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

Nya insikter om inflammatoriskt störd benremodellering

Bone is a dynamic tissue that is continuously remodelled, a process that requires equal amounts of osteoclastic bone resorption and osteoblastic bone formation. Inflammation may disturb the equilibrium and result in local and/or systemic bone loss. Negative bone mass balance occurs in several chronic inflammatory diseases, e.g. periodontitis and rheumatoid arthritis (RA). The aetiology of periodontitis is infectious, while RA is an autoimmune disease. Despite aetiological differences, an association between the two diseases has been established but it is not known if they are causally related. Periodontitis may develop when the inflammatory process, initially restricted to the gingiva (gingivitis), further invades the periodontium and causes bone resorption. The cellular and molecular mechanisms underlying the transition from gingivitis to periodontitis are not fully elucidated. Osteoclast formation is dependent on receptor activator of nuclear factor kappa B ligand (RANKL), but how osteoclast precursors are recruited to the jawbone is poorly understood. A family of cytokines named chemokines has been reported to possess such properties and increasing evidence points towards their involvement in the pathogenesis of chronic inflammatory diseases. The overall aim of this thesis was to gain extended knowledge about the role of chemokines and a newly discovered family of leukocytes named innate lymphoid cells (ILCs) in periodontitis and concomitant inflammatory disturbed bone remodelling. Furthermore, the aim was also to study the association between periodontitis and RA. We identified increased serum levels of monocyte chemoattractant protein (MCP)-1 and CCL11 in individuals with periodontitis. Moreover, a robust correlation between the two chemokines and periodontitis was detected in a weighted analysis of inflammatory markers, subject characteristics and periodontitis parameters. We detected higher MCP-1 levels in periodontitis tissue compared to non-inflamed. Furthermore we demonstrated that human gingival fibroblasts express MCP-1 and CCL11 in response to pro-inflammatory cytokines through NF-κB signalling. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that the expression increased under inflammatory conditions. Osteoclasts did not express CCL11, but its high affinity receptor CCR3 was upregulated during osteoclast differentiation and found to co-localise with CCL11 on the surface of osteoclasts. Exogenous CCL11 was internalised in osteoclasts, stimulated the migration of osteoclast precursors and increased bone resorption in vitro. To analyse if periodontitis precedes RA we analysed marginal jawbone loss in dental radiographs taken in pre-symptomatic RA cases and matched controls. The prevalence of jawbone loss was higher among cases, and the amount of jawbone loss correlated with plasma levels of RANKL. In the search of the newly discovered ILCs, we performed flow cytometry analyses on gingivitis and periodontitis tissue samples. We detected twice as many ILCs in periodontitis as in gingivitis. In addition we found RANKL expression on ILC1s (an ILC subset). In conclusion, we demonstrated that CCL11 is systemically and locally increased in periodontitis and that the CCL11/CCR3 axis may be activated in inflammatory disturbed bone remodelling. We also found that marginal jawbone loss correlated with plasma levels of RANKL and preceded clinical onset of symptoms of RA. Furthermore, we demonstrated that ILCs are present in periodontitis and represent a previously unknown source of RANKL. Skelettet har flera viktiga funktioner i kroppen såsom att möjliggöra en upprätt hållning, utgöra fäste för muskler och mediera rörelse, skydda benmärgen och de inre organen samt reglera mängden av lösligt mineral i blodet. Med tiden uppstår mikroskador i skelettet vilket innebär att benvävnaden måste byggas om för att vara fortsatt funktionell. Ombyggnaden kallas remodellering och är en kontinuerlig process som huvudsakligen utförs av benbildande celler kallade osteoblaster och bennedbrytande celler kallade osteoklaster. Remodelleringen är strikt reglerad av olika signalmolekyler och under friska förhållanden råder jämvikt mellan mängden ben som bryts ner och mängden ben som bildas, vilket innebär att benmassan hålls konstant. Vid sjukdomar som medför långvariga inflammationsprocesser i benvävnad eller i närheten av benvävnad, exempelvis parodontit (tandlossningssjukdom) och ledssjukdomen reumatoid artrit (RA), kan den rådande jämvikten rubbas, vilket oftast resulterar i minskad benmängd. Vid