Formation of Alcohol Motivation in Monkeys (Macaca mulatta) Kept in Isolation.

The effects of environmental factors (flavors, different ethanol concentration, alcoholic deprivation, and food reinforcement) on the formation of alcohol motivation was studied in rhesus macaques (Macaca mulatta, n=6). Motivation for alcohol intake was induced in two stages: initiation (sessions 1-160) and formation of motivation (sessions 161-516). Monkeys preferred multifruit flavor and 4% ethanol solution, while ethanol deprivation did not stimulate alcohol consumption. The pronounced individual differences in the pattern of alcohol motivation were revealed: the intake of 4% ethanol solution ranged from 0.21±0.03 to 0.43±0.06 g/kg without food reinforcement and increased from 0.78±0.03 to 1.22±0.09 g/kg with food reinforcement. The results suggest that the proposed method is valid and can be used as an experimental model of alcohol dependence in non-human primates.

Natural infection of captive cynomolgus monkeys (Macaca fascicularis) with hepatitis E virus genotype 4.

Data on natural HEV infection of infection in monkeys are limited. We report a case of hepatitis E virus genotype 4 infection in captive non-human primates (cynomolgus monkeys) imported from Vietnam. Phylogenetic analysis demonstrated that HEV infection was not the result of spillover from single source of infection, but rather the persistent circulation of HEV-4 among cynomolgus monkeys or multiple infections by related strains from a human or swine reservoir.

Water Immersion as a Model of Hypogravity.

We studied the effects of hypogravity modeled by water immersion on cognitive functions and physiological parameters of monkeys. Cognitive capacities of monkeys were evaluated using computer-controlled joystick task with food reward in case of target hit. Water immersion (3 days for 3 h) affected in cognitive functions, body temperature, and blood parameters. The intensity of changes depended on the type of monkey behavior. In animals with non-aggressive behavior, the number of target hits did not decrease after water immersion, and even slightly increased. On the contrary, aggressive monkeys showed poorer test performance. Body temperature after each cycle of water immersion was decreased slightly in non-aggressive monkeys, while in aggressive animals, the changes were significant. At the same time, changes in the erythrocyte count, hemoglobin concentration, and hematocrit were significant in non-aggressive monkeys. Our results are in line with previous data performed on BION biosatellites and correspond to changes of physiological parameters in astronauts during space flights.

Development of a motor and somatosensory evoked potentials-guided spinal cord Injury model in non-human primates.

Background Nonhuman primates (NHP) may provide the most adequate (in terms of neuroanatomy and neurophysiology) model of spinal cord injury (SCI) for testing regenerative therapies, but bioethical considerations exclude their use in severe SCI. New Method A reproducible model of SCI at the lower thoracic level has been developed in Rhesus macaques. The model comprises surgical resection of 25% of the spinal cord in the projection of the dorsal funiculus and dorsolateral corticospinal pathways, controlled via registration of intraoperative evoked potentials (EPs). The animals were evaluated using the modified Hindlimb score, MRI, SSEP, and MEP over a time period of 8-12 weeks post-SCI, followed by histological examination. Results Complete disappearance of intraoperative EPs from distal hindlimb muscles without restoration within two weeks post-SCI was an indicator for irreversible disruption of the abovementioned pathways. As a result, controlled damage to the spinal cord was achieved in three NHPs, clinically manifested as irreversible lower monoplegia. No significant functional restoration was observed in these NHPs up to 12 weeks post-SCI. Demyelination of the damaged ascending tracts was detected. Disturbances in pelvic organ function were not observed in all animals. Comparison with existing methods The new method of EPs-guided SCI allows a more controlled and irreversible damage to the spinal cord compared with contusion and other transection approaches. Conclusions This method to induce complete SCI in NHP is well tolerated, reproducible and ethically acceptable: these are valuable attributes in a preclinical model that will hopefully help advance testing of new regenerative therapies in SCI.

[Markers of hepatitis A in the monkeys of the Adlers primate center.]

