[Correlation Between Emotional-Affective Disorders and Gut Microbiota Composition in Patients with Parkinson's Disease].

Background: Despite the efforts of scientific community the data available on the correlation between emotional-affective symptoms of Parkinson's disease and changes in microbiome is still scarce. Deeper studies of nonmotor symptoms evident in premotor stages of the disease and the reciprocal influence of microbiota may help to understand the etiology and pathogenesis of PD neurodegeneration better. Aim of the Study: Discover the relations between emotional-affective disorders prevalent in PD population and changes in gut microbiota composition. Methods: 51 patient diagnosed with PD participated in the study. Every participant's emotional-affective state was examined using Beck's Depression Inventory (BDI) and Hospital Anxiety and Depression Scale (HADS). Taxonomic richness of microbiome was studied using 16S ribosomal RNA gene sequencing, bioinformatics, and statistical analysis. Results: Anxiety and depression are prevalent affective disorders in patients with PD. In our study, most of the subjects demonstrated certain anxiety and depression. Taxonomic diversity of gut microbiota in BP was increasing with the increase in anxiety levels, reaching the maximum in the group with subclinical anxiety, and decreasing in the group with clinically significant anxiety disorder. At the species level, patients with clinically significant anxiety had higher abundance of Clostridium clariflavum compared to the anxiety-free patients. Patients with moderate depression were characterized by the higher prevalence of Christensenella minuta, Clostridium disporicum, and Oscillibacter valericigenes compared to subjects without depression or with mild depression. Conclusion: The data we received in our study allow better understanding of PD pathogenesis.

[Serum neurofilament light chains in assessing the course of multiple sclerosis]. / Syvorotochnye legkie tsepi neirofilamentov v otsenke techeniya rasseyannogo skleroza.

OBJECTIVE: To study a role of serum neurofilament light chains (sNFL) in assessment of course and progression of multiple sclerosis (MS) in the population of patients with MS in the Tomsk region. MATERIAL AND METHODS: The study involved 93 patients with relapsing-remitting MS (RRMS) and secondary progressive MS (SPMS) (nRRMS=75, nSPMS=18). The study was carried out in a two-stage design: the first stage was a cross-sectional study for the entire sample; the second stage was a prospective observation with two visits for patients with relapse. sNFL concentration was determined by solid-phase ELISA. RESULTS: There was no statistically significant difference between RRMS and SPMS, and relapse and remission groups in terms of sNFL levels. Patients with a MS duration exceeding 14 years had higher rates of sNFL than those with a shorter duration (p=0.02). The subjects of the second study stage showed a decrease in sNFL from 2.05 (1.86; 2.19) pg/ml to 1.92 (1.87; 2.04) pg/ml (p=0.005), and slowdown in sNFL reduction correlated with the severity of cognitive impairment (k=0.52; p<0.05). CONCLUSION: Dynamic monitoring of sNFL allows the evaluation of the activity of the disease, as well as making an assumption about the compensatory possibilities of subsequent recovery.

[Clinical factors and response to therapy with disease-modifying drugs for multiple sclerosis: the experience of the Tomsk region]. / Klinicheskie faktory i otvet na terapiyu preparatami, izmenyayushchimi techenie rasseyannogo skleroza: opyt Tomskoi oblasti.

OBJECTIVE: To investigate a disease-modifying drugs (DMD) response in multiple sclerosis (MS) in the Tomsk region population and detect clinical factors associated with the treatment response. MATERIAL AND METHODS: A 5-year prospective clinical study included 363 MS patients of the Tomsk region taking DMDs of the «first-line¼ and «second-line treatments¼. The response to DMDs therapy and the impact of MS clinical characteristics on response to treatment were assessed. RESULTS: Clinical factors associated with resistance to DMD are male gender, partial reduce of the MS onset symptoms, short period of the first remission, severe neurological impairment, high relapse rate and disease progression rate. CONCLUSION: Clinical features of MS are crucial factors associated with DMD response and should be used to prescribe DMD. This factor assessment can improve efficacy of the personalized MS treatment.

