Levels of selected inflammatory parameters in gingival crevicular fluid of patients with stage III and IV periodontitis

Levels of selected inflammatory parameters in gingival crevicular fluid of patients with stage III and IV periodontitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Levels of selected inflammatory parameters in gingival crevicular fluid of patients with stage III and IV periodontitis Vladimíra Radochová, Ondřej Heneberk, Ondřej Souček, Ctirad Andrýs This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6956664/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background: The pathogenesis of periodontitis involves complex interactions within the host immune system. This study aimed to characterize the immune profile of gingival crevicular fluid (GCF) in patients with stage III and IV periodontitis. Methods: Patients diagnosed with stage III or IV periodontitis were included, with age- and sex-matched individuals serving as healthy controls. GCF samples were analyzed for soluble CD14 (sCD14), Galectin-3, interleukin-6 (IL-6), interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-8 (MMP-8), neutrophil gelatinase-associated lipocalin (NGAL), and thymic stromal lymphopoietin (TSLP) using a multiplex microbead-based assay on the XMAP/Luminex system. Results: A total of 25 patients and 25 controls were included. Clinical parameters—pocket probing depth, gingival index, and plaque index—were significantly elevated in the periodontitis group. Cytokine levels in GCF were generally higher in patients with periodontitis. Post-treatment, levels of IL-6, TSLP, sCD14, MCP-1, and MMP-8 significantly decreased and were lower in controls. In contrast, Galectin-3 levels increased following treatment, while NGAL levels remained unchanged. IL-10 levels were frequently undetectable and did not change significantly after therapy. Conclusions: This study highlights the involvement of multiple immune mediators, including those linked to neutrophil activity, in periodontal tissue destruction. Following successful periodontal therapy, local inflammatory markers decreased, reflecting a reduction in periodontal inflammatory activity. Periodontitis inflammatory cytokines host immune response interleukin-6 MMP-8 Figures Figure 1 Figure 2 Clinical Relevance Background: This study investigated the cytokine profile in the gingival crevicular fluid of patients with advanced periodontitis to understand its role in the disease's pathogenesis. Added value of the study: By analyzing key inflammatory markers, the research revealed significant elevations in IL-6, MMP-8, and other cytokines associated with neutrophilic and T cell-mediated responses in periodontitis patients. After treatment, a marked reduction in these inflammatory markers was observed, suggesting a return to near-normal levels, comparable to those in healthy individuals. Clinical implications: The findings highlight the role of neutrophilic inflammation and the immune system's complex interplay in periodontal disease and its resolution post-treatment and provide the possible options for disease activity monitoring. Introduction Periodontitis a chronic inflammatory disease associated with bacterial dysbiosis and is characterized by progressive destruction of the periodontal tissues around the tooth 1 . The causal role of pathogenic bacteria in the biofilm has been shown numerous times. The bacteria initiate inflammatory reactions in the gingival tissues and are responsible for further progression. Interactions of immunity of the host and inflammatory response are major determinants of susceptibility to this disease where also various environmental and genetic factors play a role 2 . The cellular component of immunity, whether innate or adaptive, is the basis for all immune processes 3 . The different components of immunity play a role in the development of periodontal inflammation. As the periodontal inflammation increases, more damage to the tissues is done and periodontitis advances from initial stages to more severe forms such as stage III and IV especially defined as extensive loss of periodontal tissues and alveolar bone. The initial inflammatory response to bacteria present in dental plaque is caused by leukocytes and endothelial cells. The products of bacterial metabolism stimulate the cells of the epithelium to produce cytokines and stimulate the production of vasoactive peptides that cause vasodilation of local blood vessels. Neutrophils migrate towards the site of inflammation in response to chemokines. Increasing numbers of neutrophils especially in the connective tissue is followed by the appearance of macrophages, lymphocytes, plasma cells and mast cells. The production of gingival fluid increases due to activation of complement proteins. Macrophages, plasma cells and T and B cells dominate the later stages 4 . Cytokines play an important role in the progression of periodontitis 5 . They also play a role in maintaining either protective or destructive inflammatory processes 6,7 . A balance between pro-inflammatory and anti-inflammatory cytokines can protect periodontal tissue from destruction and their respective imbalance leads to disease progression 8 . The pathogenesis of periodontal pocket involves a whole plethora of immune system cells. The dominant cells are plasma cells and lymphocytes, with B lymphocytes predominating. While the dominance of T lymphocytes is associated with relative stability, the predominance of B lymphocytes leads to the development of periodontal pocket 9 . Polymorphonuclear leukocytes migrate through the epithelial lining of the periodontal pocket, where they form a barrier between the microbial biofilm and the epithelium. The periodontal epithelium shows increased permeability and ulcerations, which allows bacterial toxins to penetrate the periodontal tissues and stimulates the inflammatory immune response and the production of pro-inflammatory cytokines such as IL-1, IL-6, TNF-α, prostaglandin E (2) ( PGE 2 ) 10 . This leads to connective tissue destruction and resorption of adjacent alveolar bone. 11 Connective tissue remodeling is the result of intercellular interaction and interaction of cells with the extracellular matrix. This includes the production of enzymes, their activators and inhibitors, cytokines and growth factors 11 . The most important enzymes degrading various components of the extracellular matrix are matrix metalloproteinases (MMPs), which are produced by monocytes/macrophages, neutrophils, fibroblasts, as well as epithelial cells. An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) is thought to be the main cause of connective tissue destruction. Increased proteolytic activity associated with periodontal tissue destruction has been described for matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 12–15 . Bone resorption in periodontitis is induced by bacterial toxins, including lipopolysaccharides (LPS) or lipopeptides, which stimulate pattern recognition receptors , in this case various types of Toll-like receptors (TLRs). This leads to the secretion of pro-inflammatory cytokines (e.g. IL-1, IL-6, TNF-α, PGE 2) , which in turn affect osteoblasts and osteoclasts, or these cells can be stimulated directly 16–18 . Both IL-1 and bacterial LPS directly stimulate osteoblasts to express more RANKL and IL-6 and reduce OPG production. In osteoclasts, LPS and IL-1 stimulate their activation and play an important role in their survival 19,20 . Epithelial cells of the gingiva do not only perform a barrier function to the external environment, hence to microorganisms and their products. They express a whole set of receptors for dangerous PPR patterns on their membrane, with the help of which the epithelia are able to identify pathogens in their vicinity. Upon stimulation by bacterial products, they secrete pro-inflammatory cytokines, including IL-1α, IL-1β, IL-6, IL-8, TNF-α. They also produce substances that have bactericidal effects, called defensins, and also activate immature dendritic cells 21 . Antigen presenting cells play an important regulatory role in controlling the inflammatory immune response. They are widely distributed in peripheral tissues, including the gingiva, where they phagocytose microorganisms whose components are subsequently degraded. Fragments of microbial molecules are then presented to T lymphocytes by major histocompatibility complex II, together with costimulatory signals and appropriate cytokine microenvironments. This process is a key regulatory mechanism and leads to the initiation of an inflammatory immune response or tolerance 22 . Dendritic cells also produce polarizing cytokines with pro-inflammatory (TNF-α, IL-1β) or anti-inflammatory (transforming growth factor β, IL-10) effects 23 . The recognition of microbes by antigen-presenting cells, but also by other cells of non-specific immunity, occurs via pathogen-associated molecular pattern receptors, PAMPs, such as LPS. They can also recognize tissue damage signals (damage-associated molecular patterns, DAMPs), i.e. intracellular proteins or molecules derived from the extracellular matrix. Recognition of PAMPs and DAMPs by cells of nonspecific immunity provides information about damage to the body's tissues or the presence of an infectious agent 24 . Danger pattern receptors include TLR receptors or CD14 22 . Neutrophils translocate from the bloodstream to peripheral tissues at sites where resident cells recognize danger signals (PAMP/DAMP) and produce mediators such as nitric oxide, IL-1β, TNF-α, and IL-17. Increased production of pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α by neutrophils circulating in the vasculature was also demonstrated in subjects with periodontitis and persisted after successful periodontal therapy. This indicates a possible "training" of neutrophils during myelopoiesis 25 . Neutrophils also form so-called neutrophil extracellular traps. This is a specific type of programmed cell death where, following massive stimulation of neutrophil surface receptors, nuclear remodeling and excretion of chromatin in the form of filaments outside the cell occurs along with secretion of granule contents, hence enzymes, antibacterial agents and reactive oxygen species 26,27 . Macrophages in tissues can perform different functions, so-called M1 macrophages, classically activated macrophages stimulating the inflammatory immune response and acquired immunity. The dominant factors stimulating polarization in M1 macrophages in periodontitis are IFN-γ produced by Th1 cells, stimulation of TLRs and other danger pattern receptors, including CD14, by bacterial LPS 28 . Other activating stimuli include reactive oxygen species, granulocyte-macrophage colony-stimulating factor, and micro ribonucleic acid miRNA 29 . M1 macrophages produce a number of pro-inflammatory cytokines (e.g. IL-1β, IL-6, IL-12, TNFα), prostaglandins (PGE 2) , chemokines (Mononuclear chemoattractant 1), adhesion molecules, reactive oxygen species and hydrolytic enzymes (e.g. various types of MMPs) 29 . The production of proinflammatory cytokines M1 by macrophages leads to further activation of the inflammatory immune response, including secretion of MMPs by other cell types (e.g. fibroblasts) and production of the soluble form of RANKL by T and B lymphocytes 30–32 . Lymphocytes are a major component of the specific immune response. As already mentioned, lymphocytes are dominant cells in early, advanced/progressive and advanced periodontal lesions 33 . CD4 T lymphocytes have an important regulatory function: Dominance of helper Th2 lymphocytes leads to higher numbers of B lymphocytes and plasma cells, leading to lesion progression 34 . In contrast, dominance of the Th1 subset is associated with stable periodontal lesions 34 . Treg are responsible for suppression of the immune response by producing anti-inflammatory cytokines such as IL-10 and expressing CD 152 35 . Th17 produce pro-inflammatory cytokines IL-6, IL-17, IL-23 36 . The imbalance between Th17 and Treg lymphocytes affects bone metabolism and bone resorption in periodontitis via RANKL/RANK/OPG 35 . The aim of our study is to map multiple components of the cytokine profile of GCF collected from the site of inflammation (periodontal pocket) in patients with stage III and IV periodontitis. Furthermore, to compare this cytokine profile in the same patients after periodontal therapy and to compare the profile with a control group of individuals with healthy periodontium. As described above, key regulators of the various components of the immune system were selected for analysis to evidence the expected activity of immune system components involved in the pathogenesis of periodontitis: soluble CD14 (sCD14), Galectin3, Interleukin-6 (IL-6), Interleukin-10 (IL-10), Monocyte chemoattractant protein-1 (MCP-1), Matrix metalloproteinase 8 (MMP-8), Neutrophil gelatinase associated lipocalin (NGAL) and Thymic stromal lymphopoietin (TSLP). The null hypothesis was that there is no difference in cytokine concentration in patients with stage III or IV periodontitis before and after therapy and compared with control group of individuals with healthy periodontium. Methods The study participants were recruited from patients of the Periodontology Division of the Department of Dentistry of the University Hospital in Hradec Kralove, Czech Republic. Data collection took place from December 2019 to January 2022. All individuals who visited the periodontology department and met the exclusion and inclusion criteria were approached to enter the experimental cohort study. Patients aged 18 years and older participated in the study. The primary inclusion criterion was the presence of stage III or IV periodontitis. Inclusion in stage III or IV was performed based on the current 2018 European Federation of Periodontology recommendations 37 . In brief, the classification into stage III or IV based primarily on interdental CAL at site of greatest loss ≥ 5 mm and other criteria listed in Table 1 37 . The healthy periodontium was classified on the basis of gingival bleeding in all examined sites BOP < 10% Probing pocket depths ≤4 mm (no site ≥ 4 mm with BOP) in reduced periodontium, in non-reduced Probing pocket depths ≤3 mm 38 . The control group included 25 patients matched in sex and age to those in the experimental group who met the criteria for a healthy periodontium according to the same recommendations. These were patients of the Department of Dentistry in Hradec Kralove. All study subjects had to be non-smokers who were generally healthy without systemic disease. Individuals meeting the inclusion criteria were added to both groups throughout the data collection period until the desired sample size was reached. One patient in the experimental group did not complete periodontal therapy and was excluded from the posttreatment evaluation. All subjects underwent a complete examination of the dentition, periodontal and oral mucosa. Periodontal examination was performed on all teeth in the oral cavity except the 3rd molars using a PCPUNC-15 manual dental probe (Hu-Friedy, Chicago, IL, USA), including recording of the following periodontal parameters: probing depth, clinical attachment level, and bleeding on probing (BOP). Parameters were recorded at 6 sites (distobuccal, distoral, buccal, oral, inter-buccal, and non-buccal). The examination was performed by two experienced physicians, V.R. and O.H., who were trained to perform this study. Inter-examiner reliability was assessed using kappa coefficients before the study. To ensure consistency and reliability in the clinical measurements taken during the study—specifically probing depth and clinical attachment level—inter-examiner agreement was assessed. Inter-examiner agreement refers to the level of consistency between different examiners (e.g., dentists or clinicians) when measuring the same parameters on the same subjects. In this study, the kappa values for inter-examiner agreement exceeded 90%, indicating very high agreement between different examiners. Kappa values range from 0 to 1, with values above 0.80 generally considered to reflect excellent agreement beyond what would be expected by chance. X-rays were also taken to detect bone loss namely orthopantomogram. Ethical considerations The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University Hospital Hradec Králové (reference number 201909 S13R). All participants were informed about the study objectives and signed an informed consent before inclusion into the study. All rights of the participants were maintained throughout. Statistical considerations To ensure the study had sufficient statistical strength to detect meaningful differences, a sample size calculation was performed prior to data collection. The calculation was based on a target significance level (alpha) of 0.05 , which indicates a 5% risk of concluding that a difference exists when there is none (i.e., a Type I error). Additionally, the study aimed for a statistical power of 0.80 (or 80%) , meaning there was an 80% chance of correctly detecting a true effect (i.e., avoiding a Type II error) if one exists. The calculation was designed to detect a minimum difference of 20% in the observational parameters (such as clinical measurements or biomarker levels) between the groups. This threshold represents the smallest difference that the researchers considered to be clinically or scientifically relevant. Based on these assumptions—a significance level of 0.05, power of 0.80, and a minimum effect size of 20%—the required sample size was determined to be 25 individuals per group . The study thus included three groups : a control group (without periodontitis), an experimental group (likely receiving a specific intervention or condition), and a periodontitis treatment group. This design allows for meaningful comparisons across different conditions while maintaining adequate statistical power. Continuous variables are presented as median and interquartile range (IQR). Categorical variables were compared using Chi-square and Fisher's exact test and are presented as counts and percentages (%). Variables that did not show a normal distribution were analyzed using the Mann-Whitney U test and Wilcoxon test and results are presented as median and IQR. Differences were considered significant at p < 0.05. All p values were obtained from two-tailed tests and all statistical analyses were performed using GraphPadPrism v. 10.2.3 software (GraphPad Software, USA) and MS Excel 365 (Microsoft, USA). Therapy of patients with periodontitis Patients with periodontitis were treated according to the guidelines for periodontitis therapy 39 . All underwent the active phase of periodontitis treatment. The first phase of therapy focused on behavior changes by motivating the patient to successfully remove supragingival dental biofilm, control of supragingival plaque, interventions to improve oral hygiene, professional mechanical plaque removal and calculus as well as any factors that impair oral hygiene practices, and