Development and validation of an assay for measurement of leptin in pig saliva

Leptin has been measured in human in saliva samples. However, the low leptin concentration found in this biological fluid makes necessary the use of high sensitive methods. To the authors' knowledge, leptin has not been measured in porcine saliva. This study aimed to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for salivary leptin measurements in pigs, using a species-specific antibody, and to evaluate how salivary leptin changes with body weight, food ingestion, and in experimental models of stress and inflammation. Polyclonal antibodies were produced in rabbits immunized with recombinant porcine leptin and used to develop a sandwich TR-IFMA. The method had intra-assay and inter-assay coefficients of variation lower than 10 and 16 %, respectively. The assay was accurate and the low limit of detection allowed detection of leptin in all analyzed samples. Salivary leptin concentration was positively correlated to body weight (r = 0.58, P = 0.01) and increased after food ingestion (P < 0.001) and after 24 h of applying a model of experimental inflammation by turpentine injection (P < 0.05). However, it did not significantly change after a model of acute stress consisting of a nose snare restraining. These results indicate that the developed assay can measure leptin in porcine saliva in a reliable way and that leptin in saliva is influenced by body weight, food ingestion and inflammation

Measurement of urea and creatinine in saliva of dogs : a pilot study

Urea and creatinine in saliva have been reported to be possible markers of chronic kidney disease (CKD) in humans. The aim of this study was to assess if urea and creatinine could be measured in canine saliva, and to evaluate their possible changes in situations of CKD

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

Use of saliva for diagnosis and monitoring the SARS-CoV-2: a general perspective

In this report, updated information and future perspectives about the use of saliva as a sample for laboratory analysis of the Covid-19 are highlighted. Saliva can be used for the direct detection of the SARS-CoV-2 virus, the quantification of the specific immunoglobulins produced against it, and for the evaluation of the non-specific, innate immune response of the patient. Moreover, a deeper knowledge of potential changes in the saliva proteome in this disease may allow the identification of new diagnostic and prognostic biomarkers, or even help our understanding of the mechanisms associated with the disease. With the development of appropriate sample collection and processing methods and the use of adequate assays, saliva can provide useful clinical information about the disease and could be potentially included in guidelines for sample collection for the diagnosis, disease management, and control of Covid-19

Application of the NEOH Framework for Self-Evaluation of One Health Elements of a Case-Study on Obesity in European Dogs and Dog-Owners

Obesity is a malnutrition disorder of global concern with increasing prevalence driven by underlying societal, economic and environmental mechanisms leading to changed physical activity patterns, eating behaviors and diet compositions in both humans and in their pet-dogs. A questionnaire-based study was carried out as a joint effort across 11 European countries. It was considered a One Health (OH) initiative between scientists from human and animal health sectors aiming to identify factors associated with obesity in dog owners and their dogs. Expected outcomes of this approach included new insights unachievable by single-sector research initiatives, and hence potentially leading to new cross-sectorial solutions. We performed an internal evaluation among the actors of the obesity initiative using the framework for evaluation developed by the “Network for Evaluation of One Health” (NEOH). It served as a case-study for the NEOH consortium to illustrate the application and provide feedback on the utility of the framework. The evaluation was performed by a subgroup of scientists also involved in the obesity study group, and it consisted of: (1) the definition of the initiative and its context, (2) the description of the theory of change, and (3) the qualitative and quantitative process evaluation of operations and supporting infrastructures scored on a scale from 0 to 1. In the One Health operations, the obesity study initiative scored medium high on OH-thinking (0.5) and OH-planning (0.45), and relatively high on OH-working (0.7). The supporting infrastructure score was high for systemic organization (0.8), but low for sharing (0.45) and learning (0.28). The calculated OH-index was 0.29 (on scale 0 to 1) indicating that the full potential of health integration and collaboration was not exploited in the initiative, and the main issue identified was a lack of stakeholder engagement. The OH-ratio of 1.1 indicated equal focus on operations and supporting infrastructures. Hence, the evaluation identified potentially counterproductive as well as beneficial characteristics, which are further discussed in this paper in relation to the expected outcomes. The NEOH framework for evaluation requires that the evaluators have a good understanding of systems thinking and the mechanisms of the health issue targeted by the initiative

Effect of thermal and chemical treatments used for SARS-COV-2 inactivation in the measurement of saliva analytes

The present study aims to assess the effects of thermal and chemical inactivating procedures, that can be used for SARS-CoV-2 inactivation, on different salivary analytes. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profile and a panel of 25 specific biomarkers of oxidative status, stress, metabolism and tissue damage were evaluated in samples subjected to different treatments: thermal (65 °C or 92 °C) and chemical with detergents [sodium dodecyl sulphate (SDS), Triton X-100 or NP-40]. Salivary SDS-PAGE profile was most affected by heating at 92 °C, with three and two protein bands decreasing and increasing their expression levels, respectively. This treatment also affected the results of several enzymes, with some of them being also affected by heating at 65 °C and incubation with SDS. The use of Triton X-100 or NP-40 resulted in increased values of cortisol, triglycerides and glucose, not affecting the other tested biomarkers. The present results will help researchers and clinicians to select the best protocols to work in safe conditions with saliva, taking into account the target analyte planned to be measured

Analytical validation of an automated assay for the measurement of adenosine deaminase (ADA) and its isoenzymes in saliva and a pilot evaluation of their changes in patients with SARS-CoV-2 infection

