[Influence of the HLDF differentiation factor on the production of cytokines by bio-tissues of breast tissue in its non-malignant diseases and in invasive carcinoma of a non-specific type]. / Vliianie faktora differentsirovki HLDF na produktsiiu tsitokinov bioptatami tkani molochnoi zhelezy pri ee nezlokachestvennykh zabolevaniiakh i pri invazivnoi kartsinome nespetsificheskogo tipa.

We studied the effect of the HLDF differentiation factor on production of cytokines by biopsy samples of nonmalignant breast diseases (ND) and invasive breast carcinoma of no special type (IBC-NST), in the absence and presence of lymphogenic metastasis: IBC-NST patients werw subdivided into groups on the prognostic protocol of the 8th edition of the AJCC committee. Group IA consisted of patients with T1-T2 tumor sizes, and predominantly with positive expression of estrogen and progesterone receptors (ER+/PR+/HER2-); it also included one patient with the HER2+ (ER-/PR-/HER2+) molecular subtype. The IB group was mainly composed of patients with T2 tumor size, with the presence of lymphogenic metastasis (in 8 out of 10) patients and with positive expression of estrogen and progesterone receptors (ER+/PR+/HER2-) and it also included three patients with the HER2+ (ER-/PR-/HER2+) molecular subtype. Group IIA consisted of patients with T1-T2 tumor sizes, mainly with no metastases in the lymph nodes (in 11 out of 12 patients) and with a triple negative molecular subtype. Group IIB included patients with T2 tumor size, the presence of nodal metastasis and the expression of markers of ER-/PR-/HER2 - and ER-/PR-/HER2+. Group IIIA consisted of patients with tumor size T1-T3, with the presence of nodal metastasis and the expression of markers of ER-/PR+/HER2+ and ER-/PR-/HER2+. Group IIIC consisted of patients with T3 tumor size, lymphogenic metastasis, and expression of ER-/PR-/HER2-markers (triple negative molecular subtype). Due to a limited number of patients in the groups IIB, IIIA and IIIC, as well as due to more severe clinical and pathological stages, according to the prognostic Protocol of the 8th edition of the AJCC Committee, they were pooled into group III. Concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1ß, IL-1Ra, TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF and MCP-1 were assayed in supernatants of biopsy specimens of breast tissue. Results have shown that with IBC-NST, a statistically significantly higher level of spontaneous production (SP) by biopsy specimens of IL-17, IL-18, IFN-γ and VEGF, and a lower level of SP IL-6 as compared with ND. Patients of all clinical and pathological groups showed a high VEGF spontaneous production as compared with ND, while statistically significant differences from patients with ND were not found in IL-17 spontaneous production in group IB patients, and IL-18 spontaneous production were absent in group IA. Only in patients with IA and IB, the IL-6 spontaneous production was lower as compared to ND, and the IL-8 spontaneous production was lower in the IA group. IFN-γ spontaneous production was higher in patients with IBC-NST group IIA as compared with ND. Under the influence of the HLDF differentiation factor, it was found that the parameters of IBC-NST patients were statistically significantly higher in the production of IL-1Ra, IL-17, IL-18 and VEGF, and statistically significantly lower in the production of IL-6 as compared to ND. HLDF had a higher impact on the content of IL-18 in IBC-NST patients than in ND. After HDLF sublimation IL-6 values were lower in patients of groups IA and IB, and HLDF-induced IL-17 production was higher only in patients of group IA. Statistically significant differences in the index of influence of HLDF (IVHLDF), representing ratio of the cytokine concentration in the supernatants of a biopsy specimen stimulated by HLDF to spontaneous cytokine production, were found between ND and IBC-NST in the case of on IFN-γ production, and also in the case of IL-4 production (between patients in the absence and presence of lymphogenic metastasis). IVHLDF for production of IL-6, IL-8 and TNF-α was lower in group IIA patients compared to group IA, and IVHLDF for production of GM-CSF and MCP-1 was lower in group IIA as compared to group III, in addition IVHLDF for MCP-1 products was lower in group IIA as compared to ND. The HLDF effect on the cytokine production by the tumor and its microenvironment was different in ND patients and IBC-NST patients. HDLF suppressed IFN-γ production in the pooled group of IBC-NST patients; HLDF mainly had a suppressive effect on the production of IL-6, IL-8, TNF-α, GM-CSF and MCP-1 in IBC-NST patients of group IIA.

Influence of HLDF Differentiation Factor on Nonspecific Invasive Breast Carcinoma in vitro.