adjunctive treatment for gingivitis. At the beginning of the treatment, chlorhexidine 0.12% was used for 14 days. In the second step of the therapy, we focused on the control (reduction/removal) of subgingival plaque and calculus (scaling and root planning). In patients initially presenting with significant purulent exudation from the periodontal pocket after the first step of therapy, additional systemic antimicrobials (antibiotics Amoxicillin and Metronidazole) were used as adjunctive therapy 40 Response to the second step of therapy was assessed at least 8 weeks after subgingival treatment. Periodontal healing was achieved in all but 1 patient (here a periodontal pocket 7 mm without bleeding). This means no periodontal pockets >4 mm with bleeding on probing or no deep periodontal pockets ≥6 mm. However, in some situations extractions were chosen at the beginning of therapy. These included teeth with significant attachment loss, grade III mobility, pulpoperiodontal involvement without good prognosis for therapy, 3 rd molars, and both solitary and further prosthodontically unusable teeth. Gingival fluid sampling Gingival crevicular fluid collection was performed 3 days after periodontal examination in all participants. In the experimental group, in addition to the collection at the beginning of treatment, GCF sampling was also performed 3 months after the completion of nonsurgical treatment of periodontitis. GCF samples were obtained in patients with periodontitis from the most affected site, i.e., the deepest periodontal pocket. the same tooth was selected if it was present in the mouth after therapy (in the experimental group). If this tooth was extracted, the next tooth with the deepest pocket before therapy was chosen. In subjects with a healthy periodontium, the highest probing site was also sampled. Prior to gingival crevicular fluid sampling, all sites were isolated with cotton rollers to prevent contamination with saliva. Supragingival plaque was removed, and the tooth was gently air dried. Sterile ISO 40 paper pins were inserted into the gingival sulcus/pocket until slight resistance was felt and were left in place for 30 seconds to allow absorption of gingival crevicular fluid from the site and to prevent bleeding. Pins visually contaminated with blood were not used for analysis. The paper pin was then placed in a 5.0 ml CryoPure tube with 0.5 ml of PBS solution. The tube with the paper pin was "shaken" on a shaker (1400/min) for 20 minutes. The paper plug was then removed from the tube using sterile tweezers. The tube was centrifuged for 15 minutes at 1800 rpm. The supernatant was pipetted into 200 µl aliquots into 0.5 ml micro tube tubes. Aliquots were stored and frozen at -70°C. Quantification of immune markers from GCF The Human cytokine premixed magnetic Luminex® performance assay was chosen for the analysis of selected cytokines in gingival fluid samples: CD14, Galectin-3, IL-6, IL-10, MCP-1, MMP-8, NGAL, and TSLP (R&D systems, Minneapolis, MN, USA). Cytokine levels were determined using Luminex xMAP® magnetic technology by Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA), analysis of the results was performed using xPONENT® 4.2. analysis software (Luminex Corporation, Austin, TX, USA). Briefly, Luminex xMAP® is a multiplex bead-based immunoassay platform that enables the simultaneous detection and quantification of up to 500 different analytes (such as proteins, cytokines, or nucleic acids) in a single sample. At the core of the system are microspheres (or beads) that are internally dyed with distinct ratios of red and infrared fluorophores. Each unique ratio produces a specific spectral signature, also known as a bead region, allowing the system to differentiate between up to 500 bead types in one assay. Each bead region is coated with a specific capture antibody that binds only to a particular analyte of interest. When a sample is added to the beads, the target analytes bind to their respective antibodies on the corresponding bead types. After unbound substances are washed away, the beads are incubated with: Biotinylated detection antibodies, which bind to a different epitope on the captured analyte. Streptavidin-phycoerythrin (PE), a fluorescent molecule that binds to biotin and acts as a reporter signal. The bead-analyte-detection antibody-reporter complex is then analyzed using a Luminex instrument equipped with two lasers, the first identifies the bead region by detecting the internal fluorophore ratio—this tells the system which analyte is being measured. Laser 2 measures the intensity of the phycoerythrin (PE) fluorescence, which is directly proportional to the amount of analyte bound to the bead. Multiple readings (typically 50–100) are collected for each bead region, ensuring high sensitivity, accuracy, and reproducibility in quantification. Gingival fluid samples were diluted 2-fold. The assay was done according to the manufacturer’s instructions. All values were expressed in nanograms (ng) per milliliter of gingival fluid. Results A total of 25 patients with periodontitis who met the entry criteria listed above were included in the study. The median age was 50 years (IQR 47-55 years), 13 were male (52%) and 12 were female (48%). One patient did not complete periodontitis therapy, and 24 patients were then analyzed for the final evaluation. The control group included 25 healthy controls with a median age of 46 years (IQR 44 - 54 years), 13 (52%) were male and 12 were female (48%). The groups did not differ in age or gender. The periodontitis group had a statistically significantly different (p<0.0001) median attachment loss of 4.36 (IQR 3.9-5.15) compared to the control group of 1.20 (IQR 1.17-1.26). After therapy, patients in the periodontitis group showed a statistically significant improvement in attachment loss to a median value of 1.88 (IQR 1.6-2.55, p<0.0001). After therapy, the median attachment loss remained significantly higher in the periodontitis group compared to the healthy periodontitis group (p<0.Similar results could be observed for other monitored parameters, i.e., dentition depth, gingival index and plaque index. The GI and PLI parameters were comparable in the posttreatment group with those of the control group. The clinical characteristics of the patients are described in detail in Table 2 and Figure 1. The levels of individual cytokines in the initial crevicular fluid samples showed significantly higher concentrations than in the control group in most cases. All results are described below and summarized in Table 3 and Figure 2. Cytokine concentration did not differ between patients with stage III compared to stage IV periodontitis (Table 4). IL-6 concentrations in the periodontitis group were with a median of 31.5 ng/ml (IQR 26.9-47) and 19.6 ng/ml (IQR 12.1-27.3, p=0.0001) in the control group. After treatment, IL-6 concentrations significantly decreased to a median of 6.45 ng/ml (IQR 0.85-11.6, p<0.0001). IL-6 concentration was significantly lower in the treated group compared to the control group after therapy (p<0.0001). IL-10 concentrations in the periodontitis group were very often below the detection limit, complicating the evaluation, with a median of 0 ng/ml (IQR 0-4.3) and 0.43 ng/ml (IQR 0.43-13.7, p=0.0001) in the control group. After therapy, IL-10 concentrations remained unchanged with a median of 0.23 ng/ml (IQR 0.23-03, p=0.9385). IL-10 concentrations after therapy were comparable in the treatment group compared to the control group (p=0.8139). The sCD14 concentrations in the periodontitis group were with a median of 4848 ng/ml (IQR 2090-9759) and 904 ng/ml (IQR 399-1286, p<0.0001) in the control group. sCD14 concentrations significantly decreased after treatment to a median of 832 ng/ml (IQR 297-2578, p<0.0001). sCD14 concentration after therapy compared to the control group was comparable in the treatment group compared to the control group (p=0.9881). The median galectin-3 concentrations in the periodontitis group were 663 ng/ml (IQR 353-1456) and 2096 ng/ml (IQR 1042-2775, p<0.0001) in the control group. After therapy, the galectin-3 concentration significantly increased to a median of 2253 ng/ml (IQR 519-10955, p=0.0007). The galectin-3 concentration after therapy was comparable in the treatment group compared to the control group (p=0.7177). The median MCP-1 concentrations in the periodontitis group were 9.2 ng/ml (IQR 6.2-15.2) and 6.2 ng/ml (IQR 3.42-15.2, p=0.2292) in the control group. After therapy, MCP-1 concentration significantly decreased to a median of 5.14 ng/ml (IQR 4.52-5.14, p<0.0001). MCP-1 concentration was lower in the treatment group compared to the control group after therapy (p=0.0338). The median MMP-8 concentrations in the periodontitis group were 383828 ng/ml (IQR 116360-662430) and 32454 ng/ml (IQR 23830-52250, p<0.0001) in the control group. After therapy, MMP-8 concentrations significantly decreased to a median of 19855 ng/ml (IQR 4963-47000, p<0.0001). MMP-8 concentrations after therapy were comparable in the treatment group compared to the control group (p=0.0766). The median NAGL concentrations in the periodontitis group were 23864 ng/ml (IQR 22563-24249) and 22696 ng/ml (IQR 22039-23407, p=0.022) in the control group. After treatment, NGAL concentrations remained unchanged with a median of 23809 ng/ml (IQR 21240-25561, p=0.9441). NGAL concentrations after therapy compared to the control group were comparable in the treatment group compared to the control group (p=0.4099). The median TSLP concentrations in the periodontitis group were 0.532 ng/ml (IQR 0.099-0.79) and 0.396 ng/ml (IQR 0.29-0.532, p=0.0339) in the control group. After therapy, TSLP concentrations decreased to a median of 0.12 ng/ml (IQR 0.12-0.25, p=0.0009). TSLP concentrations were lower in the treatment group compared to the control group after therapy (p<0.0001). There was no correlation between initial cytokine concentration and clinical parameters in periodontitis patients (Table 5). Discussion This study confirms that gingival crevicular fluid (GCF) serves as a reliable medium for assessing immune responses in periodontal disease. Key findings of our results reveal: Elevated IL-6, MCP-1, MMP-8, and sCD14 concentrations in active periodontitis. Decreased IL-10 and Galectin-3 levels in diseased states, with post-treatment normalization suggesting a regulatory role. TSLP levels strongly associated with disease activity and significantly decreased following therapy. NGAL did not show significant differences, implying limited involvement in periodontitis pathology. Our findings reinforce the potential utility of GCF biomarkers for monitoring disease progression and treatment outcomes. Our study aimed at broad view of the involvement of individual parts of the immune system in the pathogenesis of active severe periodontitis. We have shown that even in clinically healthy periodontium, a certain amount of pro- and anti-inflammatory cytokines are present, and their concentrations are in some cases even higher than in patients after successful periodontal treatment (IL-6, MCP-1, TSLP). During inflammation, the flow of fluid into the gingival crevice increases, accompanied by a rise in cellular infiltrate. Neutrophils serve as the primary cellular mechanism in the gingival crevice, with most appearing as viable cells capable of eliminating microorganisms. As this fluid passes through the junctional epithelium into the gingival crevice, it contributes to an elevated flow of gingival crevicular fluid 33 . A variety of cytokines are released by sulcular and epithelial cells, dendritic cells, connective tissue fibroblasts, macrophages, and neutrophils. Additionally, these cells produce enzymes, including matrix metalloproteinases, which contribute to the breakdown of connective tissue collagen and alveolar bone 41 . Host susceptibility is an important factor in the pathogenesis of periodontal disease, giving the level of inflammatory mediators present in the gingival fluid. Various cytokine-mediated inflammatory pathways and microbial stimuli may contribute to the development of periodontal disease. However, these pathways are likely to converge, resulting in similar metabolite profiles. For instance, one microbial community structure may trigger distinct inflammatory responses, while a different microbial community may elicit an alternative inflammatory signature. Additionally, variations in host genetics can influence microbial colonization and inflammatory responses 42 . Available studies suggest that gingival crevicular fluid is an excellent source of periodontal disease biomarkers that reliably reflect immune interactions 42 . Clinical parameters of periodontitis Our study clearly demonstrates that periodontal therapy improves the status of periodontal tissues. Our study demonstrates extremely significant differences between all clinical variables between patients with periodontitis and healthy controls. However, the differences between periodontal status in patients after therapy compared to healthy individuals are still seen. Although GI and PLI improves after therapy to comparable levels as healthy individuals, the differences in PPD, GR and CAL remain, although significantly lower compared to values before the therapy. IL-6 and IL-10 IL-6 plays a role especially in the initiation and acute phase of periodontitis. IL-6 is expressed by a variety of cells, including phagocytic cells, T cells, B cells, fibroblasts, endothelial cells, keratinocytes and also other tissues such as hepatocytes or bone marrow cells. It is also important in the secretion of acute phase proteins, which is done in cooperation with IL-1β 43 . IL-6 is also considered an osteoclastogenic cytokine because higher levels lead to increased bone loss 44 . IL-6 plays a key role in triggering acute phase responses while also facilitating specific cellular and humoral immune reactions by promoting B-cell differentiation, immunoglobulin secretion, and T-cell activation. As a result, IL-6 serves as a crucial regulator in the transition from acute to chronic inflammation 45 . Periodontitis is characterized by elevated levels of IL-6, which have been observed in GCF, oral fluid, and serum 46,47 . Our study came to the similar conclusions. Non-surgical treatment of periodontitis led to a decrease in IL-6 levels, which indicates a decrease in the intensity of the inflammatory immune response and is consistent with the results of previous studies. 48,49 . De Lima Oliveira found increased IL-6 levels in 25 healthy and 24 subjects with aggressive generalized periodontitis. Periodontal therapy also improved IL-10 levels 50 . Reis et al. evaluated IL-6 and IL-10 concentrations in 52 patients with periodontitis. Nonsurgical periodontal therapy resulted in a statistically significant reduction in total GCF IL-6 levels but not in IL-10 levels. The main function of IL-10 is to regulate the inflammatory response, and it is well known anti-inflammatory cytokine. IL-10 is produced by a broad spectrum of leukocytes including both innate and acquired immune responses, i.e. monocytes, anti-inflammatory M2 macrophages, dendritic cells, mast cells, eosinophils, NK cells, B and T lymphocytes 51,52 . IL-10 is an important regulatory/anti-inflammatory cytokine that prevents periodontal tissue damage by maintaining immune homeostasis 53 . Blocking the gene for IL-10 results in increased alveolar bone resorption and decreased alveolar bone formation 54 . Multiple variants of the IL-10 gene are found in the population based on single nucleotide polymorphisms, with some variants associated with a higher risk of periodontitis or its rapid progression 55 . The immunosuppressive effects of IL-10 are mediated by affecting macrophage function, including their polarization into anti-inflammatory M2 macrophages, leading to a decrease in secretion of pro-inflammatory IL-1, IL-6, TNF-α 53,56 . This is consistent with our results. IL-10 also induces a decrease in the number of Th17 lymphocytes, which produce the aforementioned IL-17 and play an important role in the etiopathogenesis of periodontitis by secreting a number of pro-inflammatory cytokines 53 and chemokines CXCL1/2/5, IL-8/CXCL8, MCP-1, granulocyte colony-stimulating factor and antimicrobial peptides in cells of innate immunity and epithelia 56–58 . IL-10 has been shown to play an important role in preventing alveolar bone resorption by regulating the activation of pro-inflammatory M1 macrophages in mouse models of Porphyromonas gingivalis-induced periodontitis 56 . Our data are consistent with the results of previous studies 59,60 . IL-10 plays an important role in periodontal lesion healing and maintenance of periodontal health through its anti-inflammatory action and inhibition of osteoclastogenesis 53,56,61 . Taiete et al. 59 have also shown that low levels of IL-10 in aggressive forms of periodontitis before treatment are associated with poorer periodontal outcomes and greater depth of PPD after treatment, respectively. In the study by Telese et al. a significant difference in IL-10 was found between 25 healthy patients and 31 patients with generalized aggressive periodontitis. Patients with aggressive periodontitis had a significantly lower IL-10 (p<0.001) compared to subjects with healthy periodontium 60 . Levels of IL-4 and IL-10 in GCF (in rheumatoid arthritis patients and periodontitis patients) was comparable, but lower than in controls 62,63 . Thus, IL-10 may be involved in the control of the inflammatory process. The results support the theory that pro-inflammatory cytokines may be used as markers for success of nonsurgical therapy in patients with periodontitis 64 . Our data generally support the expected behavior of both inflammatory regulators, even with the comment that IL-10 concentrations were very low in our cohort, making interpretation of the results difficult, but were often below the limit of detection in patients with periodontitis, suggesting suppression of the anti-inflammatory immune response. Gamonal et al. determined IL-10 concentrations in GCF of patients with periodontitis. IL-10 was detected in only 43% of patients with periodontitis. Eliminating bacterial plaque decreases antigenic stimulation, which in turn may influence the levels of cytokines present in gingival crevicular fluid 65 . MCP-1 Monocyte chemoattractant protein-1 (MCP-1) is a key chemokine that plays a crucial role in initiating, regulating, and directing the migration of monocytes to sites of severe periodontal inflammation 66 . MCP-1 is produced by various cell types in response to signals such as tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), and interferon-gamma (IFN-γ). These pro-inflammatory cytokines induce MCP-1 expression in human periodontal ligament fibroblasts, promoting monocyte infiltration into inflamed sites 67 . Pradeep et al. found changes in the concentration of MCP-1 in GCF. Elevated MCP-1 levels were detected in patients with periodontitis before treatment, compared to both post-treatment levels and those observed in healthy controls 67 . In a study by Gündogar in 25 periodontitis patients and 24 gingivitis patients and 24 healthy controls, there was a correlation between IL-6, IL-10 and MCP-1 marker levels and the severity of periodontal involvement. The values were always higher in patients with periodontitis 68 . A number of previous studies have confirmed the contribution of MCP-1 to the etiopathogenesis of periodontitis 66,69–75 . In periodontitis, MCP-1 levels are significantly increased compared to healthy individuals, with higher levels of MCP-1 observed in gingival fluid, oral fluid, and serum 69–75 . Successful periodontal therapy then led to a decrease in MCP-1 levels 70 . This was consistent with our results. MCP-1 is thought to be a major chemotactic factor in periodontitis 66 . A study using a mouse model of Porphyromonas gingivalis-induced periodontitis demonstrated that MCP-1 leads to increased expression of the IL-1 receptor antagonist and decreased expression of RANKL and reduced alveolar bone resorption 76 . Kawamoto et al. 47 found no significant difference in oral fluid MCP-1 levels in subjects with severe periodontitis (stage III according to the new classification of periodontal disease) and moderate progression, while MCP-1 levels were statistically significantly lower in subjects with rapid progression. The author hypothesized that it is the lower MCP-1 levels that lead to a reduction in M2 macrophage numbers and ultimately contribute to more pronounced alveolar bone resorption 47,76 . sCD14 The role of CD14 in periodontitis appears to be the activation of CD14-positive cells by interaction with bacterial LPS via TLR 2 and 4 and subsequent activation of the nuclear factor NF kappa B cascade. Immunocompetent cells are not the only cell type in the periodontal region that produce sCD14. It has been shown that gingival fibroblasts stimulated by IFN-γ and LPS can also secrete it . 