CC BY
11
DOI 10.29254/2077-4214-2018-1-2-143-351-355 UDC 616.314.17-008.1-022.7-085-091.8]-092.9 Demkovych A. Ye., Bondarenko Yu. I., Hasiuk P. A.
THE HUMORAL LINK OF IMMUNE DEFENSE IN EXPERIMENTAL
BACTERIAL-IMMUNE PERIODONTITIS I. Horbachevsky Ternopil State Medical University (Ternopil)
p.gasyuk@gmail.com
Publication relation to planned scientific research projects. This work is an integral part of the complex interdepartmental research work "Systemic and organic violations due to the actions of extraordinary factors on the body, mechanisms of their development and pathogenetic correction" (registration number 0116 U003390) and research work of the department of Prosthetic Dentistry: "Pathogenetic approaches to treatment the main dental diseases on the basis of studying the mechanisms of damage of the oral cavity tissues against the background of accompanying somatic pathology" (state registration number 0116 U005076).
Introduction. The investigation of the mechanisms inflammatory processes in the tissues of the periodontal complex is one of the important problems of modern dentistry due to the relatively high wide spread and unfavourable prognosis, because frequency of periodontal disease in the all world oscillate within 5-20% and increases with age to 75% [1,2]. Perfectining of the known and elaboration of new methods of periodontitis treatment is one of the important task and requires extraordinary approaches to their solutions. The ascertainsion of the character of immunological processes disorders will permit to establish the role of one the important links to damage of the periodontal complex and formation of an inflammatory process development of various degrees [3,4]. The response of humoral adaptive immunity mediated by immunoglobulins Ig A, Ig M, Ig G classes, relative to tissuel and bacterial antigens [5], is dynamic and does not always have a standard and strictly defined character due to the specificity of the action to the antigenic stimulus and the helper signal [6]. One of the important biological functions of immunoglobulin is the binding of the antigen and formation of the circulating immune complex (CIC), depends on their properties and localization, as well as, inclusion the cascade of signal mediators [7].
The purpose of this study was to elucidate the pathogenetic role of the humoral link immune reactivity in the dynamics of the inflammatory response in experimental bacterial-immune periodontitis.
Research methods. The investigation was performed with use of white clinically healthy rats, 150-200 g weight in conditions of vivarium. The animals were supported on standard diet, balanced by nutritional elements. The investigations was performed according to the general rules and regulations of the European Convention for the Protection of vertebrate animals that use for experimental and other scientific purposes (Strasbourg, 1986), the General ethical animal experimentation (Kyiv, 2001).
The animals were divided into 4 groups: I - intact animals, control (n=10); II - animals with experimental periodontitis on the 7th day study (n=8); III - animals with experimental periodontitis on the 14th day study (n=8); IV - animals with experimental periodontitis on the 30th day study (n=8). Experimental bacterial-immune periodontitis in experimental animals was caused by injection into the tissue of the periodontal complex of the microorganisms mixture diluted with egg protein
[8]. To enhance the immune response, an injection of complete Freund's adjuvant was simultaneously injected into the rats paw. When studies were performed with animals of group IV on the 14th day, repeated introduction of the pathogen and injection of adjuvant were carried out. For conducting studies with animals group IV, on the 14th day, the pathogen and adjuvant was introduced repeatedly.
On the 7th and 14th day experimental animals were sacrificed by blood-letting under thiopental anesthesia. For further research the blood serum was selected. In blood serum the level of Ig A, Ig M, Ig G and the concentration of circulating immune complexes (CIC) was determined. The principle of determining immunoglob-ulins in the blood serum is based on their ability to precipitate blood serum at different pH and ionic strength
[9]. Protein-buffer complexes, which are formed, change the optical density of the medium. Photometer on the photoelectric colorimeter with a blue light filter against water. The calculation of the immunoglobulins number was carried out according to the calibration table and expressed in g/l. The number of circulating immune complexes in the blood serum was determined by precipitation with their solution of polyethylene gly-col-6000 [10]. The samples were also incubated with photometric methods on a SF-46 spectrophotometer at 450 nm. The concentration of CIC was expressed in conditioned. units. The results were statistically analysed with using nonparametric indexes [11] in the Excel software (Microsoft, USA) and STATISTICA 10.0 (Statsoft, USA). The reliability of the differences in values between independent quantitative values was determined with a normal distribution according to the Mann-Whitney U criterion [12].
