Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients

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Methods This study recruited male subjects with an age range of (20-60) years; 60 were stage III and stage IV periodontitis patients, 30 were cigarette smoker, and 30 were nonsmokers. After applying the inclusion and exclusion criteria to evaluate their eligibility for recruitment, 25 control subjects with a healthy periodontal status and good oral hygiene maintenance were included. Unstimulated saliva was obtained and evaluated using an enzyme-linked immunosorbent assay, and the following periodontal parameters were documented: [plaque index, bleeding on probing, periodontal pocket depth, and clinical attachment loss]. Results The mean levels of all salivary NETosis biomarkers citH3, ELA, CALPRO, and MPO were elevated in the periodontitis groups (smokers and nonsmokers) than in controls. Moreover, the mean NETosis biomarker‘s mean levels were significantly higher in smoker than in nonsmokers. In addition, the correlations were significant between CALPRO and CitH3 in smokers and between ELA and CitH3 in nonsmokers. Conclusions The results of this study showed that the chosen salivary biomarkers of NETosis revealed elevated clinical accuracy in differentiating the studied periodontitis groups (smokers and nonsmokers) from controls. In addition, cigarette smoking increases the risk of periodontitis, and neutrophils in smokers with periodontitis exhibited more susceptibility to form neutrophil extracellular traps when compared with nonsmokers. 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F1000Research 2025, 13 :907 ( https://doi.org/10.12688/f1000research.152917.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Revised Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] Rasha Abdul Jabbar Najim https://orcid.org/0009-0008-7020-8006 1 , Batool Hassan Al Ghurabi 2 Rasha Abdul Jabbar Najim https://orcid.org/0009-0008-7020-8006 1 , Batool Hassan Al Ghurabi 2 PUBLISHED 07 Apr 2025 Author details Author details 1 Master's student, Basic Science Department, University of Baghdad, Baghdad, Baghdad Governorate, Iraq 2 Microbiological/Immunological Professor, Basic Science Department, University of Baghdad, Baghdad, Baghdad Governorate, Iraq Rasha Abdul Jabbar Najim Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Writing – Original Draft Preparation Batool Hassan Al Ghurabi Roles: Supervision, Validation, Visualization, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background To evaluate the NETosis biomarkers citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) as indicators of inflammation in the severe stages of periodontitis III and IV in both (smokers and nonsmokers) patients, and to determine the correlation between NETosis biomarkers and clinical periodontal parameters. Methods This study recruited male subjects with an age range of (20-60) years; 60 were stage III and stage IV periodontitis patients, 30 were cigarette smoker, and 30 were nonsmokers. After applying the inclusion and exclusion criteria to evaluate their eligibility for recruitment, 25 control subjects with a healthy periodontal status and good oral hygiene maintenance were included. Unstimulated saliva was obtained and evaluated using an enzyme-linked immunosorbent assay, and the following periodontal parameters were documented: [plaque index, bleeding on probing, periodontal pocket depth, and clinical attachment loss]. Results The mean levels of all salivary NETosis biomarkers citH3, ELA, CALPRO, and MPO were elevated in the periodontitis groups (smokers and nonsmokers) than in controls. Moreover, the mean NETosis biomarker‘s mean levels were significantly higher in smoker than in nonsmokers. In addition, the correlations were significant between CALPRO and CitH3 in smokers and between ELA and CitH3 in nonsmokers. Conclusions The results of this study showed that the chosen salivary biomarkers of NETosis revealed elevated clinical accuracy in differentiating the studied periodontitis groups (smokers and nonsmokers) from controls. In addition, cigarette smoking increases the risk of periodontitis, and neutrophils in smokers with periodontitis exhibited more susceptibility to form neutrophil extracellular traps when compared with nonsmokers. READ ALL READ LESS Keywords Biomarkers; Calprotectin; Citrullinated histone H3; Myeloperoxidase; NETosis; Neutrophil elastase; Periodontitis Corresponding Author(s) Rasha Abdul Jabbar Najim ( [email protected] ) Close Corresponding author: Rasha Abdul Jabbar Najim Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2025 Jabbar Najim RA and Al Ghurabi BH. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Jabbar Najim RA and Al Ghurabi BH. Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.12688/f1000research.152917.2 ) First published: 08 Aug 2024, 13 :907 ( https://doi.org/10.12688/f1000research.152917.1 ) Latest published: 07 Apr 2025, 13 :907 ( https://doi.org/10.12688/f1000research.152917.2 ) Revised Amendments from Version 1 There is a little change in the title of the manuscript in the new version by illustrating the stages of the periodontitis patients that included in this study. There is a little change in the title of the manuscript in the new version by illustrating the stages of the periodontitis patients that included in this study. See the authors' detailed response to the review by Zerina Hadzic See the authors' detailed response to the review by Amal Bouziane READ REVIEWER RESPONSES Introduction Periodontitis is a multifactorial chronic, irreversible inflammation; however, the primary cause of periodontitis has been found to be the bacterial biofilm that develops on tooth surfaces, the host’s reaction, in addition to local elements such as plaque, calculus, external factors, and the health of the patients in general, all of which influence disease progression. 