Experimental studies

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Introduction

Treatment of infl ammatory periodontal diseases (IPD) still represents a lot of issues. Th e global IPD prevalence among adults, according to the WHO data, is up to 90-95% without any trends for decrease [1, 2]. Medical treatment of IPD patients should be performed in combined, purposeful and personalized manner. Both local and general treatment should be applied, using effi cient methods of conservative, surgical and prosthetic treatment [1, 2].
Moreover, some novel treatments are recently introduced, based on cellular engineering, aiming for partial or total replacement of the damaged tissues. Such techniques presume modeling and construction of biocompatible (scaff old-type) carrier containing medical drugs. Th e main requirements for the modern matrix materials are as follows: complete biological compatibility, sustained viability of the cells seeded in the matrix, ability for biodegradation and replacement by normal tissues, changes in structure and properties fi tting the environmental eff ects [3].
Th e tissue engineering techniques are widely used in the IPD surgical treatment, especially when using the so-called directed tissue regeneration (DTR), i.e., a surgical approach which mechanically prevents epithelial migration to the apical side, thus promoting periodontal recovery without usage of natural and synthetic materials for the bone plastics. Th e method is aimed for creation of physical barrier between the graft and treated dental root surface resulting into preferential migration of slowly regenerating periodontal and bone cells to the aff ected site [4].
Both biodegradable and non- biodegradable membranes are now used at the present time when performing the directed tissue regeneration (DTR). Th ey may be classifi ed by their origin and composition, as shown in Table 1. Polytetrafl uorethylene (PTFE) is oft en used for manufacturing the non-biodegradable synthetic membranes. Th e PFTE non-biodegradable membranes are considered a “golden standard” for the DTR methodology. Th e results obtained with other membranes are usually compared to this PFTE material which has several advantages: essential mechanical resistance, no bone fi ller is required, and suffi cient barrier properties are present. PFTE drawbacks include a need for repeated surgical intervention 4 to 6 weeks for its extraction, complete closure of the membrane when sealing the flap with reliable fi xation; a need for regular weekly or biweekly inspections [4, 5, 6].

Table 1. Listing of the main membrane types [5]

66-71 Table 1. Listing of the main membrane.png
Natural membranes represent structures consisting of animal collagen (xenogeneic), or allogeneic membranes which contain allogeneic collagen supplied by lyophilized demineralized bone, or membranes consisting of xenogeneic collagen and reduced amounts of mineral matrix obtained aft er partial electrolytic plate demineralization. All these membranes are based on, mainly, type I collagen. Th eir biodegradation continues for 5 to 6 months, barrier functions retain for 4 months, with good adhesion properties and only rare complications upon their exposure. Such membrane plate is elastic, thus requiring bone plastic materials in order to retain the space. Given long resorption rates, this material should be chosen for defects with presumed slow regeneration. Synthetic membranes are produced as polymeric and gel-like structures. Th e gel membranes are subject to biodegradation within 9 to 12 months, with their barrier properties kept for 6 months. However, it is more diffi cult to work with these materials which are rather hard upon polymerization. This type of membranes is now less common applied in dentistry due to problems when handling them. Th e procedure needs much time in cases of indirect usage, when the membrane is shaped beyond surgical area, followed by introduction to the damaged area [4, 5, 6].
There are no clear benefi ts revealed for either biodegradable, or non-biodegradable membranes. Th erefore, some authors, when comparing parameters of diff erent materials for the membrane fabrication, have considered chitosan to be the most promising substance which exhibits chondro- and osteoconductive properties, high degree of biocompatibility and complete biodegradation of the polymer, as well as expressed antibacterial activity and homeostatic effi ciency [7, 8, 9].
Chitosan is a desialylated form of chitin polymer which is widely spread in living nature. It seems to be a promising raw material for matrices and biomimetics of bone and cartilageous tissues. At the present time, chitosan is increasingly used in dentistry since it meets the requirements for potential matrix base [8, 9, 10].
Chitosan applied as the matrix scaff old allows prevention of immune-related synthesis, thus increasing biocompatibility of the composite matrices. Chemical properties of chitosan provide diff erent modifi cations of its polymer structure, incorporation of various biologically active compounds (both organic and inorganic substances), thus being an important factor. [11, 12, 13, 14]. One should also note that electrostatic and hydrophobic interaction of chitosan with some modifying agents may even enhance its biological activity. Th e aim of this study was to investigate the eff ectiveness of using chitosan membranes for the treatment of infl ammatory periodontal diseases.

Materials and methods

At the present time, we perform a joint study by the Department of Preventive Stomatology, Department of Biotechnology at the R. Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantology (1st St. Petersburg State I. Pavlov Medical University), and the Research Institute of Macromolecular Compounds (Russian Academy of Sciences), concerning development and implementation of an original approach to surgical treatment of infl ammatory periodontal diseases using a novel chitosan-based matrix (Fig. 1), as described elsewhere [6].
66-71 Figure 1. A micrograph of chitosan-based.png

Figure 1. A micrograph of chitosan-based matrix was obtained employing Carl Zeiss Supra 55 VP microscopy, magnification 500X. Micrographs (HC. 500X) of chitosan A and B-based membranes were obtained with a Carl Zeiss Supra 55 VP scanning electron microscope

Micrographs A and B are made by P. Popryadukhin
To produce porous membranes, we purchased Chitosan from Fluka Chemie, BioChemika line (molecular mass, 255 kD, deacetylation degree, 80%; ash content, 0.5%). Porous 3-D matrices were obtained by lyophilization of chitosan solution by means of Heto-Holten PowerDry PL9000 -50 device. Before drying, chitosan was dissolved in 2% aqueous solution of acetic acid, at 4 wt%. Th e solvent sublimation in the lyophilizer proceeded for 48 hours. As seen in Fig. 1, pores in the chitosan matrices look like channels, with the diameter of 100 to 150 nm [4].
Th e in vivo experiments with porous chitosan membranes were carried out in rabbits (n=10). Experimental studies were scheduled and performed according to the Guidelines from the Order No.1179 of 10.10.1983, and No.267 of 19.06.2003 issued by the Russian Ministry of Healtcare, European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientifi c Purposes (Strasbourg, 1986), as well as with World Medical Association Declaration of Helsinki (1996).
Table 2 presents the design of experimental study. All surgical interventions proceeded under identical conditions. Th e surgical manipulations were performed under general anesthesia, i.e., Zoletil100, 0.1 mL, and Rometerum (20 mg/mL), 0.0125 mL intravenously.
Two diff erent surgical procedures were performed in rabbits under sterile conditions, aiming for studies of chitosan matrix biocompatibility with adjacent tissues. The surgery was performed in extraoral mode. The operation field was prepared in the right maxillar area. A 5-cm incision was made in maxillar projection, while dissecting the tissue in sharp or blunt way. Aft er accessing the maxilla, an artifi cial bone defect was inflicted, then overlaid by the tested material. Blood coagulation was controlled, and the wound was closed with suture materials layer-by-layer and treated by a tincture of iodine.
Eight rabbits were subjected to the skin and soft tissue incision at the anterior lateral chest surface followed by a rib exposure and infl iction of a bone defect (10 to 12 mm long). The resulting cavity was filled with a biodegradable porous chitosan-based matrix.
Systemic antibacterial therapy was performed during early postsurgical period (3 days) using Gentamycin (single injections of 2.0 mL daily for 3 days). After surgical procedures, the rabbits were placed to individual cages. Th e animals had free access to water and standard diet, being regularly observed. The animals were kept out of experiment, according to the schedule (1 to 6 months aft er the surgical intervention), followed by histological studies of gums and ribs, i.e., the areas of artifi cial bone defects with introduced synthetic materials.

Results

Three months aft er surgery, histological assessment of gingival tissues did not reveal any pathological changes of oral mucosa; the multilayer fl at non-squamous epithelium and underlying connective tissue did not show any signs of inflammatory response (Fig. 2).

Table 2. Experimental study design

66-71 Table 2. Experimental study design.png
Morphological examination of artifi cially damaged ribs with implanted material has revealed multi-component changes of bone tissue and porous matrix. Morphological analysis of the matrix/bone border area has shown a lot of osteoclasts at the site of bone defect 30 days aft er surgery (Fig. 3)
Fibrous connective tissue penetrated by blood vessels is developing between the bone structures and porous template. The matrix is repopulated by fi broblasts and macrophages from local blood vessels and connective tissue, as well as from the developing periosteal layer. The entire porous matrix is occupied by connective tissue cells by 30 days of experiment. The intra-matrix fi broblasts are actively producing intercellular components of connective tissue. Due to biodegradation of the matrix and phagocytic activity of macrophages, the microcavities are formed which become larger than initial pores. Hence, the matrix pores and newly developing cavities become surrounded by connective tissue structures containing big amounts of collagen fi bers and cell composition typical to the fi brous connective tissue. Moreover, the implant areas adjacent to newly formed periosteum, are more rich in connective tissue, as compared to the more centrally located matrix sites containing only fi broblasts and macrophages. Any signs of infl ammatory reaction are observed in connective tissue around the matrix, in developing periosteum, or matrix itself. Th e porous template is invaded by the blood vessels, and loose connective tissue also develops around them. Arteries and veins are detectable both in central and peripheral areas of the porous matrix. Perivascular cells are invading the matrix, along with connective tissue and vessels [4].
Morphological study of the bone/matrix at 3 months post-surgery has shown the entirely organized periosteum consisting of loose connective tissue and dense fi brous tissue. Over this period, the coarse-fi bred connective tissue further expands and fi lls the bone defect. As a next step of bone regeneration, the coarse-fi bred bone tissue and connective tissue periosteum are formed at the 4th month of experiment. At 6 months, the coarse-fibred bone tissue is replaced by a lamellar bone structure. By this term, the regenerating bone has the entirely formed periost represented by loose fibrous and coarse-fi bred connective tissue. The latter is gradually replaced by lamellar bone tissue. However, the repair osteogenesis seems to be incomplete due to predominating coarse-fibred bone tissue. Osteons with narrow Haversian canals are observed in the developing lamellar bone structures.
66-71 Figure 2-3.png

Conclusion

In the course of experimental study, the porous chitosan matrix proved to be biocompatible, bioinert, and bioresorbable material, thus meeting the requirements applicable to the materials suitable for production of the bone matrices. Th ese properties determine some unique characteristics of chitosan, thus enabling its applications in various areas of medicine, especially in dentistry, when performing surgical treatment for infl ammatory periodontal diseases.

Acknowledgements

We are much appreciated to Vladimir E. Yudin and Irina P. Dobrovolskaya for assisting in preparation of this article.

Conflict of interests

The authors have no confl icts of interests to declare.

