PANCREATIC PATHOMORPHOLOGY IN SUPERINVASIVE OPISTHORCHIASIS

Superinvasive opisthorchiasis involves many body systems in the pathological process. The parasite, due to its unique characteristics, changes numerous genes, and contributes to proliferative processes. Stem cells are initiated and subsequently differentiated along various cell lineages. The liver and pancreas are econiches, where parasites vegetate for a long time. However, there is no available information on the dynamics of proliferative pancreatic changes and various histogenetic pathways. The aim of the paper is to reveal the proliferative activity of pancreatic stem cells in superinvasive opisthorchiasis and their differentiation. Materials and Methods. Fragments of pancreatic tissue were selected and analyzed in departments of morbid anatomy of medical institutions located on the territory of the hyperendemic focus (Tyumen region, Omsk region, Khanty-Mansiysk Autonomous Okrug). The following groups were formed: Group 1 – patients with a history of superinvasive opisthorchiasis with a more than 10-year invasion (n=70); Group 2 – patients without invasion with all-cause mortality (n=10). The material was subjected to routine histological examination. The authors used histochemical and immunohistochemical staining methods and performed statistical analysis. Results. The authors observed marked proliferative reactions in the epithelial layer of the duct system, the secretory section of the pancreas, as well as changes in the endocrine apparatus. The histological types of pancreatic endocrine formations were revealed, namely, normal, hyperplastic and newly formed islets of Langerhans. The difference between the newly formed islets and the preexisting forms was in a disorderly arrangement, an indefinite shape, significantly larger sizes, and in the absence or presence of a forming capsule. The newly formed islets contained cells similar to preexisting endocrinocytes. Conclusion. Under the influence of the Opisthorchis felineus secretome, there occurs initiation and differentiation of pancreatic stem cells. Newly formed clusters of endocrine cells are formed, but they have the characteristics of the preexisting endocrine apparatus and secretory cells with exocrine properties. Key words: pancreas, opisthorchiasis, secretion, superinvasion, stem cells, differentiation. Суперинвазионный описторхоз – заболевание, при котором в патологический процесс вовлекаются многие системы организма. Паразит вследствие своих уникальных свойств изменяет многочисленные гены, дает начало пролиферативным процессам. Происходит инициация стволовых клеток с последующей дифференцировкой их по линиям различных дифферонов. Печень и поджелудочная железа являются эконишами, где длительное время вегетируют паразиты, однако сведений о динамике пролиферативных изменений в железе с рассмотрением различных гистогенетических путей в литературе не представлено. Цель работы. Выявить пролиферативную активность стволовых клеток поджелудочной железы при суперинвазионном описторхозе и их дифференцировку. Материалы и методы. Фрагменты ткани поджелудочной железы были отобраны для изучения в патологоанатомических отделениях медицинских учреждений, расположенных на территории гиперэндемического очага (Тюменская, Омская области, Ханты-Мансийский автономный округ). Сформированы следующие группы: 1 группа – лица, в анамнезе которых имелся суперинвазионный описторхоз с длительностью инвазии более 10 лет (n=70); 2 группа – лица без инвазии, умершие от случайных причин (n=10). Материал подвергался рутинному гистологическому исследованию, использовались гистохимические и иммуногистохимические методы окрашивания, проводился статистический анализ. Результаты. Обнаружены выраженные пролиферативные реакции в эпителиальном пласте протоковой системы, секреторном отделе поджелудочной железы и изменения структуры эндокринного аппарата. Выявлены гистологические типы эндокринных образований поджелудочной железы: островки Лангерганса обычного строения, гиперплазированные и новообразованные островки. Отличие последних от предсуществующих форм состояло в беспорядочном расположении, неопределенной форме, существенно более крупных размерах, отсутствии или наличии формирующейся капсулы. Новообразованные островки содержали аналогичные клетки, обладающие идентичными свойствами предсуществующих эндокриноцитов. Выводы. При воздействии секретома Opisthorchis felineus происходит инициация и дифференцировка стволовых клеток поджелудочной железы с формированием новообразованных скоплений эндокринных клеток со свойствами предсуществующего эндокринного аппарата и секреторных клеток с экзокринными свойствами. Ключевые слова: поджелудочная железа, описторхоз, секретом, суперинвазия, стволовые клетки, дифференцировка.

