Chronic Opisthorchiasis Research Articles

Investigation of a Serine Protease Inhibitor Active in the Infectious Stage of the Human Liver Fluke Opisthorchis viverrini.

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process.

Immune system parameters in patients with chronic opistorchiasis based on genetic polymorphism associated with carbohydrate and lipid metabolism disorders

A study assessing differences in the immunopathogenesis of chronic opisthorchiasis with or without genetic polymorphisms in patients associated with predisposition to type II diabetes mellitus and lipid metabolism disorders was carried out. Venous blood samples from 89 patients were collected to analyze immunological parameters and genetic polymorphisms by using pyrosequencing. In the presence of mutations affecting regulation of carbohydrate metabolism, patients with opisthorchiasis had less pronouncedly increased phagocytic activity, a lower monocyte count, higher total T-lymphocyte count, lower T-cytotoxic cell and B-lymphocyte counts, lower IgA, IgG but higher IgM concentration. This indicates a moderately compromised nonspecific resistance, imbalanced effector T- and B-immunity, but in most cases (PPARG, TCF7L2 rs12255372, CDKAL1, CDKN2A/2B, SLC30A8) aggravated humoral immune reaction to invasion particularly revealed by lower B-lymphocyte count. The presence of polymorphisms that alter lipid metabolism regulation bidirectionally affects the parameters of immune response. An increased B-lymphocyte count and other indicators of humoral immune activation to invasion, which are detected in groups with mutations in the APOE (rs429358), PCSK9, ABCA1, APOC3 rs2854117 genes, can contribute to a more effective response to sustained antigenic stimulation. Changes in the T-lymphocytes subpopulations, characteristic of opisthorchiasis invasion, are aggravated in the presence of mutations in the PCSK9, ABCA1, and APOC3 rs2854117 genes. Mutations in the genes APOC3 rs5128, LPL rs268 activate patients’ nonspecific resistance, although this effect may also be associated with exhaustion of neutrophil bactericidal reserve. In general, minor alleles of the APOE (rs429358), APOC3 rs2854117, LPL rs268, LPL rs328, PON1 rs662 genes can be considered “protective” for the immunopathogenesis of chronic opisthorchiasis. Thus, patients with chronic opisthorchiasis with different genotypes predisposing to carbohydrate and lipid metabolism disorders had significant differences in immune system parameters, which can also affect the disease course. Mutations in different loci of the PCSK9, ABCA1, APOE, APOC3, and PON1 genes have opposite effects on the analyzed immune parameters. In the presence of mutations in other genes (PPARG, TCF7L2 rs12255372, CDKAL1, CDKN2A/2B, SLC30A8, LPL), opisthorchiasis invasion leads to more pronounced alterations in immune response; mutations in the lipoprotein lipase gene may have some “protective” immune-related effect in opisthorchiasis. The effect of all studied genetic polymorphisms associated with a predisposition to developing type II diabetes mellitus was predominantly negative.

Chronic opisthorchiasis - as a systemic pathology and issues of comorbidity. Clinic, diagnosis, treatment

The article shows that chronic opisthorchiasis is a systemic and comorbid disease caused by the trematode Opistorhis felineus when consuming fish of the carp family that is not sufficiently salted or heat-treated. The systemic nature of the disease lies in the fact that with opisthorchiasis, the organs of the helminth’s permanent habitat are affected; organs located on the paths of its migration, as well as intact organs and systems. The described syndromes and symptoms indicate the comorbidity of the pathology. The author describes a three-stage system for the treatment of chronic opisthorchiasis, including gentle specific therapy.

