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Abstract Background COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. Methods A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. Results The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p

Abstract Background Diagnosis of dementia with Lewy bodies (DLB) is challenging, especially in the earlier stages of the disease, owing to the clinical overlap with other neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s disease (PD). We aimed to identify the transcranial sonography (TCS) parameters that can help us to detect early DLB patients. Methods In this cross-sectional study, we prospectively recruited newly diagnosed DLB patients with less than 3 years from the onset of cognitive symptoms. For comparison purposes, we also included AD and PD patients, with a disease duration of less than 3 years, and a control group. TCS was performed to assess the substantia nigra (SN) echogenicity, the width of the third ventricle, and the frontal horns of the lateral ventricles. Subsequently, TCS images were analyzed with the medical image viewer Horos in order to quantify the intensity of the echogenicity of the SN. Univariate analysis and a logistic regression model were used to identify which variables can predict the diagnosis of DLB. Results One hundred and seven participants were included (23 DLB, 26 AD, 27 PD and 31 controls). The median age of DLB patients was 75(72–77) years, with a disease duration of 2 years. DLB and PD patients showed higher SN hyperechogenicity rates (72.73% and 81.82%, respectively) and a greater area of the SN compared to AD patients and controls (p

Abstract Background The architecture and dynamics of T cell populations are critical in orchestrating the immune response to SARS-CoV-2. In our study, we used T Cell Receptor sequencing (TCRseq) to investigate TCR repertoires in 173 post-infection COVID-19 patients. Methods The cohort included 98 mild and 75 severe cases with a median age of 53. We amplified and sequenced the TCR β chain Complementary Determining Region 3 (CDR3b) and performed bioinformatic analyses to assess repertoire diversity, clonality, and V/J allelic usage between age, sex and severity groups. CDR3b amino acid sequence inference was performed by clustering structural motifs and filtering validated reactive CDR3b to COVID-19. Results Our results revealed a pronounced decrease in diversity and an increase in clonal expansion in the TCR repertoires of severe COVID-19 patients younger than 55 years old. These results reflect the observed trends in patients older than 55 years old (both mild and severe). In addition, we identified a significant reduction in the usage of key V alleles (TRBV14, TRBV19, TRBV15 and TRBV6-4) associated with disease severity. Notably, severe patients under 55 years old had allelic patterns that resemble those over 55 years old, accompanied by a skewed frequency of COVID-19-related motifs. Conclusions Present results suggest that severe patients younger than 55 may have a compromised TCR repertoire contributing to a worse disease outcome. Graphical Abstract

Abstract Background Cortisone is derived from cortisol through the action of the enzyme 11β-hydroxysteroid dehydrogenase type II, and it has gained importance in recent years as a biomarker of stress. This study aimed to develop and validate an assay for the measurement of cortisone in pig saliva and evaluate whether its concentration varies in stressful situations. For this purpose, a specific immunoassay was developed and validated analytically, and a study was performed to evaluate whether cortisone concentrations in saliva can vary under heat stress conditions. Results The assay proved to be accurate, reliable, and sensitive for the measurement of cortisone in pig saliva. The limit of detection of the assay was set at 0.006 ng/ml, and the lower limit of quantification was 0.023 ng/ml. It also correlated significantly with the results obtained by LC‒MS/MS (P = 0.003; r = 0.64). In addition, the cortisone concentration in animals subjected to prolonged heat stress decreased significantly 15 days after treatment (P

Abstract The aim of this study was to evaluate the changes in the serum and salivary inflammatory markers induced by Diabetes mellitus (DM) in dogs and to assess the possible confounding effect of gingivitis. A panel of 13 cytokines was measured in the serum and saliva of dogs diagnosed with DM and compared with healthy dogs without gingivitis (control group 1; CG1) and dogs with gingivitis but otherwise healthy (control group 2; CG2). The results of the present study showed statistically significantly higher levels of IL-8, KC-like and MCP1 in the serum of dogs with DM compared to CG1 dogs. In the case of saliva, the DM group presented statistically higher GM-CSF, IL6, IL15, and MCP1 levels compared to CG1, and lower KC-like chemokine compared to CG2. Finally, gingivitis produced changes in saliva, with salivary levels of GM-CSF, IL-6, IL-7, IL-15, IP-10, KC-like, IL-10, IL-18, MCP1, TNFα being statistically significantly higher in the saliva of CG2 dogs compared to CG1. The results of the present study indicate that dogs with DM have altered cytokine levels in serum and saliva compared to healthy dogs. In addition, this study highlights the importance of taking oral health into account when determining cytokines in dogs, as gingivitis can significantly alter their concentrations. .

Abstract Background Pallister-Killian syndrome (PKS) is a rare genetic disorder caused by mosaic tetrasomy of 12p with wide neurological involvement. Intellectual disability, developmental delay, behavioral problems, epilepsy, sleep disturbances, and brain malformations have been described in most individuals, with a broad phenotypic spectrum. This observational study, conducted through brain MRI scan analysis on a cohort of patients with genetically confirmed PKS, aims to systematically investigate the neuroradiological features of this syndrome and identify the possible existence of a typical pattern. Moreover, a literature review differentiating the different types of neuroimaging data was conducted for comparison with our population. Results Thirty-one individuals were enrolled (17 females/14 males; age range 0.1–17.5 years old at first MRI). An experienced pediatric neuroradiologist reviewed brain MRIs, blindly to clinical data. Brain abnormalities were observed in all but one individual (compared to the 34% frequency found in the literature review). Corpus callosum abnormalities were found in 20/30 (67%) patients: 6 had callosal hypoplasia; 8 had global hypoplasia with hypoplastic splenium; 4 had only hypoplastic splenium; and 2 had a thin corpus callosum. Cerebral hypoplasia/atrophy was found in 23/31 (74%) and ventriculomegaly in 20/31 (65%). Other frequent features were the enlargement of the cisterna magna in 15/30 (50%) and polymicrogyria in 14/29 (48%). Conversely, the frequency of the latter was found to be 4% from the literature review. Notably, in our population, polymicrogyria was in the perisylvian area in all 14 cases, and it was bilateral in 10/14. Conclusions Brain abnormalities are very common in PKS and occur much more frequently than previously reported. Bilateral perisylvian polymicrogyria was a main aspect of our population. Our findings provide an additional tool for early diagnosis.Further studies to investigate the possible correlations with both genotype and phenotype may help to define the etiopathogenesis of the neurologic phenotype of this syndrome.

