Influence of monostrain and multistrain probiotics on immunity, intestinal ultrastructure and microbiota in experimental dysbiosis.

The biological effects of three probiotic strains Lactobacillus rhamnosus K32, Bifidobacterium longum GT15, Enterococcus faecium L3 and their mixture were studied using a model of dysbiosis induced in rats by antibiotics. It was found that after taking different probiotics intestinal microbiota changed in a strain-specific manner. The maximal activity against pathogens was revealed after the administration of a mixture of bacterial strains under study or a single strain of enterococci. The strain E. faecium L3 showed the most activity against both Klebsiella spp. and Bacteroides fragilis. It helped to restore the original content of Faecalibacterium prausnitzii. The number of Klebsiella spp. was the same in the group receiving L. rhamnosus K32 and the group of animals, which was not consuming probiotics. Different probiotic strains included in the composition had various immunological effects. Probiotic bifidobacteria, enterococci and the mixture of three probiotics stimulated of mRNA expression of interleukin (IL)-10 in mesenteric lymph nodes. The changes in microbiota after consuming an enterococcal probiotic correlated with an increase in transforming growth factor (TGF)-ß and IL-10 content in blood serum and an increase of the intestinal mucus layer. Consumption of L. rhamnosus K32 led to the stimulation of IL-8 expression in mesenteric lymph nodes. Control group not receiving probiotics was characterised by expression of pro-inflammatory cytokines, damage of epithelial cells and the destruction of their tight junctions. The damage to the ultrastructure of the mucosa was prevented in all the groups taking probiotics.

[The intestinal microbiota composition in patients with multiple sclerosis receiving different disease-modifying therapies DMT]. / Osobennosti sostava mikrobioty kishechnika u patsientov s rasseiannym sklerozom, poluchaiushchikh preparaty, izmeniaiushchie techenie rasseiannogo skleroza.

AIM: To describe characteristics of the intestinal microbiota in patients with multiple sclerosis (MS) treated with glatiramer acetate (GA) or fingolimode (FG) for understanding causal relationships between gut microbiota and autoimmune processes in MS patients. MATERIAL AND METHODS: The study included 34 patients treated with GA (n=17) or FG (n=17). GA was used in a dose of 20 mg/kg subcutaneously once a day, FG in a dose of 0.5 mg daily. All patients were examined during remission. To assess the composition of gut microbiota, bacteriological and real-time PCR techniques were used. DNA was extracted from feces using DNA-EXPRESS kit. RESULTS AND CONCLUSION: There was a decrease in numbers of Escherichia coli with normal enzymatic activity, which was replaced by atypical forms of E. coli, Enterobacter spp. and fungi of the genus Candida, and, during treatment with GA, by atypical forms of E. coli, Proteus spp., Parvimonas micra. These differences indicate the effect of the therapy on the intestinal microbiota composition.

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

Impairment of non-associative learning in a rat experimental model of preclinical stage of Parkinson's disease.

An experimental model of the preclinical stage of Parkinson's disease was induced by double intranasal administration of the proteasome inhibitor lactacystin. The results demonstrated signs of cognitive impairments expressed as impaired non-associative learning. This was related to degeneration of one-third of dopaminergic neurons in the ventral tegmental area of the midbrain and their axons in the dorsolateral prefrontal cortex. Impairment of non-associative learning may be an early non-motor marker of Parkinson's disease indicating the start of neurodegenerative processes in the dopaminergic mesocortical system of the brain.

[Antiamnesic effects divaza and its component model ß-amyloid amnesia]. / Antiamnesticheskoe deistvie divazy i ee komponentov na modeli ß-amiloidnoi amnezii u krys.