Hepatic vascular changes associated with Opisthorchis felineus infection in Syrian hamsters and humans

The liver fluke Opisthorchis felineus is a foodborne zoonotic pathogen endemic to Russia, Kazakhstan, and several European countries. The adult flukes affect the hepatobiliary system of piscivorous mammals and humans, thereby causing numerous complications, including liver fibrosis. Detailing the mechanisms of progression of the fibrotic complications is a hot topic in the field of research on opisthorchiasis pathogenesis. Pathologic angiogenesis appears to be associated with the fibrogenic progression due to active participation in the recruitment of inflammatory cells and many factors involved in the modulation of the extracellular matrix. The aim of the study was to evaluate neoangiogenesis and amyloid deposits in liver tissues of model animals and patients with confirmed chronic opisthorchiasis. In addition, we assessed a possible correlation of neoangiogenesis with liver fibrosis. We found a significant increase in the number of newly formed vessels and amyloid deposits in the liver of people with chronic opisthorchiasis compared to that of uninfected ones. Thus, for the first time we have demonstrated neoangiogenesis and amyloid deposits during O. felineus infection in a Mesocricetus auratus model. Regression analysis showed that CD34+ newly formed vessels correlate with fibrosis severity in the course of the infection. Our results indicate the potential contribution of angiogenesis to the progression of liver fibrosis, associated with O. felineus infection.

ПОКАЗАТЕЛИ ФУНКЦИЙ ГЕПАТОБИЛИАРНОЙ СИСТЕМЫ У БОЛЬНЫХ ХРОНИЧЕСКИМ ОПИСТОРХОЗОМ С ГЕНЕТИЧЕСКОЙ ПРЕДРАСПОЛОЖЕННОСТЬЮ К НАРУШЕНИЯМ ЛИПИДНОГО ОБМЕНА

To assess the influence of genetic features of metabolic processes on the reactivity to opisthorchiasis invasion, biochemical parameters of the hepatobiliary system functions were studied in chronic opisthorchiasis patients with and without mutant alleles of polymorphisms associated with lipid metabolism disorders were studied. It was found that the presence of mutations in the loci rs2854117 APOC3, rs7412 APOE enhances the negative impact of chronic opisthorchiasis invasion on the state and function of hepatocytes. The presence of the rare allele rs11206510 PCSK9 prevents an increase in ALP activity. In subgroups with mutations in individual polymorphisms of the APOE, APOC3I, ABCA1, lipoprotein lipase, and paraoxanase-1 genes, differences were found in the change in parameters after anthelmintic therapy, possibly associated with their role in the response of metabolic processes not only to invasion, but also to the treatment process.

IImmunological parameters in patients with chronic opisthorchiasis bearing gene mutations associated with osteoporosis-predisposition

To identify the features of the immune system functioning in patients with chronic opisthorchiasis bearing mutations in loci associated with predisposition to developing osteoporosis, comprehensive studies of cellular and humoral arms were carried out. The state of the phagocytic system was assessed by assessing absorption, metabolic activity and reactive oxygen species formation to restore nitrosine tetrazolium (spontaneous and stimulated NBT test). The phenotype of lymphocytes was determined by flow cytometry. The humoral immune arm was evaluated by the number of immunoglobulin classes M, G, A and E. Differences in the functional state of various arms of the immune system were revealed. In patients with chronic opisthorchiasis in the presence of rs1544410 polymorphism of the gene encoding the intracellular vitamin D receptor, the relative number of T-helper cells is significantly lower than in the group with the normal allele. In the presence of rs1800012 polymorphism of the gene encoding the α1-chain of type I collagen, the absolute lymphocyte count is significantly higher, spontaneous and stimulated NBT test were lower, the number of DN-T lymphocytes is significantly lower (both in relative and absolute values). In the presence of the rs3736228 mutation of the gene encoding the transmembrane low-density lipoprotein receptor, the level of myeloperoxidase and the neutrophil stimulation index are lower, the absorption activity of neutrophils is higher. The presence of the rs2234693 mutation for estrogen receptor gene leads to significantly increased level of stimulated NBT test and IgG concentration. Thus, patients with chronic opisthorchiasis bearing mutations in the COL1 A1, LRP5, ESR1(rs2234693) genes, have altered both nonspecific innate reactions and parameters of the adaptive immune arm; mutation of the VDR gene solely affects adaptive immunity. Analysis of the results suggests that the presence of mutations associated with the development of osteoporosis has a modulating effect on the immune response in chronic opisthorchiasis invasion. The identification of polymorphic genes associated with metabolic disorders of bone tissue and the study of the immunological profile in patients with chronic opisthorchiasis invasion will allow to implement an individual approach in the treatment of such patients.

