Hemangiosarcoma Cells Promote Conserved Host-derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors. SIGNIFICANCE: We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression.

A bioengineered artificial interstitium supports long-term islet xenograft survival in nonhuman primates without immunosuppression.

Cell-based therapies hold promise for many chronic conditions; however, the continued need for immunosuppression along with challenges in replacing cells to improve durability or retrieving cells for safety are major obstacles. We subcutaneously implanted a device engineered to exploit the innate transcapillary hydrostatic and colloid osmotic pressure generating ultrafiltrate to mimic interstitium. Long-term stable accumulation of ultrafiltrate was achieved in both rodents and nonhuman primates (NHPs) that was chemically similar to serum and achieved capillary blood oxygen concentration. The majority of adult pig islet grafts transplanted in non-immunosuppressed NHPs resulted in xenograft survival >100 days. Stable cytokine levels, normal neutrophil to lymphocyte ratio, and a lack of immune cell infiltration demonstrated successful immunoprotection and averted typical systemic changes related to xenograft transplant, especially inflammation. This approach eliminates the need for immunosuppression and permits percutaneous access for loading, reloading, biopsy, and recovery to de-risk the use of "unlimited" xenogeneic cell sources to realize widespread clinical translation of cell-based therapies.

Population screening shows risk of inherited cancer and familial hypercholesterolemia in Oregon.

The Healthy Oregon Project (HOP) is a statewide effort that aims to build a large research repository and influence the health of Oregonians through providing no-cost genetic screening to participants for a next-generation sequencing 32-gene panel comprising genes related to inherited cancers and familial hypercholesterolemia. This type of unbiased population screening can detect at-risk individuals who may otherwise be missed by conventional medical approaches. However, challenges exist for this type of high-throughput testing in an academic setting, including developing a low-cost high-efficiency test and scaling up the clinical laboratory for processing large numbers of samples. Modifications to our academic clinical laboratory including efficient test design, robotics, and a streamlined analysis approach increased our ability to test more than 1,000 samples per month for HOP using only one dedicated HOP laboratory technologist. Additionally, enrollment using a HIPAA-compliant smartphone app and sample collection using mouthwash increased efficiency and reduced cost. Here, we present our experience three years into HOP and discuss the lessons learned, including our successes, challenges, opportunities, and future directions, as well as the genetic screening results for the first 13,670 participants tested. Overall, we have identified 730 pathogenic/likely pathogenic variants in 710 participants in 24 of the 32 genes on the panel. The carrier rate for pathogenic/likely pathogenic variants in the inherited cancer genes on the panel for an unselected population was 5.0% and for familial hypercholesterolemia was 0.3%. Our laboratory experience described here may provide a useful model for population screening projects in other states.

Development of an exosomal gene signature to detect residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning.

Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups ("healthy", "osteosarcoma", "other bone tumor", or "non-neoplastic disease"). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as "osteosarcoma detected" or "osteosarcoma-NOT detected". Dogs in a validation set whose post-treatment samples were classified as "osteosarcoma-NOT detected" had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.

A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumour growth of feline oral squamous cell carcinoma.

Monocarboxylate transporters (MCTs) support tumour growth by regulating the transport of metabolites in the tumour microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistant malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 reduced the viability of feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumour growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC.

Interspecies Organogenesis for Human Transplantation.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Lung Cancer: One Disease or Many.

OBJECTIVE: Lung cancer is classified as a single entity comprised of multiple histological subtypes. But how similar are these subtypes on a genetic level? This paper aims to address this question through a concise overview of germline and somatic differences between small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma. METHODS: We reveal the weak overlap found between these 3 lung cancer subtypes using published data from one of the largest germline genetic studies on lung cancer to date and somatic mutation data from Catalogue of Somatic Mutations in Cancer (COSMIC). RESULTS: These data indicate that these 3 subtypes share very little with each other at the genetic level. At the germline SNP level, only 24 independent SNPs from 2 chromosomes were shared across all 3 subtypes. We also demonstrate that only 30 unique cancer-specific mutations overlap the 3 subtypes from COSMIC, and that this is fewer than overlapping mutations chosen at random. Finally, we show that only 3 somatic mutational signatures are shared between these 3 subtypes. CONCLUSION: This paper highlights that these 3 lung cancer subtypes may be distinct diseases at the genetic level. In the era of precision medicine, we feel that these genomic differences will be of utmost importance in the choice of lung cancer therapy in the future.

