Anna Andaluz

Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy

For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA-affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.

Treatment of Diabetes and Long-Term Survival After Insulin and Glucokinase Gene Therapy

Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a “glucose sensor” in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.

Comparison of subcutaneous and transdermal administration of buprenorphine for pre-emptive analgesia in dogs undergoing elective ovariohysterectomy.

The clinical efficacy of a 70 microg/h transdermal buprenorphine patch and of 20 microg/kg of buprenorphine administered subcutaneously (SC) for the relief of post-operative pain was determined in 24 healthy female dogs undergoing elective ovariohysterectomy (OHE). Dogs were randomly assigned to three groups: (1) a control group that received no analgesics, (2) a BSC group that received buprenorphine SC (20 microg/kg), and (3) a BP group that received buprenorphine by a 70 microg/h transdermal patch. Dogs were scored for signs of pain at 0, 2, 4, 6, 8, 10, 14, 20, 26, 32 and 38 h after extubation using the Numerical Rating Scale (NRS) and a modified University of Melbourne Pain Scale (UMPS). Mean NRS and UMPS scores for dogs in the BSC group (2.56 ± 0.23 and 3.05 ± 0.27, respectively) and the BP group (2.02 ± 0.24 and 2.67 ± 0.23, respectively) were significantly lower (P<0.05) compared with dogs in the control group (5.42 ± 0.38 and 7.89 ± 0.44, respectively), whereas differences between the two buprenorphine treatment groups were not significant. The results indicated that the analgesia produced by the 70 microg/h patch was similar to that induced by SC administration of 20 microg/kg of buprenorphine in dogs undergoing OHE, suggesting that the transdermal buprenorphine patch may be a useful alternative for pain management in dogs.

Effects of platelet-rich plasma on intestinal wound healing in pigs

Anastomotic dehiscence and leakage remain a major problem in gastrointestinal surgery. Recently, attention has been focused on cellular acceleration of the wound healing process using platelet-rich plasma (PRP) and this study reports the effects of PRP on intestinal wound healing in pigs. Autologous PRP was obtained from 35 pigs that were then subjected to two intestinal anastomosis procedures. Activated PRP was applied at one of the anastomosis sites, while the other site served as control. After 24, 48, 72 and 96 h and then at 7 days, histology was performed and wound breaking strength was measured at 72 h and 7 days. PRP application appeared to increase granulation tissue and fibrosis, but did not influence anastomotic breaking strength.

A Multi-Antigenic Adenoviral-Vectored Vaccine Improves BCG-Induced Protection of Goats against Pulmonary Tuberculosis Infection and Prevents Disease Progression

The “One world, one health” initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.

The effects on cardio-respiratory and acid-base variables of the anaesthetic alfaxalone in a 2-hydroxypropyl-β-cyclodextrin (HPCD) formulation in sheep

The objective of this study was to determine the pharmacodynamic effects in sheep of the anaesthetic alfaxalone in a 2-hydroxypropyl-β-cyclodextrin formulation. Seven Ripollesa sheep, weighing 43.0±6.6 kg, were used in the study. Twenty-four hours after instrumentation, the sheep were anesthetised with alfaxalone (2 mg/kg bodyweight IV) in cyclodextrin. Heart rate, arterial blood pressure, respiratory rate and arterial blood gases were recorded. Alfaxalone administration resulted in minimal cardio-respiratory depression. Time to standing from anaesthesia was 22.0±10.6 min. Apnoea was not observed in any of the sheep. Significant differences from baseline were not observed in respiratory rate or arterial blood pressure. Heart rate increased significantly (P<0.05) immediately after administration, returning to control values at 20 min. The calculated haemoglobin saturation (SO2) decreased significantly during the first 15 min after alfaxalone administration. The arterial pH decreased significantly during the first 30 min of the study, although no significant differences from basal values were observed in the arterial partial pressure of carbon dioxide (PaCO2). The results showed that alfaxalone in 2-hydroxypropyl-β-cyclodextrin administered as an IV bolus at 2 mg/kg produced minimal adverse effects and an uneventful recovery from anaesthesia in sheep.

Pharmacokinetics of buprenorphine after intravenous administration of clinical doses to dogs.

The purpose of this study was to evaluate plasma concentrations and pharmacokinetic parameters of buprenorphine in dogs following intravenous (IV) administration of clinical doses of the opioid. An IV bolus of 0.02mg/kg buprenorphine was administered to six healthy Beagles and blood samples were collected through a jugular catheter before and at 1, 5, 10, 15, 20, 30 and 45 min, and 1, 2, 4, 6, 8 and 12h after administration. Plasma buprenorphine concentrations, measured using a commercial radioimmunoassay (RIA), decreased following a three-exponential curve. The two distribution and the elimination half-lives were 2.9+/-1.8min, 16.5+/-3.7min, and 266.6+/-82.0min, respectively; the clearance was 329.6+/-62.2mL/min, and the steady state volume of distribution was 83.7+/-26.5L. The results demonstrated the feasibility of the RIA assay to analyse buprenorphine in dog plasma samples. Following IV administration buprenorphine showed a three-compartment kinetic profile, as has been described previously in humans, rabbits and cats. The relationship between plasma concentrations and dynamic effects in dogs remains to be established.

Plasma iron, C-reactive protein, albumin, and plasma fibrinogen concentrations in dogs with systemic inflammatory response syndrome.

