Changes in the Serum Metabolome in an Inflammatory Model of Osteoarthritis in Rats.

Osteoarthritis (OA) is a pathology of great impact worldwide. Its physiopathology is not completely known, and it is usually diagnosed by imaging techniques performed at advanced stages of the disease. The aim of this study was to evaluate early serum metabolome changes and identify the main metabolites involved in an inflammatory OA animal model. This study was performed on thirty rats. OA was induced in all animals by intra-articular injection of monoiodoacetate into the knee joint. Blood samples were taken from all animals and analyzed by mass spectrometry before OA induction and 28, 56, and 84 days following induction. Histological evaluation confirmed OA in all samples. The results of this study allow the identification of several changes in 18 metabolites over time, including organic acids, benzenoids, heterocyclic compounds, and lipids after 28 days, organic acids after 56 days, and lipid classes after 84 days. We conclude that OA induces serological changes in the serum metabolome, which could serve as potential biomarkers. However, it was not possible to establish a relationship between the identified metabolites and the time at which the samples were taken. Therefore, these findings should be confirmed in future OA studies.

Yeast-Derived Nucleotides Enhance Fibroblast Migration and Proliferation and Provide Clinical Benefits in Atopic Dermatitis.

Nucleotides, glycosaminoglycans, and omega-3 essential fatty acids (O3s) could be used for improving skin health, although their modes of action, alone or in combination, are not yet fully understood. To gain some insight into these mechanisms, we performed two in vitro tests and one in vivo pilot trial. The effects on human dermal fibroblast proliferation and migration were evaluated with the following compounds and combinations: 0.156 mg/mL O3s, 0.0017 mg/mL hyaluronic acid (HA), 0.0004 mg/mL dermatan sulfate (DS), 0.0818 mg/mL nucleotides, and [O3s + HA + DS] and [O3s + HA + DS + nucleotides] at the same concentrations. In both in vitro assays, adding nucleotides to [O3s + HA + DS] provided significant improvements. The resulting combination [O3s + HA + DS + nucleotides] was then tested in vivo in dogs with atopic dermatitis by oral administration of a supplement providing a daily amount of 40 mg/kg nucleotides, 0.9 mg/kg HA, 0.18 mg/kg DS, 53.4 mg/kg EPA, and 7.6 mg/kg DHA. After 30 days, the pruritus visual analog scale (pVAS) score was significantly reduced, and no adverse effects were observed. In conclusion, the combination of nucleotides plus glycosaminoglycans and O3s could serve as a useful therapeutic alternative in skin health applications.

Gastroprotective Effects of Oral Glycosaminoglycans with Sodium Alginate in an Indomethacin-Induced Gastric Injury Model in Rats.

The gastrointestinal (GI) mucosal barrier is often exposed to inflammatory and erosive insults, resulting in gastric lesions. Glycosaminoglycans (GAGs), such as hyaluronic acid (HA), chondroitin sulfate (CS), and N-acetylglucosamine (NAG) have shown potential beneficial effects as GI protectants. This study aimed to evaluate the gastroprotective effects of oral GAGs in rats with indomethacin-induced GI lesions. Forty-five Sprague-Dawley rats (8-9 weeks-old, 228 ± 7 g) were included in the study, divided into five study groups, and given, administered orally, either sucralfate (positive control group; PC), NAG (G group), sodium alginate plus HA and CS (AHC group), sodium alginate plus HA, CS, and NAG (AHCG group), or no treatment (negative control group; NC). Animals were administered 12.5 mg/kg indomethacin orally 15 min after receiving the assigned treatment. After 4 h, stomach samples were obtained and used to perform a macroscopic evaluation of gastric lesions and to allow histological assessment of the gastric wall (via H/E staining) and mucous (via PAS staining). The AHCG group showed significant gastroprotective improvements compared to the NC group, and a similar efficacy to the PC group. This combination of sodium alginate with GAGs might, therefore, become a safe and effective alternative to prescription drugs for gastric lesions, such as sucralfate, and have potential usefulness in companion animals.

Sphingomyelin-Rich Lipid Extract Collar for Canine Atopic Dermatitis.

