Timothy D. O’Brien

Molecular Profiling Reveals Prognostically Significant Subtypes of Canine Lymphoma

We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.

Comparison of exendin‐4 on beta‐cell replication in mouse and human islet grafts

Exendin‐4 can stimulate β‐cell replication in mice. Whether it can stimulate β‐cell replication in human islet grafts remains unknown. Therefore, we compared the effects of exendin‐4 on β‐cell replication in mouse and human islet grafts. Islets, isolated from mouse and human donors at different ages, were transplanted into diabetic mice and/or diabetic nude mice that were given bromodeoxyuridine (BrdU) with or without exendin‐4. At 4 weeks post‐transplantation, islet grafts were removed for insulin and BrdU staining and quantification of insulin+/BrdU+ cells. Although diabetes was reversed in all mice transplanting syngeneic mouse islets from young or old donors, normoglycemia was achieved significantly faster in exendin‐4 treated mice. Mouse islet grafts in exendin‐4 treated mice had significantly more insulin+/BrdU+β cells than in untreated mice (P < 0.01). Human islet grafts from ≤22‐year‐old donors had more insulin+/BrdU+β cells in exendin‐4 treated mice than that in untreated mice (P < 0.01). However, human islet grafts from ≥35‐year‐old donors contained few insulin+/BrdU+β cells in exendin‐4 treated or untreated mice. Our data demonstrated that the capacity for β‐cell replication in mouse and human islet grafts is different with and without exendin‐4 treatment and indicated that GLP‐1 agonists can stimulate β‐cell replication in human islets from young donors.

Stimulating beta cell replication and improving islet graft function by GPR119 agonists

G protein‐coupled receptor 119 (GPR119) is predominantly expressed in β cells and intestinal L cells. In this study, we investigated whether oleoylethanolamide (OEA), a GPR119 endogenous ligand, and PSN632408, a GPR119 synthetic agonist, can stimulate β‐cell replication in vitro and in vivo and improve islet graft function in diabetic mice. We found that OEA and PSN632408 significantly increased numbers of insulin+/5‐bromo‐2′‐deoxyuridine (BrdU)+β cells in cultured mouse islets in a dose‐dependent manner. All diabetic recipient mice, given marginal syngeneic islet transplants with OEA or PSN632408 or vehicle, achieved normoglycemia at 4 weeks after transplantation. However, normoglycemia was achieved significantly faster in OEA‐ or PSN632408‐treated diabetic mice than in vehicle‐treated diabetic mice (P < 0.05). The percentage of insulin+/BrdU+β cells in islet grafts in OEA‐ and PSN632408‐treated mice was significantly higher than in vehicle‐treated mice (P < 0.01). Our data demonstrated that OEA and PSN632408 can stimulate β‐cell replication in vitro and in vivo and improve islet graft function. Targeting GPR119 is a novel therapeutic approach to increase β‐cell mass and to improve islet graft function by stimulating β‐cell replication.

Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium

Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10–14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube‐like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse‐transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Relationship of Nutritional Factors to the Cause, Dissolution, and Prevention of Feline Uroliths and Urethral Plugs

Feline lower urinary tract disease is often associated with uroliths and urethral plugs. Uroliths and urethral plugs are composed of variable quantities of matrix and a variety of minerals (including struvite, calcium oxalate, ammonium urate, calcium phosphate, and cystine). Knowledge of nutritional factors associated with the pathophysiology of uroliths and urethral plugs facilitates the modification of diets for the dissolution of several minerals contained within them.

Urologic disorders of immature cats.

Numerous inherited and congenital disorders may affect the urinary system of cats. Those associated with clinical signs include renal dysplasia, renal hypoplasia, polycystic kidneys, ectopic ureters, urachal anomalies, and fistulas between the lower urinary tract and other structures. Acquired disorders of the urinary system of immature cats have been uncommonly recognized. Further studies are warranted to better define their prevalence in immature cats.

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.

Dietary Management of Canine and Feline Chronic Renal Failure

Nutritional therapy is the mainstay of management of chronic renal failure in dogs and cats. Diets designed for use in renal failure are typically reduced in protein, phosphorus, and sodium content. These and other dietary modifications are designed to prevent or ameliorate clinical signs of uremia, minimize disturbances associated with excesses or losses of electrolytes and minerals, arrest or retard progression of renal failure, and maintain adequate nutrition.

Etiopathogenesis and Biological Behavior of Feline Vesicourachal Diverticula

Asymptomatic microscopic urachal remnants located at the vertex of the urinary bladder are common in cats. Because they reduce the tensile strength of the bladder wall at this location, they represent risk factors for development of acquired macroscopic diverticula in cats with lower urinary tract disorders. Elimination of the underlying cause of lower urinary tract disease is frequently associated with resolution of acquired macroscopic vesicourachal diverticula.

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.