Experimental Antiphospholipid Syndrome Research Articles

Human umbilical cord-derived mesenchymal stem cells and nitric oxide modulators attenuate the proinflammatory cytokine pattern in experimental antiphospholipid syndrome in mice

Antiphospholipid syndrome is an autoimmune disease characterized by vascular thrombosis and/or obstetric pathology and the presence of antibodies against membrane phospholipids or certain phospholipid-related proteins. Objective. The aim of the research is to study the effect of mesenchymal stem cells, nitric oxide modulators (L-arginine and aminoguanidine) on the level of cytokines in bronchoalveolar lavage in experimental antiphospholipid syndrome in mice. Material and methods. Antiphospholipid syndrome was modeled on female BALB/c mice by intramuscular injections of cardiolipin 1.2 mg/kg 4 times with a 14-day interval. Cryopreserved human umbilical cord-derived mesenchymal stem cells (5×106 cells/kg) were injected once intraperitoneally, L-arginine (25 mg/kg) and aminoguanidine (10 mg/kg) were administered intraperitoneally 1 time per day during 10 days after APS had developed. The cytokines concentration in bronchoalveolar lavage from the lungs was assessed by means of ELISA in 10 days after APS development. Results. In the bronchoalveolar lavage of the BALB/c mice with experimental APS, an increased level of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and decreased level of anti-inflammatory IL-4 and IL-10 were found. It was established that in cases of APS and administration of stem cells the concentration of proinflammatory cytokines decreased: IL-1β by 32.4 %, IL-6 by 30.6 % and TNF-α by 36.1 %, respectively, compare to the APS animals. At the same time the level of IL-4 increased by 50.5 % and IL-10 – by 57.5 % in the group of animals administered with stem cells compare to those with APS. Conclusion. In cases of correction of modeled antiphospholipid syndrome in mice using mesenchymal stem cells and combined application of mesenchymal stem cells and nitric oxide modulators (L-arginine and aminoguanidine), a decrease in the level of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α ) and an increase in the level of anti-inflammatory cytokines (IL-4 and IL-10) in bronchoalveolar lavage has been established.

Impact of modulators of nitric oxide synthesis on the level of cytokines in bronchoalveolar lavage in experimental antiphospholipid syndrome.

Aim: The aim of the research is to study the cytokine prof i le (IL-1β, IL 6, TNF-α, IL-4, IL-10) in bronchoalveolar lavage of lungs in experimental APS and its correction with L-arginine and aminoguanidine. Materials and Methods: Antiphospholipid syndrome was modeled on white female BALB/c mice. L-arginine (25 mg/kg) and aminoguanidine (10 mg/kg) were used for its correction. The concentration of cytokines in bronchoalveolar lavage from the lungs was assessed using the ELISA test. Results: It was established that in cases of APS the concentration of proinf l ammatory cytokines IL-1β, IL-6 and TNF-a increased in 1.9, 2.3 and 6.6 times, respectively, compare to the control. At the same time a decrease of the IL-4 in 1.7 and IL-10 in 1.8 times was found in the APS group compare to the control. L-arginine reduced the level of proinf l ammatory cytokines IL-1β by 22%, IL-6 - by 36%, and TNF-α - by 23% compare to the animals with APS. At the same time, the level of anti-inf l ammatory cytokines increased: IL-4 - by 46%, IL-10 - by 57% compare to the APS animal group. Aminoguanidine, a selective iNOS inhibitor, did not cause any signif i cant decrease in pro-inf l ammatory cytokines but the level of anti-inf l ammatory cytokines IL-4 increased by 44% and IL-10 - by 49%. Conclusions: The precursor of the NO synthesis L-arginine leads to a decrease in the concentrations of IL-1β, IL-6, TNF-a and an increase of IL-4 and IL-10 compare to the group of BALB/c mice with APS.

Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development

AbstractAntiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. Although autoimmune aPL cross react with various coagulation regulatory proteins, lipid-reactive aPL, including those derived from patients with COVID-19, recognize the endolysosomal phospholipid lysobisphosphatidic acid presented by the cell surface–expressed endothelial protein C receptor. This specific recognition leads to complement-mediated activation of tissue factor (TF)–dependent proinflammatory signaling and thrombosis. Here, we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of aPL derived from patients with COVID-19 in mice and the aPL–induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and interferon-α secretion from dendritic cells. Latent infection with murine cytomegalovirus causes TF cytoplasmic domain–dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short-term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic-cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells, and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

Impact of nitric oxide synthesis modulators on the state of humoral immune system in experimental antiphospholipid syndrome

Background: Antiphospholipid syndrome is an autoimmune disease of multiple venous and/or arterial thrombosis and/or pregnancy loss. Oxidative stress only enhances the body’s immune response. In pathological conditions, the formation of nitric oxide is disrupted, which can be manifested by vasoconstriction, increased coagulation, and endothelial dysfunction. Objective: The aim of the research was to study the level of immunoglobulins and circulating immune complexes (CICs) in experimental antiphospholipid syndrome and its correction with L-arginine and aminoguanidine. Materials and methods: Antiphospholipid syndrome was modeled on white female BALB/c mice. L-arginine (25 mg/kg) and aminoguanidine (10 mg/kg) were used for its correction. The content of immunoglobulins and CICs was studied. Results: It was established that the level of immunoglobulins (Ig) and circulating immune complexes increased in the group of animals with antiphospholipid syndrome compare to the control. The levels of IgA and CICs decreased significantly, and the levels of IgM and IgG did not change in the mice with antiphospholipid syndrome and L-arginine correction. In cases of aminoguanidine administration, decreased IgM and IgG levels and no significant decrease in IgA and CICs was evidenced compare to the animals with antiphospholipid syndrome. In cases of using a combination of L-arginine and aminoguanidine agents, only IgM did not change, all other parameters decreased compare to the animals with APS. Conclusion: The parameters of the humoral immunity in female mice with experimental antiphospholipid syndrome increase. The level of immunoglobulins and circulating immune complexes decrease depending on the chosen correction agents or their complex administration. Thus, L-arginine and aminoguanidine has a positive effect on various immunity responses by decreasing the negative impact of pathobiochemical alterations.

Study of immunological activity and hemostasiological features in rats with experimental antiphospholipid syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, which is characterized by thrombotic or obstetric events characterized by the presence in the blood of patients of a high titer of antibodies to natively charged membrane phospholipids, as well as to glycoproteins associated with them. The role of etiological factors, triggering mechanisms, especially at the initial stages of APS formation, is not clearly defined that’s why the aim of our work was to study of immunological activity and hemostasiological features in rats with experimental antiphospholipid syndrome. In animals with simulated APS, a positive microprecipitation reaction with cardiolipin antigen was observed, which indicated the development of APS. The development of hypercoagulation due to both the vascular and platelet and coagulation links of the hemostasis system was also noted. Established changes in hemocoagulation indicators in APS occur due to the interaction of blood clotting factors with APA.

Вплив аміногуанідину на вміст гліального фібрилярного кислого протеїну у мозочку при експериментальному антифосфоліпідному синдромі на фоні вагітності

Вплив аміногуанідину на вміст гліального фібрилярного кислого протеїну у мозочку при експериментальному антифосфоліпідному синдромі на фоні вагітності

Contents of Myelin Basic Protein and Autoantibodies against Brain Proteins in the Experimental Antiphospholipid Syndrome

We examined the content of autoantibodies against brain proteins, content of myelin basic protein (MBP), and level of NO synthesis in the cerebellum and cerebral hemispheres on day 18 of pregnancy in BALB/c mice with the experimental antiphospholipid syndrome (APS); the effects of L-arginine on the above indices were also evaluated. As was found, the contents of autoantibodies against brain proteins having the molecular masses 120, 150, and > 170 kDa were greater than in the control. Under APS conditions, the amount of eNOS-produced NO was relatively insufficient; this was observed against the background of total hyperproduction of NO synthesized by iNOS in blood serum. In APS mice, the contents of stable NO metabolites, NO2– and NO3–, in the cerebellum were higher, while these levels in the cerebral hemispheres were lower with respect to the control. There were reasons to believe that the effects of L-arginine under APS conditions of and cerebral dysfunction are provided not only at the expense of influences upon the NO system, but also via antioxidant and cytoprotective properties of this amino acid. In pregnant APS mice, the content of MBP (95–110 kDa) in the cerebellum and that of MBP (18.4 kDa) in the cerebral hemispheres were greater than in the control. In APS animals, administration of L-arginine provided increase in the content of MBP (18.4 kDa) in the cerebral hemispheres compared with the respective index with no treatment. Our results show that the remyelination processes in animals with the APS are activated; this may be interpreted as a compensatory response to the injury.