1 Smoking is one of the main ways in which individuals are exposed to harmful chemicals (cigarette smoke contains at least 4,500 different chemicals), and can cause epigenetic changes that increase inflammation. In addition, smoking generally increases oxidative stress, with excess free oxygen radicals causing cell damage. As a result, smoking exacerbates periodontal disease and negatively affects nearly all forms of periodontal therapy. 2 , 3 Several studies have indicated that smokers are more likely to have periodontal disease than nonsmokers, including greater gingival recession, bone attachment, and tooth loss. 4 – 7 Reactive oxygen species (ROS) released by periodontal infections and their byproducts activate several phagocytes, including neutrophils, monocytes, gingival fibroblasts, and cells found in the periodontal tissue. In patients with periodontitis, there is an increase in the recruitment of polymorph nuclear neutrophils (PMNs), which results in the breakdown of bone and oral tissues, 1 , 8 – 10 this is because the periodontium is a special habitat where oral bacteria are constantly in contact with the host immune system. Neutrophils serve as the initial line of defense of the human innate immune system and have the ability to eliminate infections extracellularly, connect innate and adaptive immune responses, and support tissue healing and inflammatory resolution. 9 , 11 , 12 However, severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful because it maintains the body in an inflammatory state, which destroys tissue. Maintaining a balance between neutrophils and other macrophages is crucial for maintaining periodontal homeostasis, as an imbalance can result in periodontitis. 8 , 13 According to these recent studies, 3 , 14 smoking cigarettes has several potential detrimental effects on periodontal health, including a decrease in wound healing time, vascular constriction, impaired defenses against plaque bacteria, and interference with oxidative stress processes. Because they disturb redox equilibrium, reactive oxygen species (ROS) are toxic to humans, and exposure to cigarette smoke increases their levels. However, sex hormones may also affect the gingival microcirculation, which can affect the global spread of the disease. Men are substantially more likely than women to show vasodilation in case of inflammation or when a wound is healing and when the disease is actively progressing. 3 , 15 – 19 Neutrophil equilibrium is important in periodontal homeostasis because periodontitis can develop when there are too many or too few neutrophils in the tissue. 8 One means by which Neutrophils aid in homeostasis and defense by producing neutrophil extracellular taps (NETs), which allow them to capture bacteria in the extracellular space by releasing decondensed chromatin adorned with histones and granule content. NETosis is defined as the occurrence of neutrophil death and NET production. The primary defense of periodontal tissues is provided by NETs, and a deficiency in NETs can result in severe early-onset periodontitis. 9 , 18 , 19 Smoking cigarettes accelerates the production of NETs and PMN-derived proteases, which are responsible for breaking down connective tissue. 2 , 14 NETs are composed of DNA and histones, along with associated neutrophil granule proteins such as citH3, Neutrophil Elastase, Calprotectin and Myeloperoxidase. 12 Histone H3 is the most abundant protein in NETs, and citrullination of histones is a specific marker of NETosis. 20 , 21 ELA is a serine protease maintained in abundance in azurophilic granules of neutrophils, together with other proteins involved in anti-microbial defense, and high levels of ELA in saliva can be associated with the loss of active periodontal attachment. 22 CALPRO belongs to the S100 family, which accounts for 5–40% of the cytosolic protein content of neutrophils. It acts as a new warning system by demonstrating pro-inflammatory activities primarily induced by activated granulocytes in a calcium-dependent manner. It contributes by binding to specific cell surface receptors, which sets off further signal transductions that contribute to the recruitment of white blood cells and production of inflammatory cytokines in the affected regions. 23 One of the many mediators released in tissues as inflammation advances is MPO, whose expression is highly correlated with gingival tissue in periodontitis. 