LITHIUM SALTS IN EXPERIMENTAL ONCOLOGY (REVIEW)

Recently, lithium salts have been considered as potential compounds for targeted therapy that can reduce tumor growth. There are a large number of publications indicating the effects of lithium on the signaling pathways used by tumor cells for growth and development, and have demonstrated that lithium can be used as antitumor agent in experimental oncology. The promise of using lithium salts to develop anticancer drugs is related to the fact that lithium has 2 main intracellular targets: glycogen synthase kinase-3β (GSK-3β) and inositol monophosphatase (IMPase), the inhibition of which by lithium can induce cancer cell death by apoptosis or autophagy. Lithium has been shown to block the proliferation of cancer cells by cell cycle arrest in the G2 /M phase, and also stimulates apoptosis and autophagy in cancer cells. This review summarizes data on the transport of lithium across cell membranes, characterizes its main intracellular targets and presents the results of studies in which lithium was used in experimental cancer therapy of various localization with an emphasis on signaling pathways used by cancer cells for growth and metastasis

ЛИМФАТИЧЕСКИЕ СТРУКТУРЫ ГЛАЗА И УВЕОЛИМФАТИЧЕСКИЙ (МЕТАБОЛИЧЕСКИЙ) ПУТЬ ОТТОКА ВНУТРИГЛАЗНОЙ ЖИДКОСТИ. Часть 1

PURPOSE: To identify the lymphatic structures of the human eye, study their ultrastructural organization and morphological changes in the ciliary body and the choroid in patients with primary open-angle glaucoma.METHODS: Fragments of eye tissues enucleated for medical indications (n=28) were studied. The main group included 17 eyes of patients diagnosed with terminal stage primary open-angle glaucoma. The tissues underwent an immunohistochemical study using monoclonal antibodies to markers of the following agents: blood vessel endotheliocytes CD31 and CD34 («Novocasra», Germany), lymphatic endotheliocytes LYVE-1 («Abcam», England), Podoplanin («Monosan», The Netherlands) and Prox-1 («Covance», Germany), fibroblast growth factor receptor marker FGFR («Abcam», England). Obtained eye tissues were studied by means of Leica DME light microscope. Electron microscopy of the eye tissues was carried out using JEM 1400 electron microscope (Japan).RESULTS: The imunohistochemical and ultrastructural analysis of the ciliary body revealed the presence of lymphatic channels and structured interstitial spaces (tissue slits) formed by collagen fibers and fibroblasts. Lymphatic canals and lacunae were also found in the choroid. Lymphatic channels were located in the vascular capillary layer and were confined to cells similar to fibroblasts and pigment cells, while lymphatic lacunae were found in the suprachoroid layer and were lined with cells similar to fibroblasts. For the first time lymphatic structures were found on the border between the sclera and the lamina cribrosa and in the optic nerve sheath. The ciliary body in patients with terminal primary open-angle glaucoma shows the following structural signs of edema and stromal swelling: interstitial space widening, venous vessels lumen enlargement, reduction of the lymphatic endothelial cells marker expression. Similar processes were found in the choroid during the terminal stage of primary open-angle glaucoma: enlarged blood and lymphatic vessels lumens, pericapillary spaces swelling and enlargement, choriocapillary layer stroma swelling as well as the disruption of the anchoring collagen fibrils binding to myofibroblasts and pigment cells. The study revealed an associated with swelling significant increase in choroid thickness and volume density of the epithelium, interstitial spaces and vessels, which indicated inflammation.CONCLUSION: The new fundamental data obtained during the study broadens the current understanding of lymphatic system elements presence in the human eye and their changes associated with primary open-angle glaucoma. This allows us to postulate the existence of a lymphatic (uveolymphatic) pathway of intraocular (tissue) fluid outflow, aimed at utilizing and excreting metabolic and cellular destruction products. Structural disturbances of the lymphatic outflow components play an important role in the mechanisms of primary open-angle glaucoma development.ЦЕЛЬ. Выявить лимфатические структуры в органе зрения человека, изучить их ультраструктурную организацию и морфологические изменения в цилиарном теле и хориоидее при первичной открытоугольной глаукоме (ПОУГ).