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Shomuradova Shakhnoza Shavkatovna, Tashkent Pediatric Medical Institute, Senior scientific applicant-researcher at the Department of neonatology, E-mail: shaxnoza.shavkatovna@mail.ru
The state of hepatobiliary system in juvenile rheumatoid arthritis
Abstract: The Article is dedicated to the results of the clinical-biochemical, biophysical and pathomorphological study of liver in the patients with juvenile rheumatoid arthritis. Liver injury was found in 64.8% of patients with JRA., showing the signs of mesenchymal inflammation, hypoalbuminemia, hyperbilirubinemia, hyperenzymemia. The importance of elastography in the early diagnosis of liver fibrosis in patients with JRA was determined. The results of morphological studies of liver of the deceased JRA patients treated with methotrexate showed that, in contrast to patients who did not receive methotrexate, the development of more severe disorganized, dystrophic and immunopathological processes with transition to the sclerotic and fibromatous changes was noted.
Keywords: biochemistry of liver, hepatotoxicity, juvenile rheumatoid arthritis, methotrexate, pathomorphology, fibrosis, elastography of liver, juvenile rheumatoid arthritis.
Relevancy. Diffuse disease of connective tissue, which includes juvenile rheumatoid arthritis (JRA), is one of the most severe and socially important forms of chronic pathology in children. The tendency to early disablement and possibility of system manifestations with involvement of internal organs in the pathological process necessitate timely diagnostics of complications and choice of adequate therapy. Hepatobiliary system is vulnerable in JRA patients. Its reasons include autoimmune processes on the one hand, and effect of drugs on the other hand.
Hepatotoxic reactions appearing during the application of background therapy ofJRA depend on the duration of use and dose of drugs [4]. It is known that the possibility of adverse reactions increases with the increase of the amount of simultaneously used drugs. It is established that if a patient takes five or six drugs simultaneously, the possibility of an adverse effect reaches 80% [1, 12].
The analysis of published information in respect of most often used non-steroidal anti-inflammatory drugs (NAID) in JRA and golden standard of treatment — methotrexate (MTX), certifies about high possibility of liver injury. Hepatotoxicity of drugs used in rheumatology leads to the slow-down of the processes of bio-transformation of exo- and endobiotics, their accumulation in circulating blood and development of endogenous intoxication, worsening of pathological process and toxicity of used drugs [2; 3; 6]. Methotrexate suppresses the activity of methylenetetrahydro-folate reductase, which leads to the increase of the level of homocysteine and, in the future, to the enhancement of fatty infiltration of hepatocytes, development of inflammation, Ito-cells activity and liver fibrosis [7, 14]. Methotrexate can cause the increase of liver ferments activity, development of fibrosis and cirrhosis of
liver during long-term treatment [11]. However, the data about the frequency and severity of fibrosis and cirrhosis of liver during the use of methotrexate in doses used in rheumatic diseases is ambiguous [10].
As a result of the progress of pathological process, consecutive stages offibrosis develop in the liver, for the diagnostics ofwhich the elastography of liver, a non-invasive method, has recently become the safest and most informative [5; 9; 13]. With regard to the above stated, the problem of early diagnostics ofliver injury, enhancement of the efficiency ofJRA therapy with the simultaneous ensuring of the minimum of adverse effects of drugs is very relevant both, from the point of science and practical pediatrics.
Aim of research: to study clinical-biochemical, biophysical and pathomorphological characteristics of injury of hepatobiliary system in juvenile rheumatoid arthritis.
Material and methods of research. 91 patients with JRA aged from 1,5 to 17 were examined, out of which, there were 18 patients oligo- and 73 patients with poly-arthritic variants of disease.
Out of 91 patients, there were 50 (54,9%) boys and 41 (45,0%) girls. The duration of disease was from 1 to 10 years.
59 children showed clinical signs of liver injury (main group), and 32 children with JRA. without liver injury were included in the experimental group. The criteria of inclusion were the absence of earlier diseases of hepatobiliary system, absence of anti-bodies to the viruses of hepatitis B, C and D, which were determined by immune-ferment method. Ultra-sound (US) examinations of hepatobiliary system was conducted on the device SSD-630 «Alo-ka» (Japan). Elastography of liver was conducted with the help of the device «FibroScan 502 TOUCH» («EchoSens», France).
