Unified Methodology for the Primary Preclinical In Vivo Screening of New Anticoagulant Pharmaceutical Agents from Hematophagous Organisms.

The development of novel anticoagulants requires a comprehensive investigational approach that is capable of characterizing different aspects of antithrombotic activity. The necessary experiments include both in vitro assays and studies on animal models. The required in vivo approaches include the assessment of pharmacokinetic and pharmacodynamic profiles and studies of hemorrhagic and antithrombotic effects. Comparison of anticoagulants with different mechanisms of action and administration types requires unification of the experiment scheme and its adaptation to existing laboratory conditions. The rodent thrombosis models in combination with the assessment of hemostasis parameters and hematological analysis are the classic methods for conducting preclinical studies. We report an approach for the comparative study of the activity of different anticoagulants in vivo, including the investigation of pharmacodynamics and the assessment of hemorrhagic effects (tail-cut bleeding model) and pathological thrombus formation (inferior vena cava stenosis model of venous thrombosis). The reproducibility and uniformity of our set of experiments were illustrated on unfractionated heparin and dabigatran etexilate (the most common pharmaceuticals in antithrombic therapy) as comparator drugs and an experimental drug variegin from the tick Amblyomma variegatum. Variegin is notorious since it is a potential analogue of bivalirudin (Angiomax, Novartis AG, Basel, Switzerland), which is now being actively introduced into antithrombotic therapy.

Anti-smoking drugs cytisine and varenicline reduce cardiac reperfusion injury in rat model of myocardial ischemia.

Evidence to date indicates that activation of nicotinic acetylcholine receptors (nAChRs) can reduce cardiac injury from ischemia and subsequent reperfusion. The use of nAChR agonists in various animal models leads to a reduction in reperfusion injury. Earlier this effect was shown for the agonists of α7 nAChR subtype. In this work, we demonstrated the expression of mRNA encoding α4, α6 and ß2 nAChR subunits in the left ventricle of rat heart. In a rat model of myocardial ischemia, we studied the effect of α4ß2 nAChR agonists cytisine and varenicline, medicines used for the treatment of nicotine addiction, and found them to significantly reduce myocardium ischemia-reperfusion injury, varenicline manifesting a higher protection. Dihydro-ß-erythroidine, antagonist of α4ß2 nAChR, as well as methyllycaconitine, antagonist of α7 and α6ß2-containing nAChR, prevented protective effect of varenicline. This together with the presence of α4, α6 and ß2 subunit mRNA in the left ventricule of rat heart raises the possibility that the varenicline effect is mediated by α4ß2 as well as by α7 and/or α6ß2-containing receptors. Our results point to a new way for the use of cytisine and varenicline as cardioprotective agents.

C-Terminal Region of Caveolin-3 Contains a Stretch of Amino Acid Residues Capable of Diminishing Symptoms of Experimental Autoimmune Encephalomyelitis but Not Rheumatoid Arthritis Modeled in Rats.

A short synthetic peptide from the C-terminal part of the caveolin-3 structure was tested for experimental autoimmune encephalomyelitis (EAE) treatment in rats. The structure-function similarity established between the novel synthetic peptide of pCav3 and the well-known immunomodulator immunocortin determined pCav3's ability to reduce EAE symptoms in Dark Agouti (DA) rats injected with pCav3 (500 µg/kg). pCav3 was found to interfere with the proliferation of lymphocytes extracted from the LNs of DA rats primed with homogenate injection, with IC50 = 0.42 µM (2.35 mcg/mL). pCav3 affected EAE in a very similar manner as immunocortin. The high degree of homology between the amino acid sequences of pCav3 and immunocortin corresponded well with the therapeutic activities of both peptides, as demonstrated on EAE. The latter peptide, possessing a homologous structure to pCav3, was also tested on EAE to explore whether there were structural restrictions between these peptides implied by the MHC-involved cell machinery. Consequently, immunocortin was further examined with a different autoimmune disease model, collagen-induced arthritis (CIA), established in Sprague-Dawley rats. CIA was established using an intentionally different genetic platform than EAE. Based on the results, it was concluded that the effectiveness of pCav3 and immunocortin peptides in EAE rat model was almost identical, but differed in the rat model of rheumatoid arthritis; thus, efficacy may be sensitive to the MHC type of animals used to establish the autoimmune disease model.