The study was carried out on samples of invasive breast carcinoma of no special type from 36 patients aged 48.0 to 62.8 years. The effect of HLDF on nonspecific invasive breast carcinoma was a decrease in the relative content of low-differentiated cells and an increase in the relative content of highly differentiated cells. HLDF did not have a cytotoxic effect leading to the death of low-differentiated cells but promoted promotes the acquisition of a higher degree of differentiation by them. A more pronounced effect of HLDF was observed in more aggressive metastasizing forms of neoplasia, which allows us to consider this differentiation factor as a candidate for use in the differentiation therapy of malignant neoplasms.

Influence of Internal and External Factors on the Production of Cytokines by Peripheral Blood Cells in Breast Cancer.

The article focuses on the influence of human leukemia differentiation factor (HLDF), carcinoembryonic antigen (CEA), and polyclonal activators (PA) on cytokine production by peripheral blood cells in breast cancer and benign breast diseases. It was found that the influence of internal factors on the production of cytokines by the peripheral blood cells is associated with lymphatic metastasis (CEA: IL-10; HLDF: IL-6, IL-1ß, TNF-α, and G-CSF). One special circumstance was that there were no differences between the production of cytokines by peripheral blood cells in the patients with breast cancer compared to the patients with benign breast diseases with a high risk of malignant transformation. This is evidence of the functional similarity of peripheral blood cells in patients with these conditions. Cytokine production under the influence of PA was different only in case of TNF-α in all study groups.

Topological Features of Histone H2A Monoubiquitination.

The cellular response to DNA damage protects the essential information stored in the genome. This mechanism is crucial in terms of the cancer prevention and aging progression. The DNA damage response (DDR) consists of a complex network controlling the cell cycle and multiple mechanisms of the DNA repair. The DDR disruption is a cornerstone feature of the tumor cells, which allows them to enhance beneficial mutations that prevent successful disease treatment. The important checkpoints of the DDR are currently poorly understood due to the complexity and diversity of the DNA repair machinery. Histone ubiquitination is intensively involved in the repair of the double-stranded DNA breaks. This post-translational modification is known to be a key factor in the recruitment of the repair factors to the DNA damage sites. Here, the crucial role of the ubiquitin lysine residue K27 in the process of histone H2A monoubiquitination mediated by the ubiquitin ligase RNF168 has been showed. The presented data suggest forced and intensive diffusion of ubiquitin from the cytoplasm to the nucleus, which is characterized by the dynamic equilibrium less than 10 min. The comparison of the turnover rate of the wild-type ubiquitin and its variant with a single functional lysine residue K27 suggests an important role of the ubiquitin deposition as a covalent conjugate with histone H2A in terms of the stability of the entire ubiquitinome.

[Proteins and immunohistochemical markers of breast diseases]. / Belkovye i immunogistokhimicheskie markery zabolevanii molochnoi zhelezy.

In this work, we have compared malignant and non-malignant diseases of the mammary gland using 8 proteins: HRG, MUC1, PAI-1, HSP90αA1, CDH1, ERα, PGR and IL-12. Their concentrations in the supernatants of blood cells and breast biopsies were compared in terms of spontaneous production, induced by a polyclonal activator and after exposure to biopsy samples of the HLDF differentiation factor, as well as the indices of the effect of the polyclonal activator and HLDF on the protein production. In addition, the correlation relationships of the above indicators with the expression of markers of the epithelial-mesenchymal transition: collagen type II (CII), ß-1 integrin (CD29) and cadherin-E (CDH1) were studied. The study revealed statistically significant differences in the concentration of HRG in the supernatant of blood cells, IL-12 during spontaneous production by biopsy specimens, PGR production of biopsy specimens induced by the polyclonal activator, CDH1 and IL-12 production biopsy specimens exposed to HLDF. According to the influence index of the polyclonal activator and HLDF, statistically significant differences were found for CDH1production. Comparison of non-specific invasive carcinoma biopsy specimens and non-malignant breast diseases by means of the markers of the epithelial-mesenchymal transition revealed statistically significant differences in CD29 expression and the lack of differences in the expression of CDH1 and CII. This indicates the presence of cell atypia in samples of non-malignant breast diseases; it is confirmed by the recognized correlation between the production of certain proteins and the expression of the epithelial-mesenchymal transition markers.

Studying the Specific Activity of the Amide Form of HLDF-6 Peptide using the Transgenic Model of Alzheimer's Disease.

