The Impact of the Global Pandemic on Veterans with Serious Mental Illness (SMI): Healthcare Utilization and Mortality.

BACKGROUND: Individuals with serious mental illness (SMI) experience barriers to accessing and engaging with healthcare, which may have been exacerbated during the emergence of the global pandemic and the rapid shift to telemedicine platforms, substantially decreasing healthcare utilization for non-COVID-19 disorders. Important repercussions on morbidity and mortality may be seen in the months and years to come, which may disproportionately affect high-risk populations, such as patients with SMI, with reduced access to technology platforms. In this study, we explored the impact of the pandemic on healthcare utilization and all-cause mortality rate in SMI compared to non-SMI individuals for the months of March-September 2020 and the same two quarters in 2019. METHODS: Data were obtained from the VA Corporate Data Warehouse (CDW), a data repository from clinical and administrative VA systems. The sample included veterans with ≥1 outpatient clinical encounter nationally between 1 January 2019 and 31 December 2020. RESULTS: The cohort for this study included 1,018,047 veterans receiving care through the Veterans Health Administration between 2019 and 2020. Of those, 339,349 had a diagnosis of SMI. Patients with SMI had a significantly larger pre-post-pandemic decrease in outpatient (49.7%, p < 0.001), inpatient (14.4%, p < 0.001), and ED (14.5%, p < 0.001) visits compared to non-SMI patients. Overall, 3752 (1.59%) veterans without SMI and 4562 (1.93%) veterans with SMI died during our observation period. Veterans without SMI who died during the observation period were more likely to have had a positive COVID-19 test compared to veterans with SMI. Unadjusted analyses showed that veterans with SMI were approximately 2.5 times more likely to die than veterans without SMI during the first 6 months of the pandemic, compared to the same two quarters of the previous year. However, after adjustment by pertinent covariates, the predictors associated with an increased risk of death from SMI were older age, being male, a higher CAN score, more inpatient stays in the pre period compared to post, and a positive COVID-19 test. DISCUSSION: Consistent with our initial hypothesis, all the indices of healthcare utilization, namely the number of outpatient, inpatient, and ED visits, significantly decreased between pre- and post-pandemic and did more so for veterans with SMI, despite having more chronic medical illnesses and being prescribed more medications than veterans without SMI. On the other hand, while mortality was greater post-pandemic, factors such as age, morbidity, and having a positive COVID-19 test predicted mortality above and beyond having an SMI diagnosis.

Clinical efficacy of prebiotics and glycosaminoglycans versus placebo In dogs with food responsive enteropathy receiving a hydrolyzed diet: A pilot study.

Induction of remission is easily achieved with dietary treatment in dogs diagnosed with Food Responsive Chronic Diarrhea (FRD). Administration of prebiotics and glycosaminoglycans (GAGs) may improve epithelial cell integrity and therefore be useful as adjunct treatment. This study evaluated whether the relapse rate of FRD dogs that are switched back to a normal diet can be influenced using supplemental treatment with prebiotics and GAGs. A randomized, controlled clinical trial (RCCT) was performed in dogs diagnosed with FRD. Dogs were diagnosed based on clinical exclusion diagnosis, endoscopic biopsies showing predominantly lymphoplasmacytic infiltration, and response to dietary treatment. Dogs were randomized to be fed a combination of prebiotics and GAGs (group 1) or placebo (group 2) in addition to a hydrolyzed diet. At week 10, a second endoscopy was performed and dogs were switched back to normal diet. Relapse rate was monitored every 2 weeks after that until week 18. Statistical analysis was performed for each outcome (Canine Chronic Enteropathy Clinical Activity Index (CCECAI), clinicopathological data, endoscopic scoring, mWSAVA histological scoring index (mWSAVA), and number of relapses following switch to normal diet) using a linear mixed effects model for group comparison. Time, group, and their interactions were included as a fixed effect, whereas each dog was treated as a random effect. Of the 35 dogs enrolled into the clinical trial, 10 in each group reached the point of second endoscopy. A total of 13 dogs (n = 8 in group 1 and n = 5 in group 2) reached the trial endpoint of 18 weeks. After switching back to normal diet, none of the dogs in either group relapsed. No significant differences were found over time or between groups for CCECAI, endoscopy scoring and histological scoring. Although there was a clinical worsening in the placebo group after switching back to the original diet, this was not statistically significant (CCECAI p = 0.58). Post-hoc power calculation revealed that 63 dogs per group would have been needed to detect statistically significant differences in CIBDAI between treatment groups. Standard dietary treatment induced rapid clinical response in all cases, however, additional supplementation with prebiotics and GAGs did not significantly improve clinical outcome within 4 months after switching back to normal diet. Since there are very few RCCT published in CE in dogs, this pilot study provides important power analyses for planning of further studies.