Role of peptidyl arginine deiminase 4 (PAD4) in anti-β2GP1/β2GP1 complex-mediated neutrophil extracellular trap formation

Objective To investigate the role of peptidyl arginine deiminase 4 (PAD4) in anti-β2GP1/β2GP1 complex-induced formation of neutrophil extracellular trapping networks (NETs). Methods Peripheral blood neutrophils were isolated from healthy humans by density gradient centrifugation. PAD4 expression was detected by Western blot after the neutrophils were incubated with anti-β2GP1/β2GP1 complex (100 μg/ml) for a certain period of time. PAD4 inhibitor Cl-amidine (10 μmol/L) was used to pretreat neutrophils. Changes in the expression of citrullinated histone 3 (CitH3) at protein level and the relative content of myeloperoxidase (MPO)-DNA were detected by Western blot and ELISA, respectively. A mouse thrombus model of antiphospholipid syndrome (APS) was established by inferior vena cava stenosis. Intervention experiments were performed by intraperitoneal injection of Cl-amidine (50 mg/kg). The expression of CitH3 at protein level in plasma was detected by Western blot. The concentration of circulating free DNA (cf-DNA) in plasma was measured with fluorescent staining. Thrombus in inferior vena cava was collected and weighted to evaluate whether inhibiting the activity of PAD4 would suppress the APS-IgG-induced formation of NETs and thrombosis. Differences among groups were analyzed by t test or one-way analysis of variance (ANOVA). Results The expression of PAD4 induced by anti-β2GP1/β2GP1 complex was significantly down-regulated in the cytoplasm, but increased in the nucleus [(3.67±0.32) vs (1.47±0.19), t=10.22, P<0.05; (0.57±0.19) vs (2.97±0.31), t=11.49, P<0.05]. Cl-amidine significantly inhibited the anti-β2GP1/β2GP1 complex-induced expression of CitH3 protein by neutrophils [(2.46±0.47) vs (0.46±0.13), t=12.24, P<0.01], and reduced the MPO-DNA content in the culture supernatants [(4.09±0.94) vs (2.80±0.57), t=4.23, P<0.05]. In vivo, Cl-amidine significantly inhibited the expression of CitH3 protein [(3.97±0.56) vs (1.09±0.45), t=11.83, P<0.01] and decreased the content of cf-DNA [(2 685.0±735.8) vs (1 784.0±577.0), t=3.93, P<0.05] in plasma of APS mice. Compared with the experimental APS mice in the control group, the weight of thrombus in the APS mice pretreated with Cl-amidine was significantly reduced [(8.22±3.06) vs (4.89±1.90), t=2.27, P<0.05]. Conclusions PAD4 was involved in the formation of NETs induced by anti-β2GP1/β2GP1 complex, which might play an important role in APS thrombosis. Key words: Antiphospholipid syndrome; Anti-β2GP1/β2GP1 complex; Neutrophil extracellular traps; Peptidyl arginine deiminase 4