Objectives The aim of the present study was to validate a commercially available automated assay for the measurement of total adenosine deaminase (tADA) and its isoenzymes (ADA1 and ADA2) in saliva in a fast and accurate way, and evaluate the possible changes of these analytes in individuals with SARS-CoV-2 infection. Methods The validation, in addition to the evaluation of precision and accuracy, included the analysis of the effects of the main procedures that are currently being used for SARS-CoV-2 inactivation in saliva and a pilot study to evaluate the possible changes in salivary tADA and isoenzymes in individuals infected with SARS-CoV-2. Results The automated assay proved to be accurate and precise, with intra- and inter-assay coefficients of variation below 8.2%, linearity under dilution linear regression with R2 close to 1, and recovery percentage between 80 and 120% in all cases. This assay was affected when the sample is treated with heat or SDS for virus inactivation but tolerated Triton X-100 and NP-40. Individuals with SARS-CoV-2 infection (n=71) and who recovered from infection (n=11) had higher mean values of activity of tADA and its isoenzymes than healthy individuals (n=35). Conclusions tADA and its isoenzymes ADA1 and ADA2 can be measured accurately and precisely in saliva samples in a rapid, economical, and reproducible way and can be analyzed after chemical inactivation with Triton X-100 and NP-40. Besides, the changes observed in tADA and isoenzymes in individuals with COVID-19 open the possibility of their potential use as non-invasive biomarkers in this disease

Targets for COVID-19 symptomatology and disease control

Funding Information: This study was supported by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación, Spain and EU‐FEDER [Grant BIOGAL PID2020‐116761GB‐I00]. M.C. was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain, grant IJC2020‐042710‐I. RV‐R was funded by Universidad de Castilla‐La Mancha (UCLM), Spain and the European Social Fund (ESF) [grant 2022‐PRED‐20675]. Funding Information: This study was supported by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación, Spain and EU-FEDER [Grant BIOGAL PID2020-116761GB-I00]. M.C. was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain, grant IJC2020-042710-I. RV-R was funded by Universidad de Castilla-La Mancha (UCLM), Spain and the European Social Fund (ESF) [grant 2022-PRED-20675]. Publisher Copyright: © 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still poses a challenge for biomedicine and public health. To advance the development of effective diagnostic, prognostic, and preventive interventions, our study focused on high-throughput antibody binding epitope mapping of the SARS-CoV-2 spike RBD protein by IgA, IgM and IgG antibodies in saliva and sera of different cohorts from healthy uninfected individuals to SARS-CoV-2-infected unvaccinated and vaccinated asymptomatic, recovered, nonsevere, and severe patients. Identified candidate diagnostic (455-LFRKSNLKPFERD-467), prognostic (395-VYADSFVIRGDEV-407-C-KLH, 332-ITNLCPFGEV-342-C-KLH, 352-AWNRKRI-358-C-KLH, 524-VCGPKKSTNLVKN-536-KLH), and protective (MKLLE-487-NCYFPLQSYGFQPTNGVG-504-GGGGS-446-GGNYNYLYRLFRKSNLKPFERD-467) epitopes were validated with sera from prevaccine and postvaccine cohorts. The results identified neutralizing epitopes and support that antibody recognition of linear B-cell epitopes in RBD protein is associated with antibody isotype and disease symptomatology. The findings in asymptomatic individuals suggest a role for anti-RBD antibodies in the protective response against SARS-CoV-2. The possibility of translating results into diagnostic interventions for the early diagnosis of asymptomatic individuals and prognosis of disease severity provides new tools for COVID-19 surveillance and evaluation of risks in hospitalized patients. These results, together with other approaches, may contribute to the development of new vaccines for the control of COVID-19 and other coronavirus-related diseases using a quantum vaccinomics approach through the combination of protective epitopes.publishersversionpublishe

Oral chondroitin sulfate and prebiotics for the treatment of canine Inflammatory Bowel Disease: a randomized, controlled clinical trial

BACKGROUND Canine inflammatory bowel disease (IBD) is a chronic enteropathy of unknown etiology, although microbiome dysbiosis, genetic susceptibility, and dietary and/or environmental factors are hypothesized to be involved in its pathogenesis. Since some of the current therapies are associated with severe side effects, novel therapeutic modalities are needed. A new oral supplement for long-term management of canine IBD containing chondroitin sulfate (CS) and prebiotics (resistant starch, β-glucans and mannaoligosaccharides) was developed to target intestinal inflammation and oxidative stress, and restore normobiosis, without exhibiting any side effects. This double-blinded, randomized, placebo-controlled trial in dogs with IBD aims to evaluate the effects of 180 days administration of this supplement together with a hydrolyzed diet on clinical signs, intestinal histology, gut microbiota, and serum biomarkers of inflammation and oxidative stress. RESULTS Twenty-seven client-owned biopsy-confirmed IBD dogs were included in the study, switched to the same hydrolyzed diet and classified into one of two groups: supplement and placebo. Initially, there were no significant differences between groups (p > 0.05) for any of the studied parameters. Final data analysis (supplement: n = 9; placebo: n = 10) showed a significant decrease in canine IBD activity index (CIBDAI) score in both groups after treatment (p < 0.001). After treatment, a significant decrease (1.53-fold; p < 0.01) in histologic score was seen only in the supplement group. When groups were compared, the supplement group showed significantly higher serum cholesterol (p < 0.05) and paraoxonase-1 (PON1) levels after 60 days of treatment (p < 0.01), and the placebo group showed significantly reduced serum total antioxidant capacity (TAC) levels after 120 days (p < 0.05). No significant differences were found between groups at any time point for CIBDAI, WSAVA histologic score and fecal microbiota evaluated by PCR-restriction fragment length polymorphism (PCR-RFLP). No side effects were reported in any group. CONCLUSIONS The combined administration of the supplement with hydrolyzed diet over 180 days was safe and induced improvements in selected serum biomarkers, possibly suggesting a reduction in disease activity. This study was likely underpowered, therefore larger studies are warranted in order to demonstrate a supplemental effect to dietary treatment of this supplement on intestinal histology and CIBDAI