Results and discussion. Introduction of complex mixtures of microorganisms diluted egg protein in peri-odontal tissues had caused hyperergic inflammatory process with expressed changes in the soft tissue of the lower jaw, which accompanied by edema and hyperemia of the mucous membrane and for the characteristic of the manifestation was same to changes in humans [13,14].
Indices of immune system in the blood serum rats for the experimental periodontitis (M ± m)
Indices Control, intact animals Animals with experimental periodontitis
Experiment duration (days) - 7 14 30
Number of animals 10 8 8 8
Immunoglobulin A (g / l) 1.301±0.002 1.495±0.003 p,<0.01 1.463±0.010 p1<0.01, p2<0.01 1.435±0.012 p1<0.01, p2<0.01, p3<0.05
Immunoglobulin M (g / l) 1.196±0.020 1.434±0.018 p,<0.01 1.688±0.006 p<0.01, p2<0.01 1.665±0.006 p1<0.01, p2<0.01, p3<0,05
Immunoglobulin G (g / l) 1.315±0.001 2,475±0,004 p,<0.01 1.990±0.002 p1<0.01, p2<0.01 2.472±0.004 p1<0.01, p23<0.01
Circulating immune complexes (conditioned. units.) 56.800±1.731 103.375±2.203 p,<0.01 86.250±1.962 p<0.01, p2<0.01 75.500±1.722 p1<0.01, p23<0.01
Notes: 1. p1 - significant of differences relatively to intact animals; 2. p2 - significant of differences relatively to animals with experimental periodontitis on the 7th day of the research; 3. p3 - significant of differences relatively to animals with experimental periodontitis on the 14th day of the research.
In the study of the humoral link of immune protection, changes were found in indices of immune status [15,16]. In particular, the studies concerned of humoral factors of local defense, which includes immunoglobulins A, M, G classes and indirectly indicate the state of the humoral link immune system. Ig A, M, G classes, as well as, circulating immune complexes, at the same time activate the complement system and is one of the mediator of endogenous intoxication. Activation of the complement system is part of the pathological process in experimental periodontitis. The state of the humoral immunity was determined in I-IV groups of the experimental animals (table). It was found that the contents of these immunoglobulins and circulating immune complexes varied in different directions depending on the duration of pathogenic factors action.
Thus, in the rats of the second experimental group (on the 7th day of the experimental periodontitis), the Ig A contents in the blood serum was increased by 1.15 times (p<0.01) relatively to the control group of the rats. A similar direction of
Table. tively, as compared with indices of intact animals (table, fig. 1).
In the later period, on the 14th day, the contents of immunoglobulins A and G classes was changed in the opposite direction, that is, they were decreased by 1.02 times (p<0.01) and by 1.24 times (p<0.01) respectively, as compared with the animals on the 7th day of the experiment, but were higher relatively to the intact group of animals (by 1.13 times, p<0.01 and by 1.51 times, p<0.01) respectively.
After determination of Ig M concentration in the blood serum of experimental animals with periodontitis, it should be noted that this index also continued to increase on the 14th day of the experiment, as compared with the indices on the 7th day (by 1.18 times, p<0.01) and exceeded it in the animals of the control group (by 1.41 times, p<0.01). As Ig M (antibody M class) primary is produced in response to an acute infection, and increase of immunoglobulin M class concentration in blood serum reflects a predominantly acute infectious process.
At the more later period, on the 30th day of experimental periodontitis, there was a slight decrease of the Ig A class (by 1.02 times; p<0.05), but less than in the animals with periodontitis on the 14th day of the experiment and by 1.04 times (p<0.01) as compared to the second group of experimental animals (7th day). This index was kept at the high level in comparison with the control group (by 1.10 times, p<0.01). It's evidenced about high activity of the inflammatory reaction.
The determination of Ig M class concentration in the blood serum, as immune response to the inflammatory process in the periodontal complex, showed that on the
Fig. 1. Changes in the concentration of the immunoglobulins A, M, G in the rats blood serum in the experimental periodontitis (% of control).
changes for this period of observation Notes: * - significant of differences relatively to the intact animals (p<0.01);
were occurred relatively of the contents # - significant differences relatively of the animals with periodontitis on the 7th day
of immunoglobulins M and G classes, (p<0;01); na" significant diffe^nces relatively to thhe animals with p*riodontitis °n thet7*
° , , day (p>0.05); • - significant differences relatively to the animals with periodontitis on the
that is, they were increased by 1.20 ti'mes 14th day (p<0.01); x - significant differences relatively to the animals with periodontitis on
(p<0.01) and 1.88 times (p<0.01) respec- the 14th day (p<0.05).