IMPACT OF NITRIC OXIDE SYNTHESIS MODULATORS ON THE CYTOKINES PROFILE IN EXPERIMENTAL ANTIPHOSPHOLIPID SYNDROME

Background. Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of specific antibodies. Objective. The aim of the study was to investigate the effect of combined use of L-arginine and aminoguanidine on cytokine profile (IL-1β, IL-6, TNF-α, IL-4, IL-10) in experimental APS. Methods. The study was performed on BALB/c female mice. L-arginine (25 mg/kg) and aminoguanidine (10 mg/kg) were used for correction. Serum cytokines concentrations were assessed using an ELISA test. Results. It was found that in APS the concentration of proinflammatory cytokines IL-1β, IL-6 and TNF-a increases in 3.2, 2.3 and 4.5 times respectively, compare to the control. At the same time a decrease of the IL-4 and IL-10 in 1.9 and 2.2 times was evidenced. Aminoguanidine, a selective iNOS inhibitor, caused a significant decrease of TNF-α by 57% (p&lt;0.001), but there were no changes in IL-1β, IL-6, IL-4 and IL-10 compare to the APS-group. L-arginine combined with aminoguanidine caused a significant decrease in the concentration of IL-1β by 30% (p&lt;0.01), IL-6 – by 16% (p&lt;0.05), TNF-a – by 59% (p&lt;0.001) compare to the control. At the same time, the concentration of IL-4 increased by 35% (p &lt;0.01), IL-10 – by 25% (p&lt;0.005). Conclusions. Combined use of the precursor of the NO synthesis L-arginine and aminoguanidine, a selective iNOS inhibitor, leads to a decrease in the concentrations of IL-1β, IL-6, TNF-a and an increase of IL-4 and IL-10 compare to the group of the BALB/c mice with APS and the group of animals administered with aminoguanidine.

Indexes of nitric oxide system in experimental antiphospholipid syndrome

Indexes of nitric oxide system in experimental antiphospholipid syndrome

Impact of Nitric Oxide Synthesis Modulators on the Mechanisms of Apoptosis Development and Production of Reactive Oxygen Species in the Blood Leukocytes in Experimental Antiphospholipid Syndrome

Impact of Nitric Oxide Synthesis Modulators on the Mechanisms of Apoptosis Development and Production of Reactive Oxygen Species in the Blood Leukocytes in Experimental Antiphospholipid Syndrome

Content of GFAP in the Brain of BALB/C Mice with the Antiphospholipid Syndrome: Effects of L-Arginine and Aminoguanidine

We investigated the influence of the nitric oxide precursor L-arginine and selective inhibitor of inducible NOS aminoguanidine on the content of glial fibrillary acidic protein (GFAP) in the cerebellum and cerebral hemispheres of BALB/c mice with the experimental antiphospholipid syndrome (APS). In the APS, the total amount of GFAP and amount of its component GFAP 49–37 kDa in the cerebellum were 280 and 640%, as compared with the control, while the respective figures in the cerebral cortex were 118% and 155%. Injections of L-arginine led to a further increase in the GFAP content in the cerebellum of animals with the APS. Administration of aminoguanidine alone and of aminoguanidine together with L-arginine provided decreases in the GFAP content in the cerebellum of APS mice. Aminoguanidine also reduced the GFAP content in the cerebral hemispheres in APS animals. The increased GFAP content in the cerebellum and cerebral hemispheres in mice with the APS is indicative of the development of reactive astrogliosis. Changes in the NO synthesis due to introduction of its modulators significantly affect the GFAP content in the above brain regions in animals with the APS. These facts indirectly confirm the role of NO in the regulation of the GFAP expression in astrocytes under conditions of this pathology. The presence of GFAP 49–37 kDa (mainly in the cerebellum) in BALB/c mice with APS appears to be a sign of proteolytic degradation of GFAP, suggesting alterations in the astrocyte cytoskeleton (intermediate filaments) in the respective brain regions.

ВПЛИВ L-АРГІНІНУ НА РІВЕНЬ СИНТЕЗУ ОКСИДУ АЗОТУ ТА ВМІСТ ГЛІАЛЬНОГО ФІБРИЛЯРНОГО КИСЛОГО ПРОТЕЇНУ У ГОЛОВНОМУ МОЗКУ ПРИ ЕКСПЕРИМЕНТАЛЬНОМУ АНТИФОСФОЛІПІДНОМУ СИНДРОМІ

