Вирусный маркетинг в туристской индустрии: проблемы и перспективы

Материалы XII Междунар. науч.-техн. конф. (науч. чтения, посвящ. П. О. Сухому), Гомель, 22–23 нояб. 2018 г

Plasminogen in periodontitis and wound repair

The plasminogen activator (PA) system plays a critical role in many physiological and pathological processes, such as fibrinolysis, extracellular matrix (ECM) degradation, wound healing, inflammation, and cancer. The key component of the PA system is plasmin, a broad-spectrum serine protease that is derived from its inactive form, plasminogen. The first aim of this thesis research was to determine the role of plasminogen in periodontitis, an inflammatory oral disease. The second aim was to explore the molecular mechanism by which plasminogen contributes to wound healing in the skin. Finally, the third aim was to investigate the possibility of using plasminogen as a treatment for skin wounds, especially for chronic wounds, such as diabetic wounds. Periodontitis is an oral disease that involves a bacterial infection, the inflammation of the periodontium, and the degradation of gum tissue and alveolar bone. This disease is irreversible and, in severe cases, can lead to loss of teeth due to the degradation of the periodontal ligament and alveolar bone. To study the effects of the PA system on oral health, we monitored the development of periodontitis in plasminogen-deficient mice and plasminogen activator-deficient mice. In control wild-type mice, periodontitis did not occur. However, in plasminogen-deficient mice, periodontitis developed rapidly within 20 weeks after birth. The morphological studies of plasminogen-deficient mice showed the detachment of gingival tissues, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. Immunohistochemical staining showed the massive infiltration of neutrophils into the periodontal tissues. Interestingly, doubly deficient mice lacking both tissue-type plasminogen activator (tPA) and urokinase-type PA (uPA) developed periodontitis at a similar rate as the plasminogen-deficient mice, but mice lacking only tPA or uPA remained healthy. The intravenous injection of human plasminogen for 10 days into plasminogen-deficient mice led to the absorption of necrotic tissue, the diminution of inflammation, and the full regeneration of gum tissues. Notably, there was also partial re-growth of degraded alveolar bone. The wound healing process consists of three overlapping phases: inflammatory, proliferative, and remodeling. It has been postulated that the PA system plays an integral role in this process, and a lack of plasminogen leads to delayed wound healing in mice. To study the role of the PA system in wound healing, we monitored the responses of wild-type, plasminogen-deficient and diabetic mice to incision and burn wounds. We found that in addition to being delayed, the wound healing process in plasminogen-deficient mice was only superficial in nature. The plasminogen-deficient mice were unable to clear the provisional matrix after the formation of granulation tissue, and an extensive fibrin deposition. In addition, persistent inflammation was still present subcutaneously in these mice 60 days after introduction of the wound. The essential role of plasminogen in burn and incision wounds healing was further confirmed by reconstitution experiments. Both intravenous and subcutaneous administrations of human plasminogen to plasminogen-deficient mice led to a restored healing rate and wound maturation that was comparable to those of wild-type mice. We also demonstrated that plasminogen supplementation of plasminogen to wild-type and diabetic mice significantly improved the healing of cutaneous wounds. Plasminogen levels were not only temporally increased during the inflammation phase but also spatially concentrated at the site of the wound. The wound-specific accumulation of plasminogen after systemic supplementation is mainly due to the transportation of plasminogen by neutrophils and macrophages. Furthermore, the increased expression of interleukin 6 and the enhanced phosphorylation of STAT3 were observed in the wound after plasminogen treatment. These data indicate that plasminogen acts as a key pro-inflammatory regulator. It enhances pro-inflammatory cytokines and activates intracellular signaling events during wound healing. Taken together, the data obtained during the course of this project indicate that plasminogen is crucial for oral health in mice. We also demonstrate that supplementation of plasminogen to mice with periodontitis results in healing of gum tissues and significant re-growth of alveolar bone. Therefore, plasminogen may be a new drug that will be competitive to currently used oral health-related procedures, such as implantations and surgeries. Furthermore, we demonstrate for the first time that, in addition to its role in extracellular matrix degradation, plasminogen is a key pro-inflammatory factor that accumulates at the wound and potentiates the early inflammatory response during wound healing. Based on our findings, we propose the administration of plasminogen as a novel therapeutic strategy for the treatment of different types of wounds, including chronic diabetic wounds