References

1. Antipova AV, Suslov DN, Yukina GYu, Popryadukhin PV. Use of resorbed membranes for surgical treatment of inflammatory diseases of the periodontium. Dental Scientific and Educational Journal. 2014; 1-2:16-17. (In Russian) 2. Antipova AV, Suslov DN, Yukina GYu, Popryadukhin
PV. Development of a new method of surgical treatment of infl ammatory periodontal diseases. Dental Scientifi c and Educational Journal. 2014; 3-4:14. (In Russian)
3. Roach P, Eglin D, Rohde K, Perry CC. Modern biomaterials: a review-bulk properties and implications of surface modifi cations. J Mater Sci Mater Med. 2007; 18(7); 1263-1277.
4. Ulitovskiy S.B., Galibin O.V., Th omson V.V., Antipova A.V. et al. Th e use of surgical techniques in the treatment of infl ammatory periodontal diseases. Scientifi c Notes. 2014;1 (21):71-74. (In Russian).
5. Ulitovskiy S.B., Galibin O.V., Th omson V.V., Antipova A.V. et al. Th e use of diff erent materials in the process of surgical treatment of periodontal disease. Institute of dentistry. 2014; 2(63):100-101. (In Russian).
6. Ulitovskiy S.B., Galibin O.V., Antipova A.V. et al. Application of new technologies in the treatment of periodontal diseases. Dental scientifi c and educational journal. 2013;1/2: 2-5. (In Russian).
7. Muzzarelli RA, Mattioli-Belmonte M, Pugnaloni A, Biagini G. Biochemistry, histology and clinical uses of chitins and chitosans in wound healing. EXS. 1999;87:251-264.
8. Di Martino A, Sittinger M, Risbud MV. Chitosan: a versatile biopolymer for orthopaedic tissue-engineering. Biomaterials. 2005; 26(30): 5983-5990.
9. Yang TL Chitin-based materials in tissue engineering: applications in soft tissue and epithelial organ. Int. J. Mol. Sci. 2011; 12(3):1936-1963.
10. Shi C, Zhu Y, Ran X, Wang M, Su Y, ChengT. Th erapeutic potential of chitosan and its derivatives in regenerative medicine. J Surg Res. 2006;133(2):185-192.
11. Swetha M, Sahithi K, Moorthi A, Srinivasan N, Ramasamy K, Selvamurugan N. Biocomposites containing natural polymers and hydroxyapatite for bone tissue engineering. Int J Biol Macromol. 2010; 47(1):1-4.
12. Chen JP, Chen SH, Lai GJ. Preparation and characterization of biomimetic silk fi broin/chitosan composite nanofi bers by electrospinning for osteoblasts culture. Nanoscale Res Lett. 2012; 7(1):170-178.
13. Biagini G, Pugnaloni A, Damadei A, Bertani A, Belligolli A, Bicchiega V, Muzzarelli R. Morphological study of the capsular organization around tissue expanders coated with N-carboxybutyl chitosan. Biomaterials. 1991; 12(3): 287-291.
14. Venkatesan J, Kim SK. Chitosan composites for bone tissue engineering – an overview. Mar Drugs. 2010; 8(8):2252-2266.

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Introduction

Table 1. Listing of the main membrane types [5]

Materials and methods

Figure 1. A micrograph of chitosan-based matrix was obtained employing Carl Zeiss Supra 55 VP microscopy, magnification 500X. Micrographs (HC. 500X) of chitosan A and B-based membranes were obtained with a Carl Zeiss Supra 55 VP scanning electron microscope

Results

Table 2. Experimental study design

Conclusion

Acknowledgements

We are much appreciated to Vladimir E. Yudin and Irina P. Dobrovolskaya for assisting in preparation of this article.

Conflict of interests

The authors have no confl icts of interests to declare.

References

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Улитовский 1**, Анна В. Антипова 1*, Александр Д. Вилесов 1**, 2, Галина Ю. Юкина 1***, Дмитрий Н. Суслов 1****, 3, Павел В. Попрядухин 1****, 2, Олег В. Галибин 1** * Кафедра стоматологии профилактической ** Отдел биотехнологии Института детской онкологии, гематологии и трансплантологии им. Р. М. Горбачевой *** Лаборатория патоморфологии НИЦ **** Лаборатория инвазивных технологий НИЦ, ПСПбГМУ" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(1134) "Сергей Б. Улитовский ¹^*^*, Анна В. Антипова ¹^*, Александр Д. Вилесов ¹^*^*, ², Галина Ю. Юкина ¹^*^*^*, Дмитрий Н. Суслов ¹^*^*^*^*, ³, Павел В. Попрядухин ¹^*^*^*^*, ², Олег В. Галибин ¹^*^*^* Кафедра стоматологии профилактической
^*^* Отдел биотехнологии Института детской онкологии, гематологии и трансплантологии им. Р. М. Горбачевой
^*^*^* Лаборатория патоморфологии НИЦ
^*^*^*^* Лаборатория инвазивных технологий НИЦ, ПСПбГМУ" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(12) "Авторы" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["ORGANIZATION_RU"]=> array(36) { ["ID"]=> string(2) "26" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:01:20" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(22) "Организации" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(15) "ORGANIZATION_RU" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "26" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20905" ["VALUE"]=> array(2) { ["TEXT"]=> string(687) "1 Первый Санкт-Петербургский Государственный Медицинский Университет им. акад. И. П. Павлова МЗ РФ (ПСПбГМУ), Санкт-Петербург, Россия 2 Институт высокомолекулярных соединений РАН, Санкт-Петербург, Россия 3 ФГБУ «Российский научный центр радиологии и хирургических технологий им. акад. А.М. Гранова» МЗ РФ, Санкт-Петербург, Россия" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(639) "¹ Первый Санкт-Петербургский Государственный Медицинский Университет им. акад. И. П. Павлова МЗ РФ (ПСПбГМУ), Санкт-Петербург, Россия
² Институт высокомолекулярных соединений РАН, Санкт-Петербург, Россия
³ ФГБУ «Российский научный центр радиологии и хирургических технологий им. акад. А.М. Гранова» МЗ РФ, Санкт-Петербург, Россия" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(22) "Организации" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["SUMMARY_RU"]=> array(36) { ["ID"]=> string(2) "27" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:01:20" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(29) "Описание/Резюме" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(10) "SUMMARY_RU" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "27" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20906" ["VALUE"]=> array(2) { ["TEXT"]=> string(3603) " На сегодняшний день существует большое количество методов лечения воспалительных заболеваний пародонта, являющихся самыми распространенными стоматологическими заболеваниями в мире. Описывается метод хирургического лечения воспалительных заболеваний пародонта, приводится классификация природных и синтетических мембран, использующихся при хирургическом методе лечения, приводится новая технология с использованием природного полимера хитозана. <h2 style="text-align: justify;">Материалы и методы</h2> Пористые трехмерные матрицы получали путем лиофилизации хитозана из 2% раствора уксусной кислоты. Полученные матрицы из хитозана содержали микропоры размером 100-150 нм. Эксперименты in vivo с пористыми хитозановыми мембранами проводили на кроликах. Животным наносили искусственные дефекты максиллярной кости и покрывали их испытуемым материалом. Некоторым животным наносили повреждение ребра, которое потом заполняли биодеградируемой матрицей из пористого хитозана. <h2 style="text-align: justify;">Результаты</h2> Морфологическое исследование искусственно поврежденных ребер с имплантированным материалом выявило разнообразные изменения костной ткани и пористой матрицы без существенных признаков воспаления. Спустя 1 мес., на границе кости и матрицы отмечены остеокластическая реакция наряду с неоангиогенезом в зоне костного дефекта. Через 3-6 мес. после хирургического вмешательства образовались периостальные структуры, а также зоны локального фиброза <h2 style="text-align: justify;">Выводы</h2> Пористые хитозановые матрицы оказались биосовместимым, биоинертным и биорезорбируемым материалом, что соответствует критериям, применимым к материалам для производства костных матриц. <h2 style="text-align: justify;">Ключевые слова</h2> Пародонт, регенерация, мембрана, хитозан. " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(3405) "

На сегодняшний день существует большое количество методов лечения воспалительных заболеваний пародонта, являющихся самыми распространенными стоматологическими заболеваниями в мире. Описывается метод хирургического лечения воспалительных заболеваний пародонта, приводится классификация природных и синтетических мембран, использующихся при хирургическом методе лечения, приводится новая технология с использованием природного полимера хитозана.

Материалы и методы

Пористые трехмерные матрицы получали путем лиофилизации хитозана из 2% раствора уксусной кислоты. Полученные матрицы из хитозана содержали микропоры размером 100-150 нм. Эксперименты in vivo с пористыми хитозановыми мембранами проводили на кроликах. Животным наносили искусственные дефекты максиллярной кости и покрывали их испытуемым материалом. Некоторым животным наносили повреждение ребра, которое потом заполняли биодеградируемой матрицей из пористого хитозана.

Результаты

Морфологическое исследование искусственно поврежденных ребер с имплантированным материалом выявило разнообразные изменения костной ткани и пористой матрицы без существенных признаков воспаления. Спустя 1 мес., на границе кости и матрицы отмечены остеокластическая реакция наряду с неоангиогенезом в зоне костного дефекта. Через 3-6 мес. после хирургического вмешательства образовались периостальные структуры, а также зоны локального фиброза

Выводы

Пористые хитозановые матрицы оказались биосовместимым, биоинертным и биорезорбируемым материалом, что соответствует критериям, применимым к материалам для производства костных матриц.

Ключевые слова

Пародонт, регенерация, мембрана, хитозан.

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¹^*^* Biotechnology Department, R.Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation
¹^*^*^* Laboratory of Pathomorphology, Th e University Research Center
¹^*^*^*^* Laboratory of Invasive Technologies, Th e University Research Center" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(6) "Author" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["ORGANIZATION_EN"]=> array(36) { ["ID"]=> string(2) "38" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:02:59" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(12) "Organization" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(15) "ORGANIZATION_EN" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "38" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20909" ["VALUE"]=> array(2) { ["TEXT"]=> string(311) "1 First Petersburg State I. Pavlov Medical University 2 Institute of Macromolecular Compounds, Russian Academy of Sciences 3 Russian Research A.Granov Center of Radiology and Surgical Technologies, St. Petersburg, Russia" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(263) "¹ First Petersburg State I. Pavlov Medical University
² Institute of Macromolecular Compounds, Russian Academy of Sciences
³ Russian Research A.Granov Center of Radiology and Surgical Technologies, St. Petersburg, Russia" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(12) "Organization" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["SUMMARY_EN"]=> array(36) { ["ID"]=> string(2) "39" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:02:59" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(21) "Description / Summary" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(10) "SUMMARY_EN" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "39" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20910" ["VALUE"]=> array(2) { ["TEXT"]=> string(2191) " A variety of medications is applied nowadays for treatment of infl ammatory periodontal diseases (IPD) which are the prevalent dental disorders worldwide. A method of surgical treatment is described for IPD. We present a classifi cation of natural and synthetic membranes used in surgical interventions, and describe a novel treatment technology using a natural chitosan polymer. <h2 style="text-align: justify;">Materials and methods</h2> Porous 3-D matrices were obtained by lyophilization of chitosan solution from the 2% solution of acetic acid. Th e resulting chitosan matrices had micropores of 100 to 150 nm in size. Th e in vivo experiments with porous chitosan membranes were performed in rabbits. Artificial maxillar bone defects were infl icted, being overlaidby the tested material. Some animals were subjected to rib exposure and infl iction of a bone defect, then filled with a biodegradable porous chitosan-based matrix. <h2 style="text-align: justify;">Results</h2> Morphological examination of artifi cially damaged ribs with implanted material has revealed various changes of bone tissue and porous matrix, without suffi cient inflammation signs. At 1 month, the matrix/bone border has shown osteoclasts at the site of bone defect 30 days aft er surgery, along with neoangiogenesis at the site of repair. At 3 to 6 months post-surgery, periosteal structures were organized, as well as local fi brosis was developed. <h2 style="text-align: justify;">Conclusion</h2> Porous chitosan matrix proved to be biocompatible, bioinert, and bioresorbable material, thus meeting the requirements applicable to the materials suitable for production of the bone matrices. <h2 style="text-align: justify;">Keywords</h2> Periodontium, regeneration, membrane, chitosan. " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(1993) "

A variety of medications is applied nowadays for treatment of infl ammatory periodontal diseases (IPD) which are the prevalent dental disorders worldwide. A method of surgical treatment is described for IPD. We present a classifi cation of natural and synthetic membranes used in surgical interventions, and describe a novel treatment technology using a natural chitosan polymer.

Materials and methods

Porous 3-D matrices were obtained by lyophilization of chitosan solution from the 2% solution of acetic acid. Th e resulting chitosan matrices had micropores of 100 to 150 nm in size. Th e in vivo experiments with porous chitosan membranes were performed in rabbits. Artificial maxillar bone defects were infl icted, being overlaidby the tested material. Some animals were subjected to rib exposure and infl iction of a bone defect, then filled with a biodegradable porous chitosan-based matrix.

Results

Morphological examination of artifi cially damaged ribs with implanted material has revealed various changes of bone tissue and porous matrix, without suffi cient inflammation signs. At 1 month, the matrix/bone border has shown osteoclasts at the site of bone defect 30 days aft er surgery, along with neoangiogenesis at the site of repair. At 3 to 6 months post-surgery, periosteal structures were organized, as well as local fi brosis was developed.

Conclusion

Porous chitosan matrix proved to be biocompatible, bioinert, and bioresorbable material, thus meeting the requirements applicable to the materials suitable for production of the bone matrices.

Keywords

Periodontium, regeneration, membrane, chitosan.