A Multi-Institutional Retrospective Analysis of Toceranib Phosphate for Presumed or Confirmed Canine Aortic Body Chemodectomas.

Aortic body tumors, specifically chemodectomas, are the second most common type of canine cardiac tumor; however, information about treatment is currently lacking. This study included dogs with a presumptive or definitive diagnosis of an aortic body chemodectoma that underwent treatment with toceranib phosphate. Cases were solicited via the American College of Veterinary Internal Medicine Cardiology, Internal Medicine, and Oncology listservs using an electronic survey. Cox multivariate analysis of factors potentially impacting survival time was completed. Twenty-seven (27) cases were included in analysis. The clinical benefit rate (complete remission, partial remission, or stable disease >10 weeks) was 89%. A median survival time of 478 days was found for those receiving toceranib alone (n = 14), which was not statistically different from those treated with additional modalities (521 days). No factors evaluated statistically impacted outcome. Further, prospective studies are warranted to evaluate the use of toceranib for the treatment of canine aortic body chemodectomas.

Non-Linear Mixed-Effects Pharmacokinetic Modeling of the Novel COX-2 Selective Inhibitor Vitacoxib in Cats.

The objective of this study was to develop a non-linear mixed-effects (NLME) model to describe the disposition kinetics of vitacoxib in cats following intravenous (I.V) and oral (P.O) (single and multiple) dosing. Data from six consecutive studies with 16 healthy neutered domestic short hair cats were pooled together to build a pharmacokinetic (PK) model using NLME. Population PK parameters were estimated using the stochastic approximation expectation maximization (SAEM) algorithm implemented in Monolix 2019R2. A two-compartment mammillary disposition model with simultaneous zero- and first-order absorption best described the PK of vitacoxib in plasma after oral dosing. The systemic CL of vitacoxib was found to be low (110 ml/h), with a steady-state volume of distribution (VSS) of 3.42 L in cats. Results from the automated covariate search in Monolix 2019R2 showed that bodyweight had a significant effect on the central volume of distribution of vitacoxib. Lastly, using Monte Carlo simulations, we investigated the time course of several dosages of vitacoxib from 0.01 to 8 mg/kg. Using this simulation set, we found a range of reasonable dosages that produce therapeutic plasma concentrations of vitacoxib for 24 h or more in cats.

Evaluation of a Pasteurella multocida Respiratory Disease Induction Model for Goats (Capra aegagrus hircus).

Infectious respiratory diseases are a serious health concern worldwide. However, few models describe the experimental induction of lung infection, or the effect of experimental infection on clinical pathologic parameters in goats. Goats offer benefits compared to cattle because of size and tractability and compared to sheep with regard to specific features of their anatomy. In previous experimental models of infection in goats, coadministration of an immunosuppressive dose of a corticosteroid is common; however, protocols that use corticosteroid often note mortality as an adverse effect. We therefore investigated an infection protocol that did not use immunosuppression but instead relied on 2 intratracheal inoculations of Pasteurella multocida in healthy meat goats to induce clinical and hematologic changes associated with respiratory infection. Healthy Boer or Boer-Kiko cross goats (n = 6; age, 10 mo) were inoculated with Pasteurella multocida and were monitored over a 312-h period for clinical and hematologic parameters of infection. After induction of pneumonia, the goats had a significant 1.2 °C rise in rectal temperature and auscultatable rales for up to 96 h. Lymphocyte counts, serum amyloid A values, and respiratory scores were significantly different before and after induction of disease and were consistent with respiratory infection. No mortality was associated with this experimental infection, and minimal gross pathologic changes were noted at study termination. The clinical and pathologic findings of this study suggest a potentially reproducible method of establishing clinical respiratory infection in goats. The repeated intratracheal inoculation method of inducing caprine respiratory disease can be used to produce experimental respiratory disease in goats when the use of corticosteroid is not desirable. With the feasibility of this method established, additional research evaluating the optimal dose and frequency of P. multocida administration is needed.