24 Based on the available evidence, this study evaluated the NETosis biomarkers as indicators of inflammation in stage III and stage IV (smokers and nonsmokers) periodontitis groups and compared the findings with those of the controls. Furthermore, we determined the relationship between the NETosis biomarkers and clinical periodontal parameters. Methods Study design This was a case-control study based on observational data. The study was carried out in the Periodontics Department/College Dentistry, University of Baghdad, Al-Baladiat specialized center for dentistry, Al Sader specialized center for dentistry, and Nawar Mousa specialized center for dentistry in Baghdad/Iraq, from October 18, 2023, to January 2024. Ethical considerations were essential during the conduct of this study, introduced by the World Medical Association’s Helsinki Declaration (WMA), after submission to the ethical committee at the Dentistry College/University of Baghdad to obtain ethical approval (Reference Number: 844, project number: 844823, date: 15-10-2023). Study population The 85 male subjects recruited in the study and selected according to their oral and periodontal health status, the type of this study was case control study not clinical trial study, Depending on the classification criteria by Tonetti, 25 the selected patients were classified into: • Periodontal healthy control group with intact periodontium. • Stage III periodontitis (loss of bone reaches the root’s midpoint). • Stage IV periodontitis (loss of bone reaches the apical third of the root). Each periodontitis group was further subcategorized into two groups: • Smokers (stage III and stage IV). • Non-smokers (stage III and stage IV). Inclusion criteria All males recruited in the study had at least 20 teeth and no systemic disease with no antibiotic consumption for the last 3 months. The control group with healthy periodontium exhibiting good oral hygiene without any periodontal disease history or symptoms with intact periodontium (BOP<10% and PPD≤3), 26 On the other hand, cases of periodontitis were chosen based on the following criteria, 25 which defined the disease as having visible interdental CAL at two or more teeth that are not adjacent, CAL measuring more than 3 mm along with pocketing measuring at least 3 mm, can be found at two or more teeth, stage III and stage IV, in which the percentage of the loss of bone reaches the midpoint and apical third of roots, as well as unstable with the depth of the pocket of more than 5 mm or when bleeding occurs upon probing at a depth of 4 mm. Exclusion criteria These criteria included subjects with the following: systemic diseases such as autoimmune diseases and diabetes mellitus, history of smoking or alcohol drinking, use of anti-inflammatory drugs and/or antibiotics within the previous three months, use of any periodontal treatment that was used in the past or at present, orthodontic appliances or prosthodontics, consumption of any type of smoke other than cigarettes (electric cigarettes and vapes), oral lesions such as lichen planus, and other diseases unrelated to periodontitis; Females excluded from the study. Sample size The G power 3.1.9.7 program (developed by Franz-Faull, Kiel University/Germany) was employed to compute the size of the sample. Under these conditions, the sample size is around 85 participants, with a power of 90%, an alpha error of 0.05 on both sides’ results in, a significant effect size of F at 0.4, and three groups. 27 Effect size F (Small =0.1, Medium=0.25, Large=0.4). Saliva sample collection The patients were advised to clean and floss their teeth to maintain good oral hygiene. Unstimulated saliva was collected prior to clinical examination. Salivary samples were conducted between 9 am and 12 pm. The participants’ saliva was drooled passively into a plastic cup without any stimulation or spitting, and approximately 3 mL of saliva was collected from each subject. Using a micropipette, saliva was drawn from a disposable cup, transferred to a sterile plain tube, centrifuged at 4000 rpm for 3 min, placed into an uncontaminated Eppendorf tube, 28 and then frozen at -20 °C immediately before ELISA analysis for citH3, ELA, CALPRO, and MPO biomarkers. Screening of NETosis biomarkers in saliva Using manual ELISA technology, the levels of citH3 (EPIGEN TEK), ELA, CALPRO, and MPO (Cloud-Clone Corp) biomarkers in saliva were determined for each participant. Clinical periodontal parameters Following saliva sampling, the clinical parameters (PLI, BOP, PPD, and CAL) and periodontal status was determined using William’s periodontal probe. Six distinct areas of each tooth (Distobuccal, Buccal, Mesiobuccal, Distolingual, Lingual, and Mesiolingual) were inspected to perform clinical parameters BOP/PPD and CAL only, while the scores of PLI were recorded by examining only four surfaces (mesial, distal, labial/buccal, lingual/palatal). The 3 rd molar was excluded from the examination of all parameters, except PLI. PLI was recorded using disclosing agents to detect the presence or absence of dental plaque, 29 while the percentage of BOP was noted as 1 present 0 absent. 30 The distance from the free gingival margin to the base of the pocket is known as PPD, whereas the distance from the cemento-enamel junction (CEJ) to the base of the pocket in the absence of gingival recession is called CAL. If the margin of the gingiva is at the CEJ, then the CAL was considered to be equal the PPD; when there is gingival recession, CAL was determined by adding the recession depth and PPD; and when the margin was higher than CEJ, in order to determine the CAL, the distance from gingival margin to CEJ was subtracted from the PPD. 25 Staging was performed by calculating the CAL plus 2 mm of biological width per root length multiplied by 100 for the most affected tooth in the dentition. Calibration The calibrated periodontal evaluations were completed and performed on five patients not included in the study at the Periodontics Department/Dentistry College/Baghdad. The kappa-coefficient assay was used to examine categorical variables to prevent false-positive results. Clinical periodontal data (PPD and CAL) were recorded every two hours for each of the six sites per tooth (except for the third molars). Measurement reliability was confirmed by the coefficients of intra-class correlation (ICC) of 0.950 for PPD and 0.860 for CAL, while those for PLI and BOP were 0.917 and 0.890, respectively. Statistical analysis Data description, analysis, and presentation were performed using the Statistical Package for Social Sciences (SPSS) (version -22/Chicago/Illinois/USA) ( https://research-repository.uwa.edu.au/en/publications/spss-statistics-version-22-a-practical-guide ). 