МЕТОДЫ. Исследованы фрагменты энуклеированных по медицинским показаниям глаз больных (n=28). Основная группа — 17 глаз пациентов с диагнозом «терминальная стадия ПОУГ». Проведено иммуногистохимическое исследование с использованием моноклональных антител к маркерам эндотелиоцитов кровеносных сосудов CD31 и CD34 («Novocasra», Германия), к маркерам эндотелиоцитов лимфатических сосудов LYVE-1 («Abcam», Англия), Podoplanin («Monosan», Нидерланды) и Prox-1 («Covance», Германия), к маркеру рецептора фактора роста фибробластов FGFR — («Abcam», Англия). Полученные препараты тканей глаза изучали в световом микроскопе Leiсa DMЕ. Электронно-микроскопическое исследование тканей глаза проводили на электронном микроскопе JEM 1400 (Япония).РЕЗУЛЬТАТЫ. Иммуногистохимический и ультраструктурный анализ выявил наличие в цилиарном теле лимфатических каналов и структурированных интерстициальных пространств (тканевые щели), сформированных коллагеновыми волокнами и фибробластами. Установлено, что в структуре хориоидеи присутствуют лимфатические каналы и лимфатические лакуны. Лимфатические каналы располагаются в сосудистокапиллярной пластинке и ограничены фибробластоподобными и пигментными клетками, а лимфатические лакуны расположены в надсосудистой пластинке и выстланы фибробластоподобными клетками. Впервые лимфатические структуры выявлены на границе между склерой и решетчатой пластинкой зрительного нерва и в его оболочках. При терминальной стадии ПОУГ в цилиарном теле отмечены структурные признаки отека и набухания стромы: расширение интерстициальных пространств, увеличение просветов венозных сосудов, уменьшение степени экспрессии маркеров эндотелия лимфатических сосудов. Сходные процессы при терминальной стадии ПОУГ обнаружены в хориоидее. Показано расширение просветов кровеносных сосудов и лимфатических каналов, набухание и увеличение размеров перикапиллярных пространств, набухание стромы хориокапиллярной пластинки и нарушение связи якорных коллагеновых волокон с миофибробластами и пигментными клетками. Выявлено достоверное увеличение толщины хориоидеи, возрастание объемных плотностей эпителия, интерстициальных пространств и сосудов, связанное с отеком и набуханием стромы хориоидеи, что указывает на развитие воспалительного процесса.ЗАКЛЮЧЕНИЕ. Полученные в исследовании новые фундаментальные данные расширяют современные представления о наличии элементов лимфатической системы в органе зрения человека и изменении их структуры при первичной открытоугольной глаукоме. Указанное позволяет сформулировать концепцию о существовании лимфатического (увеолимфатического) пути оттока внутриглазной (тканевой) жидкости, направленного на утилизацию и выведение продуктов метаболизма и клеточной деструкции. Структурные нарушения компонентов лимфатического пути играют важную роль в механизмах развития ПОУГ

Investigation of Cytotoxic Effects of Recombinant Human Interferon Lambda-1 and Its Pegylated Form on Human Conjunctival Epithelial Cells

Currently, there are no efficacious, all-purpose antiviral medicines for the treatment of ocular surface infections caused by viruses. At the same time, type III interferons demonstrate high potency for histological barriers, such as the conjunctiva. Modification of protein molecules in native products can significantly improve their pharmacodynamic properties. Thus, it seems reasonable to develop antiviral medicines based on interferon lambda (IFN-λ1) and its pegylated form (PEG IFN-λ1).The aim of the study was to evaluate the in vitro cytotoxic effect of recombinant human IFN-λ1 and its pegylated form on Chang conjunctiva clone 1-5c-4 human conjunctival cells.Materials and methods: PEG IFN-λ1 was obtained by the electron beam immobilisation method. A normal human conjunctival cell line Chang conjunctiva clone 1-5c-4 was used for cell cultivation. The MTT test was used to assess the cytotoxic effect. Cell proliferative activity was studied by measuring microelectrode impedance. Ultrastructural changes were assessed by electron microscopy. Statistical processing was performed using the Statistica 10.0 software package.Results: IFN-λ1 (37 μg/mL) and PEG IFN-λ1 (42 μg/mL) had no significant cytotoxic effect on the human conjunctiva cell culture and the cell proliferative activity. The analysis of ultrastructural changes demonstrated that IFN-λ1 activated metabolic processes in the cells, and PEG IFN-λ1 promoted differentiation and keratinisation of epithelial cells and led to modification of the cell membrane. A ten-fold increase in IFN-λ1 and PEG IFN-λ1 concentration (to 370 μg/mL and 420 μg/mL, respectively) reduced the cell viability by 15–20% as compared to the intact control.Conclusions: the study results demonstrated that IFN-λ1 and PEG IFN-λ1 could be used as active pharmaceutical ingredients in the development of medicines for the treatment of conjunctival viral infections