CCL11, a novel mediator of inflammatory bone resorption

Normal bone homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts is perturbed by inflammation. Inchronic inflammatory disease with disturbed bone remodelling, e.g. rheumatoid arthritis, patients show increased serum levels of the chemokine eotaxin-1 (CCL11). Herein, we demonstrate an inflammatory driven expression of CCL11 in bone tissue and a novel role of CCL11 in osteoclast migration and resorption. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that expression increased during inflammatory conditions. Osteoclasts did not express CCL11, but the high affinity receptor CCR3 was significantly upregulated during osteoclast differentiation and found to colocalise with CCL11. Exogenous CCL11 was internalised in osteoclast and stimulated the migration of pre-osteoclast and concomitant increase in bone resorption. Our data pinpoints that the CCL11/CCR3 pathway could be a new target for treatment of inflammatory bone resorption

Cloning and analysis of Nkx6.3 during CNS and gastrointestinal development

International audienceMembers of the Nkx family of homeodomain proteins are involved in a variety of developmental processes such as cell fate determination in the CNS and in the pancreas. Here we describe the cloning and developmental expression pattern of Nkx6.3, a new member of the Nkx6 subfamily of homeodomain proteins. Nkx6.3 is expressed in the developing CNS and gastro-intestinal tract. In contrast to Nkx6.1 and Nkx6.2 that are broadly expressed in ventral positions of the developing CNS, Nkx6.3 shows a remarkably selective expression in a subpopulation of differentiating V2 neurons at caudal hindbrain levels. The expression of Nkx6.3 at this level depends on the activity of other Nkx6 proteins. In the gut, Nkx6.3 is expressed in duodenal and glandular stomach endoderm and at the end of gestation Nkx6.3 became restricted to the base of the gastric units in the glandular stomach. The expression of Nkx6.3 overlapped with the expression of Nkx6.2 both in the CNS and in the gut. Transient Nkx6.2 expression was also detected in the developing pancreas. However, analysis of Nkx6.2(-/-) mice did not display any obvious aberrations of pancreatic or stomach development

Lack of CCR3 leads to a skeletal phenotype only in male mice

We recently showed that adult male mice that lacked the C–C-chemokine receptor 3 (CCR3) exhibited disturbed bone remodeling, which resulted in a cortical bone phenotype of thin femoral cortical bone. However, it remains unknown whether this phenotype would be present during bone modeling, or it affects female mice. Here, we analyzed juvenile and adolescent CCR3-deficient mice to determine when bone modeling was affected in the absence of CCR3 signaling. To investigate whether the CCR3 bone phenotype was sex-related, we analyzed both young female and male mice, and adult females. Micro-computed tomography (μCT) and histomorphometric analyses in adolescent CCR3-deficient male mice revealed reduced cortical bone volume and thickness, and an increase in periosteal mineralization. Interestingly, no skeletal phenotype was observed in adolescent or adult female CCR3-deficient mice. Among juvenile CCR3-deficient mice, neither males nor females showed a skeletal phenotype, which indicated that bone modeling was not affected by the CCR3 deficiency. In summary, adolescent and adult male mice that lacked CCR3 receptors exhibited a cortical phenotype that was not present in female mice, probably due to an estrogen protective mechanism. Based on these and our previous results, we suggest that the importance of CCR3 in cortical bone turnover is related to sex hormones. Because only a few molecules are known to control cortical bone turnover, our novel finding that CCR3 regulated cortical bone thickness only in males suggested that CCR3 is a novel target for controlling cortical bone morphology in male individuals, and perhaps, in post-menopausal women

Increased Eotaxin and MCP-1 Levels in Serum from Individuals with Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines

Periodontitis is a chronic inflammatory disease of tooth supporting tissues resulting in periodontal tissue destruction, which may ultimately lead to tooth loss. The disease is characterized by continuous leukocyte infiltration, likely mediated by local chemokine production but the pathogenic mechanisms are not fully elucidated. There are no reliable serologic biomarkers for the diagnosis of periodontitis, which is today based solely on the degree of local tissue destruction, and there is no available biological treatment tool. Prompted by the increasing interest in periodontitis and systemic inflammatory mediators we mapped serum cytokine and chemokine levels from periodontitis subjects and healthy controls. We used multivariate partial least squares (PLS) modeling and identified monocyte chemoattractant protein-1 (MCP-1) and eotaxin as clearly associated with periodontitis along with C-reactive protein (CRP), years of smoking and age, whereas the number of remaining teeth was associated with being healthy. Moreover, body mass index correlated significantly with serum MCP-1 and CRP, but not with eotaxin. We detected higher MCP-1 protein levels in inflamed gingival connective tissue compared to healthy but the eotaxin levels were undetectable. Primary human gingival fibroblasts displayed strongly increased expression of MCP-1 and eotaxin mRNA and protein when challenged with tumor necrosis factor-alpha (TNF-alpha and interleukin-1 beta (IL-1 beta), key mediators of periodontal inflammation. We also demonstrated that the upregulated chemokine expression was dependent on the NF-kappa B pathway. In summary, we identify higher levels of CRP, eotaxin and MCP-1 in serum of periodontitis patients. This, together with our finding that both CRP and MCP-1 correlates with BMI points towards an increased systemic inflammatory load in patients with periodontitis and high BMI. Targeting eotaxin and MCP-1 in periodontitis may result in reduced leukocyte infiltration and inflammation in periodontitis and maybe prevent tooth loss

Requirement of non-hematopoietic SIRPα to maintain normal numbers of blood and BM B cells.