Abstract Background Celiac disease (CeD) is an immune-mediated disorder that develops in genetically predisposed individuals upon gluten consumption. HLA risk alleles explain 40% of the genetic component of CeD, so there have been continuing efforts to uncover non-HLA loci that can explain the remaining heritability. As in most autoimmune disorders, the prevalence of CeD is significantly higher in women. Here, we investigated the possible involvement of the X chromosome on the sex bias of CeD. Methods We performed a X chromosome-wide association study (XWAS) and a gene-based association study in women from the CeD Immunochip (7062 cases, 5446 controls). We also constructed a database of X chromosome cis-expression quantitative trait loci (eQTLs) in monocytes from unstimulated (n = 226) and lipopolysaccharide (LPS)-stimulated (n = 130) female donors and performed a Summary-data-based MR (SMR) analysis to integrate XWAS and eQTL information. We interrogated the expression of the potentially causal gene (TMEM187) in peripheral blood mononuclear cells (PBMCs) from celiac patients at onset, on a gluten-free diet, potential celiac patients and non-celiac controls. Results The XWAS and gene-based analyses identified 13 SNPs and 25 genes, respectively, 22 of which had not been previously associated with CeD. The X chromosome cis-eQTL analysis found 18 genes with at least one cis-eQTL in naïve female monocytes and 8 genes in LPS-stimulated female monocytes, 2 of which were common to both situations and 6 were unique to LPS stimulation. SMR identified a potentially causal association of TMEM187 expression in naïve monocytes with CeD in women, regulated by CeD-associated, eQTL-SNPs rs7350355 and rs5945386. The CeD-risk alleles were correlated with lower TMEM187 expression. These results were replicated using eQTLs from LPS-stimulated monocytes. We observed higher levels of TMEM187 expression in PBMCs from female CeD patients at onset compared to female non-celiac controls, but not in male CeD individuals. Conclusion Using X chromosome genotypes and gene expression data from female monocytes, SMR has identified TMEM187 as a potentially causal candidate in CeD. Further studies are needed to understand the implication of the X chromosome in the higher prevalence of CeD in women.

Abstract Background The management of respiratory distress syndrome (RDS) in premature newborns is based on different types of non-invasive respiratory support and on surfactant replacement therapy (SRT) to avoid mechanical ventilation as it may eventually result in lung damage. European guidelines currently recommend SRT only when the fraction of inspired oxygen (FiO2) exceeds 0.30. The literature describes that early SRT decreases the risk of bronchopulmonary dysplasia (BPD) and mortality. Lung ultrasound score (LUS) in preterm infants affected by RDS has proven to be able to predict the need for SRT and different single-center studies have shown that LUS may increase the proportion of infants that received early SRT. Therefore, the aim of this study is to determine if the use of LUS as a decision tool for SRT in preterm infants affected by RDS allows for the reduction of the incidence of BPD or death in the study group. Methods/design In this study, 668 spontaneously-breathing preterm infants, born at 25+0 to 29+6 weeks’ gestation, in nasal continuous positive airway pressure (nCPAP) will be randomized to receive SRT only when the FiO2 cut-off exceeds 0.3 (control group) or if the LUS score is higher than 8 or the FiO2 requirements exceed 0.3 (study group) (334 infants per arm). The primary outcome will be the difference in proportion of infants with BPD or death in the study group managed compared to the control group. Discussion Based on previous published studies, it seems that LUS may decrease the time to administer surfactant therapy. It is known that early surfactant administration decreases BPD and mortality. Therefore, there is rationale for hypothesizing a reduction in BPD or death in the group of patients in which the decision to administer exogenous surfactant is based on lung ultrasound scores. Trial registration ClinicalTrials.gov identifier NCT05198375 . Registered on 20 January 2022.

Abstract Background and aims Monocytes and dendritic cells (DC) are both key inflammatory cells, with recognized effects on cardiac repair. However, there are distinct subsets of monocytes with potential for beneficial or detrimental effects on heart failure (HF) pathogenesis. The connection between reverse cardiac remodelling, the potential anti-inflammatory effect of cardiac resynchronization therapy (CRT) and monocytes and DC homeostasis in HF is far from being understood. We hypothesized that monocytes and DC play an important role in cardiac reverse remodelling and CRT response. Therefore, we aimed to assess the potential role of baseline peripheral levels of blood monocytes and DC subsets and their phenotypic and functional activity for CRT response, in HF patients. As a secondary objective, we aimed to evaluate the impact of CRT on peripheral blood monocytes and DC subsets, by comparing baseline and post CRT circulating levels and phenotypic and functional activity. Methods Forty-one patients with advanced HF scheduled for CRT were included in this study. The quantification and phenotypic determination of classical (cMo), intermediate (iMo) and non-classical monocytes (ncMo), as well as of myeloid (mDC) and plasmacytoid DC (pDC) were performed by flow cytometry in a FACSCanto™II (BD) flow cytometer. The functional characterization of total monocytes and mDC was performed by flow cytometry in a FACSCalibur flow cytometer, after in vitro stimulation with lipopolysaccharide from Escherichia coli plus interferon (IFN)-γ, in the presence of Brefeldina A. Comparisons between the control and the patient group, and between responders and non-responders to CRT were performed. Results Compared to the control group, HF population presented a significantly lower frequency of pDC at baseline and a higher proportion of monocytes and mDC producing IL-6 and IL-1β, both before and 6-months after CRT (T6). There was a remarkable decrease of cMo and an increase of iMo after CRT, only in responders. The responder group also presented higher ncMo values at T6 compared to the non-responder group. Both responders and non-responders presented a decrease in the expression of CD86 in all monocyte and DC populations after CRT. Moreover, in non-responders, the increased frequency of IL-6-producing DC persisted after CRT. Conclusion Our study provides new knowledge about the possible contribution of pDC and monocytes subsets to cardiac reverse remodelling and response to CRT. Additionally, CRT is associated with a reduction on CD86 expression by monocytes and DC subsets and in their potential to produce pro-inflammatory cytokines, contributing, at least in part, for the well described anti-inflammatory effects of CRT in HF patients.