Secreted and surface proteome and transcriptome of Opisthorchis felineus

IntroductionOpisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis are the most medically important species of fish-borne zoonotic trematodes. O. felineus is endemic to the river plains of Western Siberia and Eastern Europe, and it is estimated that more than 1.6 million people could be infected with this parasite. Chronic opisthorchiasis may lead to significant gastrointestinal and hepatobiliary pathology. This study aimed to identify and characterize proteins from the secreted and tegumental proteomes of O. felineus. MethodsAdult flukes were collected from experimentally infected hamsters and cultured in vitro in serum-free media. We extracted proteins from different compartments of the O. felineus secretome, including (i) soluble excretory/secretory (ES) products; (ii) secreted 15K-extracellular vesicles (EVs); and (iii) tegument. ResultsWe also generated a transcriptome using long-read sequencing, and when this was combined with high-resolution mass spectrometry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation, and protein digestion, we identified 686, 894, 389, 324, and 165 proteins from the ES, 15K-EV, and the three sequentially extracted tegument (TEG) protein fractions, respectively. We conducted in-depth gene ontology and protein family analyses on the identified proteins and discussed comparisons against similar proteome data sets acquired for the Southeast Asian liver fluke O. viverrini and the Chinese liver fluke C. sinensis. DiscussionThe information from this study will form a biologically relevant data set of O. felineus proteins that could be used to develop diagnostic and therapeutic tools to manage the human cost of O. felineus infection and its associated comorbidities.

Functional gastrointestinal disorders in patients with chronic opisthorchiasis and rosacea

Functional gastrointestinal disorders in patients with chronic opisthorchiasis and rosacea

Differences in immune response in patients with chronic opisthorchiasis invasion related to clinical manifestations of hepatobiliary system diseases

The aim of the study was to identify differences in the immune response in chronic opisthorchiasis with or without clinical manifestations of hepatobiliary system diseases. A comparison of laboratory parameters of immune system functioning (lymphocyte phenotypes detected by flow cytometry, concentrations of immunoglobulins and cytokines, indicators of nonspecific resistance) in patients with chronic opisthorchiasis (caused by Opisthorchis felineus) lacking clinical manifestations and with manifestations of cholecystitis, cholangitis, pancreatitis, gastritis, gastroduodenitis, confirmed by ultrasound and FGDS. It was found that the immunological parameters of the innate immune response were increased in all patients with chronic opisthorchiasis compared with the control group of apparently healthy subjects, which indicates activated macrophage-phagocytic arm in patients with opisthorchiasis invasion. The indicator of the stimulated NBT test is maximal in chronic opisthorchiasis without hepatobiliary system diseases, in case of comorbidity with such diseases it declines, but remains significantly higher than the values of the control group. This may be due to the exhaustion in the reserve capabilities of bactericidal neutrophil systems in patients with symptoms of damage to the hepatobiliary system due to prolonged inflammation. Indicators of the cellular arm in patients with chronic opisthorchiasis without clinical manifestations of hepatobiliary system diseases demonstrate a decrease in the absolute and relative level of cytotoxic T-lymphocytes. In the group with picture of hepatobiliary system disorders, an even deeper imbalance of indicators is revealed: the relative and absolute level of lymphocytes, the absolute count of T-lymphocytes are reduced, which may indicate about involvement of cellular immunity in the pathogenesis of complications of chronic opisthorchiasis. In all examined patients with chronic opisthorchiasis, the humoral immune response was also activated the total immunoglobulin E was significantly increased; at the same time, in patients with hepatobiliary system lesions, this parameter increases even more and a decrease in B-lymphocytes is detected. In all patients with chronic opisthorchiasis, there is also an increased level of proinflammatory cytokine IL-8, which rises even more in the group with hepatobiliary system diseases. The data obtained indicate the involvement of all arms of the immune response in a prominent inflammatory process when chronic opisthorchiasis is complicated by clinically overt hepatobiliary system diseases: the degree of deviation in the indicators of the innate and adaptive immune response from the norm and from those in uncomplicated opisthorchiasis increases.

