Antimicrobial and toxicological evaluation of Origanum vulgare: an in vivo study / Avaliação antimicrobiana e toxicológica de Origanum vulgare: um estudo in vivo

Origanum vulgare has been of great interest in academia and pharma industry due to its antioxidant, antifungal and antitumor properties. The present study aimed to find the anti-MRSA potential and in vivo toxicity assessments of O. vulgare. O. vulgare extract was used to monitor anti-MRSA activity in mice. Following MRSA established infection in mice (Mus musculus), treatment with O. vulgare was continued for 7 days. Autopsies were performed and re-isolation, gross lesion scoring and bacterial load in various organs were measured. Additionally, blood sample was analysed for hematological assays. Toxicity assessment of O. vulgare potential as medicine was done at 200 mg/kg and 400 mg/kg by evaluating liver and kidney functions. Bacterial load and gross lesion in lungs and heart were significantly low compared to positive control following O. vulgare treatment. Likewise, O. vulgare treated groups had hematological, neutrophil and TLC values similar to control groups. Increased AST, ALP and total bilirubin along with marked hepatocellular degeneration and distortion around the central vein, inflammatory cell infiltration, and cytoplasmic vacuolization of hepatic cells was observed at higher dose. It is concluded that crude extract of O. vulgare may contain beneficial secondary metabolites and in future may be explored for curing infectious diseases.

Origanum vulgare tem despertado grande interesse na academia e na indústria farmacêutica devido às suas propriedades antioxidantes, antifúngicas e antitumorais. O presente estudo teve como objetivo encontrar o potencial anti-MRSA e avaliações de toxicidade in vivo de O. vulgare. O extrato de O. vulgare foi usado para monitorar a atividade anti-MRSA em camundongos. Após infecção estabelecida por MRSA em camundongos (Mus musculus), o tratamento com O. vulgare foi continuado por 7 dias. As autópsias foram realizadas e o reisolamento, pontuação das lesões grosseiras e carga bacteriana em vários órgãos foram medidos. Além disso, a amostra de sangue foi analisada para ensaios hematológicos. A avaliação da toxicidade do potencial de O. vulgare como medicamento foi feita com 200 mg / kg e 400 mg / kg, avaliando as funções hepática e renal. A carga bacteriana e as lesões graves nos pulmões e no coração foram significativamente baixas em comparação com o controle positivo após o tratamento com O. vulgare. Da mesma forma, os grupos tratados com O. vulgare apresentaram valores hematológicos, de neutrófilos e de TLC semelhantes aos grupos de controle. Aumento de AST, ALP e bilirrubina total juntamente com degeneração hepatocelular marcada e distorção ao redor da veia central, infiltração de células inflamatórias e vacuolização citoplasmática de células hepáticas foram observados em doses mais altas. Conclui-se que o extrato bruto de O. vulgare pode conter metabólitos secundários benéficos e, no futuro, pode ser explorado para a cura de doenças infecciosas.

The rapid Bethesda assay is equivalent to the standard Bethesda assay for detection of factor IX inhibitors in patients with severe haemophilia B.

INTRODUCTION: The time-dependent nature of factor VIII (FVIII) inhibitors is well described, and the standard FVIII Bethesda assay used to measure inhibitors incorporates a 2-hour incubation. Despite case reports and reviews describing the immediate-acting nature of factor IX (FIX) inhibitors, many coagulation laboratories continue to use a traditional prolonged incubation for FIX Bethesda assays. To our knowledge, a comprehensive evaluation of the FIX Bethesda assay without incubation has not been reported. AIM: The goal of this study was to evaluate the performance of a rapid FIX Bethesda (ie no incubation) compared with the standard Bethesda assay (2-hour incubation). METHODS: The analysis used a Bethesda assay configured for either immediate testing or a 2-hour incubation. Samples from 14 haemophilia B patients with inhibitors and 9 non-human controls were tested. RESULTS: The two assays yielded similar performance overall. The average per cent difference in inhibitor titre between the rapid and standard FIX Bethesda assay was -3% (range -15% to +13%; P = .175) for patient samples and -2% (range -17% to +14%; P = .376) for controls. CONCLUSION: The rapid Bethesda assay showed good agreement with the standard Bethesda assay for determination of inhibitor levels in patients with severe haemophilia B. The rapid assay allows for faster assessment of inhibitors in patients with severe haemophilia B and has the potential to improve the ability of the coagulation laboratory to perform testing from a logistical viewpoint. Further studies involving larger numbers of patients would be important to confirm our findings.