[BDNF gene RS6265 polymorphism in patients with multiple sclerosis of Tomsk region]. / Polimorfizm RS6265 gena BDNF v populyatsii bol'nykh rasseyannym sklerozom Tomskoi oblasti.

OBJECTIVE: To study the effect of the RS6265 polymorphism of BDNF gene on the risk of development, main clinical characteristics and DMT response in MS patients in Tomsk region. MATERIAL AND METHODS: The study group included 321 patients, the control group consisted of 266 healthy volunteers. Deoxyribonucleic acid (DNA) was isolated from venous blood using the standard phenol-chloroform method. Genotyping was carried out by real-time polymerase chain reaction (PCR) using competing TaqMan probes complementary to the polymorphic nucleotide sequence. RESULTS: Carriage of the C allele and CC genotype of the RS6265 polymorphism of the BDNF gene was found to be a factor determining a more favorable MS course. CONCLUSION: Carriers of the indicated genotype had a low rate of MS progression, a lower frequency of relapses and a less pronounced degree of disability with a comparable MS duration, and significantly more often demonstrated a more optimal response to first and second line of DMT.

[C-kit is a marker of human pancreatic endocrinocyte stem cells].

The aim of our study was to analyze the expression of one of the markers of progenitor cell of different cell types - CD 117 (C-kit) - in human pancreas during prenatal development. The pancreas of human embryos and fetuses at 4-28 weeks of gestation as well as of infants aged up to 2nd postnatal month, was studied. In histological sections, the immunocytochemical reactions were performed with the antibodies against C-kit, insulin and glucagon. In situ hybridization was used for detection of proinsulin mRNA. First cells expressing C-kit were found in human pancreas at 8.5 weeks of gestation among ductal epithelial cells. At 11.5 weeks of gestation these cells were found to segregate from the ductal epithelium and start to form islets. From 8.5 weeks of gestation C-kit positive cells started to express glucagon and proinsulin mRNA, and after 11.5 weeks they also expressed insulin. Islet C-kit positive cells coexpressing both glucagon and insulin, were also found after the birth. It may be concluded that C-kit positive endocrinocyte progenitor cells are common for pancreatic islet A- and B-cells and they are preserved in he islets after the birth.

[Brain-derived neurotrophic factor in multiple sclerosis]. / Mozgovoi neirotroficheskii faktor pri rasseiannom skleroze.

The review presents data on brain-derived neurotrophic factor (BDNF), its structure and functions, the effect on the pathogenesis of experimental autoimmune encephalomyelitis and multiple sclerosis (MS). The correlation of BDNF level with clinical manifestations of MS and the changes of its level during disease-modifying therapy is considered.

[Production of monoclonal antibodies to the prion protein and their characterization].

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25-36 of PrP corresponds to one antibody, and epitopes located in region 222-229, to the other two. The antibodies to fragment 222-229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.

[Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B].

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306-332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.

[New approaches to the immunotherapy of Alzheimer's disease with the synthetic fragments of alpha7 subunit of the acetylcholine receptor].

The effect of immunization with the synthetic fragments of the alpha7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neuro-degenetrative disease of Alzheimer's type, was studied. Mice of the NMRI line were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the alpha7 subunit extraxcellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenetrative disease of Alzheimer's type, and then the state of the spartial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with the N-terminal 1-23 fragment exhibited good spatial memory after training. Immunization with the peptide construct (159-167)-(179-188) consisting of two hydrophilic exposed regions of alpha7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of the animals were educated. Thus, the development of immunotherapy with peptide (159-167)-(179-188) seems to be a promising approach to prophylaxis and treatment of Alzheimer's disease.

[Antitumor immunotherapy with the use of synthetic fragments of survivin].