Treatment With Hydrolyzed Diet Supplemented With Prebiotics and Glycosaminoglycans Alters Lipid Metabolism in Canine Inflammatory Bowel Disease.

Canine inflammatory bowel disease (IBD) is a chronic, immunologically mediated intestinal disorder, resulting from the complex interaction of genetic, environmental and immune factors. Hydrolyzed diets are used in dogs with food-responsive diarrhea (FRD) to reduce adverse responses to immunostimulatory proteins. Prebiotics (PRBs) and glycosaminoglycans (GAGs) have previously been demonstrated to show anti-inflammatory activity in the intestinal mucosa. Notably, hydrolyzed diets combined with the administration of PRBs and GAGs offer a promising approach for the treatment of canine IBD. Our aim was to investigate the effects of hydrolyzed diet and GAG+PRB co-treatment on the serum metabolomic profile of IBD dogs. Dogs with IBD randomly received either hydrolyzed diet supplemented with GAGs and PRBs (treatment 1) or hydrolyzed diet alone (treatment 2) for 10 weeks. A targeted metabolomics approach using mass spectrometry was performed to quantify changes in the serum metabolome before and after treatment and between treatment 1 and 2. Principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA) and univariate statistics were used to identify differences between the treatment groups. PCA, PLS-DA, and HCA showed a clear clustering of IBD dogs before and after hydrolyzed diet, indicating that the treatment impacted the serum metabolome. Univariate analysis revealed that most of the altered metabolites were involved in lipid metabolism. The most impacted lipid classes were components of cell membranes, including glycerophospholipids, sphingolipids, and di- and triglycerides. In addition, changes in serum metabolites after GAG+PRB co-treatment suggested a possible additional beneficial effect on the lipid metabolism in IBD dogs. In conclusion, the present study showed a significant increase in metabolites that protect gut cell membrane integrity in response to hydrolyzed diet alone or in combination with GAG+PRB co-treatment. Administration of such treatment over 70 days improved selected serum biomarkers of canine IBD, possibly indicating improved intestinal membrane integrity.

Evaluation of dose-response effects of short-term oral prednisone administration on clinicopathologic and hemodynamic variables in healthy dogs.

OBJECTIVE: To determine whether a dose-response relationship exists between short-term oral prednisone administration and common clinicopathologic variables, cardiovascular biomarkers, and systolic arterial blood pressure (SAP) in healthy dogs. ANIMALS: 8 healthy Beagles. PROCEDURES: Dogs underwent five 5-day experiments (no prednisone treatment [control condition] and prednisone administration at 0.5, 1, 2, and 4 mg/kg, PO, q 24 h), with a 9-day washout period between protocols. Analyses performed before and after treatments included a CBC, serum biochemical analysis, and determination of SAP, fractional excretion of electrolytes, urine protein-to-creatinine ratio, glomerular filtration rate (GFR), serum N-terminal pro B-type natriuretic peptide (NT-proBNP) and plasma cortisol concentrations, and plasma renin activity. Linear mixed-effects modeling was used to compare changes in variables from baseline (day 1 for the same experiment) among treatment conditions. RESULTS: Changes in serum glucose concentration and GFR were significantly greater after administration of prednisone at 4 mg/kg than for the control condition. Fractional excretion of sodium was decreased from baseline when dogs received 0.5, 1, or 4 mg of prednisone/kg, compared with results for the control condition. Several expected changes in clinicopathologic values were observed after prednisone administration at any dose. Changes in serum NT-proBNP concentration, plasma renin activity, and SAP did not differ from changes for the control condition at any prednisone dose. CONCLUSIONS AND CLINICAL RELEVANCE: Oral prednisone administration did not affect SAP, NT-proBNP concentration, or measures of renin-angiotensin-aldosterone system activation in healthy laboratory-housed dogs but was associated with relative increases in GFR and serum glucose concentration.

Glucocorticoid and dietary effects on mucosal microbiota in canine inflammatory bowel disease.

