E.coli type II L-asparaginase is widely used for treatment of acute lymphoblastic leukemia. However, serious side effects such as allergic or hypersensitivity reactions are common for L-asparaginase treatment. Methods for minimizing immune response on L-asparaginase treatment in human include bioengeneering of less immunogenic version of the enzyme or utilizing the homologous enzymes of different origin. To rationalize these approaches we compared immunogenicity of L-asparaginases from five bacterial organisms and performed sequence-structure analysis of the presumable epitope regions. IgG and IgM immune response in C57B16 mice after immunization with Wollinella succinogenes type II (WsA), Yersinia pseudotuberculosis type II (YpA), Erwinia carotovora type II (EwA), and Rhodospirillum rubrum type I (RrA) and Escherichia coli type II (EcA) L-asparaginases was evaluated using standard ELISA method. The comparative bioinformatics analysis of structure and sequence of the bacterial L-asparaginases presumable epitope regions was performed. We showed different immunogenic properties of five studied L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin as a second-line treatment. Studied L-asparaginases might be placed in the following order based on the immunogenicity level: YpA > RrA, WsA ≥ EwA > EcA. Most significant cross-immunogenicity was shown between EcA and YpA. We propose that a long N-terminus of YpA enzyme enriched with charged aminoacids and tryptophan could be a reason of higher immunogenicity of YpA in comparison with other considered enzymes. Although the recognized structural and sequence differences in putative epitope regions among five considered L-asparaginases does not fully explain experimental observation of the immunogenicity of the enzymes, the performed analysis set the foundation for further research in this direction. The performed studies showed different immunogenic properties of L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin. The preferable enzymes for the second line treatment are WsA, RrA, or EwA.

Резюме. Для исследования влияния микроокружения на функциональную активность В-лимфоцитов использовали модель адоптивного переноса клеток мышей CBA конгенным мышам CBA/N, не имеющим CD5+ В-1 клеток, и модель кокультивирования спленоцитов мышей CBA/N с клетками селезенки или перитонеальной полости мышей СВА. Об активности В-лимфоцитов судили по числу IgM-продуцентов и клеток, образующих антитела к Т-независимому антигену 2-го типа – поливинилпирролидону. За распределением введенных клеток следили, определяя количество клеток, окрашенных витальным красителем CDFA-SE, в селезенке и брюшной полости реципиентов. Внутривенный перенос спленоцитов CBA приводил к значительному (в 3-4 раза) увеличению числа иммуноглобулин-продуцентов в селезенке xid-реципиентов, достигавшему уровня, характерного для мышей СВА. Перенос CBA/N мышам xid-клеток увеличения количества иммуноглобулин-продуцентов не вызывал. Это свидетельствует о том, что увеличение количества IgM-продуцентов в селезенке реципиентов обусловлено клетками донора, предположительно, CD5+ В-1 лимфоцитами. Вместе с тем восстановить у xid-мышей иммунный ответ на поливинилпирролидон не удалось. В брюшной полости интактных мышей (и CBA, и CBA/N) продуценты иммуноглобулина не выявлялись. Внутрибрюшинный перенос СВА спленоцитов к появлению иммуноглобулин-продуцентов в брюшной полости реципиентов также не приводил. Это означает, что микроокружение брюшной полости тормозит активность В-лимфоцитов. В то же время внутривенное введение «молчащих» перитонеальных клеток мышей CBA мышам CBA/N, приводило к значительному возрастанию числа IgM-продуцентов в селезенке реципиента, т.е. в другом микроокружении работа перитонеальных В-клеток «разрешалась». Данные опытов in vivo согласуются с результатами, полученными in vitro. Добавление к спленоцитам CBA/N 10-50% спленоцитов или перитонеальных клеток мышей СВА приводило к резкому повышению числа IgM-продуцентов в культурах. Полученные данные свидетельствуют об определяющей роли микроокружения в функциональной активности В-лимфоцитов мыши.

In contrast to B-splenocytes, murine peritoneal B-cells do not produce or secrete immunoglobulins (Ig). Twenty-four hours after intraperitoneal transfer of splenocytes containing Ig-forming cells (IFC), IFC content in the peritoneal cavity of recipient mice decreases dramatically. This decrease does not depend on the migration of transferred IFC to the spleen (homing); splenectomy has no effect on this process. In order to check whether the lack of IFC in the peritoneal cavity is due to inhibition of synthesis or secretion of Ig, peritoneal cells (contained 4360 IFC/10⁶ cells) of CBA mice were intraperitoneally transferred to CBA/N mice after a 4-day preincubation in vitro. On the next day, ∼30% of in vitro transferred IFC were detected in the peritoneal cavity of recipient mice, but on day 4 the IFC content in peritoneum returned to the background level. Repeated in vitro incubation of peritoneal cells of recipient mice restored the IFC number in cultured cells. It means that peritoneal microenvironment inhibits functional activity of murine B-lymphocytes.

Introduction: Currently, the need to expand knowledge about the mechanisms of impaired immune tolerance in autoimmune adrenal insufficiency (AAI) remains relevant. It is not excluded that in AAI there are disturbances in the system of regulatory B-lymphocytes (Breg).Objective: To assess the state of the B-regulatory cells system of immunity in AAI.Material and methods: the content of Breg was evaluated in patients with AAI, including isolated AAI and AAI as part of autoimmune polyglandular syndrome type 2 (APS-2) and as part of APS-1, in patients with primary adrenal insufficiency (1-AI) of non-autoimmune genesis, conditionally healthy individuals without AI and autoimmune diseases.Results: A decrease in the content of Breg in vivo was revealed in patients with isolated AAI and AAI in the composition of APS-2 compared with conditionally healthy participants. The content of Breg in vivo in patients with 1-AI of non-autoimmune genesis did not statistically differ from the indicators of conditionally healthy participants.Conclusion: for the first time in the world, a tendency to a decrease in the content of Breg in AAI, caused by a disturbance of peripheral immune tolerance, was found. Thus, these cells can be considered as promising markers for the prognosis, early diagnosis, and differential diagnosis of AAI.

Medical article медицинская статья

Aim: search for new genetic predictors of autoimmune adrenal insufficiency (AAI). Materials and methods: In n=54 patients with AAI (isolated and as part of type 2 autoimmune polyglandular syndrome (APS-2; group 1)) and n=32 healthy individuals (group 2) we analyzed polymorphisms in IL28B (rs12979860, rs8099917), TLR9 (rs5743836, rs352140), TLR2 (rs5743708). Results: In group 1, compared with group 2, a predominance of CT genotype of rs12979860 polymorphism of IL28B (p=0.024), and T allele of rs5743836 polymorphism of TLR9 (p=0.044) was revealed. The allele C of rs5743836 polymorphism of TLR9 (p=0.044) was more common in group 2 than in group 1. With respect to other genotypes, alleles and haplotypes, no significant differences (or differences at the level of statistical trend) were found between groups 1 and 2. Conclusion: Thus, it is possible that the CT genotype according to the polymorphic locus rs12979860 of the IL28B gene and the allele T of the rs5743836 polymorphism of the TLR9 gene are prognostic markers that increase the likelihood of developing AAI due to violation the peripheral immune tolerance (IT), whereas the allele C of the rs5743836 polymorphism of the TLR9 gene performs a protective role in this disease in the Russian population.