Abstract Background Aortic stenosis is the most common valvulopathy in Western countries. The treatment of choice had been surgery aortic valve replacement (SAVR), but the improvement in endovascular approaches as transcatheter aortic valve implantation (TAVI), initially reserved for patients with very high surgical risk, has been extended to high and intermediate, and recently also to low-risk patients. Stroke and vascular cognitive impairment are the most important complications. It is not entirely clear which technique is best to avoid these complications as well as their impact. Our goal is to evaluate changes in cognitive performance in the early (1-month) and late (1-year) postoperative period in patients undergoing SAVR or TAVI, by extensive neuropsychological study (NRP) and advanced Magnetic Resonance Imaging (MRI). Specifically, to compare early and late cognitive changes after the intervention between both groups, the occurrence of stroke during follow-up and to compare the appearance of silent vascular lesions and changes in brain activity and functional connectivity with functional MRI during follow-up between both groups. Methods/design Prospective longitudinal cohort study. A non-selected representative sample of 80 subjects, 40 SAVR and 40 TAVI to obtain a final sample of 36 eligible subjects in each group, ranging from 70 to 85 years old, with indication for aortic replacement and intermediate or high surgical risk will be studied. At baseline, within one month before the treatment, all individuals will undergo an extensive NRP and advanced MRI study. These studies will also be performed 1-month and 1-year after treatment, to assess the appearance of new vascular lesions, as well as changes in cognitive performance with respect to baseline. Discussion This study aims to evaluate changes in cognitive performance as well as both clinical and silent vascular events occurring in the early (1-month) and late (1-year) periods after SAVR and TAVI. We will also analyze the correlation between neuropsychological and neuroimaging approaches in order to evaluate cognition. Therefore, it may provide high-quality data of cognitive changes and vascular events for both techniques, and be useful to tailor interventions to individual characteristics and ultimately aiding in decision-making. Trial registration This study is register in Clinicaltrials.gov (NCT05235529) on 11th February 2022.

Abstract Background Streptococcus suis (S. suis) is a Gram-positive bacteria that infects pigs causing meningitis, arthritis, pneumonia, or endocarditis. This increases the mortality in pig farms deriving in severe economic losses. The use of saliva as a diagnostic fluid has various advantages compared to blood, especially in pigs. In this study, it was hypothesized that saliva could reflect changes in different biomarkers related to stress, inflammation, redox status, and muscle damage in pigs with S. suis infection and that changes in these biomarkers could be related to the severity of the disease. Results A total of 56 growing pigs from a farm were selected as infected pigs (n = 28) and healthy pigs (n = 28). Results showed increases in biomarkers related to stress (alpha-amylase and oxytocin), inflammation (haptoglobin, inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), total protein, S100A8-A9 and S100A12), redox status (advanced oxidation protein producs (AOPP)) and muscle damage (creatine kinase (CK), CK-MB, troponin I, lactate, aspartate aminotransferase, and lactate dehydrogenase). An increase in adenosine deaminase (ADA), procalcitonin, and aldolase in infected animals were also observed, as previously described. The grade of severity of the disease indicated a significant positive correlation with total protein concentrations, aspartate aminotransferase, aldolase, and AOPP. Conclusions This report revealed that S. suis infection caused variations in analytes related to stress, inflammation, redox status, and muscle damage in the saliva of pigs and these can be considered potential biomarkers for this disease.

Abstract Oxytocin has traditionally been known for its physiological effects on muscle contraction associated with birth and lactation, but in the last years is widely used as a biomarker of “positive experiences” in psychology and behavior. Different types of samples have been used for oxytocin measurements with saliva samples having the particular advantage of an easy and non-stressful collection. However, the low concentration of oxytocin in saliva can represent a limitation for its use. For this reason, sensitive assays and even a previous sample treatment in some cases are required for saliva oxytocin quantification. In addition, the lack of standardized and generally agreed-upon approach to peripheral oxytocin measurement leads to large discrepancies between different laboratories, that use different sample treatment protocols and different assays. The main objectives of this review are to describe the current status of the use of saliva for oxytocin measurement, provide details of the different sample processing techniques that can be applied and inform about the analytical techniques and assays available in different animal species, and also in humans for comparative purposes. It is expected that this information can contribute to an increase in the knowledge about the measurements of oxytocin in saliva and to its wider use in the future.

Abstract The main aim of this report was to investigate and compare the response of serum C-reactive protein (CRP) and ferritin, two positive acute phase proteins (APPs) which usually show an increase in inflammatory processes, in dogs with pyometra. For this purpose, two different studies were made. In the first one , both proteins were measured together in an APPs profile in 25 dogs with pyometra, 25 dogs with pancreatitis (as an example of a positive inflammatory control group), and in 25 healthy dogs. In the second study, to advance the knowledge of the changes and evolution of serum ferritin and CRP in dogs with pyometra after treatment, the concentrations of both APPs were analyzed in 30 dogs with pyometra at diagnosis and after ovariohysterectomy and in 10 clinically healthy female dogs before and after elective spaying. In both studies, bitches with pyometra showed significant increases in serum CRP, indicating an inflammatory condition, but not in serum ferritin despite being a moderate positive APP. This divergence between the dynamics of these APPs could be a useful tool for the suspicion of cases of canine pyometra.