31 For quantitative variables, the descriptive analysis produced the mean and standard deviation (SD), while the percentage and frequency were qualitative variables. The kappa statistical test was employed to evaluate the two readings for dichotomous variables. The linear connection between the two quantitative variables was assessed using the Pearson’s correlation parametric test. Normality of distribution was verified using the Shapiro-Wilk test. Homogeneity was verified using Levene’s test. Two independent sample T test were used to determine how the two groups differed from one another. Two qualitative variables, distributional correlations, were tested using the chi-square test. Receiving operating characteristic curves (ROC) were used to calculate the sensitivity and specificity of each salivary biomarker. Pilot study Ten participants from the dental teaching hospital at the Dentistry College/Baghdad University participated in a pilot study to investigate the association between NETosis biomarkers (citH3, ELA, CALPRO, and MPO) as indicators of an inflammatory response in smokers and nonsmokers with periodontitis. Results A total of 150 patients with periodontitis were screened to evaluate their eligibility for recruitment, and 75 people were excluded from the study after applying the inclusion and exclusion criteria to patients undergoing periodontal care clinics. Ultimately, only 60 male patients with periodontitis and 25 controls were included in the study ( Figure 1 ). Figure 1. Flow Chart of the study design. Clinical periodontal parameters of the study groups When comparing the periodontitis groups (smokers and nonsmokers) to the control group, the periodontal parameters of all 85 male study participants increased considerably ( Table 1 ). The mean values of the parameters (PLI/BOP/CAL) were compared among the study groups and showed a significant difference (p<0.05); smokers periodontitis patients had the highest mean levels of PLI and CAL, while the nonsmokers’ group had the highest elevation of the mean percentage of BOP. The smokers group also had a slightly higher mean PPD than the nonsmokers group; however, the results showed non-significant differences in both smokers and nonsmokers stage III and stage IV periodontitis groups. Table 1. Mean of the clinical periodontal parameters of the study groups. Clinical Parameters and age Controls (n= 25) Smokers periodontitis (SP)(n=30) Nonsmoker periodontitis (NSP)(n=3) Study groups P value Multiple pairwise comparison (mean difference) SP VS controls P value NSP Vs. controls P value SP vs. NSP P value Age — Mean ±SD 36.72±11.24 40.47±11.27 43.90±9.77 0.053 NS — — — — — PLI Mean ±SD 22.58±10.03 60.84±27.58 54.11±24.59 * 0.000 38.258 * 0.000 31.530 * 0.000 6.727 0.582NS BOP Mean ±SD 5.28±2.41 34.29±20.08 42.29±18.31 * 0.000 29.011 * 0.000 37.016 * 0.000 8.004 0.248 NS PPD — — Mean ±SD — 0.4777±0.859 4.500±0.543 0.140 NS — — — — CAL — — — Mean ±SD — 5.695±1.179 5.045±0.957 * 0.022 — — — * , significant p<0.05; n, number; PLI, plaque index; BOP, bleeding on probing; PPD, probing pocket depth; CAL, clinical attachment loss. SP: Smokers periodontitis, NSP: nonsmokers periodontitis. According to multiple pairwise comparisons, as shown in Table 1 between smokers and nonsmokers periodontitis groups, the difference in the average levels of PLI, BOP, and PPD was not statistically significant; however, there was a significant difference in the average CAL of smokers and nonsmokers with periodontitis. The difference in the mean levels of clinical periodontal parameters between the research groups is shown in Figure 2 . Figure 2. Clinical parameter comparison between research groups; (a) Mean of PLI, (b) Mean of BOP, (c) Mean of PPD, (d) Mean of CAL. Salivary biomarkers levels Table 2 illustrates that the average levels of salivary biomarkers were higher in the periodontitis groups (smokers and nonsmokers) than in the controls, while the highest level was in the smokers group and the multiple pairwise comparison between the groups was statistically significant. Table 2. Salivary biomarkers’ mean levels among groups. Salivary biomarkers Controls (n= 25) Smokers periodontitis (SP) (n=30) Nonsmoker periodontitis (NSP) (n=30) Study groups P value Multiple pairwise comparison (mean difference) SP Vs. controls P value NSP Vs. controls P value SP Vs. NSP P value citH3 Mean ± SD 0.49 ± 0.59 2.95 ± 0.64 2.24 ± 0.55 * 0.000 2.533 * 0.000 1.818 * 0.000 0.715 * 0.000 ELA Mean ± SD 0.63 ± 0.18 Β 3.12 ± 0.81 1.83 ± 0.45 * 0.000 2.488 * 0.000 1.193 * 0.000 1.295 * 0.000 CALPRO Mean ± SD 153.12 ± 93.38 382.96±88.50 308.36 ± 117.23 * 0.000 229.839 * 0.000 155.244 * 0.000 74.595 * 0.016 MPO Mean ± SD 93.68 ± 22.0 369.06 ± 92.49 302.67 ± 94.23 * 0.000 275.382 * 0.000 208.995 * 0.000 66.386 * 0.021 * Significant, p<0.05; Figure 3 demonstrates the comparison of the mean level of the NETosis salivary biomarkers (citH3, ELA, CALPRO, and MPO) among the study groups and that the higher elevation is in the smokers’ periodontitis group than in nonsmokers’ periodontitis and controls. Figure 3. Comparison