Improving the Stability and Effectiveness of Immunotropic Squalene Nanoemulsion by Adding Turpentine Oil.

Turpentine oil, owing to the presence of 7-50 terpenes, has analgesic, anti-inflammatory, immunomodulatory, antibacterial, anticoagulant, antioxidant, and antitumor properties, which are important for medical emulsion preparation. The addition of turpentine oil to squalene emulsions can increase their effectiveness, thereby reducing the concentration of expensive and possibly deficient squalene, and increasing its stability and shelf life. In this study, squalene emulsions were obtained by adding various concentrations of turpentine oil via high-pressure homogenization, and the safety and effectiveness of the obtained emulsions were studied in vitro and in vivo. All emulsions showed high safety profiles, regardless of the concentration of turpentine oil used. However, these emulsions exhibited dose-dependent effects in terms of both efficiency and storage stability, and the squalene emulsion with 1.0% turpentine oil had the most pronounced adjuvant and cytokine-stimulating activity as well as the most pronounced stability indicators when stored at room temperature. Thus, it can be concluded that the squalene emulsion with 1% turpentine oil is a stable, monomodal, and reliably safe ultradispersed emulsion and may have pleiotropic effects with pronounced immunopotentiating properties.

Differences in bioavailability and cognitive-enhancing activity exerted by different crystal polymorphs of latrepirdine (Dimebon®).

Introduction: Pharmacokinetic characteristics as well as cognitive-enhancing nootropic activity of latrepirdine (Dimebon®) in relationship with its polymorphic forms have been studied in SD and Wistar rats. Methods: The pharmacokinetics of six polymorphs (A, B, C, D, E, F) of latrepirdine were studied in male SD rats after 7 days of oral administration in corn oil at a dose of 10 mg/kg once a day. Blood and brain samples were taken on the 7th day of administration at 15 min, 30 min, 60 min and 120 min after administration and analyzed for latrepirdine content by LC-MS. The cognitive-enhancing nootropic effect was studied in male and female Wistar rats after 9 days of oral administration in corn oil at a dose of 10 mg/kg, after prior administration of scopolamine, an agent that causes memory impairment similar to that in Alzheimer's disease. The animals' cognitive function was studied in the passive avoidance test. Results: When studying the pharmacokinetics, the highest bioavailability both in the blood and in the brain was demonstrated by polymorph E, whose AUC was the highest relative to other polymorphs. In the study of the cognitive-enhancing nootropic effect, polymorph E also showed the highest activity, whose values of the latent period of entering the dark chamber did not differ from control animals, and differed from other polymorphs. Conclusion: Thus, the crystal structure has been shown to play a key role in the bioavailability and efficacy of latrepirdine, and polymorph E has also been shown to be a promising drug for the treatment of neurodegenerative diseases associated with memory impairment, such as Alzheimer's disease.

AICAR Improves Outcomes of Metabolic Syndrome and Type 2 Diabetes Induced by High-Fat Diet in C57Bl/6 Male Mice.