Exposure to organic and inorganic traffic-related air pollutants alters haematological and biochemical indices in albino mice Mus musculus.

The relationship between air pollution exposure and haematology remains controversial. Evidences in the effect of trace organic air pollutants and in the impact of such exposure on lipid and protein levels are scarce. This work investigated the health effects of medium-term exposure to traffic-related air pollution on both haematological and biochemical indices in animal models. Two groups of albino mice (Mus musculus) were exposed to ambient air polluted by vehicle exhaust for three and six months, and one group was kept as control. Results found significant depletions (p < 0.05) in red blood cells, packed cell volume, neutrophils, eosinophils, monocytes, and total cholesterol after air pollution exposure. On the contrary, significant elevations (p < 0.05) were observed in platelet, lymphocytes, and serum albumin compared to control condition. Correlation data suggested that significant changes in blood parameters may be altered by the synergistic effect of several organic and inorganic air pollutants.

Social hierarchy position in female mice is associated with plasma corticosterone levels and hypothalamic gene expression.

Social hierarchies emerge when animals compete for access to resources such as food, mates or physical space. Wild and laboratory male mice have been shown to develop linear hierarchies, however, less is known regarding whether female mice have sufficient intrasexual competition to establish significant social dominance relationships. In this study, we examined whether groups of outbred CD-1 virgin female mice housed in a large vivaria formed social hierarchies. We show that females use fighting, chasing and mounting behaviors to rapidly establish highly directionally consistent social relationships. Notably, these female hierarchies are less linear, steep and despotic compared to male hierarchies. Female estrus state was not found to have a significant effect on aggressive behavior, though dominant females had elongated estrus cycles (due to increased time in estrus) compared to subordinate females. Plasma estradiol levels were equivalent between dominant and subordinate females. Subordinate females had significantly higher levels of basal corticosterone compared to dominant females. Analyses of gene expression in the ventromedial hypothalamus indicated that subordinate females have elevated ERα, ERß and OTR mRNA compared to dominant females. This study provides a methodological framework for the study of the neuroendocrine basis of female social aggression and dominance in laboratory mice.

Regeneration-Competent and -Incompetent Murids Differ in Neutrophil Quantity and Function.

Regeneration is rare in mammals, but spiny mice (Acomys spp.) naturally regenerate skin and ear holes. Inflammation is thought to inhibit regeneration during wound healing, but aspects of inflammation contribute to both regeneration and pathogen defense. We compared neutrophil traits among uninjured, regeneration-competent (Acomys: A. cahirinus, A. kempi, A. percivali) and -incompetent (Mus musculus: Swiss Webster, wild-caught strains) murids to test for constitutive differences in neutrophil quantity and function between these groups. Neutrophil quantity differed significantly among species. In blood, Acomys had lower percentages of circulating neutrophils than Mus; and in bone marrow, Acomys had higher percentages of band neutrophils and lower percentages of segmented neutrophils. Functionally, Acomys and Mus neutrophils did not differ in their ability to migrate or produce reactive oxygen species, but Acomys neutrophils phagocytosed more fungal zymosan. Despite this enhanced phagocytosis activity, Acomys neutrophils were not more effective than Mus neutrophils at killing Escherichia coli. Interestingly, whole blood bacteria killing was dominated by serum in Acomys versus neutrophils only or neutrophils and serum in Mus, suggesting that Acomys primarily rely on serum to kill bacteria whereas Mus do not. These subtle differences in neutrophil traits may allow regeneration-competent species to offset damaging effects of inflammation without compromising pathogen defense.

Assessing Blood Clotting and Coagulation Factors in Mice.