ПОКАЗАТЕЛИ ФУНКЦИЙ ГЕПАТОБИЛИАРНОЙ СИСТЕМЫ У БОЛЬНЫХ ХРОНИЧЕСКИМ ОПИСТОРХОЗОМ С ГЕНЕТИЧЕСКОЙ ПРЕДРАСПОЛОЖЕННОСТЬЮ К РАЗВИТИЮ САХАРНОГО ДИАБЕТА 2 ТИПА

To assess the influence of genetic features of metabolic processes on the reactivity to opisthorchiasis invasion, biochemical parameters of the hepatobiliary system functions were studied in chronic opisthorchiasis patients with and without mutant alleles of polymorphisms associated with the type 2 diabetes mellitus. It was found that the presence of mutations in the genes PPARG and TCF7L2 (rs12255372), HHEX, IGF2BP2 and SLC30A8 enhances the opisthorchiasis negative impact on the activity of alpha-amylase and indicators of impaired bile secretion. The presence of a rare allele of the IGF2BP2 gene prevents an increase in GGT activity, and the presence of a rare allele of the CDKAL1 and SLC30A8 genes reduces the degree of increase in glucose concentration in opisthorchiasis. Key words: chronic opisthorchiasis, polymorphisms, type 2 diabetes mellitus, biochemical parameters, disorders of bile secretion.

The role of endoscopic draining in the treatment of mechanical jaundice of different etiology on the background of chronic opistorchiasis

Purpose of the study: to conduct a comparative analysis of endoscopic retrograde transpapillary interventions in patients with chronic opisthorchiasis and obstructive jaundice of various etiologies.Material and Methods. The results of treatment of 129 patients with chronic opisthorchiasis and obstructive jaundice of various etiologies who underwent transpapillary interventions to correct bile duct, were analyzed. In order to assess the condition of patients, general clinical methods and a set of laboratory and instrumental methods were used. The study was carried out in two groups, in the first endoscopic drainage of the bile ducts was carried out, in the second – either endoscopic papillosphincterotomy with choledocholitholite extraction, or endoscopic papillosphincterotomy with bougienage of the ducts. Each group of patients was divided into subgroups according to nosology: 1 – diseases of the biliary tract; 2 – oncological diseases of the hepatopancreatic zone; 3 – pancreatitis. Laboratory indicators before and after the application of minimally invasive interventions were compared.Results. The use of various methods of transpapillary interventions normalizes indicators of bilirubin, and aminotransferases in patients by 5–10 days. Endoscopic stenting of the ducts was the method of choice with cancers of the parasitic organs. With concomitant choledocholithiasis, the endoscopic papillosphincterotomy technique with subsequent lithotripsy and lithoextraction were used. Draining operations showed a good efficacy in the treatment of jaundice in the case of impossibility of removing the calculus. Endoscopic papillosphincterotomy with ductal bougienage was the operation of choice for streak tours of the common bile duct. With purulent cholangitis, endoscopic papillotomy with the installation of nasobiliary drainage and subsequent sanitation of the bile ducts was used.Conclusion. The use of endoscopic drainage has proven itself well in patients with obstructive jaundice of tumor etiology, as well as in patients with choledocholithiasis when it is impossible to remove the calculus. This surgery allows for quick normalization of the bilirubin level.