The study aims to investigate the infuence of L-arginine on the content of nitrite anions (NO2¯) and nitrate anions (NO3¯) and the content of glial fibrillary acidic protein (GFAP) in the cerebellum and cerebral hemispheres of BALB/c mice with antiphospholipid syndrome.&#x0D; The studies were performed on 30 female BALB/c mice. The experimental animals were divided into 3 groups: 1 – control (intact) animals; 2 – animals with experimental antiphospholipid syndrome (APS), 3 – animals with APS, which were injected with L-arginine at a dose of 25 mg/kg, intraperitoneally once a day, for 10 consecutive days after the development of APS.&#x0D; The increase in glial fibrillary acidic protein and stable metabolites of nitric oxide NO2¯ and NO3¯ in the cerebellum and cerebral hemispheres, relative to the control, was observed in APS-developed BALB/c mice. In case of injection of the precursor of NO synthesis, L-arginine, animals with APS were found to have a further significant increase in the content of NO2¯ and NO3¯ in the cerebellum and the cerebral hemispheres. The introduction of L-arginine did not cause significant changes in GFAP (total) in cerebral hemispheres. However, GFAP content (49-37 kDa) was decreasing. The cerebellum showed an increase in GFAP (total) and GFAP (49-37 kDa) content, compared to the performance of animals with APS.&#x0D; Therefore, the increase in the content of GFAP in the cerebellum and the cerebral hemispheres of BALB/c mice under APS indicates the development of reactive astrogliosis. The introduction of the precursor of NO synthesis, L-arginine, is accompanied by an increase in the content of stable metabolites of nitric oxide (NO2¯, NO3¯) and GFAP in the cerebellum of BALB/c mice, which can indirectly confirm the role of NO in regulating of GFAP expression in astrocytes under APS.

Mitigating placental injuries through up-regulating DAF in experimental APS mice: new mechanism of progesterone.

Anti-phospholipid syndrome (APS) is characterized by recurrent pathological pregnancy, arterial or venous thrombosis in the presence of anti-phospholipid antibody (aPL). Complement activation is recognized as an intermediate link leading to placental thrombosis and placental inflammation in APS model mice. Decay accelerating factor (DAF, CD55), MAC-inhibitory protein (MAC-IP, CD59) and membrane co-factor protein (MCP, CD46) are important complement inhibitory proteins (CIPs) highly expressed in normal placenta to curb excessive complement activation and its mediated injuries. Anti-β2 glycoprotein I (anti-β2GPI) antibody is an important aPL. We found that placental DAF and CD46 decreased in β2GPI passively immunized APS model mice, accompanied by C3 deposition, neutrophil infiltration and increased proinflammatory cytokine levels detected in its placenta. Progesterone supplement can up-regulate DAF but not CD46 expression, curb C3 activation and decrease proinflammatory cytokines levels to reduce fetal loss frequency. Progesterone receptor antagonist (mifepristone) or knock-down DAF with specific siRNA, above the protective effects of progesterone, were significantly weakened. Another sex hormone, oestrogen, has no significant effect on placental DAF and C3 contents and fetal loss frequency in the APS mice model. This may be an important mechanism by which progesterone induces maternal-fetal immune tolerance. At the same time, it may provide evidence for the use of progesterone in APS abortion patients.

Сочетанная тромбофилия по данным гематологического консультирования

Введение. Диагностическая значимость гомозиготной мутации генов факторов свертывания крови FV Leiden и FII, а также их гетерозиготного полиморфизма в сочетании с маркерами первичного антифосфолипидного синдрома как предикторов тромбоза остается неоднозначной. Цель исследования: представить клинические случаи сочетанных тромбофилий, выявленных в процессе амбулаторного гематологического консультирования. Материалы и методы. В базе данных приема гематолога с января 2012 г. по апрель 2017 г. выявлен 891 пациент (837 женщин и 54 мужчины), которым было проведено обследование и консультирование на предмет тромбофилии и/или наличия активных факторов тромбогенного риска. Результаты. Сочетанная тромбофилия выявлена у 7 (0,8%) из 891 обследованного или у 7 (2,8%) из 246 пациентов с факторами тромбогенного риска. В 4 случаях у 2 мужчин (44 и 28 лет) и у 2 женщин (29 и 33 лет) диагноз установлен в связи с тяжелой и/или рецидивирующей венозной и артериальной тромбоэмболией и у 3 женщин (27, 36 и 39 лет) в связи с синдромом потери плода. Заключение. Анализ наблюдений подтверждает целесообразность поиска сочетанной тромбофилии в случае рецидивирующего тромбоза для выбора оптимальной антикоагулянтной терапии. Introduction. The diagnostic signifi cance of homozygous mutation of FV Leiden and FII clotting factor genes, as well as their heterozygous polymorphism in combination with the markers of the primary antiphospholipid syndrome as predictors of thrombosis remains ambiguous. Aim: to present clinical cases of combined thrombophilia revealed during outpatient hematology counseling. Materials and methods. In hematologist database from January 2012 to April 2017 we found 891 patients (837 women and 54 men) who were examined and consulted for thrombophilia and/or presence of active factors of thrombogenic risk. Results. Combined thrombophilia was identifi ed in 7 (0.8%) of 891 patients or in 7 (2.8%) of 246 patients with thrombogenic risk factors. In 4 cases — 2 men (44 and 28 years) and 2 women (29 and 33 years old) had a diagnosis due to severe and/or recurrent venous and arterial thromboembolism and 3 women (27, 36 and 39 years) had a diagnosis due to fetal loss syndrome. Conclusion. The observations confi rm the practicability of researching for combined thrombophilia in case of recurrent thrombosis for the choice of optimal anticoagulant therapy.