The pathogenesis of canine inflammatory bowel disease (IBD) involves complex interactions between mucosal immunity and the intestinal microbiota. Glucocorticoids are commonly administered to reduce mucosal inflammation and gastrointestinal signs. The study objective was to evaluate the effects of diet and oral prednisone on the spatial distribution of mucosal bacteria in IBD dogs. Eight dogs diagnosed with IBD were treated with immunosuppressive doses of prednisone. The mucosal microbiota from endoscopic biopsies of IBD dogs and healthy controls (HC; n = 15 dogs) was evaluated by fluorescence in situ hybridization (FISH) targeting the 16S rRNA genes of total bacteria and bacterial species relevant in canine/human IBD. Apicaljunction protein (AJP) expression using immunohistochemistry investigated the effect of medical therapy on intestinal barrier integrity. All IBD dogs had a reduction in GI signs following diet and prednisone therapy compared with baseline CIBDAI scores (P < 0.05). The mucosal microbiota of HC and diseased dogs was most abundant in free and adherent mucus. Only Lactobacilli were increased (P < 0.05) in the adherent mucus of IBD dogs compared to HC. The spatial distribution of mucosal bacteria was significantly different (P < 0.05) in IBD dogs following prednisone therapy, with higher numbers of Bifidobacteria and Streptococci detected across all mucosal compartments and increased numbers of Bifidobacterium spp., Faecalibacterium spp., and Streptococcus spp. present within adherent mucus. Differences in intestinal AJPs were detected with expression of occludin increased (P < 0.05) in IBD dogs versus HC. The expressions of occludin and E-cadherin were increased but zonulin decreased (P < 0.05 for each) in IBD dogs following prednisone therapy. In conclusion, the spatial distribution of mucosal bacteria differs between IBD and HC dogs, and in response to diet and glucocorticoid administration. Medical therapy was associated with beneficial changes in microbial community structure and enhanced mucosal epithelial AJP expression.

In vitro Cytotoxicity and Pharmacokinetic Evaluation of Pharmacological Ascorbate in Dogs.

Background: High-dose, pharmacological ascorbate (P-AscH-) is preferentially cytotoxic to human cancer cells in vitro. Investigations on the efficacy of P-AscH- as an adjuvant treatment for multiple human cancers are on-going, but has yet to be formally investigated in dogs. The primary objectives of this study were to determine the pharmacokinetic (PK) profile of P-AscH- in healthy Beagle dogs and the effects of P-AscH- on canine osteosarcoma cells in vitro. Methods: Eight purpose-bred, healthy, spayed female Beagle dogs, between 20 and 21 months old, and 8-10 kg were administered two doses of P-AscH- (550 or 2,200 mg/kg) via intravenous infusion over 6 h, on separate days. Plasma ascorbate concentrations were measured at 12 time points during and after infusion for PK analysis using nonlinear mixed-effects (NLME) and non-compartmental analysis (NCA). Clonogenic assays were performed on 2 canine osteosarcoma cell lines (D-17 and OSCA-8) and canine primary dermal fibroblasts after exposure to high concentrations of ascorbate (75 pmoles/cell). Results: Plasma ascorbate levels in the dogs peaked at approximately 10 mM following 2,200 mg/kg and returned to baseline 6-8 h after dosing. Minor adverse effects were seen in two dogs. Ascorbate (75 pmoles/cell) significantly decreased survival in both the osteosarcoma cell lines (D-17 63% SD 0.010, P = 0.005; OSCA-8 50% SD 0.086, P = 0.026), compared to normal fibroblasts (27% SD 0.056). Conclusions: Pharmacological ascorbate is preferentially cytotoxic to canine-derived cancer cells. High levels of ascorbate can be safely administered to dogs. Further studies are needed to determine the effects of P-AscH- on canine patients.

Nonlinear mixed-effects pharmacokinetic modeling of the novel COX-2 selective inhibitor vitacoxib in dogs.