Состояние вегетативной адаптации у пациентов с хроническим описторхозом

Aim: to assess autonomic regulation by analyzing heart rate variability in patients with chronic opisthorchiasis (CO). Patients and Methods: the study involved 31 patients with CO and 30 healthy volunteers (control group). Clinical parameters of the biliary system and autonomic nervous system were as-sessed in all participants. The Baevsky method of mathematical analysis of heart rhythm was used to study autonomic nervous system functions. Parameters characterizing the activity of various branches of the autonomic nervous system were calculated. The results obtained were processed using the original CORVEG program. Results and Discussion: frequency analysis in patients with CO revealed significant deviations of SD, AMo, and CV in the supine position and EV, MSD, Mo, and AMo in active orthostatic position, thereby illustrating an increase in the effect of the sympathetic branch and a reduction in the effect of the parasympathetic branch of the autonomic nervous system (a decrease in heart rate variability in the CO group). Values of the HF and LF waves were significantly higher in the CO group (p<0.05) in the supine position, thereby illustrating an impairment of humoral regulation and a decrease in the activity of intersystem regulation. Changes in the parameters of heart rate regulation significantly (p<0.05) showed increased sympathetic regulation at rest and orthostasis in CO. Their autonomic homeostasis shifted toward the sympathetic nervous system both at rest and during exercise. Moreover, in some patients (31%), central dysregulation was detected. Autonomic tone and autonomic reactivity in CO were also significantly (p<0.05) shifted toward sympathicotonia and hypersympathicotonia. Conclusion: in CO, deviations at all levels of autonomic regulation were detected, which manifested as increased activity of the sympathetic branch and decreased parasympathetic regulation, vasoreflex and baroreflex mechanisms, and humoral regulation. KEYWORDS: chronic opisthorchiasis, Opisthorchis felineus, autonomic nervous system, parasympathetic nervous system, sympathetic nervous system, heart rate variability, cardiointervalography. FOR CITATION: Plotnikova E.Yu., Karbysheva N.V., Nikonorova M.A., Beskhlebova O.V., Ivanova M.E., Sinkov M.A., Kanner E.V., Kanner I.D. Vegetative adaptation in patients with chronic opisthorchiasis. Russian Medical Inquiry. 2023;7(11):725–732 (in Russ.). DOI: 10.32364/2587- 6821-2023-7-11-4.

ВЕГЕТАТИВНАЯ ДИСФУНКЦИЯ ПРИ ХРОНИЧЕСКОМ ОПИСТОРХОЗЕ

Introduction. Altai Krai is one of the most significant natural foci of opisthorchiasis not only in Western Siberia, but also on the territory of the Russian Federation as a whole, where the incidence of opisthorchiasis ranges from 44 to 34 per 100,000 people. The primary habitat of opisthorchiasis is the hepatobiliary system; the polymorphism of long-term invasion polymorphism is characterized by a violation of the functions of many organs and systems: liver, immune and cardiovascular systems, digestive organs, etc. The control center responsible for these vital functions of the body is the autonomic nervous system, which regulates them in the body. Analysis of heart rate variability can give an idea of sympathetic-parasympathetic vegetative balance. The purpose of the study. A comprehensive study of the state of autonomic regulation by analyzing heart rate variability in patients with chronic opisthorchiasis. Materials and methods. During the course of the study, 31 patients with chronic opisthorchiasis (CO) were observed, a comparison was made with 30 healthy individuals of similar age and gender. When studying the function of the autonomic nervous system, the method of mathematical analysis of the heart rate according to R.M. Baevsky was used with the help of the original program "CORVEG". The level of critical significance of the null statistical hypothesis was assumed to be 0.05. Results and Discussion. In the study of the nature of vegetative changes, methods of frequency and spectral analysis of heart rate were used. Changes in heart rate regulation indicators were statistically significantly (p<0.05) indicated an increase in sympathetic regulation at rest and orthostasis in patients with SC, their autonomic homeostasis was shifted toward the sympathetic nervous system both at rest and under load, and in some patients (31%) dysregulation of the central type was detected. Conclusions. Dysregulation of autonomic nervous system in patients with chronic opisthorchiasis was revealed at all levels of its regulation and was characterized by increased sympathetic tone and a decrease in parasympathetic and humoral regulation, as well as vasoreflective and baroreflective mechanisms.