THE ELEMENTS OF INTRAOCULAR FLUID LYMPHATIC OUTFLOW PATHWAYS IN CHOROID IN NORM AND IN GLAUCOMA PATIENTS

Purpose. To detect and study the structure of aqueous humor lymphatic outflow pathways in choroid in normotensive patients and patients with primary open-angle glaucoma (POAG).Material and methods. Choroid fragments of seven human eyes (including two eyes with terminal glaucoma) enucleated on medical indications were studied. The structure of the choroid was investigated using immunohistochemistry and electron microscopy.Results. Lymphatic channels and lymphatic lacunae were revealed in the choroid structure. Lymphatic channels were detected in choriocapillar and vascular layers and were limited by Podoplanin+, Prox-1+, LYVE-1+ endothelium-like cells, fibroblasts and pigment cells. Lymphatic lacunae were located in suprachoroid layer and covered with endothelium-like cells and fibroblasts. Morphometric study showed an increase of the volume density of epithelium, interstitial spaces and choroid vessels related to edema and swelling of the choroidal stroma in the terminal stage of glaucoma.Conclusions. Human choroid contains lymphatic structures that are probably a part of the ocular lymphatic drainage system and participate in the aqueous humor outflow. Choroidal edema and swelling, the increase of the volume density of epithelium, interstitial spaces and choroid vessels in the terminal POAG stage indicate the lymphatic drainage dysfunction beginning from initial stages of glaucoma and lead to an aqueous humor outflow alteration through the protective lymphatic system

Местный воспалительный процесс как возможное проявление нарушений увеолимфатического оттока внутриглазной жидкости при глаукоме. Часть 2