The management of canine atopic dermatitis (CAD) is complex, and it needs to be multimodal, combining topical and systemic therapies. Given that the currently available options are not always totally effective and might have some associated adverse effects, novel alternatives are needed. For this reason, a new collar for CAD was developed with 2.5% of a sphingomyelin-rich lipid extract (LE) with proven benefits for skin health. The release of the active ingredient when incorporated into the collar was tested in vitro, showing an adequate kinetic profile. Then, the efficacy and safety of the collar were assessed in 12 client-owned dogs with CAD in a pilot study. After eight weeks, the dogs experienced significant clinical improvements on the Canine Atopic Dermatitis Extent and Severity Index (CADESI)-4, Pruritus Index for Canine Atopic Dermatitis (PCAD) and Pruritus Visual Analogue Scale (PVAS) scores, without any adverse effects. Additionally, further in vitro studies were performed, indicating that this LE collar should be compatible with antiparasitic collars (with deltamethrin or imidacloprid/flumethrin) if worn simultaneously. Given the observed benefits of this LE collar, combining it with other CAD therapies could potentially allow for drug sparing, reduction in adverse effects, enhanced owner compliance, and reduced treatment costs.

Sphingolipids and DHA Improve Cognitive Deficits in Aged Beagle Dogs.

Canine cognitive dysfunction syndrome (CDS) is a disorder found in senior dogs that is typically defined by the development of specific behavioral signs which are attributed to pathological brain aging and no other medical causes. One way of objectively characterizing CDS is with the use of validated neuropsychological test batteries in aged Beagle dogs, which are a natural model of this condition. This study used a series of neuropsychological tests to evaluate the effectiveness of supplementation with a novel lipid extract containing porcine brain-derived sphingolipids (Biosfeen®) and docosahexaenoic acid (DHA) for attenuating cognitive deficits in aged Beagles. Two groups (n = 12), balanced for baseline cognitive test performance, received a daily oral dose of either test supplement, or placebo over a 6-month treatment phase. Cognitive function was evaluated using the following tasks: delayed non-matching to position (DNMP), selective attention, discrimination learning retention, discrimination reversal learning, and spatial discrimination acquisition and reversal learning. The effect of the supplement on brain metabolism using magnetic resonance spectroscopy (MRS) was also examined. A significant decline (p = 0.02) in DNMP performance was seen in placebo-treated dogs, but not in dogs receiving the supplement, suggesting attenuation of working memory performance decline. Compared to placebo, the supplemented group also demonstrated significantly improved (p = 0.01) performance on the most difficult pattern of the spatial discrimination task and on reversal learning of the same pattern (p = 0.01), potentially reflecting improved spatial recognition and executive function, respectively. MRS revealed a significant increase (p = 0.048) in frontal lobe glutamate and glutamine in the treatment group compared to placebo, indicating a physiological change which may be attributed to the supplement. Decreased levels of glutamate and glutamine have been correlated with cognitive decline, suggesting the observed increase in these metabolites might be linked to the positive cognitive effects found in the present study. Results of this study suggest the novel lipid extract may be beneficial for counteracting age-dependent deficits in Beagle dogs and supports further investigation into its use for treatment of CDS. Additionally, due to parallels between canine and human aging, these results might also have applicability for the use of the supplement in human cognitive health.

Knee Cartilage and Subchondral Bone Evaluations by Magnetic Resonance Imaging Correlate with Histological Biomarkers in an Osteoarthritis Rabbit Model.

OBJECTIVE: To evaluate pathological changes in cartilage and subchondral bone MRI biomarkers in a rabbit model of osteoarthritis (OA) and correlate these with histological variations. DESIGN: Transection of the anterior cruciate ligament was performed on the right knee of eighteen 12-week-old New Zealand white rabbits to induce OA. 3-Tesla MR images were obtained from 18 healthy control knees (left) and 18 knees with OA (right). Imaging biomarkers included volume, thickness, T1 and T2* cartilage parametric maps, and several subchondral bone features: bone volume to total volume ratio, trabecular thickness, trabecular spacing, trabecular number (TbN), 2D and 3D fractal dimensions, and quality of trabecular score (QTS). Microscopic analysis of the lateral femoral condyles was set as the ground truth. RESULTS: When healthy and osteoarthritic knees were compared, significant differences were seen in the T1 and T2* values of the femur and tibia cartilage and in the subchondral bone volume to total volume, TbN, and QTS of both the lateral and medial aspects of the femur and tibia. Histological findings revealed significant osteoarthritic changes between healthy and osteoarthritic knees in stain, structure, chondrocyte density, total score, and subchondral bone biomarker levels. A positive correlation was found between histological staining, structure, chondrocyte density, and total score variables in T1 and T2* cartilage biomarkers. A negative correlation was observed between histological subchondral bone variables and magnetic resonance D2D and QTS biomarkers. CONCLUSION: Quantification of several cartilage and subchondral bone imaging biomarkers in a rabbit model of OA allows the detection of significant changes, which are correlated with histological findings.