ЭНДОТЕЛИАЛЬНАЯ ДИСФУНКЦИЯ ПРИ ХРОНИЧЕСКОМ ОПИСТОРХОЗЕ

Relevance. The basis of the complex, in some cases, of interrelated pathological processes in opisthorchiasis invasion may be endothelial dysfunction, which provokes the risks of cardiovascular events, and is an important aspect in establishing pathogenetic connections between diseases of the liver and the cardiovascular system. When assessing the inflammatory process in the pathogenesis of invasion, there is increasing interest in the contribution of not only cellular elements and circulating inflammatory markers, but also in intercellular communication and regulation through microvesicles. Purpose of the study. To study the circulation of extracellular vesicles, which are associated with blood and endothelial cells, in patients with chronic opisthorchiasis. Materials and methods. The study was conducted on two groups of people (men and women aged 18 to 55 years (34.3±1.3 years)). The experimental group included patients with chronic opisthorchiasis (n=26). There was the control group (n=14) matched by gender and age with experimental group. The qualitative composition and quantity of extracellular vesicles (EVs) was determined by flow cytometry. Results. In the experimental group, there was a significant increase in the number of CD41+ events (platelet EVs) 2 times (from 120 to 245 events/μl (p = 0.030), CD45+ events (panleukocyte EVs) - more than 120 times (from 95 to 11713 events/μl (p=0. 0330), as well as a trend towards an increase in the rate for CD31+ events by 19.8 times (from 495 to 9818 events/μl (p=0.133) in comparison with control group. Screening assessment of the CD9+ population of platelet EVs (CD41+31+CD9+), endothelial EVs (CD41-31+9+) and leukocyte EVs (CD45+CD9+) showed a significant (p=0.0330) increase in events, while their content corresponded to the data obtained by CD31+41+ events in quantitative terms. For endothelial EVs, there was a significant increase in the proportion of CD9+ events from 23.3% in the control to 46.2% in the experimental group, while for leukocyte EVs there was no statistically significant change in the percentage ratio, and it was 9.9% in the control and 12.9% in the experimental group. Conclusions. These results can be considered from the perspective of platelet activation, as well as the development of the inflammatory process and endothelial dysfunction, where microvesicles can act as potential highly sensitive markers for opisthorchiasis invasion.

О микробном пейзаже небных миндалин у больных хроническим тонзиллитом, ассоциированным с хронической описторхозной инвазией

The palatine tonsils, both in normal conditions and in chronic tonsillitis (CT), can be carriers of a diverse, including pathogenic, flora, which becomes virulent under the influence of various adverse factors of the external and internal environment. Bacteriological studies carried out by CT patients show a wide variety of vegetative flora in the palatine tonsils. However, according to many researchers, the leading microbial agent is group A B-hemolytic streptococcus. Objective. To study the microbial landscape of palatine tonsils in patients with chronic tonsillitis associated with chronic opisthorchiasis and without the latter. Patients and methods. The authors studied the microbial landscape in 106 patients with chronic tonsillitis aged 20 to 55 years who were randomized into 2 groups (the main 56 people with CT associated with chronic opisthorchiasis invasion (COI)) and the control group (50 patients without the presence of the latter). Conclusions. It was established that the microbial landscape of the palatine tonsils in patients with chronic tonsillitis in association with COI is characterized by significant polymorphism with the predominant role of staphylococci and streptococci, with the lacunar microflora being more virulent, while the microflora of the palatine tonsils in patients without COI did not significantly differ from the same as in the control group, but it was less virulent.

Risk Factors of Cholangiocarcinogenesis in Parasitization of a Natural Focal Ecopathogen Helminth <i>Opisthorchis felineus</i>