The activity of ferments of serum glutamic pyruvic transaminase (SGPT), gamma glutaminetransferase (GGTP) and alkaline phosphatase (ALP), content of total protein, albumins, bilirubin and its fractions, total cholesterol, thymol test were determined on the automated multiple-unit apparatus "Autohumolizer F1" ("Human", Germany) with the help of special sets of chemicals. The results of12 autopsy examinations of children and teenagers who died of rheumatoid arthritis during the last 10 years (2005-2014) were studied at the Republic pathologicoanatomic center. Histological sections from the liver were stained with hematoxylin and eosin for general morphological study; to identify collagen fibers, the sections were stained in accordance with van Gieson's method and mucopolysaccharides were defined according to Schick test.
The statistical processing of the obtained data was conducted on personal computer with the help of the applied programs package « Microsoft Office» and « Statistica 6.0».
Results of research. It was found from the anamnesis that in 64 (70,3%) children, the development ofJRA was promoted by trigger factors, among which acute respiratory diseases take the first place.
Burdened perinatal anamnesis was found in 70 (76,9%) children and 21 (23,1%) patients had burdened anamnesis on rheumatic diseases. 35 (38,5%) children were considered almost healthy before the appearance ofJRA.
The performed studies showed that 59 (64,8%) out of 91 patients had the symptoms of hepatobiliary system injury. Reactive hepatitis was detected in 37 patients and other 22 patients showed the signs of chronic hepatitis. According to the data of ultrasound diagnostics, the growth of liver (+1cm-2,5cm), slight increase of parenchymal echogenicity of the liver and enhancement ofvascular pattern. Primarily, the liver injuries were characterized during US by diffuse changes of parenchyma of the liver, increased echogenicity, reactive hepatatitis and hepatomegalia. Liver injuries related to drugs usually manifest themselves with the increase of liver ferments without symptoms, i. e. take place sub-clinically being a «biochemical finding» (anicteric variant of acute drug-induced hepatitis). The patients with the duration of the disease of 1-3 years showed functional disorders from the side of the liver manifested in the disruption of enzymatic status. 57,9% reported complaints about nausea, vomiting and unstable stool. Classic clinical signs of hepatitis were not observed in them. The increase of the level of aminotransferase without symptoms can be observed during the use of non-steroidal anti-inflammatory drugs, cytostatic agents, immunodepressants, which are major drugs in JRA. treatment. During long-term use of listed drugs, severe hepatitis can develop. Hence, attention should be paid to the isolated increase of aminotransferase activity, because it can certify about the development of drug-induced pathology of the liver.
The diagnostics of drug-induced hepatitises poses a complex problem. Several criteria allowing clarifying the diagnosis and confirming that originated symptoms are actually drug-induced are proposed: chronology of appearance of complications; regress of clinical symptoms after the discontinuation of treatment; relapse of a complication after repeated drug administration; absence of other possible etiology; results of laboratory-instrumental studies.
During the diagnostics of drug-induced hepatitises, we relied upon the chronological criteria, absence of other possible etiology and results of laboratory-instrumental studies. We couldn't use the regress of the clinical signs of complication after the discontinuation of treatment because long-term cancellation of background
therapy will lead to the aggravation of the underlying disease (JRA). The signs of hepatitis with all typical clinical-laboratory manifestations and confirmed with US were observed in the patients in the course of progress of the disease and further use of drugs, often in increased doses. The latter was noted in the patients with the duration of disease of 3-5 years and more. The study of clinical manifestations of liver injury shows that the complaints about the pain in the right hypochondrium and stomach were made by 2/3 of the patients with the duration of the disease of 3-5 years and more; the reduction of appetite was observed in more than half of the patients; icterus of skin cover was revealed in half of the patients; all patients showed the increase of liver size. Biochemical studies established the increase of aminotransferase activity in 9 (15,2%), GGTP and ALP — in 21 (35,6%), hyperbilirubinemia — in 38 (64,4%) and direct bilirubin — in 10 (16,9%) children. 44 (74,6%) children show the signs of hepatodepression manifested with hypoalbuminemia and partly, in 10 (16,9%), — hypoproteinemia. The increase of thymol test was noted in all 59 (100%) patients, which certifies about the presence of the signs of mesenchymal inflammation. This coincided with high frequency of increased echogenicity during US of liver, diffuse parenchymal changes.