The aim of the study was to investigate the effect of AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on the consequences of metabolic syndrome and type 2 diabetes induced by the consumption of a high-fat diet (HFD) in male C57Bl/6 mice. Additionally, the animals from group 6 were administered Methotrexate (MTX) at a dose of 1 mg/kg in parallel with AICAR, which slows down the metabolism of AICAR. The animals were recorded with signs of metabolic syndrome and type 2 diabetes mellitus by recording their body weights, glucose and insulin levels, and the calculating HOMA-IRs. At the end of the study, at the end of the 13th week, during necropsy, the internal organs were assessed, the masses of the organs were recorded, and special attention was paid to visceral fat, assessing its amount and the mass of the fat surrounding epididymis. The biochemical parameters and histology of the internal organs and tissues were assessed. The animals showed signs of metabolic syndrome and type 2 diabetes, namely, weight gain, hyperglycemia, hyperinsulinemia, an increase in the amount and mass of abdominal fat, and metabolic disorders, all expressed in a pathological change in biochemical parameters and pathological changes in internal organs. The AICAR treatment led to a decrease in body weight, a decrease in the amount and mass of abdominal fat, and an improvement in the pathomorphological picture of internal organs. However, some hepatotoxic effects were observed when the animals, on a received standard diet (STD), were treated with AICAR starting from the first day of the study. The additional administration of MTX, an AICAR metabolic inhibitor, did not improve its efficacy. Thus, AICAR has therapeutic potential for the treatment of metabolic syndrome and type 2 diabetes.

Efficacy of Ficus tikoua Bur. extract in ethylene glycol-induced urolithiasis model in SD rats.

The development of new herbal preparations for the treatment of urolithiasis is an urgent task of medical science. Ficus have attracted the attention of pharmacologists due to a wide range of biological properties, including antioxidant, anti-inflammatory, antibacterial and antifungal activity. We studied the effectiveness of Ficus tikoua Bur. in SD rats in which urolithiasis was induced by 6 weeks of oral administration of ethylene glycol 0.5% ad libitum instead of drinking water. Administration of the extract of Ficus tikoua Bur., as well as comparative drug Cystone® after modeling of urolithiasis lead to the restoration of diuresis and the concentration of inorganic phosphates starting from the 6th week of the experiment. The use of the Ficus tikoua Bur. extract for 6 weeks, both during the modeling of urolithiasis and during the recovery period, led to the restoration of the percentage of lymphocytes in the blood, content of sodium, chlorine and inorganic phosphates in the blood to the control level. Thus, the extract of Ficus tikoua Bur. seems to be a promising drug for effective treatment of the initial stages of the development of urolithiasis.

Derinat® has an immunomodulatory and anti-inflammatory effect on the model of acute lung injury in male SD rats.

To simulate acute lung injury (ALI) in SD male rats they we administered intratracheally with lipopolysaccharide (LPS) followed by hyperventilation of the lungs (HVL), which lead to functional changes in the respiratory system and an increase in the blood serum concentration of inflammatory cytokines. LPS + HVL after 4 h lead to pronounced histological signs of lung damage. We have studied the effectiveness of Derinat® when administered intramuscularly at dose of 7.5 mg/kg for 8 days in the ALI model. Derinat® administration lead to an increase in the concentration of most of the studied cytokines in a day. In the ALI model the administration of Derinat® returned the concentration of cytokines to its original values already 48 h after LPS + HVL, and also normalized the parameters of pulmonary respiration in comparison with animals without treatment. By the eighth day after LPS + HVL, respiratory parameters and cytokine levels, as well as biochemical and hematological parameters did not differ between groups, while histological signs of residual effects of lung damage were found in all animals, and were more pronounced in Derinat® group, which may indicate stimulation of the local immune response. Thus, the administration of Derinat® stimulates the immune response, has a pronounced protective effect against cytokinemia and respiratory failure caused by ALI, has immunomodulatory effect, and also stimulates a local immune response in lung tissues. Thus, Derinat® is a promising treatment for ALI.

Delta Sleep-Inducing Peptide Recovers Motor Function in SD Rats after Focal Stroke.