The mammalian blood coagulation system was designed to restrict blood loss due to injury as well as keep the blood fluid within the blood vessels of the organism. Blood coagulation activity in inbred mouse strains varies widely among strains, suggesting that many genomic variants affect hemostasis. Some of these molecules have been discovered and characterized; however, many are still unknown. Genetically modified mouse technologies are providing a plethora of new mouse models for investigating the regulation of blood coagulation. Here we provide a protocol for the tail bleeding time as a primary assessment of in vivo blood coagulation, as well as in vitro methods such as the prothrombin time, activated partial thromboplastin time, and thrombin generation assay. We also provide protocols for the assessment of the activities of specific known factors involved in blood coagulation. © 2019 by John Wiley & Sons, Inc.

Systematic evaluation of graphene quantum dot toxicity to male mouse sexual behaviors, reproductive and offspring health.

Graphene quantum dots (GQDs) have attracted considerable attention across multiple fields, particularly biomedical research. However, the effects of GQDs on reproductive and offspring health in mammals are unclear. Here, we show that GQD exposure via oral gavage or intravenous injection had no effect on the frequency and timing of sexual behaviors in male mice. GQD-exposed male mice retained healthy structural and functional reproductive physiology (e.g., production and storage of healthy sperm, maintenance of normal total protein and key enzyme concentrations in testes) of the testes and epididymides, as well as normal testosterone levels. Female mice housed with GQD-exposed males produced first, second, and subsequent litters of healthy pups without obvious differences to females housed with buffer-treated males. These findings may be explained by the low toxicity of GQDs in germ cells and their rapid excretion after exposure in mice, mainly via the urine and/or feces; GQDs, even at high doses, are virtually undetectable in male mouse testis, epididymis, and brain. Our findings reveal the short- and long-term effects of GQD exposure on male mouse sexual behaviors, reproductive activity, and offspring development and indicate the potential mechanisms of action of GQDs to provide further insight into their bio-safety.

The mitochondria-targeting peptide elamipretide diminishes circulating HtrA2 in ST-segment elevation myocardial infarction.

BACKGROUND: The extent of myocardial damage in patients with ST-segment elevation myocardial infarction (STEMI) depends on both the time to reperfusion as well as injury induced by ischaemia-reperfusion resulting in a cascade of cellular and humoral reactions. As a consequence of ischaemia-reperfusion in the heart, the high-temperature requirement serine peptidase 2 (HtrA2) is translocated from the mitochondria to the cytosol, whereupon it induces protease activity-dependent apoptosis mediated via caspases. Myocardial damage induced by reperfusion cannot be monitored due to a current lack in specific biomarkers. We examined the serum level of HtrA2 as a potentially novel biomarker for mitochondrial-induced cardiomyocyte apoptosis. METHODS: After informed consent, peripheral blood was obtained from patients (n=19) with first-time acute anterior STEMI after percutaneous coronary intervention. Within this group, 10 of the patients received the mitochondria-targeting peptide elamipretide (phase 2a clinical study EMBRACE (NCT01572909)). Blood was also obtained from a control group of healthy donors (n=16). The serum level of HtrA2 was measured by an enzyme-linked immunosorbent assay (ELISA). In a murine model of myocardial ischaemia-reperfusion injury, HtrA2 was determined in plasma by ELISA after left anterior descending artery occlusion. RESULTS: HtrA2 median was significantly increased in patients with STEMI compared to healthy controls 392.4 (240.7-502.8) pg/mL vs. 1805.5 (981.3-2220.1) pg/mL (P⩽0.05). Elamipretide significantly reduced the HtrA2 median serum level after myocardial infarction 1805.5 (981.3-2220.1) pg/mL vs. 496.5 (379.4-703.8) pg/mL (P⩽0.05). Left anterior descending artery occlusion in mice significantly increased HtrA2 mean in plasma (117.4 fg/ml±SEM 28.1 vs. 525.2 fg/ml±SEM 96; P⩽0.05). CONCLUSION: Compared to healthy controls, we found significantly increased serum levels of HtrA2 in patients with STEMI. The result was validated in a murine model of myocardial ischaemia-reperfusion injury. In humans the increased serum level was significantly reduced by the mitochondria-targeting peptide elamipretide. In conclusion, HtrA2 is detectable in serum of patients with STEMI and might present a novel biomarker for mitochondrial-induced cardiomyocyte apoptosis. Consequently, HtrA2 may also show promise as a biomarker for the identification of ischaemia-reperfusion injury. However, this must be validated in a lager clinical trial.