Effects of Dietary Nucleotide Supplementation on Performance, Profitability, and Disease Resistance of Litopenaeus vannamei Cultured in Indonesia under Intensive Outdoor Pond Conditions.

This study evaluated the effects of dietary nucleotide supplementation in Pacific white shrimp, Litopenaeus vannamei, cultured in Indonesia. A total of 22,500 shrimp receiving diets in which fish meal (FM) had been partially replaced with vegetable protein sources were classified into five study groups (4500 shrimp/group) and received different diets for 110 days: 10FM (control group; 10% FM), 6FM (6% FM-low FM and no nucleotide supplementation), 10FMN (10% FM; 0.1% nucleotides), 8FMN (8% FM; 0.1% nucleotides) and 6FMN (6% FM; 0.1% nucleotides). Growth performance, body composition, total hemocyte count (THC), lysozyme activity, and hepatopancreas histopathology were assessed. Organoleptic evaluation and profitability assessments were also performed. In addition, shrimp resistance to a Vibrio harveyi challenge was studied in shrimps after having received the diets for 30 days. Results showed that reducing FM had a negative impact on growth performance and hepatopancreas morphology. Adding nucleotides resulted in better performance and profitability, a healthier histomorphological appearance of the hepatopancreas, and significantly higher survival rates upon challenge with V. harveyi, while it did not negatively affect organoleptic parameters. In conclusion, nucleotide supplementation could be useful for optimizing performance, profitability, and disease resistance in shrimp cultured under intensive outdoor pond conditions.

Enhanced In Vitro Expression of Filaggrin and Antimicrobial Peptides Following Application of Glycosaminoglycans and a Sphingomyelin-Rich Lipid Extract.

Filaggrin is an epidermal protein involved in skin barrier formation and hydration, whose expression is altered in canine atopic dermatitis (CAD). CAD patients also present an abnormal immune response with an altered expression of antimicrobial peptides (AMPs), such as ß-defensins and cathelicidins. Sphingolipids and glycosaminoglycans (GAGs) have been reported to improve the skin barrier in several animal species, including dogs. Our objective was to evaluate the in vitro effects of a sphingomyelin-rich lipid extract (LE), a hyaluronic acid-rich GAG matrix, and their combination, on the expression of filaggrin and human ß-defensin 2 (hBD-2). Filaggrin expression was quantified in a reconstructed human epidermis (RHE), and hBD-2 in normal human epidermal keratinocyte (NHEK) cultures. LE and GAGs were tested at 0.02 mg/mL, with or without adding a cytokine mix. A significant increase in mean hBD-2, compared to the control (99 pg/mL) was achieved with LE (138 pg/mL) and LE+GAGs (165 pg/mL). Filaggrin increased with GAGs (202% ± 83) and LE (193% ± 44) vs. the stimulated control, but this difference was statistically significant (p < 0.05) only with LE+GAGs (210% ± 39). In conclusion, the tested GAGs and LE enhance filaggrin and AMP expression in vitro, which might benefit CAD patients if applied in vivo.

Improved Joint Health Following Oral Administration of Glycosaminoglycans with Native Type II Collagen in a Rabbit Model of Osteoarthritis.