77 Human oral epithelial cells (GMSM-K lineage) secrete the pro-inflammatory cytokines IL-6 and IL-8 and the chemokine Regulated on Activation of Normal T Cells Expressed and Secreted (RANTES) upon stimulation with sCD14 and may contribute to the pathogenesis of periodontitis, as IL-8 and RANTES affect chemotaxis and neutrophil activity and IL-6 plays an important role in osteoclast activation and formation 78 . sCD14 also stimulates periodontal ligament stem cells, with activation of TLR 2 and 4. Research has shown increased expression and production of IL-6, CXCL8 and MCP-1 chemokines 79,80 by these cells. Human monocytes and macrophages activated by modified low-density lipoprotein secreted IL-10 via CD14 81 . MCP-1 secretion was also demonstrated by umbilical vein endothelial cells following stimulation with inactivated Porphyromonas gingivalis , and MCP-1 production was mediated via sCD14 82 . Previous studies have shown that there is an increase in plasma sCD14 concentrations in periodontitis 83–85 , which is consistent with our results. Bacterial LPS stimulate higher expression of mCD14 by human monocytes and macrophages and consequently there is also an increased release of sCD14 86 . Thus, it is hypothesized that sCD14 originates from periodontal tissues and accumulates in the vasculature by virtue of its increased production rather than reduced clearance 83,85 . In their study, Vijaya et al. 83 published results where sCD14 levels in GCF were statistically significantly lower in individuals with periodontitis compared to healthy individuals. The same results were published by Jin and Darveau 87 . In addition, they found that sCD14 concentrations in GCF were higher in shallower pockets, which may indicate that sCD14 plays a protective role in periodontal tissues. On the other hand, the decrease in sCD14 concentration in deep periodontal pocets may be due to the presence of more microorganisms 87 . It has been shown that gingipains, a cysteine protease produced by Porphyromonas gingivalis , are able to degrade mCD14 on the surface of macrophages or macrophage-like cells (U937 lineage), with proteolysis of CD14 rather than cleavage into a soluble form 88–90 . On the other hand, serum levels of sCD14 showed a weak but statistically significant negative correlation with the level of IgG class antibodies to Aggregatibacter actinomycetemcomitans. Thus, sCD14 appears to be effective in the uptake of LSP Aggregatibacter actinomycetemcomitans 84 . The levels of sCD14 in oral fluid were also monitored. Isaza-Guzman et al. showed statistically significantly higher levels in subjects with periodontitis compared to healthy controls 91 . Non-surgical treatment of periodontitis led to a decrease in sCD14 levels in GCF. The same results were observed in serum 84,85 . The exact function of CD14 in periodontal health and the progression of periodontitis remains unclear. Data in patients with periodontitis are scarce. In our cohort of patients and controls, we clearly demonstrated a significant elevation of sCD14 concentration in patients with periodontitis and its subsequent decrease after successful therapy to values comparable to the control group. MMP-8 Matrix metalloproteinase 8 or is an enzyme present in connective tissue responsible for collagen cleaving 92,93 . The primary function of MMP-8 is the degradation collagen. In periodontitis, MMP-8 is the major collagenase in the gingival connective tissues 94 . MMP-8 is the major collagenase of periodontal tissues, as it is responsible for more than 90% of the collagenolytic activity in gingival fluid, oral fluid and oral rinses 12 . MMP-8 plays a key role in the etiopathogenesis of periodontitis 95 , and it is the disparity between the levels of matrix metalloproteinases and their inhibitors that is responsible for the destruction of periodontal tissues 96 . Monitoring MMP-8 levels in oral fluids has even been recommended as a diagnostic marker of periodontitis 12,97,98 . Although elevated MMP-8 levels in oral fluids are considered a sign of periodontitis, the primary role of this enzyme is defensive. This was demonstrated, for example, in mice infected with Porphyromonas gingivalis , where MMP-8 deficient mice showed significantly higher alveolar bone resorption compared to mice with a functional MMP-8 gene 99,100 . Elevated MMP-8 levels in periodontitis occur not only in gingival fluid but also in oral fluid, as confirmed by several previous studies 96,100–102 . Serum MMP-8 levels also correlate with oral fluid MMP-8 levels and clinical signs of periodontitis 96 . Elevated levels of MMP-8 in gingival and oral fluid also occur in gingivitis, with levels significantly different between healthy subjects and subjects with periodontitis 96,103 . Increased collagenase activity in gingival crevicular fluid and MMP-8 levels correlate with collagen degradation in patients with periodontitis 104 . In a study of 30 patients with periodontitis and 21 healthy controls, Konopka et al. found a significant difference in MMP-8 as well as in MMP-8 levels after treatment in GCF 105 . In Romer's study, 11 patients with periodontitis had a significant decrease in MMP-8 after therapy 106 . Escalona et al. reported elevated levels of cytokines and metalloproteinases in patients with periodontitis, which were significantly associated with the severity of the disease in their study 107 . Our data suggest clear contribution of MMP-8 to the destructive processes of periodontitis, given the orders of magnitude higher MMP-8 concentrations in active inflammation and their decline to healthy tissue-like concentrations after therapy. NGAL Neutrophil gelatinase associated lipocalin (NGAL) is part of the lipocalin protein family. It is mostly expressed by neutrophils and oral epithelial cells. It can bind to both prostaglandins and matrix metalloproteinases 108 . It is also a chemoattractant of neutrophils and increases their capacity as phagocytes as wee as it binds iron ions effectively blocking bacterial metabolism. Lipopolysaccharide (LPS) is a potent inducer of NGAL 109 . Data on the importance of NGAL in periodontitis are scarce. Significant increases in NGAL concentrations occur in association with stimulation by periopathogens such as Porphyromonas gingivalis 110 , and NGAL in periodontal tissues is mainly produced by polymorphonuclears 110,111 . It has also been shown that NGAL is produced by different epithelial cell types, with IL-1β being the stimulus for NGAL expression via the nuclear factor kappa B signaling pathway 112 . Paradoxically, TNF-α does not induce NGAL expression 112 . Westerlund et al. (1996) 111 demonstrated NGAL expression in vitro in oral epithelial cells, including gingival keratinocytes. In vivo , however, NGAL production in oral keratinocytes including gingival epithelial junction cells has not been demonstrated 111 . Bondy-Carey et al. (2013) 110 demonstrated NGAL secretion by gingival keratinocytes following stimulation of Porphyromonas gingivalis cells in vivo. However, significantly higher levels of NGAL were produced by gingival fibroblasts 110 . We are not aware of extensive previous studies on NGAL in periodontitis. Positive associations between NGAL and inflammatory periodontal disease have been scarcely published: an increase in NGAL in the oral fluid occurs in gingivitis, but higher levels are found in individuals with periodontitis 109 . A similar increase in NGAL levels also occurs in serum 109 . Increased levels of NGAL have also been observed in oral fluid and urine in periodontitis 113,114 . Tan et al. then examined NGAL in saliva and serum of patients with periodontitis 109 . Our data showed no significant differences in NGAL between healthy periodontium and pretreatment patients, suggesting a low involvement of NGAL in periodontal inflammation. TSLP Thymic stromal lymphopoietin (TSLP) is crucial for T-cell maturation by activating antigen-presenting cells. Primarily produced by non-hematopoietic cells like fibroblasts and epithelial cells, TSLP facilitates the differentiation of naive CD4+ T cells into Th2 lymphocytes 115 . The role of TSLP in the etiopathogenesis of periodontitis is not entirely clear. Betancur et al. 116 demonstrated increased TSLP expression by oral keratinocytes and macrophages stimulated by Porphyromonas gingivalis. In contrast, application of an alginate gel enriched with TSLP and granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in an increase in FOXP3+ regulatory T-lymphocytes at the application site and in the overlying lymph nodes. These regulatory T-lymphocytes play an important role in the inhibition of the inflammatory immune response in periodontal tissues 117 . Our results demonstrate increased TSLP production in hinge tissues in periodontitis and are consistent with results published by Betancur et al. 116 . Since an increase in B lymphocyte numbers is associated with the progression of periodontal lesions 9 , stimulation of the immune system by TSLP to mount a Th2 immune response 118 may contribute to the progression of periodontitis. Also, Jimenez et al. suggested the presence of TSLP in GCF in periodontitis. Here, in our cohort, we originally demonstrated a clear relationship between TSLP concentrations and periodontal inflammation as well as a significant decrease to below healthy controls after successful therapy. GAL-3 Galectin-3 is a β-galactoside-binding lectin produced by various cell types, including immune cells, epithelial cells, and fibroblasts. It plays a regulatory role in immune cell function and exhibits both pro- and anti-inflammatory activities, depending on factors such as its intracellular or extracellular localization and the specific target cells involved 119 . Galectin-3 directly stimulates the migration and production of inflammatory mediators in the cells of innate immunity 120 . Data regarding galectin-3 in periodontitis are very scarce. In our work, the concentrations of galectin-3 increased after therapy to the level of healthy controls after tissue healing, suggesting an opposite role of galectin-3 as an anti-inflammatory cytokine. There is little information available in the literature regarding Gal-3 and periodontal health. Tamai and Kiyoura 121 demonstrated increased Gal-3 secretion by human gingival epithelia and gingival fibroblasts, but due to stimulation by the yeasts Candida albicans and Candida parapsilosis . Hendek et al. 122 observed significantly higher Gal-3 levels in subjects with gingivitis and periodontitis compared to subjects with healthy periodontium. In both cases, Gal-3 levels decreased following periodontal treatment 122 . Increased Gal-3 levels in oral fluid were observed in subjects with periodontitis compared to healthy controls 123 . Afacan et al. observed elevated concentrations of galectin-3 in GCF in patients with periodontitis and an association with higher grades of periodontitis 124 . Similar conclusions were reached in a study by Ali et al. 125 . Studies overall provide conflicting results. Our results suggest a positive association of Gal-3 with healthy periodontium or healing of periodontal lesions. This is consistent with the work published by Zhang et al., who demonstrated that Mac 2-binding protein together with Gal-3 induces the differentiation of human periodontal ligament stem cells into osteoblasts 126 . The association with the anti-inflammatory effect of Gal-3 is supported by a similar trend of IL-10 and Gal-3 levels, where significantly higher levels were observed in subjects with untreated periodontitis compared to healthy controls. IL-10 has been shown to upregulate Gal-3 expression in M2 macrophages via the STAT3 signaling pathway and is thus involved in the degradation of necrotic tissues in myocardial infarction 127 . Gal-3 also induces IL-10 production in monocytes. The C-terminus of Gal-3, i.e., its lectin domain CRD, is responsible for the increase in secretion; binding of lactose to CRD results in a decrease in IL-10 secretion by monocytes 128 . Limitations of the study The sample size can be considered as a limitation of the study as this number of patients may underepresent general population so the applicability may be limited. Although it may serve as the pilot study for further biomarker research. Another limitation is the selection of molecular marker panel. Obviously, it is not possible with the amount of samples to examine all possible combinations of cytokines. Again, this may aid the future research in the field. Collecting gingival crevicular fluid can be challenging, particularly in individuals with a healthy periodontium. The protein yield from GCF samples cav vary depending on the type of filter paper used for collection 129 . For this reason, our results may not be clearly transferable to other sites. The protocol for analyzing gingival crevicular fluid has not been optimized based on the specific mediator being studied, as a uniform approach is typically used. Cytokines are believed to play a crucial role in the pathogenesis of periodontal disease. However, since periodontal disease is unevenly distributed across the dentition, it remains uncertain to what extent cytokine detection at various sites correlates with disease presence. Strengths of the study One of the strengths of the study is the fact that this is a human study including the control group as well as comparison after therapy in individual patients. Despite the limitations above we feel this study may provide a basis for future biomarker research Multiplex immunoassays provide several advantages over conventional monoplex immunoassays, including greater efficiency and higher data output per sample volume. Given the limited amounts of gingival crevicular fluid obtained from patients and the typically low cytokine concentrations, serial dilution is often not feasible, restricting analysis to a single ELISA per sample. In contrast, multiplex immunoassays enable the simultaneous quantification of multiple cytokines within a single sample, allowing for a comprehensive analysis of various analytes without requiring multiple collections. Conclusion Our study highlights the pivotal role of several major immune systém regulators, especially the IL-6 axis with MMP-8 as a key effector—in the pathogenesis and tissue destruction in periodontitis. It also indicates involvement of other immune components, including potential T cell activation, underscoring the interplay between innate and adaptive immunity. Additionally, we found that successful periodontal treatment markedly reduces inflammatory activity to levels comparable to clinically healthy tissue. Declarations Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University Hospital Hradec Králové (reference number 201909 S13R). All participants were informed about the study objectives and signed an informed consent before inclusion into the study. All rights of the participants were maintained throughout. Consent for publication: Not applicable Availability of data and materials Data available upon reasonable request at corresponding author. Competing interests The authors declare no conflict of interest. Funding Supported by MH CZ - DRO (UHHK, 00179906) and by the Cooperatio Program, research area STOM. Authors' contributions V.R. conceptualized the work, performed the clinical evaluations, statistics, wrote manuscript. O.H. performed clinical evaluations. O.S. performed laboratory analysis, wrote manuscript. C.A. performed laboratory analysis, wrote manuscript. All authors reviewed and accepted manuscript. Acknowledgements None References Papapanou PN, Sanz M, Buduneli N, et al. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J Clin Periodontol . 2018;45(S20):S162-S170. doi:10.1111/jcpe.12946 Slots J. Periodontology: past, present, perspectives. Periodontol 2000 . 2013;62(1):7-19. doi:10.1111/prd.12011 Dentino A, Lee S, Mailhot J, Hefti AF. Principles of periodontology. 