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Ulitovskiy 1*, Anna V. Antipova 1*, Alexander D. Vilesov 1**, 2, GalinaYu. Yukina 1***, Dmitry N. Suslov 1****, 3, Pavel V. Popryadukhin 1****, 2, Oleg V. Galibin 1** 1* Department of Preventive Stomatology 1** Biotechnology Department, R.Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation 1*** Laboratory of Pathomorphology, Th e University Research Center 1**** Laboratory of Invasive Technologies, Th e University Research Center" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(957) "Sergey B. Ulitovskiy ¹^*, Anna V. Antipova ¹^*, Alexander D. Vilesov ¹^*^*, ², GalinaYu. Yukina ¹^*^*^*, Dmitry N. Suslov ¹^*^*^*^*, ³, Pavel V. Popryadukhin ¹^*^*^*^*, ², Oleg V. Galibin ¹^*^*¹^* Department of Preventive Stomatology
¹^*^* Biotechnology Department, R.Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation
¹^*^*^* Laboratory of Pathomorphology, Th e University Research Center
¹^*^*^*^* Laboratory of Invasive Technologies, Th e University Research Center" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(6) "Author" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["DISPLAY_VALUE"]=> string(957) "Sergey B. Ulitovskiy ¹^*, Anna V. Antipova ¹^*, Alexander D. Vilesov ¹^*^*, ², GalinaYu. Yukina ¹^*^*^*, Dmitry N. Suslov ¹^*^*^*^*, ³, Pavel V. Popryadukhin ¹^*^*^*^*, ², Oleg V. Galibin ¹^*^*¹^* Department of Preventive Stomatology
¹^*^* Biotechnology Department, R.Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation
¹^*^*^* Laboratory of Pathomorphology, Th e University Research Center
¹^*^*^*^* Laboratory of Invasive Technologies, Th e University Research Center" } ["SUMMARY_EN"]=> array(37) { ["ID"]=> string(2) "39" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:02:59" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(21) "Description / Summary" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(10) "SUMMARY_EN" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "39" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20910" ["VALUE"]=> array(2) { ["TEXT"]=> string(2191) " A variety of medications is applied nowadays for treatment of infl ammatory periodontal diseases (IPD) which are the prevalent dental disorders worldwide. A method of surgical treatment is described for IPD. We present a classifi cation of natural and synthetic membranes used in surgical interventions, and describe a novel treatment technology using a natural chitosan polymer. <h2 style="text-align: justify;">Materials and methods</h2> Porous 3-D matrices were obtained by lyophilization of chitosan solution from the 2% solution of acetic acid. Th e resulting chitosan matrices had micropores of 100 to 150 nm in size. Th e in vivo experiments with porous chitosan membranes were performed in rabbits. Artificial maxillar bone defects were infl icted, being overlaidby the tested material. Some animals were subjected to rib exposure and infl iction of a bone defect, then filled with a biodegradable porous chitosan-based matrix. <h2 style="text-align: justify;">Results</h2> Morphological examination of artifi cially damaged ribs with implanted material has revealed various changes of bone tissue and porous matrix, without suffi cient inflammation signs. At 1 month, the matrix/bone border has shown osteoclasts at the site of bone defect 30 days aft er surgery, along with neoangiogenesis at the site of repair. At 3 to 6 months post-surgery, periosteal structures were organized, as well as local fi brosis was developed. <h2 style="text-align: justify;">Conclusion</h2> Porous chitosan matrix proved to be biocompatible, bioinert, and bioresorbable material, thus meeting the requirements applicable to the materials suitable for production of the bone matrices. <h2 style="text-align: justify;">Keywords</h2> Periodontium, regeneration, membrane, chitosan. " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(1993) "

Materials and methods

Results

Conclusion

Porous chitosan matrix proved to be biocompatible, bioinert, and bioresorbable material, thus meeting the requirements applicable to the materials suitable for production of the bone matrices.

Keywords

Periodontium, regeneration, membrane, chitosan.

Materials and methods

Results

Conclusion

Porous chitosan matrix proved to be biocompatible, bioinert, and bioresorbable material, thus meeting the requirements applicable to the materials suitable for production of the bone matrices.

Keywords

Periodontium, regeneration, membrane, chitosan.

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² Institute of Macromolecular Compounds, Russian Academy of Sciences
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Материалы и методы

Результаты

Выводы

Ключевые слова

Пародонт, регенерация, мембрана, хитозан.

Материалы и методы

Результаты

Выводы

Ключевые слова

Пародонт, регенерация, мембрана, хитозан.

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Introduction

The Major Histocompatibility Complex (MHC) is among the most polymorphic genetic systems in humans. Over last decade, extensive research in HLA (Human Leukocyte Antigens) has revealed hundreds of new HLA alleles through intensive application of immunogenetic sequencing methods, including monoallelic Sanger-sequencing method, or, more recently, next-generation sequencing. In September 2018, the database of the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System (IPD-IMGT/HLA Database) contained information on the nucleotide sequences of 20272 diff erent HLA alleles, of which 14800 were HLA class I and 5288 founded for the HLA class II alleles [1-3].
During last 20 years, the automated Sanger technique has become a prevalent approach to genome sequencing in humans, animals, bacteria, and viruses. However, a need for more rapid routine genome screening required some novel technologies of multiplex DNA sequencing. It depicts these modern methods as the second-generation approaches (Next-Generation Sequencing, NGS). Th ese technological platforms based on diff erent strategies, regarding unique preparations of DNA templates, their sequencing, registration, retrieval and evaluation of the nucleotide sequences with novel bioinformatics approaches [4]. A principal benefit of the new-generation sequencing is an opportunity get large databases of multiple defi ned oligonucleotide sequences within a short time period with low costs.
Out of all known HLA loci, the relatively important and most commonly used for transplantation of hematopoietic cells are HLA – A, B, C, DRB1 and DQB1 (Fig. 1). The American Society for Histocompatibility and Immunogenetics (ASHI) established a catalogue of common and well‐documented (CWD) HLA. It is very commonly used now around the world as a great tool for resolving typing ambiguities in tissue transplantation or for checking the universality of any HLA allele in the world [5]. There established catalogues (database). Th e total number of CWD alleles is similar in the EFI (N = 1048) and ASHI (N = 1031) catalogues [6] (http://igdawg.org/cwd.html).
The importance of only Exons 2 and 3 for the Class I and Exon 2 for Class II is very well-known and designated as coding proteins involved in antigen presentation in the major histocompatibility complex (MHC) receptor grove in-between the two helices accommodates peptides and interaction between an alloantibody IgG complex.
HLA alleles having nucleotide sequences that encode the same protein sequence for the peptide binding domains (exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles) designated by an upper case ‘P’ which follows the allele designation of the lowest numbered allele in the group. HLA alleles that have identical nucleotide sequences for the exons encoding the peptide binding domains (exon 2 and 3 for HLA class I and exon 2 only for HLA class II alleles) designated by an upper case ‘G’ which follows the allele designation of the lowest numbered allele in the group.
The first two digits describe the allele family, which oft en corresponds to the serological antigen carried by the allotype. The third and fourth digits assigned in the order in which the sequences have been determined. Alleles whose numbers diff er in the fi rst four digits must diff er by one or more nucleotide substitutions that change the amino-acid sequence of the encoded protein. Alleles that differ only by synonymous nucleotide substitutions within the coding sequence distinguished by the use of the fifth and sixth digits. Alleles that only differ by sequence polymorphisms in introns or in the 5’ and 3’ untranslated regions that fl ank the exons and introns distinguished by the use of the seventh and eight digits [7].

72-82 Figure 1. Current mapping of HLA loci.png

Figure 1. Current mapping of HLA loci on the chromosome 6 [Robinson J et al.] http://www.hla.alleles.org/alleles/index.html

No wonder that the general NGS approach adapted for HLA typing proved to be a breakthrough in molecular biology applications being quite promising to the transplantation clinics and bone marrow donor registries. However, to promote the NGS implementation, we need specialized typing strategies and digital program algorithms. The sequencing costs per single run sharply decreased with NGS approach, which may be accessible to the small size tissue typing laboratories in a sooner time [8].
However, despite higher resolution of NGS [9], it was necessary to conduct a comparative analysis of control samples with "rare" genotypes. It is also important to understand the cost-eff ectiveness of diff erent methods. Hence, the aim of this pilot study was to evaluate the comparative advantages of using NGS and the Sanger sequencing approach, to identify rare HLA alleles, and to estimate the costs for the both different methods.

Materials and methods

The potential donor's test samples were obtained from the Bone Marrow Donor Registry at the First I. P. Pavlov State Medical University of St. Petersburg, Russia, Raisa Gorbacheva Memorial Institute of Children's Hematology, Oncology, and Transplantation, and various hematology patients undergoing HLA SBT testing for planned allogeneic hematopoietic stem cell transplantation.
Genomic DNA was isolated from peripheral blood leukocytes using MagNA Pure System (Roche Life Science). The target DNA concentration was from 10 to 140 ng/μL. Quantity and quality estimation of the isolated DNA was performed with Quantus Fluorometer TM (Promega Corp., USA). The main steps of the NGS as performed with Illumina platform (MiSeq, USA) using NGSgo protocol were as follows:
1. HLA locus-specifi c amplifi cation: the complete sequences of HLA genes are amplifi ed with allele-specifi c primers in a single reaction for each locus using Long-Range DNA polymerase;
2. DNA quantifi cation by Quantus Fluorometer and pooling of amplicons according to the volumes calculated by the NGSgo Pooling Calculation Sheet (provided by GenDx, Netherlands);
3. Double-stranded DNA fragmentation by means of specific fragmentase optimized by its size for the specifi c HLA locus, end repair, 5’ phosphorylation of poly-A and poly-T ends and adapter ligation (Fig. 2);
4. DNA cleanup and size selection with 0.45x SPRI beads using 80% ethanol (Beckman Coulter, AMPure XP);
5. Indexing PCR products using a unique combination of i5 and i7 primers for each sample;
6. Plate-based DNA cleanup and size selection with 0.6x SPRI beads using 80% ethanol (Beckman Coulter, AMPure XP);
7. Plate-based library pooling, library quantifi cation performed using Qubit Fluorometer and loading to the NGS sequencer (MiSeq, Illumina, USA);
8. Next-generation sequencing by MiSeq and data analysis.
The libraries are sequenced on an Illumina NGS platform. The FastQ data can be analyzed with an HLA typing soft ware package to determine the HLA typing (for example, NGS engine). To assign the HLA alleles, the soft ware allows communicating with updated IMGT database (Fig. 3).
The NGS method allows performing sequencing of all exons in the A, B, and C HLA loci and three exons (from second to fourth) of DQB1 and DRB1 (Fig. 4) which, however, has its limitations. Th e allele imbalances can be observed in some
rare cases:
• NGSgo HLA-DRB1: allele imbalances for DRB1*01, DRB*04, and DRB1*14 alleles can occur in case of imbalanced amplifi cation.
• NGSgo HLA-DRB4: allele imbalances for DRB2 exon 2 and exon 3 can occur in case of imbalanced DRB4 amplifi cation. In the case of an HLA-DRB4 exon 3 amplicon dropout, limit the analysis to exon 2 only.
• NGSgo HLA-DRB3/4/5: allele imbalances for heterozygous DRB3/4/5 samples can occur in case of imbalanced amplicon pooling. Analysis of DRB3/4/5 has been optimized in NGSengine v2.1 (and higher), which applies a split-analysis of the individual DRB3/4/5 loci to improve HLA typing.
Th e main steps of Sanger sequencing when performed with Applied Biosystems Genetic Analyzer (USA) 3500xl genetic analyzer using PROTRANS HLA SBT Class I and Class II S4 (Hockenheim, Germany, http://www.protrans.info/nano.cms/en/products/MainCatID/9/). Single Allele, Allele- Group and Locus Specific Sequencing. Fourteen specific primer mixes pre-pipetted in 8 and 16 well strip, in order of sequencing the Exons 1, 2, 3 and 4 for Class I, Exon 2 for DRB1 and Exon 2, 3 for DQB1, according to the manufacturers’ recommendations.
72-82 Figure 2. Gene library preparation with.png

Figure 2. Gene library preparation with NGSgo – LibX and NGSgo – IndX referred from https://www.gendx.com [10]

72-82 Figure 3. Data analysis software.png

Figure 3. Data analysis software (NGSengine, https://www.gendx.com)

72-82 Figure 4. Target generation with NGSgo.png

Figure 4. Target generation with NGSgo – AmpX [10]

Direct automated fl uorescent DNA sequencing was performed by a 24-channel automated capillary electrophoresis system, and fl uorescent detection of DNA fragments using an Applied Biosystems GA3500xl Genetic Analyzer. Capillary electrophoresis proceeded in the POP-7 polymer under denaturation conditions. Th e data on nucleotide sequences were retrieved at a stationary computer in the Data Collection program, then having been analyzed by Protrans SEQUENCE PILOT soft ware (Hockenheim, Germany).
DNA amplification kits for Sanger sequencing are designed to provide high-resolution identifi cation of alleles of the human HLA-A, -B, -C, -DRB1, -DQB1 genes.