30th day the concentration it changed similarity Ig A, that is, was decreased, but in the smaller degree (by 1.01 times, p<0.05) as compared with the 14th day of experiment. Comparing the Ig M indices in this observation period, it should be noted that they were higher than the indices on the 7th day of the experiment (by 1.16 times, p<0.01), exceeding the control by 1.39 times (p<0.01).
By comparison of the above indicated indices of the humoral defense of organism, on the 30th day of experimental periodontitis development, were found an increase in the Ig G contents (by 1.24 times, p<0.01) as compared with the 14th day, however by comparing with the 7th day of the experiment was slightly less, but the changes were unsignificant (p>0.05), although they were significantly higher relatively to the control group animals (by 1.88 times, p<0.01).
One of the important physiological functions of immunoglobulins is the neutralization of the antigens, including autoantigens, with the formation of the CIC and their subsequent elimination from the organism, directed to supporting immunobiological homeostasis. Therefore, studies of humoral factors of local protection were also associated with an assessment of circulating immune complexes contents in the blood serum. For all that indices in the blood serum on the 7th day of the experiment were foud increased in comparison with the intact group (by 1.82 times, p<0.01), and on the 14th day - by 1.52 times (p<0.01). It should be note that concentration of the CIC in the blood serum was decreased on the 14th day as compared to the 7th day by 1.20 times (p<0.01) (table, figure 2).
Characterizing the changes circulating immune complexes contents in the blood serum of experimental animals with periodontitis, it should be noted that on the 30th day of the experiment, their concentration also significantly exceeded (by 1.33 times, p<0.01) as compared with the indices in the animals control group. However, these data were lower than those that were in rats on the 7th (1.37 times, p<0.01) and 14th day of the experiment (by 1.14 times, p <0.01), in accordance.
It should be noted that an increase in the concentration of circulating immune complexes in the serum is mainly an indirect sign of activation of complement,
Fig. 2. Changes in the indices of circulating immune complexes in the rats blood serum in
the experimental periodontitis (% of control). Notes: * - significant of differences relatively to the intact animals (p<0.01); # - significant differences relatively to the animals with periodontitis on the 7th day (p<0.01); • - significant differences relatively to the animals with periodontitis on the 14th day (p<0.01).
which lead to tissue damage. Formed CICs interact with all cells of the blood, and activate complement, as well as cellular receptors of many organs and tissues. Modulation of the immune response occur when the circulating immune complexes interact with immuno-competent cells. Thus, in a result of interaction with monocyte-macrophage cells proteolytic enzymes realising and activation of complement components, increase production of the kinines, anaphylatoxin, opsonines, chemotaxines, which display damaging effect of the tissues [17,18]. Conclusions
1. The disturbances of humoral chain of the immu-nological reactivity in the experimental animals with bacterial-immune periodontitis is an essential pathogenetic link that forms the character of the development and course of the inflammatory process.
2. The course of experimental periodontitis is accompanied by an increase in the contents in blood serum of the immunoglobulins A, M, G classes and increase contents of circulating immune complexes, that is associated with increased intensity of their formation in response to antigenic stimulation and may indicate about chronicity of inflammation in the periodontal complex.
The prospect of further scientific research in this direction is to study the best ways of pharmacological corrections of humoral link of immune defense changes in animals with experimental bacterial-immune peri-odontitis.
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ГУМОРАЛЬНА ЛАНКА 1МУННОГО ЗАХИСТУ ОРГАН1ЗМУ ПРИ ЕКСПЕРИМЕНТАЛЬНОМУ БАКТЕР1АЛЬНО-1МУННОМУ ПАРОДОНТИТ1
Демкович А. €., Бондаренко Ю. I., Гасюк П. А.