Background and Objectives: Mutual effect of the preliminary and therapeutic intranasal treatment of SD rats with DSIP (8 days) on the outcome of focal stroke, induced with intraluminal middle cerebral occlusion (MCAO), was investigated. Materials and Methods: The groups were the following: MCAO + vehicle, MCAO + DSIP, and SHAM-operated. DSIP or vehicle was applied nasally 60 (±15) minutes prior to the occlusion and for 7 days after reperfusion at dose 120 µg/kg. The battery of behavioral tests was performed on 1, 3, 7, 14, and 21 days after MCAO. Motor coordination and balance and bilateral asymmetry were tested. At the end of the study, animals were euthanized, and their brains were perfused, serial cryoslices were made, and infarction volume in them was calculated. Results: Although brain infarction in DSIP-treated animals was smaller than in vehicle-treated animals, the difference was not significant. However, motor performance in the rotarod test significantly recovered in DSIP-treated animals. Conclusions: Intranasal administration of DSIP in the course of 8 days leads to accelerated recovery of motor functions.

DSIP-Like KND Peptide Reduces Brain Infarction in C57Bl/6 and Reduces Myocardial Infarction in SD Rats When Administered during Reperfusion.

A structural analogue of the DSIP, peptide KND, previously showed higher detoxification efficacy upon administration of the cytotoxic drug cisplatin, compared to DSIP. DSIP and KND were investigated using the model of acute myocardial infarction in male SD rats and the model of acute focal stroke in C57Bl/6 mice. A significant decrease in the myocardial infarction area was registered in KND-treated animals relative to saline-treated control animals (19.1 ± 7.3% versus 42.1 ± 9.2%). The brain infarction volume was significantly lower in animals intranasally treated with KND compared to the control saline-treated animals (7.4 ± 3.5% versus 12.2 ± 5.6%). Injection of KND in the first minute of reperfusion in the models of myocardial infarction and cerebral stroke reduced infarction of these organs, indicating a pronounced cardioprotective and neuroprotective effect of KND and potentiality for the treatment of ischemia-reperfusion injuries after transient ischemic attacks on the heart and brain, when administered during the reperfusion period. A preliminary pilot study using the model of myocardial infarction with the administration of DSIP during occlusion, and the model of cerebral stroke with the administration of KND during occlusion, resulted in 100% mortality in animals. Thus, in the case of ischemia-reperfusion injuries of the myocardium and the brain, use of these peptides is only possible during reperfusion.

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro.

The success in treatment of venous thromboembolism and acute coronary syndromes using direct thrombin inhibitors has stimulated research aimed at finding a new anticoagulant from haematophagous organisms. This study deals with the comparison between hirudin-1 from Hirudomedicinalis(desirudin), being the first-known and most well-studied natural anticoagulant, along with recombinant analogs of haemadin from the leech Haemadipsa sylvestris, variegin from the tick Amblyomma variegatum, and anophelin from Anopheles albimanus. These polypeptides were chosen due to their high specificity and affinity for thrombin, as well as their distinctive inhibitory mechanisms. We have developed a universal scheme for the biotechnological production of these recombinant peptides as pharmaceutical substances. The anticoagulant activities of these peptides were compared using the thrombin amidolytic activity assay and prolongation of coagulation time (thrombin time, prothrombin time, and activated partial thromboplastin time) in mouse and human plasma. The preliminary results obtained suggest haemadin as the closest analog of recombinant hirudin-1, the active substance of the medicinal product Iprivask (Aventis Pharmaceuticals, USA) for the prevention of deep venous thrombosis in patients undergoing elective hip or knee replacement surgery. In contrast, variegin can be regarded as a natural analog of bivalirudin (Angiomax, The Medicines Company), a synthetic hirudin-1 derivative certified for the treatment of patients undergoing percutaneous coronary intervention and of patients with unstable angina pectoris after percutaneous transluminal coronary angioplasty.

Novel Bradykinin-Potentiating Peptides and Three-Finger Toxins from Viper Venom: Combined NGS Venom Gland Transcriptomics and Quantitative Venom Proteomics of the Azemiops feae Viper.

Feae's viper Azemipos feae belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From A. feae venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other A. feae toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae, which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.

First-In-Human Trials of GamTBvac, a Recombinant Subunit Tuberculosis Vaccine Candidate: Safety and Immunogenicity Assessment.

