary T cell‐based index correlated moderately with renal function decline in a small cohort of ADPKD patients. Together, these data suggest that similar to innate immune responses, T cells participate in ADPKD pathogenesis. They also point to urinary T cells as a novel candidate marker of the disease activity in ADPKD.

A variety of inflammatory conditions of unknown cause (meningoencephalomyelitis of unknown etiology-MUE) and neoplastic diseases can affect the central nervous system (CNS) of dogs. MUE can mimic intracranial neoplasia both clinically, radiologically and even in some cases, histologically. Serum immunosignature protein microarray assays have been used in humans to identify CNS diseases such as Alzheimer's and neoplasia, and in dogs, to detect lymphoma and its progression. This study evaluated the effectiveness of immunosignature profiles for distinguishing between three cohorts of dogs: healthy, intracranial neoplasia, and MUE. Using the learned peptide patterns for these three cohorts, classification prediction was evaluated for the same groups using a 10-fold cross validation methodology. Accuracy for classification was 100%, as well as 100% specific and 100% sensitive. This pilot study demonstrates that immunosignature profiles may help serve as a minimally invasive tool to distinguish between MUE and intracranial neoplasia in dogs.

Pet guinea pigs are presented to veterinary clinics for routine care and treatment of clinical diseases. In addition to obtaining clinical history and exam findings, diagnostic testing may be required, including hematological assessments. This article describes common blood collection methods, including venipuncture sites, the volume of blood that can be safely collected, and handling of the blood. Hematological parameters for normal guinea pigs are provided for comparison with in-house or commercial test results. A description of the morphology of guinea pig leukocytes is provided to assist in performing a differential count.

Pet rabbits are presented to veterinary clinics for routine care and treatment of clinical diseases. In addition to obtaining clinical history, additional diagnostic testing may be required, including hematological assessments. This article describes common blood collection methods, including venipuncture sites, volume of blood that can be safely collected, and handling of the blood. Hematological parameters for normal rabbits are provided for comparison with in-house or commercial test results. A description of the morphology of rabbit leukocytes is provided to assist in performing a differential count. Differential diagnoses are provided for abnormal values identified in the hemogram.

Background/Aims: The endoplasmic reticulum (ER) stress protein, calreticulin (CRT), is required for the production of TGF-β-stimulated extracellular matrix (ECM) by fibroblasts. Since TGF-β regulates vascular fibroproliferative responses and collagen deposition, we investigated the effects of CRT knockdown on vascular smooth-muscle cell (VSMC) fibroproliferative responses and collagen deposition. Methods: Using a carotid artery ligation model of vascular injury, Cre-recombinase-IRES-GFP plasmid was delivered with microbubbles (MB) to CRT-floxed mice using ultrasound (US) to specifically reduce CRT expression in the carotid artery. Results: In vitro, Cre-recombinase-mediated CRT knockdown in isolated, floxed VSMCs decreased the CRT transcript and protein, and attenuated the induction of collagen I protein in response to TGF-β. TGF-β stimulation of collagen I was partly blocked by the NFAT inhibitor 11R-VIVIT. Following carotid artery ligation, CRT staining was upregulated with enhanced expression in the neointima 14-21 days after injury. Furthermore, Cre-recombinase-IRES-GFP plasmid delivered by targeted US reduced CRT expression in the neointima of CRT-floxed mice and led to a significant reduction in neointima formation and collagen deposition. The neointimal cell number was also reduced in mice, with a local, tissue-specific knockdown of CRT. Conclusions: This work establishes a novel role for CRT in mediating VSMC responses to injury through the regulation of collagen deposition and neointima formation.