A comparative study on effects of static electric field and power frequency electric field on hematology in mice.

With the development of the ultra high voltage transmission technology, the voltage level of transmission line rised. Accordingly, the strength of electric field in the vicinity of transmission line increased, thus possible health effects from electric field have caused many public attentions. In this study, in order to compare effects induced by static electric field (SEF) and power frequency electric field (PFEF) on immune function, Institute of Cancer Research (ICR) mice were exposed to 35 kV/m SEF (0 Hz) and PFEF (50 Hz),respectively. Several indicators of white blood cell, red blood cell as well as hemoglobin in peripheral blood were tested after exposure of 7, 14 and 21 days, respectively. There was no significant difference in any indicators under SEF exposure of 35 kV/m for 7d, 14d and 21d between experimental group and control group. Under the PFEF exposure of 35 kV/m, white blood cell count significantly reduced after exposure of 7d, 14d and 21d. Meanwhile, red blood cell count significantly reduced after exposure of 7d, and returned to normal level through the compensatory response of organism after exposure of 14d and 21d. Hemoglobin concentration significantly decreased only after exposure of 21d. Based on tested results of hematological indicators, SEF exposure of 35 kV/m did not affect immune functions in mice but PFEF exposure of 35 kV/m could cause a decline of immune function. This difference of effects from SEF and PFEF on immune function was possibly caused by the difference of the degree of molecular polarization and ion migration in organism under exposure of two kinds of electric fields.

Effect of dimerized melittin on gastric cancer cells and antibacterial activity.

Melittin is the peptide toxin found in bee venom and is effective against cancer cells. To enhance its activity, a branched dimeric form of melittin was designed. The monomeric form of the peptide was more cytotoxic against gastric cancer cells at low concentrations (1-5 µM) than the dimer form, while the cytotoxic effect was comparable at higher concentrations (10 µM). Confocal microscopy showed that both the monomer and dimer forms of melittin with fluorescent label at the C terminus penetrated the cytoplasm and localized at the cell nucleus and disrupted the cell membrane. The results indicated that both peptides localized in the nucleus and no significant difference in penetration was observed between monomer and dimer of melittin. Although the C and N termini are important for melittin activity, using C terminus for dimerization of the peptide resulted in similar activity for the monomer and dimer against bacteria and gastric cancer cells.

Assessment of endogenous 25-hydroxyvitamin D serum concentrations by liquid chromatography-tandem mass spectrometry in various animal species.

BACKGROUND: Mass spectrometry (MS) has become the preferential method for the analysis of vitamin D in the clinic, yet no single platform is utilized for preclinical species in drug development studies. For vitamin D, the MS platform can provide certain benefits such as applicability of a single assay for multiple species, low cost, and high specificity. OBJECTIVES: A quantitative liquid chromatography-tandem MS (LC-MS/MS) assay for 25-hydroxyvitamin D3 (25OHD3 ) and D2 (25OHD2 ) was validated for rat, dog, mouse, and monkey, and suitability for drug development studies was assessed. METHODS: Standards were used to determine intra- and inter-assay accuracy and precision for LC-MS/MS. Extraction recovery and carryover due to instrumentation were determined. Repeat analyses of pooled serum samples from rat, dog, mouse, and monkey were assessed for precision, and other serum samples were used to determine the normal range in each species and detect biologically relevant changes. RESULTS: For both 25OHD3 and 25OHD2 , inaccuracy was ≤ 6%, and imprecision was ≤ 13%. Extraction recovery was 75% for 25OHD3 and 72% for 25OHD2 , and carryover was ≤ 0.1%. Measurable concentrations of 25OHD3 were recorded in serum samples from all species tested, but no 25OHD2 as diets were only fortified with 25OHD3 . This dataset provides preliminary information for the determination of RIs for 25OHD3 in rat, dog, mouse, and monkey with the LC-MS/MS platform. CONCLUSIONS: The LC-MS/MS assay was accurate and precise for determination of endogenous concentrations of 25OHD3 in serum samples from drug development studies in rat, dog, mouse, and monkey.

Effects of activity, genetic selection and their interaction on muscle metabolic capacities and organ masses in mice.

