Cross-Polarization Optical Coherence Tomography for Clinical Evaluation of Dermal Lesion Degrees in Vulvar Lichen Sclerosus.

The aim of the study was to identify different degrees of dermal lesions in vulvar lichen sclerosus (VLS) using cross-polarization optical coherence tomography (CP OCT) based on attenuation coefficient to detect disease early manifestations and to monitor the effectiveness of treatment. Materials and Methods: The study included 10 patients without pathology and 39 patients with VLS diagnosed histologically. CP OCT was performed in vivo on the inner surface of the labia minora, in the main lesion area. From each scanning point, a 3.4×3.4×1.25-mm3 3D data array was obtained in 26 s. CP OCT examination results were compared with histological examination of specimens stained with Van Gieson's picrofuchsin.Quantitative analysis of OCT images was performed by measuring the attenuation coefficient in co-polarization and cross-polarization. For visual analysis, color-coded charts were developed based on OCT attenuation coefficients. Results: According to histological examination, all patients with VLS were divided into 4 groups as per dermal lesion degree: initial (8 patients); mild (7 patients); moderate (9 patients); severe (15 patients). Typical features of different degrees were interfibrillary edema up to 250 µm deep for initial degree, thickened collagen bundles without edema up to 350 µm deep for mild degree, dermis homogenization up to 700 µm deep for moderate degree, dermis homogenization and total edema up to 1200 µm deep for severe degree.Pathological processes in dermis during VLS like interfibrillary edema and collagen bundles homogenization were visualized using CP OCT method based on values of attenuation coefficient in co- and cross-polarization channels. However, CP OCT method appeared to be less sensitive to changes of collagen bundles thickness not allowing to distinguish thickened collagen bundles from normal ones with enough statistical significance. The CP OCT method was able to differentiate all degrees of dermal lesions among themselves. OCT attenuation coefficients differed from normal condition with statistical significance for all degrees of lesions, except for mild. Conclusion: For the first time, quantitative parameters for each degrees of dermis lesion in VLS, including initial degree, were determined by CP OCT method allowing to detect the disease at an early stage and to monitor the applied clinical treatment effectiveness.

[Possibilities of multiphoton microscopy for the diagnosis of the vulvar lichen sclerosus]. / Vozmozhnosti mul'tifotonnoi mikroskopii dlya diagnostiki skleroticheskogo likhena vul'vy.

BACKGROUND: Vulvar lichen sclerosus (VLS) is a chronic and recurrent dermatosis of an inflammatory nature with severe focal atrophy of the skin. Connective tissue changes are polymorphic and are still not taken into account in histological diagnostics due to the difficulty of interpreting routine histological methods. In this work, we use multiphoton microscopy (MPM) as a new imaging technique that provides detailed information about the organization of collagen fibers in the dermis based on a non-linear second harmonic generation (SHG) process. OBJECTIVE: To determine the degree of connective tissue damage in lichen sclerosus using standard histological techniques and to reveal the diagnostic capabilities of multiphoton microscopy. MATERIAL AND METHODS: We studied 42 biopsies with a histopathological diagnosis of VLS and 10 biopsies of normal vulvar skin. Histological, histochemical and immunohistochemical evaluation was used in comparison with MPM data. Quantitative analysis included the determination of the thickness, length of collagen fibers and the average intensity of the SHG signal. RESULTS: A comprehensive study of the skin showed 4 groups of changes that can be regarded as the degree of the dermis damage: initial, mild, moderate, severe. The affected area at the initial and mild degree has subtle changes, however, it is reliably identified by quantitative analysis of the SHG signal. So, the initial degree is characterized by thin (1.3-1.8 µm) long (56-69 µm) collagen fibers, with a moderate degree, the fibers are thickened (3.4-4.3 µm) and fragmented (22-37 µm). The affected area in moderate and severe cases undergoes homogenization, which is associated with the deposition of extremely thin (0.6-0.9 µm) short (16-28 µm) collagen fibers and the expression of type V collagen. CONCLUSION: Multiphoton microscopy in the second harmonic generation mode is a reliable method for identifying collagen fibers in tissues. The study made it possible to identify 4 degrees of the dermis damage in vulvar lichen sclerosus.

[Investigation of the role of polymorphic loci RS2299941, RS1903858, RS10490920, RS2735343 of the PTEN gene in patients with prostate cancer].