Oral administration of Domain-I of beta-2glycoprotein-I induces immunological tolerance in experimental murine antiphospholipid syndrome

It is well established that the humoral immunity in antiphospholipid syndrome (APS) is presented by circulating pathogenic anti-β2GPI autoantibodies targeting mainly domain I of the β2GPI protein, playing a major role in the disease pathogenesis. Previously, we have demonstrated that treatment of experimental APS mice with tolerogenic dendritic cells loaded with domain-I was more efficient in tolerance induction than with the whole molecule or domain-V. In the current study we had orally administered a domain-I derivative of the β2GPI molecule, as a new therapeutic approach to induce oral tolerance in this mouse model of APS. BALB/c mice immunized with β2GPI, were fed with either domain-I, domain-V derivative or the complete β2GPI protein. β2GPI immunized mice developed experimental APS which were fed with domain-I significantly had decreased fetal loss (p < 0.004), a lower size of thrombi (p < 0.001) and lower circulating anti-β2GPI Abs in comparison to mice fed with domain-V or PBS (p < 0.002). Likewise, Domain-I fed mice had a lowered inflammatory response, exhibited by decreased expression of inflammatory cytokines (IFNγ, IL-6, IL-17) and elevated production of IL-10 anti-inflammatory cytokine by splenocytes. Moreover, the anti-inflammatory response in the domain-I fed APS mice was associated with increased circulating miRNA variations (155, 146, 182, 98) by RT-PCR, which are associated with immunomodulation of the immune network.We propose that oral tolerance with domain-I can be a novel therapy for patients with APS.

Decreased hippocampal cell proliferation in mice with experimental antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, which may trigger vascular thrombosis with consecutive infarcts. However, cognitive dysfunctions representing one of the most commonest neuropsychiatric symptoms are frequently present despite the absence of any ischemic brain lesions. Data on the structural and functional basis of the neuropsychiatric symptoms are sparse. To examine the effect of APS on hippocampal neurogenesis and on white matter, we induced experimental APS (eAPS) in adult female Balb/C mice by immunization with β2-glycoprotein 1. To investigate cell proliferation in the dentate gyrus granular cell layer (DG GCL), eAPS and control mice (n = 5, each) were injected with 5-bromo-2'-deoxyuridine (BrdU) once a day for 10 subsequent days. Sixteen weeks after immunization, eAPS resulted in a significant reduction of BrdU-positive cells in the DG GCL compared to control animals. However, double staining with doublecortin and NeuN revealed a largely preserved neurogenesis. Ultrastructural analysis of corpus callosum (CC) axons in eAPS (n = 6) and control mice (n = 7) revealed no significant changes in CC axon diameter or g-ratio. In conclusion, decreased cellular proliferation in the hippocampus of eAPS mice indicates a limited regenerative potential and may represent one neuropathological substrate of cognitive changes in APS while evidence for alterations of white matter integrity is lacking.