Hepatitis A is a widespread viral infection. The HAV strains of "human" and "monkey" origin are similar in their morphological and antigenic properties, but differ genotypically. OBJECTIVES: The aim of this research was a comparative study of serological and molecular-genetic markers of HAV infection in monkeys born at the Adler Primate Center and in those imported from different countries. MATERIAL AND METHODS: Fecal samples (n = 313) and serum (n = 266) from various species of monkey using ELISA and RT-PCR were studied. RESULTS AND DISCUSSION: The frequency of anti-HAV-IgG was high (78.9%) in imported animals (vervet monkeys from Tanzania and cynomolgus monkeys from Vietnam) and as well as in various species of monkeys (rhesus monkeys, cynomolgus monkeys, green monkeys and papio hamadryas) of the Center (88.6%). At the same time, in the imported monkeys, the markers of "fresh" HAV infection (IgM-27.2%, Ag-HAV-16.7%, RNA-22.0%) were detected significantly more often (p> 0.05) than in monkeys kept at the Colony (IgM-7.5%, HAV-Ag - 5.2%, RNA - 3.6%). In general, anti-IgG reactivity ranged from 1.064 to 2.073 OD450, anti-IgM ranged from 0.546 to 1.059 OD450. The number of HAV-Ag was 0.496 - 1.995 OD450. RNA HAV only in rhesus monkeys and cynomolgys monkeys born at the Colony, as well as in imported vervet monkeys was detected. CONCLUSIONS: The data obtained indicate a wide circulation of HAV among monkeys born in the Adler Primate Center and among the imported animals. Markers of "fresh" HAV infection varied depending on the species of monkeys and their origin.

Functional Parameters of Physiological Systems of Laboratory Primates after Administration of Doxorubicin and Transplantation of Mesenchymal Stem Cells.

We studied physiological parameters of rhesus monkeys after administration of anthracycline antibiotic doxorubicin. Intravenous administration of the drug caused intoxication manifested in in an abrupt body weight loss, baldness, vomiting, and exicosis. Intoxication in monkeys determined by the damaging effects of doxorubicin on organs and tissues is also characterized by significant changes in the blood: leukopenia, thrombocytopenia, neutropenia, monocytopenia, lymphocytosis, and a sharp drop of CD20+ B cell content. The total protein and albumin content in the blood significantly decreased. A sharp increase in C-reactive protein was also accompanied by an increase in activity of proinflammatory cytokine IL-6. Transplantation of mesenchymal stem cells in some cases can significantly alleviate doxorubicin-induced damage to organs and maintain normal clinical status of monkeys after two injections of the drug. Late transplantation of stem cells does not have a protective effect and does not protect the animals from the damaging effects of doxorubicin. We found that the protective effect of mesenchymal stem cells depends on the dose of the drug, total number of cells, and the time of their transplantation. It should be noted that human and monkey mesenchymal stem cells produce similar regenerative effects, at least in the doxorubicin toxicity model.

Use of Mesenchymal Stem Cells for Possible Repair of Doxorubicin-Damaged Organs and Tissues in Experimental Monkeys.

Three injections of doxorubicin to rhesus macaques cause severe intoxication, characterized by anemia, cachexia, and degeneration of the viscera. The life span of monkeys injected with the drug and receiving after 24 h mesenchymal stem cell transplantation varied from 96 to 120 days in comparison with 50-74 days in animals receiving stem cells before doxorubicin. Controls received doxorubicin and saline; the lifespan of one monkey was 24 days, of the other - 1 year and 8 months. The increase in activity of proinflammatory cytokine IL-6 was paralleled by an increase in the level of C-reactive protein.

In Vivo Experimental Study of Biological Compatibility of Tissue Engineered Tracheal Construct in Laboratory Primates.

Biological compatibility of a tissue engineered construct of the trachea (synthetic scaffold) and allogenic mesenchymal stem cells was studied on laboratory Papio hamadryas primates. Subcutaneous implantation and orthotopic transplantations of tissue engineered constructs were carried out. Histological studies of the construct showed chaotically located filaments and mononuclear cells fixed to them. Development of a fine connective tissue capsule was found at the site of subcutaneous implantation of the tissue engineered construct. The intact structure of the scaffold populated by various cell types in orthotopic specimens was confirmed by expression of specific proteins. The results indicated biological compatibility of the tissue engineered construct with the mesenchymal stem cells; no tissue rejection reactions were recorded; simulation of respiratory disease therapy on Papio hamadryas proved to be an adequate model.

Transplantation of Simian Mesenchymal Stem Cells to Baboons with Experimentally Induced Myocardial Infarction.

Culture of mesenchymal stem cells isolated from the bone marrow of primates by their characteristics met the requirements of stem cells. It was shown that transplantation of allogeneic mesenchymal stem cells (2 million cells per 1 kg body weight) immediately after ligation of the left anterior descending coronary artery between the middle and upper thirds led to neovascularization and capillarization of the ischemic myocardium.

Markers of hepatitis E among the population of the Greater Sochi and in monkeys of the Adler primate center.