Weak sharing of genetic association signals in three lung cancer subtypes: evidence at the SNP, gene, regulation, and pathway levels.

BACKGROUND: There are two main types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC has many subtypes, but the two most common are lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). These subtypes are mainly classified by physiological and pathological characteristics, although there is increasing evidence of genetic and molecular differences as well. Although some work has been done at the somatic level to explore the genetic and biological differences among subtypes, little work has been done that interrogates these differences at the germline level to characterize the unique and shared susceptibility genes for each subtype. METHODS: We used single-nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS) of European samples to interrogate the similarity of the subtypes at the SNP, gene, pathway, and regulatory levels. We expanded these genotyped SNPs to include all SNPs in linkage disequilibrium (LD) using data from the 1000 Genomes Project. We mapped these SNPs to several lung tissue expression quantitative trait loci (eQTL) and enhancer datasets to identify regulatory SNPs and their target genes. We used these genes to perform a biological pathway analysis for each subtype. RESULTS: We identified 8295, 8734, and 8361 SNPs with moderate association signals for LUAD, LUSC, and SCLC, respectively. Those SNPs had p < 1 × 10- 3 in the original GWAS or were within LD (r2 > 0.8, Europeans) to the genotyped SNPs. We identified 215, 320, and 172 disease-associated genes for LUAD, LUSC, and SCLC, respectively. Only five genes (CHRNA5, IDH3A, PSMA4, RP11-650 L12.2, and TBC1D2B) overlapped all subtypes. Furthermore, we observed only two pathways from the Kyoto Encyclopedia of Genes and Genomes shared by all subtypes. At the regulatory level, only three eQTL target genes and two enhancer target genes overlapped between all subtypes. CONCLUSIONS: Our results suggest that the three lung cancer subtypes do not share much genetic signal at the SNP, gene, pathway, or regulatory level, which differs from the common subtype classification based upon histology. However, three (CHRNA5, IDH3A, and PSMA4) of the five genes shared between the subtypes are well-known lung cancer genes that may act as general lung cancer genes regardless of subtype.

Comparative Transcriptome Analysis Quantifies Immune Cell Transcript Levels, Metastatic Progression, and Survival in Osteosarcoma.

Overall survival of patients with osteosarcoma (OS) has improved little in the past three decades, and better models for study are needed. OS is common in large dog breeds and is genetically inducible in mice, making the disease ideal for comparative genomic analyses across species. Understanding the level of conservation of intertumor transcriptional variation across species and how it is associated with progression to metastasis will enable us to more efficiently develop effective strategies to manage OS and to improve therapy. In this study, transcriptional profiles of OS tumors and cell lines derived from humans (n = 49), mice (n = 103), and dogs (n = 34) were generated using RNA sequencing. Conserved intertumor transcriptional variation was present in tumor sets from all three species and comprised gene clusters associated with cell cycle and mitosis and with the presence or absence of immune cells. Further, we developed a novel gene cluster expression summary score (GCESS) to quantify intertumor transcriptional variation and demonstrated that these GCESS values associated with patient outcome. Human OS tumors with GCESS values suggesting decreased immune cell presence were associated with metastasis and poor survival. We validated these results in an independent human OS tumor cohort and in 15 different tumor data sets obtained from The Cancer Genome Atlas. Our results suggest that quantification of immune cell absence and tumor cell proliferation may better inform therapeutic decisions and improve overall survival for OS patients.Significance: This study offers new tools to quantify tumor heterogeneity in osteosarcoma, identifying potentially useful prognostic biomarkers for metastatic progression and survival in patients. Cancer Res; 78(2); 326-37. ©2017 AACR.

Constitutive activation of alternative nuclear factor kappa B pathway in canine diffuse large B-cell lymphoma contributes to tumor cell survival and is a target of new adjuvant therapies.