Epidemiological researches, carried out by professor A.A. Shain in Ob-Irtysh basin of Tyumen region, have allowed to consider chronic opisthorchiasis as facultative liver precancer and develop the original concept of cholangiocarcinogenesis on the background of long-term invasion by trematode Opisthorchis felineus of hepatobiliary system of the body. According to this concept, the initiating role in cholangiocyte transformation belongs to endogenous factors secondary bile acids, having mutagenic/carcinogenic properties, which formation is mediated by helminthosis-related exogenous intestinal microflora in intrahepatic bile ducts. Obtained data confirmed content of endogenous carcinogenic factors secondary bile acids in duct bile of chronic opistorchosis patients. Cholestasis, caused by long-term parasitization of helminth Opisthorchis felineus in hepatobiliary system of the body, mediates formation of the host promoter factors, contributing to cholangiocarcinoma progression. These include: high concentration in duct bile of bile acids, products of helminths activity and lipid peroxidation of somatic cell membranes. Promoter factors of cholangiocanerogenesis are also disorders of cholangiocyte membranes structure, tissue regulation of their regeneration, DNA repair and anti-tumorigenic protection of the body. The results of the study confirm the concept of occurrence and development of the process of cholangiocarcinogenesis in chronic opistorchosis infestation, proposed by Professor A.A. Shain and allow to reasonably develop approaches to the correction of homeostasis regulation systems of the organism in the specified helminthiasis. Which also determines the secondary prevention of malignant process in ductal epithelium of hepatobiliary system in posthelminthic period.

Jagged-1/Notch Pathway and Key Transient Markers Involved in Biliary Fibrosis during Opisthorchis felineus Infection.

Chronic opisthorchiasis associated with Opisthorchis felineus infection is accompanied by severe fibrotic complications. It is of high practical significance to elucidate the mechanisms of hepatic fibrosis in chronic infection dynamics. The goal of the study is to investigate the temporal profile of key markers and the Jagged1/Notch signaling pathway in the implementation of fibrosis in a chronic O. felineus infection. For the first time, using histological methods and real-time PCR analysis, we demonstrated the activation of the Jagged1/Notch pathway in liver fibrogenesis, including the activation of the Hes1 and Hey1 target genes during experimental opisthorchiasis in Mesocricetus auratus. Cluster analysis followed by regression analysis of key markers during the infection showed that Jagged1 and Mmp9have the greatest contribution to the development of cholangiofibrosis and periductal fibrosis. Moreover, we detected a significant increase in the number of Jagged1-positive cells in the liver of chronic opisthorchiasis patients compared to that of the control group without infection. The results of the study are extremely informative both in terms of investigation both diverse fibrosis mechanisms as well as potential targets in complex antihelmintic therapy.

THE COMPARATIVE ANALYSIS OF BLOOD CELL MORPHOLOGY AND ITS BIOCHEMICAL FEATURES IN RABBITS WITH CHRONIC OPISTHORCHIASIS

There are many preconditions for changes in erythropoiesis and leukopoiesis, changes in biochemical parameters of blood, hemostatic system in animals and humans under conditions of opisthorchiasis invasion. The purpose of the study was to analyze morphological and biochemical changes in the blood system in chronic opisthorchiasis using an experimental model of Oryctolagus cuniculus. The experiment was carried out on rabbits (Oryctolagus cuniculus) with modeling of chronic opisthorchiasis. The invasive dose was 50 Opisthorchis felineus metacercariae. The animals were divided into a control group (n=10) and an observation group (n=10). The morphological blood picture wich was studied by the standard method was compared with the previously obtained results of its biochemical analysis for the content of lactate dehydrogenase, muscle creatine kinase, aspartate aminotransferase, alanine aminotransferase and uric acid. It was established that 1,5 years after infection of rabbits in the observation group, structural heteromorphism of leukocytes is manifested. Pseudo-eosinophils had toxic granularity in the cytoplasm. In 19,5% of pseudoeosinophils, both stab and segmented, vacuolization of the cytoplasm was noted. A significant number of forms of lympholysis (shadows of Botkin-Gumprecht-Klein) was found – 9,6% per 100 leukocytes. In the study of erythrocytes, the presence of poikilocytosis was established; the most common were acanthocytes, dacryocytes. In rabbits of the control group and the observation group, the differences in blood pH are non-significant. Microscopic observation in a thick blood drop revealed that in the control group erythrocytes in vitro retained passive mobility for 59,8±15,2 seconds, in the observation group this figure was 42,6±7,5 seconds, erythrocytes formed aggregates. The blood clotting time in the control group is 101,9±32,1 seconds, and in the observation group – 194,8±35,2 seconds, the differences are significant. A change in the tinctorial properties of the platelet chromomere was noted. Thus, in rabbits with experimentally induced chronic opisthorchiasis, morphological changes in blood cells are observed, a decrease in the activity of the coagulation system with associated changes in its biochemical parameters, which contributes to a more detailed understanding of the pathogenetic mechanisms of changes in the blood system in chronic opisthorchiasis.