Abstract Background The use of molecular biomarkers for COVID-19 remains unconclusive. The application of a molecular biomarker in combination with clinical ones that could help classifying aggressive patients in first steps of the disease could help clinician and sanitary system a better management of the disease. Here we characterize the role of ACE2, AR, MX1, ERG, ETV5 and TMPRSS2 for trying a better classification of COVID-19 through knowledge of the disease mechanisms. Methods A total of 329 blood samples were genotyped in ACE2, MX1 and TMPRSS2. RNA analyses were also performed from 258 available samples using quantitative polymerase chain reaction for genes: ERG, ETV5, AR, MX1, ACE2, and TMPRSS2. Moreover, in silico analysis variant effect predictor, ClinVar, IPA, DAVID, GTEx, STRING and miRDB database was also performed. Clinical and demographic data were recruited from all participants following WHO classification criteria. Results We confirm the use of ferritin (p

Abstract Background T cells have been implicated in the development and progression of inflammatory processes in chronic heart failure (CHF). Cardiac resynchronization therapy (CRT) has beneficial effects on symptoms and cardiac remodeling in CHF. However, its impact on the inflammatory immune response remains controversial. We aimed to study the impact of CRT on T cells in heart failure (HF) patients. Methods Thirty-nine HF patients were evaluated before CRT (T0) and six months later (T6). Quantification of T cells, their subsets, and their functional characterization, after in vitro stimulation, were evaluated by flow cytometry. Results T regulatory (Treg) cells were decreased in CHF patients (healthy group (HG): 1.08 ± 0.50 versus (heart failure patients (HFP)-T0: 0.69 ± 0.40, P = 0.022) and remaining diminished after CRT (HFP-T6: 0.61 ± 0.29, P = 0.003). Responders (R) to CRT presented a higher frequency of T cytotoxic (Tc) cells producing IL-2 at T0 compared with non-responders (NR) (R: 36.52 ± 12.55 versus NR: 24.71 ± 11.66, P = 0.006). After CRT, HF patients presented a higher percentage of Tc cells expressing TNF-α and IFN-γ (HG: 44.50 ± 16.62 versus R: 61.47 ± 20.54, P = 0.014; and HG: 40.62 ± 15.36 versus R: 52.39 ± 18.66, P = 0.049, respectively). Conclusion The dynamic of different functional T cell subpopulations is significantly altered in CHF, which results in an exacerbated pro-inflammatory response. Even after CRT, it seems that the inflammatory condition underlying CHF continues to evolve with the progression of the disease. This could be due, at least in part, to the inability to restore Treg cells levels. Trial registration: Observational and prospective study with no trial registration.

Abstract Background ALL is the most frequent hematological tumor in children, so during remission induction chemotherapy protocol (RICP) adverse events (AEs) may appear. The public program in Mexico in charge of financial support to oncologic children without social security delivered a fix amount for ALL chemotherapy, but additional money needed to treat any other unexpected condition should be taken from the budget of the oncologic healthcare providers. So the purpose of our study was to estimate and evaluate the direct medical costs associated to EAs during RICP in children with ALL. Methods This study was retrospective, longitudinal, and observational based on medical records review of patients in RICP. The CTCAE was used to identify and classify AEs according to a SOC category. We focused on extracting resources data that were consumed both for inpatients and outpatients AEs. A micro-costing approach was adopted which involve quantification of each healthcare resource consumed by the hospital multiplying them by unit cost. The probability distributions of data were evaluated to identify the appropriated statistical tests to be used for comparisons between groups that were performed with Wilcoxon rank sum test. Generalized linear models (GLM) were adjusted to evaluate the effects of patient characteristics on total cost. Results Forty patients accumulated 204 inpatient and 81 outpatient AEs during RICP. Comparison of total costs between groups showed an incremental cost of $7,460.23 likewise attributable to AEs. The total cost of a pediatric patient undergoing RICP without adverse events was $3,078.36 and the total cost of a patient with AEs exceeds it threefold. Conclusions The costs associated with AEs during RICP in Mexican children with ALL representing a high burden for the healthcare provider. Generalized linear models showed that variables such as sex, risk category and alive status are associated with the total costs of AEs. This is the first study aiming to analyze the effect of ALL-related AEs on health care costs in pediatric population, so our results may help not only to local decision making but also it may contribute to the research agenda in this field.

Abstract Objective The Department of Veterans Affairs’ (VA) electronic health records (EHR) offer a rich source of big data to study medical and health care questions, but patient eligibility and preferences may limit generalizability of findings. We therefore examined the representativeness of VA veterans by comparing veterans using VA healthcare services to those who do not. Methods We analyzed data on 3051 veteran participants age ≥ 18 years in the 2019 National Health Interview Survey. Weighted logistic regression was used to model participant characteristics, health conditions, pain, and self-reported health by past year VA healthcare use and generate predicted marginal prevalences, which were used to calculate Cohen’s d of group differences in absolute risk by past-year VA healthcare use. Results Among veterans, 30.4% had past-year VA healthcare use. Veterans with lower income and members of racial/ethnic minority groups were more likely to report past-year VA healthcare use. Health conditions overrepresented in past-year VA healthcare users included chronic medical conditions (80.6% vs. 69.4%, d = 0.36), pain (78.9% vs. 65.9%; d = 0.35), mental distress (11.6% vs. 5.9%; d = 0.47), anxiety (10.8% vs. 4.1%; d = 0.67), and fair/poor self-reported health (27.9% vs. 18.0%; d = 0.40). Conclusions Heterogeneity in veteran sociodemographic and health characteristics was observed by past-year VA healthcare use. Researchers working with VA EHR data should consider how the patient selection process may relate to the exposures and outcomes under study. Statistical reweighting may be needed to generalize risk estimates from the VA EHR data to the overall veteran population.

Abstract Background Procalcitonin (PCT) is a widely used biomarker of sepsis in human medicine and can have potential applications in the veterinary field. This study aimed to explore whether PCT could be measured in the saliva of pigs and whether its concentration changes in sepsis. Therefore, a specific assay was developed and analytically validated, and changes in PCT concentration were evaluated in two conditions: a) in an experimental model of sepsis produced by the administration of lipopolysaccharide (LPS) to pigs (n = 5), that was compared with a model of non-septic inflammation induced by turpentine oil (n = 4), and b) in healthy piglets (n = 11) compared to piglets with meningitis (n = 20), a disease that usually involves sepsis and whose treatment often requires large amounts of antibiotics in farms. Results The assay showed coefficients of variation within the recommended limits and adequate linearity after serial sample dilutions. The method's detection limit was set at 68 μg/L, and the lower limit of quantification was 414 μg/L. In the LPS experiment, higher concentrations of PCT were found after 24 h in the animals injected with LPS (mean = 5790 μg/L) compared to those treated with turpentine oil (mean = 2127 μg/L, P = 0.045). Also, animals with meningitis had higher concentrations of PCT (mean = 21515 μg/L) than healthy pigs (mean = 6096 μg/L, P value