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Periodontitis Stage I Stage II Stage III Stage IV Severity Interdental CAL 1-2 mm 3-4 mm ≥5 mm ≥5 mm RBL Coronal third (<15%) Coronal third (15-33%) Extending to middle third of root and beyond Extending to middle third of root and beyond Tooth loss No tooth loss ≤4 teeth ≥5 teeth Complexity Local Max. probing depth ≤4 mm Max. probing depth ≤5 mm In addition to stage II: In addition to stage III: Mostly horizontal bone loss Mostly horizontal bone loss Probing depths ≥6 mm Need for complex rehabilitation due to: Vertical bone loss ≥3 mm Masticatory dysfunction Furcation class II or III Secondary occlusal trauma (mobility ≥2) Moderate ridge defects Severe ridge defects Bite collapse, drifting, flaring <20 remaining teeth (10 opposing pairs) Extent and distribution Add to stage as descriptor For each stage, describe extent as: Localized (<30% of teeth involved) Generalized or Molar/incisor pattern Table 2. Basic characteristics of the patients and the control group Perio group Control group p median IQR median IQR Age 50 (47-55) 46 (44-54) 0.1852 Males 13 13 >0.9999 Females 11 12 DMF 15 (11-20) 13 (8-17) 0.2219 Pretreatment perio group Posttreatment perio group Control group p p p Parameter median IQR median IQR median IQR (pre vs post) (pre vs control) (post vs control) Mean PPD 3.98 (3.58-5.1) 1.7 (1.31-1.96) 1.17 (1.12-1.23) <0.0001 <0.0001 <0.0001 Mean GR 0.163 (0-0.374) 0.307 0.0514-0.443) 0.0357 (0.00606-0.0774) 0.0153 0.0761 0.0003 Mean CAL 4.36 (3.9-5.15) 1.88 (1.6-2.55) 1.2 (1.17-1.26) <0.0001 <0.0001 <0.0001 Mean GI 2.6 (1.84-2.99) 0.0817 (0.0476-0.167) 0.0897 (0.0586-0.119) <0.0001 0.9999 Mean PLI 2.4 (1.04-2.97) 0.0931 (0.0498-0.134) 0.0179 (0-0.0863) <0.0001 <0.0001 0.1345 DMF decay missing filling; PPD probing pocket depth; GR gingival recession; CAL Clinical attachment loss; GI gingival index; PLI plaque index; IQR interquartile range. Table 3. Results of cytokine levels in gingival crevicular fluid Cytokine Pretreatment perio group Posttreatment perio group Control group p p p median IQR median IQR median IQR (pre vs post) (pre vs control) (post vs control) Galectin-3 663 (353-1456) 2253 (519-10955) 2096 (1042-2775) 0.0007 <0.0001 0.7177 IL-10 0 (0-4.3) 0.23 (0.22-0.3) 0.43 (0.43-13.7) 0.9385 0.0001 0.8139 IL-6 31.5 (26.9-47.7) 6.45 (0.85-11.6) 19.6 (12.1-27.3) <0.0001 0.0001 <0.0001 MCP-1 9.2 (6.2-15.2) 5.14 (4.52-5.14) 6.2 (3.42-15.2) <0.0001 0.2292 0.0338 MMP-8 383828 (116360-662430 19855 (4963-47000) 32454 (23830-52250) <0.0001 <0.0001 0.0766 NGAL 23864 (22563-24249) 23809 (21240-25561) 22696 (22039-23407) 0.9441 0.022 0.4099 sCD14 4848 (2090-9759) 832 (297-2578) 904 (399-1286) <0.0001 <0.0001 0.9881 TSLP 0.532 (0.099-0.79) 0.12 (0.12-0.25) 0.396 (0.29-0.532 0.0009 0.0339 <0.0001 IL interleukin; Monocyte chemoatractant protein MCP, MMP matrix metalloproteinase; Neutrophil gelatinase associated lipocalin NGAL ; sCD soluble cluster of differentiation; Thymic stromal lymphopoietin TSLP. Table 4. Comparison of cytokine levels in stage III versus IV periodontitis Dunn's multiple comparisons test Mean rank diff, Summary Adjusted P Value IL-6 Stage 3 vs. IL-6 Stage 4 3,625 ns >0,9999 sCD14 stage 3 vs. sCD14 stage 4 0,25 ns >0,9999 Galectin-3 stage 3 vs. Galectin-3 stage 4 1,531 ns >0,9999 IL-10 stage 3 vs. IL-10 stage 4 -5,781 ns >0,9999 MCP-1 stage 3 vs. MCP-1 stage 4 -2,281 ns >0,9999 MMP-8 stage 3 vs. MMP-8 stage 4 -0,1875 ns >0,9999 NGAL stage 3 vs. NGAL stage 4 0,6563 ns >0,9999 TSLP stage 3 vs. TSLP stage 4 0,5938 ns >0,9999 Table 5. Correlation of clinical characteristics and cytokine concentration in periodontitis patients Average CAL paro pre Average PLI paro pre Average GI paro pre p value p value p value IL-6 0,614 0,950 0,262 sCD14 0,533 0,229 0,303 Galectin-3 0,801 0,994 0,858 IL-10 0,462 0,195 0,188 MCP-1 0,968 0,555 0,550 MMP-8 0,330 0,260 0,098 NGAL 0,305 0,467 0,252 TSLP 0,419 0,914 0,708 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6956664","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493346579,"identity":"9ae40a64-c408-4e48-a860-bbd2981b2d6d","order_by":0,"name":"Vladimíra Radochová","email":"data:image/png;base64,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","orcid":"","institution":"University Hospital Hradec Kralove, Charles University","correspondingAuthor":true,"prefix":"","firstName":"Vladimíra","middleName":"","lastName":"Radochová","suffix":""},{"id":493346580,"identity":"373bd50c-bb6b-4a44-9bc9-138bd336e720","order_by":1,"name":"Ondřej Heneberk","email":"","orcid":"","institution":"University Hospital Hradec Kralove, Charles University","correspondingAuthor":false,"prefix":"","firstName":"Ondřej","middleName":"","lastName":"Heneberk","suffix":""},{"id":493346581,"identity":"a6d50b92-4713-4212-92f8-7376083fefe5","order_by":2,"name":"Ondřej Souček","email":"","orcid":"","institution":"University Hospital Hradec Kralove, Charles University","correspondingAuthor":false,"prefix":"","firstName":"Ondřej","middleName":"","lastName":"Souček","suffix":""},{"id":493346582,"identity":"6196cc2c-2bbd-4b66-8e3f-be335f72141d","order_by":3,"name":"Ctirad Andrýs","email":"","orcid":"","institution":"University Hospital Hradec Kralove, Charles University","correspondingAuthor":false,"prefix":"","firstName":"Ctirad","middleName":"","lastName":"Andrýs","suffix":""}],"badges":[],"createdAt":"2025-06-23 12:08:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6956664/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6956664/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88096361,"identity":"08438322-91c9-4abc-af58-abb06e1ebd5d","added_by":"auto","created_at":"2025-08-01 10:56:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1033684,"visible":true,"origin":"","legend":"\u003cp\u003eClinical parameters of patients and controls\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6956664/v1/bc12900b53bd2ee066c3d86c.png"},{"id":88094502,"identity":"9cc45ed8-9953-4595-9ed5-beeffcf9e1b5","added_by":"auto","created_at":"2025-08-01 10:48:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1676084,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine levels of patients and controls\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6956664/v1/8b4757975a218d9a2e78864c.png"},{"id":88097498,"identity":"17765961-7394-43de-ad1f-456bcefbd4ee","added_by":"auto","created_at":"2025-08-01 11:04:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4729264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6956664/v1/a7365601-0a1a-4de3-8f51-eaa2ce509959.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Levels of selected inflammatory parameters in gingival crevicular fluid of patients with stage III and IV periodontitis","fulltext":[{"header":"Clinical Relevance","content":"\u003cp\u003eBackground:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study investigated the cytokine profile in the gingival crevicular fluid of patients with advanced periodontitis to understand its role in the disease\u0026apos;s pathogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdded value of the study: By analyzing key inflammatory markers, the research revealed significant elevations in IL-6, MMP-8, and other cytokines associated with neutrophilic and T cell-mediated responses in periodontitis patients. After treatment, a marked reduction in these inflammatory markers was observed, suggesting a return to near-normal levels, comparable to those in healthy individuals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinical implications: The findings highlight the role of neutrophilic inflammation and the immune system\u0026apos;s complex interplay in periodontal disease and its resolution post-treatment and provide the possible options for disease activity monitoring.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003ePeriodontitis a chronic inflammatory disease associated with bacterial dysbiosis and is characterized by progressive destruction of the periodontal tissues around the tooth\u003csup\u003e1\u003c/sup\u003e. \u0026nbsp;The causal role of pathogenic bacteria in the biofilm has been shown numerous times. The bacteria initiate inflammatory reactions in the gingival tissues and are responsible for further progression. Interactions of immunity of the host and inflammatory response are major determinants of susceptibility to this disease where also various environmental and genetic factors play a role\u003csup\u003e2\u003c/sup\u003e. The cellular component of immunity, whether innate or adaptive, is the basis for all immune processes\u003csup\u003e3\u003c/sup\u003e. The different components of immunity play a role in the development of periodontal inflammation. As the periodontal inflammation increases, more damage to the tissues is done and periodontitis advances from initial stages to more severe forms such as stage III and IV especially defined as extensive loss of periodontal tissues and alveolar bone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe initial inflammatory response to bacteria present in dental plaque is caused by leukocytes and endothelial cells. The products of bacterial metabolism stimulate the cells of the epithelium to produce cytokines and stimulate the production of vasoactive peptides that cause vasodilation of local blood vessels. Neutrophils migrate towards the site of inflammation in response to chemokines. Increasing numbers of neutrophils especially in the connective tissue is followed by the appearance of macrophages, lymphocytes, plasma cells and mast cells. The production of gingival fluid increases due to activation of complement proteins. Macrophages, plasma cells and T and B cells dominate the later stages\u003csup\u003e4\u003c/sup\u003e. Cytokines play an important role in the progression of periodontitis\u0026nbsp;\u003csup\u003e5\u003c/sup\u003e. They also play a role in maintaining either protective or destructive inflammatory processes\u003csup\u003e6,7\u003c/sup\u003e. A balance between pro-inflammatory and anti-inflammatory cytokines can protect periodontal tissue from destruction and their respective imbalance leads to disease progression\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe pathogenesis of periodontal pocket involves a whole plethora of immune system cells. The dominant cells are plasma cells and lymphocytes, with B lymphocytes predominating. \u0026nbsp;While the dominance of T lymphocytes is associated with relative stability, the predominance of B lymphocytes leads to the development of periodontal pocket\u0026nbsp;\u003csup\u003e9\u003c/sup\u003e. Polymorphonuclear leukocytes migrate through the epithelial lining of the periodontal pocket, where they form a barrier between the microbial biofilm and the epithelium. The periodontal epithelium shows increased permeability and ulcerations, which allows bacterial toxins to penetrate the periodontal tissues and stimulates the inflammatory immune response and the production of pro-inflammatory cytokines such as IL-1, IL-6, TNF-\u0026alpha;, prostaglandin E\u003csub\u003e(2) (\u003c/sub\u003ePGE\u003csub\u003e2\u003c/sub\u003e)\u003csup\u003e10\u003c/sup\u003e . This leads to connective tissue destruction and resorption of adjacent alveolar bone.\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eConnective tissue remodeling is the result of intercellular interaction and interaction of cells with the extracellular matrix. This includes the production of enzymes, their activators and inhibitors, cytokines and growth factors\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e. The most important enzymes degrading various components of the extracellular matrix are matrix metalloproteinases (MMPs), which are produced by monocytes/macrophages, neutrophils, fibroblasts, as well as epithelial cells. An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) is thought to be the main cause of connective tissue destruction. Increased proteolytic activity associated with periodontal tissue destruction has been described for matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13\u0026nbsp;\u003csup\u003e12\u0026ndash;15\u003c/sup\u003e . Bone resorption in periodontitis is induced by bacterial toxins, including lipopolysaccharides (LPS) or lipopeptides, which stimulate pattern recognition receptors , in this case various types of Toll-like receptors (TLRs). This leads to the secretion of pro-inflammatory cytokines (e.g. IL-1, IL-6, TNF-\u0026alpha;, PGE\u003csub\u003e2)\u0026nbsp;\u003c/sub\u003e, which in turn affect osteoblasts and osteoclasts, or these cells can be stimulated directly\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e . Both IL-1 and bacterial LPS directly stimulate osteoblasts to express more RANKL and IL-6 and reduce OPG production. In osteoclasts, LPS and IL-1 stimulate their activation and play an important role in their survival\u003csup\u003e19,20\u003c/sup\u003e. Epithelial cells of the gingiva do not only perform a barrier function to the external environment, hence to microorganisms and their products. They express a whole set of receptors for dangerous PPR patterns on their membrane, with the help of which the epithelia are able to identify pathogens in their vicinity. Upon stimulation by bacterial products, they secrete pro-inflammatory cytokines, including IL-1\u0026alpha;, IL-1\u0026beta;, IL-6, IL-8, TNF-\u0026alpha;. They also produce substances that have bactericidal effects, called defensins, and also activate immature dendritic cells\u003csup\u003e21\u003c/sup\u003e . Antigen presenting cells play an important regulatory role in controlling the inflammatory immune response. They are widely distributed in peripheral tissues, including the gingiva, where they phagocytose microorganisms whose components are subsequently degraded. Fragments of microbial molecules are then presented to T lymphocytes by major histocompatibility complex II, together with costimulatory signals and appropriate cytokine microenvironments. This process is a key regulatory mechanism and leads to the initiation of an inflammatory immune response or tolerance\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e. Dendritic cells also produce polarizing cytokines with pro-inflammatory (TNF-\u0026alpha;, IL-1\u0026beta;) or anti-inflammatory (transforming growth factor \u0026beta;, IL-10) effects\u0026nbsp;\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe recognition of microbes by antigen-presenting cells, but also by other cells of non-specific immunity, occurs via pathogen-associated molecular pattern receptors, PAMPs, such as LPS. They can also recognize tissue damage signals (damage-associated molecular patterns, DAMPs), i.e. intracellular proteins or molecules derived from the extracellular matrix. Recognition of PAMPs and DAMPs by cells of nonspecific immunity provides information about damage to the body\u0026apos;s tissues or the presence of an infectious agent\u003csup\u003e24\u003c/sup\u003e . Danger pattern receptors include TLR receptors or CD14\u003csup\u003e22\u003c/sup\u003e . Neutrophils translocate from the bloodstream to peripheral tissues at sites where resident cells recognize danger signals (PAMP/DAMP) and produce mediators such as nitric oxide, IL-1\u0026beta;, TNF-\u0026alpha;, and IL-17. Increased production of pro-inflammatory cytokines IL-1\u0026beta;, IL-6, IL-8 and TNF-\u0026alpha; by neutrophils circulating in the vasculature was also demonstrated in subjects with periodontitis and persisted after successful periodontal therapy. This indicates a possible \u0026quot;training\u0026quot; of neutrophils during myelopoiesis\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e. Neutrophils also form so-called neutrophil extracellular traps. This is a specific type of programmed cell death where, following massive stimulation of neutrophil surface receptors, nuclear remodeling and excretion of chromatin in the form of filaments outside the cell occurs along with secretion of granule contents, hence enzymes, antibacterial agents and reactive oxygen species\u0026nbsp;\u003csup\u003e26,27\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMacrophages in tissues can perform different functions, so-called M1 macrophages, classically activated macrophages stimulating the inflammatory immune response and acquired immunity. The dominant factors stimulating polarization in M1 macrophages in periodontitis are IFN-\u0026gamma; produced by Th1 cells, stimulation of TLRs and other danger pattern receptors, including CD14, by bacterial LPS\u003csup\u003e28\u003c/sup\u003e . Other activating stimuli include reactive oxygen species, granulocyte-macrophage colony-stimulating factor, and micro ribonucleic acid miRNA\u003csup\u003e29\u003c/sup\u003e . M1 macrophages produce a number of pro-inflammatory cytokines (e.g. IL-1\u0026beta;, IL-6, IL-12, TNF\u0026alpha;), prostaglandins (PGE\u003csub\u003e2)\u0026nbsp;\u003c/sub\u003e, chemokines (Mononuclear chemoattractant 1), adhesion molecules, reactive oxygen species and hydrolytic enzymes (e.g. various types of MMPs)\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e. The production of proinflammatory cytokines M1 by macrophages leads to further activation of the inflammatory immune response, including secretion of MMPs by other cell types (e.g. fibroblasts) and production of the soluble form of RANKL by T and B lymphocytes\u003csup\u003e30\u0026ndash;32\u003c/sup\u003e. Lymphocytes are a major component of the specific immune response. As already mentioned, lymphocytes are dominant cells in early, advanced/progressive and advanced periodontal lesions\u003csup\u003e33\u003c/sup\u003e . CD4 T lymphocytes have an important regulatory function: Dominance of helper Th2 lymphocytes leads to higher numbers of B lymphocytes and plasma cells, leading to lesion progression\u0026nbsp;\u003csup\u003e34\u003c/sup\u003e. In contrast, dominance of the Th1 subset is associated with stable periodontal lesions\u003csup\u003e34\u003c/sup\u003e. Treg are responsible for suppression of the immune response by producing anti-inflammatory cytokines such as IL-10 and expressing CD 152\u0026nbsp;\u003csup\u003e35\u003c/sup\u003e. Th17 produce pro-inflammatory cytokines IL-6, IL-17, IL-23\u003csup\u003e36\u003c/sup\u003e. The imbalance between Th17 and Treg lymphocytes affects bone metabolism and bone resorption in periodontitis via RANKL/RANK/OPG\u0026nbsp;\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe aim of our study is to map multiple components of the cytokine profile of GCF collected from the site of inflammation (periodontal pocket) in patients with stage III and IV periodontitis. Furthermore, to compare this cytokine profile in the same patients after periodontal therapy and to compare the profile with a control group of individuals with healthy periodontium. As described above, key regulators of the various components of the immune system were selected for analysis to evidence the expected activity of immune system components involved in the pathogenesis of periodontitis: soluble CD14 (sCD14), Galectin3, Interleukin-6 (IL-6), Interleukin-10 (IL-10), Monocyte chemoattractant protein-1 (MCP-1), Matrix metalloproteinase 8 (MMP-8), Neutrophil gelatinase associated lipocalin (NGAL) and Thymic stromal lymphopoietin (TSLP). The null hypothesis was that there is no difference in cytokine concentration in patients with stage III or IV periodontitis before and after therapy and compared with control group of individuals with healthy periodontium.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe study participants were recruited from patients of the Periodontology Division of the Department of Dentistry of the University Hospital in Hradec Kralove, Czech Republic. Data collection took place from December 2019 to January 2022. All individuals who visited the periodontology department and met the exclusion and inclusion criteria were approached to enter the experimental cohort study.