Показники прооксидантно-антиоксидантної системи печінки при експериментальному антифосфоліпідному синдромі та застосуванні L-аргініну

Introduction. Antiphospholipid syndrome (APS) that is developed due to underlying autoimmune mechanism often causes miscarriage, vessels thrombosis of different localization, cerebrovascular accident, cardiovascular and other diseases that are accompanied by significant activation of free radical processes. Pathogenesis of liver damage in cases of APS has not been adequately studied.The aim of the study – to establish the changes in parameters of prooxidative-antioxidant system and tissue respiration in liver in cases of experimental antiphospholipid syndrome and L-arginine administration.Research Methods. Mice of BALB/c line were used in the research. The APS was modeled using cardiolipin emulsified in 75 complete Freund's adjuvant; subsequent injections were administered with incomplete Freund's adjuvant. Cardiolipin was injected intramuscularly four times at a dose of 30 µg per 1 injection with 14-day intervals between the injections. For correction, L-arginine hydrochloride (25 mg/kg) was administered once daily during 10 days after APS development.The activity and content of antioxidant system components (superoxide dismutase, catalase, reduced glutathione) were investigated in liver tissue. The level of lipid peroxidation products was determined by the content of lipid hydroperoxides and TBA-active products. The state of energy-supplying processes of mitochondria was studied due to the activity of succinate dehydrogenase and cytochrome oxidase. The concentration of proteins was determined by Lowry method.Results and Discussion. In cases of APS in liver the imbalance in functioning of prooxidative antioxidant system (increased activity of superoxide dismutase and catalase, decrease in the content of reduced glutathione, increase in the level of lipids hydroperoxides, TBA-active products), of mitochondrial electron transport chain (decrease in the activity of succinate dehydrogenase, cytochrome oxidase) in cases of experimental antiphospholipid syndrome was evidenced. In cases of experimental APS in presence of L-arginine administration, the re-establishment of activity and content of antioxidant system components, enzymes of mitochondrial electron transport chain, decrease in content of lipid hydroperoxides and TBA-active products in liver was proved.Conclusion. In cases of experimental antiphospholipid syndrome, L-arginine, in the event of its repeated administration, reduces manifestations of oxidative stress and reactivate enzymes of mitochondrial electron transport chain in liver tissue of BALB/c mice.

Cross-reactivity between annexin A2 and Beta-2-glycoprotein I in animal models of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) affects coagulation and the brain by autoimmune mechanisms. The major antigen in APS is beta-2-glycoprotein I (β2-GPI) is known to complex with annexin A2 (ANXA2), and antibodies to ANXA2 have been described in APS. We measured these antibodies in mice with experimental APS (eAPS) induced by immunization with β2-GPI. Sera of these mice reacted significantly with recombinant ANXA2 by enzyme-linked immunosorbent assay (ELISA) and the eAPS mice had significantly high levels of immunoglobulin G (IgG) in the brain by immunoblot assays compared to adjuvant immunized controls. Immunoprecipitation performed by mixing eAPS brain tissue with protein-G beads resulted in identification of two autoantigens unique to the eAPS group, one of which was ANXA2. In order to study more directly and methodically the specific role of anti-ANXA2 antibodies in APS, we immunized mice with β2-GPI which contained no ANXA2 or with ANXA2 and measured antibodies to these proteins. Levels of antibodies to ANXA2 measured by ELISA were 0.72±0.007 arbitrary units (a.u), 0.24±0.03 and 0.02±0.01a.u for sera from ANXA2, β2-GPI and control mice, respectively (p<0.0001 and p=0.037 for the comparison of the ANXA2 and β2-GPI groups to the controls). Purified IgG from β2-GPI sera did not show cross-binding with ANXA2. Antibodies to β2-GPI and phospholipids were found in the β2-GPI immunized group only. The present study suggests an immune response to the β2-GPI-ANXA2 complex in eAPS and provides a novel ANXA2 immunization model which will serve to study the role of ANXA2 antibodies in of APS.

Research of hemostasis in experimental antiphospholipid syndrome

&lt;p&gt;This study investigated changes of indicators in blood coagulation system and platelet levels in experimental&lt;br /&gt;antiphospholipid syndrome (APS), which was modeled in female mice of the line BALB/c. The results show that the&lt;br /&gt;study group of the animals with APS reduced number of platelets, reduced prothrombin time, increase of prothrombin&lt;br /&gt;index and decrease of international normalized ratio.&lt;/p&gt;