AIM: On amnesia models induced by (icv) injection of ß-amyloid fragment 25-35 peptide were evaluated antiamnestic actitity. MATERIAL AND METHODS: It was used of active antibody preparations (RA AT) to protein S100 (tenoten), to eNOS (impaza) and combinations (divaza) antiamnestic activity behavioral tests novel object conditioned response passive avoidance. RESULTS: Under the influence of RA AT S100 observed recovery of violation of the ß-amyloid short-term memory (1 hour after the initial presentation of objects), and RA AT eNOS were more effective when tested 24 hours later. Combined medication completely compensate for the simulated deflection behavior of rats did not differ from the intact control. The CRPA RA AT S100 had the greatest impact on the LP entry into the dark compartment, and RA AT eNOS influenced primarily on the emotional component of the reaction. When using the integrated product tends to increase the LP entry into the dark compartment was observed in the absence of changes in the number of boluses. Thus, tenoten had the greatest impact on cognitive impairment, impaza greater effect on the symptoms associated with surgery. CONCLUSION: Combined preparation divaza rendered more effective action, leveling and amnesia neophobia, which confirms the need for further research and prospect release of active drugs in models of neurodegenerative disorders.

Enterococcus faecium strain L-3 and glatiramer acetate ameliorate experimental allergic encephalomyelitis in rats by affecting different populations of immune cells.

The effect of probiotic Enterococcus faecium strain L-3 was studied in rats with experimental allergic encephalomyelitis (EAE). Glatiramer acetate (GA) was used as control drug. E. faecium strain L-3 and GA both were able to reduce the severity of EAE in a similar fashion. Both approaches increased the proportion of EAE resistant rats and rats with mild disease, prolonged the inductive phase of EAE and reduced the disease duration. Study of the phenotypes of immune cells in blood revealed the differences in immunoregulatory pathways that mediate the protective action of probiotic or GA treatment of EAE. The presence of pronounced protective and immunomodulating effects of the probiotic E. faecium strain L-3 opens an opportunity of its application for the treatment of multiple sclerosis.

[EFFECT OF PROBIOTIC ENTEROCOCCI AND GLATIRAMER ACETATE ON THE SEVERITY OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS IN RATS ].

Currently intestinal microbiota is considered as a potential target for influence in various pathologies which have inflammation, autoimmunity or neurodegeneration in the genesis. Multiple sclerosis (MS) combines all these processes in the pathogenesis. Furthermore, the balance of the components of intestinal microbiota is disrupted during MS and followed by disbiosis. Different probiotics - bacteria with proven beneficial properties are widely used to correct dysbisis. In this paper, was investigated the ability of probiotic strain Enterococcus faecium L-3 to reduce disease severity in multiple sclerosis model - experimental allergic encephalomyelitis (EAE). E. faecium L-3 were used alone or in combination with glatiramer acetate (GA). It is shown that administration of E. faecium L-3 reduces the severity of EAE in rats almost as same as that of GA. However, when the probiotic enterococci administered together with GA the protective effect does not observed. It is assumed that these preparations stimulates different ways of the immune system, because their action stimulate different immune cells populations. The study demonstrates the ability of E. faecium L-3 to influence on the immune system in MS, directly and indirectly (through the correction of dysbiosis). This fact allows us to consider E. faecium L-3 as a potential tool for immunomodulation in autoimmune, inflammatory and neurodegenerative diseases.

[Release-active antibodies to S100 protein are able to improve the experimental allergic encephalomyelitis].

AIM: To reveal the effects of release-active antibodies to S100 protein in an animal model of multiple sclerosis. MATERIAL AND METHODS: Sixty female Wistar rats, aged 12 weeks, were included in the study. The pathology was induced by subcutaneous injection of the spinal cord homogenate. Afterwards the rats received a water solution of release-active antibodies to S100 protein (2,5 ml/kg/day, tenoten) or distilled water intragastrically during 30 days. Intramuscular injections of glatiramer acetate (4 mg/kg/day, copaxone) were used as a positive control. RESULTS AND CONCLUSION: Release-active antibodies to S100 protein enhanced the latency period of the disease, reduced its peak intensity and compensated the loss of body weight of the animals. The experimental drug effect was similar to the results of copaxone injections.

[EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS IN ADULT RATS AFTER ADMINISTRATION OF INTERLEUKIN-1beta DURING DIFFERENT PERIODS OF EARLY LIFE].

