Tralesinidase Alfa Enzyme Replacement Therapy Prevents Disease Manifestations in a Canine Model of Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects. SIGNIFICANCE STATEMENT: This work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.

Temporospatial Development of Neuropathologic Findings in a Canine Model of Mucopolysaccharidosis IIIB.

Mucopolysaccharidosis (MPS) IIIB is a neuropathic lysosomal storage disease characterized by the deficient activity of a lysosomal enzyme obligate for the degradation of the glycosaminoglycan (GAG) heparan sulfate (HS). The pathogenesis of neurodegeneration in MPS IIIB is incompletely understood. Large animal models are attractive for pathogenesis and therapeutic studies due to their larger size, outbred genetics, longer lifespan, and naturally occurring MPS IIIB disease. However, the temporospatial development of neuropathologic changes has not been reported for canine MPS IIIB. Here we describe lesions in 8 brain regions, cervical spinal cord, and dorsal root ganglion (DRG) in a canine model of MPS IIIB that includes dogs aged from 2 to 26 months of age. Pathological changes in the brain included early microscopic vacuolation of glial cells initially observed at 2 months, and vacuolation of neurons initially observed at 10 months. Inclusions within affected cells variably stained positively with PAS and LFB stains. Quantitative immunohistochemistry demonstrated increased glial expression of GFAP and Iba1 in dogs with MPS IIIB compared to age-matched controls at all time points, suggesting neuroinflammation occurs early in disease. Loss of Purkinje cells was initially observed at 10 months and was pronounced in 18- and 26-month-old dogs with MPS IIIB. Our results support the dog as a replicative model of MPS IIIB neurologic lesions and detail the pathologic and neuroinflammatory changes in the spinal cord and DRG of MPS IIIB-affected dogs.

Prognostic value of dyslipidemia for sick dogs hospitalized in the intensive care unit of a veterinary teaching hospital.

OBJECTIVE To evaluate the lipidemia status and serum concentrations of cholesterol and triglycerides of dogs when initially examined for hospitalization in the intensive care unit (ICU) of a veterinary teaching hospital and to determine whether these variables were predictive of survival to hospital discharge. DESIGN Retrospective cohort study. ANIMALS 549 client-owned sick (n = 398) and healthy (151) dogs. PROCEDURES Medical records of sick dogs hospitalized in the ICU at a veterinary teaching hospital between January 1, 2012, and September 30, 2015, and of healthy dogs evaluated at the teaching hospital during the same time frame were reviewed. Data collection included signalment, results of initial physical and clinicopathologic examinations, treatments, diagnosis, and survival to hospital discharge. Lipidemia status and serum concentrations of cholesterol and triglycerides were compared between healthy and sick dogs and between sick dogs that did and did not survive to hospital discharge. Regression analysis was performed to determine whether these variables were predictive of survival to hospital discharge in dogs. RESULTS Factors associated with increased odds of sick dogs not surviving to hospital discharge were hypocholesterolemia (OR, 1.87; 95% confidence interval [CI], 1.04 to 3.34), hypertriglyceridemia (OR, 3.20; 95% CI, 2.00 to 5.13), and concurrent hypocholesterolemia and hypertriglyceridemia (OR, 55.7; 95% CI, 3.2 to 959.6) at the time of initial evaluation. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that hypocholesterolemia and hypertriglyceridemia, alone or in combination, at initial examination were negative prognostic indicators for survival of dogs hospitalized in the ICU and that these conditions were easily identified with routine serum clinicopathologic analyses. (J Am Vet Med Assoc 2019;254:699-709).

Age-related variation in the cellular composition of equine bronchoalveolar lavage fluid.

BACKGROUND: Previous reports reveal variation in the cellular composition of equine bronchoalveolar lavage fluid (BALF). OBJECTIVES: The purpose of this study was to compare the profiles of BALF from horses to assess age-related differences. Serial BALF samples were collected from the same individuals over a one-year period to identify changes in individual animals as they aged. METHODS: Collection of BALF was performed on horses aged one week and one, 2, 6, and 12 months. Total nucleated cell count (TNCC), protein concentration, and cytology were assessed. Longitudinal analysis was performed and compared to healthy adults. RESULTS: Foals at one week and 6 months of age had significantly higher TNCC than adults (medians: 320/µL, 285/µL, and 90/µL, respectively); no differences in total protein were found. Foals at one month had the highest proportion of macrophages (median: 87.3%), differing significantly from both yearlings and adults (medians: 45.5% and 48.7%, respectively). Foals aged one week and one month had significantly lower proportions of lymphocytes than yearlings and adults (medians: 3.2% and 4.7% vs 43.2% and 45.8%, respectively). Eosinophil percentage was lowest in foals aged one week, one month, and 2 months (median: 0.0%) and highest in foals aged 6 months (median: 2.2%). Mast cell percentages were highest in yearlings and adults (medians: 2.2% and 3.3%, respectively) and neutrophil percentage was highest in foals aged one week (13.7%). CONCLUSIONS: Cytologic profiles of BALF from foals and adult horses differed considerably. Significant changes in TNCC and percentages of lymphocytes, macrophages, and eosinophils occurred with age.