Activation of the classical nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway is a common molecular event observed in both human and canine diffuse large B-cell lymphoma (DLBCL). Although the oncogenic potential of the alternative NFκB pathway (ANFκBP) has also been recently identified in DLBCL, its precise role in tumor pathogenesis and potential as a treatment target is understudied. We hypothesized that up-regulation of the ANFκBP plays an important role in the proliferation and survival of canine DLBCL cells, and we demonstrate that the ANFκBP is constitutively active in primary canine DLBCL samples and a cell line (CLBL1). We further demonstrate that a small interfering RNA inhibits the activation of the NFκB pathway and induces apoptosis in canine DLBCL cells. In conclusion, the ANFκBP facilitates survival of canine DLBCL cells, and thus, dogs with spontaneous DLBCL can provide a useful large animal model to study therapies targeting the ANFκBP.

Eradication of Canine Diffuse Large B-Cell Lymphoma in a Murine Xenograft Model with CD47 Blockade and Anti-CD20.

Cancer immunotherapies hold much promise, but their potential in veterinary settings has not yet been fully appreciated. Canine lymphomas are among the most common tumors of dogs and bear remarkable similarity to human disease. In this study, we examined the combination of CD47 blockade with anti-CD20 passive immunotherapy for canine lymphoma. The CD47/SIRPα axis is an immune checkpoint that regulates macrophage activation. In humans, CD47 is expressed on cancer cells and enables evasion from phagocytosis. CD47-blocking therapies are now under investigation in clinical trials for a variety of human cancers. We found the canine CD47/SIRPα axis to be conserved biochemically and functionally. We identified high-affinity SIRPα variants that antagonize canine CD47 and stimulate phagocytosis of canine cancer cells in vitro When tested as Fc fusion proteins, these therapeutic agents exhibited single-agent efficacy in a mouse xenograft model of canine lymphoma. As robust synergy between CD47 blockade and tumor-specific antibodies has been demonstrated for human cancer, we evaluated the combination of CD47 blockade with 1E4-cIgGB, a canine-specific antibody to CD20. 1E4-cIgGB could elicit a therapeutic response against canine lymphoma in vivo as a single agent. However, augmented responses were observed when combined with CD47-blocking therapies, resulting in synergy in vitro and in vivo and eliciting cures in 100% of mice bearing canine lymphoma. Our findings support further testing of CD47-blocking therapies alone and in combination with CD20 antibodies in the veterinary setting. Cancer Immunol Res; 4(12); 1072-87. ©2016 AACR.

Porcine lung mesenchymal stromal cells possess differentiation and immunoregulatory properties.

INTRODUCTION: Mesenchymal stem (stromal) cells (MSCs) possess self-renewal, differentiation and immunoregulatory properties, and therefore are being evaluated as cellular therapy for inflammatory and autoimmune diseases, and for tissue repair. MSCs isolated from bone marrow are extensively studied. Besides bone marrow, MSCs have been identified in almost all organs of the body including the lungs. Lung-derived MSCs may be more effective as therapy for lung diseases as compared to bone marrow-derived MSCs. Pigs are similar to humans in anatomy, physiology and immunological responses, and thus may serve as a useful large animal preclinical model to study potential cellular therapy for human diseases. METHODS: We isolated MSCs from the lungs (L-MSCs) of 4-6-week-old germ-free pigs. We determined the self-renewal, proliferation and differentiation potential of L-MSCs. We also examined the mechanisms of immunoregulation by porcine L-MSCs. RESULTS: MSCs isolated from porcine lungs showed spindle-shaped morphology and proliferated actively in culture. Porcine L-MSCs expressed mesenchymal markers CD29, CD44, CD90 and CD105 and lacked the expression of hematopoietic markers CD34 and CD45. These cells were multipotent and differentiated into adipocytes, osteocytes and epithelial cells. Like human MSCs, L-MSCs possessed immunoregulatory properties and inhibited proliferation of T cells and interferon-γ and tumor necrosis factor-α production by T cells and dendritic cells, respectively, and increased the production of T-helper 2 cytokines interleukin (IL)-4 and IL-13 by T cells. L-MSCs induced the production of prostaglandin E2 (PGE2) in MSC-T cell co-cultures and inhibition of PGE2 significantly restored (not completely) the immune modulatory effects of L-MSCs. CONCLUSIONS: Here, we demonstrate that MSCs can be isolated from porcine lung and that these cells, similar to human lung MSCs, possess in vitro proliferation, differentiation and immunomodulatory functions. Thus, these cells may serve as a model system to evaluate the contribution of lung MSCs in modulating the immune response, interactions with resident epithelial cells and tissue repair in a pig model of human lung diseases.