Serum from humans (n = 646) and monkeys (n = 1867) collected during the period 1999-2013 was tested by enzyme immunoassay. Anti-HEV IgG was detected significantly more frequently (P ≥ 0.001) in rhesus macaques (Macaca mulatta) - 45.1 ± 1.6% (n = 1001) than in cynomolgus macaques (M. fascicularis) 16.2 ± 1.8% (n = 426). Single seropositive individuals were found among M. nemestrina - 4.0±2.8% (n = 50). Anti-HEV was not detected in the sera of green monkeys (Chlorocebus aethiops) - n = 162, Papio hamadryas (n = 124), and Papio anubis - n = 104. The presence of the anti-HEV IgM indicating the cases of fresh infection in Macaca mulatta - 2.1 ± 0.5% (n = 717) and M. fascicularis - 3.5 ± 1.3% (n = 266) is of great significance. The overall frequency of detection of the anti-HEV IgG among the staff of the Adler Primate Center - 6.8 ± 2.3% (n = 118) was significantly lower (P ≤ 0.001) than among the population of the Greater Sochi - 15.9% ± 1.6 (n = 528). It is important that only in patients of medical institutions (clinic, hospital, cancer center), anti-HEV IgM were detected (2.7-11.8%) along with anti-HEV IgG (15-23.5%), thereby indicating the presence of acute cases of HEV infection among this population. HEV RNA was not detected in the serum of anti-HEV IgM-positive people and monkeys. Seroepidemiological data do not confirm the assumption on the ability of seropositive monkeys of Macaca genus to be a natural reservoir of HEV infection for humans.

[To the 50th anniversary of the discovery of the simian hemorrhagic fever and SHF virus].

The results of the study of SHF and virus SHF for the last period since their discovery are summed up. It was established that the source of this infection fatal for Asian macaques are African monkeys--virus carriers. There is still a danger of the occurrence of epizootics in Primatological Centers at the importation of these monkeys for research. The importance of the obtained experimental SHF in macaques was emphasized. This model is unique, safe and adequate. It is necessary for further study of pathogenesis and evaluation of the means of pathogenetic therapy of HF dangerous to human health.

[Preclinical study of AV0012 early stage inhibitor of hepatitis C virus infection: I. In vitro ADME and pharmacokinetics].

In vitro immunohistochemical investigations on the human hepatoma cell line (Huh7) infected with hepatitis C virus (HCV) strain JFH-1 showed that AV0012 compound blocks the early stages of viral infection. AV0012 also blocked viral infection spread in tissue culture through the secreted virus and through tight cell-to-cell contact. AV0012 is a specific inhibitor of HCV but not of related pestivirus, flaviviruses and other RNA-containing viruses such as bovine diarrhea (BVDV), Venezuelan equine encephalitis (strain TC-83), dengue type 2 (New Guinea), yellow fever (strain 17D), west Nile fever, parainfluenza (type 3) virus, RSV (strain A2), and Rhinovirus (type 2 strain HGP). It is established that human serum does not significantly affect the antiviral activity of AV0012 in vitro. The drug combination studies with AV0012 and interferon alpha 2a in vitro showed that the two inhibitors act additively, which makes possible the use of this combination in clinical tests. AV0012 is highly soluble and stable in aqueous solutions and murine blood plasma, has limited metabolic stability, low binding to human plasma proteins, high permeability through biological membranes, and only interacts with isoenzymes 2D6 and 3A4 of human cytochrome P450. In animal pharmacokinetic studies, AV0012 was rapidly absorbed into the blood stream upon oral administration, showed sufficiently long half-elimination times, and had high oral bioavailability that reached 92% in monkeys. Further preclinical development of AV0012 is in progress.

[Preclinical study of safety of the new pharmacological substance AV0012 to treat hepatitis C].

Pharmacological safety of a new type of HCV inhibitor, AV0012, was studied including acute, subchronic and chronic toxicity in mice, rats and monkeys. Genotoxicity was assessed using the Ames test and the chromosomal aberrations assay in the bone marrow cells of mice. It is established that AV0012 has low toxicity in SHK line mice, Wistar line rats, and monkey of Rhesus macaques species. Results obtained in the study of genetic toxicity showed that AV0012 exhibits no mutagenic activity. Data on general toxicity and mutagenicity discussed in this paper, together with data on 1 the pharmacological activity, pharmacokinetics, and metabolism published previously, allow us to consider AV0012 as a candidate drug for clinical research phase I.

[Efficiency of cycloferon against slow viral infection caused by SV-40 in monkeys and man].