of the salivary biomarker among smokers’ periodontitis, nonsmokers’ periodontitis and controls, (a) citH3, (b) ELA, (c) CALPRO and (d) MPO. Table 3 demonstrates that the mean salivary biomarker levels in individuals with stage III and IV periodontitis showed a non-significant difference, regardless of whether they smoked. Table 3. Salivary biomarkers’ mean levels among stage III and IV smokers and nonsmokers. Salivary biomarkers Periodontitis groups Smokers stage III Smokers stage IV Nonsmokers stage III Nonsmokers stage IV P value smokers P value nonsmokers No. 22 8 20 10 — — citH3 Mean ± SD 2.88 ± 0.49 3.15 ± 0.95 2.27 ± 0.66 2.16 ± 0.24 0.313NS 0.611NS ELA Mean ± SD 3.02 ± 0.79 3.40 ± 0.84 1.89 ± 0.49 1.70 ± 0.47 0.266NS 0.296NS CALPRO Mean ± SD 393.65 ± 83.07 353.55 ± 101.96 305.26 ± 100.50 314.57 ± 151.30 0.280NS 0.842NS MPO Mean ± SD 378.51 ± 89. 94 343.06 ± 100.59 289.57 ± 78.75 328.87 ± 119.91 0.362NS 0.289NS According to the comparison between periodontitis smokers and nonsmokers, as illustrated in Table 4 , the mean salivary biomarker levels showed a non-significant association (p>0.05) between stage III and stage IV. Table 4. Mean of the salivary biomarkers between stage Ill and IV of smokers and nonsmokers. Group Stage No. citH3 Mean ±SD T test P Value ELA Mean ±SD T test P value CALPRO Mean ±SD T test P value MPO Mean ±SD T test P value Smokers Periodontitis III 22 2.88 ± 0.49 1.028 0.313 NS 3.02 ± 0.79 1.136 0.266 393.65 ± 83.07 1.101 .280 NS 378.51 ± 89.94 0.926 0.362 NS IV 8 3.15 ± 0.95 3.40 ± 0.84 353.55 ± 101.96 343.06 ± 100.59 Nonsmokers Periodontitis III 20 2.27 ± 0.66 0.515 0.611 NS 1.89 ± 0.44 1.066 0.296 305.26 ± 100.50 0.201 .842 NS 289.57 ± 78.75 1.080 0.289 NS IV 10 2.16 ± 0.24 1.70 ± 0.47 314.57 ± 151.30 328.87 ± 119.91 Total III 42 2.59 ± 0.65 0.053 0.962 NS 2.48 ± 0.86 0.098 0.922 NS 351.56 ± 101.05 0.634 .529 NS 336.16 ± 95.07 0.035 0.972 NS IV 18 2.60 ± 0.81 2.45 ± 1.08 331.89 ± 129.61 335.18 ± 108.77 On the other hand ( Figure 4 ), demonstrates in graphs the comparison of the mean of salivary biomarkers between stage III and IV periodontitis of both smokers and nonsmokers groups. Figure 4. Comparison of Salivary biomarker between stage III and IV of periodontitis; (a) citrullinated histone H3 (citH3), (b) neutrophil elastase (ELA), (c) calprotectin (CALPRO), (d) myeloperoxidase (MPO). Correlation between stage III and stage IV periodontal parameters with the salivary biomarkers Regarding the Pearson Correlation Coefficient between stage III and IV periodontal parameters and the salivary biomarkers, there was a significant relationship (p<0.05) in stage III periodontitis between citH3 and CAL as well as between CALPRO and each of (PLI, PPD, and CAL), whereas in stage IV periodontitis, the correlation was significant only between MPO and CAL, as shown in Table 5 . Table 5. Correlation between stage III and IV periodontal parameters and salivary biomarker. Stage Histon-H3 ELA CALPRO MPO r p r P r P r p III PLI -0.211 0.179 -0.003 0.985 0.340 * 0.027 0.017 0.915 BOP -0.156 0.325 -0.051 0.750 0.219 * 0.164 -0.157 0.319 PPD 0.127 0.422 0.210 0.182 0.327 * 0.015 0.073 0.644 CAL 0.386 * 0.012 0.178 0.260 0.312 * 0.044 0.176 0.264 IV PLI 0.333 0.177 0.378 0.122 0.074 0.771 0.264 0.289 BOP -0.288 0.246 -0.114 0.653 0.220 0.379 0.057 0.821 PPD 0.346 0.159 0.417 0.085 -0.044 0.862 0.261 0.296 CAL 0.030 0.906 0.100 0.693 0.392 0.108 0.570 * 0.013 * Significant difference (p<0.05); r: coefficient correlation between -1 and 1; p: probability. Correlation among salivary NETosis biomarkers Concerning the correlations between citH3 and CALPRO levels, a strong correlation was observed in the group of smokers with periodontitis, whereas the correlation of citH3 with ELA was strong in the group of nonsmokers with periodontitis, as illustrated in Table 6 . Table 6. Correlation among the salivary biomarkers. Groups ELA CALPRO MPO r p r p r p SP citH3 0.124 0.515 0.685 0.000 -0.034 0.857 ELA -0.266 0.156 0.231 0.219 CALPRO 1 -0.084 0.657 NSP citH3 0.535 0.002 -0.106 0.576 -0.027 0.887 ELA 0.094 0.620 0.139 0.464 CALPRO 0.215 0.254 Relationship between biomarkers and clinical parameters Based on the relationship between salivary biomarkers and clinical parameters, this study found a strong association between BOP% and CALPRO in smokers with periodontitis. Moreover, there was a strong association between BOP and ELA, as well as between CAL and MPO in the nonsmokers periodontitis group, as shown in Table 7 . Table 7. Correlation between periodontal parameters and salivary biomarkers. Clinical Parameters of Groups citH3 ELA CALPRO MPO r p r P r p r p Smokers Periodontitis PLI% -0.068 0.720 0.013 0.944 0.165 0.384 -0.121 0.524 BOP% -0.341 0.066 -0.075 0.694 0.505 * 0.004 -0.052 0.785 PPD 0.201 0.288 0.230 0.220 -0.010 0.959 -0.052 0.785 CAL 0.032 0.868 -0.215 0.255 0.097 0.608 0.057 0.765 Nonsmokers periodontitis PLI% 0.136 0.472 0.129 0.495 0.219 0.244 0.254 0.176 BOP% 0.175 0.356 0.459 * 0.011 0.119 0.530 0.046 0.808 PPD 0.005 0.981 0.002 0.992 -0.032 0.867 0.284 0.128 CAL 0.076 0.689 0.005 0.980 0.226 0.230 0.379 * 0.039 * Significant difference (p<0.05), r: correlation coefficient between -1 and 1, p: probability. Figure 5 describes the linear correlation between citH3 and CALPRO levels in smokers periodontitis group, as well as between citH3 levels and ELA levels in nonsmokers with periodontitis. On the other hand, also describes the linear correlation between CALPRO and BOP%, ELA and BOP%, and MPO and CAL. Figure 5. Correlation between; (a) CALPRO