A prospective, experimental, randomized, double blinded study was designed to evaluate the effects of glycosaminoglycans, with or without native type II collagen (NC), in an osteoarthritis model induced by cranial cruciate ligament transection. The following compounds were tested: chondroitin sulfate (CS), glucosamine hydrochloride (GlHCl), hyaluronic acid (HA) and NC. Fifty-four female 12-week-old New Zealand rabbits were classified into three groups: CTR (control-no treatment), CGH (CS + GlHCl + HA) and CGH-NC (CS + GlHCl + HA + NC). Each group was subdivided into three subgroups according to survival times of 24, 56 and 84 days. Over time, all rabbits developed degenerative changes associated with osteoarthritis. CGH-NC showed significantly improved values on macroscopic evaluation, compared to CTR and CGH. Microscopically, significantly better results were seen with CGH and CGH-NC, compared to CTR, and synovial membrane values were significantly better with CGH-NC compared to CGH. A significant improvement in magnetic resonance imaging biomarkers was also observed with CGH-NC in cartilage transversal relaxation time (T2) and subchondral bone D2D fractal dimension in the lateral condyle. In conclusion, our results show beneficial effects on joint health of CGH and CGH-NC and also supports that adding NC to CGH results in even greater efficacy.

Nutritional Modulation of the Immune Response Mediated by Nucleotides in Canine Leishmaniosis.

Leishmaniasis is an emerging, uncontrolled, and neglected zoonotic disease. Climate change is contributing to its ongoing global expansion. The dog is the main reservoir; hence the importance of implementing effective treatment, prevention, and control measures in this animal species to protect public health. However, although the standard treatment for canine leishmaniosis (CanL) is effective, it does not provide full parasitological clearance, and side effects and drug resistance have been described. The host's immune system plays a key role in the establishment and evolution of leishmaniasis. Dietary nucleotides modulate the immune response and, given their reported efficacy and safety in sick and clinically healthy Leishmania-infected dogs and because they represent a sustainable option with no associated side effects or resistance, they could be included within the prevention, treatment, and control strategies for leishmaniasis. This article briefly summarizes the scientific literature on CanL management, including unresolved issues, and reviews the scientific evidence on immunomodulatory effects of dietary nucleotides in different animal species. It also proposes a CanL management algorithm, including nucleotides. It is concluded that nutritional modulation of the immune response with nucleotides can contribute to better management of leishmaniasis following a One Health approach, especially in the COVID-19 era.

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.

Effects of Oral Hyaluronic Acid Administration in Dogs Following Tibial Tuberosity Advancement Surgery for Cranial Cruciate Ligament Injury.

Hyaluronic acid (HA) intraarticular injection is used in the management of osteoarthritis in veterinary medicine. However, HA oral administration is less common given the scarce currently available scientific evidence. This study was aimed at evaluating the effects of oral HA administration on synovial fluid concentrations of several selected biomarkers in dogs with cranial cruciate ligament (CCL) injury operated on using the tibial tuberosity advancement (TTA) technique. Fifty-five dogs were included in this prospective, randomized, double-blind, clinical study; they were randomly assigned to receive either a placebo (group A; n = 25) or HA (group B; n = 30) orally for 10 weeks. Synovial fluid samples were obtained before surgery, and at 10 weeks postoperatively to measure concentrations of HA, haptoglobin, nitric oxide, and paraoxonase-1. After 10 weeks, group HA showed a significant increase in HA concentration (p = 0.0016) and a significant decrease in PON-1 concentration (p = 0.011) compared to baseline. In conclusion, post-op oral HA administration in canine patients with CCL injury leads to improvements in osteoarthritis biomarkers, namely higher synovial fluid HA concentrations and reduced synovial fluid paraoxonase-1 concentrations. These findings support the bioavailability of orally-administered HA and its usefulness in improving biomarkers of osteoarthritis.

Nucleotides and AHCC Enhance Th1 Responses In Vitro in Leishmania-Stimulated/Infected Murine Cells.