Abstract Background Congenital brain tumors are extremely rare in the neonatal population, and often associated with a poor prognosis. The diagnostic suspicion is often aroused at antenatal scans or postnatally, if clinical signs and symptoms of increased intracranial pressure become evident. We present a case of definitely congenital glioblastoma multiforme incidentally diagnosed in a preterm infant, aiming to raise clinical awareness on this condition and to highlight the challenges of the related diagnostic work-up. Case presentation This female infant was born at 31 weeks’ gestation after an uneventful pregnancy. No abnormalities were detected at antenatal ultrasound scans and genetic tests. Head circumference at birth was on the 25th centile. A routine brain ultrasound scan performed on day 1 revealed a large, inhomogeneous lesion in the right cerebral hemisphere, with contralateral midline shift, which was confirmed by brain magnetic resonance imaging (MRI). Eye fundus and routine blood exams, including platelets count, coagulation screening and C-reactive protein, were normal. Given the high risk of complications, surgical biopsy of the lesion was temporarily hold and a daily sonographic follow-up was undertaken. Although head circumference growth was steady on the 25th centile, progressive changes of the lesion were detected by cranial ultrasound. The repeat MRI scans showed a significant enlargement of the mass, with contralateral midline shift and signs of intralesional and intraventricular bleeding. In view of this worsening, surgical resection was performed. The histological examination of the lesion biopsy documented a GFAP+ highly cellular neoplasm, with no mutation on SMARCB1 gene. At the molecular analysis, mutations on IDH and H3F3A genes were absent, whereas MGMT promoter was unmethylated. The diagnosis was grade IV glioblastoma IDH wild-type. Conclusions Congenital glioblastoma multiforme is an extremely rare but highly aggressive neoplasm. Since intralesional biopsy is not often feasible in affected neonates, knowledge of the associated clinical and neuroradiological features is particularly important, as they can also add useful information on the neoplasm behavior. Specimens from open surgical resection allow to perform a definite histological analysis and an extended molecular characterization, with relevant prognostic implications.

Abstract Background Identification of relevant in vivo biomarkers for fertility remains a challenge for the livestock industry. Concentrations of the small peptide hormone oxytocin (OXT), involved in male reproductive function and present in the seminal plasma (SP) of several species could be a robust one. This study characterized concentrations of SP-OXT in ejaculates from boars used in artificial insemination (AI) programs aiming to evaluate its relationship with sperm quality variables and in vivo fertility of their liquid-stored AI-semen. Seminal OXT concentrations (ng/mL) were measured in 169 ejaculates from 61 boars of the Duroc, Pietrain, Landrace and Large White breeds using a direct competitive immunoassay test based on AlphaLISA® technology. Ejaculate (ejaculate volume, sperm concentration, total sperm count) and sperm parameters (motility, viability, intracellular generation of reactive oxygen species, plasma membrane fluidity) were assessed at 0 h and 72 h in AI-semen samples stored at 17 °C. In vivo fertility included only 18 Large White and Landrace boars whose AI-semen was used to inseminated > 100 sows and evaluated both farrowing rate and litter size of 3,167 sows. Results The results showed that SP-OXT differed between boars and between ejaculates within boar (P

Abstract Background The possible use of oxytocin in saliva as an indicator of positive emotions in bovine species has been poorly investigated. In the present study, two new assays (one using a monoclonal antibody and the other using a polyclonal antibody) for the measurement of oxytocin in bovine saliva were developed and validated. Also, the changes in oxytocin in saliva were explored in two different situations. One was around parturition, and for this purpose, saliva samples from 13 cows were collected at three different times: 7 days before the parturition, the day of parturition and 7 days after the parturition. The second situation was weaning and grouping of calves, and for this purpose, saliva from 25 calves was collected at three different times: 1 day before weaning, 2 days after weaning or milk withdrawal and 4 days after grouping calves. Results In cows, oxytocin concentrations showed an increase on the day of parturition with both assays, while in calves, oxytocin concentrations showed a decrease 4 days after the grouping. Conclusions The assays validated in this report could be used for the measurement of oxytocin in bovine saliva and detect changes in this analyte that can occur in different physiological or productive situations such as parturition and weaning.

Abstract Background At present, the Americas report the largest number of cases of COVID-19 worldwide. In this region, Mexico is the third country with most deaths (20,781 total deaths). A sum that may be explained by the high proportion of people over 50 and the high rate of chronic diseases. The aim of this analysis is to investigate the risk factors associated with COVID-19 deaths in Mexican population using survival analysis. Methods Our analysis includes all confirmed COVID-19 cases contained in the dataset published by the Epidemiological Surveillance System for Viral Respiratory Diseases of the Mexican Ministry of Health. We applied survival analysis to investigate the impact of COVID-19 on the Mexican population. From this analysis, we plotted Kaplan-Meier curves, and constructed a Cox proportional hazard model. Results The analysis included the register of 16,752 confirmed cases of COVID-19 with mean age 46.55 ± 15.55 years; 58.02% (n = 9719) men, and 9.37% (n = 1569) deaths. Male sex, older age, chronic kidney disease, pneumonia, hospitalization, intensive care unit admission, intubation, and health care in public health services, were independent factors increasing the risk of death due to COVID-19 (p

Abstract Background Captive and free-ranging wild mammals have been recognized as potential reservoirs of Leishmania infantum infection. The aim of this study was to describe the first clinical case of leishmaniosis in the Eurasian otter. Case presentation A case of clinical leishmaniosis is reported in a 4-year-old male Eurasian otter housed at a wildlife park (Murcia, South Eastern Spain). The Eurasian otter showed bilateral epistaxis, anorexia, apathy, and weight loss. A complete blood cell count and biochemical analyses revealed hyperproteinemia, hyperglobulinemia, decreases of paraoxonase-1, increases of haptoglobin and ferritin, and proteinuria. Bilateral nephropathy with hydronephrosis, mesenteric lymphadenomegaly, and ascites were also observed. L. infantum infection was confirmed by microscopy (amastigotes were detected in macrophages from spleen aspirate), molecular diagnosis (L. infantum DNA was detected by real-time polymerase chain reaction), and serology (anti-Leishmania IgG2 antibodies were detected by time-resolved immunofluorometry). The animal was treated with allopurinol for 3 months and gained weight, the epistaxis disappeared, and the ferritin concentration decreased. Conclusions This is the first report of clinical leishmaniosis in the Eurasian otter. Our results suggest that Eurasian otters are susceptible to infection with L. infantum and can develop clinical leishmaniosis in endemic areas.