Results

The aim of our pilot study was a comparison of two methods and an evaluation of their eff ectiveness. To achieve our purpose, we selected a group of 35 persons (see Materials and methods), and conducted analysis by Sanger and NGS method in parallel. Th e NGS method allowed detecting rare variants of alleles when performing data analysis with NGSengine soft ware (Fig. 5).
We have conducted two sets of experiments. Mean coverage in the fi rst experiment was 881x – (1010x, 897x, 768x, 807x, 923x, respectively for A-, B-, C-, DRB-, DQB- loci), and 992x in a second experiment (1194x, 856x, 698x, 1001x, 1346x, respectively for A-, B-, C-, DRB-, DQB- loci).
Mean percentage of aligned reads to the total read number was 96.5% in the fi rst set (DRB locus, 92.6%, other loci, >97.2%). In the second set, an appropriate percentage of aligned reads to the total read number was 95.0% (DRB locus, 91.6%, other loci, >95.5%) (Fig. 6). This metrics shows a high quality of the sequencing that was performed according to the manufacturer’s instructions.
To perform a more detailed analysis of each sample, the NGSengine soft ware contains the sections of «typing results» and «visualization», where the coverage for diff erent regions may be registered in more details, or a nucleotide position of interest should be found (Fig. 7).
72-82 Figure 5. A typical data evaluation table pres.png

Figure 5. A typical data evaluation table presented by NGSengine software (Genome Diagnostics, Netherlands)

It presents information for each locus (HLA-A, -B, -C, -DRB1, -DQB1) for single samples. Data on total read number and percentage of aligned reads for the given locus, mean read length, mean coverage, alleles identifi ed and presence of synonymous substitutions in coded [Ex] and it also displays non-coding [In] regions.

72-82 Figure 6. Statistics for samples (percentage.png

Figure 6. Statistics for samples (percentage aligned reads from total number of reads and number of reads mapped to the reference per strand) in NGSengine software

72-82 Figure 7. The results of the NGS.png

Figure 7. The results of the NGS sequencing all alleles for sample “3” and visualization of HLA-A locus for sample “3” in NGSengine software

It displays typing results for all HLA loci assayed. Th e cases of ambiguous results shown as Allele Ambiguities. In our series, no ambiguities were detectable for any locus. The figure visualization allows us to look at the visual segment (it shows exons in yellow). It indicates the sequencing coverage of the given locus below (marked gray). The vertical ticks seen at appropriates points of HLA loci in cases of synonymous nucleotide substitutions.

Table 1. Comparison of allele sequenced by NGS and Sanger’s method – 100% homology results (there are no differences in 2nd and 3rd exons sequences)

72-82 Table 1. Comparison of allele sequenced.png

Factors contributing to the costs arising for the in-depth sequencing

The reagents for the entire HLA-sequencing process include those used for routine pre-analytic steps (e.g., DNA extraction, quality assessment, and initial low-resolution typing step). Additional expenditures are subject to some ambiguities, due to diff erent prices for reagents and equipment off ered by distinct manufacturers. Moreover, it should be addressed that all the commercial NGS platforms off er their closed-type systems, thus causing broad variations in prices for the entire NGS procedure per single DNA sample, strongly depending on the annual capacity of the given HLA typing laboratory.
However, even considering maintenance costs (about 10% equipment cost), enrolling third-party core facilities or shared equipment, the Sanger sequencing (220 K) proves to be twice more expensive than NGS (variable, but still less than Sanger technique), as shown elsewhere [11]. Hence, the sample preparation costs remain the same whereas the sequencing tends to decrease as discussed in [9]. Calculations of economic efficiency for HLA typing in Russia by Sanger technique versus NGS were among our major tasks. Therefore, we have performed a pricing for the sequencing kits at a company providing reagents to this purpose. Th e request was made twice (February 2017 and August 2018). The reagent price in Euros did not change suffi ciently. Of note, the sequencing kits by Sanger are produced for 25 or 100 tests, whereas NGS kits are off ered for 24 and 96 tests.
Clear benefit of Sanger approach is that a single locus may be sequenced in the sample, being, however, economically ineff ective when using NGS technology.
Hence, we have compared the panels for 100 tests covering fi ve main HLA loci, i.e., AlleleSEQR HLA-A PCR/Sequencing Mix, AlleleSEQR HLA-B PCR/Sequencing Mix, AlleleSEQR HLA-C Plus PCR/Sequencing Mix, AlleleSEQR HLA-DRB1 PCR/Sequencing Mix, AlleleSEQR HLA-DQB1 PCR/Sequencing Mix). Each set was purchased for 6250 Euros. Hence, the total cost of locus-specifi c reagents for 5 loci was 31250 Euros, thus providing 312.50 Euros per 1 human DNA sample (ca. 24,000 roubles as per October 2018). The prime costs should also include disposables for the core sequencing procedure. E.g., if performing 100 tests for 5 HLA loci, we run 2500 reactions with a 24-channel Applied Biosystems GA3500xl Genetic Analyzer ABI 3500. To start the process, the following general items are needed: 26 plates for the gene analyzer (MicroAmp 96 Well Reaction Plate); three universal polymers. For capillary electrophoresis, POP-7 for 960 samples, fi ve Formamide packs (25 mL each), containers with anode and cathode buff ers etc., at a total price of 4500 Euros. Hence, the prime costs of Sanger reagents, when sequencing 5 main loci at the full-load regimen and usage of a 100-test kit, makes about 35750 Euros (ca. 2681250 roubles), excluding costs for pipette tips, microtubes, gloves and other inexpensive disposables. Th at means ca. 27000 roubles per one human DNA sample.
When applying NGS approach, the number of samples taken into analysis is quite suffi cient, since even a high-throughput MiSeq machine may perform sequencing of up to 269 samples in parallel, using a standard 4.5-Gb cartridge.
To calculate costs of comparable NGS analysis, we have chosen a reagent set for sequencing of 96 samples which incuded the following items: NGSgo®-AmpX HLA-A, B, C, DRB1, DQB1, NGSgo®-LibrX Library Preparation (2 kits), NGSgo ®-IndX Adapter & Indices (4x24) RUO Illumina, Agencourt AMPure XP 5 mL Kit, GenDx LongRange polymerase (3 kits), MiSeq Reagent Kit v2. A total sum for typing 5 loci made ca. 14 800 Euros, thus comprising 155 Euros (>10000 Roubles) per sample.
However, in case of spared use of the reagents for sample preparation by two-fold decrease in reaction volume (as proven by our experience), the prime costs per a single test dropped to 90 Euros. Th e self-cost is here provided without accessory disposables.
Hence, the costs of sequencing reagents, even without their sparing, is suffi ciently cheaper when using NGS technologies. Moreover, the procedure takes 2-fold less time for its performance than the Sanger technique.

Conclusion

Comparison of allele sequenced by NGS and Sanger’s method yielded 100% homology results. Hence, our work is in accordance with previously published data [9], which demonstrate the advantage and effi ciency of NGS, as compared to Sanger sequencing.
NGS-based HLA analysis is performed with a 100% reliability, and well fi ts the tasks of HLA typing in unrelated donors, in concordance with EFI and ASHI policies. Th is work process well corresponds to the working schedules for medium- and high-capacity laboratories, thus being potentially attractive to the donor registries. Recently introduced next-generation sequencing techniques have a facilitating potential for the high-resolution genotyping via a decrease of general ambiguity of end results, like as due to more extended sequencing regions. In near future, the NGS approaches will be an eff ective and cost-eff ective technology when evaluating histocompatibility parameters and immunogenetic interactions.

Conflict of interest

The authors have declared no conflicting interests.

Funding

This research was funded by Russian Science Foundation grant №14-50-00069.

References

1. Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE. Th e IPD and IMGT/HLA database: allele variant databases. Nucleic Acids Research. 2015; 43:D423-431.
2. Marsh SGE, Albert ED, Bodmer W, Bontrop RE, Dupont B, Erlich HA, Fernández-Viña M, Geraghty DE, Holdsworth R, Hurley CK, Lau M, Lee KW , Mach B, Maiers M, Mayr WR, Müller CR, Parham P, Petersdorf EW, Sasazuki T, Strominger JL, Svejgaard A, Terasaki PI, Tiercy JM, Trowsdale J. Nomenclature for factors of the HLA system, 2010. Tissue Antigens. 2010; 75(4):291-455.
3. Marsh SGE. Nomenclature for factors of the HLA system, update July 2017. Human Immunol. 2017; 78(11-12):758- 761.
4. Holcomb CL, Höglund B, Anderson MW, Blake LA, Böhme I, Egholm M, Ferriola D, Gabriel C, Gelber SE, Goodridge D, Hawbecker S, Klein R, Ladner M, Lind C, Monos D, Pando MJ, Pröll J, Sayer DC, Schmitz-Agheguian G, Simen BB, Th iele B, Trachtenberg EA, Tyan DB, Wassmuth R, White S, Erlich HA.A multi-site study using high-resolution HLA genotyping by next generation sequencing. // Tissue Antigens. 2011;77(3):206-217. doi: 10.1111/j.1399-0039.2010.01606.x.
5. Mack SJ1, Cano P, Hollenbach JA, He J, Hurley CK, Middleton D, Moraes ME, Pereira SE, Kempenich JH, Reed EF, Setterholm M, Smith AG, Tilanus MG, Torres M, Varney MD, Voorter CE, Fischer GF, Fleischhauer K, Goodridge D, Klitz W, Little AM, Maiers M, Marsh SG, Müller CR, Noreen H, Rozemuller EH, Sanchez-Mazas A, Senitzer D, Trachtenberg E, Fernandez-Vina M. Common and well-documented HLA alleles: 2012 update to the CWD catalogue. Tissue Antigens. 2013 Apr;81(4):194-203. doi: 10.1111/tan.12093.
6. A. Sanchez‐Mazas, J. M. Nunes, D. Middleton, J. Sauter, S. Buhler, A. McCabe, J. Hofmann, D. M. Baier, A. H. Schmidt, G. Nicoloso, M. Andreani, Z. Grubic, J.‐M. Tiercy, K. Fleischhaue.r Common and well‐documented HLA alleles over all of Europe and within European sub‐regions: A catalogue from the European Federation for Immunogenetics. HLA Volume 89, Issue2.February 2017 Pages 104-113.
7. S. G. E. Marsh, E. D. Albert, W. F. Bodmer, R. E. Bontrop, B. Dupont, H. A. Erlich, M. Ferna´ndez-Vin˜ a, D. E. Geraghty, R. Holdsworth, C. K. Hurley, M. Lau, K. W. Lee, B. Mach, M. Maiers, W. R. Mayr, C. R. Mu¨ ller, P. Parham, E. W. Petersdorf, T. Sasazuki, J. L. Strominger, A. Svejgaard, P. I. Terasaki, J. M. Tiercy & J. Trowsdale. Nomenclature for factors of the HLA system, 2010 Tissue Antigens 75, 291-455.
8. Kuzmich EV, Alyanskiy AL, Tyapushkina SS, Nasredinova AA, Ivanova NE, Zubarovskaya LS, Afanasyev BV. Identifi cation of the new HLA-B*44:02:45, DQB1*02:85, DQB1*06:210, DRB1*01:01:30 alleles by monoallelic Sanger sequencing. Cell Th er Transplant. 2018. 7(1):62-66. 9. Serov YA, Barkhatov IM, Klimov AS, Berkos AS. Current methods and opportunities of next-generation sequencing (NGS) for HLA typing // Cell Th er Transplant. 2016; 5(4): 63-70. doi: 10.18620/ctt-1866-8836-2016-5-4-63-70.
10. https://www.gendx.com
11. Baxter-Lowe LA. Tailoring NGS for smaller volume labs. Proc. 42nd ASHI Annual Meeting. Abstract: Sept 28, 2016.

