[Therapeutic monitoring and prediction of the efficacy of neurotrophic treatment in patients with amnestic type of mild cognitive impairment]. / Terapevticheskii monitoring i prognoz éffektivnosti neirotroficheskoi terapii u patsientov s sindromom miagkogo kognitivnogo snizheniia amnesticheskogo tipa.

AIM: To perform therapeutic monitoring and prediction of the neurotrophic therapy efficacy in patients with amnestic type of mild cognitive impairment (aMCI) in a model of course cerebrolysin therapy. MATERIAL AND METHODS: The study involved a group of 19 elderly patients who met the diagnostic criteria of aMCI. All patients received a course of neurotrophic therapy consisting of 20 intravenous infusions of cerebrolysin (30 ml of cerebrolysin in 100 ml of isotonic sodium chloride solution). To assess the therapy efficacy, psychometric scales (CGI, MMSE, MoCA-test, МDRS, FAB, Clock Drawing Test, BNT, Word Recall test, delayed reproduction of 10 words, naming digits in a direct and reverse order) were used at 0, 4, 10 and 26 weeks of the study. Antibodies to p75 neurotrophin receptor (NTR) were measured by ELISA in blood serum of 19 patients before cerebrolysin therapy and after 10 and 26 weeks of treatment. RESULTS AND CONCLUSION: The study showed that аMCI patients had an increased level of antibodies against P75NTR that was significantly decreased after 5.5 month of cerebrolysin treatment. Therefore, it can be a potential biomarker of long-term therapeutic effect of cerebrolysin treatment in aMCI patients. The modified fragment 155-164 of P75 NTR determined in the serum of patients can be an effective indicator for monitoring and predicting the efficacy of long-term neurotrophic therapy.

[Protective Activity of Prion Protein Fragments after Immunization of Annimals with Experimentally Induced Alzheimer's Disease].

The prion protein is considered as one of the membrane targets of neurotoxic beta-amyloid during Alzheimer's disease development. We have chosen and synthesized 17-33, 23-33, 95-110 and 101-115 prion fragments involved in beta-amyloid binding. The effect of immunization with the peptides on the features of Alzheimer's disease was investigated in animals with an experimentally induced form of the disease. It was shown that immunization either with peptide 17-33 or with protein conjugates of peptides 23-33 and 101-115 increases the level of brain beta-amyloid and improves morfofunctional state of the brain.

[The reduced level of antibodies to acetylcholine receptor alpha 7 fragment in the blood serum of patients with Alzheimer's disease].

OBJECTIVE: Determination of antibodies to neuronal membrane proteins in the blood serum of patients is of interest for diagnosis and optimization of treatment of Alzheimer's disease (AD). Authors studied the level of antibodies to acetylcholine receptor alpha 7 protein fragment (AChR), prion protein (РrР) and glycation end-products (RAGE) as well as to intracellular proteins nucleophosmin (Nuc) and survivin (Sur) in the serum of AD patients. MATERIAL AND METHODS: Serum samples of 26 patients with probable AD and 13 healthy people were studied. Exposed sections of each protein were used for the choice of peptides for antibody visualization. ELIZA was a main method in this study. RESULTS AND CONCLUSION: Antibodies to several proteins were identified but significant differences were found only for AChR-(173-193). The results demonstrated the involvement of AChR and AChR-antibodies in the development of AD. Determination of antibodies to AChR-(173-193) may be a marker of AD and a method for specifying the diagnosis of AD.

[Fragment of Receptor for Advanced Glycation End Products Improves Memory State in a Model of Alzheimer's Disease].

A number of synthetic peptides corresponding to potentially important regions in the sequence of the four membrane proteins known as beta-amyloid cell receptors have been investigated on their ability to improve memory state in experimental model of Alzheimer's disease. Nine fragments repeating all the exposed nonstructural regions of the RAGE protein according to X-ray data, have been synthesized. The activity of these peptides and synthesized earlier immunoprotective fragments of other three proteins (acetylcholine receptor alpha7-type, prion protein and neurotrophin receptor p75) has been investigated under intranasal administration, without immune response to the peptide. Only one fragment RAGE (60-76) was shown to have a therapeutic activity improving the memory state of bulbectomized mice and leads to decreasing in the level of brain beta-amyloid.

Immunization with either prion protein fragment 95-123 or the fragment-specific antibodies rescue memory loss and neurodegenerative phenotype of neurons in olfactory bulbectomized mice.