PURPOSE: To study the ultrastructure of human trabecular zone by electron and light microscopy and inflammatory cytokines in intraocular fluid in primary open-angle glaucoma (POAG). METHODS: Fragments of eye tissues enucleated for medical indications (n=28) were studied. The main group included 17 eyes of patients diagnosed with terminal stage primary open-angle glaucoma. Obtained eye tissues were studied by means of Leica DME light microscope. Electron microscopy of the eye tissues was carried out using JEM 1400 electron microscope (Japan). 45 samples of intraocular fluid from patients with advanced POAG and 30 samples from patients with uncomplicated cataract were studied. Concentrations of 17 cytokines were detected by flow fluorimetry in double-beam laser analyzer (Bio-Plex 200, «Bio-Rad», USA) using Bio-Plex Pro™ Human Cytokine 17-plex Assay set («Bio-Rad», USA). RESULTS: Inflammatory cytokines IL-6, IL-8, and IL-17 were found to be significantly and interdependently increased in intraocular fluid of patients with advanced POAG. These results allow making a conclusion on the importance of local inflammation in POAG pathogenesis. Morphological study using light and electron microscopy detected the following changes in trabecular zone and Schlemm’s canal in terminal POAG: inflammatory infiltration, destruction, multidirectionality, swelling and merging of connected tissue fibers, a big amount of extracellular material in the basal membrane of Schlemm’s canal wall, an increase of lysosomes number, mitochondrial swelling in endothelium, and inter-endothelial connections density increase. These results could be considered as evidence of local inflammation and destructive processes. CONCLUSION: The study provided new basic science data obtained by light and electron microscopy, as well as multiplex assay, helps expand modern understanding of the role of local inflammation in POAG pathogenesis.ЦЕЛЬ. Изучить структурные изменения в трабекулярной зоне глаза человека с использованием методов световой и электронной микроскопии и дисбаланс провоспалительных цитокинов во внутриглазной жидкости при первичной открытоугольной глаукоме (ПОУГ).МЕТОДЫ. Исследованы фрагменты энуклеированных по медицинским показаниям глаз больных (n=28).Основная группа — 17 глаз пациентов с диагнозом «терминальная стадия ПОУГ». Полученные препараты тканей глаза изучали в световом микроскопе Leiсa DMЕ.Электронно-микроскопическое исследование тканей глаза проводили на электронном микроскопе JEM 1400 (Япония). Проведено исследование 45 образцов внутриглазной жидкости от пациентов с развитой стадией ПОУГ и 30 образцов от пациентов с неосложненной катарактой. Концентрацию 17 цитокинов определяли с использованием набора фирмы «Bio Rad» (США) — Bio-Plex Pro™ Human Cytokine 17-plex Assay методом проточной флюориметрии на двухлучевом лазерном анализаторе — Bio-Plex 200, «Bio-Rad», США.РЕЗУЛЬТАТЫ. В результате проведенного исследования во внутриглазной жидкости пациентов с развитой стадией ПОУГ установлено достоверное и взаимосвязанное повышение концентраций провоспалительных цитокинов ИЛ-6, ИЛ-8 и ИЛ-17А, что позволяет сделать заключение о значимости местного воспалительного процесса в механизмах развития ПОУГ. Проведенное морфологическое исследование образцов трабекулярной зоны при терминальной стадии ПОУГ выявило воспалительную инфильтрацию, деструкцию, разнонаправленность, набухание и слияние соединительнотканных пластинок, большое содержание депозитов в субэндотелиальном слое шлеммова канала, увеличение количества лизосом, набухание митохондрий в эндотелии шлеммова канала, возрастание плотности межэндотелиальных контактов, что свидетельствует о наличии местного воспалительно-деструктивного процесса.ЗАКЛЮЧЕНИЕ. Полученные в исследовании новые фундаментальные данные с использованием световой, электронной микроскопии и мультиплексного анализа расширяют современные представления о роли местного воспалительного процесса в механизмах развития ПОУГ

Serum adipokine concentrations in patients with type 2 diabetes: the relationships with distribution, hypertrophy and vascularization of subcutaneous adipose tissue

Academy of Sciences, Novosibirsk, Russia 2Novosibirsk State Medical University, Novosibirsk, Russia BACKGROUND: Adipose tissue (AT) dysfunction plays an important role in metabolic disorders in obesity and type 2 diabetes. The role of distribution, hypertrophy and vascularization of AT in adipokine secretion disturbances remain to be clarified. AIMS: To determine the relationships between serum concentrations of adipokines and the mass and distribution of AT, diameter of adipocytes and vascularization of subcutaneous AT in patients with type 2 diabetes. MATERIALS AND METHODS: A total of 125 patients were examined, including 82 subjects with obesity. Thirty persons without diabetes and obesity, matched by sex and age, were acted as control. Concentrations of leptin, resistin, visfatin, adipsin and adiponectin in fasting serum were determined using multiplex analysis. Mass and distribution of AT was assessed by dual-energy X-ray absorptiometry. Samples of SAT were obtained from umbilical region using a knife biopsy in 25 patients and in 15 individuals who died in accidents. Blood and lymphatic vessels in SAT were revealed with immunohistochemistry, using antibody to CD-34 and podoplanin respectively. The volume and numerical density, ultrastructure of blood and lymphatic vessels, and mean diameter of subcutaneous adipocytes were evaluated. RESULTS: Patients with diabetes, as compared to control, had significantly higher levels of leptin, resistin, adipsin and visfatin (all p<0.001). Adiponectin showed no differences. Concentrations of leptin, resistin, visfatin, adipsin and adiponectin correlated positively with gynoid fat mass. Additionally, leptin and adipsinshowed positive correlations with truncal and central abdominal fat mass. Concentration of leptin, but not other adipokines, was associated with hypertrophy of subcutaneous adipocytes. A decrease in volumetric density of microvessels(р=0.01) and increase in volume and numerical density of lymphatic vessels (both р=0.02) was observed in subcutaneous AT from diabetic subjects. The swelling of cytoplasm, mitochondria, cisterns of granular endoplasmic reticulum and reduced content of micropinocytotic vesicles was revealed in lymphatic capillaries. Resistin and visfatin showed inverse associations with density of microvessels. CONCLUSION: Endocrine dysfunction of AT in patients with type 2 diabetes, manifested by elevation of serum concentrations of leptin, resistin, visfatin and adipsin, is associated with mass and distribution of AT, hypertrophy of subcutaneous adipocytes and vascularization abnormalities of subcutaneous AT