<p>(A-C) Blood and BM B cell numbers in Ly5.2 wild-type (Wt recip) or SIRPα-mutant (Mut recip) chimeric mice at 20 weeks after lethal irradiation and reconstitution with Ly5.1 wild-type BM cells. (A) Numbers of total CD19⁺, immature, F-II, or mature B cells in blood of wild-type or SIRPα-mutant recipient mice were quantified based on their expression levels of IgM and CD23, as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134113#pone.0134113.g001" target="_blank">Fig 1</a>. (B) Absolute numbers of CD4/8⁺ T cells, CD11b^hi Gr1^hi neutrophils and CD11b^hi Gr1^low monocytes per microliter of blood in wild-type or SIRPα-mutant recipient mice. (C) Immature (IgD^-IgM⁺), F-II (IgD⁺IgM^hi) and F-I (IgD⁺IgM^lo) BM B cells were quantified as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134113#pone.0134113.g002" target="_blank">Fig 2D</a>. Data are means±SEM for 5 mice per genotype. (D-F) Blood and BM cell numbers in Ly5.1 wild-type mice reconstituted with Ly5.2 wild-type (Wt donor) or SIRPα-mutant (Mut donor) BM. (D) Blood B cell numbers, (E) blood T cell, neutrophil or monocyte numbers, and (F) BM B cell numbers in wild-type recipients of wild-type or SIRPα-mutant BM were quantified as described above. Data are means±SEM for 6 mice per genotype. *P<0.05, **P<0.01 and ***P<0.001, using Student’s t-test for unpaired analyses (n.s. = not significant).</p

Lack of SIRPα signaling <i>in vivo</i> results in reduced numbers of blood B cells.

<p>(A) Fraction of CD19⁺ blood B cells in 12 weeks old wild-type (Wt–open bars) or SIRPα-mutant mice (Mut–black bars). (B) CD19⁺ blood B cell subsets were separated based on their expression levels of sIgM and CD23 to identify sIgM^hiCD23^-/lo immature B cells (Immature B), sIgM^hiCD23^hi semi-mature/follicular type-II (F-II B) B cells and sIgM^lo/intCD23^hi mature/F-I (Mature B) B cells. Data shown are the numbers of total CD19⁺, immature, F-II, or mature B cells per microliter of blood in wild-type (Wt–open bars) or SIRPα-mutant mice (Mut–black bars) at (C) 12 weeks or (D) 20 weeks of age. Data are means±SEM for 9 mice/group at 12 weeks and 4–7 mice/group at 20 weeks of age. (E) Absolute numbers of CD4/8⁺ T cells, CD11b^hi Gr1^hi neutrophils and CD11b^hi Gr1^low monocytes per microliter of blood in 16 weeks old wild-type (Wt–open bars) or SIRPα-mutant mice (Mut–black bars). Data are means±SEM for 5 mice/group. *P<0.05 and **P<0.01, as compared with that in wild-type mice, using Student’s t-test for unpaired analyses.</p

Impaired B cell maturation in the bone marrow of SIRPα-mutant mice.

<p>(A) Absolute number of B220⁺ B cells in BM of 12 weeks old wild-type (open bar) or SIRPα-mutant mice (black bar). Data are means±SEM for 9 mice/group. (B) Representative flow cytometry analyses of BM B cell maturation based on expression of IgD and IgM in B220⁺ BM cells of wild-type or SIRPα-mutant mice. (C) Absolute numbers, or (D) relative numbers, of IgD^-IgM⁺ immature (Imm B), IgD^loIgM^hi transitional 1 (T1), IgD⁺IgM^hi follicular type-II (F-II) and IgD⁺IgM^lo follicular type-I (F-I) B cells in BM of 12 weeks old wild-type (open bars) or SIRPα-mutant mice (black bars). Data are means±SEM for 9 mice/group. (E) The relative expression of BIM protein, and (F) the relative increase in annexin V⁺ apoptotic cells, were quantified among BM B cell subsets defined as described in panels C-D. Data are means±SEM for 5 mice/group. *P<0.05, **P<0.01 and ***P<0.001, using Student’s t-test for unpaired analyses.</p