\u003c/p\u003e\n\u003cp\u003ePatients aged 18 years and older participated in the study. The primary inclusion criterion was the presence of stage III or IV periodontitis. Inclusion in stage III or IV was performed based on the current 2018 European Federation of Periodontology recommendations\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e. In brief, the classification into stage III or IV based primarily on interdental CAL at site of greatest loss ≥ 5 mm and other criteria listed in Table 1\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e. The healthy periodontium was classified on the basis of gingival bleeding in all examined sites BOP \u0026lt; 10% Probing pocket depths ≤4 mm (no site ≥ 4 mm with BOP) in reduced periodontium, in non-reduced Probing pocket depths ≤3 mm\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e. The control group included 25 patients matched in sex and age to those in the experimental group who met the criteria for a healthy periodontium according to the same recommendations. These were patients of the Department of Dentistry in Hradec Kralove. All study subjects had to be non-smokers who were generally healthy without systemic disease.\u003c/p\u003e\n\u003cp\u003eIndividuals meeting the inclusion criteria were added to both groups throughout the data collection period until the desired sample size was reached. One patient in the experimental group did not complete periodontal therapy and was excluded from the posttreatment evaluation.\u003c/p\u003e\n\u003cp\u003eAll subjects underwent a complete examination of the dentition, periodontal and oral mucosa. Periodontal examination was performed on all teeth in the oral cavity except the 3rd molars using a PCPUNC-15 manual dental probe (Hu-Friedy, Chicago, IL, USA), including recording of the following periodontal parameters: probing depth, clinical attachment level, and bleeding on probing (BOP). Parameters were recorded at 6 sites (distobuccal, distoral, buccal, oral, inter-buccal, and non-buccal). The examination was performed by two experienced physicians, V.R. and O.H., who were trained to perform this study. Inter-examiner reliability was assessed using kappa coefficients before the study. To ensure consistency and reliability in the clinical measurements taken during the study—specifically probing depth and clinical attachment level—inter-examiner agreement was assessed. Inter-examiner agreement refers to the level of consistency between different examiners (e.g., dentists or clinicians) when measuring the same parameters on the same subjects. In this study, the kappa values for inter-examiner agreement exceeded 90%, indicating very high agreement between different examiners. Kappa values range from 0 to 1, with values above 0.80 generally considered to reflect excellent agreement beyond what would be expected by chance. X-rays were also taken to detect bone loss namely orthopantomogram.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthical considerations\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University Hospital Hradec Králové (reference number 201909 S13R). All participants were informed about the study objectives and signed an informed consent before inclusion into the study. All rights of the participants were maintained throughout. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure the study had sufficient statistical strength to detect meaningful differences, a sample size calculation was performed prior to data collection. The calculation was based on a \u003cstrong\u003etarget significance level (alpha) of 0.05\u003c/strong\u003e, which indicates a 5% risk of concluding that a difference exists when there is none (i.e., a Type I error). Additionally, the study aimed for a \u003cstrong\u003estatistical power of 0.80 (or 80%)\u003c/strong\u003e, meaning there was an 80% chance of correctly detecting a true effect (i.e., avoiding a Type II error) if one exists.\u003c/p\u003e\n\u003cp\u003eThe calculation was designed to detect a \u003cstrong\u003eminimum difference of 20% in the observational parameters\u003c/strong\u003e (such as clinical measurements or biomarker levels) between the groups. This threshold represents the smallest difference that the researchers considered to be clinically or scientifically relevant.\u003c/p\u003e\n\u003cp\u003eBased on these assumptions—a significance level of 0.05, power of 0.80, and a minimum effect size of 20%—the required \u003cstrong\u003esample size was determined to be 25 individuals per group\u003c/strong\u003e. The study thus included \u003cstrong\u003ethree groups\u003c/strong\u003e: a control group (without periodontitis), an experimental group (likely receiving a specific intervention or condition), and a periodontitis treatment group. This design allows for meaningful comparisons across different conditions while maintaining adequate statistical power.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Continuous variables are presented as median and interquartile range (IQR). Categorical variables were compared using Chi-square and Fisher's exact test and are presented as counts and percentages (%). Variables that did not show a normal distribution were analyzed using the Mann-Whitney \u003cem\u003eU\u0026nbsp;\u003c/em\u003etest and Wilcoxon test and results are presented as median and IQR. Differences were considered significant at p \u0026lt; 0.05. All p values were obtained from two-tailed tests and all statistical analyses were performed using GraphPadPrism v. 10.2.3 software (GraphPad Software, USA) and MS Excel 365 (Microsoft, USA).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTherapy of patients with periodontitis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients with periodontitis were treated according to the guidelines for periodontitis therapy\u003csup\u003e39\u003c/sup\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eAll underwent the active phase of periodontitis treatment. The first phase of therapy focused on behavior changes by motivating the patient to successfully remove supragingival dental biofilm, control of supragingival plaque, interventions to improve oral hygiene, professional mechanical plaque removal and calculus as well as any factors that impair oral hygiene practices, and adjunctive treatment for gingivitis. At the beginning of the treatment, chlorhexidine 0.12% was used for 14 days. In the second step of the therapy, we focused on the control (reduction/removal) of subgingival plaque and calculus (scaling and root planning). In patients initially presenting with significant purulent exudation from the periodontal pocket after the first step of therapy, additional systemic antimicrobials (antibiotics Amoxicillin and Metronidazole) were used as adjunctive therapy\u003csup\u003e40\u003c/sup\u003eResponse to the second step of therapy was assessed at least 8 weeks after subgingival treatment. Periodontal healing was achieved in all but 1 patient (here a periodontal pocket 7 mm without bleeding). \u0026nbsp;This means no periodontal pockets \u0026gt;4 mm with bleeding on probing or no deep periodontal pockets ≥6 mm. However, in some situations extractions were chosen at the beginning of therapy. These included teeth with significant attachment loss, grade III mobility, pulpoperiodontal involvement without good prognosis for therapy, 3\u003csup\u003erd\u003c/sup\u003e molars, and both solitary and further prosthodontically unusable teeth.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eGingival fluid sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGingival crevicular fluid collection was performed 3 days after periodontal examination in all participants. In the experimental group, in addition to the collection at the beginning of treatment, GCF sampling was also performed 3 months after the completion of nonsurgical treatment of periodontitis. GCF samples were obtained in patients with periodontitis from the most affected site, i.e., the deepest periodontal pocket. the same tooth was selected if it was present in the mouth after therapy (in the experimental group). If this tooth was extracted, the next tooth with the deepest pocket before therapy was chosen. In subjects with a healthy periodontium, the highest probing site was also sampled. Prior to gingival crevicular fluid sampling, all sites were isolated with cotton rollers to prevent contamination with saliva. Supragingival plaque was removed, and the tooth was gently air dried. Sterile ISO 40 paper pins were inserted into the gingival sulcus/pocket until slight resistance was felt and were left in place for 30 seconds to allow absorption of gingival crevicular fluid from the site and to prevent bleeding. \u0026nbsp;Pins visually contaminated with blood were not used for analysis. The paper pin was then placed in a 5.0 ml CryoPure tube with 0.5 ml of PBS solution. The tube with the paper pin was \"shaken\" on a shaker (1400/min) for 20 minutes. The paper plug was then removed from the tube using sterile tweezers. The tube was centrifuged for 15 minutes at 1800 rpm. The supernatant was pipetted into 200 µl aliquots into 0.5 ml micro tube tubes. Aliquots were stored and frozen at -70°C. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of immune markers from GCF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Human cytokine premixed magnetic Luminex® performance assay was chosen for the analysis of selected cytokines in gingival fluid samples: CD14, Galectin-3, IL-6, IL-10, MCP-1, MMP-8, NGAL, and TSLP (R\u0026amp;D systems, Minneapolis, MN, USA). Cytokine levels were determined using Luminex xMAP® magnetic technology by Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA), analysis of the results was performed using xPONENT® 4.2. analysis software (Luminex Corporation, Austin, TX, USA). Briefly, Luminex xMAP® is a multiplex bead-based immunoassay platform that enables the simultaneous detection and quantification of up to 500 different analytes (such as proteins, cytokines, or nucleic acids) in a single sample. At the core of the system are microspheres (or beads) that are internally dyed with distinct ratios of red and infrared fluorophores. Each unique ratio produces a specific spectral signature, also known as a bead region, allowing the system to differentiate between up to 500 bead types in one assay. Each bead region is coated with a specific capture antibody that binds only to a particular analyte of interest. When a sample is added to the beads, the target analytes bind to their respective antibodies on the corresponding bead types. After unbound substances are washed away, the beads are incubated with: Biotinylated detection antibodies, which bind to a different epitope on the captured analyte. Streptavidin-phycoerythrin (PE), a fluorescent molecule that binds to biotin and acts as a reporter signal. The bead-analyte-detection antibody-reporter complex is then analyzed using a Luminex instrument equipped with two lasers, the first identifies the bead region by detecting the internal fluorophore ratio—this tells the system which analyte is being measured. Laser 2 measures the intensity of the phycoerythrin (PE) fluorescence, which is directly proportional to the amount of analyte bound to the bead. Multiple readings (typically 50–100) are collected for each bead region, ensuring high sensitivity, accuracy, and reproducibility in quantification. Gingival fluid samples were diluted 2-fold. The assay was done according to the manufacturer’s instructions. All values were expressed in nanograms (ng) per milliliter of gingival fluid.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 25 patients with periodontitis who met the entry criteria listed above were included in the study. The median age was 50 years (IQR 47-55 years), 13 were male (52%) and 12 were female (48%). One patient did not complete periodontitis therapy, and 24 patients were then analyzed for the final evaluation. The control group included 25 healthy controls with a median age of 46 years (IQR 44 - 54 years), 13 (52%) were male and 12 were female (48%). The groups did not differ in age or gender. The periodontitis group had a statistically significantly different (p\u0026lt;0.0001) median attachment loss of 4.36 (IQR 3.9-5.15) compared to the control group of 1.20 (IQR 1.17-1.26). After therapy, patients in the periodontitis group showed a statistically significant improvement in attachment loss to a median value of 1.88 (IQR 1.6-2.55, p\u0026lt;0.0001). After therapy, the median attachment loss remained significantly higher in the periodontitis group compared to the healthy periodontitis group (p\u0026lt;0.Similar results could be observed for other monitored parameters, i.e., dentition depth, gingival index and plaque index. The GI and PLI parameters were comparable in the posttreatment group with those of the control group. The clinical characteristics of the patients are described in detail in Table 2 and Figure 1.\u003c/p\u003e\n\u003cp\u003eThe levels of individual cytokines in the initial crevicular fluid samples showed significantly higher concentrations than in the control group in most cases. All results are described below and summarized in Table 3 and Figure 2. Cytokine concentration did not differ between patients with stage III compared to stage IV periodontitis (Table 4).\u003c/p\u003e\n\u003cp\u003eIL-6 concentrations in the periodontitis group were with a median of 31.5 ng/ml (IQR 26.9-47) and 19.6 ng/ml (IQR 12.1-27.3, p=0.0001) in the control group. After treatment, IL-6 concentrations significantly decreased to a median of 6.45 ng/ml (IQR 0.85-11.6, p\u0026lt;0.0001). IL-6 concentration was significantly lower in the treated group compared to the control group after therapy (p\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003eIL-10 concentrations in the periodontitis group were very often below the detection limit, complicating the evaluation, with a median of 0 ng/ml (IQR 0-4.3) and 0.43 ng/ml (IQR 0.43-13.7, p=0.0001) in the control group. After therapy, IL-10 concentrations remained unchanged with a median of 0.23 ng/ml (IQR 0.23-03, p=0.9385). IL-10 concentrations after therapy were comparable in the treatment group compared to the control group (p=0.8139).\u003c/p\u003e\n\u003cp\u003eThe sCD14 concentrations in the periodontitis group were with a median of 4848 ng/ml (IQR 2090-9759) and 904 ng/ml (IQR 399-1286, p\u0026lt;0.0001) in the control group. sCD14 concentrations significantly decreased after treatment to a median of 832 ng/ml (IQR 297-2578, p\u0026lt;0.0001). sCD14 concentration after therapy compared to the control group was comparable in the treatment group compared to the control group (p=0.9881).\u003c/p\u003e\n\u003cp\u003eThe median galectin-3 concentrations in the periodontitis group were 663 ng/ml (IQR 353-1456) and 2096 ng/ml (IQR 1042-2775, p\u0026lt;0.0001) in the control group. After therapy, the galectin-3 concentration significantly increased to a median of 2253 ng/ml (IQR 519-10955, p=0.0007). The galectin-3 concentration after therapy was comparable in the treatment group compared to the control group (p=0.7177).\u003c/p\u003e\n\u003cp\u003eThe median MCP-1 concentrations in the periodontitis group were 9.2 ng/ml (IQR 6.2-15.2) and 6.2 ng/ml (IQR 3.42-15.2, p=0.2292) in the control group. After therapy, MCP-1 concentration significantly decreased to a median of 5.14 ng/ml (IQR 4.52-5.14, p\u0026lt;0.0001). MCP-1 concentration was lower in the treatment group compared to the control group after therapy (p=0.0338).\u003c/p\u003e\n\u003cp\u003eThe median MMP-8 concentrations in the periodontitis group were 383828 ng/ml (IQR 116360-662430) and 32454 ng/ml (IQR 23830-52250, p\u0026lt;0.0001) in the control group. After therapy, MMP-8 concentrations significantly decreased to a median of 19855 ng/ml (IQR 4963-47000, p\u0026lt;0.0001). MMP-8 concentrations after therapy were comparable in the treatment group compared to the control group (p=0.0766).\u003c/p\u003e\n\u003cp\u003eThe median NAGL concentrations in the periodontitis group were 23864 ng/ml (IQR 22563-24249) and 22696 ng/ml (IQR 22039-23407, p=0.022) in the control group. After treatment, NGAL concentrations remained unchanged with a median of 23809 ng/ml (IQR 21240-25561, p=0.9441). NGAL concentrations after therapy compared to the control group were comparable in the treatment group compared to the control group (p=0.4099).\u003c/p\u003e\n\u003cp\u003eThe median TSLP concentrations in the periodontitis group were 0.532 ng/ml (IQR 0.099-0.79) and 0.396 ng/ml (IQR 0.29-0.532, p=0.0339) in the control group. After therapy, TSLP concentrations decreased to a median of 0.12 ng/ml (IQR 0.12-0.25, p=0.0009). TSLP concentrations were lower in the treatment group compared to the control group after therapy (p\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003eThere was no correlation between initial cytokine concentration and clinical parameters in periodontitis patients (Table 5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study confirms that gingival crevicular fluid (GCF) serves as a reliable medium for assessing immune responses in periodontal disease. Key findings of our results reveal: Elevated IL-6, MCP-1, MMP-8, and sCD14 concentrations in active periodontitis. Decreased IL-10 and Galectin-3 levels in diseased states, with post-treatment normalization suggesting a regulatory role. TSLP levels strongly associated with disease activity and significantly decreased following therapy. NGAL did not show significant differences, implying limited involvement in periodontitis pathology. Our findings reinforce the potential utility of GCF biomarkers for monitoring disease progression and treatment outcomes.\u003c/p\u003e\n\u003cp\u003eOur study aimed at broad view of the involvement of individual parts of the immune system in the pathogenesis of active severe periodontitis. We have shown that even in clinically healthy periodontium, a certain amount of pro- and anti-inflammatory cytokines are present, and their concentrations are in some cases even higher than in patients after successful periodontal treatment (IL-6, MCP-1, TSLP). During inflammation, the flow of fluid into the gingival crevice increases, accompanied by a rise in cellular infiltrate. Neutrophils serve as the primary cellular mechanism in the gingival crevice, with most appearing as viable cells capable of eliminating microorganisms. As this fluid passes through the junctional epithelium into the gingival crevice, it contributes to an elevated flow of gingival crevicular fluid\u003csup\u003e33\u003c/sup\u003e. A variety of cytokines are released by sulcular and epithelial cells, dendritic cells, connective tissue fibroblasts, macrophages, and neutrophils. Additionally, these cells produce enzymes, including matrix metalloproteinases, which contribute to the breakdown of connective tissue collagen and alveolar bone\u003csup\u003e41\u003c/sup\u003e. Host susceptibility is an important factor in the pathogenesis of periodontal disease, giving the level of inflammatory mediators present in the gingival fluid. Various cytokine-mediated inflammatory pathways and microbial stimuli may contribute to the development of periodontal disease. However, these pathways are likely to converge, resulting in similar metabolite profiles. For instance, one microbial community structure may trigger distinct inflammatory responses, while a different microbial community may elicit an alternative inflammatory signature. Additionally, variations in host genetics can influence microbial colonization and inflammatory responses \u003csup\u003e42\u003c/sup\u003e. Available studies suggest that gingival crevicular fluid is an excellent source of periodontal disease biomarkers that reliably reflect immune interactions\u0026nbsp;\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eClinical parameters of periodontitis\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study clearly demonstrates that periodontal therapy improves the status of periodontal tissues. Our study demonstrates extremely significant differences between all clinical variables between patients with periodontitis and healthy controls. However, the differences between periodontal status in patients after therapy compared to healthy individuals are still seen. \u0026nbsp; Although GI and PLI improves after therapy to comparable levels as healthy individuals, the differences in PPD, GR and CAL remain, although significantly lower compared to values before the therapy. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eIL-6 and IL-10\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-6 plays a role especially in the initiation and acute phase of periodontitis. IL-6 is expressed by a variety of cells, including phagocytic cells, T cells, B cells, fibroblasts, endothelial cells, keratinocytes and also other tissues such as hepatocytes or bone marrow cells. It is also important in the secretion of acute phase proteins, which is done in cooperation with IL-1\u0026beta;\u003csup\u003e43\u003c/sup\u003e. IL-6 is also considered an osteoclastogenic cytokine because higher levels lead to increased bone loss\u0026nbsp;\u003csup\u003e44\u003c/sup\u003e. IL-6 plays a key role in triggering acute phase responses while also facilitating specific cellular and humoral immune reactions by promoting B-cell differentiation, immunoglobulin secretion, and T-cell activation. As a result, IL-6 serves as a crucial regulator in the transition from acute to chronic inflammation\u0026nbsp;\u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePeriodontitis is characterized by elevated levels of IL-6, which have been observed in GCF, oral fluid, and serum\u0026nbsp;\u003csup\u003e46,47\u003c/sup\u003e. Our study came to the similar conclusions. Non-surgical treatment of periodontitis led to a decrease in IL-6 levels, which indicates a decrease in the intensity of the inflammatory immune response and is consistent with the results of previous studies.\u0026nbsp;\u003csup\u003e48,49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDe Lima Oliveira found increased IL-6 levels in 25 healthy and 24 subjects with aggressive generalized periodontitis. Periodontal therapy also improved IL-10 levels\u0026nbsp;\u003csup\u003e50\u003c/sup\u003e. Reis et al. evaluated IL-6 and IL-10 concentrations in 52 patients with periodontitis. Nonsurgical periodontal therapy resulted in a statistically significant reduction in total GCF IL-6 levels but not in IL-10 levels.\u003c/p\u003e\n\u003cp\u003eThe main function of IL-10 is to regulate the inflammatory response, and it is well known anti-inflammatory cytokine. IL-10 is produced by a broad spectrum of leukocytes including both innate and acquired immune responses, i.e. monocytes, anti-inflammatory M2 macrophages, dendritic cells, mast cells, eosinophils, NK cells, B and T lymphocytes\u0026nbsp;\u003csup\u003e51,52\u003c/sup\u003e .\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIL-10 is an important regulatory/anti-inflammatory cytokine that prevents periodontal tissue damage by maintaining immune homeostasis\u0026nbsp;\u003csup\u003e53\u003c/sup\u003e. Blocking the gene for IL-10 results in increased alveolar bone resorption and decreased alveolar bone formation\u0026nbsp;\u003csup\u003e54\u003c/sup\u003e. Multiple variants of the IL-10 gene are found in the population based on single nucleotide polymorphisms, with some variants associated with a higher risk of periodontitis or its rapid progression\u0026nbsp;\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe immunosuppressive effects of IL-10 are mediated by affecting macrophage function, including their polarization into anti-inflammatory M2 macrophages, leading to a decrease in secretion of pro-inflammatory IL-1, IL-6, TNF-\u0026alpha;\u003csup\u003e53,56\u003c/sup\u003e . This is consistent with our results.\u003c/p\u003e\n\u003cp\u003eIL-10 also induces a decrease in the number of Th17 lymphocytes, which produce the aforementioned IL-17 and play an important role in the etiopathogenesis of periodontitis by secreting a number of pro-inflammatory cytokines \u003csup\u003e53\u003c/sup\u003e and chemokines CXCL1/2/5, IL-8/CXCL8, MCP-1, granulocyte colony-stimulating factor and antimicrobial peptides in cells of innate immunity and epithelia\u003csup\u003e56\u0026ndash;58\u003c/sup\u003e. IL-10 has been shown to play an important role in preventing alveolar bone resorption by regulating the activation of pro-inflammatory M1 macrophages in mouse models of \u003cem\u003ePorphyromonas gingivalis-induced\u003c/em\u003e periodontitis \u003csup\u003e56\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur data are consistent with the results of previous studies \u003csup\u003e59,60\u003c/sup\u003e. IL-10 plays an important role in periodontal lesion healing and maintenance of periodontal health through its anti-inflammatory action and inhibition of osteoclastogenesis \u003csup\u003e53,56,61\u003c/sup\u003e. Taiete et al. \u003csup\u003e59\u003c/sup\u003e have also shown that low levels of IL-10 in aggressive forms of periodontitis before treatment are associated with poorer periodontal outcomes and greater depth of PPD after treatment, respectively. In the study by Telese et al. a significant difference in IL-10 was found between 25 healthy patients and 31 patients with generalized aggressive periodontitis. Patients with aggressive periodontitis had a significantly lower IL-10 (p\u0026lt;0.001) compared to subjects with healthy periodontium\u003csup\u003e60\u003c/sup\u003e. Levels of IL-4 and IL-10 in GCF (in rheumatoid arthritis patients and periodontitis patients) was comparable, but lower than in controls \u003csup\u003e62,63\u003c/sup\u003e. Thus, IL-10 may be involved in the control of the inflammatory process. The results support the theory that pro-inflammatory cytokines may be used as markers for success of nonsurgical therapy in patients with periodontitis \u003csup\u003e64\u003c/sup\u003e. Our data generally support the expected behavior of both inflammatory regulators, even with the comment that IL-10 concentrations were very low in our cohort, making interpretation of the results difficult, but were often below the limit of detection\u0026nbsp;in patients with periodontitis, suggesting suppression of the anti-inflammatory immune response. Gamonal et al. determined IL-10 concentrations in GCF of patients with periodontitis. IL-10 was detected in only 43% of patients with periodontitis.\u0026nbsp;Eliminating bacterial plaque decreases antigenic stimulation, which in turn may influence the levels of cytokines present in gingival crevicular fluid\u003csup\u003e65\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eMCP-1\u003c/u\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMonocyte chemoattractant protein-1 (MCP-1) is a key chemokine that plays a crucial role in initiating, regulating, and directing the migration of monocytes to sites of severe periodontal inflammation\u003csup\u003e66\u003c/sup\u003e. MCP-1 is produced by various cell types in response to signals such as tumor necrosis factor-alpha (TNF-\u0026alpha;), interleukin 1-beta (IL-1\u0026beta;), and interferon-gamma (IFN-\u0026gamma;). These pro-inflammatory cytokines induce MCP-1 expression in human periodontal ligament fibroblasts, promoting monocyte infiltration into inflamed sites\u003csup\u003e67\u003c/sup\u003e. Pradeep et al. found changes in the concentration of MCP-1 in GCF. Elevated MCP-1 levels were detected in patients with periodontitis before treatment, compared to both post-treatment levels and those observed in healthy controls \u003csup\u003e67\u003c/sup\u003e. In a study by G\u0026uuml;ndogar in 25 periodontitis patients and 24 gingivitis patients and 24 healthy controls, there was a correlation between IL-6, IL-10 and MCP-1 marker levels and the severity of periodontal involvement. The values were always higher in patients with periodontitis \u003csup\u003e68\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA number of previous studies have confirmed the contribution of MCP-1 to the etiopathogenesis of periodontitis\u0026nbsp;\u003csup\u003e66,69\u0026ndash;75\u003c/sup\u003e. In periodontitis, MCP-1 levels are significantly increased compared to healthy individuals, with higher levels of MCP-1 observed in gingival fluid, oral fluid, and serum\u0026nbsp;\u003csup\u003e69\u0026ndash;75\u003c/sup\u003e . Successful periodontal therapy then led to a decrease in MCP-1 levels\u0026nbsp;\u003csup\u003e70\u003c/sup\u003e . This was consistent with our results. MCP-1 is thought to be a major chemotactic factor in periodontitis\u0026nbsp;\u003csup\u003e66\u003c/sup\u003e. A study using a mouse model of \u003cem\u003ePorphyromonas gingivalis-induced\u003c/em\u003e periodontitis demonstrated that MCP-1 leads to increased expression of the IL-1 receptor antagonist and decreased expression of RANKL and reduced alveolar bone resorption\u0026nbsp;\u003csup\u003e76\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eKawamoto et al.\u0026nbsp;\u003csup\u003e47\u003c/sup\u003e found no significant difference in oral fluid MCP-1 levels in subjects with severe periodontitis (stage III according to the new classification of periodontal disease) and moderate progression, while MCP-1 levels were statistically significantly lower in subjects with rapid progression. The author hypothesized that it is the lower MCP-1 levels that lead to a reduction in M2 macrophage numbers and ultimately contribute to more pronounced alveolar bone resorption\u0026nbsp;\u003csup\u003e47,76\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003esCD14\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe role of CD14 in periodontitis appears to be the activation of CD14-positive cells by interaction with bacterial LPS via TLR 2 and 4 and subsequent activation of the nuclear factor NF kappa B cascade. Immunocompetent cells are not the only cell type in the periodontal region that produce sCD14. It has been shown that gingival fibroblasts stimulated by IFN-\u0026gamma; and LPS can also secrete it .\u0026nbsp;\u003csup\u003e77\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHuman oral epithelial cells (GMSM-K lineage)\u0026nbsp;secrete the pro-inflammatory cytokines IL-6 and IL-8 and the chemokine Regulated on Activation of Normal T Cells Expressed and Secreted (RANTES) upon stimulation with sCD14 and may contribute to the pathogenesis of periodontitis, as IL-8 and RANTES affect chemotaxis and neutrophil activity and IL-6 plays an important role in osteoclast activation and formation\u0026nbsp;\u003csup\u003e78\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003esCD14 also stimulates periodontal ligament stem cells, with activation of TLR 2 and 4. Research has shown increased expression and production of IL-6, CXCL8 and MCP-1 chemokines\u0026nbsp;\u003csup\u003e79,80\u003c/sup\u003e by these cells. Human monocytes and macrophages activated by modified low-density lipoprotein secreted IL-10 via CD14\u0026nbsp;\u003csup\u003e81\u003c/sup\u003e . MCP-1 secretion was also demonstrated by umbilical vein endothelial cells following stimulation with inactivated \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, and MCP-1 production was mediated via sCD14\u0026nbsp;\u003csup\u003e82\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that there is an increase in plasma sCD14 concentrations in periodontitis\u0026nbsp;\u003csup\u003e83\u0026ndash;85\u003c/sup\u003e, which is consistent with our results. Bacterial LPS stimulate higher expression of mCD14 by human monocytes and macrophages and consequently there is also an increased release of sCD14\u0026nbsp;\u003csup\u003e86\u003c/sup\u003e. Thus, it is hypothesized that sCD14 originates from periodontal tissues and accumulates in the vasculature by virtue of its increased production rather than reduced clearance\u0026nbsp;\u003csup\u003e83,85\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn their study, Vijaya et al. \u003csup\u003e83\u003c/sup\u003e published results where sCD14 levels in GCF were statistically significantly lower in individuals with periodontitis compared to healthy individuals. The same results were published by Jin and Darveau \u003csup\u003e87\u003c/sup\u003e . In addition, they found that sCD14 concentrations in GCF were higher in shallower pockets, which may indicate that sCD14 plays a protective role in periodontal tissues. On the other hand, the decrease in sCD14 concentration in deep periodontal pocets may be due to the presence of more microorganisms \u003csup\u003e87\u003c/sup\u003e. It has been shown that gingipains, a cysteine protease produced by \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, are able to degrade mCD14 on the surface of macrophages or macrophage-like cells (U937 lineage), with proteolysis of CD14 rather than cleavage into a soluble form \u003csup\u003e88\u0026ndash;90\u003c/sup\u003e. On the other hand, serum levels of sCD14 showed a weak but statistically significant negative correlation with the level of IgG class antibodies to \u003cem\u003eAggregatibacter actinomycetemcomitans.\u0026nbsp;\u003c/em\u003eThus, sCD14 appears to be effective in the uptake of LSP \u003cem\u003eAggregatibacter actinomycetemcomitans\u0026nbsp;\u003c/em\u003e\u003csup\u003e84\u003c/sup\u003e. The levels of sCD14 in oral fluid were also monitored. Isaza-Guzman et al. showed statistically significantly higher levels in subjects with periodontitis compared to healthy controls \u003csup\u003e91\u003c/sup\u003e. Non-surgical treatment of periodontitis led to a decrease in sCD14 levels in GCF. The same results were observed in serum \u003csup\u003e84,85\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe exact function of CD14 in periodontal health and the progression of periodontitis remains unclear. Data in patients with periodontitis are scarce. In our cohort of patients and controls, we clearly demonstrated a significant elevation of sCD14 concentration in patients with periodontitis and its subsequent decrease after successful therapy to values comparable to the control group. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eMMP-8\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMatrix metalloproteinase 8 or is an enzyme present in connective tissue responsible for collagen cleaving\u003csup\u003e92,93\u003c/sup\u003e. The primary function of MMP-8 is the degradation collagen. In periodontitis, MMP-8 is the major collagenase in the gingival connective tissues \u003csup\u003e94\u003c/sup\u003e. \u0026nbsp;MMP-8 is the major collagenase of periodontal tissues, as it is responsible for more than 90% of the collagenolytic activity in gingival fluid, oral fluid and oral rinses \u003csup\u003e12\u003c/sup\u003e . MMP-8 plays a key role in the etiopathogenesis of periodontitis \u003csup\u003e95\u003c/sup\u003e, and it is the disparity between the levels of matrix metalloproteinases and their inhibitors that is responsible for the destruction of periodontal tissues\u003csup\u003e96\u003c/sup\u003e. Monitoring MMP-8 levels in oral fluids has even been recommended as a diagnostic marker of periodontitis \u003csup\u003e12,97,98\u003c/sup\u003e . Although elevated MMP-8 levels in oral fluids are considered a sign of periodontitis, the primary role of this enzyme is defensive. This was demonstrated, for example, in mice infected with \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e, where MMP-8 deficient mice showed significantly higher alveolar bone resorption compared to mice with a functional MMP-8 gene \u003csup\u003e99,100\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElevated MMP-8 levels in periodontitis occur not only in gingival fluid but also in oral fluid, as confirmed by several previous studies \u003csup\u003e96,100\u0026ndash;102\u003c/sup\u003e . Serum MMP-8 levels also correlate with oral fluid MMP-8 levels and clinical signs of periodontitis\u003csup\u003e96\u003c/sup\u003e . Elevated levels of MMP-8 in gingival and oral fluid also occur in gingivitis, with levels significantly different between healthy subjects and subjects with periodontitis \u003csup\u003e96,103\u003c/sup\u003e. Increased collagenase activity in gingival crevicular fluid and MMP-8 levels correlate with collagen degradation in patients with periodontitis \u003csup\u003e104\u003c/sup\u003e. In a study of 30 patients with periodontitis and 21 healthy controls, Konopka et al. found a significant difference in MMP-8 as well as in MMP-8 levels after treatment in GCF \u003csup\u003e105\u003c/sup\u003e. In Romer\u0026apos;s study, 11 patients with periodontitis had a significant decrease in MMP-8 after therapy \u003csup\u003e106\u003c/sup\u003e. Escalona et al. reported elevated levels of cytokines and metalloproteinases in patients with periodontitis, which were significantly associated with the severity of the disease in their study\u003csup\u003e107\u003c/sup\u003e. Our data suggest clear contribution of MMP-8 to the destructive processes of periodontitis, given the orders of magnitude higher MMP-8 concentrations in active inflammation and their decline to healthy tissue-like concentrations after therapy. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eNGAL\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeutrophil gelatinase associated lipocalin (NGAL) is part of the lipocalin protein family. It is mostly expressed by neutrophils and oral epithelial cells. It can bind to both prostaglandins and matrix metalloproteinases\u003csup\u003e108\u003c/sup\u003e. It is also a chemoattractant of neutrophils and increases their capacity as phagocytes as wee as it binds iron ions effectively blocking bacterial metabolism. Lipopolysaccharide (LPS) is a potent inducer of NGAL\u0026nbsp;\u003csup\u003e109\u003c/sup\u003e. Data on the importance of NGAL in periodontitis are scarce.