OSOBENNOSTI STRUKTURNOY ORGANIZATsIISUSTAVNOGO KhRYaShchA v zavisimosti ot stadii gonartroza

The purpose - revealing of features of the structural organization of the articulate cartilage of a knee joint at patients with various stages of arthrosis. Materials and methods. Morphological researches of an articulate cartilage are visited at 52 patients with various stages of gonarthrosis. The control group was made by 5 men and 5 women in the age of from 18 till 25 years with traumatic damages of components of a knee joint. The basic group was made by 42 persons. On a stage of gonarthrosis patients have been divided into 2 subgroups: 23 patients with 1-2 stage and 19 patients with 2-3 stage of gonarthrosis. The histological material for research has been received at performance medical-diagnostic arthroscopies a knee joint. Results. It is noted, that morphological changes in an articulate cartilage at gonarthrosis are connected with change of density intercellular matrix and, depending on a stage of process, of chondrocyte dystrophy, necrosis and apoptosis are took place. In cytoplasm of chondrocyte there is a decrease in concentration of cytoplasmic organelles, lipids accumulation and electron density inclusions. Conclusion. The most expressed structural changes in the articulate cartilage, connected with local destruction of chondrocytes, lipids and electron density inclusions accumulation are noted at patients with 2-3 stage of gonarthrosis

A87

Cutaneous melanoma is one of the most aggressive human neoplasms that can quickly metastasize to regional lymph nodes. Currently, prognosis is determined by measuring tumor thickness but more reliable markers for metastatic spread are urgently needed. It is well known that tumors require a microvasculature development in order to grow and metastasize. Angiogenesis and lymphangiogenesis play an important role not only in the tumor growth, but also in the tumor metastasis. As malignancy is a disorder of cellular growth control, the assessment of cell proliferation rates in tumors has intuitive appeal as a prognostic marker. One such biomarker is Ki-67, a cell cycle dependent protein. Recent reports related to its role as a prognostic factor have been contradictory. The purpose of this study was to investigate lymphangiogenesis, angiogenesis and tumor tissue proliferative activity depending on the clinical stages of melanoma. The objects of the study were the samples of tumor tissue obtained during surgical treatment of cutaneous malignant melanoma. To analyze tumor angiogenesis, lymphangiogenesis and proliferation, we performed immunostains of the primary melanomas for the vascular marker CD34, for the lymphatic-specific markers LYVE-1, D2 -40 (Podoplanin) and marker of proliferation – Ki-67. Samples of tumor tissue from 40 patients with IA, IB, IIA, IIB, IIIC melanoma stages were fixed in 10% neutral formalin, processed by standard histological techniques and embedded in paraffin. All steps of the immunohistochemical reaction were performed by using BENCHMARK/XT slide stainer (Ventana). Determination of blood vessel volume density revealed its growth an average of 2 times in peritumoral areas. Similar data were obtained about the location lymphatic vessels. Greater content peritumoral blood and lymphatic vessels, then intratumoral could be detected in all stages of melanoma. In addition more significantly greater volume density both intratumoral and peritumoral blood vessels, than lymphatic vessels was found. A greater degree expression of endothelium lymphatic vessels markers Podoplanin, compared with the LYVE-1 was shown. It was noted an increasing volume density of the peritumoral blood and lymphatic vessels in primary tumors with expansion clinical stage melanoma. Three levels of proliferative activity of tumor tissue were determined (low, an average degree and high Ki-67 expression). Results of the analysis have shown, that the high proliferative activity corresponded to significant content of blood and lymphatic vessels. High level of these markers was observed in patients with regional lymph nodes metastases. In recent years there have been appeared publications suggesting that lymphatic vessel density (particularly in a peritumoral location) and lymphatic vessel invasion are predictors of sentinel node metastasis and poorer survival. It was noted, that “larger, carefully conducted and well-reported studies that confirm these preliminary findings are required before it would be appropriate to recommend the routine application of costly and time-consuming immunohistochemistry for lymphatic markers in the routine clinical assessment of primary cutaneous melanomas”. In our opinion, one must consider not only the lymphatic vessels density, but also blood vessels, and the tumor tissue proliferative activity. This study has shown that the complex markers of lymphangiogenesis (Podoplanin), angiogenesis (CD34) and proliferation (Ki-67) may be predictors of high risk early melanoma metastasis