Prostate cancer is a clinically heterogeneous disease, and accurate risk stratification of patients is becoming a key clinical task. This is the most common malignant neoplasm and the leading cause of cancer death in men worldwide. Genomic markers include tools and technologies that can predict the probability of an initial positive biopsy, reduce the number of unnecessary repeated biopsies, identify tumors with low, medium and high risk, classify the degree of disease, as well as predict and monitor the clinical response to intervention. Variants of the PTEN gene are of great interest as genetic markers of the risk of developing prostate malignancies.

Description of two new alleles: HLA-B*50:79 and HLA-DRB1*04:332.

Two novel HLA alleles HLA-B*50:79 and -DRB1*04:332 have non-synonymous mutations in exon 4 and exon 3, respectively.

Characterization of the novel HLA-C*01:195 allele.

The new allele HLA-C*01:195 showed one nucleotide difference with HLA-C*01:02:01:01.

Two novel HLA alleles, HLA-DRB1*14:223 and HLA-DQB1*03:01:49, detected in a Buryat individual.

Two new alleles were characterized by next generation sequencing in a Buryat individual.

[Features of distribution HLAalleles and haplotypes in Kalmyks.]

Conducted high-resolution HLA-typing loci HLA-A, -B, -C, -DRB1 and -DQB1 by massively parallel sequencing of 150 potential donors of hematopoietic stem cells from the Republic of Kalmykia. In the studied population, four new alleles identified that not previously registered by the International Committee on the Nomenclature of Factors of the HLA-system of WHO. During the HLA-typing identified: 29 alleles at the HLA-A locus, 44 - at the HLA-B locus, 26 - at the HLA-C locus, 15 - at the DQB1 locus, 37 - at the HLA-DRB1 locus. The following alleles have a frequency of more than 10%: HLA-A*02:01 (11,7%), HLA-A*01:01 (11%), HLA-B*51:01 (10,3%), HLA-B*58:01 (10,3%), HLA-C*06:02 (17,7%), HLA-C*03:04 (10,3%), HLA-C*03:02 (10%), HLA-DQB1*03:01 (26,7%), HLA-DQB1*02:02 (10%), HLA-DRB1*07:01 (11,7%). The most common HLA-A-B-C-DQB1-DRB1 haplotype is A*02:05-B*50:01-C*06:02-DQB1*02:02-DRB1*07:01 (3,7%). Deviations from the Hardy - Weinberg equilibrium not identified.

[Expirience introduction of quantitative analysis of chimerism after allogenic stem cell transplantation by real-time PCR with InDel polymorphism.]

Using data obtained from domestic and foreign sources, we formed a set of primers and fluorogenic probes for analyzing twentysix specific sequence polymorphisms and one reference gene. In the course of evaluating the effectiveness of real-time PCR, using the example of one of the markers (S01a), we obtained the optimal amount of DNA per reaction (70 ng), providing a resolution of at least 0.1% of the method with the ability to estimate linear chimerism. Formed panel of primers for genetic polymorphisms - InDel has a high degree of informational content for donor-recipient pairs of Russia. From January 2018 to June 2019, a quantitative assessment of the level of linear (CD3 +, CD34 +) and general chimerism was carried out for 28 patients of the clinic of the Institution. Finally, we analyzed patients who received allografts and present 4 different clinical situations that illustrate the informativity level of this method.

[Design and verification of the reagent kit for high-productive HLA-typing of potential donors of hemopoietic stem cells.]

The work describes the development of a reagent kit for high-performance HLA-typing using the Illumina MiSeq System platform. The developed reagent kit contains all the necessary components for target enrichment of five HLA genes, preparation of libraries, and software for automatic data analysis. The reagent kit verified on a 93 DNA samples with known genotypes. During the verification, the sensitivity and specificity of the reagent kit for each of the HLA loci were determined - for A and B they were 1.0 and 1.0, respectively, for the C-1.0 and 0.99 locus, for the DRB1 locus 0.98 and 0.99, for the DQB1 locus 0.98 and 0.93.

Description of a new HLA-A*02 allele, A*02:658, in a Russian individual.

Sequence of the novel allele, HLA-A*02:658, differs from HLA-A*02:01:01:01 by one-nucleotide exchange in exon 2.

[The genetic characteristics of Novosibirsk donors of hematopoietic stem cells.]