Tuberculosis is known to be the biggest global health problem, causing the most deaths by a single infectious agent. Vaccine-development efforts are extremely important. This paper represents the results of the first-in-human trial of recombinant subunit tuberculosis vaccine GamTBvac in a Phase I study. GamTBvac is a new BCG booster candidate vaccine containing dextran-binding domain modified Ag85a and ESAT6-CFP10 MTB antigens and CpG ODN adjuvant, formulated with dextrans. Safety and immunogenicity of GamTBvac were estimated in an open-label clinical trial on 60 Mycobacterium tuberculosis uninfected (MTB-uninfected) volunteers previously-vaccinated with Bacillus Calmette-Guérin vaccine (BCG). The candidate vaccine had an acceptable safety profile and was well-tolerated. Three different vaccine doses with a double-immunization scheme were assessed for immunogenicity and induced a significant increase in IFN-γ in-house IGRA response and IgG ELISA analysis. Among them, the half dose vaccine group (containing DBD-ESAT6-CFP10, 12.5 µg; DBD-Ag85a, 12.5 µg; CpG (ODN 2216), 75 µg; DEAE-Dextran 500 kDa, 250 µg; and Dextran 500 kDa, 5 mg) provided high, early and stable in time immune response specific to both protein antigen fusions and is proposed for the further studies.

Efficacy of Synthetic Peptide Corresponding to the ACTH-Like Sequence of Human Immunoglobulin G1 in Experimental Autoimmune Encephalomyelitis.

Peptide immunocortin sequence corresponds to the amino acid residues 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. Since immunocortin was shown previously to inhibit phagocytosis in peritoneal macrophages and ConA-induced T-lymphocytes proliferation in culture, we suggested that immunocortin administering may be of use for patients with self-immune syndrome. Immunocortin in concentration 10 µM inhibited proliferation of both antigen (myelin)-induced and ConA-induced LN lymphocytes isolated from the lymph nodes of Dark Agouti (DA) rats immunized with chorda shear. The biological trials of the synthetic immunocortin were carried out on the DA rats with induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. These in vivo experiments have shown that intraperitoneal injections of immunocortin in a daily dosage 100 µg per animal reduced symptoms of EAE in DA rats.

Immunosuppressant Peptide Abu-TGIRIS-Abu-NH2 and its Application for Treatment of Multiple Sclerosis.

Immunosuppressant peptide immunocortin for the first time was described in 1993. It corresponds to residues 11-20 of human Ig heavy chain (conserved motif of VH domain). There are no data about production of immunocortin by proteolysis of Ig in vivo. Synthetic immunocortin in concentration ~ 10-9 M suppresses phagocytosis in peritoneal macrophages, ConA-dependent blast transformation of rat lymphocytes, exhibits ACTH-like neurotropic activity and was suggested as a potential drug for treatment of a multiple sclerosis (MS). Here, we report a sequence and method of synthesis of Abu-TGIRIS-Abu-NH2 (Abu, alpha-aminobutyric acid), an artificial analogue of immunocortin. Biological trials of peritoneally injected Abu-TGIRIS-Abu-NH2 gave an evidence of its better efficacy versus immunocortin in a test for suppression of the experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats.

Hepatic and Aortic Arch Expression and Serum Levels of Syndecan-1 in ApoE-/- Mice.