Epidemiological studies demonstrated association between head injury (HI) and the subsequent development of Alzheimer's disease (AD). Certain hallmarks of AD, e.g. amyloid-ß (Aß) containing deposits, may be found in patients following traumatic BI (TBI). Recent studies uncover the cellular prion protein, PrP(C), as a receptor for soluble polymeric forms of Aß (sAß) which are an intermediate of such deposits. We aimed to test the hypothesis that targeting of PrP(C) can prevent Aß related spatial memory deficits in olfactory bulbectomized (OBX) mice utilized here to resemble some clinical features of AD, such as increased level of Aß, memory loss and deficit of the CNS cholin- and serotonin-ergic systems. We demonstrated that immunization with the a.a. 95-123 fragment of cellular prion (PrP-I) recovered cortical and hippocampus neurons from OBX induced degeneration, rescued spatial memory loss in Morris water maze test and significantly decrease the Aß level in brain tissue of these animals. Affinity purified anti-PrP-I antibodies rescued pre-synaptic biomarker synaptophysin eliciting similar effect on memory of OBX mice, and protected hippocampal neurones from Aß25-35-induced toxicity in vitro. Immunization OBX mice with a.a. 200-213 fragment of cellular prion (PrP-II) did not reach a significance in memory protection albeit having similar to PrP-I immunization impact on Aß level in brain tissue. The observed positive effect of targeting the PrP-I by either active or passive immunization on memory of OBX mice revealed the involvement of the PrP(C) in AD-like pathology induced by olfactory bulbectomy. This OBX model may be a useful tool for mechanistic and preclinical therapeutic investigations into the association between PrP(C) and AD.

[Antibodies for detection of E/K amino acid substitution in 129 position of the survivin sequence].

Survivin is an oncofetal protein involved both in inhibiting of apoptosis and in cell cycle regulation. The functions of survivin are defined by its structural state. Due to nature polymorphism, survivin cancontain either E or K amino acid in 129 residue, and K129 is commonly acetylated. Only the protein having acetylated K129 tends to form dimeric structure. Thus, antibodies detecting the amino acid substitution can be a useful tool for structural and functional research of the protein. To obtain the antibodies specific to amino acid substitution E129/K129 peptide fragments overlapping 129 amino acid residue were synthesized, rabbits were immunized with the peptides and affinity purification of the antibodies on sepharose conjugated with the peptides was carried out. The data of ELISA and western blot showed that antibodies obtained were able to detect amino acid substitution E129/K129 in the recombinant and endogenous survivin.

[Immunization witha synthetic fragment 155-164 of neurotrophin receptor p75 prevents memory loss and decreases beta-amyloid level in mice with experimentally induced Alzheimer's disease].

Neurotoxic beta-amyloid peptide plays an important role in the pathology of Alzheimer's disease. In aggregated form it binds to several proteins on the surface of the brain cells leading to their death. p75 receptor in- volved in supporting of cell balance is one of the targets for toxic beta-amyloid. We proposed that induction of antibodies against potential binding sites of p75 with beta-amyloid can be a promising approach towards new drug development for Alzheimer's disease therapy. Four potentially immunoactive fragments of p75 were chosen and chemically synthesized. Investigation of immunoprotective effect of the peptide fragments carried out in mice with experimentally induced form of Alzheimer's disease helped to reveal two fragments effectively preserving murine memory from impairment. Results obtained by ELISA biochemical analysis showed that only immunization with fragment p75 155-164 led to significant decrease in beta-amyloid level in the brain of the experimental mice. Thus, immunization with both fragments of p75 receptor is believed to be an effective tool for the development of new drugs against Alzheimer's disease.

[Antibodies to synthetic fragment 95-123 of the prion protein protect neurons and astrocytes from beta-amyloid toxicity].

Toxic effect of beta-amyloid on brain cells during Alzheimer's disease pathology is known to be associated with oxidative stress, intracellular Ca2+ increase in neurons and astrocytes and activation of reactive oxygen species production. Prion protein is involved in beta-amyloid toxicity. Here we investigated an effect of affinity purified antibodies to synthetic fragment 95-123 of the prion protein on beta-amyloid induced cell death, Ca(2+)-signal, reactive oxygen species production and caspase 3 activation. We have shown that antibodies to fragment 95-123 are able to protect neurons and astrocytes from beta-amyloid induced cell death with no effect on the intracellular Ca2+ concentration altered by beta-amyloid treatment. However, the antibodies significantly reduced Abeta induced reactive oxygen species production in astrocytes and decreased caspase 3 activation in neurons and astrocytes. Thus, induction of antibodies to synthetic fragment 95-123 of the prion protein provides a new approach to anti-AD vaccine design.

[Obtaining of the affinity purified antibodies against survivin for the structure functional study of the protein].