The HLA-typing was carried out concerning of 200 residents of Novosibirsk, potential donors of hematopoietic stem cells on loci (HLA)-A, -B, -C, -DRB1. The study detected in mentioned population two new alleles non-registered previously by the WHO International Committee on nomenclature of factors of HLA-system. The analysis of distribution rates of HLA-alleles and haplotype revealed 16 alleles alternatives of locus HLA-A, 24-HLA-B, 13-HLA-C, 13-HLA-DRB1. The rate of frequency more than 10% is intrinsic to following allele alternatives: HLA-A*02 (29.25%), 01 (14%), 03 (13.5%), 24 (10.75%), HLA-B*35 (12.25%), 07 (12%), HLA-C*07 (29.75%), 06 (13%), 04 (12.5%), 12 (11.5%), 03 (10.75%), HLA-DRB*13 (15.25%), 07 (13.75%), 01 (13%), 11 (12.75%), 15 (12.75%), 04 (10.5%). The application of software Arlequin v. 3.1 established 239 possible gaplotypes HLA-AB-C-DRB1. The gaplotypes A*01-B*08-C*07-DRB1*03, A*02-B*13-C*06-DRB1*07, A*03-B*35-C*04-DRB1*01 with rate of frequency 4.5; 2.75 and 2,75% correspondingly. The The distribution of alleles and analysis of galotypes permitted to compare analyzed population with other Russian populations.

Identification of a new HLA-B*27 allele, B*27:133, in a Russian individual.

The sequence of HLA-B*27:133 differs from HLA-B*27:05:02 by one nucleotide change within exon 3.

[The analysis of detection rate of ambiguity of loci under HLA-typing by SSO technology].

The article deals with the results of HLA-typing of 1829 patients on loci HLA-A, HLA-B, HLA-DRB1 using kits of LABType SSO reagents (One Lambada, USA). The indicators of each locus ambiguity are determined: locus HLA-A - 4.43%, locus HLA-B--4.43%, locus HLA-DRB1--0.16%. The list of "rare" alleles was applied to analyze 13 types of determined ambiguities on locus HLA-A, 27 types on locus HLA-B, 3 types on locus HLA-DRB1.

[Mechanisms of radioresistance in terminally differentiated cells of mature retina].

Retinopathy of animals is induced by many agents damaging DNA. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation, and methylnitrosourea (MNU) on mice retina. We evaluated morphological changes, DNA damage and repair in retina, and expression of 5 proteins participating in apoptosis: p53, ATM, FasR, PARP and caspase 3 active. Dose of 14 Gy is equitoxic in terms of induction of DNA single strand breaks by both gamma and proton irradiation. But protons were 2 fold more effective than gamma-rays in induction of DNA double strand breaks. All breaks were repaired within < or =10 h. Irradiation resulted in increased expression of p53 and ATM. But no sings of cell death and retinal degeneration were observed during 7 days after irradiation. Proton irradiation in dose of 25 Gy resulted in increasing over time destructive changes localized mainly in photoreceptor layer of retina. These changes were followed by increased expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, caspase 3 active, which was followed by destructive changes in retina with sings of apoptosis of photoreceptors. As in the case of irradiation, the 2-fold dose reduction of MNU abrogated cytotoxic effect of MNU on retina. High level of spontaneous DNA damage such as apurine and apyrimidine sites were observed in mouse retina. The results of our study demonstrate the occurrence of genotoxic threshold in the initiation of retinal cell death in vivo. Topoisomerase 2 of retina is suggested to translate primary DNA damage to cytotoxic effect.

[Experience in using the LABType SSO reagent kits in the practice of a HLA typing laboratory].

HLA was typed from HLA-A, HLA-B, HLA-DRB1 loci (to the second place) in 443 patients, by applying the LABType SSO reagent kits (One Lambda, USA). The findings were analyzed using the software Arlequin version 3.1. There were no deviations from the Hardy-Weinberg law. Overall, the authors identified 16, 27, and 13 allelic variants ofHLA-A, HLA-B, and HLA-DRB1 loci, respectively. There were also 4 most common haplotypes of HLA-A-B-DRBII: HLA-A *03-B*35-DRB1 *01 (4.29%), HLA-A *01-B*08-DRBl *03 (3.5%), HLA-A*03-B*07-DRBI *15 (3.37%), and HLA-A *02-B*07-DRBI *15 (2.93%).

[Methylnitrosourea as challenge mutagen in assessment of the DNA mismatch repair (MMR) activity: association with some types of cancer].