The endogenous protein survivin is present in tumor cells and inhibits apoptosis. The influence of vaccination of mice by survivin fragments on growth of various types of tumors was studied to examine the possibility of creation of an antitumor vaccinating agent on its basis. Two peptides corresponding to the (118-144) and (80-88)-(153-165) sequences of survivin 2B were chosen and synthesized on the basis of literature data and theoretical calculations. Their ability to stimulate antibody production in mice of the C57BL/6J line (b haplotype) and in BDF1 hybrids (b x d haplotype) was investigated. Both peptides were shown to stimulate production of antibodies that bound the recombinant survivin in the BDF1 mice. Immunization of the BDF1 and C57BL/6J mice with the recombinant survivin resulted in the formation of antibodies that reacted with the (118-144) peptide. The effect of preventive vaccination with the peptides and the recombinant protein on the dynamics of growth of several species of tumors was studied. Vaccination with the (80-88)-(153-165) peptide was found to cause an antitumor effect in BDF1 mice suffering from sarcoma S-37. Thus, the creation of an antitumor agent on the basis of this peptide is a promising area of further studies.

[Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect].

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16- to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.

[Induction of antibodies to particular sites of the alpha7 subunit of the nicotinic acetylcholine receptor in mice of different lines].

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).

[Cell sources of liver development].

The work is devoted to consequent expression of different cell types' protein markers such as vimentin, desmin, cytokeratins 7, 18, 19, stem cell markers CD34 and Bcl-2 at early stages of human prenatal development. Desmin was revealed in sinusoidal liver cells on 3.5-12 weeks of gestation, in mesenchymal cells of ventral mesentery and hepatoblasts on the 4-7 accordingly. During hepatic period of blood formation such desmin positive sinusoidal cells were found to be located close to blood cells. So called "cholangio-" cytokeratins 7 and 19 showed different expression, the first one was found only in cholangiocytes, while cytokeratin 19 existed in hepatoblasts as well until week 15-16 of prenatal development. Mesenchymal cells of ventral mesentery are positive for cytokeratins 18 and 19 even brighter than hepatoblasts in the 4-7 weeks of gestation. Bcl-2 expression was seen in the same periods in most sinusoidal and mesenchymal cells of ventral mesentery. CD34 positive cells are strongly depicted in liver sinusoids from 4th until 9th weeks of gestation, but probably they are not a source of hepatocytes' development in embryonic ontogenesis. Ventral mesentery mesenchyme was negative for this very marker. These results let us suppose that hepatocytes and cholangiocytes may develop from quite different embryonic sources: cholangyocytes grow exceptionally from duodenum epithelial cells, while there is a strong possibility that hepatoblasts formation occurs with participation of mesenchymal cells.

[Production of antibodies to the alpha7-subunit of human acetylcholine nicotinic receptor with the use of immunoactive synthetic peptides].

Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the alpha7-subunit of human acetylcholine receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four alpha7-subunit regions containing 16-23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR alpha7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR alpha7-subunit and do not react with the analogous domain of the alpha1-subunit of the ray Torpedo californica AChR.

[Familial multiple sclerosis in Tomsk region]. / Semeinyi rasseyannyi skleroz v Tomskoi oblasti.

AIM: Multiple sclerosis (MS) has a multifactorial etiology. To explore a role of genetic factors in the pathogenesis of MS, the authors studied familial cases of MS. MATERIAL AND METHODS: For an analysis of familial cases, a database of 320 MS patients registered in Tomsk region since 1980 till 2014 was used. The following items for each patient were recorded: disease onset, manifestation of disease, duration of first remission, rate of progression of disease. RESULTS AND CONCLUSION: Nine families with several members with MS were identified. In 2014, the frequency of familial cases in the MS population of Tomsk region was 4.7%. The prevalence of familial MS was 1.4 cases per 100.000 of people. The younger age at disease onset and higher rate of disease progression measured with the EDSS in parent-child pairs were identified. The most families with several members with MS were characterized by clinical polymorphism of onset, duration and rate of disease progression.