A stronger Th1 (cellular) immune response in canine leishmaniosis (CanL) leads to a better prognosis. Dietary nucleotides plus AHCC® have shown beneficial effects in dogs with clinical leishmaniosis and in clinically healthy Leishmania-infected dogs. The potential leishmanicidal activity of nucleotides and AHCC was assessed by quantifying nitric oxide (NO) production and replication of parasites. Their effects on lymphocyte proliferation were studied with and without soluble Leishmania infantum antigen (SLA) stimulation. Cytokine level variations were assessed using naïve and L. infantum-infected macrophages/lymphocytes cocultures. Promastigotes and amastigotes proliferation and NO macrophage production were not directly affected. Lymphocyte proliferation was significantly enhanced by nucleotides, AHCC, and their combinations only after SLA stimulation. Nucleotides and AHCC significantly increased the production of IL-1ß, IL-2, IL-5, IL-9, IL-10, and IL-12 by naïve immune cells. In naïve and L. infantum-infected macrophage/lymphocyte cocultures, nucleotides with or without AHCC led to significant increases in IFN-γ and TNF-α. Given that these cytokines are involved in the effective Th1 immune response against Leishmania parasites, these mechanism of action could explain the previously reported in vivo clinical efficacy of such combination and further support the use of nucleotides with or without AHCC in the management of CanL patients.

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.

Topical treatment with SPHINGOLIPIDS and GLYCOSAMINOGLYCANS for canine atopic dermatitis.

BACKGROUND: Skin barrier dysfunction plays a key role in atopic dermatitis (AD). This impairment is related to altered composition and metabolism of epidermal sphingolipids and a deficiency of ceramides. Glycosaminoglycans (GAGs), and especially hyaluronic acid, could be useful in the management of AD. This study aimed to evaluate the effects of a novel topical treatment consisting of sphingolipids and GAGs extracts in dogs with AD. This formulation is different from previously tested products because the sphingolipid extract contained high amounts of sphingomyelin, a precursor of ceramides, and this has been shown to enhance endogenous synthesis of ceramides and to increase lamellar-related structures in vitro. Thus, it was hypothesized that this formulation could improve clinical disease and skin barrier function in patients with AD. RESULTS: Twelve house dust mite (HDM) allergic atopic beagle dogs were randomized into two groups: control (n = 6; no treatment) or treatment (n = 6; topical sphingolipids and GAGs twice weekly for 8 weeks). Dogs were challenged with allergen twice weekly and the severity of dermatitis was scored using the canine atopic dermatitis and extent severity index (CADESI-03) once weekly. Skin barrier function (measurement of transepidermal water loss) and severity of pruritus (both pruritus visual analog scale [PVAS] and pruritus timed episodes) were assessed at 0, 4 and 8 weeks of treatment. Assessments were done by personnel unaware of group allocation. Complete blood count, serum biochemistry and stratum corneum (SC) lipidomics analyses were done at baseline and at week 8. Compared to baseline, significant increases in CADESI (P = 0.0003) and PVAS (P = 0.041) were observed only in the control group, and SC polyunsaturated fatty acids increased significantly only with treatment (P = 0.039). Compared to control, treatment group had a significantly lower CADESI after 1 week (P = 0.0078) and a significantly lower PVAS after 8 weeks (P = 0.0448). Treatment was well tolerated. CONCLUSIONS: In this study in dogs with AD, a new topical formulation containing sphingomyelin-rich sphingolipids plus GAGs extracts attenuated the clinical worsening induced by HDM, supporting its use in atopic patients, either as an adjunctive treatment or used as monotherapy in certain cases.

Changes in serum anti-Leishmania antibody concentrations measured by time-resolved immunofluorometric assays in dogs with leishmaniosis after treatment.

The aim of this study was to evaluate the changes in anti-Leishmania IgG2 and IgA antibodies measured by two time-resolved immunofluorometric assays (TR-IFMAs) recently validated and by means of a commercially available ELISA test in dogs with leishmaniosis after treatment. Serum samples from 16 dogs with clinical leishmaniosis were obtained on days 0, 30 and 180 of treatment. In addition, these serological changes were compared with the clinical signs and selected analytes (total proteins, albumin, globulins and urinary protein:creatinine ratio). Concentrations of IgG2 and IgA by TR-IFMA were significantly lower on days 30 (p < 0.05) and 180 of treatment (p < 0.0001) compared to day 0 in dogs that showed a positive response to treatment. Magnitudes of decrease of IgG2 (1.66 and 20.4-fold) and IgA (1.3 and 11.43-fold) concentrations on days 30 and 180 were greater than those of the commercially available ELISA test (1.29 and 2.06-fold), and that of other analytes (total proteins: 1.11 and 1.25-fold; globulins: 1.22 and 1.74-fold; and albumin: 0.93 and 0.8-fold). This study shows that serum IgG2 and IgA anti-Leishmania antibodies measured by TR-IFMAs were useful for treatment monitoring in dogs with leishmaniosis, showing a significant reduction in antibody concentrations earlier than the commercial ELISA assay. Results suggest that the method used for antibody measurements greatly influences the results and, consequently, the usefulness for measuring anti-Leishmania antibodies to monitor the treatment of canine leishmaniosis.