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Introduction

Figure 1. Current mapping of HLA loci on the chromosome 6 [Robinson J et al.] http://www.hla.alleles.org/alleles/index.html

Materials and methods

Figure 2. Gene library preparation with NGSgo – LibX and NGSgo – IndX referred from https://www.gendx.com [10]

72-82 Figure 3. Data analysis software.png

Figure 3. Data analysis software (NGSengine, https://www.gendx.com)

72-82 Figure 4. Target generation with NGSgo.png

Figure 4. Target generation with NGSgo – AmpX [10]

Results

Figure 5. A typical data evaluation table presented by NGSengine software (Genome Diagnostics, Netherlands)

72-82 Figure 6. Statistics for samples (percentage.png

Figure 6. Statistics for samples (percentage aligned reads from total number of reads and number of reads mapped to the reference per strand) in NGSengine software

72-82 Figure 7. The results of the NGS.png

Figure 7. The results of the NGS sequencing all alleles for sample “3” and visualization of HLA-A locus for sample “3” in NGSengine software

Table 1. Comparison of allele sequenced by NGS and Sanger’s method – 100% homology results (there are no differences in 2nd and 3rd exons sequences)

72-82 Table 1. Comparison of allele sequenced.png

Factors contributing to the costs arising for the in-depth sequencing

Conclusion

Conflict of interest

The authors have declared no conflicting interests.

Funding

This research was funded by Russian Science Foundation grant №14-50-00069.

References

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На протяжении последних 20 лет при секвенировании генома человека, животных, бактерий и вирусов преобладает автоматизированная технология Сэнгера. Однако необходимость более быстрого скрининга генома стимулировало развитие новых технологий мультиплексного секвенирования ДНК. Эти современные методы обозначаются как подходы следующего поколения (Next-Generation Sequencing, NGS). Целью нашего исследования было сравнение двух этих методов и оценка их эффективности. Чтобы достичь этой цели, мы выбрали группу из 35 образцов ДНК, в основном – потенциальных доноров гемопоэтических клеток, и провели сравнительный анализ по Сэнгеру и методом NGS. Метод NGS позволяет выявлять редкие или новые варианты аллелей. 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Глотов 1,2, Ольга В. Романова 1,2, Юрий А. Эйсмонт 1, Андрей М. Сарана 1,2, Сергей Г. Щербак 1,2, Елена В. Кузьмич 3, Александр Л. Алянский 3, Наталья Е. Иванова 3, Вера В. Тепляшина 3, Юрий А. Серов 3, Людмила С. Зубаровская 3, Борис В. Афанасьев 3" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(600) "Олег С. Глотов ¹,², Ольга В. Романова ¹,², Юрий А. Эйсмонт ¹, Андрей М. Сарана ¹,², Сергей Г. Щербак ¹,², Елена В. Кузьмич ³, Александр Л. Алянский ³, Наталья Е. Иванова ³, Вера В. Тепляшина ³, Юрий А. Серов ³, Людмила С. Зубаровская ³, Борис В. Афанасьев ³" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(12) "Авторы" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["ORGANIZATION_RU"]=> array(36) { ["ID"]=> string(2) "26" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:01:20" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(22) "Организации" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(15) "ORGANIZATION_RU" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "26" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20915" ["VALUE"]=> array(2) { ["TEXT"]=> string(733) "1 Городская больница №40, Сестрорецк, Санкт-Петербург, Россия 2 Институт трансляционной биомедицины, Санкт-Петербургский государственный университет, Санкт-Петербург, Россия 3 НИИ детской онкологии, гематологии и трансплантологии им. Р. М. Горбачевой, Первый Санкт-Петербургский государственный медицинский университет, Санкт-Петербург, Россия" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(679) "¹ Городская больница №40, Сестрорецк, Санкт-Петербург, Россия
² Институт трансляционной биомедицины, Санкт-Петербургский государственный университет, Санкт-Петербург, Россия
³ НИИ детской онкологии, гематологии и трансплантологии им. Р. М. Горбачевой, Первый Санкт-Петербургский
государственный медицинский университет, Санкт-Петербург, Россия" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(22) "Организации" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } } ["SUMMARY_RU"]=> array(36) { ["ID"]=> string(2) "27" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:01:20" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(29) "Описание/Резюме" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(10) "SUMMARY_RU" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "27" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20916" ["VALUE"]=> array(2) { ["TEXT"]=> string(3069) " База данных Всемирной организации здравоохранения (ВОЗ) Комитета по номенклатуре факторов системы HLA (база данных IPD-IMGT/HLA) на сентябрь 2018 г. содержала информацию о нуклеотидных последовательностях 20272 различных аллелей HLA, из которых 14800 были аллелями HLA класса I, а 5288 – класса II. На протяжении последних 20 лет при секвенировании генома человека, животных, бактерий и вирусов преобладает автоматизированная технология Сэнгера. Однако необходимость более быстрого скрининга генома стимулировало развитие новых технологий мультиплексного секвенирования ДНК. Эти современные методы обозначаются как подходы следующего поколения (Next-Generation Sequencing, NGS). Целью нашего исследования было сравнение двух этих методов и оценка их эффективности. Чтобы достичь этой цели, мы выбрали группу из 35 образцов ДНК, в основном – потенциальных доноров гемопоэтических клеток, и провели сравнительный анализ по Сэнгеру и методом NGS. Метод NGS позволяет выявлять редкие или новые варианты аллелей. Этот подход подтвержден в качестве более чувствительного и более экономичного, особенно в больших лабораториях по HLA-типированию. <h2 style="text-align: justify;">Ключевые слова</h2> Главный комплекс гистосовместимости, новые аллели HLA, технологические решения, секвенирование следующего поколения, NGS, секвенирование по Сэнгеру, трансплантация гемопоэтических клеток, типирование по сиквенсам ДНК. " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(2859) "

База данных Всемирной организации здравоохранения (ВОЗ) Комитета по номенклатуре факторов системы HLA (база данных IPD-IMGT/HLA) на сентябрь 2018 г. содержала информацию о нуклеотидных последовательностях 20272 различных аллелей HLA, из которых 14800 были аллелями HLA класса I, а 5288 – класса II.

На протяжении последних 20 лет при секвенировании генома человека, животных, бактерий и вирусов преобладает автоматизированная технология Сэнгера. Однако необходимость более быстрого скрининга генома стимулировало развитие новых технологий мультиплексного секвенирования ДНК. Эти современные методы обозначаются как подходы следующего поколения (Next-Generation Sequencing, NGS).

Целью нашего исследования было сравнение двух этих методов и оценка их эффективности. Чтобы достичь этой цели, мы выбрали группу из 35 образцов ДНК, в основном – потенциальных доноров гемопоэтических клеток, и провели сравнительный анализ по Сэнгеру и методом NGS. Метод NGS позволяет выявлять редкие или новые варианты аллелей. Этот подход подтвержден в качестве более чувствительного и более экономичного, особенно в больших лабораториях по HLA-типированию.

Ключевые слова

Главный комплекс гистосовместимости, новые аллели HLA, технологические решения, секвенирование следующего поколения, NGS, секвенирование по Сэнгеру, трансплантация гемопоэтических клеток, типирование по сиквенсам ДНК.

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² Institute of Translation Biomedicine, St. Petersburg State University, St. Petersburg, Russia
³ Raisa Gorbacheva Memorial Research Institute for Pediatric Oncology, Hematology and Transplantation, The First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russia
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The database of the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System (IPD-IMGT/HLA Database) contained information on the nucleotide sequences of 20272 diff erent HLA alleles in September 2018, of which 14800 were HLA class I and 5288 were found for the HLA class II alleles. Over the last 20 years, the automated Sanger technique is a prevalent approach to genome sequencing in humans, animals, bacteria, and viruses. However, a need for more rapid routine genome screening stimulated novel technologies of multiplex DNA sequencing. These modern methods are depicted as the second-generation approaches (Next-Generation Sequencing, NGS). The aim of our research was a comparison of two methods and their effi ciency evaluation. To achieve our purpose, we selected a group of 35 DNA samples, mainly from potential hematopoietic cells donors, and conducted a comparative analysis by Sanger and NGS method. NGS method allowed detecting rare or novel variants of alleles. This approach is confirmed to be more sensitive and more cost-eff ective, especially in large HLA-typing laboratories.

Keywords

Major histocompatibility complex, novel HLA alleles, technological solutions, next-generation sequencing, NGS, Sanger sequencing, hematopoietic cells transplantation, Sequence-Based Typing (SBT).

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Keywords

Major histocompatibility complex, novel HLA alleles, technological solutions, next-generation sequencing, NGS, Sanger sequencing, hematopoietic cells transplantation, Sequence-Based Typing (SBT).

Keywords

Major histocompatibility complex, novel HLA alleles, technological solutions, next-generation sequencing, NGS, Sanger sequencing, hematopoietic cells transplantation, Sequence-Based Typing (SBT).

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² Institute of Translation Biomedicine, St. Petersburg State University, St. Petersburg, Russia
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² Institute of Translation Biomedicine, St. Petersburg State University, St. Petersburg, Russia
³ Raisa Gorbacheva Memorial Research Institute for Pediatric Oncology, Hematology and Transplantation, The First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russia
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Ключевые слова

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³ НИИ детской онкологии, гематологии и трансплантологии им. Р. М. Горбачевой, Первый Санкт-Петербургский
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³ НИИ детской онкологии, гематологии и трансплантологии им. Р. М. Горбачевой, Первый Санкт-Петербургский
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Introduction

Erythropoietin (EPO) was initially known as a physiological growth factor which is produced, mainly, by renal glomeruli, macrophages and some other cell types. EPO is shown to support survival and mitotic activity, as well as inhibit apoptosis of late erythroid precursor cells in bone marrow [1]. Th erefore, recombinant erythropoietins-alpha (Eprex, Epobioicrine, Epostim etc.) are widely used to correct anemias in diff erent diseases and posttransplant conditions [2].

However, biological mechanisms of EPO action upon immune cells are still not clear. E.g., antioxidant in vivo eff ects of EPO (reduced lipid peroxidation in blood lymphocytes) are shown by Osikov et al. [3]. Immunotropic eff ects of EPO are recently studied, due to its immunomodulatory eff ects under clinical and experimental conditions [4-6]. Response of lymphocytes and macrophages to EPO seems to be mediated by the EPO receptors on their surface [7]. In vitro biological eff ects of EPO towards T-lymphoid cells were previously shown by Hisatomi et al. [8] who demonstrated suppression of IL-2 gene expression in TLC cells after their short-term (6 h) incubation with EPO. Hence, EPO is able to exert fast immediate action upon T-lymphoid cells over short incubation terms. Indeed, we have also revealed modulatory effect of EPO upon rat thymocytes using a specifi c potential-sensitive chemical probe [9]. EPO was found to exert activating eff ect upon the TLC by changing total transmembrane potential of plasmatic (Δφp) and mitochondrial membranes (Δφm). This activation correlated with increased numbers of the probe-labeled fl uorescent mitochondria in exposed cells [10]. To discern these mechanisms, we used specifi c inhibitors of oxydative phosphorylation, in order to assess the type of potential responsible for these EPO eff ects. Hence, the aim of this work was to evaluate the role of mitochondrial functions in EPO eff ects upon thymic lymphocytes in order to specify this response to EPO, either Δφm, or Δφp. To address this issue, we used specifi c inhbitors of phosphorylation in the respiratory chain which serve as important tools for studying energy supply in the living cells.
To address this issue, we used specifi c inhibitors of oxydative phosphorylation. In order to evaluate the role of mitochondrial functions for energy supply in thymic lymphocytes exposed to EPO. Hence, the aim of this study was to assess a restoring EPO eff ect upon the in vitro response of thymocytes aft er treatment with diff erent specifi c inhibitors of oxidative phosphorylation.