IMBALANCED IMMUNOBIOLOGICAL REACTIVITY, INFECTION AND MUTAGENESIS FACTORS IN THE HEPATOBILIARY SYSTEM AFFECTED BY A NATURAL FOCAL HABITAT FACTOR – A TREMATODE INVASION BY OPISTHORCHIS FELINEUS

An interaction between decreasing host anti-infective defense due to long-term invasion with Opisthorchis felineus in the hepatobiliary system, duct bile colonization by microflora and revealing the endogenous mutagenesis (carcinogenesis) factor - secondary bile acids - in bile is considered in the article. The role of organism genotype in the pathogen-related immune response to Opisthorchis felineus trematode and helminth development in the hepatobiliary system has been shown. The role of dysregulated mechanisms of tissue homeostasis in induction of compensatory chronic homeostatic proliferation and somatic cell oncogenesis is discussed. The study results evidence that disturbed functioning of the regulatory T-cells, inhibition of the NK cell effector function and very high functional activity of the memory B-cells are of great importance in imbalanced host immunobiological reactivity, caused by chronic opistorchosis invasion. Decreased host anti-infective protection causes intrahepatic bile duct infection with different bacterial species. Presence of secondary bile acids in hepatobiliary system was associated with biliary bacterial strains inhabiting intestinal tract: Proteus vulgaris*, Proteus mirabilis*, Citrobacter freundii*, Bacteroides alcaligues faecalis*, Clostridium*, Streptococcus faecalis*, Еscherichia coli* (*gut microflora). Participation of microbiota in bile acid biotransformation immediately in the duct bile has been confirmed in in vitro experiments. Experimental methods on Drosophila melanogaster and Salmonella tiphimurium strains: TA 100, TA 98 allowed to find out that bile from chronic opistorchosis patients exerts higher mutagenic activity compared with control groups. Mutational events in somatic and bacterial cells depend on the presence of secondary bile acids (deoxycholic, lithocholic) in duct bile, as well as the level of total bile acid concentration. The study data confirm the concept by Professor A.A. Shain about presence of endogenous risk factor for developing primary cholangiocellular liver cancer such as secondary bile acids in the bile of chronic opistorchosis patients. A concept of cholangiocarcinogenesis, based on mutational events, is added up with disturbance of generative cycle in tissue cells and their differentiation due to decreased kylon factor activity, as well as sensitivity threshold to it. Level of investigation and understanding of mechanisms underlying cholangiocarcinogenesis during chronic opisthorchiasis invasion will allow to develop pathogenetic approaches to correct homeostasis regulation and prevention of cholangiocarcinomas.

Effects of single and 7-fold administration of a complex of albendazole with disodium salt of glycyrrhizic acid to hamsters infected with Opisthorchis felineus

The purpose of the research is to evaluate the effect of albendazole as part of the supramolecular complex with disodium salt of glycyrrhizic acid obtained by solid-phase mechanical treatment.Materials and methods. The anthelmintic activity of the complex and its effect on the host organism was assessed on hamsters infected with Opisthorchis felineus by single and 7-fold administration at 45 days after infection. After 21 days, we counted the number of helminthes in the liver, and conducted a morphometric analysis of the liver and spleen, and detected biochemically the activity of alanine aminotransferase and aspartate aminotransferase in the animals’ blood serum.Results and discussion. The number of O. felineus significantly decreased after 7-fold, but not a single, administration of albendazole (ABZ) and ABZ-Na2GA complex (1 : 10). The administrated substances had no effect on the weight gain of the animals and the daily consumption of the pellets. At the same time, ABZ only as part of the complex normalized the weight of the liver and spleen in hamsters infected with O. felineus and reduced the alanine aminotransferase activity. Consequently, a longer administration of ABZ as part of the complex with disodium glycyrrhizinate has not only a pronounced anthelmintic effect, but also improves some of the physiological parameters of hamsters to a greater extent than a pure substance.

On the Helminth Fauna of the Muskrat (Ondatra zibethicus (Linnaeus, 1766)) in the Barnim District of Brandenburg State/Germany

The muskrat is a neozoon species that has occupied many countries of continental North Europe after its introduction from north America as fur animals. Due to its burrowing activity it damages river and canal banks and structures of flood control. For this reason, the eradication of this alien species is recommended. Muskrats are also of parasitological interest since they can act as suitable intermediate hosts for Echinococcus multilocularis. On the other hand, little is known on the other helminths that infect muskrats. A total of 130 muskrats of different age groups trapped in different habitats in the Barnim district of the Brandenburg state by a professional hunter were examined for parasites and seven trematodes (Echinostoma sp., Notocotylus noyeri, Plagiorchis elegans, Plagiorchis arvicolae, Psilosostoma simillimum, P. spiculigerum, Opisthorchis felineus and four larval cestode species (Hydatigera taeniaeformis, Taenia martis, Taenia polyacantha, Taenia crassiceps) were detected. Larval stages of E. multilocularis were not found. O. felineus was found for the first time in muskrats in Germany. All the named parasites were present in Europe prior to the introduction of muskrats. With a prevalence of 48.9%, Strobilocercus fasciolaris, the larval stage of the cat tapeworm, H. taeniaeformis, was the most frequent parasite found in adult muskrats.