Резюме. Мета роботи - з'ясування патогенетичноТ ролi гуморальноТ ланки iiwyHHoi реактивност в дина-мщ запальноТ реакцп за умови експериментального бам^альночмунного пародонтиту. На ранньому етап розвитку експериментального пародонтиту, тобто в перюд гострих проявiв, на 7-му добу, вщбуваеться шд-вищення концентрацп у сироватц кровi основних клаав iмуноглобулiнiв (А, М, G), але тзшше, на 14-ту добу спостереження, концентращя lg А та G змшилася в протилежному напрямку, почала зменшуватися, а рiвeнь iмуноглобулiну М, навпаки, шдвищився. На 30-ту добу спостереження рiвeнь lg А та М пщвищувався, порiв-няно з 14-ою добою, нaтомiсть концeнтрaцiя lg G змшилася у протилежному напрямку, тобто знижувалася. Паралельно проводилось визначення вмiсту Ц1К в сироватц кровi, як важливоТ ланки патогенезу iмунного ураження за умови експериментального запалення в тканинах пародонта. Динамк показнимв рiвня Ц1К були подiбною до динамти lg, тобто на 7-му добу експерименту концентращя Тх була значно бшьшою, а в по-дальшому почала знижуватися. Дослiдивши функцiонaльний стан iмунного захисту у бiлих щурiв iз змодельо-ваним бaктeрiaльно-iмунним пародонтитом було виявлено порушення гуморальноТ ланки iмунологiчноТ ре-aктивностi, що проявлялися вщповщно до пeрiоду розвитку запальноТ реакци в пародонтальному комплeксi.
K^40Bi слова: бaктeрiaльно-iмунний пародонтит, iмуноглобулiни, циркулюючi iмуннi комплекси, iмун-ний статус.
ГУМОРАЛЬНОЕ ЗВЕНО ИММУННОЙ ЗАЩИТЫ ОРГАНИЗМА ПРИ ЭКСПЕРИМЕНТАЛЬНОМ БАКТЕРИАЛЬНО-ИММУННОМ ПАРОДОНТИТЕ
Демкович А. Е., Бондаренко Ю. И., Гасюк П. А.
Резюме. Цель работы - выяснение патогенетической роли гуморального звена иммунной реактивности в динамике воспалительной реакции при экспериментальном бактериально-иммунном пародонтите. На раннем этапе развития экспериментального пародонтита, то есть в период острых проявлений, на 7-е сутки, происходит повышение концентрации в сыворотке крови основных классов иммуноглобулинов (А, М, G), но позже, на 14-е сутки наблюдения, концентрация lg А и G изменилась в противоположном направлении, начала уменьшаться, а уровень иммуноглобулина М, наоборот, повысился. На 30-е сутки наблюдения уровень lg А и М повышался по сравнению с 14-ми сутками, зато концентрация lg G изменилась в противоположном направлении, то есть снижалась. Параллельно проводилось определение содержания ЦИК в сыворотке крови, как важного звена патогенеза иммунного поражения при условии экспериментального воспаления в тканях пародонта. Динамика показателей уровня ЦИК были подобной динамики lg, то есть на 7-е сутки эксперимента концентрация их было значительно больше, а в дальнейшем начала снижаться. Исследовав функциональное состояние иммунной защиты у белых крыс с смоделированным бактериально-иммунным пародонтитом были выявлены нарушения гуморального звена иммунологической реактивности, проявлявшиеся в соответствии с периодом развития воспалительной реакции в пародонтальном комплексе.
Ключевые слова: бактериально-иммунный пародонтит, иммуноглобулины, циркулирующие иммунные комплексы, иммунный статус.
THE HUMORAL LINK OF IMMUNE DEFENSE IN EXPERIMENTAL BACTERIAL-IMMUNE PERIODONTITIS Demkovych A. Ye., Bondarenko Yu. I., Hasiuk P. A.
Abstract. Objective - to elucidate the pathogenetic role of the humoral link immune reactivity in the dynamics of the inflammatory response in experimental bacterial-immune periodontitis. ln blood serum the level of lg A, lg M, lg G and the concentration of circulating immune complexes (ClC) was determined. The results were statistically
analysed with using nonparametric indexes. At the early stage of experimental periodontitis development, that is, in the period of acute manifestations, on the 7th day, the concentration of the main classes of immunoglobulins (A, M, G) in the blood serum was increased, however, on the 14th day of observation, the concentration of Ig A and G was changed in the opposite direction, that is, to decrease, and the level of immunoglobulin M class, on the contrary, was increased. On the 30th day of observation, Ig A and Ig M levels was increased as compared to the 14th day, however, Ig G concentration changed in the opposite direction, that is, decreased. At the same time the contents of the CIC in blood serum was measured as an important element in the pathogenesis of immune damage for experimental inflammation in periodontal tissues. The dynamic of the CIC concentration were similar to the dynamic of the immunoglobulins, that is, on the 7th day of the experiment, their concentration was more larger and subsequently began to decline. It's investigated of the functional state of immune defense in white rats with simulated bacterial-immune periodontitis, disorders of the humoral element of immunological reactivity were revealed, which were manifested in accordance to the period of the inflammatory reaction development in the periodontal complex.