Background: Biopsy of the liver evaluates a small portion of tissue, with inferences made to the entire organ. The method and number of biopsies obtained are tempered by consideration of the risks and benefits. Recommendations often include biopsy of more than one liver lobe, although the consistency of histopathology among lobes in dogs is unknown. Hypothesis/Objectives: To describe the distribution of histopathologic abnormalities between liver lobes. We hypothesized that discordant results would be evenly distributed among all liver lobes. Animals: Seventy dogs undergoing necropsy. Methods: Prospective study. Liver samples were obtained from all lobes. A primary diagnosis was assigned to each liver sample based on the predominant histopathologic abnormality. Results: In this population of dogs, biopsy of at least 2 liver lobes identified the predominant histologic abnormality in 98.6% of the cases. Ten ( 14%) of the dogs had = 3 lobes in agreement and could not be assigned a predominant diagnosis. The same diagnosis was present in 6/ 6 lobes in 39 ( 56.5%) dogs, 5/ 6 lobes in 10 ( 14.5%) dogs, 4/ 6 lobes in 10 ( 14.5%) dogs, 3/ 6 lobes in 7 ( 10.1%) dogs, and 2/ 6 in 3 ( 4.3%) dogs. The number of discordant results did not differ between the liver lobes. Conclusion and Clinical Importance: The likelihood of obtaining a sample that is reflective of the predominant histologic abnormality in the liver is increased when multiple liver lobes are biopsied. Virginia Veterinary Memorial Fund The study was funded by the Virginia Veterinary Memorial Fund.

Hamsters, gerbils, rats, and mice are presented to veterinary clinics and hospitals for prophylactic care and treatment of clinical signs of disease. Physical examination, history, and husbandry practice information can be supplemented greatly by assessment of hematologic parameters. As a resource for veterinarians and their technicians, this article describes the methods for collection of blood, identification of blood cells, and interpretation of the hemogram in mice, rats, gerbils, and hamsters.

Endoplasmic reticulum (ER) stress is a key factor contributing to fibrotic disease. Although ER stress is a short-term adaptive response, with chronic stimulation, it can activate pathways leading to fibrosis. ER stress can induce TGF-β signaling, a central driver of extracellular matrix production in fibrosis. This review will discuss the role of an ER protein, calreticulin (CRT), which has both chaperone and calcium regulatory functions, in fibrosis. CRT expression is upregulated in multiple different fibrotic diseases. The roles of CRT in regulation of fibronectin extracellular matrix assembly, extracellular matrix transcription, and collagen secretion and processing into the extracellular matrix will be discussed. Evidence for the importance of CRT in ER calcium release and NFAT activation downstream of TGF-β signaling will be presented. Finally, we will summarize evidence from animal models in which CRT expression is genetically reduced or experimentally downregulated in targeted tissues of adult animals and discuss how these models define a key role for CRT in fibrotic diseases.

Polycystic kidney disease (PKD) is a commonly inherited disorder characterized by cyst formation and fibrosis (Wilson, N Engl J Med 350:151–164, 2004) and is caused by mutations in cilia or cilia-related proteins, such as polycystin 1 or 2 (Oh and Katsanis, Development 139:443–448, 2012; Kotsis et al., Nephrol Dial Transplant 28:518–526, 2013). A major pathological feature of PKD is the development of interstitial inflammation and fibrosis with an associated accumulation of inflammatory cells (Grantham, N Engl J Med 359:1477–1485, 2008; Zeier et al., Kidney Int 42:1259–1265, 1992; Ibrahim, Sci World J 7:1757–1767, 2007). It is unclear whether inflammation is a driving force for cyst formation or a consequence of the pathology (Ta et al., Nephrology 18:317–330, 2013) as in some murine models cysts are present prior to the increase in inflammatory cells (Phillips et al., Kidney Blood Press Res 30:129–144, 2007; Takahashi et al., J Am Soc Nephrol JASN 1:980–989, 1991), while in other models the increase in inflammatory cells is present prior to or coincident with cyst initiation (Cowley et al., Kidney Int 43:522–534, 1993, Kidney Int 60:2087–2096, 2001). Additional support for inflammation as an important contributor to cystic kidney disease is the increased expression of many pro-inflammatory cytokines in murine models and human patients with cystic kidney disease (Karihaloo et al., J Am Soc Nephrol JASN 22:1809–1814, 2011; Swenson-Fields et al., Kidney Int, 2013; Li et al., Nat Med 14:863–868, 2008a). Based on these data, an emerging model in the field is that disruption of primary cilia on tubule epithelial cells leads to abnormal cytokine cross talk between the epithelium and the inflammatory cells contributing to cyst growth and fibrosis (Ta et al., Nephrology 18:317–330, 2013). These cytokines are produced by interstitial fibroblasts, inflammatory cells, and tubule epithelial cells and activate multiple pathways including the JAK-STAT and NF-κB signaling (Qin et al., J Am Soc Nephrol JASN 23:1309–1318, 2012; Park et al., Am J Nephrol 32:169–178, 2010; Bhunia et al., Cell 109:157–168, 2002). Indeed, inflammatory cells are responsible for producing several of the pro-fibrotic growth factors observed in PKD patients with fibrosis (Nakamura et al., Am J Nephrol 20:32–36, 2000; Wilson et al., J Cell Physiol 150:360–369, 1992; Song et al., Hum Mol Genet 18:2328–2343, 2009; Schieren et al., Nephrol Dial Transplant 21:1816–1824, 2006). These growth factors trigger epithelial cell proliferation and myofibroblast activation that stimulate the production of extracellular matrix (ECM) genes including collagen types 1 and 3 and fibronectin, leading to reduced glomerular function with approximately 50% of ADPKD patients progressing to end-stage renal disease (ESRD). Therefore, treatments designed to reduce inflammation and slow the rate of fibrosis are becoming important targets that hold promise to improve patient life span and quality of life. In fact, recent studies in several PKD mouse models indicate that depletion of macrophages reduces cyst severity. In this chapter, we review the potential mechanisms of interstitial inflammation in PKD with a focus on ADPKD and discuss the role of interstitial inflammation in progression to fibrosis and ESRD.