Mesenchymal stromal cells inhibit murine syngeneic anti-tumor immune responses by attenuating inflammation and reorganizing the tumor microenvironment.

The potential of mesenchymal stromal cells (MSCs) to inhibit anti-tumor immunity is becoming increasingly well recognized, but the precise steps affected by these cells during the development of an anti-tumor immune response remain incompletely understood. Here, we examined how MSCs affect the steps required to mount an effective anti-tumor immune response following administration of adenovirus Fas ligand (Ad-FasL) in the Lewis lung carcinoma (LL3) model. Administration of bone marrow-derived MSCs with LL3 cells accelerated tumor growth significantly. MSCs inhibited the inflammation induced by Ad-FasL in the primary tumors, precluding their rejection; MSCs also reduced the consequent expansion of tumor-specific T cells in the treated hosts. When immune T cells were transferred to adoptive recipients, MSCs impaired, but did not completely abrogate the ability of these T cells to promote elimination of secondary tumors. This impairment was associated with a modest reduction in tumor-infiltrating T cells, with a significant reduction in tumor-infiltrating macrophages, and with a reorganization of the stromal environment. Our data indicate that MSCs in the tumor environment reduce the efficacy of immunotherapy by creating a functional and anatomic barrier that impairs inflammation, T cell priming and expansion, and T cell function-including recruitment of effector cells.

Inconsistency and features of single nucleotide variants detected in whole exome sequencing versus transcriptome sequencing: A case study in lung cancer.

Whole exome sequencing (WES) and RNA sequencing (RNA-Seq) are two main platforms used for next-generation sequencing (NGS). While WES is primarily for DNA variant discovery and RNA-Seq is mainly for measurement of gene expression, both can be used for detection of genetic variants, especially single nucleotide variants (SNVs). How consistently variants can be detected from WES and RNA-Seq has not been systematically evaluated. In this study, we examined the technical and biological inconsistencies in SNV detection using WES and RNA-Seq data from 27 pairs of tumor and matched normal samples. We analyzed SNVs in three categories: WES unique - those only detected in WES, RNA-Seq unique - those only detected in RNA-Seq, and shared - those detected in both. We found a small overlap (average ∼14%) between the SNVs called in WES and RNA-Seq. The WES unique SNVs were mainly due to low coverage, low expression, or their location on the non-transcribed strand in RNA-Seq data, while the RNA-Seq unique SNVs were primarily due to their location out of the WES-capture boundary regions (accounting ∼71%), as well as low coverage of the regions, low coverage of the mutant alleles or RNA-editing. The shared SNVs had high locus-specific coverage in both WES and RNA-Seq and high gene expression levels. Additionally, WES unique and RNA-Seq unique SNVs showed different nucleotide substitution patterns, e.g., ∼55% of RNA-Seq unique variants were A:T→G:C, a hallmark of RNA editing. This study provides an important evaluation on the inconsistencies of somatic SNVs called in WES and RNA-Seq data.

A chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells.

Three-dimensional (3D) cell culture platforms are increasingly utilized due to their ability to more closely mimic the in vivo microenvironment compared to traditional two-dimensional methods. Limitations of currently available 3D materials include lack of cell attachment, long polymerization times, and inclusion of undefined xenobiotics, and cytotoxic cross-linkers. Evaluated here is a unique hydrogel comprised of polyelectrolytic complex (PEC) fibers formed by hyaluronic acid and chitosan (CT). When hydrated with fetal bovine serum containing human mesenchymal stem/stromal cells (hMSCs), a hydrogel with an elastic modulus of 264±38 Pa formed in seconds with cells distributed throughout the matrix. Scanning electron microscopy showed a lattice-like meshwork of PEC fibers forming irregular compartments. hMSCs showed 48% viability during the first 24 h, with cell populations thereafter reaching a steady state for 14 days. hMSCs in the matrix were induced to differentiate to chondrogenic, osteogenic, and adipogenic phenotypes. Emergent features, at days 56 and 70, consisted of chondrogenesis on the surface of hydrogels induced to osteogenic and adipogenic phenotypes. Results indicate that this matrix may be useful for tissue engineering and disease modeling applications.

Stimulation with Concanavalin-A Induces IL-17 Production by Canine Peripheral T Cells.