Key words: bacterial-immune periodontitis, immunoglobulins, circulating immune complexes, immune status.
Рецензент - проф. Ткаченко I. М.
Стаття надшшла 27.03.2018 року
DOI 10.29254/2077-4214-2018-1-2-143-355-358
УДК 616.311.2+616.314.17+616.314.19)-072:616-005]:613.84
Льчишин М. П., Хороз Л. М., Виноградова О. М.
ДОСЛ1ДЖЕННЯ СТАНУ СУДИН ПАРОДОНТУ ТЮТЮНОЗАЛЕЖНИХ ХВОРИХ 13 ГЕНЕРАЛ1ЗОВАНИМ ПАРОДОНТИТОМ, ЗА ДАНИМИ ПОКАЗНИК1В
РЕОПАРОДОНТОГРАФП Льв1вський нацюнальний медичний ушверситет 1мен1 Данила Галицького (м. Льв1в)
immartella@gmail.com
Зв'язок публшацп з плановими науково-дослщ-ними роботами. Дане дослщження е фрагментом науково-дослщноТ роботи кафедри терапевтичноТ стоматологи Львiвського нацюнального медичного ушверситету iменi Данила Галицького, № державноТ реестрацп - 0115и000036.
Вступ. Тютюнопалшня через значну поширешсть та шкщливий вплив на здоров'я людей являеться найважлившою проблемою краТни. У курщв ус ви-явлеш ознаки хвороби набувають виражешшого характеру, вони зумовлеш хiмiчними, фiзичними, термiчними чинниками, i залежать вщ штенсивносп та тривалосп дм, а також вщ шдивщуальних морфо-функцюнальних особливостей тканин порожнини рота. Численш дослщження довели, що палшня мае багатофакторний вплив на оргашзм людини. Ступшь ризику розвитку захворювання сильно змшюеться залежно вщ тривалосп палшня цигарок, штенсивносл впливу i типу тютюнового диму, генетично зу-мовленою чутливiстю оргашзму курця до них. Тютюнопалшня негативно впливае на будову та структуру стшок судин: артерп втрачають еластичшсть; АТ пщ-вищуеться; прискорюеться вiдкладення холестерину на стшках i формування атеросклеротичних бляшок; просвiт ще бiльше звужуеться; розвиваеться артери альна гiпертензiя [1-8].
Мета дослщження. Визначити стан судин у паци ентiв-курцiв, хворих на генералiзований пародонтит, за даними показникiв реопародонтографи.
Об'ект i методи дослiдження. Дослщження стану кровообiгу у тканинах пародонта проводили за до-
помогою сершного реографа Р4-02. При тетраполяр-нш методицi був використаний метод розташуван-ня двох електродiв (круглi пластинки з нержавшчоТ сталi площею 0,5 см2, вмонтоваш в основу з еластич-ноТ пластмаси), якi розташовували на вестибуляр-нiй та оральнш поверхнях альвеолярного вiдростку щелепи.
Яшсш показники РПГ визначали вiзуально, звер-таючи увагу на фронти кривоТ, ТТ верхiвку, наявнiсть та розташування дикротичноТ iнцiзури. З кiлькiсних показнишв РПГ визначали: реографiчний iндекс (Р1)
- спiввiдношення амплiтуди реограми до амплп"уди калiбровочного сигналу; показник тонусу судин (ПТС)
- стввщношення часу, за яке вщбуваеться макси-мальне розтягнення судин при проходженш по ним пульсового об'ему кровi (а) до часу всього перюду проходження цього об'ему (Т); шдекс еластичносп (1Е) - спiввiдношення амплiтуд швидкого (а) та по-вiльного (с) кровонаповнення; шдекс периферично-го опору (1ПО) - вiдношення амплiтуди нижчоТ точки iнцiзури реограми (d) до амплп"уди швидкого крово-наповнення (а).
Амплiтуднi i часовi вiдрiзки реопародонтогра-фiчних (РПГ) кривих вимiрювали за допомогою зви-чайного трикутника i циркуля. Цифровi значення амплiтуд виражали в мшметрах, часовi промiжки
- в секундах. При аналiзi РПГ враховували кшьккш характеристики, базованi на описi форми РПГ кривоТ i вимiрюваннi амплiтудних i часових вiдрiзкiв. При розшифров^ елементiв РПГ кривоТ двi нижш точки (перед початком висхiдноТ частини кривоТ i пiсля за-