Materials and Methods

We have studied lymphoid cells isolated from thymuses of white Wistar rats (200-300 g), aft er gentle mincing of thymus glands [9]. Th e cells were resuspended in standard Hank’s solution at 2 to 3x107 cells/mL. Percentage of viable (dye-excluding) cells was determined by routine Trypan Blue staining.
To determine possible points for EPO actions, we used different inhibitors of the oxidative phosphorylation, i.e., dinitrophenol (DNP, Sigma, USA), an inhibitor of respiratory chain and uncoupler of oxidative phosphorylation; pentachlorophenol (PCP, Sigma USA), an uncoupler of oxidative phosphorylation; dicyclohexyl carbodiimide (DCCD, Sigma, USA), an inhibitor of mitochondrial membrane-bound ATP-ase domain. Erythropoietin (EPO) was purchased from Cilag (Eprex) was dissolved in Hank’s solution. A potential- sensitive fl uorescent probe [4-(p-dimethylaminostyryl)- 1-methylpyridinium] (DSM) was used to test the energy potential of cells. DSM was produced and purchased from the Latvian Institute of Organic Synthesis [9]. Th e thymocyte suspensions in Eppendorf-type tubes were pre-incubated with inhibitors for 10…20…40 min at 37°С, then EPO was added at the fi nal concentration of 2U/ml), followed by incubation for 30 min, addition of the DSM probe (1.5 μM), and post-incubated for 20 min. Final concentrations of inhibitors were as follows: DNP, 0.1 mM; PCP, 1.5 μM; DCCD, 0.1 mM, at a v/v ratio of <5% to the initial suspension volume. The control samples of cells were supplied with equal volumes of Hanks’ solution, and, aft er 10…30 min. at 37°С, were incubated with DSM and EPO, as described above. Control and experimental TLC samples were then studied at the luminescent microscope LUMAM – R8 (LOMO, St. Petersburg, Russia) at a 900x magnifi cation, using a temperature-controlled stage for the count chamber. Th e fl uorescence was excited by a mercury lamp (λ= 405-436 nm). To measure light emission, a FMEL-1 photometric device was used, with an interference fi lter with a maximum transmission at 585 nm. Fluorescence intensity was manually measured for single cells localized in the vision fi eld, then transformed to a digital signal. Th e numbers of DSM-stained bright mitochondria, looking as intracellular yellow granules, were counted per each single cell (the nm/c values) [9]. Fift y to seventy cells were studied per sample, and the mean fl uorescence intensity was calculated as conventional units (F̃, arbitrary units). The fluorescent signal did not quench over the measurement time. Photomicrographs of the DSM-stained cells were performed with a TSA 5.0 camera mounted in the LOMO R8 microscope, and analyzed by a Microanalysis View soft ware (from the same manufacturer). Statistical evaluation of the data was performed by the Spearmen range correlation criterion. A total of 8,000 cells have been studied in 160 samples. Each independent experiment included 3 to 5 measure points.

Results

Initial amount of intact thymic lymphoid cells in cell suspensions was 92 to 96%. Several control experiments with have shown a decrease of nm/c and F̃ values for these cells aft er incubation with either inhibitor. Th e degree of such decrease depended on the type of inhibiting substance, and incubation terms (Table 1, Fig. 1 A, B, C). Th e most pronounced and faster eff ect was observed with DNP, i.e., fl uorescent mitochondria became virtually absent in DNP-treated cells as soon as aft er 10 min of exposure. F̃ values were also decreased by 20 min, with both nm/c and F̃ reduction. Fluorescent mitochondria disappeared by 40 min, with F̃ at the background levels. EPO addition did not restore F̃ and nm/c- parameters. Th e dynamics of thymocytes with absent mitochondrial fl uorescence aft er DNP exposure shown in Fig. 1A (NC-EM,%), like as absence of recovery aft er EPO addition. Th is eff ect may be caused by classical protonophore properties of DNP which irreversibly reduces both mentioned components of electrochemical gradient, thus causing the mitochondrial membrane depolarization.

Table 1. Time-dependent changes of mean fluorescence (F, arb. units) and number of fluorescent mitochondria per one cell (nm/c) upon short-term exposure of rat TLC to respiratory chain inhibitors followed by EPO treatment

83-88 Table 1. Time-dependent changes.png

Note: * – P< 0.01; ** – P< 0.025; nm/c – mean number of fl uorescent mitochondria per cell; F̃, arb. units – mean fl uorescence levels for all the cell measured at the given time points. Controls: TLC incubated without inhibitors at 37°С, with EPO supplied aft er 40 min. of incubation. Experimental samples: cells with addition of DNP, or PCP, or DCCD followed by incubation with EPO at 37°С. Mean values (M) and mean error (m) are shown for each time point

Similar, but less pronounced decrease was observed 10 min aft er treatment with PCP, i.e., n m/c dropped by 37%, and F̃, by ~ 35%. Following 20-min incubation, we observed only ~ 26% nm/c and ~ 25% F̃ of control values. At 40 min., no fl uorescent mitochondria are seen.
Meanwhile, Fig. 1B shows increase of non-fl uorescent cell numbers (NC-EM, %) induced by PCP, followed by a recovery induced by EPO supplement. The inhibitory effect of PCP upon thymocytes proved to be partially reversible aft er addition of EPO (nm/c recovery by ~23%, and F̃ values by ~20%).
A 10-min. incubation of thymocytes with DCCD again retains only a part of fl uorescent cells (nm/c, 30%, and ~33% F̃). Only 18% nm/c and ~ 20% F̃ remain aft er 20 min. with DCCD, a mitochondrial membrane-bound ATP-ase. At 40 min. with DCDD, no fl uorescence was observed in the cells, with background F̃ values. However, 30-min. incubation with EPO has resulted into recovery of mitochondria-associated fl uorescence to 42% for nm/c and 38% for F̃ levels, as compared with control samples. Fig. 1C illustrates the dynamics (NC-EM, %) of de-energized thymocytes induced by DCCD followed by restoration of potential-coupled fl uorescence aft er EPO treatment.
Typical patterns of DSM-stained cells before and aft er treatment with mitochondrial ATP-ase inhibitor (DCCD) are shown in Fig. 2 A-C.
83-88 Figure 1. Changing amounts.png
Figure 1. Changing amounts of rat thymic lymphocytes devoid of fluorescent mitochondria (NC-EM, %) from initial
time points (0 min.), following incubation with different inhibitors, and after EPO addition. Incubation at 37°C with
DNP (Fig. 1A); PCP (Fig. 1B); DCCD (Fig. 1C) was followed by uniform exposure to EPO. Abscissa: incubation terms (min);
ordinate, mean values of TLC without fluorescent mitochondria (NC-EM, %) for each time point. Vertical bars show
appropriate confidence intervals for P<0.05 as compared to initial values

83-88 Figure 2. Rat thymocytes stained.png
Figure 2. Rat thymocytes stained with a fluorescent DSM probe. 2A, an original sample after incubation with EPO,
F=52,0 arb. units; 2B, cells after 40 min of incubation with DCCD, F=3,0 arb. units; 2C, thymocytes after exposure of
DCCD-treated cells to EPO, F=20,0 arb. units

Discussion

In our works, we have used inhibitor analysis, in order to assess the mechanisms which regulate anti-apoptotic eff ects of erythropoietin. Th e transmembrane potential of membranes in thymocytes was determined as fl uorescence intensity of DSM probe. Total DSM fl uorescence depends on a summary potentials of plasmatic and mitochondrial membranes. Earlier we have found that EPO acts upon thymic lymphoid cells by changing electric charge of cellular membranes. Th e EPO stimulatory eff ect is accompanied by increased nm/c, due to proton potential (Δφm), and/or external membrane potential (Δφp) [9].
Our data suggest that some metabolic eff ects of EPO are exerted via mitochondrial respiratory pathways. EPO was shown to reverse the de-energizing eff ects of DCCD, thus presuming functional changes of F0F1 membrane ATP which is specifi cally inhibited by the DCCD.

The mitochondrial F0F1-ATP-ase is a complex lipoprotein containing of hydrophilic catalytic center (F1), and a membrane domain (F0) [11]. DCDD used in this work is a specific proton translocation inhibitor in the F0F1 ATP-ase [12], causing decrease in nm/c and general F̃ shown in our experiments, thus refl ecting a critical role of ATP-ase for sustaining the mitochondrial membrane potential. Th e reduced fl uorescence of DSM-induced mitochondria could be considered as mitochondrial de-energization, which proved to be reversible by EPO treatment. Th is fi nding may refl ect ability of EPO to restore functional integrity of mitochondrial membranes as a component of their eff ects upon immune system. The restored mitochondrial functions in the target cells for EPO allow to perform regulatory signaling in lymphoid cells, e.g., via phosphorylation of some transcription factors, e.g., STAT5 in lymphoid cells and tissues [13]. Further studies in other lymphoid cell models could further elucidate the role of EPO as a regulator of mitochondrial function.

Conclusions

1. Thymocytes may represent a non-usual, but suitable model for evaluation of growth factor eff ects upon dividing, apoptosis- prone immune cells.

2. The in vitro testing of modifying EPO eff ects in thymocytes exposed to inhibitors of mitochondrial functions has revealed irreversible deletorious eff ect upon energetic potential of these cells. Erythropoietin (EPO) does not reverse the DNP-induced damage, but partially restores mitochondrial damage induced by DCDD, a specifi c inhibitor of the F0F1 mitochondrial membrane ATP-ase.

3. More extended studies are required, in order to screen positive eff ects of EPO in other non-erythroid cell types.

Conflict of interests

No potential confl icts of interests are reported.

References

1. Jelkmann W., Gross A.J.(Eds.). Erythropoietin, Springer-Verlag Berlin Yeidelberg New York, 1989,180 p.
2. Biggar P, Kim GH. Treatment of renal anemia: Erythropoiesis stimulating agents and beyond. Kidney Res Clin Pract. 2017; 36(3):209-223.
3. Osikov M.V., Simonyan E.V., Saedgalina O.T., Fedosov A.A. Mechanisms of erythropoietin infl uence on the quantitative composition of blood lymphocytes in experimental thermal injury. Modern Problems of Science and Education. 2016 (2). URL: https://elibrary.ru/download/elibrary_25869790_84106576.pdf. (In Russian).
4. Lifshitz L., Avneon M., Prutchi-Sagiv S., Katz O.,- Gassmann M., Mittelman M.,Neuman. Immunomodulatory functions of erythropoietin. Focus uni-luebeck. 8th Luebeck Conference «Pathophysiology and Pharmacology of Erythropoietin and other Hemopoietic Growth Factors».Suppl. 2009, P. 28.
5. Todosenko NM, Shmarov VA, Malashchenko VV, Meniailo ME, Melashchenko OB, Gazatova ND, Goncharov AG, Seledtsov VI. Erythropoietin exerts direct immunomodulatory eff ects on the cytokine production by activated human T-lymphocytes. Int Immunopharmacol. 2016; 36:277-281.
6. Wang S, Zhang C, Li J, Niyazi S, Zheng L, Xu M, Rong R, Yang C, Zhu T. Erythropoietin protects against rhabdomyolysis- induced acute kidney injury by modulating macrophage polarization. Cell Death Dis. 2017; 8(4):e2725.
7. Lisowska KA, Bryl E, Witkowski JM. Erythropoietin receptor is detectable on peripheral blood lymphocytes and its expression increases in activated T lymphocytes. Haematologica. 2011;96(3):e12-3
8. Hisatomi K, Nakao M, Isomura T, Kosuga K, Itoh K. Effect of recombinant erythropoietin on peripheral T lymphocytes. J Th orac Cardiovasc Surg. 1995 109(4):809.
9. Morozova G.I., Parkhomenko T.V., Klitsenko O.A., Tomson V.V. Stimulating eff ect of erythropoietin on thymocyte energetics established in vitro with a potential-sensitive fl uorescent probe in the mitochondria. Biochem. Suppl. Series A: Membr Cell Biology. 2007; 1 (4):325-330.
10. Parkhomenko T.V., Morozova G.I., Klytsenko O.A., Tomson V.V. Quantitative evaluationof erythropoietin (EPO) infl uence on rat T-lymphocytes. Annals of Hematology. 2000; 79 (5): B8.
11. Futai M., Noumi T., Maeda M. ATP – synthase (H+-ATP- ASE) – results by combined biochemical and molecular biological approaches. Annual Review of Biochemistry. 1989; 58:111-136.
12. Clejan L., Bosch C.G., Beattie D.S. Inhibition by dicyclohexylcarbodiimide of proton ejection but not electron-transfer in rat-liver mitochondria. Journal of Biological Chemistry. 1984; 259 (21):13017-13020.
13. Lisowska KA, Dębska-Ślizień A, Jasiulewicz A, Jóźwik A, Rutkowski B, Bryl E, Witkowski JM. Flow cytometric analysis of STAT5 phosphorylation and CD95 expression in CD4+ T lymphocytes treated with recombinant human erythropoietin. J Recept Signal Transduct Res. 2011; 31(3):241-246.