Therapeutic use of a receptor mimic probiotic reduces intestinal Shiga toxin levels in a piglet model of hemolytic uremic syndrome.

BACKGROUND: Hemolytic uremic syndrome (HUS) is a systemic and potentially fatal complication of gastroenteritis secondary to Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infection characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal damage. Shiga toxin (Stx), the toxin principle in HUS, is produced locally within the gut following EHEC colonization and is disseminated via the vasculature. Clinical development of HUS currently has no effective treatment and is a leading cause of renal failure in children. Novel post-exposure therapies are currently needed for HUS; therefore, the purpose of this study was to investigate the efficacy of a Stx receptor mimic probiotic in a porcine model of HUS. Edema disease, an infection of swine caused by host adapted Shiga toxin-producing Escherichia coli (STEC) and mediated by Shiga toxin 2e (Stx2e), shares many pathogenic similarities to HUS. In this study, three-week old piglets were inoculated with STEC and 24 hours later treated twice daily with a probiotic expressing an oligosaccharide receptor mimic for Stx2e to determine if the probiotic could reduce intestinal toxin levels. METHODS: Piglets were orally inoculated with 10(10) CFU of STEC strain S1191 eight days after weaning. Beginning day 1 post-inoculation, piglets were treated orally twice daily with 5 × 10(11) CFU of either the receptor mimic probiotic or a sham probiotic for 10 days. Intestinal Stx2e levels were assessed daily via Vero cell assay. The efficacy of the probiotic at reducing intestinal Stx2e, vascular lesions, and clinical disease was evaluated with repeated measures ANOVA and Fisher's exact test as appropriate. RESULTS: The probiotic significantly reduced intestinal Stx2e, as reflected by decreased fecal toxin titers on days 3-8 post-inoculation (p < 0.01). Despite this reduction in intestinal toxin levels, however, the probiotic failed to reduce the incidence of vascular necrosis in target organs and had no effect on clinical disease. CONCLUSIONS: The data suggest that post-exposure treatment with a Stx-binding probiotic is effective in reducing intestinal toxin burden. Future studies could target this approach for possible development of post-exposure interventions.

Sucrose stabilization of Respiratory Syncytial Virus (RSV) during nebulization and experimental infection.

BACKGROUND: Respiratory syncytial virus (RSV) is a common respiratory pathogen that can cause severe pneumonia. In vivo studies of RSV can be difficult due to variation in viral infection and disease severity in some animal models. Factors that may contribute to the variation are decreases in viral titer due to preparation and storage and method of virus administration. Nebulization is one method of RSV administration that provides even distribution of virus to all lung lobes; however, the exact quantity of the virus killed by nebulization is not defined. To test the hypothesis that sucrose enhances RSV stability and infectivity, a series of in vitro experiments were conducted with RSV strain Memphis 37 stored at varying concentrations (0%, 3%, 5%, 8%, 10%, 15%, and 20%) of sucrose as a possible cryo- and nebulization protectant. The optimal in vitro concentration was then assessed in vivo in a lamb model. METHODS: Prior to titering the virus on HEp-2 cells, the various virus solutions were subjected to one freeze-thaw cycle and one nebulization cycle. Forty-eight hours after viral plating, infectious foci were detected and counted using immunofluorescent imaging. Titers were determined after freeze-thaw and after freeze-thaw followed by nebulization, then compared to the stock titers (before freezing) as well as to one another to determine the loss of infectivity. To further test this in vivo, lambs 2 to 3-days-old were infected via nebulization with RSV using inoculate containing either 20% sucrose or no sucrose followed by assessments of infection severity. RESULTS: Nebulization of virus in 0% sucrose resulted in a 0.580 log reduction in infectivity while virus in 20% sucrose exhibited a 0.297 log reduction. In vivo studies demonstrated that 20% sucrose enhanced RSV lesions and antigen distribution. CONCLUSIONS: The data suggests that both nebulization and freeze-thawing of RSV in the absence of sucrose cause unacceptable losses in viral infectivity and that sucrose acts as a RSV protectant in both regards.