Multiple sclerosis is a chronic disease of the CNS that affects people of working age, in which the targets of aggressive immune cells become the myelin and myeline producing cells, as well as neurons. It is assumed that a predisposition to MS is forming in childhood, due to common infections. In this paper the experimental allergic encephalomyelitis (EAE) was examined in rats administered IL-1beta at different periods of the early postnatal ontogenesis. EAE was induced in rats at the age of 3 months by single subcutaneous immunization with a homologous homogenate of spinal cord in complete Freund's adjuvant. The number of sick animals were evaluated, as well as the severity of the disease and its duration. It was shown that in rats after administration of IL-1beta on 1st and on 4th week of life EAE is more severe than corresponding control groups of rats. Discusses the damaging or protective effects of injections of IL-1beta during different periods of early postnatal ontogenesis, role of stress reactivity and communication with the "hygiene hypothesis".

Modification of Anxious Behavior after Psychogenic Trauma and Treatment with Galanin Receptor Antagonist.

Effects of blockage of central galanin receptors on anxiety manifestations were studied in rats with psychogenic trauma. Psychogenic trauma was modeled by exposure of a group of rats to the situation when the partner was killed by a predator. Antagonist of galanin receptors was intranasally administered before stress exposure. Animal behavior was evaluated using the elevated-plus maze test, free exploratory paradigm, and open-field test. Psychogenic trauma was followed by an increase in anxiety level and appearance of agitated behavior. Blockage of galanin receptors aggravated behavioral impairment, which manifested in the pathological anxious reactions - manifestations of hypervigilance and hyperawareness. The results suggest that endogenous pool of galanin is involved into prevention of excessive CNS response to stressful stimuli typical of posttraumatic stress disorder.

[Effects of activation of kappa-opioid receptors on the behavior at the postnatal development of the stress reactivity systems].

It is known that stress changes state and reactivity of humoral systems of stress, particularly the hypothalamic-pituitary-adrenal system (HPA) and the dynorphin-K-opioid system (DKOS) in any age periods, including ones of early postnatal development. Supposedly these changes are underlying some disorders. Difference in state and reactivity of the HPA system is well established. But the role of DKOS is not clear. Further study of this requires summarizing of the literature data on physiology of DKOS activation and ethological features of the activation in different periods of postnatal development. It is possible to conclude that the mode of reaction to stimulation of the DKOS differs in the early development in contrast to adult animals. The mode of reaction can be changed in relation to the periods of development of the system of stress-reactivity and can depend on prior activation of the stress system in a particular period.

[CHANGES IN THE QUALITATIVE AND QUANTITATIVE COMPOSITION OF GUT MICROBIOTA IN RATS DURING EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS].

In this study, on the model of multiple sclerosis - experimental allergic encephalomyelitis (EAE), the dynamics of changes in the qualitative and quantitative composition of the intestinal microbiota in rats with symptoms of the disease and asymptomatic course were compared. It was found that the composition of the intestinal microbiota in rats with the clinical symptoms of EAE is shifted towards gram-negative opportunistic microorganisms of the genus Citrobacter, Prote- us, Klebsiella and enteropathogenic Escherichia coli. It has been shown that rats without clinical signs of EAE have higher levels of Faecalibacteriumprausnitzii. The significance of the complex changes in the composition of the intestinal microbiota, indicating long-lasting dysbiosis in rats during the development of EAE is discussing.

[Heterogeneity of the mechanisms of damaging nervous cells in demyelinating autoimmune diseases of the CNS].

A review of modern state of the neurodegeneration problem in demyelinating autoimmune diseases. Experimental and clinical proofs of heterogeneity of the mechanisms of destruction of the nervous tissue are given. Interconnection among the processes of neuroinflammation, excitotoxicity and oxidative stress are considered under damage of oligodendrocytes (myelin) and neurons (axons).

[Analysis of morphological manifestations of the inflammatory reaction in wistar rat spinal cord in experimental model].