parodontit är den bakomliggande orsaken till inflammationen bakterier som finns i placket på tänderna, men vid RA tros anledningen vara att immunförsvaret attackerar kroppsegna celler. Trots olikheterna delar de två sjukdomarna flera gemensamma drag med avseende på riskfaktorer, vilka signalmolekyler som återfinns i blodet samt hur inflammationsprocessen fortskrider. Parodontit föregås av gingivit (tandköttsinflammation). Hos vissa individer övergår gingivit till parodontit, en process som inkluderar nedbrytning av tandens stödjevävnader inklusive käkben. Det är inte helt klarlagt vilka celler och molekyler som finns närvarande vid gingivit respektive parodontit eller vilka mekanismer som ligger bakom skiftet mellan de två tillstånden. Det är sedan tidigare känt att molekylen RANKL är viktig för osteoklastbildning, men det är delvis okänt hur osteoklastförstadieceller rekryteras från blodcirkulationen till käkbenet. En grupp av molekyler kallade kemokiner, som även finns i förhöjda nivåer i blod vid parodontit och RA, har visat sig ha sådana egenskaper. För att finna läkemedel som kan förhindra bennedbrytning till följd av den inflammatoriskt störda benremodellering som sker vid både parodontit och RA är det viktigt att studera sambandet mellan sjukdomarna och få en tydlig bild av vilka celler som är närvarande vid inflammationsprocessen. Det är även av betydelse att kartlägga vilka celler och molekyler som främjar rekrytering av osteoklastförstadieceller och bidrar till bennedbrytning. Syftet med den här avhandlingen var att undersöka betydelsen av kemokiner vid inflammatoriskt störd benremodellering och vid parodontit samt att undersöka sambandet mellan parodontit och RA. För att skapa en tydligare bild av vilka cellertyper som är närvarande vid inflammationsprocessen vid parodontit undersöktes även förekomsten av en nyligen upptäckt celltyp vid namn ILCimmunceller (ILCs) samt om dessa celler uttrycker RANKL. Först analyserades förekomsten av olika inflammatoriska signalmolekyler i blod från individer med parodontit samt från friska kontroller. Individer med parodontit hade förhöjda nivåer av kemokinerna MCP-1 och CCL11. Genom att använda en statistisk analysmetod som utöver inflammatoriska signalmolekyler även inkluderade kliniska variabler kunde ett samband mellan de två kemokinerna och parodontit påvisas. Vidare undersöktes möjliga ursprung till de i blodet förhöjda kemokinnivåerna genom att analysera tandkött från tänder med parodontit samt friskt tandkött. Vid parodontit uppmättes högre nivåer av MCP-1. Gingivala fibroblaster (en celltyp som producerar bindväv och ansvarar för tandköttets uppbyggnad) från människa bildade MCP-1 och CCL11 när de stimulerats med inflammationsfrämjande substanser, vilket krävde aktivering en intracellulär signaleringsväg kallad NF-κB. För att utreda betydelsen av CCL11 vid inflammatoriskt störd benremodellering analyserades bencellers bildning av CCL11 in vivo i skalltak från möss samt in vitro i cellodlingar. Osteoblaster bildade CCL11 in vivo och in vitro och bildningen ökade under inflammatoriska förhållanden. Osteoklaster bildade inte CCL11, men däremot fanns ett uttryck av receptorn CCR3, vilket är en mottagarmolekyl till CCL11. I vävnadssnitt från skalltak visades att CCL11 och CCR3 ser ut att binda till varandra på osteoklasternas yta. Dessutom hade CCL11 en positiv effekt på rekrytering av osteoklastförstadieceller och CCL11 som tillsattes till cellodlingar togs upp av osteoklaster och stimulerade benresorption. För att studera sambandet mellan parodontit och RA analyserades käkbensförlust vid tänder med hjälp av röntgenbilder tagna på individer som senare utvecklade RA (pre-symptomatiska) samt matchade kontroller. De presymptomatiska individerna hade en högre grad av käkbenförlust och det fanns också ett samband mellan käkbensförlust och nivåer av RANKL i blodet. Förekomsten av ILCs i tandkött från tänder med gingivit respektive parodontit analyserades med flödescytometri. Dubbelt så många ILCs återfanns vid parodontit än vid gingivit, varav majoriteten bestod av ILC1 (en undergrupp till ILCs). Vidare analyser visade att ILC1 cellerna bildar RANKL. Sammanfattningsvis, vid parodontit finns förhöjda nivåer av CCL11 i vävnaden och i blodet, och interaktionen mellan CCL11 CCR3 kan vara av betydelse vid inflammatoriskt störd benremodellering. Käkbensförlust föregår RA och korrelerar med nivåer av den osteoklaststimulerande molekylen RANKL i blodet, vilket stödjer teorin om att det finns ett samband mellan de två sjukdomarna. De nyligen upptäckta ILCs återfinns vid både gingivit och parodontit och utgör dessutom en tidigare okänd källa till RANKL