The analysis of biochemical indicators of blood serum ofJRA patients without involvement in the pathological process of the liver showed significant reduction of the level of albumins by 1,17, the increase of SGPT by 1,17 times against the background of preservation of the guideline values of the activity of GGTP (Table 1). The level of direct bilirubin significantly increased by 1,63 times against the background of preserved normal values of the total and indirect bilirubin. The increase of thymol test vy 1,58 times should be especially noted.
At the same time, more expressed changes of biochemical indicators of blood serum were observed in JRA patients with liver injury. Thus, albumin content significantly decreased by 1,5 and 1,27 times, the activity of SGPT increased by 1,54 and 1,32 times, GGTP — by 1,4 and 1,51 times, corresponding to the values of almost healthy people and JRA groups without liver injuries. Herewith, the content of total, direct and indirect bilirubin increased significantly by 1,56, 1,63 and 1,55 times relative to normal values; total and indirect bilirubin — by 1,37 and 1,55 times relative to the indicators JRA. children without liver injury. The indicator of thymol test was increasing sharply, exceeding the values of almost healthy people and group of patients without liver injury by 2,77 and 1,75 times respectively.
A wide scatter of researched indicators was observed, which, in our opinion, is related to the range of used drugs. The most changes were typical for patients with the use of NSAID complex, prednisolone, plaquenil and methotrexate.
The data obtained by us coincides with the evidence of clinical manifestations in children with hepatitis. Children of this group complained about headaches, weakness, reduction of appetite more often; asthenovegetative syndrome was expressed more distinctly etc. They were manifested with high frequency in children, who were receiving a combination of several drugs, especially in combination with methotrexate.
According to the data of conducted liver elastography, 19 (71%) of patient out of 25 JRA patients had no signs of fibrosis (F0). 4 (16%) patients were diagnosed with minimal fibrosis (F1) and 2 (8%) — with moderate fibrosis (F2). Severe fibrosis and cirrhosis were not found.
Table 1. - Biochemical indicators of blood serum of JRA patients, M±m
Indicators Almost healthy JRA patients
Experimental group Main group
Total protein, g/l 68,30±0,61 68,62±0,88 63,24±1,03
Albumins, g/l 43,28±2,14 36,84±1,98 a 28,93±0,61 a, b
SGPT, u/l 25,34±1,31 29,54±1,66 a 38,93±0,61 a, b
GGTP, u/l 31,42±2,21 29,04±1,48 43,92±2,52 a, b
ALP, u/l 127,3±9,8 158,9±10,3 224,17±16,6 a, b
Total bilirubin, mmol/l 10,80±0,92 12,32±0,81 16,91±0,46 a, b
Direct, mmol/l 1,73±0,09 2,83±0,11 a 2,82±0,21 a
Indirect, mmol/l 9,07±0,63 9,49±0,65 14,09±0,43 a, b
Total cholesterol, mmol/l 3,79±0,16 3,87±0,12 4,24±0,16 a, b
Thymol test, rel. u. 2,22±0,11 3,51±0,28 a 6,16±0,27 a, b
Remarks: a — differences between the indicators of almost healthy people and JRA patients, statistically significant, b — differences between the indicators of children with JRA without and with liver injury, statistically significant.
The assessment of fibrosis was conducted according to META-VIR scale. The mean indicator of elasticity of liver was 3,5±0,5 kPa for F0, 5,8±0,5 kPa for F1 and 6,5±1,5 kPa for F2 stages of fibrosis respectively. The indicators offibrosis stage in the group ofJRA patients with liver injury were spread in equal ration between F0, F1 and F2.
The results of pathomorphological studies showed that the development of disorganized, dystrophic and immune-pathological processes were noted in the liver of deceased JRA children. Disorganized changes of vascular walls and interstice of liver develop first, which are manifested in the edema of inter-cellular substance, shredding of fiber structures, collapse of the elements of connective tissue. These changes are more expressed in the wall of the central vein and Disse's space. The disorganization of the central vein wall and sinusoids was accompanied with the development of discirculation of the liver in the form of diapedetic bleeding. Venous dis-circulation led to the development of dystrophic changes from the side of parenchyma of the liver and they were manifested in the form of hyaline-drop and vacuolar dystrophy of hepatocytes. The histochemical study designed to reveal mucopolysaccharides in the composition of stroma-vascular components and glycogen in the cytoplasm of hepatocytes by the method of Schick reaction found that the content of mucopolysaccharides increased in the stroma in the form of more intensive staining of inter-cellular substance in pink-red color, which certifies about the accumulation of glycoza-minoglycans typical for disorganized processes of connective tissue. From the side of the parenchyma of the liver, the reduction of Schick positive substance in cytoplasm was noted, which proves the enhanced disintegration of glycogen and prevailing of protein and vacuolar dystrophy of hepatocytes.