The objective of this study was to develop a nonlinear mixed-effects model of vitacoxib disposition kinetics in dogs after intravenous (I.V.), oral (P.O.), and subcutaneous (S.C.) dosing. Data were pooled from four consecutive pharmacokinetic studies in which vitacoxib was administered in various dosing regimens to 14 healthy beagle dogs. Plasma concentration versus time data were fitted simultaneously using the stochastic approximation expectation maximization (SAEM) algorithm for nonlinear mixed-effects as implemented in Monolix version 2018R2. Correlations between random effects and significance of covariates on population parameter estimates were evaluated using multiple samples from the posterior distribution of the random effects. A two-compartment mamillary model with first-order elimination and first-order absorption after P.O. and S.C. administration, best described the available pharmacokinetic data. Final parameter estimates indicate that vitacoxib has a low-to-moderate systemic clearance (0.35 L hr-1  kg-1 ) associated with a low global extraction ratio, but a large volume of distribution (6.43 L/kg). The absolute bioavailability after P.O. and S.C. administration was estimated at 10.5% (fasted) and 54.6%, respectively. Food intake was found to increase vitacoxib oral bioavailability by a fivefold, while bodyweight (BW) had a significant impact on systemic clearance, thereby confirming the need for BW adjustment with vitacoxib dosing in dogs.

Microbiota-Related Changes in Unconjugated Fecal Bile Acids Are Associated With Naturally Occurring, Insulin-Dependent Diabetes Mellitus in Dogs.

Diabetes mellitus (DM) in humans has recently been associated with altered intestinal microbiota. The consequences of intestinal dysbiosis, such as increased intestinal permeability and altered microbial metabolites, are suspected to contribute to the host inflammatory state and peripheral insulin resistance. Human diabetics have been shown to have changes in bile acid (BA) metabolism which may be detrimental to glycemic control. The purpose of this study was to examine BA metabolism in dogs with naturally-occurring, insulin-dependent DM and to relate these findings to changes in the intestinal microbiota. A prospective observational study of adult dogs with a clinical diagnosis of DM (n = 10) and healthy controls (HC, n = 10) was performed. The fecal microbiota were analyzed by 16S rRNA gene next-generation (Illumina) sequencing. Concentrations of fecal unconjugated BA (fUBA) were measured using gas chromatography and mass spectrometry. Analysis of bacterial communities showed no significant difference for any of the alpha-diversity measures between DM vs. HC dogs. Principal coordinate analysis based on unweighted Unifrac distance metric failed to show significant clustering between dog groups (ANOSIMUnweighted: R = 0.084; p = 0.114). However, linear discriminate analysis effects size (LEfSe) detected differentially abundant bacterial taxa (α = 0.01, LDA score >2.0) on various phylogenetic levels. While Enterobacteriaceae was overrepresented in dogs with DM, the proportions of Erysipelotrichia, Mogibacteriaceae, and Anaeroplasmataceae were increased in HC dogs. Dogs with DM had increased concentration of total primary fUBA compared to HC dogs (p = 0.028). The concentrations of cholic acid and the cholic acid percentage of the total fUBA were increased (p = 0.028 and p = 0.035, respectively) in the feces of DM dogs relative to HC dogs. The levels of lithocholic acid (both absolute value and percentage of the total fUBA) were decreased (p = 0.043 and p < 0.01, respectively) in DM dogs vs. HC dogs. Results indicate that dogs with DM have both intestinal dysbiosis and associated fUBA alterations. The pattern of dysbiosis and altered BA composition is similar to that seen in humans with Type 2 DM. The dog represents a novel large animal model for advancing translational medicine research efforts (e.g., investigating pathogenesis and therapeutics) in DM affecting humans.

Clinicopathologic, hemodynamic, and echocardiographic effects of short-term oral administration of anti-inflammatory doses of prednisolone to systemically normal cats.

OBJECTIVE: To evaluate the clinicopathologic, hemodynamic, and echocardiographic effects of short-term administration of anti-inflammatory dosages of prednisolone to systemically normal cats. ANIMALS: 10 cats with allergic dermatitis and 10 healthy control cats. PROCEDURES: Cats with allergic dermatitis were randomly allocated to 2 groups and received 2 dosages of prednisolone (1 and 2 mg/kg/d, PO, for 7 days) in a crossover design followed by 9-day tapering and 14-day washout periods. Each prednisolone-treated cat was matched to a healthy control cat on the basis of sex, neuter status, age (± 1 year), and body weight (± 10%). Control cats received no treatment during the 35-day observation period. Clinicopathologic, echocardiographic, and hemodynamic variables were measured at baseline (day 0) and predetermined times during and after prednisolone administration and compared within and between the 2 treatment groups. RESULTS: Prednisolone-treated cats had expected clinicopathologic alterations (mild increases in neutrophil and monocyte counts and serum concentrations of albumin, cholesterol, and triglycerides) but systolic arterial blood pressure; blood glucose, serum potassium, and cardiac biomarker concentrations; urinary sodium excretion; and echocardiographic variables did not differ significantly from baseline at any time. Statistically significant, albeit clinically irrelevant, increases in blood glucose and N-terminal pro-B-type natriuretic peptide concentrations were observed between baseline and the prednisolone pharmacokinetic steady state (7 days after initiation) only when the 2-mg/kg dosage was administered. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated short-term oral administration of anti-inflammatory dosages of prednisolone did not cause relevant hemodynamic, echocardiographic, or diabetogenic effects in systemically normal cats with allergic dermatitis.