Abstract Background Most ovarian cancer patients are diagnosed at a late stage with 85% of them relapsing after surgery and standard chemotherapy; for this reason, new treatments are urgently needed. Ovarian cancer has become a candidate for immunotherapy by reason of their expression of shared tumor-associated antigens (TAAs) and private mutated neoantigens (NeoAgs) and the recognition of the tumor by the immune system. Additionally, the presence of intraepithelial tumor infiltrating lymphocytes (TILs) is associated with improved progression-free and overall survival of patients with ovarian cancer. The aim of active immunotherapy, including vaccination, is to generate a new anti-tumor response and amplify an existing immune response. Recently developed NeoAgs-based cancer vaccines have the advantage of being more tumor specific, reducing the potential for immunological tolerance, and inducing robust immunogenicity. Methods We propose a randomized phase I/II study in patients with advanced ovarian cancer to compare the immunogenicity and to assess safety and feasibility of two personalized DC vaccines. After standard of care surgery and chemotherapy, patients will receive either a novel vaccine consisting of autologous DCs pulsed with up to ten peptides (PEP-DC), selected using an agnostic, yet personalized, epitope discovery algorithm, or a sequential combination of a DC vaccine loaded with autologous oxidized tumor lysate (OC-DC) prior to an equivalent PEP-DC vaccine. All vaccines will be administered in combination with low-dose cyclophosphamide. This study is the first attempt to compare the two approaches and to use NeoAgs-based vaccines in ovarian cancer in the adjuvant setting. Discussion The proposed treatment takes advantage of the beneficial effects of pre-treatment with OC-DC prior to PEP-DC vaccination, prompting immune response induction against a wide range of patient-specific antigens, and amplification of pre-existing NeoAgs-specific T cell clones. Trial registration This trial is already approved by Swissmedic (Ref.: 2019TpP1004) and will be registered at http://www.clinicaltrials.gov before enrollment opens.

Abstract Background The biochemical components of saliva can change in certain pathologies in horses, for example in acute abdominal disease. The aim of this study was (1) to evaluate if a panel of biochemical analytes usually used in serum can be measured in saliva of horses and (2) to study the possible changes of these biochemical analytes in saliva of horses affected by acute abdominal disease. A panel of 23 analytes was analytically validated in saliva of horses and possible changes in these analytes in a pilot study with six healthy horses and six horses with acute abdominal disease were evaluated. The analytes with significant changes were then evaluated in a larger population of 20 healthy and 37 diseased horses. Results Seven analytes showed significant increases in the pilot study which were confirmed in the larger population. The analytes which showed significant changes, and their median fold increase and significance shown in the larger population were salivary γ-glutamyl transferase (gGT, 2.3 fold, P = 0.001), creatine kinase (CK, 6.2 fold, P

Abstract Background Biomarkers of oxidative stress in pigs have been measured in serum/plasma samples. However, blood collection in pigs can be highly stressful to the animals. Saliva is a biological fluid with several advantages in pigs over blood, since it can be easily collected without stress to the animals, being therefore an ideal sample in this species. The objective of this study was the validation of assays for the evaluation of oxidative stress status in saliva of pigs. For this purpose, three assays commonly used to evaluate the total antioxidant capacity (TAC): trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing ability of plasma (FRAP)), one individual antioxidant (uric acid) and two assays to evaluate oxidant concentrations (advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2)) were measured and validated in porcine saliva. In addition, the possible changes of these assays in sows’ saliva during lactation were be studied. Results The methods had intra- and inter-assays coefficient of variation lower than 15%. They also showed an adequate linearity and recovery, and their detection limits were low enough to detect the analytes in saliva of pigs. Overall the analytical validation tests showed that the assays used in our study are valid and reliable for the evaluation of oxidative stress in porcine saliva. In addition, it was observed that these salivary biomarkers can change in a situation of oxidative stress such as lactation in sows. Conclusions All assays for salivary biomarkers of oxidative stress evaluated in this study have demonstrated a high analytical accuracy and low imprecision. In addition, it has been observed that these biomarkers showed significant changes in a situation of oxidative stress such as lactation in sows. Therefore, this study opens a new possibility of using saliva as a non-invasive sample to evaluate oxidative stress in pigs.

Abstract Background The present work is aimed at evaluating the effect of different inclusion levels of a partially defatted black soldier fly (Hermetia illucens, L.; HI) larva meal for ducks. A total of 192 female 3-day-old Muscovy ducklings (Cairina moschata domestica, Canedins R71 L White, Grimaud Freres Selection, France) were divided into 4 groups, assigned 4 different dietary treatments (6 replicates/treatment and 8 birds/replicate) and reared from 3 to 50 days of age. HI larva meal was included at increasing levels (0, 3%, 6% and 9%, HI0, HI3, HI6 and HI9, respectively) in isonitrogenous and isoenergetic diets formulated for 3 feeding phases: starter (3–17 days of age), grower (18–38 days of age) and finisher (39–50 days of age). The growth performance and apparent total tract digestibility were evaluated during the trial using titanium dioxide as an inert marker (0.3% of inclusion). At 51 days of age, two birds per pen were slaughtered and histomorphological investigations were performed. Results The live weight and average daily gain showed a quadratic response to increasing HI meal in the grower period (minimum corresponding to the HI6 group). No effects of dietary inclusion levels were observed for the daily feed intake or feed conversion ratio. The apparent dry matter and organic matter digestibility were not affected by the dietary treatment. A linear decrease was observed for the crude protein apparent digestibility in the starter period (minimum for the HI9 groups). The ether extract apparent digestibility increased linearly during the grower and finisher periods (minimum for the HI0 group). The morphometric indices were not influenced by the dietary treatments. Conclusions The inclusion of up to 9% of HI partially defatted larva meal in the diet of ducks did not cause any effect on growth performance, as well as the apparent digestibility. Moreover, dietary HI inclusion preserved the physiological intestinal development.