Serotonin Signalling in Flatworms: An Immunocytochemical Localisation of 5-HT7 Type of Serotonin Receptors in Opisthorchis felineus and Hymenolepis diminuta

The study is dedicated to the investigation of serotonin (5-hydroxytryptamine, 5-HT) and 5-HT7 type serotonin receptor of localisation in larvae of two parasitic flatworms Opisthorchis felineus (Rivolta, 1884) Blanchard, 1895 and Hymenolepis diminuta Rudolphi, 1819, performed using the immunocytochemical method and confocal laser scanning microscopy (CLSM). Using whole mount preparations and specific antibodies, a microscopic analysis of the spatial distribution of 5-HT7-immunoreactivity(-IR) was revealed in worm tissue. In metacercariae of O. felineus 5-HT7-IR was observed in the main nerve cords and in the head commissure connecting the head ganglia. The presence of 5-HT7-IR was also found in several structures located on the oral sucker. 5-HT7-IR was evident in the round glandular cells scattered throughout the larva body. In cysticercoids of H. diminuta immunostaining to 5-НТ7 was found in flame cells of the excretory system. Weak staining to 5-HT7 was observed along the longitudinal and transverse muscle fibres comprising the body wall and musculature of suckers, in thin longitudinal nerve cords and a connective commissure of the central nervous system. Available publications on serotonin action in flatworms and serotonin receptors identification were reviewed. Own results and the published data indicate that the muscular structures of flatworms are deeply supplied by 5-HT7-IR elements. It suggests that the 5-HT7 type receptor can mediate the serotonin action in the investigated species and is an important component of the flatworm motor control system. The study of the neurochemical basis of parasitic flatworms can play an important role in the solution of fundamental problems in early development of the nervous system and the evolution of neuronal signalling components.

Experimental study on the proliferating activity and differentiation of renal stem cells in superinvasive opisthorchiasis

AIM: This study aimed to identify the replication potential of the kidneys in different forms of opisthorchiasis in laboratory animals. MATERIALS AND METHODS: An experiment was conducted on 60 Syrian male hamsters. The first group was set as the control (n = 10), the second group (n = 25) was infected with metacercariae (Opisthorchis felineus), and the third group (n = 25) was a model of a superinvasive form of opisthorchiasis infection with 50 O. felineus larvae and repeated infection with 50 metacercariae in 14 and 25 days. The hamsters were withdrawn from the experiment on days 7, 15, and 30 via an overdose of narcosis and decapitation. The kidneys were isolated and histologically examined through histochemical and immunohistochemical staining methods. Microscopy was conducted, and results were statistically analyzed. RESULTS: The quantitative characteristics, proliferation tendencies, and differentiation of regional stem cells were identified. In the cortical and medullary substance of the kidneys, CD117, Oct4, and CD34 markers were expressed, and CD31-positive stem cells further differentiated to progenitor cells. Epithelial structures developed in the form of tubules. In the glomeruli, vasculogenesis occurred, and the number of vascular loops increased. CONCLUSION: O. felineus secretome initiated the activation of stem cells in the renal tubules and pericytes of a microcirculatory network. The transitional epithelium of the renal pelvis and the initial parts of the ureter proliferated. Under the action of the secretome of parasites, stem cells proliferated directly in glomerular loops.

Liver pathology in spontaneous superinvasive opistorchiasis in synanthropic animals in hyperendemic hotbed

Opisthorchiasis is a parasitic disease that represents a serious medical and social problem for the population living in the area of vegetation of opisthorchis larval stages. The parasitic disease significantly reduces the activity and potential of the indigenous and newcomer population of hyperendemic hotbeds. Wild and domestic animals are actively involved in maintaining the epidemiological tension of the hotbeds, among the latter, cats and dogs infected with opisthorchiasis, in which the pathomorphology of the liver and oncogenesis have not been studied, are of great importance. On the basis of sufficient factual material, this gap has been partially filled in this work. Aim. To determine the features of pathomorphological transformations of the liver and pancreas in spontaneous opisthorchiasis in cats and dogs, to identify the possibility of oncogenesis against the background of superinvasive opisthorchiasis (SO) in synanthropic animals of a hyperendemic hotbed. Material and methods. The pathomorphology of the liver and pancreas in 48 animals was studied. The specimens were processed by histological, histochemical methods, the indices of the area of inflammatory infiltrates, granulomas, necroses were calculated, the indicators were subjected to statistical processing. Results. In superinvasive opisthorchiasis, cats and dogs develop a complex of structural proliferating transformations characteristic of other hosts of the parasite – humans, laboratory animals. In synanthropic animals, against the background of SO, the development of malignant neoplasms is possible. Conclusion. Superinvasive opisthorchiasis in synanthropic animals causes permanent proliferation of progenitor cells of the liver and pancreas, which provides the promotor effect of oncogenesis in the liver – the main econiche of Opisthorchis felineus vegetation.