LIGHT protein : a novel gingival crevicular fluid biomarker associated with increased probing depth after periodontal surgery

Aim: To evaluate the protein profiles in gingival crevicular fluid (GCF) in relation to clinical outcomes after periodontal surgery and examine if any selected proteins affect the mRNA expression of pro-inflammatory cytokines in human gingival fibroblasts. Materials and Methods: This exploratory study included 21 consecutive patients with periodontitis. GCF was collected, and the protein pattern (n = 92) and clinical parameters were evaluated prior to surgery and 3, 6 and 12 months after surgery. Fibroblastic gene expression was analysed by real-time quantitative polymerase chain reaction. Results: Surgical treatment reduced periodontal pocket depth (PPD) and changed the GCF protein pattern. Twelve months after surgery, 17% of the pockets showed an increase in PPD. Levels of a number of proteins in the GCF decreased after surgical treatment but increased with early signs of tissue destruction, with LIGHT being one of the proteins that showed the strongest association. Furthermore, LIGHT up-regulated the mRNA expression of pro-inflammatory cytokines interleukin (IL)-6, IL-8 and MMP9 in human gingival fibroblasts. Conclusions: LIGHT can potentially detect subjects at high risk of periodontitis recurrence after surgical treatment. Moreover, LIGHT induces the expression of inflammatory cytokines and tissue-degrading enzymes in gingival fibroblasts

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

Are Increased in Patients with Severe Periodontitis, and Associate with Presence of Specific Autoantibodies and Myocardial Infarction

There is accumulating data suggesting that periodontitis is associated with increased risk of systemic and autoimmune diseases, including cardiovascular disease, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and there is an unmet need to identify these individuals early. With the periodontal bacteria Porphyromonas gingivalis (Pg) as one of the key drivers of periodontitis, we set out to investigate whether antibodies to Pg virulence factor arginine gingipain (Rgp) could serve as a biomarker for periodontitis patients at increased risk of autoimmunity and systemic disease. We measured serum anti-Rgp IgG in three study populations: PAROKRANK (779 individuals with myocardial infarction (MI); 719 controls), where 557 had periodontitis, and 312 were positive for autoantibodies associated with RA/SLE; the PerioGene North pilot (41 periodontitis; 39 controls); and an SLE case/control study (101 SLE; 100 controls). Anti-Rgp IgG levels were increased in severe periodontitis compared to controls (p < 0.0001), in individuals positive for anti-citrullinated protein antibodies (p = 0.04) and anti-dsDNA antibodies (p = 0.035), compared to autoantibody-negative individuals; and in MI patients versus matched controls (p = 0.035). Our data support longitudinal studies addressing the role of anti-Rgp antibodies as biomarkers for periodontitis patients at increased risk of developing autoimmunity linked to RA and SLE, and mechanisms underpinning these associations

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

CCR3 deficiency is associated with increased osteoclast activity and reduced cortical bone volume in adult male mice

Increasing evidence emphasizes the importance of chemokines and chemokine receptors as regulators of bone remodeling. The C–C chemokine receptor 3 (CCR3) is dramatically upregulated during osteoclastogenesis, but the role of CCR3 in osteoclast formation and bone remodeling in adult mice is unknown. Herein, we used bone marrow macrophages derived from adult male CCR3-proficient and CCR3-deficient mice to study the role of CCR3 in osteoclast formation and activity. CCR3 deficiency was associated with formation of giant hypernucleated osteoclasts, enhanced bone resorption when cultured on bone slices, and altered mRNA expression of related chemokine receptors and ligands. In addition, primary mouse calvarial osteoblasts isolated from CCR3-deficient mice showed increased mRNA expression of the osteoclast activator–related gene, receptor activator of nuclear factor kappa-B ligand, and osteoblast differentiation–associated genes. Microcomputed tomography analyses of femurs from CCR3-deficient mice revealed a bone phenotype that entailed less cortical thickness and volume. Consistent with our in vitro studies, the total number of osteoclasts did not differ between the genotypes in vivo. Moreover, an increased endocortical osteoid mineralization rate and higher trabecular and cortical bone formation rate was displayed in CCR3-deficient mice. Collectively, our data show that CCR3 deficiency influences osteoblast and osteoclast differentiation and that it is associated with thinner cortical bone in adult male mice

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

Pearson correlation coefficients between serum CRP, MCP-1, eotaxin, and BMI in periodontitis (PD) and in periodontally healthy (PH) subjects.

<p>1) Log10 values</p><p>Pearson correlation coefficients between serum CRP, MCP-1, eotaxin, and BMI in periodontitis (PD) and in periodontally healthy (PH) subjects.</p

Characteristics of study participants describing periodontitis (PD) and periodontally healthy (PH) subjects.

<p>a) Distribution of numbers was tested with Chi² test.</p><p>b) Differences between means were tested with Student´s <i>t</i>-test.</p><p>c) Standardized for sex, age and education using general linear modeling.</p><p>d) Standardized for sex, age, education and number of teeth using general linear modeling.</p><p>Characteristics of study participants describing periodontitis (PD) and periodontally healthy (PH) subjects.</p