and citH3, (b) ELA and citH3, (c) CALPRO and BOP%, (d) ELA and BOP%, (e) MPO and CAL. Diagnostic accuracy of NETosis biomarkers Periodontitis smokers and nonsmokers, according to receiver operating curve (ROC) data, had good accuracy in estimating salivary biomarker sensitivity and specificity in differentiating periodontitis from healthy periodontium, as shown in Figure 6 . Figure 6. Receiver operating curves (ROC) for salivary biomarkers (a) Smokers periodontitis vs. control, (b) Nonsmokers periodontitis vs. control, (c) Smokers vs. nonsmokers. Additionally, the area under the curve (AUC) values between smokers periodontitis and control for salivary citH3, ELA, CALPRO, and MPO were 0.991-1,000, and the AUC between periodontitis nonsmokers and control for the selected markers was 0.964-1,000 also between smokers and nonsmokers periodontitis group was 0.878-0.750, as shown in Table 8 . Table 8. Comparison of the diagnostic properties of NETosis biomarkers among the study groups. Comparison Test result variables (s) AUC P value Optimal cut of point Sensitivity Specificity Smokers Periodontitis Vs. Controls citH3 0.991 * 0.000 2.041 96.7 96 ELA 1.000 * 0.000 1.43 100 100 CALPRO 0.963 * 0.000 266.67 93 96 MPO 1.000 * 0.000 62.008 100 100 Nonsmokers Periodontitis Vs. Controls citH3 0.964 * 0.000 1.804 93 96 ELA 1.000 * 0.000 1.004 100 100 CALPRO 0.903 * 0.000 163.920 93.3 72 MPO 1.000 * 0.000 62.008 100 100 Smokers periodontitis Vs. Nonsmokers Periodontitis citH3 0.878 * 0.000 2.472 83.3 86.7 ELA 0.937 * 0.000 2.158 93.3 83.3 CALPRO 0.700 * 0.008 348.468 60 66.7 MPO 0.750 * 0.001 304.025 70 70 * Significant difference, AUC: area under the curve. Discussion This study evaluated NETosis biomarkers in smokers and nonsmokers with periodontitis. The benefit of our case-control study was the use of multiple salivary biomarkers (citH3, ELA, CALPRO, and MPO), thus distinguishing between the biomarkers with the greatest impact on periodontitis groups. There are different factors that may increase the severity of periodontitis, one of which is cigarettes smoking. 2 – 4 According to our findings, CALPRO was significantly elevated in the saliva of male smokers in the periodontitis group. Cardoso et al. 32 found that the male sex plays a role in CALPRO in predisposing individuals to periodontitis. The current finding is in agreement with Wei et al. 23 who attributed elevated CALPRO to the recruitment of neutrophils in the inflammatory periodontitis site, which acts as an innate immune defense, and also found that CALPRO is used as an antibacterial agent released highly from neutrophils in response to periodontal disorders. Brembach et al. 33 reported that nicotine induced changes in the release of ROS, which is one of the defense mechanisms of neutrophils against inflammation, in addition to NET formation. Smoking has an enormous effect on the host response. However, the present study was consistent with a previous study, 33 and showed an elevation of CALPRO in smokers. However, D’Haens et al. 34 showed that the salivary level of S100A protein, one of the CALPRO components, is decreased in individuals with inflammatory disease, which leads to a decline in the host’s oral defense. Herein, our data revealed that there is a higher elevation of ELA in smokers with periodontitis than in nonsmokers with periodontitis and controls; the part of our study of increasing ELA production in smokers with periodontitis group was consistent with the study of Özkaca et al. 35 Our study also agrees with another recent study by Tseng et al., 36 who found that ELA promotes the progression and pathogenesis of periodontal disease. Pauletto et al. 37 disagreed with our findings, and found that ELA showed no significant change in smokers’ periodontitis and the level of ELA is increased in nonsmokers with periodontitis; therefore, more studies are needed to focus on the level of ELA in smokers with periodontitis. The other biomarker identified in this study was MPO. The current study detected higher elevation of the level of MPO in smokers with periodontitis; the current results were consistent with those of Omer et al., 38 who found that compromised periodontal inflammation in smokers may have increased the expression of MPO. Gattani et al. 39 also showed the same increase in MPO level in smokers with periodontitis than in nonsmokers with periodontitis, as it is considered as an indicator of oxidative stress caused by cigarette smoke and increased level of inflammation in the progression of periodontitis. However, a pilot study conducted by Magán-Fernández et al. 40 discovered that, without statistical significance, MPO expression was greater in periodontitis than in gingivitis. Additionally, this study found a significant elevation in citH3 levels in smoker with periodontitis compared with nonsmokers. This was consistent with a study by Okamato et al. 20 which found that citH3 is increased in individuals at risk than in control subjects. Kim et al. 9 agreed with our findings of histone elevation, which is highly associated with the presence of NETs and bone destruction, and that citH3 bears NET-associated posttranslational modification. Few studies have examined the effect of smoking on citH3 in periodontitis patients. A study performed by Hosseinzadeh et al. 41 demonstrated that smoking-related diseases may be aggravated by nicotine in cigarette smoke, which then activates the production of NETs. In contrast, Magán-Fernández et al. 40 reported that the expression of citH3 is higher in gingivitis