A dose-escalation ex vivo study on the effects of intracameral benzalkonium chloride in rabbits.

BACKGROUND: Rabbits are currently not a good model for studying diseases of the corneal endothelium because their corneal endothelial cells (CECs) maintain a high proliferative capacity throughout almost all their life. Addressing this particular feature might allow the use of this species for such a purpose. The aim of this study was to evaluate the corneal endothelial injury after intracameral benzalkonium chloride (BAC) injection into rabbit eyes ex vivo, and to establish the most suitable starting dose for an in vivo study aimed at developing an animal model of corneal endothelial disease. RESULTS: Forty rabbit eyes obtained postmortem by transconjunctival enucleation were divided into 8 groups according to the injected compound: Control (no injection), BSS, and increasing BAC concentrations (0.005%, 0.01%, 0.025%, 0.05%, 0.1% and 0.2%). At 0, 6, 24 and 48 h, ophthalmologic examination of the anterior segment, pachymetry and specular microscopy were performed, and corneas were finally vital-stained and observed under the light microscope to assess the CECs morphology and mortality rate. When compared to BSS, CECs density started to decrease significantly at 0.025% BAC concentration, while mean cell area, corneal edema and corneal thickness began to increase significantly at 0.05%, 0.005% and 0.1% BAC concentrations, respectively. Concentrations of 0.05% BAC and above caused significant increases in CECs pleomorphism (decreased hexagonality) and mortality, compared to control and BSS. CONCLUSIONS: Ex vivo intracameral BAC injection induces corneal endothelial toxicity in rabbits. However, confirmatory in vivo studies are required to develop the desired model, with 0.05% BAC being a suggested starting point.

Prevention of disease progression in Leishmania infantum-infected dogs with dietary nucleotides and active hexose correlated compound.

BACKGROUND: The prevalence of Leishmania infantum infection in clinically healthy dogs can be several times higher than that of clinical disease in endemic areas. Although treatment is not recommended in dogs with subclinical infection, these animals should be managed to prevent disease progression and parasite transmission to human beings or to other dogs. Dietary nucleotides and active hexose correlated compound (AHCC) have been shown to modulate the immune response. A recent study in dogs with clinical leishmaniosis receiving an initial 28-day course of methylglucamine antimoniate showed that six-month administration of a dietary supplement containing nucleotides plus AHCC achieves similar efficacy to allopurinol. Since the type of immune response plays a key role in the evolution of patients with leishmaniosis, the present study was aimed at evaluating the preventive effect of this supplement in avoiding or delaying disease progression in clinically healthy Leishmania-infected dogs. METHODS: Forty-six dogs were included in this multicenter, randomized, double-blind, placebo-controlled trial. Dogs received once-daily oral administration of a placebo or a dietary supplement containing nucleotides plus AHCC. Disease progression was monitored throughout the study in both groups. At 0, 60, 180 and 365 days of treatment, clinical signs were evaluated using a validated clinical scoring system, and several analytes were measured from blood, urine, and bone marrow samples. RESULTS: During the study, a significantly lower (P = 0.047) proportion of dogs changed their clinical status and became sick in the supplement group (3/20; 15%), compared to the placebo group (10/22; 45.5%). ELISA-determined antibody titers were significantly reduced compared to baseline at all time points with the supplement (P < 0.01), but not with the placebo. The mean clinical score of disease severity was significantly lower in the supplement group after 180 days (P = 0.014). No significant differences were observed for the other parameters. The dietary supplement was well tolerated. CONCLUSIONS: Oral administration of nucleotides plus AHCC for 365 days in clinically healthy L. infantum-infected dogs is safe, allows a significant reduction in anti-Leishmania antibodies, and leads to a lower disease progression rate, hence exerting a preventive effect.