Chronic voluntary exercise elevates total daily energy expenditure and food consumption, potentially resulting in organ compensation supporting nutrient extraction/utilization. Additionally, species with naturally higher daily energy expenditure often have larger processing organs, which may represent genetic differences and/or phenotypic plasticity. We tested for possible adaptive changes in organ masses of four replicate lines of house mice selected (37 generations) for high running (HR) compared with four non-selected control (C) lines. Females were housed with or without wheel access for 13-14 weeks beginning at 53-60 days of age. In addition to organ compensation, chronic activity may also require an elevated aerobic capacity. Therefore, we also measured hematocrit and both citrate synthase activity and myoglobin concentration in heart and gastrocnemius. Both selection (HR versus C) and activity (wheels versus no wheels) significantly affected morphological and biochemical traits. For example, with body mass as a covariate, mice from HR lines had significantly higher hematocrit and larger ventricles, with more myoglobin. Wheel access lengthened the small intestine, increased relative ventricle and kidney size, and increased skeletal muscle citrate synthase activity and myoglobin concentration. As compared with C lines, HR mice had greater training effects for ventricle mass, hematocrit, large intestine length and gastrocnemius citrate synthase activity. For ventricle and gastrocnemius citrate synthase activity, the greater training was quantitatively explainable as a result of greater wheel running (i.e. 'more pain, more gain'). For hematocrit and large intestine length, differences were not related to amount of wheel running and instead indicate inherently greater adaptive plasticity in HR lines.

Discovery of natural mouse serum derived HIV-1 entry inhibitor(s).

Among rationally designed human immunodeficiency virus 1 (HIV-1) inhibitors, diverse natural factors have showed as potent anti-HIV activity in human blood. We have discovered that the boiled supernatant of healthy mouse serum could suppress HIV-1 entry, and exhibited reduced inhibitory activity after trypsin digestion. Further analysis demonstrated that only the fraction containing 10-25 K proteins could inhibit HIV-1 mediated cell-cell fusion. These results suggest that the 10-25 K protein(s) is novel natural HIV-1 entry inhibitor(s). Our findings provide important information about novel natural HIV entry inhibitors in mouse serum.

Murine Monocytes: Origins, Subsets, Fates, and Functions.

Monocytes are short-lived mononuclear phagocytes that circulate in the bloodstream and comprise two main subpopulations that in the mouse are best defined by the Ly6C marker. Intravascular functions of "classical" Ly6C+ monocytes and their interactions with other lymphoid and myeloid leukocytes in the circulation remain poorly understood. Rather, these cells are known to efficiently extravasate into tissues. Indeed, Ly6C+ monocytes and their descendants have emerged as a third, highly plastic and dynamic cellular system that complements the two classical, tissue-resident mononuclear phagocyte compartments, i.e., macrophages and dendritic cells, on demand. Following recruitment to injured tissue, Ly6C+ monocytes respond to local cues and can critically contribute to the initiation and resolution of inflammatory reactions. The second main murine monocyte subset, Ly6C- cells, derive in steady state from Ly6C+ monocytes and remain in the vasculature, where the cells act as scavengers. Moreover, a major fraction of Ly6C- monocytes adheres to the capillary endothelium and patrols the vessel wall for surveillance. Given the central role of monocytes in homeostasis and pathology, in-depth study of this cellular compartment can be highly informative on the health state of the organism and provides an attractive target for therapeutic intervention.

Effects of Embryo Transfer on Emotional Behaviors in C57BL/6 Mice.

Microbiologic standardization plays a key role in the management of animal facilities because contamination of stock could affect the health status and wellbeing of animals and thereby induce artifacts in biomedical research. One common method to avoid the dissemination of pathogens is embryo transfer (ET). Although disturbances in the perinatal environment may cause long-lasting effects on the behavior and physiology of mouse offspring, the influences of ET during this sensitive phase have not yet been addressed. Our study investigated the effects of various components of ET (anesthesia, surgery, recipient strain) on the behavior of dams (exploration, nest-building) and offspring (nest-building, exploration, anxiety, and social and depressive-like behaviors). For ET, the donor strain C57BL/6N and a standard protocol were used. Whereas treatment with anesthesia-analgesia did not affect maternal behavior, female offspring demonstrated overall effects on weight gain and corticosterone levels. Compared with naturally delivered female offspring, dams obtained through ET demonstrated decreased exploration and nest-building. In addition, female ET-derived offspring had enhanced levels of anxiety and increased social interest. Furthermore, ET-derived dams obtained by using NMRI as the recipient strain showed increased exploratory behavior compared with that of dams obtained by using C57 mice as recipients. Compared with using C57 as recipients, both sexes of offspring transferred into NMRI recipients weighed more, and female mice showed a depressive-like phenotype. Our findings suggest that ET, now considered to be a routine procedure in animal husbandry, bears the risk of introducing artifacts.