Background: The liver sampling technique in dogs that consistently provides samples adequate for accurate histopathologic interpretation is not known. Hypothesis/ Objectives: To compare histopathologic results of liver samples obtained by punch, cup, and 14 gauge needle to large wedge samples collected at necropsy. Animals: Seventy dogs undergoing necropsy. Methods: Prospective study. Liver specimens were obtained from the left lateral liver lobe with an 8 mm punch, a 5 mm cup, and a 14 gauge needle. After sample acquisition, two larger tissue samples were collected near the center of the left lateral lobe to be used as a histologic standard for comparison. Histopathologic features and numbers of portal triads in each sample were recorded. Results: The mean number of portal triads obtained by each sampling method were 2.9 in needle samples, 3.4 in cup samples, 12 in punch samples, and 30.7 in the necropsy samples. The diagnoses in 66% of needle samples, 60% of cup samples, and 69% of punch samples were in agreement with the necropsy samples, and these proportions were not significantly different from each other. The corresponding kappa coefficients were 0.59 for needle biopsies, 0.52 for cup biopsies, and 0.62 for punch biopsies. Conclusion and Clinical Importance: The histopathologic interpretation of a liver sample in the dog is unlikely to vary if the liver biopsy specimen contains at least 3- 12 portal triads. However, in comparison large necropsy samples, the accuracy of all tested methods was relatively low. Virginia Veterinary Memorial Fund The authors thank Dr Stephen R. Werre for statistical assistance. The study was funded by the Virginia Veterinary Memorial Fund.

Fine needle aspiration and tissue biopsy samples were taken from a subcutaneous mass in the cranial stifle region of a 10-yr-old horse. Key cytologic features included numerous mildly to moderately pleomorphic histiocytic cells admixed with mononuclear inflammatory cells, occasional fibroblasts, multinucleated cells, and hemosiderin-laden macrophages. Based on these features, cytologic differentials included granulomatous inflammation with reactive fibroplasia, histiocytic sarcoma, giant cell tumor of soft parts (GCTSPs), and giant cell tumor of bone. The tissue biopsy supported a diagnosis of superficial GCTSPs. This neoplasm is of controversial origin but generally considered to be a soft tissue sarcoma and classified in the World Health Organization classification of tumors under fibrous histiocytic tumors as a giant cell tumor of soft tissue with low risk of malignancy. When found in the subcutis, this neoplasm rarely metastasizes in the horse and has a low rate of local reoccurrence. In this case, there was no local recurrence of the tumor after 6 mo, after which the horse was lost to follow-up.Keywords: Cytology, Equine, Giant cell tumor of soft parts, Histopathology.