Tumor-associated protein survivin is the bifunctional protein which can participate either in cell division regulation or in apoptosis inhibition depending on its localization and structure state. The aim of this work was to obtain monospecific antibodies useful for investigation of protein structure and functional features. Six affinity purified antibodies directed to different protein regions were obtained. The ability of antibodies obtained to detect survivin in tumor cells and breast cancer tissues was studied. It was shown that antibodies to (1-22) and (95-105) survivin fragments have the highest specific activity. In western-blot antibodies to (1-22) region predominantly binds with survivin-containing complex, which may be the survivin dimer as we suppose, while antibodies to (95-105) region detects only monomeric form of the protein. Breast cancer tissues study demonstrated that survivin monomer presents only in the tumor core tissues, while survivin-containing complex is expressed both in tumor core and tumor periphery tissues. It was shown that antibodies to (1-22) fragment detect predominantly nuclear survivin, which participates in mitosis regulation, while antibodies to (95-105) fragment gave nucleoplasm and cytoplasm staining at all stages of cell cycle. Thereby antibodies obtained are the useful tool for structure-functional study of survivin.

[The apoptosis inhibitor survivin in transitional cell carcinoma of the urinary bladder].

Whether the expression of the apoptosis inhibitor survivin was correlated with the degree of differentiation and the stage of transitional cell carcinoma of the urinary bladder was studied. Sixty samples of surgical specimens from patients with urothelial carcinomas of various degrees of differentiation and different stages were examined. An immunohistochemical study using the monoclonal antibodies obtained by the authors was conducted. The high expression of survivin was shown to be correlated with the lower-grade differentiation of a tumor and its higher stage.

[Autoantibodies to α7-subunit of neuronal acetylcholine receptor in children with traumatic brain injury].

An objective of the study was to search for new biologically significant markers of brain damage. Levels of blood serum autoantibodies (aAB) to different fragments of α7-subunit of acetylcholine receptor (ACR) were studied in children with traumatic brain injury of different severity. The more severe was trauma, the higher was the level of aAB to fragments of α7-subunit of ACR in the first week after trauma. The data obtained suggest that α7-subunits of ACR and aAB to them are involved in the pathogenesis of traumatic brain lesions and, probably, play a significant role in the course of post traumatic period.

[Antibodies to synthetic peptides for the detection of survivin in tumor tissues].

Survivin, an endogenous protein, is a promising marker for the diagnosis of cancer. The aim of the present work was to obtain antibodies specific to survivin and capable of detecting this protein in tumor tissues. Four peptides corresponding to fragments (1-22), (54-74), (80-88)-(153-165), and (118-144) of the survivin-2B sequence were selected and synthesized. Rabbits were immunized with the synthetic peptides. It has been shown that all peptides in a free state, without conjugation with a high-molecular-weight carrier, stimulate the production of antibodies capable of binding with recombinant survivin. Antipeptide antibodies were isolated from sera and their performance in the immunohistochemical detection of survivin in human tumor tissues was studied. It was shown that only antibodies to the (80-88)-(153-165) peptide bind to the survivin present in breast and bladder tumors. The ability of antibodies to this peptide to detect survivin in tumor tissue lysates was demonstrated by immunoblotting. The part of the sequence targeted by the antibodies against the (80-88)-(153-165) peptide was localized using truncated peptide fragments.

[Antibodies to synthetic fragments of nucleophosmin for the specific detection of its monomeric and oligomeric forms].

Immunoactive fragments corresponding to the N-terminal (19-36) and C-terminal (283-294) regions of the NPM1.1 isoform of nucleophosmin and their shortened fragments were chosen and synthesized. Rabbits were immunized with free full-size peptides and their protein conjugates. Antibodies produced against the 19-36 and 283-294 peptides were purified by affinity chromatography on bromocyanogen-activated sepharose that was preliminary conjugated with the synthetic peptides. An analysis of immunoblots of lysates of the HeLa and Ramos cells demonstrated that the antibodies produced against the 19-36 peptide detected the monomeric form of nucleophosmin, whereas the antibodies against the 283-294 peptide predominantly revealed its oligomeric form. It was established by immunocytochemical analysis that the antibodies induced by the 19-36 peptide stained the nucleoplasm and perinuclear space of the cytoplasm of the HeLa and Ramos cells, but did not stain the nucleoli, while the antibodies against the 283-294 peptide stained only the nucleoli of the same cells. On the basis of these results, one could propose that the monomeric and oligomeric forms of nucleophosmin were located in the nucleoplasm and nucleoli of the examined cells, respectively. Thus, antibodies which can predominantly detect monomeric and oligomeric forms of nucleophosmin were produced for the first time. An analysis of the monomeric-oligomeric state and the location of the nucleophosmin in tumor cells could be performed using these antibodies.

[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.

[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.

[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.

[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).

[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.

Mechanisms of defense formation against meningococcal infection in mice immunized with synthetic peptides.

Immunization of mice with synthetic peptide fragments of conservative sites of meningococcal outer membrane proteins led to defense formation against infection with virulent serogroup A and B Meningococci. The role of cellular immunity in the formation of defense against meningococcal infection after immunization with the peptides and the possibility of stimulating lymphocyte population with these peptides were demonstrated.