Total repair capability is a widely used phenotypic marker of predisposition to cancer. Evaluation of this parameter implies using a challenge mutagen in an in vitro system to unmask latent genetic instability and repair insufficiency in the target cells. Traditionally, these investigations involve two tests, evaluation of mutagenic susceptibility (chromosomal aberrations) and genotoxic effect (DNA comet assay). The present study was focused on analysis of the effect of methylnitrosourea (MNU) on resting and PHA-stimulated lymphocytes from healthy donors and patients with gynecological cancer. Cytotoxic effect of MNU (apoptotic lymphocyte death) was estimated using two parameters, interaction of the cells with the annexin V-FITC complex, and morphological changes of the nuclei after their staining with the mixture of two DNA tropic dyes. The genotoxic effect of MNU, namely, secondary double-strand DNA breaks, was scored using the neutral comet assay, modified for the calculation of the comets produced exclusively by BrUdr-labeled proliferating lymphocytes. The proportion of these comets was represented as the proliferative cell index. It was shown that resting lymphocytes were resistant to genotoxic and cytotoxic effects of MNU. The response of proliferating cells to the action of MNU was expressed as the development of secondary DNA breaks (P <0.01), along with the increased frequency of apoptosis (P <0.05). The genotoxic effect of MNU on stimulated lymphocytes of gynecological cancer patients was fourfold lower compared to healthy donor lymphocytes. In response to the MNU action, patient lymphocytes did not change their proliferative index, while in healthy donor lymphocytes proliferative index was two times decreased in response to the MNU action. The data obtained pointed to the association between the cytotoxic response of the lymphocytes to the action of MNU and gynecological cancer. Since only proliferating lymphocytes response to the genotoxic effect of MNU, and the effect is revealed a day after the mutagen action, it is suggested that this phenomenon is associated with postreplicative repair, MMR, the substrate of which is O6-methylguanin. The MMR deficiency in patient lymphocytes determines their tolerance to the action of MNU. Genotoxic effect of lymphocytes to the action of MNU can serve as a marker of MMR, as well as of the MMR deficiency-associated gynecological cancer.

Light damaging action of all-trans-retinal and its derivatives on rhodopsin molecules in the photoreceptor membrane.

We have reproduced the model system containing A2-rhodopsin, NR-PE, A2-PE, and ATR-dimer-PE in order to study photosensitized damage of rhodopsin within photoreceptor membranes of rod outer segments. We have demonstrated that irradiation of such a system with visible light (400-700 nm) distorts the most important functional property of native visual pigment--its ability to regenerate after addition of 11-cis-retinal in the dark. We have also shown that all-trans-retinal bound to membrane phospholipids and rhodopsin has less photosensitizing activity that free all-trans-retinal.

[Retina radioresistance: whole-body exposure of mice to gamma-irradiation induces the retina DNA damage, accumulation of p53 protein followed by DNA repair rather than the apoptosis].

Whole-body irradiation of mice with gamma-rays at 14 Gy causes DNA single and double strand breaks effectively repaired later. p53 is accumulated during the repair period. There is still some amount of DNA breaks 48-72 hours after the irradiation. Despite p53 accumulation and residual DNA lesions in the cells, mice retina demonstrated no morphological destructive changes or apoptosis signs. Retina resistance to apoptotic signals could derive from efficient repair of radiation-induced lesions in transcriptionally active regions of the genome of differentiated cells.

[Neurologic complications of multiple myeloma]. / Nevrologicheskie oslozhneniia pri mnozhestvennoi mielome.

The investigation was concerned with concomitant nervous disorders involved in multiple myeloma (MM) and evaluation of the role of neurologic symptoms in primary diagnosis of myeloma. The study included 37 patients in most of whom pain was the first syndrome of MM as well as various neurologic symptoms. The latter were caused by compression of the reticular cells of the roots of spinal nerves, separate nerves and cerebral and spinal cord tissue by malignant infiltrates. Pronounced and moderate changes in the bioelectrical activity of the cerebral cortex were identified by electroencephalography in 14 patients. Brain compression by direct contact or due to metastasis formation was shown to be mostly responsible for said differences which was confirmed by X-ray examination of skull bones and pathological liquor studies. The brain appeared to be particularly susceptible to that pathology in those patients who had not received special therapy for a long time. Among other factors were paraproteinemia and uremia-related intoxication. Early diagnosis of neurologic complications in MM is absolutely key in selecting adequate therapy.

[Energy metabolism in the brain during experimental hemorrhagic shock]. / Sostoianie énergeticheskogo obmena mozga pri éksperimental'nom gemorragicheskom shoke.

State of energy metabolism was studied in brain of rats at agonal steps caused by bleeding during severe and long-term hypotension (30 mmHg, up to 30 hrs). Fast loss of high energy-containing phosphates, cell energy potential of adenine nucleotides and an increase in the lactate/pyruvate ratio were found in non-resistant rats. At the same time, in the most resistant rats normal content of creatine phosphate, ATP was maintained; energy potential was unaltered and anaerobic glycolysis was not activated. Content of glucose and glycogen did not alter considerably in all the animals studied.