Histamine-Induced Conjunctivitis and Breakdown of Blood-Tear Barrier in Dogs: A Model for Ocular Pharmacology and Therapeutics.

Conjunctival inflammation disturbs the blood-tear barrier and thus affects the tear film stability and composition. We aimed to develop a non-invasive and reliable method to induce conjunctivitis in dogs, a large animal model for translational work on ocular surface disease in humans. Six beagle dogs underwent a randomized, vehicle-controlled, balanced crossover trial-on six separate days, one eye received topical artificial tears (vehicle), while the other eye received one of six concentrations of histamine solution (0.005-500 mg/ml). At sequential times after eyedrop administration, a conjunctivitis score was given to each eye based on the degree of palpebral and bulbar conjunctival hyperemia and chemosis, ocular pruritus, and discharge. Total protein content (TPC) and serum albumin were quantified in tear fluid at baseline and 20 min. Additionally, 13 dogs presenting for various ophthalmic diseases with associated conjunctivitis were examined. Experimentally induced conjunctivitis developed rapidly (<1 min) following topical histamine administration and lasted for 1-3 h (four lowest doses) to 6-8 h (two highest doses). The severity of conjunctivitis was dose-dependent. Histamine was overall well tolerated, although transient blepharitis, aqueous flare, and ocular hypertension occurred in a few dogs receiving histamine ≥375 mg/ml. TPC and serum albumin levels increased in tears of eyes receiving histamine ≥1.0 mg/ml, being significantly higher than vehicle and baseline in eyes receiving histamine ≥375 mg/ml. Lacrimal albumin levels were also increased in 13 dogs with naturally acquired conjunctivitis, up 2.7-14.9 fold compared to contralateral healthy eyes. Histamine-induced conjunctivitis represents a robust model for translational work on the ocular surface given the low cost, non-invasiveness, self-resolving nature, ability to adjust the duration and severity of the disease, and shared features with naturally occurring ocular diseases. Histamine solutions of 1, 10, and 375 mg/ml induce mild, moderate, and severe conjunctivitis in dogs, respectively. Leakage of serum albumin in tear fluid of eyes with conjunctivitis suggests a breakdown of the blood-tear barrier.

Interleukin-13 and interleukin-33 mRNA are underexpressed in the duodenal mucosa of German Shepherd dogs with chronic enteropathy.

BACKGROUND: A recent genome-wide association study in German Shepherd dogs (GSDs) with chronic enteropathy (CE) has identified polymorphisms in the Th2 cytokine genes. HYPOTHESIS/OBJECTIVE: To determine if the expression of the Th2 cytokines, interleukin-13 (IL-13) and interleukin-33 (IL-33), is altered in the duodenal mucosa of GSDs with CE compared to non-GSDs with CE and healthy dogs. ANIMALS: Twenty client-owned dogs diagnosed with CE (10 GSDs and 10 non-GSDs) at the Bristol Veterinary School and 8 healthy Beagle dogs from the Iowa State University Service Colony. METHODS: Retrospective study using archived paraffin-embedded duodenal biopsy samples. A novel RNA in situ hybridization technology (RNAscope) was used to hybridize IL-13 and IL-33 mRNA probes onto at least 10 sections from duodenal biopsy samples for each dog. RNAscope signals were visualized using a microscope and semi-quantitative assessment was performed by a single operator. RESULTS: Based on duodenal villus, subvillus, epithelial, and lamina propria average expression scores, GSDs with CE had significantly lower IL-13 and IL-33 mRNA expression compared to non-GSDs with CE (IL-13, P < .04; IL-33, P < .02) and healthy Beagle dogs (IL-13, P < .02; IL-33, P < .004). CONCLUSIONS AND CLINICAL IMPORTANCE: Similar to human patients with ulcerative colitis, a subtype of human inflammatory bowel disease, these data indicate that Th2 cytokines may be involved in the pathogenesis of CE in GSDs.

Indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-1) mRNA is over-expressed in the duodenal mucosa and is negatively correlated with serum tryptophan concentrations in dogs with protein-losing enteropathy.

INTRODUCTION: Dogs with protein-losing enteropathy (PLE) have decreased serum tryptophan concentrations, which may contribute to disease pathogenesis. Indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-1) expression is associated with low serum tryptophan concentrations and is increased in the gastrointestinal tract of humans with inflammatory bowel disease (IBD). Therefore, the objective of our study was to determine if the mRNA expression of IDO-1 is increased in the duodenal mucosa of dogs with PLE as compared to dogs with chronic enteropathy (CE) and healthy dogs, and whether this expression is correlated with changes in serum tryptophan concentration. METHODS: Our study was a retrospective study using archived paraffin-embedded duodenal biopsy specimens from 8 healthy Beagle dogs from the Iowa State University Canine Service Colony and 18 and 6 client-owned dogs diagnosed with CE and PLE, respectively at the Bristol Veterinary School. A novel RNA in situ hybridization (ISH) technology, RNAscope, was used to identify IDO-1 mRNA mucosal expression in duodenal tissues. An IDO-1 specific probe was hybridized onto 10 duodenal biopsy sections from each dog whereby RNAscope signal (mRNA expression) was quantified by a single operator using light microscopy. RESULTS: Dogs with PLE had significantly higher mRNA expression of IDO-1 in the duodenal mucosa compared to healthy dogs (mucosal percentage IDO-1 positive: P = 0.0093, (mean ± S.D) control: 19.36 ± 7.08, PLE: 34.12 ± 5.98, average fold difference: 1.76 and mucosal IDO-1 H-score: P = 0.0356, (mean ± S.D) control: 45.26 ± 19.33, PLE: 84.37 ± 19.86, average fold difference: 1.86). The duodenal mucosal mRNA expression of IDO-1 was negatively correlated with serum tryptophan concentrations in dogs with PLE (mucosal IDO-1 H-score: Spearman's rank correlation coefficient = -0.94, P = 0.0048). CONCLUSIONS: In conclusion, our study suggests that decreased serum tryptophan concentrations in dogs with PLE is associated with increased intestinal IDO-1 expression. Further studies are needed to determine potential inflammatory pathways responsible for increased expression of IDO-1 in the intestinal tract of dogs with PLE.

Aptamer-enabled uptake of small molecule ligands.

The relative ease of isolating aptamers with high specificity for target molecules suggests that molecular recognition may be common in the folds of natural RNAs. We show here that, when expressed in cells, aptamers can increase the intracellular concentrations of their small molecule ligands. We have named these aptamers as DRAGINs (Drug Binding Aptamers for Growing Intracellular Numbers). The DRAGIN property, assessed here by the ability to enhance the toxicity of their ligands, was found for some, but not all, aminoglycoside aptamers. One aptamer protected cells against killing by its ligand. Another aptamer promoted killing as a singlemer and protected against killing as a tandemer. Based on a mathematical model, cell protection vs. killing is proposed as governed by aptamer affinity and access to the inner surface of the cell membrane, with the latter being a critical determinant. With RNA molecules proposed as the earliest functional polymers to drive the evolution of life, we suggest that RNA aptamer-like structures present in primitive cells might have selectively concentrated precursors for polymer synthesis. Riboswitches may be the evolved forms of these ancient aptamer-like "nutrient procurers". Aptamers with DRAGIN capability in the modern world could be applied for imaging cells, in synthetic cell constructs, or to draw drugs into cells to make "undruggable" targets accessible to small molecule inhibitors.

A computational study of alternate SELEX.