The latent infection caused by Simian virus (SV-40) defeats monkeys and others animals. Currently there are not the prophylactic and therapeutic measures against this disease. We showed the infringement of immunity of M. mulatta infected by SV-40. The use of the preparation Cyclopheron for the treatment of this infection led to the normalization of the functions of immunity of monkeys and to disappearance of the virus from the organism. We suggested the new method for prophylaxis and treatment of latent SV-40 infection by Cyclopheron, may be used for the correction of the immunity.

Blood glucose levels in rhesus monkeys (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) under moderate stress and after recovery.

We evaluated normal blood glucose levels in two species of monkeys, namely rhesus monkeys (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) kept in the Breeding Center of the Institute of Medical Primatology. The animals were either under moderate stress due to catching or after recovery of defensive and orienting reactions by habituation to the blood sampling. Based on these data, seasonal, gender- and age-dependent variations in glucose levels were found. Glucose levels in rhesus monkeys accustomed to blood sampling procedures were shown to be substantially lower compared with those in non-adapted gender- and age-matched animals.

Possible aspects of using cultured stem cell of laboratory primates in experimental medicine.

We describe the methods of isolation and culturing of mesenchymal stem cells from 3 monkey species Macaca mulatta, Papio hamadryas, and Macaca fascicularis. Flow cytofluorometry showed that the cells do not express CD34, CD45, and HLA-DR, but most of them (78-98%) express CD90 marker. The cardioprotective effects of cultured mesenchymal stem cells in cardiomyopathy induced by administration of antitumor anthracycline drugs (doxorubicin).

[The phylogenetic position of Chlamydia strains isolated from monkeys and humans with Chlamydial pathology in the family Chiamydiaceae. Genotypic and phenotypic properties of this pathogen].

Based on the results of the comparative analysis concerning relatedness and evolutional difference of the 16S - 23S nucleotide sequences of the middle ribosomal cluster and 23S rRNA I domain, and based on identification of phylogenetic position for Chlamydophila pneumoniae and Chlamydia trichomatis strains released from monkeys, relatedness of the above stated isolates with similar strains released from humans and with strains having nucleotide sequences presented in the GenBank electronic database has been detected for the first time ever. Position of these isolates in the Chlamydiaceae family phylogenetic tree has been identified. The evolutional position of the investigated original Chlamydia and Chlamydophila strains close to analogous strains from the GenBank electronic database has been demonstrated. Differences in the 16S - 23S nucleotide sequence of the middle ribosomal cluster and 23S rRNA I domain of plasmid and non-plasmid Chlamydia trachomatis strains released from humans and monkeys relative to different genotype groups (group B- B, Ba, D, Da, E, L1, L2, L2a; intermediate group - F, G, Ga) have been revealed for the first time ever. Abnormality in incA chromosomal gene expression resulting in Chlamydia life and development cycle disorder and decrease of Chlamydia virulence can be related to probable changes in the nucleotide sequence of the gene under consideration.

[Phylogenetic analysis of cytomegaloviruses isolated from man and different primate species].

Institute of Medical Primatology, Russian Academy of Medical Sciences, Sochi The conserved regions of nucleotide sequences were found in primate cytomegaloviruses (CMV). Universal primers were designed for the consensus sequence of a conservative region of the UL56 gene of the betaherpesvirinae subfamily. Amplification, sequencing, and phylogenetic analysis of the fragments of CMV strains isolated from man and different primate species were made. Analysis of sequenced gene fragments showed that the UL56 gene area is most suitable for the phylogenetic analysis of primate CMV and could identify several groups of clusters by the degree of relationship among the viruses of this family.

[Preclinical studies of live intranasal H5N1 influenza vaccine with the deleted HS1 gene].

The paper gives the results of evaluating the efficiency of deINS1 pandemic H5N1 vaccine candidate VN1203delNS1 which was constructed by reverse genetics on the basis of influenza virus strain A/Vietnam/1203/04. The safety, immunogenicity and cross-protection of the vaccine strain against different H5N1 virus clades were demonstrated in mouse and macaque models. The results showed the possibility of designing a new-generation replication-deficient intranasal influenza vaccine, by applying an approach to deleting the NS1 pathogenicity factor, an antagonist of the interferon system.

Effects of low-dose γ-irradiation on the counts of immunocompetent cells in a model experiment on primates.

Cellular immunity was studied by flow cytometry with Becton Dickinson monoclonal antibodies in clinically healthy Macaca mulatta males before and after low-dose exposure to ionizing radiation. It was shown that T and B cells are radiosensitive, B cells being more sensitive, which is seen from a significant drop of their count. Natural killers are radioresistant. The count of immunocompetent cells recovers sooner after single compared to fractionated irradiation in the same summary dose.