Morphological changes in the spinal cord of rats with different intensity of pathological symptoms were studied at the peak of the experimental encephalomyelitis development. Light-microscopical and immunohistochemical methods were used. Distribution of proliferating cell nuclear antigen (PCNA), astrocyte marker - glial fibrillar acidic protein (GFAP), and microglia and macrophage marker Iba-1, was studied. Heterogeneity in morphological manifestations of the experimental allergic encephalomyelitis was shown. Four typical patterns of morphological manifestations of the disease were demonstrated depending on the preferential involvement of pia mater, vessels, spinal cell nuclei or conductive tracts in the pathological process.

[Activity of calpaines in spinal cord cells of the rats with experimental allergic encephalomyelitis of different grades].

In simulation of experimental allergic encephalomyelitis (EAE), a hyperactivation of calpaines occurs due to m-calpaines. The m-calpaines involvement in the process of initiation of neurons and glial cells death in the EAE development was suggested.

The role of the neuropeptide galanin in forming type-specific behavioral characteristics.

Intranasal administration of a galanin receptor blocker to rats was found to change their behavioral type on being placed in an unfamiliar environment, with decreases in movement and investigative activity and increases in the level of anxiety in the open field test. The basal level of expression of the galanin precursor mRNA in the anterior hypothalamus was significantly higher in rats with the active type of behavior in the open field test. In conditions of galanin receptor blockade, there was also a faster increase in the serum corticosterone level in response to a stress situation (forced swimming test), which was accompanied by a reduction in the immobilization time. These data support the involvement of galanin in the formation of individual-typological behavioral characteristics and demonstrate its important role in adaptation to stress.

Comparative study of preventive and therapeutic effects of IEM-1966 and memantine in rats with experimental allergic encephalomyelitis.

We compared preventive and therapeutic effects of memantine, a selective blocker of NMDA-receptors, and IEM-1966, a blocker of both NMDA- and GluR1 AMPA-receptors, on the model of acute experimental allergic encephalomyelitis. Memantine in high doses prevented the development of experimental allergic encephalomyelitis only in 10% rats, slightly (by 1.4-1.5 times) moderated the neurological disturbances, and shortened the duration of the disease. In far lower doses, IEM-1966 prevented the development of experimental allergic encephalomyelitis in 50% rats, while in the affected rats it decreased the severity of neurological disturbances and duration of the disease by 3-4 times. When applied during the clinical phase of the disease, IEM-1966 decreased the severity of neurological disturbances and duration of the disease by 2.0-2.5 times predominantly in rats with mild and moderate course of experimental allergic encephalomyelitis.

[Role of neuropeptide galanin in formation of the typical behavioural characteristics].

Blockade of central galanin receptors through intranasal treatment with specific antagonis M-35 changes the behavioral reactions to novel conditions: the animals treated with M-35 show decreased exploratory activity and increased anxiety level in open field test. The level of pre-progalanin mRNA expression in brain structures was investigated in Wistar rats with opposite strategies of behaviors in open field test. A higher level of pre-progalanin mRNA in anterior hypothalamus of "active" rats was revealed as compared with "passive" rats. The rats treated with galanin receptor antagonist showed a significant rise of serum corticosteron level immediately after forced swimming, while the control rats (without M-35) had only slight corticosterone increase under the sam conditions. The data obtained suggest involvement of brain galanin in formation of individual beha vior and indicate important role of galanin in coping with the stress.

Effects of serotonin synthesis blockade on interleukin-1 beta action in the brain of rats.

The ability of the brain serotonergic system to mediate the effects of interleukin-1beta (IL-1beta) was investigated. Intracerebroventricular administration of IL-1beta induced a significant pyrogenic reaction, depression in social behaviour, loss of body weight and reduced food intake in rats. Pre-treatment with p-chlorphenylalanine, an inhibitor of serotonin synthesis, blocked the IL-1beta-induced decrease in food intake and loss of body weight, but failed to alter the temperature increase and the decrease in communicative activity.