A94

Squamous cell carcinoma is the most common malignancy in the lower lip. Although lower lip squamous cell carcinoma is slow growing, up to 29% of tumors develop metastases to the cervical lymph nodes. Thus, identification of biological markers that could provide prognostic information about the invasive or metastatic potential of these lesions is important. It is known that angiogenesis, lymphangiogenesis and proliferation play an important role in tumor progression. Therefore, the goal of this study was to analyze the immunohistochemical expression of Ki-67, CD34 and Podoplanin in hyperkeratosis and in squamous cell carcinoma of the lower lip. It was intended to assess the possibility of using such markers as indicators of morphological aggressiveness of squamous cell carcinoma of the lower lip (LLSCC). Materials and methods: Seventy-one cases of the lower lip lesions, obtained from the files of Novosibirsk Regional Oncology Center were selected for this study. The specimens were divided into three groups: a lower lip hyperkeratosis group consisting of 22 cases; LLSCC with keratinization consisting of 34 cases and LLSCC without keratinization consisting of 15 cases. To analyze angiogenesis, lymphangiogenesis and proliferation, we performed immunostains of the lower lip biopsy material for the CD34, vascular marker, Podoplanin, lymphatic-specific markers and Ki-67, marker of proliferation. Tissue samples were fixed in 10% neutral formalin, processed by standard histological techniques and embedded in paraffin. All steps of the immunohistochemical reaction were performed by using BENCHMARK/XT slide stainer (Ventana). The lymphatic and blood vessels volume density and Ki-67 cells numerical density were morphometrically analyzed in all groups and compared using the non-parametric Mann–Whitney test and the Wilcoxon signed rank test. A level of significance of 5% (p < 0.05) was adopted for all tests. Results: All cases of a lower lip hyperkeratosis and LLSCC were positive for Ki-67, CD34 and Podoplanin. With respect to the pattern of staining, specimens exhibited a predominantly peripheral staining for CD34 and Podoplanin in inflammatory infiltrates and tumor sites. In contrast, staining for Ki-67 was predominantly central in inflammatory infiltrates and tumor sites in hyperkeratosis and LLSCC. When compared to lower lip hyperkeratosis, LLSCC (both with keratinization and without keratinization) showed a higher number of immunopositive Ki-67 cells (by 64% and 77%, respectively, p < 0.05). It was found that proliferative activity of tumor cells in LLSCC with keratinization was 2 times higher than that in LLSCC without keratinization. Comparison of the volume density of blood vessels showed that the density of CD34+ – blood vessels in hyperkeratosis was lower by 77% than in LLSCC without keratinization and lower by 64% than in LLSCC with keratinization. At the same time, volume density of blood vessels in LLSCC without keratinization was higher by 56% than that in LLSCC with keratinization. Investigation of lymphatic vessels showed that Podoplanin+ – lymphatic vessels volume density in hyperkeratosis was lower by 50% than that in LLSCC without keratinization and lower by 24% than that in LLSCC with keratinization. Whereas in LLSCC without keratinization the lymphatic vessels volume density was higher by 51% than that in LLSCC with keratinization. Conclusion: This study has shown the greater development of blood and lymphatic vessels in LLSCC without keratinization in comparison with hyperkeratosis and LLSCC with keratinization, thus contributing to the development of metastasis