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Introduction

Materials and Methods

Results

Table 1. Time-dependent changes of mean fluorescence (F, arb. units) and number of fluorescent mitochondria per one cell (nm/c) upon short-term exposure of rat TLC to respiratory chain inhibitors followed by EPO treatment

83-88 Table 1. Time-dependent changes.png

Discussion

Conclusions

1. Thymocytes may represent a non-usual, but suitable model for evaluation of growth factor eff ects upon dividing, apoptosis- prone immune cells.

3. More extended studies are required, in order to screen positive eff ects of EPO in other non-erythroid cell types.

Conflict of interests

No potential confl icts of interests are reported.

References

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Одним из основных эффектов ЭПО является снижение частоты апоптоза эритроидных клеток-предшественниц в костном мозге. Эти свойства ЭПО широко применяются при лечении различных заболеваний системы крови, в том числе – после трансплантации стволовых клеток. Ранее было установлено, что ЭПО оказывает активирующее воздействие на Т-лимфоциты (ТЛЦ), сопровождающееся увеличением количества флуоресцирующих митохондрий в клетке (nm/c) и увеличением суммарного трансмембранного потенциала на плазматической (Δφp) и митохондриальных мембранах (Δφm). Однако остается неясным, какой именно мембранный потенциал реагирует на воздействие ЭПО: Δφm, или (и) Δφp. Для ответа на этот вопрос мы использовали специфические ингибиторы окислительного фосфорилирования. Цель настоящего исследования – оценка роли митохондриальных функций в воздействии ЭПО на лимфоциты тимуса. <h2 style="text-align: justify;">Материалы и методы</h2> Исследовалось влияние ЭПО (“Eprex”, Cilag) на флуоресценцию ТЛЦ крыс in vitro после краткосрочной инкубации и воздействия несколькими ингибиторами: динитрофенолом (ДНФ) – ингибитором дыхательной цепи и разобщителем окислительного фосфорилирования; пентахлорфенолом (ПХФ) – разобщителем окислительного фосфорилирования; дициклогексилкарбодиимидом (ДЦКД) – ингибитором мембрансвязанной части АТФ-азы митохондриальной мембраны с помощью зонда DSM [4-(p-диметиламиностирилил)-1-метил пиридиний], определяющего трансмембранный градиент электрического поля. ТЛЦ выделяли из тимусов по стандартной методике. Окрашенные ДСМ клетки исследовали на люминесцентном микроскопе («Люмам – Р 8», ЛОМО, Россия) с использованием термостатированного столика. В каждом препарате измеряли флуоресценцию 50-70 клеток и рассчитывали среднюю интенсивность флуоресценции ТЛЦ (F̃). В каждой флуоресцирующей клетке подсчитывали nm/c. Статистическую обработку данных экспериментов проводили по коэффициенту корреляции рангов Спирмена. <h2 style="text-align: justify;">Результаты и обсуждение</h2> В серии экспериментов с ТЛЦ зарегистрировано снижение nm/c и F̃ после инкубации со всеми использованными ингибиторами, причем степень и скорость снижения этих параметров зависела от типа ингибитора и длительности инкубации. Максимальное снижение энергетики ТЛЦ достигалось при инкубации с ДНФ, после которого ЭПО не восстанавливает F̃ и nm/c. После инкубации с ПХФ ЭПО восстанавливает ~20-23% nm/c и F̃. Реакция ТЛЦ на ДЦКД подтверждает важную роль АТФ-азы в поддержании мембранного митохондриального потенциала. 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И. П. Павлова, Санкт-Петербург, Российская Федерация " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(348) "Лаборатория патоморфологии научно-исследовательского центра, Первый Санкт-Петербургский государственный медицинский университет им. И. П. Павлова, Санкт-Петербург, Российская Федерация
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Эритропоэтин (ЭПО) является физиологическим стимулятором эритропоэза. Одним из основных эффектов ЭПО является снижение частоты апоптоза эритроидных клеток-предшественниц в костном мозге. Эти свойства ЭПО широко применяются при лечении различных заболеваний системы крови, в том числе – после трансплантации стволовых клеток. Ранее было установлено, что ЭПО оказывает активирующее воздействие на Т-лимфоциты (ТЛЦ), сопровождающееся увеличением количества флуоресцирующих митохондрий в клетке (nm/c) и увеличением суммарного трансмембранного потенциала на плазматической (Δφp) и митохондриальных мембранах (Δφm). Однако остается неясным, какой именно мембранный потенциал реагирует на воздействие ЭПО: Δφm, или (и) Δφp. Для ответа на этот вопрос мы использовали специфические ингибиторы окислительного фосфорилирования. Цель настоящего исследования – оценка роли митохондриальных функций в воздействии ЭПО на лимфоциты тимуса.

Материалы и методы

Исследовалось влияние ЭПО (“Eprex”, Cilag) на флуоресценцию ТЛЦ крыс in vitro после краткосрочной инкубации и воздействия несколькими ингибиторами: динитрофенолом (ДНФ) – ингибитором дыхательной цепи и разобщителем окислительного фосфорилирования; пентахлорфенолом (ПХФ) – разобщителем окислительного фосфорилирования; дициклогексилкарбодиимидом (ДЦКД) – ингибитором мембрансвязанной части АТФ-азы митохондриальной мембраны с помощью зонда DSM [4-(p-диметиламиностирилил)-1-метил пиридиний], определяющего трансмембранный градиент электрического поля. ТЛЦ выделяли из тимусов по стандартной методике. Окрашенные ДСМ клетки исследовали на люминесцентном микроскопе («Люмам – Р 8», ЛОМО, Россия) с использованием термостатированного столика. В каждом препарате измеряли флуоресценцию 50-70 клеток и рассчитывали среднюю интенсивность флуоресценции ТЛЦ (F̃). В каждой флуоресцирующей клетке подсчитывали nm/c. Статистическую обработку данных экспериментов проводили по коэффициенту корреляции рангов Спирмена.

Результаты и обсуждение

В серии экспериментов с ТЛЦ зарегистрировано снижение nm/c и F̃ после инкубации со всеми использованными ингибиторами, причем степень и скорость снижения этих параметров зависела от типа ингибитора и длительности инкубации. Максимальное снижение энергетики ТЛЦ достигалось при инкубации с ДНФ, после которого ЭПО не восстанавливает F̃ и nm/c. После инкубации с ПХФ ЭПО восстанавливает ~20-23% nm/c и F̃. Реакция ТЛЦ на ДЦКД подтверждает важную роль АТФ-азы в поддержании мембранного митохондриального потенциала. После деэнергизации ТЛЦ под действием ДЦКД, ЭПО восстанавливает ~42% nm/c и ~38% F̃.

Заключение

ЭПО способен частично восстанавливать поляризацию мембран митохондрий в ТЛЦ, нарушенную в результате воздействия ингибитора АТФ-азы (ДЦКД).

Ключевые слова

Эритропоэтин, Т-лимфоциты, энергетическая активность, ингибиторы, потенциалчувствительный витальный флуоресцентный зонд-катион 4-(п-диметиламиностирил)-1-метилпиридиния (ДСМ).

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One of the main eff ects of EPO is to prevent apoptosis of erythroid progenitor cells in the bone marrow. Th ese properties of EPO are widely used for treatment of various hematopoietic disorders including posttransplant conditions. Previously, it was found that activating EPO-eff ect on T-lymphocytes (TLC) accompanied by an increase in the number of fluorescent mitochondria (n m/c) and an increase in the total transmembrane potential on plasmatic (Δφp) and mitochondrial membranes (Δφm). However, it remains unclear which membrane potential is responsible for the EPO effect. Hence, we used specifi c inhibitors of oxidative phosphorylation in the respiratory chain. The aim of the present work was to assess the role of mitochondrial functions in EPO eff ects upon thymic lymphocytes. Materials and methods We studied EPO (Eprex, Cilag) infl uence on fl uorescence of rat TLC aft er short-term incubation and treatment with some inhibitors: dinitrophenol (DNP-uncoupler of oxidative phosphorylation and inhibitor of respiratory chain), pentachlorphenol (PCP- uncoupler of oxidative phosphorylation), N,N -dicyclohexylcarbodiimide (DCCD- inhibitor of Ca2+- dependent mitochondria ATP-аse). Th e cells were then tested by electrical fi eld gradient sensitive probe DSM [4-(p-dimethylaminostyryl)- 1-methylpyridinium]. Rat TLC were isolated according to the standard method. Th e microfl uorimetric studies of DSM-stained TLC were performed by means of fl uorescent microscope “Lumam R-8”, “LOMO”, Russia) with thermostatic plate. Fift y to 70 single cells were measured per each specimen the mean fl uorescence intensity of TLC was calculated (F̃), as well as nm/c values. Statistical evaluation of the data was performed by the Spearmen range correlation. <h2 style="text-align: justify;">Results</h2> In a series of experiments with TLC, we have registered a decrease in F̃ and nm/c aft er incubation with all used inhibitors. It was found that the diff erence in decrease of nm/c rates and F̃ values depends on the type of inhibitor and on the duration of incubation. Maximal irreversible reduction of the TLC energy potential (F̃ and nm/c) after incubation was seen with DNP being not restored by EPO. Aft er incubation with PCP, EPO restores nm/c and F̃ by ca. 20-23%. Th e reaction of TLC on the DCCD confirms the important role of the ATP-ase for maintenance of mitochondrial membrane potential. After de-energization of TLC by DCCD, EPO has the maximum rescuing effect, i.e. recovery by approx. 42% for nm/c and ~38% for F̃ values. <h2 style="text-align: justify;">Conclusion</h2> EPO is able to partially recover the damage and polarization of the mitochondria membranes in TLC disturbed aft er exposure to specifi c ATP-ase inhibitor (DCCD). This in vitro approach may be used for screening other growth factors. <h2 style="text-align: justify;">Keywords</h2> Erythropoietin, T-lymphocytes, energy activity, inhibitors, electrical field gradient sensitive probe DSM [4-(p-dimethylaminostyryl)-1-methylpyridinium]. " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(3458) "

Erythropoietin (EPO) is a physiological stimulator of erythropoiesis. One of the main eff ects of EPO is to prevent apoptosis of erythroid progenitor cells in the bone marrow. Th ese properties of EPO are widely used for treatment of various hematopoietic disorders including posttransplant conditions. Previously, it was found that activating EPO-eff ect on T-lymphocytes (TLC) accompanied by an increase in the number of fluorescent mitochondria (n m/c) and an increase in the total transmembrane potential on plasmatic (Δφp) and mitochondrial membranes (Δφm). However, it remains unclear which membrane potential is responsible for the EPO effect. Hence, we used specifi c inhibitors of oxidative phosphorylation in the respiratory chain. The aim of the present work was to assess the role of mitochondrial functions in EPO eff ects upon thymic lymphocytes.