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSignificant increases in NGAL concentrations occur in association with stimulation by periopathogens such as \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e\u003csup\u003e110\u003c/sup\u003e, and NGAL in periodontal tissues is mainly produced by polymorphonuclears \u003csup\u003e110,111\u003c/sup\u003e. It has also been shown that NGAL is produced by different epithelial cell types, with IL-1\u0026beta; being the stimulus for NGAL expression via the nuclear factor kappa B signaling pathway\u003csup\u003e112\u003c/sup\u003e . Paradoxically, TNF-\u0026alpha; does not induce NGAL expression\u003csup\u003e112\u003c/sup\u003e . Westerlund et al. (1996)\u003csup\u003e111\u003c/sup\u003e demonstrated NGAL expression \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003ein oral epithelial cells, including gingival keratinocytes. \u003cem\u003eIn vivo\u003c/em\u003e, however, NGAL production in oral keratinocytes including gingival epithelial junction cells has not been demonstrated \u003csup\u003e111\u003c/sup\u003e. Bondy-Carey et al. (2013)\u003csup\u003e110\u003c/sup\u003e demonstrated NGAL secretion by gingival keratinocytes following stimulation of \u003cem\u003ePorphyromonas gingivalis\u0026nbsp;\u003c/em\u003ecells in vivo. However, significantly higher levels of NGAL were produced by gingival fibroblasts \u003csup\u003e110\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe are not aware of extensive previous studies on NGAL in periodontitis. Positive associations between NGAL and inflammatory periodontal disease have been scarcely published: an increase in NGAL in the oral fluid occurs in gingivitis, but higher levels are found in individuals with periodontitis\u003csup\u003e109\u003c/sup\u003e . A similar increase in NGAL levels also occurs in serum \u003csup\u003e109\u003c/sup\u003e. Increased levels of NGAL have also been observed in oral fluid and urine in periodontitis \u003csup\u003e113,114\u003c/sup\u003e. Tan et al. then examined NGAL in saliva and serum of patients with periodontitis \u003csup\u003e109\u003c/sup\u003e. Our data showed no significant differences in NGAL between healthy periodontium and pretreatment patients, suggesting a low involvement of NGAL in periodontal inflammation. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eTSLP\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThymic stromal lymphopoietin (TSLP) is crucial for T-cell maturation by activating antigen-presenting cells. Primarily produced by non-hematopoietic cells like fibroblasts and epithelial cells, TSLP facilitates the differentiation of naive CD4+ T cells into Th2 lymphocytes \u003csup\u003e115\u003c/sup\u003e. The role of TSLP in the etiopathogenesis of periodontitis is not entirely clear. Betancur et al. \u003csup\u003e116\u003c/sup\u003e demonstrated increased TSLP expression by oral keratinocytes and macrophages stimulated by \u003cem\u003ePorphyromonas gingivalis.\u003c/em\u003e In contrast, application of an alginate gel enriched with TSLP and granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in an increase in FOXP3+ regulatory T-lymphocytes at the application site and in the overlying lymph nodes. These regulatory T-lymphocytes play an important role in the inhibition of the inflammatory immune response in periodontal tissues \u003csup\u003e117\u003c/sup\u003e. Our results demonstrate increased TSLP production in hinge tissues in periodontitis and are consistent with results published by Betancur et al. \u003csup\u003e116\u003c/sup\u003e . Since an increase in B lymphocyte numbers is associated with the progression of periodontal lesions \u003csup\u003e9\u003c/sup\u003e, stimulation of the immune system by TSLP to mount a Th2 immune response \u003csup\u003e118\u003c/sup\u003e may contribute to the progression of periodontitis. Also, Jimenez et al. suggested the presence of TSLP in GCF in periodontitis. Here, in our cohort, we originally demonstrated a clear relationship between TSLP concentrations and periodontal inflammation as well as a significant decrease to below healthy controls after successful therapy. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eGAL-3\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGalectin-3 is a \u0026beta;-galactoside-binding lectin produced by various cell types, including immune cells, epithelial cells, and fibroblasts. It plays a regulatory role in immune cell function and exhibits both pro- and anti-inflammatory activities, depending on factors such as its intracellular or extracellular localization and the specific target cells involved\u003csup\u003e119\u003c/sup\u003e. Galectin-3 directly stimulates the migration and production of inflammatory mediators in the cells of innate immunity\u0026nbsp;\u003csup\u003e120\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData regarding galectin-3 in periodontitis are very scarce. In our work, the concentrations of galectin-3 increased after therapy to the level of healthy controls after tissue healing, suggesting an opposite role of galectin-3 as an anti-inflammatory cytokine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere is little information available in the literature regarding Gal-3 and periodontal health. Tamai and Kiyoura \u003csup\u003e121\u003c/sup\u003e demonstrated increased Gal-3 secretion by human gingival epithelia and gingival fibroblasts, but due to stimulation by the yeasts \u003cem\u003eCandida albicans\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCandida parapsilosis\u003c/em\u003e. Hendek et al. \u003csup\u003e122\u003c/sup\u003e observed significantly higher Gal-3 levels in subjects with gingivitis and periodontitis compared to subjects with healthy periodontium. In both cases, Gal-3 levels decreased following periodontal treatment \u003csup\u003e122\u003c/sup\u003e. Increased Gal-3 levels in oral fluid were observed in subjects with periodontitis compared to healthy controls \u003csup\u003e123\u003c/sup\u003e. Afacan et al. observed elevated concentrations of galectin-3 in GCF in patients with periodontitis and an association with higher grades of periodontitis \u003csup\u003e124\u003c/sup\u003e. Similar conclusions were reached in a study by Ali et al. \u003csup\u003e125\u003c/sup\u003e. Studies overall provide conflicting results. Our results suggest a positive association of Gal-3 with healthy periodontium or healing of periodontal lesions. This is consistent with the work published by Zhang et al., who demonstrated that Mac 2-binding protein together with Gal-3 induces the differentiation of human periodontal ligament stem cells into osteoblasts \u003csup\u003e126\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe association with the anti-inflammatory effect of Gal-3 is supported by a similar trend of IL-10 and Gal-3 levels, where significantly higher levels were observed in subjects with untreated periodontitis compared to healthy controls. IL-10 has been shown to upregulate Gal-3 expression in M2 macrophages via the STAT3 signaling pathway and is thus involved in the degradation of necrotic tissues in myocardial infarction \u003csup\u003e127\u003c/sup\u003e. Gal-3 also induces IL-10 production in monocytes. The C-terminus of Gal-3, i.e., its lectin domain CRD, is responsible for the increase in secretion; binding of lactose to CRD results in a decrease in IL-10 secretion by monocytes\u003csup\u003e128\u003c/sup\u003e . \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations of the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sample size can be considered as a limitation of the study as this number of patients may underepresent general population so the applicability may be limited. Although it may serve as the pilot study for further biomarker research. Another limitation is the selection of molecular marker panel. Obviously, it is not possible with the amount of samples to examine all possible combinations of cytokines. Again, this may aid the future research in the field.\u003c/p\u003e\n\u003cp\u003eCollecting gingival crevicular fluid can be challenging, particularly in individuals with a healthy periodontium. The protein yield from GCF samples cav vary depending on the type of filter paper used for collection \u003csup\u003e129\u003c/sup\u003e. For this reason, our results may not be clearly transferable to other sites. The protocol for analyzing gingival crevicular fluid has not been optimized based on the specific mediator being studied, as a uniform approach is typically used. Cytokines are believed to play a crucial role in the pathogenesis of periodontal disease. However, since periodontal disease is unevenly distributed across the dentition, it remains uncertain to what extent cytokine detection at various sites correlates with disease presence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrengths of the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the strengths of the study is the fact that this is a human study including the control group as well as comparison after therapy in individual patients. Despite the limitations above we feel this study may provide a basis for future biomarker research\u003c/p\u003e\n\u003cp\u003eMultiplex immunoassays provide several advantages over conventional monoplex immunoassays, including greater efficiency and higher data output per sample volume. Given the limited amounts of gingival crevicular fluid obtained from patients and the typically low cytokine concentrations, serial dilution is often not feasible, restricting analysis to a single ELISA per sample. In contrast, multiplex immunoassays enable the simultaneous quantification of multiple cytokines within a single sample, allowing for a comprehensive analysis of various analytes without requiring multiple collections.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study highlights the pivotal role of several major immune syst\u0026eacute;m regulators, especially the IL-6 axis with MMP-8 as a key effector\u0026mdash;in the pathogenesis and tissue destruction in periodontitis. It also indicates involvement of other immune components, including potential T cell activation, underscoring the interplay between innate and adaptive immunity. Additionally, we found that successful periodontal treatment markedly reduces inflammatory activity to levels comparable to clinically healthy tissue.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University Hospital Hradec Kr\u0026aacute;lov\u0026eacute; (reference number 201909 S13R). All participants were informed about the study objectives and signed an informed consent before inclusion into the study. All rights of the participants were maintained throughout.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available upon reasonable request at corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by MH CZ - DRO (UHHK, 00179906) and by the Cooperatio Program, research area STOM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV.R. conceptualized the work, performed the clinical evaluations, statistics, wrote manuscript. O.H. performed clinical evaluations. O.S. performed laboratory analysis, wrote manuscript. C.A. performed laboratory analysis, wrote manuscript. All authors reviewed and accepted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePapapanou PN, Sanz M, Buduneli N, et al. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. \u003cem\u003eJ Clin Periodontol\u003c/em\u003e. 2018;45(S20):S162-S170. doi:10.1111/jcpe.12946\u003c/li\u003e\n\u003cli\u003eSlots J. Periodontology: past, present, perspectives. \u003cem\u003ePeriodontol 2000\u003c/em\u003e. 2013;62(1):7-19. doi:10.1111/prd.12011\u003c/li\u003e\n\u003cli\u003eDentino A, Lee S, Mailhot J, Hefti AF. 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Validation of Periodontitis Screening Model Using Sociodemographic, Systemic, and Molecular Information in a Korean Population. \u003cem\u003eJ Periodontol\u003c/em\u003e. 2014;85(12):1676-1683. doi:10.1902/jop.2014.140061\u003c/li\u003e\n\u003cli\u003eNascimento GG, Baelum V, Sorsa T, Tervahartiala T, Skottrup PD, L\u0026oacute;pez R. Salivary levels of MPO, MMP-8 and TIMP-1 are associated with gingival inflammation response patterns during experimental gingivitis. \u003cem\u003eCytokine\u003c/em\u003e. 2019;115:135-141. doi:10.1016/j.cyto.2018.12.002\u003c/li\u003e\n\u003cli\u003eLuchian I, Goriuc A, Sandu D, Covasa M. The Role of Matrix Metalloproteinases (MMP-8, MMP-9, MMP-13) in Periodontal and Peri-Implant Pathological Processes. \u003cem\u003eInt J Mol Sci\u003c/em\u003e. 2022;23(3):1806. doi:10.3390/ijms23031806\u003c/li\u003e\n\u003cli\u003eKonopka L, Pietrzak A, Brzezińska-Błaszczyk E. Effect of scaling and root planing on interleukin-1\u0026beta;, interleukin-8 and MMP-8 levels in gingival crevicular fluid from chronic periodontitis patients. \u003cem\u003eJ Periodontal Res\u003c/em\u003e. 2012;47(6):681-688. doi:10.1111/j.1600-0765.2012.01480.x\u003c/li\u003e\n\u003cli\u003eRomero AM, Mastromatteo-Alberga P, Escalona L, Correnti M. [MMP-3 and MMP-8 levels in patients with chronic periodontitis before and after nonsurgical periodontal therapy]. \u003cem\u003eInvest Clin\u003c/em\u003e. 2013;54(2):138-148.\u003c/li\u003e\n\u003cli\u003eEscalona LA, Mastromatteo-Alberga P, Correnti M. Cytokine and metalloproteinases in gingival fluid from patients with chronic periodontitis. \u003cem\u003eInvest Clin\u003c/em\u003e. 2016;57(2):131-142.\u003c/li\u003e\n\u003cli\u003eNasioudis D, Witkin SS. Neutrophil gelatinase-associated lipocalin and innate immune responses to bacterial infections. \u003cem\u003eMed Microbiol Immunol (Berl)\u003c/em\u003e. 2015;204(4):471-479. doi:10.1007/s00430-015-0394-1\u003c/li\u003e\n\u003cli\u003eTan A, G\u0026uuml;rb\u0026uuml;z N, \u0026Ouml;zbalci Fİ, Koşkan \u0026Ouml;, Yetkin Ay Z. Increase in serum and salivary neutrophil gelatinase-associated lipocalin levels with increased periodontal inflammation. \u003cem\u003eJ Appl Oral Sci Rev FOB\u003c/em\u003e. 2020;28:e20200276. doi:10.1590/1678-7757-2020-0276\u003c/li\u003e\n\u003cli\u003eBondy-Carey JL, Galicia J, Bagaitkar J, et al. Neutrophils alter epithelial response to Porphyromonas gingivalis in a gingival crevice model. \u003cem\u003eMol Oral Microbiol\u003c/em\u003e. 2013;28(2):102-113. doi:10.1111/omi.12008\u003c/li\u003e\n\u003cli\u003eWesterlund U, Ingman T, Lukinmaa PL, et al. Human neutrophil gelatinase and associated lipocalin in adult and localized juvenile periodontitis. \u003cem\u003eJ Dent Res\u003c/em\u003e. 1996;75(8):1553-1563. doi:10.1177/00220345960750080601\u003c/li\u003e\n\u003cli\u003eCowland JB, S\u0026oslash;rensen OE, Sehested M, Borregaard N. Neutrophil Gelatinase-Associated Lipocalin Is Up-Regulated in Human Epithelial Cells by IL-1\u0026beta;, but Not by TNF-\u0026alpha;. \u003cem\u003eJ Immunol\u003c/em\u003e. 2003;171(12):6630-6639. doi:10.4049/jimmunol.171.12.6630\u003c/li\u003e\n\u003cli\u003eMorelli T, Stella M, Barros SP, et al. Salivary biomarkers in a biofilm overgrowth model. \u003cem\u003eJ Periodontol\u003c/em\u003e. 2014;85(12):1770-1778. doi:10.1902/jop.2014.140180\u003c/li\u003e\n\u003cli\u003eNakajima M, Hosojima M, Tabeta K, et al. \u0026beta;2 Microglobulin and Neutrophil Gelatinase-Associated Lipocalin, Potential Novel Urine Biomarkers in Periodontitis: A Cross-Sectional Study in Japanese. \u003cem\u003eInt J Dent\u003c/em\u003e. 2019;2019:1-10. doi:10.1155/2019/1394678\u003c/li\u003e\n\u003cli\u003eAllakhverdi Z, Comeau MR, Jessup HK, et al. Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells. \u003cem\u003eJ Exp Med\u003c/em\u003e. 2007;204(2):253-258. doi:10.1084/jem.20062211\u003c/li\u003e\n\u003cli\u003eBetancur D, Mu\u0026ntilde;oz Grez C, O\u0026ntilde;ate A. Comparative Analysis of Cytokine Expression in Oral Keratinocytes and THP-1 Macrophages in Response to the Most Prevalent Serotypes of Aggregatibacter actinomycetemcomitans. \u003cem\u003eMicroorganisms\u003c/em\u003e. 2021;9(3). doi:10.3390/microorganisms9030622\u003c/li\u003e\n\u003cli\u003eSands RW, Verbeke CS, Ouhara K, et al. Tuning cytokines enriches dendritic cells and regulatory T cells in the periodontium. \u003cem\u003eJ Periodontol\u003c/em\u003e. 2020;91(11):1475-1485. doi:10.1002/JPER.19-0411\u003c/li\u003e\n\u003cli\u003eTakai T. TSLP Expression: Cellular Sources, Triggers, and Regulatory Mechanisms. \u003cem\u003eAllergol Int\u003c/em\u003e. 2012;61(1):3-17. doi:10.2332/allergolint.11-RAI-0395\u003c/li\u003e\n\u003cli\u003eYang RY, Hsu DK, Liu FT. Expression of galectin-3 modulates T-cell growth and apoptosis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e. 1996;93(13):6737-6742. doi:10.1073/pnas.93.13.6737\u003c/li\u003e\n\u003cli\u003eBrito LNS, de Lemos Almeida MMR, de Souza LB, Alves PM, Nonaka CFW, Godoy GP. Immunohistochemical Analysis of Galectins-1, -3, and -7 in Periapical Granulomas, Radicular Cysts, and Residual Radicular Cysts. \u003cem\u003eJ Endod\u003c/em\u003e. 2018;44(5):728-733. doi:10.1016/j.joen.2018.01.008\u003c/li\u003e\n\u003cli\u003eTamai R, Kiyoura Y. Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells. \u003cem\u003eMycopathologia\u003c/em\u003e. 2014;177(1-2):75-79. doi:10.1007/s11046-013-9725-1\u003c/li\u003e\n\u003cli\u003eKarsiyaka Hendek M, Olgun E, Kisa U. The effect of initial periodontal treatment on gingival crevicular fluid galectin-3 levels in participants with periodontal disease. \u003cem\u003eAust Dent J\u003c/em\u003e. 2021;66(2):169-174. doi:10.1111/adj.12815\u003c/li\u003e\n\u003cli\u003eIsola G, Polizzi A, Alibrandi A, Williams RC, Lo Giudice A. Analysis of galectin-3 levels as a source of coronary heart disease risk during periodontitis. \u003cem\u003eJ Periodontal Res\u003c/em\u003e. 2021;56(3):597-605. doi:10.1111/jre.12860\u003c/li\u003e\n\u003cli\u003eAfacan B, Ilhan HA, K\u0026ouml;se T, Emingil G. Gingival crevicular fluid galectin-3 and interleukin-1 beta levels in stage 3 periodontitis with grade B and C. \u003cem\u003eClin Oral Investig\u003c/em\u003e. 2023;27(7):3749-3758. doi:10.1007/s00784-023-04991-7\u003c/li\u003e\n\u003cli\u003eAli M, Shemais N, Shaker O, Ghallab N. Gingival Crevicular Fluid and Salivary Levels of Galectin-3 in Patient with Gingivitis and Patient with Stage III Periodontitis: An Observational Study. \u003cem\u003eAdv Dent J\u003c/em\u003e. 2023;5(4):742-751. doi:10.21608/adjc.2023.230788.1386\u003c/li\u003e\n\u003cli\u003eZhang L, Huang Y, Lou H, Gong X, Ouyang Q, Yu H. LGALS3BP/Gal-3 promotes osteogenic differentiation of human periodontal ligament stem cells. \u003cem\u003eArch Oral Biol\u003c/em\u003e. 2021;128:105149. doi:10.1016/j.archoralbio.2021.105149\u003c/li\u003e\n\u003cli\u003eShirakawa K, Endo J, Kataoka M, et al. IL (Interleukin)-10\u0026ndash;STAT3\u0026ndash;Galectin-3 Axis Is Essential for Osteopontin-Producing Reparative Macrophage Polarization After Myocardial Infarction. \u003cem\u003eCirculation\u003c/em\u003e. 2018;138(18):2021-2035. doi:10.1161/CIRCULATIONAHA.118.035047\u003c/li\u003e\n\u003cli\u003eChung AW, Sieling PA, Schenk M, et al. Galectin-3 Regulates the Innate Immune Response of Human Monocytes. \u003cem\u003eJ Infect Dis\u003c/em\u003e. 2013;207(6):947-956. doi:10.1093/infdis/jis920\u003c/li\u003e\n\u003cli\u003eJohnson RB, Streckfus CF, Dai X, Tucci MA. Protein recovery from several paper types used to collect gingival crevicular fluid. \u003cem\u003eJ Periodontal Res\u003c/em\u003e. 1999;34(6):283-289. doi:10.1111/j.1600-0765.1999.tb02255.x\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Periodontitis staging criteria.