than in periodontitis. Furthermore, the present study showed that the biomarkers were unable to differentiate between stage III and IV periodontitis groups. However, another recent study conducted by Mohammed et al. 42 agreed with our finding that the concentrations of salivary biomarkers matrix metalloproteinase (MMP8 and 9) and tissue inhibitor of metalloproteinase (TIMP1) were not significantly different (p<0.05) among the various periodontitis stages. Interestingly, the present study noticed a strong correlation between CALPRO and most of the clinical periodontal parameters in stage III periodontitis, thus enhancing the hypothesis that this biomarker performs an essential function in the NETosis stimulation process of periodontal inflammation, the present study finding was in line with Wei et al., 23 who found that CALPRO is positively correlated with clinical periodontal parameters in generalized aggressive periodontitis, and that there is a positive correlation between citH3 and CAL in stage III periodontitis, which is consistent with the findings of Kim et al., 9 who found that citH3 is strongly associated with bone destruction. Moreover, MPO showed a positive correlation with CAL. Similarly, Polizzi et al. 24 showed that generalized chronic periodontal disease is positively correlated with salivary MPO. There was a strong and positive association between citH3 and CALPRO in smokers with periodontitis. Our data also revealed a highly significant positive relationship between citH3 and ELA in nonsmokers’ periodontitis. The results of this study indicate that the elevation of citH3 in both periodontitis groups is indicative of the process of NETosis and increases inflammatory reactions, even if periodontitis risk factors are present, which emphasizes a possible interaction between neutrophil loss and the prevalence of autoimmune diseases. Elevation of these biomarkers in periodontitis smokers and nonsmokers aids in the external breakdown of pathogens that get stuck within NETs. A limitation of the current study is the high exclusion criteria proposed in the study. However, it provides additional specifications for our data. The novelty of this study is the limitation of studies that evaluated the effect of the citH3 NETosis salivary biomarker in smokers with periodontitis. Conclusions The selected salivary biomarkers (citH3, ELA, CALPRO, and MPO) were used to identify differences between healthy periodontium and periodontitis. All the chosen biomarkers were elevated in the smoker periodontitis group, and there was a statistically significant difference between smokers and nonsmokers with periodontitis compared to the controls. Smoking appears to have harmful effects, one of which is the downregulation of immunological responses to bacterial infection. On the other hand, it reduces the ability of neutrophils to respond to periodontal infection; therefore, smoking increases the biomarker levels in the smokers periodontitis group. The citH3, ELA, CALPRO, and MPO levels could not differentiate between stage III and IV smokers and nonsmoker periodontitis groups. There was a significant correlation between the salivary biomarkers citH3 and CALPRO in the periodontitis group and between citH3 and ELA in the nonsmoker periodontitis groups. Interestingly, the salivary biomarkers citH3, ELA, and CALPRO were positively correlated. All the selected salivary biomarkers increased in cases of periodontitis and were associated with neutrophil enzymes that mediate NETosis and are connected to the onset and progression of periodontitis. Author’s contributions Conceptualization Ideas: all authors RAN and BHA designed and performed the manuscript. RAN collected the patients, samples, and provided clinical information. RAN, BHA, MG, and AAA performed the experiments and contributed to the statistical analysis, reviewing the work for clarity and originality. RAN, BHA, and supervision by BHA. The authors reviewed and provided their stamp of approval for the paper. All necessary conditions for authorship have been fulfilled, and all the authors agree that the manuscript conveys their honest work. Consent to participate Following a sequential presentation of the research purpose and methods, all participants signed an informed consent form. The samples that collected in this study were salivary samples and this study was conducted at University of Baghdad/College of Dentistry/Department of Periodontics after ethical approval with reference Number: 844, project number: 844823 in 15-10-2023 was obtained, and a written consent was taken from the participant. Ethics and consent Ethical considerations were essential during the conduct of this study, introduced by the World Medical Association’s Helsinki Declaration (WMA), after submission to the ethical committee at the Dentistry College/University of Baghdad to obtain ethical approval (Reference Number: 844, project number: 844823, date: 15-10-2023). Data availability Figshare: Evaluation of NETosis (Neutrophil Extracellular Traps) Biomarkers in Smokers and Nonsmokers Periodontitis Patients. https://doi.org/10.6084/m9.figshare.25997872.v2 43 The project contains the following underlying data: • 2=F1000=Data=Rasha Najim--InFormt.xlsx. Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgment The authors are grateful to all study participants. References 1. Gasner NS, Schure RS: Periodontal disease. StatPearls. StatPearls Publishing; 2023. 2. 