BACKGROUND: Heparan sulfate proteoglycan (HSPG) syndecan-1 (Sdc1) acts as a receptor for triglyceride-rich lipoproteins (TRLs), growth factors, chemokines and enzymes. Due to the disordered structure, its function is as diverse as its ligands. In this paper, we have analyzed hepatic and aortic arch expression of Sdc1 in ApoE-/- mice and examined their association with biochemical changes in plasma during the atheroma formation. METHODS: ApoE knockout (ApoE-/-) mice as a model of atherosclerosis were used. Plasma chemistry parameters were estimated by automatic biochemical analyzer. The ELISA test was used to detect soluble Sdc1. The mRNA level of syndecan-1 in liver cells and aortic arch was determined by real time PCR. RESULTS: The Sdc1 mRNA level in liver cells was 1.5-2.5 times higher in ApoE-/- mice compared to the wild-type species and increased with age, whereas it remained at the same level in wild-type mice upon aging. Furthermore, the plasma cholesterol level was 4-6 times higher in ApoE-/- mice compared to the wild type; in contrast, triglyceride (TG) remained at the same level. Simultaneously, the expression of Sdc1 in the aortic arch of ApoE-/- mice decreases with age; however, it increases in wild-type mice of the same age. We determined that the Sdc1 mRNA expression in liver cells is significantly higher compared to the cells of aortic arch. In addition, our research demonstrated that the level of soluble Sdc1 slightly increased with age and did not depend on mouse genotype; yet, the total amount of soluble Sdc1 was higher in ApoE-/- mice. CONCLUSION: Our data suggest that the level of soluble Sdc1 in serum of mice can be associated with chronic inflammation. In addition, we hypothesized that a compensatory increase in the Sdc1 expression in ApoE-/- mice may prevent accumulation of triglycerides in serum, yet having no effect on cholesterol accumulation.

Using Tiletamine-Zolazepam-Xylazine Anesthesia Compared to CO2-inhalation for Terminal Clinical Chemistry, Hematology, and Coagulation Analysis in Mice.

INTRODUCTION: It is important that the method of anesthesia of mice does not considerably alter the animal's physiological and metabolic status before terminal blood sampling taken in order to analyze clinical pathology parameters. METHODS: Hematology, hemostasis, and clinical chemistry parameters were compared in male and female BALB/c mice exposed to either tiletamine-zolazepam-xylazine (TZX) anesthesia or euthanasia in carbon dioxide (CO2) chamber to reveal an alternative method of anesthesia vs. the recommended CO2 inhalation. Blood samples were taken from the inferior vena cava. RESULTS: Clinical blood parameters in mice exposed to CO2 inhalation or TZX anesthesia proved to be substantially different. The TZX group had lower activated partial thromboplastin time (APTT) and fibrinogen (statistically in males and tending in females) and lower platelets (PLT), red blood cells (RBC), hemoglobin (HGB), and white blood cells (WBC) in both sexes. TZX anesthesia resulted in lower blood serum concentrations of total protein, albumin and globulins, creatinine in males (higher in females); cholesterol, triglycerides, alanine aminotransferase (АLT) and alkaline phosphatase (AP) in both sexes, and bilirubin in males. The calcium level decreased in TZX-anesthetized males and females while the phosphates decreased only in females. The volume of serum obtained from females of TZX group was approximately two times higher than in the CO2-anesthetized group, with the degree of hemolysis tending to decrease. DISCUSSION: The studied method of mouse anesthesia, followed by terminal blood sampling and analysis of clinical pathology parameters, suggests that TZX is a good alternative to CO2 inhalation in toxicological and other nonclinical studies. The differences in hemostasis, hematology, and clinical chemistry parameters between these groups are supposedly associated with alterations in physiological and metabolic status of mice under conditions of increasing hypoxia, respiratory standstill, and circulatory arrest after CO2 inhalation.

Autonomic dysfunction determines stress-induced cardiovascular and immune complications in mice.