Systematic evolution of ligands by exponential enrichment (SELEX) is a procedure for identifying nucleic acid (NA) molecules with affinities for specific target species, such as proteins, peptides, or small organic molecules. Here, we extend the work in Seo et al. (Bull Math Biol 72:1623-1665, 2010) (multiple-target SELEX or positive SELEX) and examine an alternate SELEX process with multiple targets by incorporating negative selection into a positive SELEX protocol. The alternate SELEX process is done iteratively by alternating several positive selection rounds with several negative selection rounds. At the end of each positive selection round, NAs are eluted from the bound product and amplified by polymerase chain reaction (PCR) to increase the size of the pool of NA species that bind preferentially to the given positive target vector. The enriched population of NAs is then exposed to the negative targets (undesired targets). The free NA species (instead of the bound NA species being eluted) are retained and amplified by PCR (negative selection). The goal is to minimize an enrichment of nonspecifically binding NAs against multiple targets. While positive selection alone results in a pool of NAs that bind tightly to a given target vector, negative selection results in the subset of the NAs that bind best to the nontarget vectors that are also present. By alternating the two processes, we eventually obtain a refined population of nucleic acids that bind to the desired target(s) with high "selectivity" and "specificity." In the present paper, we give formulations of the negative and alternate selection processes and define their efficiencies in a meaningful way. We study the asymptotic behavior of alternate SELEX system as a discrete-time dynamical system. To do this, we use the chemical potential to examine how alternate SELEX leads to the selection of NAs with more specific interactions when the ratio of the number of positive selection rounds to the number of negative selection rounds is fixed. Alternate SELEX is said to be globally asymptotically stable if, given the initial target vector and a fixed ratio, the distribution of the limiting NA fractions does not depend on the relative concentrations of the NAs in the initial pool (provided that all of the NA species are initially present in the initial pool). We state conditions on the matrix of NA-target affinities that determine when the alternate SELEX process is globally asymptotically stable in this sense and illustrate these results computationally.

A mathematical analysis of multiple-target SELEX.

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a procedure by which a mixture of nucleic acids can be fractionated with the goal of identifying those with specific biochemical activities. One combines the mixture with a specific target molecule and then separates the target-NA complex from the resulting reactions. The target-NA complex is separated from the unbound NA by mechanical means (such as by filtration), the NA is eluted from the complex, amplified by PCR (polymerase chain reaction), and the process repeated. After several rounds, one should be left with the nucleic acids that best bind to the target. The problem was first formulated mathematically in Irvine et al. (J. Mol. Biol. 222:739-761, 1991). In Levine and Nilsen-Hamilton (Comput. Biol. Chem. 31:11-25, 2007), a mathematical analysis of the process was given. In Vant-Hull et al. (J. Mol. Biol. 278:579-597, 1998), multiple target SELEX was considered. It was assumed that each target has a single nucleic acid binding site that permits occupation by no more than one nucleic acid. Here, we revisit Vant-Hull et al. (J. Mol. Biol. 278:579-597, 1998) using the same assumptions. The iteration scheme is shown to be convergent and a simplified algorithm is given. Our interest here is in the behavior of the multiple target SELEX process as a discrete "time" dynamical system. Our goal is to characterize the limiting states and their dependence on the initial distribution of nucleic acid and target fraction components. (In multiple target SELEX, we vary the target component fractions, but not their concentrations, as fixed and the initial pool of nucleic acids as a variable starting condition). Given N nucleic acids and a target consisting of M subtarget component species, there is an M × N matrix of affinities, the (i,j) entry corresponding to the affinity of the jth nucleic acid for the ith subtarget. We give a structure condition on this matrix that is equivalent to the following statement: For any initial pool of nucleic acids such that all N species are represented, the dynamical system defined by the multiple target SELEX process will converge to a unique subset of nucleic acids, each of whose concentrations depend only upon the total nucleic acid concentration, the initial fractional target distribution (both of which are assumed to be the same from round to round), and the overall limiting association constant. (The overall association constant is the equilibrium constant for the system of MN reactions when viewed as a composite single reaction). This condition is equivalent to the statement that every member of a certain family of chemical potentials at infinite target dilution can have at most one critical point. (The condition replaces the statement for single target SELEX that the dynamical system generated via the process always converges to a pool that contains only the nucleic acid that binds best to the target). This suggests that the effectiveness of multiple target SELEX as a separation procedure may not be as useful as single target SELEX unless the thermodynamic properties of these chemical potentials are well understood.