Global changes in gene expression related to Opisthorchis felineus liver fluke infection reveal temporal heterogeneity of a mammalian host response

The food-borne trematode Opisthorchis felineus colonizes bile ducts of the liver of fish-eating mammals including humans. Among chronically infected individuals, this opisthorchiasis involves hepatobiliary problems, including chronic inflammation, periductal fibrosis, biliary intraepithelial neoplasia, and even cholangiocarcinoma. Despite numerous studies at the pathomorphological level, the systemic response and cellular pathogenesis of these disorders are not well studied.To conduct in-depth research and to gain insights into the mechanism by which O. felineus infection causes precancerous liver lesions, we (i) applied a next-generation-sequencing–based technology (high-throughput mRNA sequencing) to identify differentially expressed genes in the liver of golden hamsters infected with O. felineus at 1 and 3 months postinfection and (ii) verified the most pronounced changes in gene expression by western blotting and immunohistochemistry.A total of 2151 genes were found to be differentially expressed between uninfected and infected hamsters (“infection” factor), whereas 371 genes were differentially expressed when we analyzed “time × infection” interaction. Cluster analysis revealed that sets of activated genes of cellular pathways were different between acute (1 month postinfection) and chronic (3 months postinfection) opisthorchiasis. This enriched KEGG pathways were “Cell adhesion molecules”, “Hippo signaling”, “ECM-receptor interaction”, “Cell cycle”, “TGF-beta”, and “P53 signaling”. Moreover, epithelial–mesenchymal transition was the most enriched (q-value = 2.2E-07) MSigDB hallmark in the set of differentially expressed genes of all O. felineus–infected animals. Transcriptomic data were supported by the results of western blotting and immunohistochemistry revealing the upregulation of vimentin, N-cadherin, and α-smooth muscle actin postinfection.Our data expand knowledge about global changes in gene expression in the O. felineus–infected host liver and contribute to understanding the biliary neoplasia associated with the liver fluke infection.

Investigation of clinical and epidemiological features of Opisthorchis felineus invasion in Voronezh region

The functioning of a natural focus of opisthorchiasis with a high level of epidemiological risk has been established within the borders of the Voronezh region, which forms the social and medical significance of the invasion. The aim of the study was study the epidemiology of opisthorchiasis and analyze the features of the clinical manifestations of the chronic opisthorchiasis invasion in the natural focus of the Voronezh region. Clinical and epidemiological data, the results of general clinical laboratory studies, as well as parasitological, serological, instrumental studies were analyzed. The epidemiological features of chronic opisthorchiasis have been studied and it has been established that the leading factor in the transmission of opisthorchiasis in the Voronezh region is the insufficiently thermally processed carp fish used for food. A detailed analysis of the clinical manifestations of helminthiasis was carried out and the main syndromes of chronic opisthorchiasis — asthenovegetative and dyspeptic — were identified. It was noted that there were no significant changes in hemogram parameters in the form of leukocytosis and eosinophilia. The biochemical parameters of the blood of patients with chronic opisthorchiasis also showed no deviations from normal values. During ultrasound of the abdominal organs, some patients showed hepatomegaly, diffuse liver changes, and biliary dyskinesia. The results of a serological blood test by ELISA revealed antibodies — immunoglobulins of class G — to the causative agent of opisthorchiasis in a small percentage of the infected, which is probably due to the significant immunosuppressive effect of the parasite. When carrying out parasitological diagnostics of helminthiasis — coproovoscopic examination by the method of acetic-ether deposition — the eggs of Opisthorchis felineus were found in 80.4 % of the infested. All patients underwent coproovoscopic examination once. It was found that the most valuable diagnostic method for verifying the diagnosis of chronic opisthorchiasis is coproovoscopy.