BACKGROUND: Clinical studies suggest that acute inflammation in patients with elevated heart rate (HR) increases morbidity and mortality. The SJL/J (SJL) inbred mouse strain is a unique genetic model that has higher HR and systemic and vascular inflammation compared with C3HeB/FeJ (C3HeB) mice. The goal of this study was to investigate the role of stress on cardiac and vascular complications between 2 strains. METHODS AND RESULTS: Radiotelemetry was used for continuous recordings of HR and blood pressure in mice. Hemodynamic differences between mouse strains were very small without stress; however, tail-cuff training generated mild stress and significantly increased HR (≈2-fold) in SJL compared with C3HeB mice. Circulating proinflammatory monocytes (CD11b(+)Ly6C(H) (i)) significantly increased in SJL mice but not in C3HeB mice after stress. Presence of Ly6C(+) cells in injured carotids was elevated only in SJL mice after stress; however, a transfer of bone marrow cells from SJL/C3HeB to C3HeB/SJL chimeras had no effect on HR or vascular inflammation following stress. Arterial inflammation (VCAM-1(+)) was greater in SJL inbred mice or SJL recipient chimeras, even without stress or injury. HR variability was reduced in SJL mice compared with C3HeB mice. CONCLUSIONS: We found that impaired parasympathetic activity is central for stress-induced elevation of HR and systemic and vascular inflammation; however, immune cells from stress-susceptible mice had no effect on HR or vascular inflammation in stress-protected mice.

Anti-inflammatory cytokine interleukin-10 increases resistance to brain ischemia through modulation of ischemia-induced intracellular Ca²âº response.

It is suggested that anti-inflammatory cytokine interleukin-10 (IL-10) mediates the delayed protective effects through activation of Jak-Stat3, PI3K-Akt and NF-κB signaling pathways. However, our previous experiments have demonstrated that IL-10 is capable to exert the rapid neuroprotective action through modulation of hypoxia-induced intracellular Ca(2+) ([Ca(2+)]i) response. The first purpose of the present study was to evaluate the neuroprotective effects of IL-10 using three models of the ischemic insults in rats: permanent middle cerebral artery occlusion, ischemia in acute hippocampal slices in vitro and ischemia in cultured hippocampal cells in vitro. The second purpose of the study was to elucidate a role of [Ca(2+)]i changes in the mechanisms underlying IL-10 elicited protection of neurons and astrocytes from ischemia-induced death in cultures of primary hippocampal cells. The data presented here shown that anti-inflammatory cytokine IL-10 is capable to induce a resistance of the brain cells to ischemia-evoked damages in in vivo and in vitro models of the ischemic insults in rats. This protective effect in cultured hippocampal cells is developed rapidly after application of IL-10 and strongly associated with the IL-10 elicited elimination of [Ca(2+)]i response to ischemia. Thus, our results provide the evidence that anti-inflammatory cytokine IL-10, in addition to an activation of the canonical signaling pathways, is capable to exert the rapid neuroprotective effects through transcription-independent modulation of ischemia-induced intracellular Ca(2+) responses in the brain cells.

Genetic locus on mouse chromosome 7 controls elevated heart rate.

Elevated heart rate (HR) is a risk factor for cardiovascular diseases. The goal of the study was to map HR trait in mice using quantitative trait locus (QTL) analysis followed by genome-wide association (GWA) analysis. The first approach provides mapping power and the second increases genome resolution. QTL analyses were performed in a C3HeB×SJL backcross. HR and systolic blood pressure (SBP) were measured by the tail-cuff plethysmography. HR was ∼80 beats/min higher in SJL compared with C3HeB. There was a wide distribution of the HR (536-763 beats/min) in N2 mice. We discovered a highly significant QTL (logarithm of odds = 6.7, P < 0.001) on chromosome 7 (41 cM) for HR in the C3HeB×SJL backcross. In the Hybrid Mouse Diversity Panel (58 strains, n = 5-6/strain) we found that HR (beats/min) ranged from 546 ± 12 in C58/J to 717 ± 7 in MA/MyJ mice. SBP (mmHg) ranged from 99 ± 6 in strain I/LnJ to 151 ± 4 in strain BXA4/PgnJ. GWA analyses were done using the HMDP, which revealed a locus (64.2-65.1 Mb) on chromosome 7 that colocalized with the QTL for elevated HR found in the C3HeB×SJL backcross. The peak association was observed for 17 SNPs that are localized within three GABA(A) receptor genes. In summary, we used a combined genetic approach to fine map a novel elevated HR locus on mouse chromosome 7.