The characteristics of canine IL-17-producing cells are incompletely understood. Expression of mRNA encoding orthologs of IL-17 and the IL-17 receptor has been documented in tissues from dogs with arthritis, inflammatory bowel disease, and lymphoma; however, no associations have been found between IL-17 gene expression and disease phenotype in these conditions. Robust assessment of the role of IL-17-producing cells in dogs will require measuring the frequency of these cells in health and disease in balance with other lymphocyte subsets. The aim of this study was to confirm that the T-cell IL-17 response in dogs is evolutionarily conserved. Canine peripheral blood mononuclear cells were stimulated with Concanavalin A with or without polarizing cytokines. We used a canine specific IL-17 ELISA and flow cytometry to identify IL-17-producing T cells. Accumulation of intracellular IL-17 was observed in stimulated CD4 and CD8 T cells. The addition of pro-inflammatory cytokines appeared to enhance polarization of canine CD4 T cells to the Th17 phenotype. Conversely, the addition of IL-2 in the presence of TGF-ß resulted in expansion of Treg cells. We conclude that canine IL-17-producing cells behave similarly to those from humans and mice when stimulated with mitogens and polarized with pro-inflammatory or immune regulatory cytokines.

Nano-engineered mesenchymal stem cells as targeted therapeutic carriers.

Poor availability in deep-seated solid tumors is a significant challenge that limits the effectiveness of currently used anticancer drugs. Approaches that can specifically enhance drug delivery to the tumor tissue can potentially improve therapeutic efficacy. In our current studies, we used nano-engineered mesenchymal stem cells (nano-engineered MSCs) as tumor-targeted therapeutic carriers. In addition to their exquisite tumor homing capabilities, MSCs overexpress efflux transporters such as P-glycoprotein and are highly drug resistant. The inherent tumor-tropic and drug-resistant properties make MSCs ideal carriers for toxic payload. Nano-engineered MSCs were prepared by treating human MSCs with drug-loaded polymeric nanoparticles. Incorporating nanoparticles in MSCs did not affect their viability, differentiation or migration potential. Nano-engineered MSCs induced dose-dependent cytotoxicity in A549 lung adenocarcinoma cells and MA148 ovarian cancer cells in vitro. An orthotopic A549 lung tumor model was used to monitor the in vivo distribution of nanoengineered MSCs. Intravenous injection of nanoparticles resulted in non-specific biodistribution, with significant accumulation in the liver and spleen while nano-engineered MSCs demonstrated selective accumulation and retention in lung tumors. These studies demonstrate the feasibility of developing nano-engineered MSCs loaded with high concentration of anticancer agents without affecting their tumor-targeting or drug resistance properties.

Proteomic analysis of highly prevalent amyloid A amyloidosis endemic to endangered island foxes.

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.

Abnormal infant islet morphology precedes insulin resistance in PCOS-like monkeys.

Polycystic ovary syndrome (PCOS) is prevalent in reproductive-aged women and confounded by metabolic morbidities, including insulin resistance and type 2 diabetes. Although the etiology of PCOS is undefined, contribution of prenatal androgen (PA) exposure has been proposed in a rhesus monkey model as premenopausal PA female adults have PCOS-like phenotypes in addition to insulin resistance and decreased glucose tolerance. PA female infants exhibit relative hyperinsulinemia, suggesting prenatal sequelae of androgen excess on glucose metabolism and an antecedent to future metabolic disease. We assessed consequences of PA exposure on pancreatic islet morphology to identify evidence of programming on islet development. Islet counts and size were quantified and correlated with data from intravenous glucose tolerance tests (ivGTT) obtained from dams and their offspring. Average islet size was decreased in PA female infants along with corresponding increases in islet number, while islet fractional area was preserved. Infants also demonstrated an increase in both the proliferation marker Ki67 within islets and the beta to alpha cell ratio suggestive of enhanced beta cell expansion. PA adult females have reduced proportion of small islets without changes in proliferative or apoptotic markers, or in beta to alpha cell ratios. Together, these data suggest in utero androgen excess combined with mild maternal glucose intolerance alter infant and adult islet morphology, implicating deviant islet development. Marked infant, but subtle adult, morphological differences provide evidence of islet post-natal plasticity in adapting to changing physiologic demands: from insulin sensitivity and relative hypersecretion to insulin resistance and diminished insulin response to glucose in the mature PCOS-like phenotype.