Background Intramedullary neoplasms of the canine spinal cord are infrequently reported. Objective To describe distribution, clinicopathologic characteristics, radiographic findings, and clinical features of canine intramedullary spinal tumors. Methods Retrospective series of histologically confirmed canine intramedullary spinal tumors. Contingency tables were generated for categorical variables (breed, sex, treatment, pain, chief complaint, localization, histology, imaging, and site). Associations were assessed by Fisher's exact, Wilcoxon rank sum test, t-test, and one-way ANOVA. Results Intramedullary spinal cord tumors comprised 16% (53/331) of all tumors of the spinal cord. Primary tumors were diagnosed in 66% (35/53) of cases, with neuroepithelial-origin tumors comprising 51% (18/35) of all primary neoplasms. Intraparenchymal metastases of transitional cell carcinoma and hemangiosarcoma accounted for 66% (6/18 each) of all secondary tumors. Primary tumors were more likely to affect younger dogs. Dogs with intramedullary metastases were most commonly presented for primary myelopathic signs (8/18, 44%). The majority of all tumors (52.8%) occurred in the T3-L3 spinal cord segments. All dogs with cervical neurolocalization had primary tumors. Dogs with metastatic lesions had a shorter duration of clinical signs before presentation, but there was no difference in survival time between dogs with primary as compared with secondary tumors. Conclusions Intramedullary spinal cord tumors are uncommon. Primary intramedullary spinal cord tumors are more common than secondary intramedullary spinal cord tumors and tend to occur in the cervical spinal cord of younger dogs. Intramedullary metastases occur in older dogs, are rarely asymptomatic, and neurologic dysfunction is a common clinical presentation. Dogs with primary tumors may have a protracted clinical course compared with those with intramedullary metastases.

Brucella is amongst the top 5 causes of zoonotic disease worldwide. Infection is through ingestion, inhalation or contact exposure. Brucella is characterized as a class B pathogen by Centers of Disease Control and Prevention (CDC). Currently, there are no efficacious vaccines available in people. Currently available USDA approved vaccines for animals include B. abortus strain RB51 and B. melitensis Rev1. Protection is mediated by a strong innate and CD4 Th1, CD8 Tc1 immune response. If protective vaccines can be developed, disease in people and animals can be controlled. While strain RB51 protects in cattle, and against intraperitoneal challenge in mice, it does not protect against respiratory challenge. Therefore, we assessed the efficacy of strain RB51 combined with different TLR agonists, and O-side chain from LPS, to enhance protection against respiratory challenge with strain 2308. We hypothesized that TLR agonists and O-side chain would enhance protection. Strains RB51 with TLR2 agonist, RB51 with TLR4 agonist and strain 19 provided significant protection in the lung. Protection using strain RB51 with TLR agonists was associated with increased IgG2a and IgG1 in the (bronchoalveolar lavage) BAL and serum, and increased IgA (serum). Splenocytes from strain RB51 with TLR2 vaccinated mice up-regulated antigen specific interferon-gamma and TNF-alpha production. Vaccination and challenge resulted in significant increases in activated dendritic cells (DCs), and increased CD4 and CD8 cells in the BAL. Overall, this study demonstrates the ability of TLR agonists 2 and 4 to up-regulate strain RB51 mediated protection in the lung to respiratory challenge against strain 2308.

Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation that could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptor to bleomycin-induced lung injury and measured efferocytosis, inflammation, and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days postinjury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days postinjury, 51% increase in lung macrophages 14 days postinjury), and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days postinjury, respectively). Reduced fibrosis in CD36(-/-) mice was associated with lower levels of profibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1, and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 h following zymosan administration in G2A(-/-) mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.