OBJECTIVE To investigate the diagnostic and prognostic value over time of plasma iron compared with the inflammatory markers albumin, C-reactive protein (CRP), and fibrinogen in dogs with systemic inflammatory response syndrome (SIRS). DESIGN Prospective observational study of sequentially enrolled dogs. SETTING ICU of a veterinary teaching hospital. ANIMALS One hundred and sixteen client-owned dogs: 54 dogs with SIRS or sepsis, 42 with focal inflammation, and 20 clinically healthy dogs. MEASUREMENTS AND MAIN RESULTS Blood samples were obtained on admission in all study groups, and then on alternate days until discharge or death in both inflammation groups. On admission, dogs with SIRS had significantly lower plasma iron (65 ± 5.8 μg/dL, P = 0.001) concentrations than dogs with focal inflammation (89.5 ± 6.2 μg/dL, P = 0.001). Plasma iron, albumin, and CRP effectively discriminated the SIRS/sepsis group from those presenting with focal inflammation with areas under the curve for the receiver operating curves of 0.679, 0.834, and 0.704, respectively. The admission values for these variables did not discriminate survivors from nonsurvivors within the SIRS/sepsis group. However, the magnitude of increase in iron concentration and the decrease in CRP concentration from admission to hospital discharge was higher in survivors than in nonsurvivors within the SIRS/septic group (22.8 vs. 2.51 μg/dL, respectively, P = 0.021 for iron; -67.1 vs. -4.1 mg/L, respectively, P = 0.002 for CRP), resulting in iron and CRP concentrations at hospital discharge for survivors similar to those in the focal inflammation group. CONCLUSION Hypoferremia is a sensitive marker of systemic inflammation in dogs. In this study, the increase in iron concentrations during the hospitalization period of SIRS/septic dogs was associated with a better prognosis, suggesting that plasma iron in combination with CRP and albumin concentrations might be used to monitor dogs with inflammatory disease processes.Objective To investigate the diagnostic and prognostic value over time of plasma iron compared with the inflammatory markers albumin, C-reactive protein (CRP), and fibrinogen in dogs with systemic inflammatory response syndrome (SIRS). Design Prospective observational study of sequentially enrolled dogs. Setting ICU of a veterinary teaching hospital. Animals One hundred and sixteen client-owned dogs: 54 dogs with SIRS or sepsis, 42 with focal inflammation, and 20 clinically healthy dogs. Measurements and Main Results Blood samples were obtained on admission in all study groups, and then on alternate days until discharge or death in both inflammation groups. On admission, dogs with SIRS had significantly lower plasma iron (65 ± 5.8 μg/dL, P = 0.001) concentrations than dogs with focal inflammation (89.5 ± 6.2 μg/dL, P = 0.001). Plasma iron, albumin, and CRP effectively discriminated the SIRS/sepsis group from those presenting with focal inflammation with areas under the curve for the receiver operating curves of 0.679, 0.834, and 0.704, respectively. The admission values for these variables did not discriminate survivors from nonsurvivors within the SIRS/sepsis group. However, the magnitude of increase in iron concentration and the decrease in CRP concentration from admission to hospital discharge was higher in survivors than in nonsurvivors within the SIRS/septic group (22.8 vs. 2.51 μg/dL, respectively, P = 0.021 for iron; −67.1 vs. −4.1 mg/L, respectively, P = 0.002 for CRP), resulting in iron and CRP concentrations at hospital discharge for survivors similar to those in the focal inflammation group. Conclusion Hypoferremia is a sensitive marker of systemic inflammation in dogs. In this study, the increase in iron concentrations during the hospitalization period of SIRS/septic dogs was associated with a better prognosis, suggesting that plasma iron in combination with CRP and albumin concentrations might be used to monitor dogs with inflammatory disease processes.

Plasma buprenorphine concentrations after the application of a 70 μg/h transdermal patch in dogs. Preliminary report

The objective of the present study was to evaluate the plasma concentrations and pharmacokinetics of buprenorphine after transdermal application in dogs (n = 4). A 70 microg/h transdermal buprenorphine patch was applied to the ventral abdomen of four healthy beagles. Blood samples were collected through a preplaced jugular catheter before and at 1, 2, 4, 8, 12, 24, 36, 48 and every 6 h until 108 h after the patch application. Plasma buprenorphine concentrations were measured using a (125)I-labelled radioimmunoassay (RIA) assay. No adverse effects were observed in any of the dogs. Concentrations of buprenorphine were detected in plasma after the application of the transdermal buprenorphine patch on the four experimental animals. Buprenorphine plasma concentrations increased during the first 36 h and then remained in the 0.7-1.0 ng/mL range during the study period. A decrease in plasma buprenorphine concentration was not observed during the study. Although analgesia could not be demonstrated the present study shows the ability of buprenorphine transdermal delivery systems developed for human use to deliver measurable concetrations of buprenorphine in dogs.

Effects of an intravenous bolus of alfaxalone versus propofol on intraocular pressure in sheep.

The objective of this prospective study was to determine the effects of a single intravenous bolus of alfaxalone in 2-hydroxypropyl-β-cyclodextrin and propofol on the intraocular pressure (IOP) in sheep. Ten Ripollesa sheep with a bodyweight of 48.5 (6.8) kg (mean [sd]) were used in the study. Twenty-four hours before the experimental procedure, a complete ophthalmic examination was performed in all animals. The day of the study, intravenous alfaxalone (2 mg/kg) or propofol (6 mg/kg) was randomly administered in a cross-over design, with a washout period of two weeks. Measurements of IOP, globe position and pupil size were obtained at basal time, before induction (time 0) and at two, five, 10, 15, 20, 30, 45, 60, 90 and 120 minutes after drug administration. Occasional side effects and time to standing were also noted. Intravenous administration of alfaxalone and propofol in sheep resulted in no alteration of IOP. Nevertheless, a decrease in the pupil size was observed in both groups. This present study shows that alfaxalone and propofol, administrated as a single intravenous bolus, are good options for maintaining IOP during anaesthesia in sheep, although marked miosis was observed after administration.