The development of deeper disorganized processes in the form of mucoid, fibrinoid swelling and myxomatosis of the connective tissue of the wall ofliver vessels was observed in the liver of children suffering from JRA. for a long time. Herewith, it was noted that the wall of the central vein of the liver was thickened at the expense of firboelastosis and myxomatosis of fiber structures, which expend towards the sinusoid wall. These changes led to paralytic expansions of sinusoids, perivascular bleeding and pigment formation. Beam location of hepatocytes is destroyed in the form of formation both, disorderly located and subjected to cytolysis and apoptosis ofhepa-tocytes. Nuclei of the latter are in the condition of karyolysis, karyo-pyknosis. Herewith, the cholestasis is manifested in the form of accumulation in the internal side of the cytoplasm of hepatocytes in the form of numerous small brown pigment inclusions.
Basic pathomorphological changes of immune-pathological character of JRA are revealed around the vessels of liver triads, which developed by appearing of expressed cellular infiltrate from lymphoid and histiocytic cells admixed with eosinophils (fig. 1, 2). Cellular infiltrate mainly encircles arterial vessels of triads and spreads towards the parenchyma of the liver along the sinusoids. Herewith, the vessel walls are in the condition of mucoid and fibrinoid swelling. Activated lymphoid cells, which are tightly born to liver cells, appear in the parenchyma, especially in the Disse's space. Hepatocytes located around the triads are subject to dystrophic changes and cholestasis. Histochemical study showed significant reduction of Schick positive substance in the cytoplasm of hepatocytes in all functional zones of the liver.
The results of morphological study showed that in the liver of deceased JRA children who didn't receive methotrexate, the development of both, general morphological changes in the form of disorganization and dystrophy of connective tissue of the wall of the vessels and immune-pathological processes in the form of periportal lympho-histiocytic infiltrate admixed with eosinophils and mucoid, fibrinoid swelling of the wall of the vessels and connective tissue, interstice of the liver were observed. The results of morphological study of deceased JRA children who received methotrexate in the total dosage from 1,5 to 3 g showed that, unlike the patients who didn't receive methotrexate, the development of more expressed disorganized, dystrophic and immune-pathological processes with the transition to sclerotic and fibromatous changes was noted. At the increase of the dose of methotrexate, proliferative activity of histiocytic cells with the accumulation of fibrillary substance in the wall of sinusoids and central vein with the development of fiber connective tissue was observed; herewith, such fibrwas more expressed in the wall of the central vein.
Based on the obtained data, one can make the following conclusions:
1. Most JRA patients (64,8%) showed the liver injury manifested in all cases with the signs of mesenchymal inflammation, hypoalbuminemea — 74,6%, hyperbilirubinemia — 64,4%, cholestasis — 35,6% and hyperensymemia — 15,2% to 2 norms.
2. Non-invasive methods of diagnostics of the liver fibrosis — ultrasound elastography, allow revealing the phenomena of fibrosis and conducting the monitoring of MTX-toxicity of the liver in JRA. patients. The advantage of elastography of the liver is the simplicity of performance, non-invasiveness, quickness in obtaining the result and possibility of repeated studies.
3. The depth and severity ofpathomorphological disorders depend on the dose and duration of used drugs of background therapy and are characterized by: disorganization and dystrophy of connective tissue and wall of the vessels, lympho-histiocytic infiltrate of the wall of the vessels and connective tissue, interstice of the liver,
Fig. 1. 14 year old child with JRA. Myxomatosis of the wall of the central vein and sinusoids, paralytic expansion of sinusoids, apoptosis of hepatocytes. Stain: G-E. Uv: oc.10,
ob.20
till fatty dystrophy of hepatocytes, expressed proliferative activity, both histiocytic and lymphoid cells with the formation of the foci of granulomatous inflammation and expressed fibromatosis in the wall of the central vein.
Fig. 2. Same 14 year old child with JRA. Lympho-histio-cytic infiltration around the triad admixed with eosinophils, lymphocytes in the Disse's space, cholestasis. Stain: G-E. Uv: oc.10, ob.20
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