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeriodontitis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage I\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage II\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage III\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStage IV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSeverity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003eInterdental CAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e1-2 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e3-4 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026ge;5 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026ge;5 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003eRBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003eCoronal third (\u0026lt;15%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003eCoronal third (15-33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eExtending to middle third of root and beyond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eExtending to middle third of root and beyond\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003eTooth loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003eNo tooth loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026le;4 teeth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026ge;5 teeth\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"7\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eComplexity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"7\" valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003eLocal\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMax. probing depth \u0026le;4 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMax. probing depth \u0026le;5 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eIn addition to stage II:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eIn addition to stage III:\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMostly horizontal bone loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003eMostly horizontal bone loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eProbing depths \u0026ge;6 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eNeed for complex rehabilitation due to:\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eVertical bone loss \u0026ge;3 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eMasticatory dysfunction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eFurcation class II or III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eSecondary occlusal trauma (mobility \u0026ge;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eModerate ridge defects\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eSevere ridge defects\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003eBite collapse, drifting, flaring\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026lt;20 remaining teeth (10 opposing pairs)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExtent and distribution\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003eAdd to stage as descriptor\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003eFor each stage, describe extent as:\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"bottom\" style=\"width: 74px;\"\u003e\n \u003cp\u003eLocalized (\u0026lt;30% of teeth involved)\u003c/p\u003e\n \u003cp\u003eGeneralized or\u003c/p\u003e\n \u003cp\u003eMolar/incisor pattern\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2. Basic characteristics of the patients and the control group\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"417\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePerio group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 128px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e(47-55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e(44-54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e0.1852\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMales\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026gt;0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFemales\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 64px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDMF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e(11-20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 62px;\"\u003e\n \u003cp\u003e(8-17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e0.2219\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePretreatment perio group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosttreatment perio group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Parameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e(pre vs post)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;(pre vs control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;(post vs control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean PPD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e3.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e(3.58-5.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(1.31-1.96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(1.12-1.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean GR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e(0-0.374)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e0.0514-0.443)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.0357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(0.00606-0.0774)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0153\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.0761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0003\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean CAL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e4.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e(3.9-5.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(1.6-2.55)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(1.17-1.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean GI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e(1.84-2.99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.0817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(0.0476-0.167)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.0897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(0.0586-0.119)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e\u0026gt;0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean PLI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e(1.04-2.97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.0931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(0.0498-0.134)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.0179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(0-0.0863)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 13px;\"\u003e\n \u003cp\u003e0.1345\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDMF decay missing filling; PPD probing pocket depth; GR gingival recession; CAL Clinical attachment loss; GI gingival index; PLI plaque index; IQR interquartile range.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 3. Results of cytokine levels in gingival crevicular fluid \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003eCytokine\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 19px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePretreatment perio group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosttreatment perio group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIQR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e(pre vs post)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;(pre vs control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;(post vs control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGalectin-3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(353-1456)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e2253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(519-10955)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e2096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(1042-2775)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0007\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.7177\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(0-4.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(0.22-0.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(0.43-13.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.9385\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.8139\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL-6\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e31.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(26.9-47.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e6.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(0.85-11.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e19.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(12.1-27.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMCP-1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(6.2-15.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e5.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(4.52-5.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(3.42-15.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e0.2292\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0338\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMMP-8\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e383828\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(116360-662430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e19855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(4963-47000)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e32454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(23830-52250)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.0766\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNGAL\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e23864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(22563-24249)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e23809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(21240-25561)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e22696\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(22039-23407)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e0.9441\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.022\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.4099\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003esCD14\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e4848\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(2090-9759)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(297-2578)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e904\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(399-1286)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.9881\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTSLP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e(0.099-0.79)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(0.12-0.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 6px;\"\u003e\n \u003cp\u003e0.396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e(0.29-0.532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0009\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0339\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIL interleukin; Monocyte chemoatractant protein MCP, MMP matrix metalloproteinase; Neutrophil gelatinase associated lipocalin\u0026nbsp;NGAL ; sCD soluble cluster of differentiation; Thymic stromal lymphopoietin TSLP.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 4. Comparison of cytokine levels in stage III versus IV periodontitis\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"537\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003eDunn\u0026apos;s\u0026nbsp;multiple\u0026nbsp;comparisons\u0026nbsp;test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003eMean\u0026nbsp;rank\u0026nbsp;diff,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003eSummary\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003eAdjusted\u0026nbsp;P\u0026nbsp;Value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; IL-6 Stage 3 vs. IL-6 Stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e3,625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; sCD14 stage 3 vs. sCD14 stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0,25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; Galectin-3 stage 3 vs. Galectin-3 stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e1,531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; IL-10 stage 3 vs. IL-10 stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e-5,781\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; MCP-1 stage 3 vs. MCP-1 stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e-2,281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; MMP-8 stage 3 vs. MMP-8 stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e-0,1875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; NGAL stage 3 vs. NGAL stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0,6563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 260px;\"\u003e\n \u003cp\u003e\u0026nbsp; TSLP stage 3 vs. TSLP stage 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0,5938\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 66px;\"\u003e\n \u003cp\u003ens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026gt;0,9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u0026nbsp;\u003cp\u003eTable 5. Correlation of clinical characteristics and cytokine concentration in periodontitis patients\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"472\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003eAverage\u0026nbsp;CAL\u0026nbsp;paro\u0026nbsp;pre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003eAverage\u0026nbsp;PLI\u0026nbsp;paro\u0026nbsp;pre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003eAverage\u0026nbsp;GI\u0026nbsp;paro\u0026nbsp;pre\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003ep value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,950\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,262\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003esCD14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,533\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,303\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eGalectin-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,994\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,858\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eIL-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,188\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMCP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,968\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,550\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eMMP-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,098\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eNGAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,252\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 75px;\"\u003e\n \u003cp\u003eTSLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 131px;\"\u003e\n \u003cp\u003e0,419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0,914\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 130px;\"\u003e\n \u003cp\u003e0,708\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Periodontitis, inflammatory cytokines, host immune response, interleukin-6, MMP-8 ","lastPublishedDoi":"10.21203/rs.3.rs-6956664/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6956664/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: The pathogenesis of periodontitis involves complex interactions within the host immune system. This study aimed to characterize the immune profile of gingival crevicular fluid (GCF) in patients with stage III and IV periodontitis.\u003c/p\u003e\n\u003cp\u003eMethods: Patients diagnosed with stage III or IV periodontitis were included, with age- and sex-matched individuals serving as healthy controls. GCF samples were analyzed for soluble CD14 (sCD14), Galectin-3, interleukin-6 (IL-6), interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-8 (MMP-8), neutrophil gelatinase-associated lipocalin (NGAL), and thymic stromal lymphopoietin (TSLP) using a multiplex microbead-based assay on the XMAP/Luminex system.\u003c/p\u003e\n\u003cp\u003eResults: A total of 25 patients and 25 controls were included. Clinical parameters—pocket probing depth, gingival index, and plaque index—were significantly elevated in the periodontitis group. Cytokine levels in GCF were generally higher in patients with periodontitis. Post-treatment, levels of IL-6, TSLP, sCD14, MCP-1, and MMP-8 significantly decreased and were lower in controls. In contrast, Galectin-3 levels increased following treatment, while NGAL levels remained unchanged. IL-10 levels were frequently undetectable and did not change significantly after therapy.\u003c/p\u003e\n\u003cp\u003eConclusions: This study highlights the involvement of multiple immune mediators, including those linked to neutrophil activity, in periodontal tissue destruction. Following successful periodontal therapy, local inflammatory markers decreased, reflecting a reduction in periodontal inflammatory activity.\u003c/p\u003e","manuscriptTitle":"Levels of selected inflammatory parameters in gingival crevicular fluid of patients with stage III and IV periodontitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-01 10:48:32","doi":"10.21203/rs.3.rs-6956664/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-15T08:54:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"170179605937085321932097095426133841534","date":"2026-03-18T10:22:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-14T12:18:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336741156437374468888677353537129639747","date":"2026-03-13T05:52:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-22T09:10:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270597490809799139789991351520083843776","date":"2025-11-14T21:45:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T13:57:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191971189521875482272278226093930661962","date":"2025-07-30T13:37:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-28T13:22:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-28T13:21:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-18T14:46:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-17T13:50:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2025-07-17T13:49:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8efe792b-f341-42b3-b9f1-a679008113bd","owner":[],"postedDate":"August 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T15:08:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-01 10:48:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6956664","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6956664","identity":"rs-6956664","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}