Abstract Background Stroke is the most common neurological disease in the world. After the stroke, some people suffer a cognitive disability. Commercial videogames have been used after stroke for physical rehabilitation; however, their use in cognitive rehabilitation has hardly been studied. The objectives of this study were to analyze attention, processing speed, and working memory in patients with moderate stroke after an intervention with Wii Sports Resort and compared these results with a control group. Methods A pre-post design study was conducted with 30 moderate stroke patients aged 65 ± 15. The study lasted eight weeks. 15 participated in the intervention group and 15 belong to the control group. They were assessed in attention and processing speed (TMT-A and B) and working memory (Digit Span of WAIS-III). Parametric and effect size tests were used to analyze the improvement of those outcomes and compared both groups. Results At the baseline, there was no difference between TMT-A and B. A difference was found in the scalar score of TMT-B, as well as in Digit Backward Span and Total Digit Task. In TMT-A and B, the intervention group had better scores than the control group. The intervention group in the Digit Forward Span and the Total Digit obtained a moderate effect size and the control group also obtained a moderate effect size in Total Digit. In the Digit scalar scores, the control group achieved better results than the intervention group. Conclusions The results on attention, processing speed and working memory improved in both groups. However, according to the effect sizes, the intervention group achieved better results than the control group. In addition, the attention and processing speed improved more than the working memory after the intervention. Although more studies are needed in this area, the results are encouraging for cognitive rehabilitation after stroke.

Abstract Background The aim of this trial was to investigate the effects of different inclusion levels of a partially defatted black soldier fly (BSF, Hermetia illucens L.) larva meal on the growth performance, nutrient digestibility, blood profile, gut morphology and histological features of piglets. A total of 48 newly weaned piglets were individually weighed (initial body weight (IBW): 6.1 ± 0.16 kg) and randomly allocated to 3 dietary treatments (4 boxes as replicates/treatment and 4 animals/box). BSF larva meal was included at increasing levels (0% [BSF0], 5% [BSF5] and 10% [BSF10]) in isonitrogenous and isoenergetic diets formulated for two feeding phases: I (from d 1 to d 23) and II (from d 24 to d 61). The weight gain (WG), average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated for each feeding phase and for the whole trial. The haematochemical parameters and nutrient digestibility of the piglets were also evaluated. A total of 3 piglets per box were slaughtered on d 61 and the slaughtered piglets were submitted to morphometric investigations and histopathological examinations. Results No overall significant differences were observed for growth performance (P > 0.05), except for the ADFI of phase II, which showed a linear response to increasing BSF meal levels (P 0.05). Conclusions The obtained results show that a partially defatted BSF larva meal can be used as a feed ingredient in diets for weaned piglets without negatively affecting their growth performance, nutrient digestibility, blood profile, gut morphology or histological features.

Abstract Background Salivary alpha-amylase (sAA) is considered a non-invasive biomarker of acute stress that can be evaluated by changes in activity and concentration, and also by changes in its isoforms, although this last way of evaluation has never been used in veterinary medicine. This research evaluated the changes of sAA by three different ways in which sAA can be evaluated in an experimental acute stress model in six pigs based in a technique of temporarily restraining. These ways of evaluation were 1) activity by a spectrophotometric assay, 2) concentration by a fluorometric assay, and 3) isoforms of the enzyme by a Western blot. Results Although salivary cortisol significantly increased due to the stimulus of stress and all the pigs manifested signs of stress by high-pitched vocalization, sAA activity showed an increase of different degree in the six pigs after the stress stimulus, while sAA concentration showed decreases in four of the six pigs. sAA activity did not correlate with sAA concentration or salivary cortisol, and a low correlation was observed between sAA concentration and salivary cortisol (r = 0.48, p = 0.003). The inter-individual variability was higher in sAA activity than in sAA concentration and salivary cortisol. Finally, three possible isoforms of sAA at 154–160 kDa, 65–66 kDa and 59–60 kDa were observed that showed different dynamics after the stress induction. Conclusions Although this pilot study’s results should be taken with caution due to the low sample size, it reveals a different behavior between sAA activity and concentration in pig after an acute stressful stimulus leading to evident external signs of stress by high-pitched vocalization, and opens a new field for the evaluation of possible selected isoforms of sAA as potential biomarkers of stress.

Abstract Background In order to ensure sustainability of aquaculture production of carnivourous fish species such as the gilthead seabream (Sparus aurata, L.), the impact of the inclusion of alternative protein sources to fishmeal, including plants, has been assessed. With the aim of evaluating long-term effects of vegetable diets on growth and intestinal status of the on-growing gilthead seabream (initial weight = 129 g), three experimental diets were tested: a strict plant protein-based diet (VM), a fishmeal based diet (FM) and a plant protein-based diet with 15% of marine ingredients (squid and krill meal) alternative to fishmeal (VM+). Intestines were sampled after 154 days. Besides studying growth parameters and survival, the gene expression related to inflammatory response, immune system, epithelia integrity and digestive process was analysed in the foregut and hindgut sections, as well as different histological parameters in the foregut. Results There were no differences in growth performance (p = 0.2703) and feed utilization (p = 0.1536), although a greater fish mortality was recorded in the VM group (p = 0.0141). In addition, this group reported a lower expression in genes related to pro-inflammatory response, as Interleukine-1β (il1β, p = 0.0415), Interleukine-6 (il6, p = 0.0347) and cyclooxigenase-2 (cox2, p = 0.0014), immune-related genes as immunoglobulin M (igm, p = 0.0002) or bacterial defence genes as alkaline phosphatase (alp, p = 0.0069). In contrast, the VM+ group yielded similar survival rate to FM (p = 0.0141) and the gene expression patterns indicated a greater induction of the inflammatory and immune markers (il1β, cox2 and igm). However, major histological changes in gut were not detected. Conclusions Using plants as the unique source of protein on a long term basis, replacing fishmeal in aqua feeds for gilthead seabream, may have been the reason of a decrease in the level of different pro-inflammatory mediators (il1 β, il6 and cox2) and immune-related molecules (igm and alp), which reflects a possible lack of local immune response at the intestinal mucosa, explaining the higher mortality observed. Krill and squid meal inclusion in vegetable diets, even at low concentrations, provided an improvement in nutrition and survival parameters compared to strictly plant protein based diets as VM, maybe explained by the maintenance of an effective immune response throughout the assay.