PARASITOCENOSES OF FISH IN RIVERS AND RESERVOIRS OF THE RYAZAN REGION

The wide distribution of trematodes and other helminths with a complex biological cycle in the reservoirs of the Ryazan Region indicates an increase in the level of their eutrophication. We performed a complete helminthological study of the ide (25 copies), roach (21 copies), and perch (29 copies) according to K.I. Skryabin. Taking into account complex hydrological indicators (species composition of higher plants, microphytes, fauna of hydrobionts and their parasites), the Pra River is a dystrophic water body, the Pronya River is an eutrophic water body, and the Novomichurinsk Reservoir is a hypertrophic water body. Parasites of Pra River were represented by nine species, the Pronya River by four species, and the Novomichurinsky Reservoir by six species. Trematodes dominate in all reservoirs of the Ryazan Region. We found an endemic focus of opisthorchiasis in the Pra River where the infection of the ide with metacercarias Opisthorchis felineus reaches 38–50% with an invasion intensity of up to 70 specimens. A number of species of trematodes (Ichthyocotylurus spp., Paracoenogonimus ovatus, Diplostomum spp., Posthodiplostomum spp., Tylodelphys clavata) and cestodes (Ligula intestinalis) should be considered as indicators for assessing an increase in concentration of dissolved biogenic substances in the water bodies. There is a direct correlation between the infection of fish with the above species of trematodes and cestodes (tapeworms) being indicators of the invasion intensity and the degree of eutrophication of the reservoirs.

CYPRINID FISH INFECTION WITH METACERCARIAS OF TREMATODES UNDER ANTHROPOGENIC EFFECT

We presented parasitological studies of fish (Cyprinidae) in the Usman River within the Voronezhsky Biosphere Reserve. The infection of fish with metacercarias of trematodes in the river sections with various anthropogenic effect was assessed. We identified metacercarias of 6 species of trematodes: Opisthorchis felineus, Pseudamphistomum truncatum, Metorchis bilis, M. xanthosomus, Posthodiplostomum cuticola, and Paracoenogonimus ovatus. The highest occurrence and abundance of P. cuticola (95.4%, 36.4 sp.) and P. ovatus (56.9%, 8.8 sp.) was noted in the upper reaches of the River, in areas with high anthropogenic impact. An increased prevalence of infection was recorded for the group Opisthorchiidae (O. felineus, P. truncatum, and M. bilis) in areas within the Reserve with minimal anthropogenic impact. Infection rate of Opisthorchiidae metacercarias was 86.5% and 8.2 specimens in the middle and lower reaches of the River. At the same time, we noted a decrease in infection with P. cuticola (54.5%; 5.8 sp.) and P. ovatus (33.9%; 1.3 sp.). The degree of anthropogenic eutrophication of the Reservoir affects the development of different species of mollusks and the infection of fish with metacercarias. Metacercarias of trematodes can be used as indicators of water pollution in assessing anthropogenic effect on natural ecosystems.

THE PROBLEM OF BERDSKY BAY OF THE NOVOSIBIRSK RESERVOIR IN RELATION TO OPISTHORCHIDOSES

We studied the epidemic situation of opisthorchiasis in the population of Berdsk, and determined the level of infection of fish of the family Cyprinidae by larvae of opisthorchides in Berdsky Bay, as well as the abundance of the first intermediate host of the parasite, the mollusks of the family Bithyniidae in the water body. It is found that Berdsky Bay is generally unfavorable for opisthorchiasis. The incidence of opisthorchiasis in people in Berdsk was recorded annually; the average long-term incidence rate was 89.85 per 100 thousand of population with a minimum of 21.4 in 1991 and a maximum of 188.5 in 2002. The level of infection of cyprinid fish with metacercarias of opisthorchides is quite high (33.3%).The epidemic danger is posed by fish infected with larvae of Opisthorchis felineus (the prevalence 31.7%) and Metorchis bilis (the prevalence 3.3%). The ide, dace and verkhovka represent a high potential danger in infecting the population with opisthorchiasis and are indicators of the adverse situation with the reservoir as regards the invasion. In the water area of Berdsky Bay, we identified new actively functioning foci of opisthorchis invasion located in the Lebedevskaya and Elbanskaya streams, as well as in the mouth of the Sukhaya River where not only infected fish were recorded, but also the number of the first intermediate host of opisthorchides, the mollusks of the family Bithyniidae (Bithynia troscheli and B. tentaculata), is quite high.