Comparison of Submental Blood Collection with the Retroorbital and Submandibular Methods in Mice (Mus musculus).

Nonterminal blood sample collection of sufficient volume and quality for research is complicated in mice due to their small size and anatomy. Large (>100 µL) nonterminal volumes of unhemolyzed or unclotted blood currently are typically collected from the retroorbital sinus or submandibular plexus. We developed a third method-submental blood collection-which is similar in execution to the submandibular method but with minor changes in animal restraint and collection location. Compared with other techniques, submental collection is easier to perform due to the direct visibility of the target vessels, which are located in a sparsely furred region. Compared with the submandibular method, the submental method did not differ regarding weight change and clotting score but significantly decreased hemolysis and increased the overall number of high-quality samples. The submental method was performed with smaller lancets for the majority of the bleeds, yet resulted in fewer repeat collection attempts, fewer insufficient samples, and less extraneous blood loss and was qualitatively less traumatic. Compared with the retroorbital technique, the submental method was similar regarding weight change but decreased hemolysis, clotting, and the number of overall high-quality samples; however the retroorbital method resulted in significantly fewer incidents of insufficient sample collection. Extraneous blood loss was roughly equivalent between the submental and retroorbital methods. We conclude that the submental method is an acceptable venipuncture technique for obtaining large, nonterminal volumes of blood from mice.

Immunological models in high dilution research following M Bastide.

In 1994, Madeleine Bastide described experimental models in immunology that were used during the 1980s to investigate high dilution effects on several biological systems. She classified the available papers in four categories: High dilutions of antigens; High dilutions of thymus, bursa and other hormones; High dilutions of cytokines; Immunopharmacological activity of silica. The studies about high dilutions of antigens were not continued after this period, but gave rise to a long process of a series of in vitro models on antigens and histamine dilutions, that led to the demonstration of the biological modulation effects of these preparations on basophil degranulation. During this process, a multi-centre study was performed, with a high degree of reproducibility among different independent laboratories. The studies about high diluted cytokines, thymulin and other hormones opened a new line of scientific investigation, about the regulatory properties of endogenous substances prepared according to homeopathic methods. The most frequently studied substance, thymulin, when administered to mice at 5cH potency, is able to improve the activity of phagocytes in different experimental situations, such as viral, bacterial and parasitic infections. The immunopharmacological activity of silica was demonstrated, at that time, as an in vivo illustration of the homeopathic 'similia principle'. More recently, studies on silica have assumed another focus: the putative role of silica as active contaminant present in high dilutions. This paper presents a follow-up summary on these items, considering the evolution of discoveries from 1994 to 2014.

Evaluation of a novel technique for intraperitoneal injections in mice.

Intraperitoneal injection is a common technique that safely delivers a substance into the peritoneal cavity but can induce high stress in animals. The authors have developed a new method for administering intraperitoneal injections in mice, with the goal of causing less stress during handling and injection. Here, they compare their novel technique with a conventional technique in three experiments. In the first experiment, the authors administered intraperitoneal injections of contrast medium using either technique and then used micro-computed tomography to evaluate the placement and retention of the medium. In the second and third experiments, the authors administered intraperitoneal injections or control treatments, then sampled blood to determine circulating concentrations of stress-related hormones. Imaging showed that both the novel and the conventional techniques properly delivered a contrast medium into the peritoneal cavity. The novel technique was also associated with lower concentrations of stress-related hormones than was the conventional technique. These results indicate that this novel technique might be beneficial to investigators that use intraperitoneal injections with mice.