Increased concentration of iodide in airway secretions is associated with reduced respiratory syncytial virus disease severity.

Recent studies have revealed that the human and nonrodent mammalian airway mucosa contains an oxidative host defense system. This three-component system consists of the hydrogen peroxide (H2O2)-producing enzymes dual oxidase (Duox)1 and Duox2, thiocyanate (SCN(-)), and secreted lactoperoxidase (LPO). The LPO-catalyzed reaction between H2O2 and SCN(-) yields the bactericidal hypothiocyanite (OSCN(-)) in airway surface liquid (ASL). Although SCN(-) is the physiological substrate of LPO, the Duox/LPO/halide system can generate hypoiodous acid when the iodide (I(-)) concentration is elevated in ASL. Because hypoiodous acid, but not OSCN(-), inactivates respiratory syncytial virus (RSV) in cell culture, we used a lamb model of RSV to test whether potassium iodide (KI) could enhance this system in vivo. Newborn lambs received KI by intragastric gavage or were left untreated before intratracheal inoculation of RSV. KI treatment led to a 10-fold increase in ASL I(-) concentration, and this I(-) concentration was approximately 30-fold higher than that measured in the serum. Also, expiratory effort, gross lung lesions, and pulmonary expression of an RSV antigen and IL-8 were reduced in the KI-treated lambs as compared with nontreated control lambs. Inhibition of LPO activity significantly increased lesions, RSV mRNA, and antigen. Similar experiments in 3-week-old lambs demonstrated that KI administration was associated with reduced gross lesions, decreased RSV titers in bronchoalveolar lavage fluid, and reduced RSV antigen expression. Overall, these data indicate that high-dose KI supplementation can be used in vivo to lessen the severity of RSV infections, potentially through the augmentation of mucosal oxidative defenses.

Effects of formalin-inactivated respiratory syncytial virus (FI-RSV) in the perinatal lamb model of RSV.

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960's that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3-5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.

Disseminated Leishmania infantum infection in two sibling foxhounds due to possible vertical transmission.

Two sibling foxhounds born to a Leishmania seropositive bitch were presented after testing seropositive for Leishmania. Leishmania infantum infection was detected via histopathology, culture, and quantitative polymerase chain reaction (q-PCR). This is the first report of natural infection with Leishmania infantum with the possibility for vertical transmission in North America.

Surveillance of amyloidosis and other diseases at necropsy in captive trumpeter swans (Cygnus buccinator).

The purpose of this study was to characterize the incidence and diagnostic features of amyloidosis and other diseases found at necropsy in captive trumpeter swans (Cygnus buccinator). A search of Iowa State University's Department of Veterinary Pathology and Veterinary Diagnostic Laboratory databases yielded 31 trumpeter swan (C. buccinator) necropsy cases from captive swans in protected habitats. Eleven of the 31 birds had amyloid deposition most commonly in the spleen (8 of 11), liver (7 of 11), and kidney (6 of 11) and less often in the pancreas (2 of 11) and adrenal gland (2 of 11). Amyloid deposition effaced normal tissue with adjacent necrosis and hemorrhage in severe cases. Amyloidosis was most often diagnosed in February and March. Other disease diagnoses in the trumpeter swans included aspergillosis (5 of 31, 16%); bacterial infection (5 of 31, 16%); lead toxicosis (3 of 31, 10%); gout (2 of 31, 6%); parasitic infection (2 of 31, 6%); vitamin E deficiency (1 of 31, 3%); trauma (1 of 31, 3%); and ventricular foreign body (1 of 31, 3%). Histopathologic, toxicologic, and microbiologic analyses did not define an etiologic diagnosis in the deaths of 9 trumpeter swans. In these cases, necropsy lesions included emaciation (5 of 9), enteritis (1 of 9), pulmonary hemorrhage (1 of 9), and no lesions (3 of 9). The number of trumpeter swan case submissions was greatest in January and February. This study provides a reference for veterinary diagnosticians concerning incidence and diagnostic features of amyloidosis and other diseases in captive trumpeter swans of the midwestern United States.