A 6-month-old, neutered male, mixed-breed dog was examined for a 2-month persistent fever, nonhealing dermal metacarpal area wound, and leukocytosis (47.0–198.0 × 103/μl). Serum chemistry findings included hypoalbuminemia, hyperglobulinemia, hyperphosphatemia, and hyperphosphatasemia. Complete blood cell count results revealed a moderate microcytic, hypochromic nonregenerative anemia with a profound leukocytosis (198.5 × 103/μl), characterized by neutrophilia with toxicity and hypersegmentation, and significant band cells. Tick-borne disease titers (genera Anaplasma, Ehrlichia, and Borrelia) were negative, as were polymerase chain reaction for other infectious agents (genera Hepatozoon, Mycobacterium, Mycoplasma; and Canine distemper virus). No agents were identified in a deep dermal biopsy (conventional and special histochemical stains) of the chronic draining, metacarpal region lesion. Cytology of the draining tract revealed numerous mixed bacteria and a surprising lack of neutrophils. Chronic occult blood loss with iron deficiency was considered a possible cause of the anemia. Differentials for the leukon were chronic established inflammation (occult infectious agent), chronic neutrophilic leukemia, paraneoplastic leukocytosis (neoplastic source of granulocyte colony-stimulating factor [CSF] or granulocyte-macrophage CSF), and leukocyte adhesion deficiency (LAD). The possibility of a LAD disorder was further investigated because of the noted hypersegmented neutrophils, absence of neutrophils in the cytology sample, the animal’s young age, and persistence of clinical and laboratory signs. Flow cytometry of blood neutrophils showed a 60% reduction in surface expression of the β2-integrin (CD18) subunit, whereas neutrophil function tests (oxidative burst and phagocytosis) were normal. Genetic testing revealed a homozygous missense mutation in the β2-integrin subunit gene, previously recognized only in purebred Irish Setters, leading to a diagnosis of LAD type 1 disorder in this mixed-breed dog.

Objective—To analyze survival time and identify prognostic factors associated with outcome following discharge in dogs with primary brain tumors treated palliatively. Design—Prospective case series. Animals—51 dogs with 5 histopathologic types of brain tumors. Procedures—Owners with dogs examined from 2004 to 2008 were invited to participate if dogs had CT or MRI evidence of a brain mass that was histopathologically confirmed as a neoplasm upon death, dogs survived for ≥ 48 hours after hospital discharge, and treatments following discharge were limited to administration of prednisone or phenobarbital. Prognostic factors, including signalment, clinical signs (including duration), tumor type, tumor location, degree of peritumoral edema, lesion burden, and prescribed treatment, were evaluated. Survival time was estimated and animal- and tumor-specific variables evaluated as potential prognostic factors. Results—The median survival time in all dogs was 69 days (95% confidence interval [CI], 18 to 201 days). Multivariate analyses identified neuroanatomic location as the only significant prognostic variable, with the survival time of dogs with infratentorial tumors (n = 18) being significantly shorter (median, 28 days; 95% CI, 19 to 68 days) than survival time of dogs with supratentorial (33) tumors (median, 178 days; 95% CI, 119 to 270 days). Seizures were the most common clinical sign associated with supratentorial tumors (24/33 [73%]) and central vestibular dysfunction with infratentorial tumors (12/18). Conclusions and Clinical Relevance—Dogs with palliatively treated primary brain tumors, particularly those with tumors in the cerebellum, pons, or medulla, had a poor prognosis. However, dogs with supratentorial tumors had survival times > 3 months.

Inflammatory bowel disease (IBD) and alimentary lymphoma (ALA) are common gastrointestinal diseases in cats. The very similar clinical signs and histopathologic features of these diseases make the distinction between them diagnostically challenging. We tested the use of supervised machine-learning algorithms to differentiate between the 2 diseases using data generated from noninvasive diagnostic tests. Three prediction models were developed using 3 machine-learning algorithms: naive Bayes, decision trees, and artificial neural networks. The models were trained and tested on data from complete blood count (CBC) and serum chemistry (SC) results for the following 3 groups of client-owned cats: normal, inflammatory bowel disease (IBD), or alimentary lymphoma (ALA). Naive Bayes and artificial neural networks achieved higher classification accuracy (sensitivities of 70.8% and 69.2%, respectively) than the decision tree algorithm (63%, p < 0.0001). The areas under the receiver-operating characteristic curve for classifying cases into the 3 categories was 83% by naive Bayes, 79% by decision tree, and 82% by artificial neural networks. Prediction models using machine learning provided a method for distinguishing between ALA–IBD, ALA–normal, and IBD–normal. The naive Bayes and artificial neural networks classifiers used 10 and 4 of the CBC and SC variables, respectively, to outperform the C4.5 decision tree, which used 5 CBC and SC variables in classifying cats into the 3 classes. These models can provide another noninvasive diagnostic tool to assist clinicians with differentiating between IBD and ALA, and between diseased and nondiseased cats.