Materials and methods

We studied EPO (Eprex, Cilag) infl uence on fl uorescence of rat TLC aft er short-term incubation and treatment with some inhibitors: dinitrophenol (DNP-uncoupler of oxidative phosphorylation and inhibitor of respiratory chain), pentachlorphenol (PCP- uncoupler of oxidative phosphorylation), N,N -dicyclohexylcarbodiimide (DCCD- inhibitor of Ca2+- dependent mitochondria ATP-аse). Th e cells were then tested by electrical fi eld gradient sensitive probe DSM [4-(p-dimethylaminostyryl)- 1-methylpyridinium]. Rat TLC were isolated according to the standard method. Th e microfl uorimetric studies of DSM-stained TLC were performed by means of fl uorescent microscope “Lumam R-8”, “LOMO”, Russia) with thermostatic plate. Fift y to 70 single cells were measured per each specimen the mean fl uorescence intensity of TLC was calculated (F̃), as well as nm/c values. Statistical evaluation of the data was performed by the Spearmen range correlation.

Results

In a series of experiments with TLC, we have registered a decrease in F̃ and nm/c aft er incubation with all used inhibitors. It was found that the diff erence in decrease of nm/c rates and F̃ values depends on the type of inhibitor and on the duration of incubation. Maximal irreversible reduction of the TLC energy potential (F̃ and nm/c) after incubation was seen with DNP being not restored by EPO. Aft er incubation with PCP, EPO restores nm/c and F̃ by ca. 20-23%. Th e reaction of TLC on the DCCD confirms the important role of the ATP-ase for maintenance of mitochondrial membrane potential. After de-energization of TLC by DCCD, EPO has the maximum rescuing effect, i.e. recovery by approx. 42% for nm/c and ~38%
for F̃ values.

Conclusion

EPO is able to partially recover the damage and polarization of the mitochondria membranes in TLC disturbed aft er exposure to specifi c ATP-ase inhibitor (DCCD). This in vitro approach may be used for screening other growth factors.

Keywords

Erythropoietin, T-lymphocytes, energy activity, inhibitors, electrical field gradient sensitive probe DSM [4-(p-dimethylaminostyryl)-1-methylpyridinium].

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Materials and methods We studied EPO (Eprex, Cilag) infl uence on fl uorescence of rat TLC aft er short-term incubation and treatment with some inhibitors: dinitrophenol (DNP-uncoupler of oxidative phosphorylation and inhibitor of respiratory chain), pentachlorphenol (PCP- uncoupler of oxidative phosphorylation), N,N -dicyclohexylcarbodiimide (DCCD- inhibitor of Ca2+- dependent mitochondria ATP-аse). Th e cells were then tested by electrical fi eld gradient sensitive probe DSM [4-(p-dimethylaminostyryl)- 1-methylpyridinium]. Rat TLC were isolated according to the standard method. Th e microfl uorimetric studies of DSM-stained TLC were performed by means of fl uorescent microscope “Lumam R-8”, “LOMO”, Russia) with thermostatic plate. Fift y to 70 single cells were measured per each specimen the mean fl uorescence intensity of TLC was calculated (F̃), as well as nm/c values. 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Materials and methods

Results

Conclusion

Keywords

Erythropoietin, T-lymphocytes, energy activity, inhibitors, electrical field gradient sensitive probe DSM [4-(p-dimethylaminostyryl)-1-methylpyridinium].

Materials and methods

Results

Conclusion

Keywords

Erythropoietin, T-lymphocytes, energy activity, inhibitors, electrical field gradient sensitive probe DSM [4-(p-dimethylaminostyryl)-1-methylpyridinium].

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Пархоменко, Владимир В. Томсон, Олег В. Галибин" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(103) "Татьяна В. Пархоменко, Владимир В. Томсон, Олег В. Галибин" ["TYPE"]=> string(4) "HTML" } ["~DESCRIPTION"]=> string(0) "" ["~NAME"]=> string(12) "Авторы" ["~DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["DISPLAY_VALUE"]=> string(103) "Татьяна В. Пархоменко, Владимир В. Томсон, Олег В. Галибин" } ["SUMMARY_RU"]=> array(37) { ["ID"]=> string(2) "27" ["TIMESTAMP_X"]=> string(19) "2015-09-02 18:01:20" ["IBLOCK_ID"]=> string(1) "2" ["NAME"]=> string(29) "Описание/Резюме" ["ACTIVE"]=> string(1) "Y" ["SORT"]=> string(3) "500" ["CODE"]=> string(10) "SUMMARY_RU" ["DEFAULT_VALUE"]=> array(2) { ["TEXT"]=> string(0) "" ["TYPE"]=> string(4) "HTML" } ["PROPERTY_TYPE"]=> string(1) "S" ["ROW_COUNT"]=> string(1) "1" ["COL_COUNT"]=> string(2) "30" ["LIST_TYPE"]=> string(1) "L" ["MULTIPLE"]=> string(1) "N" ["XML_ID"]=> string(2) "27" ["FILE_TYPE"]=> string(0) "" ["MULTIPLE_CNT"]=> string(1) "5" ["TMP_ID"]=> NULL ["LINK_IBLOCK_ID"]=> string(1) "0" ["WITH_DESCRIPTION"]=> string(1) "N" ["SEARCHABLE"]=> string(1) "N" ["FILTRABLE"]=> string(1) "N" ["IS_REQUIRED"]=> string(1) "N" ["VERSION"]=> string(1) "1" ["USER_TYPE"]=> string(4) "HTML" ["USER_TYPE_SETTINGS"]=> array(1) { ["height"]=> int(200) } ["HINT"]=> string(0) "" ["PROPERTY_VALUE_ID"]=> string(5) "20926" ["VALUE"]=> array(2) { ["TEXT"]=> string(5899) " Эритропоэтин (ЭПО) является физиологическим стимулятором эритропоэза. Одним из основных эффектов ЭПО является снижение частоты апоптоза эритроидных клеток-предшественниц в костном мозге. Эти свойства ЭПО широко применяются при лечении различных заболеваний системы крови, в том числе – после трансплантации стволовых клеток. Ранее было установлено, что ЭПО оказывает активирующее воздействие на Т-лимфоциты (ТЛЦ), сопровождающееся увеличением количества флуоресцирующих митохондрий в клетке (nm/c) и увеличением суммарного трансмембранного потенциала на плазматической (Δφp) и митохондриальных мембранах (Δφm). Однако остается неясным, какой именно мембранный потенциал реагирует на воздействие ЭПО: Δφm, или (и) Δφp. Для ответа на этот вопрос мы использовали специфические ингибиторы окислительного фосфорилирования. Цель настоящего исследования – оценка роли митохондриальных функций в воздействии ЭПО на лимфоциты тимуса. <h2 style="text-align: justify;">Материалы и методы</h2> Исследовалось влияние ЭПО (“Eprex”, Cilag) на флуоресценцию ТЛЦ крыс in vitro после краткосрочной инкубации и воздействия несколькими ингибиторами: динитрофенолом (ДНФ) – ингибитором дыхательной цепи и разобщителем окислительного фосфорилирования; пентахлорфенолом (ПХФ) – разобщителем окислительного фосфорилирования; дициклогексилкарбодиимидом (ДЦКД) – ингибитором мембрансвязанной части АТФ-азы митохондриальной мембраны с помощью зонда DSM [4-(p-диметиламиностирилил)-1-метил пиридиний], определяющего трансмембранный градиент электрического поля. ТЛЦ выделяли из тимусов по стандартной методике. Окрашенные ДСМ клетки исследовали на люминесцентном микроскопе («Люмам – Р 8», ЛОМО, Россия) с использованием термостатированного столика. В каждом препарате измеряли флуоресценцию 50-70 клеток и рассчитывали среднюю интенсивность флуоресценции ТЛЦ (F̃). В каждой флуоресцирующей клетке подсчитывали nm/c. Статистическую обработку данных экспериментов проводили по коэффициенту корреляции рангов Спирмена. <h2 style="text-align: justify;">Результаты и обсуждение</h2> В серии экспериментов с ТЛЦ зарегистрировано снижение nm/c и F̃ после инкубации со всеми использованными ингибиторами, причем степень и скорость снижения этих параметров зависела от типа ингибитора и длительности инкубации. Максимальное снижение энергетики ТЛЦ достигалось при инкубации с ДНФ, после которого ЭПО не восстанавливает F̃ и nm/c. После инкубации с ПХФ ЭПО восстанавливает ~20-23% nm/c и F̃. Реакция ТЛЦ на ДЦКД подтверждает важную роль АТФ-азы в поддержании мембранного митохондриального потенциала. После деэнергизации ТЛЦ под действием ДЦКД, ЭПО восстанавливает ~42% nm/c и ~38% F̃. <h2 style="text-align: justify;">Заключение</h2> ЭПО способен частично восстанавливать поляризацию мембран митохондрий в ТЛЦ, нарушенную в результате воздействия ингибитора АТФ-азы (ДЦКД). <h2 style="text-align: justify;">Ключевые слова</h2> Эритропоэтин, Т-лимфоциты, энергетическая активность, ингибиторы, потенциалчувствительный витальный флуоресцентный зонд-катион 4-(п-диметиламиностирил)-1-метилпиридиния (ДСМ). " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(5701) "

Материалы и методы

Результаты и обсуждение

Заключение

Ключевые слова

Материалы и методы

Результаты и обсуждение

Заключение

Ключевые слова

Experimental studies

Surgical treatment of inflammatory periodontal diseases using chitosan matrices

Sergey B. Ulitovskiy ¹^*, Anna V. Antipova ¹^*, Alexander D. Vilesov ¹^*^*, ², GalinaYu. Yukina ¹^*^*^*, Dmitry N. Suslov ¹^*^*^*^*, ³, Pavel V. Popryadukhin ¹^*^*^*^*, ², Oleg V. Galibin ¹^*^*¹^* Department of Preventive Stomatology
¹^*^* Biotechnology Department, R.Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation
¹^*^*^* Laboratory of Pathomorphology, Th e University Research Center
¹^*^*^*^* Laboratory of Invasive Technologies, Th e University Research Center

Comparative analysis of NGS and Sanger sequencing methods for HLA typing at a Russian university clinic

Oleg S. Glotov ¹,², Olga V. Romanova ¹,², Yuri A. Eismont ¹, Andrey M. Sarana ¹,², Sergey G. Scherbak ¹,², Elena V. Kuzmich ³, Alexander L. Alyanskiy ³, Natalya E. Ivanova ³, Vera V. Teplyashina ³, Yury A. Serov ³, Ludmila S. Zubarovskaya ³, Boris V. Afanasyev ³

In vitro modifying effect of erythropoietin upon thymic lymphocytes: an inhibitor analysis

Tatyana V. Parkhomenko, Vladimir V. Tomson, Oleg V. Galibin

Experimental studies

Surgical treatment of inflammatory periodontal diseases using chitosan matrices

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¹ First Petersburg State I. Pavlov Medical University
² Institute of Macromolecular Compounds, Russian Academy of Sciences
³ Russian Research A.Granov Center of Radiology and Surgical Technologies, St. Petersburg, Russia

Materials and methods

Results

Conclusion

Porous chitosan matrix proved to be biocompatible, bioinert, and bioresorbable material, thus meeting the requirements applicable to the materials suitable for production of the bone matrices.

Keywords

Periodontium, regeneration, membrane, chitosan.

Experimental studies

Comparative analysis of NGS and Sanger sequencing methods for HLA typing at a Russian university clinic

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¹ City Hospital №40, Sestroretsk, St. Petersburg, Russia
² Institute of Translation Biomedicine, St. Petersburg State University, St. Petersburg, Russia
³ Raisa Gorbacheva Memorial Research Institute for Pediatric Oncology, Hematology and Transplantation, The First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russia

Keywords

Major histocompatibility complex, novel HLA alleles, technological solutions, next-generation sequencing, NGS, Sanger sequencing, hematopoietic cells transplantation, Sequence-Based Typing (SBT).

Experimental studies

In vitro modifying effect of erythropoietin upon thymic lymphocytes: an inhibitor analysis

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Tatyana V. Parkhomenko, Vladimir V. Tomson, Oleg V. Galibin

Research Center, Th e First St. Petersburg State I. P. Pavlov Medical University, St. Petersburg, Russian Federation

Materials and methods

Results

Conclusion

Keywords

Erythropoietin, T-lymphocytes, energy activity, inhibitors, electrical field gradient sensitive probe DSM [4-(p-dimethylaminostyryl)-1-methylpyridinium].