Randomized, allopurinol-controlled trial of the effects of dietary nucleotides and active hexose correlated compound in the treatment of canine leishmaniosis.

First-line treatment for canine leishmaniosis (CanL) is N-methylglucamine antimoniate (MGA) combined with allopurinol. However, in some dogs allopurinol may induce hyperxanthinuria leading to urolithiasis. Moreover, allopurinol resistance has recently been described in Leishmania infantum isolates from treated dogs with a relapse of the disease. Alternative treatments are thus needed. Since the type of host immune response strongly influences CanL progression and prognosis, dogs could benefit from treatments targeted at modulating such response, such as nucleotides and active hexose correlated compound (AHCC). The aim of this study was to evaluate the effects of an oral combination of nucleotides and AHCC in dogs with clinical leishmaniosis. Sixty-nine dogs with naturally-occurring clinical leishmaniosis were included in this multicenter, open-label, positively-controlled clinical trial and randomized to receive 10mg/kg allopurinol PO BID (allopurinol group) or 17mg/kg AHCC plus 32mg/kg nucleotides PO SID (supplement group) for 180 days. All dogs were also given 50mg/kg MGA SC BID during the first 28 days. At the time points 0, 30, and 180 days of the trial, dogs underwent a clinical examination, and blood, urine, and bone marrow samples were submitted for analytical tests. Final data analyses (allopurinol group: n=29; supplement group: n=24) revealed a significant improvement in both groups in clinical scores and ELISA-determined antibody titers after treatment. However, the supplement group showed a significantly lower clinical score (P=0.005) and significantly higher antibody titers (P=0.032) after 180 days, compared to the allopurinol group. RT-PCR parasite loads were reduced in groups (mean±SD supplement: 0.38±0.56 vs 5.23±18.9; allopurinol: 0.45±1.47 vs 3.09±8.36 parasites/ng of DNA), but there were no significant differences over time or between groups. During the study, 12 dogs in the allopurinol group developed xanthinuria (41%) compared to no dogs (0%) in the supplement group (P=0.000). Both treatments led to significantly increased CD4+/CD8+ ratio, and improvements in protein electrophoretic pattern and acute phase response. In conclusion, 6-month oral treatment with nucleotides and AHCC in addition to MGA showed similar efficacy to the current first-line treatment for CanL, without producing xanthinuria. This combination could be a good alternative to MGA-allopurinol combination treatment for CanL, especially for dogs suffering allopurinol-related adverse events.

Evaluation of various biomarkers for kidney monitoring during canine leishmaniosis treatment.

BACKGROUND: The objective of this study was to evaluate and compare the evolution of the profile currently recommended by the International Renal Interest Society (IRIS) (sCr, UPC and sSDMA) with a panel of other different kidney biomarkers during treatment for canine leishmaniosis. This panel included three urinary glomerular biomarkers (uIgG, uCRP and uferritin) and three urinary tubular biomarkers (uGGT, uNAG and uRBP). These biomarkers were measured in two groups of dogs with canine leishmaniosis at IRIS stage I. Group 1: dogs showing proteinuria (UPC > 0.5) before treatment which did not decrease after treatment; Group 2: dogs showing proteinuria before treatment which decreased after treatment. RESULTS: Group 1 showed no significant changes in any biomarker after treatment. In group 2, among the biomarkers recommended by the IRIS, only UPC showed a significant decrease after treatment. However all biomarkers of glomerular damage showed a significant decrease after treatment, with uIgG/Cr and uCRP/Cr showing the greater decreases. In addition uRBP/Cr and uNAG/Cr showed significant decreases after treatment. CONCLUSIONS: In dogs with leishmaniosis at IRIS stage I that reduced UPC after treatment, there were no significant changes in serum creatinine and sSDMA. However, all the urine biomarkers evaluated with exception of uGGT showed a significant decrease. These decreases were more evident in those markers related with glomerular function, being uIgG/Cr the biomarker more associated with UPC. Further studies involving a larger number of animals and histological analysis of the kidney would be recommended to confirm these findings and evaluate the routine practical use of these urine biomarkers in canine leishmaniosis.