Abstract Background 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. Results The spectrophotometric assays for urea and creatinine measurements in saliva of dogs showed intra- and inter-assay imprecision lower than 12% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed median salivary concentrations of urea of 39.6 mg/dL and creatinine of 0.30 mg/dL, whereas dogs with CKD showed median salivary urea of 270.1 mg/dL and creatinine of 1.86 mg/dL. Positive high correlations were found between saliva and serum activities of the two analytes (urea, r = 0.909; P

Abstract Background The resilience to face disease is a process of positive adaptation despite the loss of health. It involves developing vitality and skills to overcome the negative effects of adversity, risks, and vulnerability caused by disease. In Mexico, the Mexican Resilience Measurement Scale (RESI-M) has been validated with a general population and has a five-factor structure. However, this scale does not allow evaluation of resilience in specific subpopulations, such as caregivers. Method This study investigated the psychometric properties of RESI-M in 446 family caregivers of children with chronic diseases. A confirmatory factor analysis (CFA) was performed, internal consistency values were calculated using Cronbach’s alpha coefficient, and mean comparisons were determined using t-tests. Results The expected five-factor model showed an adequate fit with the data based on a maximum likelihood test. The internal consistency for each factor ranged from .76 to .93, and the global internal consistency was .95. No average difference in RESI-M and its factors was found between women and men. Conclusion The RESI-M showed internal consistency and its model of five correlated factors was valid among family caregivers of children with chronic diseases.

Abstract Background Canine heartworm infection is characterized by pulmonary endarteritis and pulmonary hypertension (PH). The aim of the present study was to evaluate the relationship between PH with the concentrations of different positive (C-reactive protein [CRP] and haptoglobin [Hp]) and negative (albumin and paraoxonase-1 [PON-1]) acute phase proteins (APP), as well as the oxidative stress, by measuring glutathione peroxidase (GPx) and total antioxidant capacity (TAC) in 27 heartworm-infected dogs on Day 0 (diagnosis) and Day 120 (1 month after the last adulticide injection). Presence/absence of PH was determined by the Right Pulmonary Artery Distensibility (RPAD) Index. Results On Day 0, 62.9% of the dogs showed PH. Concentrations of CRP and Hp were higher in dogs with PH, especially in dogs with moderate-severe PH (P

Abstract Background Muscle enzymes in saliva have been reported to be possible markers of heart and muscle damage in humans. The aim of this study was to assess if Creatine kinase (CK) and Aspartate aminotransferase (AST) activities could be measured in canine saliva, and to evaluate their possible changes in situations of muscle damage. Results The spectrophotometric assays for CK and AST measurement in saliva of dogs showed intra- and inter-assay imprecision lower than 1 and 16% and coefficients of correlation close to 1 in linearity under dilution tests. Healthy dogs showed activities in saliva of CK between 27 and 121 U/L and AST between 46 and 144 U/L, whereas in saliva of dogs with muscle damage CK ranged between 132 and 3862 U/L and AST between 154 and 4340 U/L. Positive moderate correlations were found between saliva and serum activities of the two enzymes (CK, r = 0.579; P = 0.001; AST, r = 0.674; P = 0.001). Conclusions CK and AST activities can be measured in canine saliva with commercially available spectrophotometric assays. In addition these enzymes show higher values in saliva of dogs with muscle damage and their values are moderately correlated with those of serum.

Abstract Background The study aimed to determine the apparent total tract digestibility coefficients (ATTDC) of nutrients, the apparent metabolizable energy (AME and AMEn) and the amino acid (AA) apparent ileal digestibility coefficients (AIDC) of a partially defatted (BSFp) and a highly defatted (BSFh) black soldier fly larvae meal. The experimental diets were: a basal diet and two diets prepared by substituting 250 g/kg (w/w) of the basal diet with BSFp or BSFh, respectively. Results Significant differences were found between BSFp and BSFh meals for ATTDC of the nutrients: BSFp resulted more digestible than BSFh, except for ATTDC of CP which did not differed between meals, while a statistical trend was observed for ATTDC of DM and EE. The AME and AMEn values were significantly (P

An amendment to this paper has been published and can be accessed via the original article.

Abstract Background Canine monocytic ehrlichiosis (CME) is a tick-borne disease with a global distribution, caused by Ehrlichia canis. The inflammatory response to E. canis infection includes changes in certain acute phase proteins (APP) and in biomarkers of the oxidative status. APP responses are considered part of the innate immune response to CME. The aim of this study was to evaluate the APP and oxidative marker responses in dogs vaccinated against CME with an attenuated vaccine and subsequently challenged with a wild E. canis strain. Methods The study included 3 groups of 4 beagle dogs. Group 1 dogs were inoculated subcutaneously with an attenuated E. canis vaccine on day 0, and again on day 213. Group 2 initially served as controls for group 1 during the vaccination phase and then vaccinated once on day 213. Group 3 consisted of naïve dogs which constituted the control group for the challenge phase. All 12 dogs were infected intravenously with a wild strain of E. canis on day 428 of the study. APP levels were serially measured during two periods: days 0–38 post-vaccination (groups 1 and 2) and days 0–39 post-challenge (groups 1, 2, 3). Results Changes in C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, albumin, paraoxonase-1 (PON-1) and total antioxidant capacity (TAC) were of significantly smaller magnitude in vaccinated dogs and appeared later on a time scale compared to unvaccinated dogs challenged with a wild strain. Alterations in the level of APP during the vaccination phase of the study were of lower extent compared to those in the challenged unvaccinated dogs during the post-challenge phase. Positive APP levels correlated positively with the rickettsial load, body temperature and negatively with the thrombocyte counts (p