[A role of the gastrointestinal tract microbiota in the pathogenesis of Parkinson's disease]. / Vozmozhnaia rol' mikrobioty zheludochno-kishechnogotraktavpatogenezebolezniarkinsona.

Microbiota is a community of microorganisms, viruses, protozoa, colonizing the gut. There are tight phylogenetic relationships between the gut microbiota and the human body, the disturbance of which may lead to the CNS dysfunction as well as to the development of neurodegenerative diseases. This review focuses on general and specific aspects of the influence of gut microbiota on the pathogenesis of Parkinson's disease (PD). Current theories and models of the relationship between microbiota and brain structures in PD are presented with a specific focus on neurochemical and immunological aspects of the problem.

[Induction of immune response by synthetic fragments of the bovine prion protein and their analogues in mice of various lines]. / Induktsiia immunogo otveta sinteticheskimi fragmentami prionnogo belka i ikh analogami u myshei raznykh linii.

The antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and their six analogues. The analogues contained the amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides, except for one, induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera that were effectively bound to the brain preparations from the bovine with spongiform encephalopathy were identified; it was shown that they do not interact with the preparations of normal brain. Therefore, it was shown that the immunization of mice with the synthetic fragments of a prion protein helps obtain specific antibodies suitable for the study and diagnostics of prion diseases.

[Structure prediction for peptides capable of inducing antibody formation in mice]. / Predskazanie struktury peptidov, sposobnykh indutsirovat' obrazovanie antitel u myshei.

A simple method for the sequence prediction of peptides capable of the in vivo stimulation of antibody production in mice without conjugation with protein carriers was proposed on the basis of literature data on the structure of T-helper epitopes active in vivo. According to this approach, a potentially active peptide should contain a nine-membered sequence with a hydrophobic amino acid residue in the first position and a positively charged residue in the ninth position. The efficiency of this approach was confirmed by the presence of such sequences in the previously described synthetic peptides with immune activities, by the application of this approach to the choice of immunogenic fragments within the sequences of various proteins that exhibited further the specific activity, and by the construction of immunogenic peptides on the basis of inactive natural sequences.

[Antibodies against synthetic fragments of the prion protein for the diagnosis of bovine spongiform encephalopathy]. / Antitela protiv sinteticheskikh fragmentov prionnogo belka dlia diagnostiki gubkoobraznoi éntsefalopatii krupnogo rogatogo skota.

Seven peptides matching fragments of the prion protein and containing from 17 to 31 amino acid residues were synthesized to obtain antibodies for diagnostics of bovine spongiform encephalopathy. Rabbits were immunized with either free peptides or peptide-protein conjugates to result in sera with a high level of antipeptide antibodies. Immunohistochemical assay revealed sera against four free peptides and a protein-peptide conjugate, which effectively bind to the pathogenic isoform of the prion protein in brain tissue preparations from cattle afflicted with bovine spongiform encephalopathy and do not interact with normal brain preparations. The resulting antipeptide sera can be used in developing a diagnostic kit for bovine spongiform encephalopathy.

[Induction of the anti-meningitis immunity with synthetic peptides. III. Immunoactive synthetic fragments of NspA protein from Neisseria meningitidis]. / Induktsiia protivomeningitnogo immuniteta s pomoshch'iu sinteticheskikh peptidov. III. Immunoaktivnye sinteticheskie fragmenty belka NspA iz Neisseria meningitidis.

Four potentially immunoactive peptide fragments of the NspA protein from the outer membrane of the bacterium Neisseria meningitidis were synthesized in order to create a synthetic vaccine against the meningococcal infection by the serogroup B bacterium. Mice of various lines were immunized with the free peptides nonconjugated with a protein carrier. All the synthetic peptides were shown to induce the production of the antipeptide antibodies in mice. A peptide capable of inducing a decrease in the number of bacteria in blood and the protection of infected animals from death was found in the experiments on the protection of the animals infected with two strains of the Neisseria meningitidis serogroup B. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.