The neuroprotective and nootropic activities of the amide form (AF) of the HLDF-6 peptide (TGENHR-NH2) were studied in transgenic mice of the B6C3-Tg(APPswe,PSEN1de9)85Dbo (Tg+) line (the animal model of familial Alzheimer's disease (AD)). The study was performed in 4 mouse groups: group 1 (study group): Tg+ mice intranasally injected with the peptide at a dose of 250 µg/kg; group 2 (active control): Tg+ mice intranasally injected with normal saline; group 3 (control 1): Tg- mice; and group 4 (control 2): C57Bl/6 mice. The cognitive functions were evaluated using three tests: the novel object recognition test, the conditioned passive avoidance task, and the Morris water maze. The results testify to the fact that the pharmaceutical substance (PhS) based on the AF of HLDF-6 peptide at a dose of 250 µg/kg administered intranasally efficiently restores the disturbed cognitive functions in transgenic mice. These results are fully consistent with the data obtained in animal models of Alzheimer's disease induced by the injection of the beta-amyloid (ßA) fragment 25-35 into the giant-cell nucleus basalis of Meynert or by co-injection of the ßA fragment 25-35 and ibotenic acid into the hippocampus, and the model of ischemia stroke (chronic bilateral occlusion of carotids, 2VO). According to the overall results, PhS based on AF HLDF-6 was chosen as an object for further investigation; the dose of 250 µg/kg was used as an effective therapeutic dose. Intranasal administration was the route for delivery.

Interaction of Cholera Toxin B-subunit with Human T-lymphocytes.

In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (Kd = 3.3 nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (Ki 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (Ki > 1 µM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.

Binding of Synthetic LKEKK Peptide to Human T-Lymphocytes.

The synthetic peptide LKEKK corresponding to sequence 16-20 of human thymosin-α1 and 131-135 of human interferon-α2 was labeled with tritium to specific activity 28 Ci/mol. The [3H]LKEKK bound with high affinity (Kd = 3.7 ± 0.3 nM) to donor blood T-lymphocytes. Treatment of cells with trypsin or proteinase K did not abolish [3H]LKEKK binding, suggesting the non-protein nature of the peptide receptor. The binding was inhibited by thymosin-α1, interferon-α2, and cholera toxin B subunit (Ki = 2.0 ± 0.3, 2.2 ± 0.2, and 3.6 ± 0.3 nM, respectively). Using [3H]LKEKK, we demonstrated the existence of a non-protein receptor common for thymosin-α1, interferon-α2, and cholera toxin B-subunit on donor blood T-lymphocytes.

Specific effect of the HLDF differentiation factor on the cytokine production potential of immunocompetent blood cells in stomach adenocarcinoma.

The cytokine production potential of immunocompetent cells from the blood of stomach adenocarcinoma patients was analyzed after the pretreatment of cells with the HLDF differentiation factor with subsequent exposure to polyclonal activators (HLDF+PA). IL-1ß, IL-1Ra, TNFα, IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-18BPa, IFNγ, G-CSF, and GM-CSF were quantified in the supernatants after precipitation of the cells. Specific effects of HLDF+PA were manifested as an increase in the production of IL-8, IL-17, and GM-CSF due to suppression of Th1-dependent immune reactions in a Th17-mediated mechanism that is a part of a broader functional antagonism of Th1 and Th17 lymphocyte subpopulations.

[The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

[Amyloid-like fibrils forming and fibroblasts destruction in Tenon's capsule in progressive myopia as a result of pigment epithelium derived factor resistance to restricted proteolysis].

We have shown previously the presence of full length (50 kD) and truncated proteolytic form (45 kD) of pigment epithelium derived factor (PEDF) in the eye Tenon's capsule in progressive myopia. The full length PEDF is prevalent in myopia that correlates with breach in collagen fibrils forming. Immunohistochemical analysis of Tenon's capsule with polyclonal antibodies to PEDF revealed PEDF in control group being exclusively inside fibroblasts, whereas in myopia, PEDF was distributed extracellularly as halo around blasted fibroblasts. By means of atomic force microscopy and immunodot analysis with anti amyloid fibrils antibodies the ability was studied of recombinant PEDF fragments to form fibrils. Only full length PEDF was shown to form amyloid like fibril structures, but not the truncated form. Accumulation offibrils results in fibroblasts destruction and might be the cause of changes in biochemical and morphological structure of Tenon's capsule observed in myopia.

[Classification of self-organizing peptides].

Amino acid sequences of known natural and synthetic self-assembling peptides were searched and analyzed for their characteristic patterns. The attempted formal numerical description of the repeating motifs, which have been revealed, resulted in building of general classification system embracing core-sequences of the peptides capable of nanostructure formation. Advantages and potency of the proposed rational classification were demonstrated via its comparison with the output from the earlier system described by the others.

[Identification in the rat olfactory epithelium new subgroup YM-1 chitinase-like protein].

Novel protein with a molecular mass of ~43 kDa from rat olfactory epithelium in pathophysiological conditions was discovered. Its amino acid sequence and affiliation with the family 18 glycohydrolase subgroup of chitinase-like proteins YM-1 were determined.