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D’Haens G, Ferrante M, Vermeire S, et al. : Fecal calprotectin is a surrogate marker for endoscopic lesions in inflammatory bowel disease. Inflamm Bowel Dis. 2012 Dec 1; 18 (12): 2218–2224. Publisher Full Text 35. Özçaka Ö, Bıçakcı N, Pussinen P, et al. : Smoking and matrix metalloproteinase, neutrophil elastase and myeloperoxidase in chronic periodontitis. Oral Dis. 2011 Jan; 17 (1): 68–76. PubMed Abstract | Publisher Full Text 36. Tseng HY, Chen YW, Lee BS, et al. : The neutrophil elastase-upregulated placenta growth factor promotes the pathogenesis and progression of periodontal disease. J Periodontol. 2022 Sep; 93 (9): 1401–1410. PubMed Abstract | Publisher Full Text 37. Pauletto NC, Liede K, Nieminen A, et al. : Effect of cigarette smoking on oral elastase activity in adult periodontitis patients. J Periodontol. 2000 Jan; 71 (1): 58–62. PubMed Abstract | Publisher Full Text 38. Omer-Cihangir R, Baser U, Kucukgergin C, et al. : Impact of smoking on neutrophil enzyme levels in gingivitis: a case-control study. Int J Environ Res Public Health. 2021 Jul 30; 18 (15): 8075. PubMed Abstract | Publisher Full Text | Free Full Text 39. Gattani D, Sahu J, Kar N, et al. : Salivary profile of matrix metalloproteinase-8, tissue inhibitors of matrix metalloproteinase-1, myeloperoxidase, and nitrous oxide in smokers versus nonsmokers with chronic periodontitis. J Int Clinical Dent Res Org. 2021 Jul 1; 13 (2): 118–123. Publisher Full Text 40. Magán-Fernández A, O’Valle F, Abadía-Molina F, et al. : Characterization and comparison of neutrophil extracellular traps in gingival samples of periodontitis and gingivitis: A pilot study. J Periodontal Res. 2019 Jun; 54 (3): 218–224. PubMed Abstract | Publisher Full Text 41. Hosseinzadeh A, Thompson PR, Segal BH, et al. : Nicotine induces neutrophil extracellular traps. J Leukoc Biol. 2016 Nov; 100 (5): 1105–1112. 42. Mohammed HA, Abdulkareem AA, Zardawi FM, et al. : Determination of the accuracy of salivary biomarkers for periodontal diagnosis. Diagnostics. 2022 Oct 14; 12 (10): 2485. PubMed Abstract | Publisher Full Text | Free Full Text 43. Najim R: 2=F1000=Data=Rasha Najim.xlsx. Dataset. figshare. 2024. Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 08 Aug 2024 ADD YOUR COMMENT Comment Author details Author details 1 Master's student, Basic Science Department, University of Baghdad, Baghdad, Baghdad Governorate, Iraq 2 Microbiological/Immunological Professor, Basic Science Department, University of Baghdad, Baghdad, Baghdad Governorate, Iraq Rasha Abdul Jabbar Najim Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Writing – Original Draft Preparation Batool Hassan Al Ghurabi Roles: Supervision, Validation, Visualization, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (2) version 2 Revised Published: 07 Apr 2025, 13:907 https://doi.org/10.12688/f1000research.152917.2 version 1 Published: 08 Aug 2024, 13:907 https://doi.org/10.12688/f1000research.152917.1 Copyright © 2025 Jabbar Najim RA and Al Ghurabi BH. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Jabbar Najim RA and Al Ghurabi BH. Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.12688/f1000research.152917.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 07 Apr 2025 Revised Views 0 Cite How to cite this report: Daily ZA. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r434266 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-434266 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 02 Dec 2025 Zina Ali Daily , University of Al-Ameed,Karbala, Karbala, Iraq Approved VIEWS 0 https://doi.org/10.5256/f1000research.176674.r434266 The work of this study is clear and given an information to the severity of periodontitis in smokers and nonsmokers. However, it is better to add a few words about the statistical method within the methods of the abstract and ... Continue reading READ ALL The work of this study is clear and given an information to the severity of periodontitis in smokers and nonsmokers. However, it is better to add a few words about the statistical method within the methods of the abstract and add the value of p in the results of the abstract for further clarification. Regarding the use manual ELISA technology to determine the levels of citH3, ELA, CALPRO, and MPO biomarkers in saliva but it was not clarifying how to obtain these levels, so it would be preferable to explain the processes of ELISA. It is also preferable to add a source to an existing paragraph within the article and explain the reason for that (The calibrated periodontal evaluations were performed on five patients which recorded every two hours for each of the six sites per tooth). Who did the examinations? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Immunology, genetics, periodontics, oncology, endocrine disorders, cardiovascular diseases and dental implants I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Daily ZA. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r434266 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-434266 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Javali MA. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r381719 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-381719 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 28 May 2025 Mukhatar Ahmed Javali , King Khalid University,, Abha, Saudi Arabia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176674.r381719 Major Comments: Objective of the study is novel, the manuscript needs a proper english language for better understanding of what the authors are willing to prove by this study. Many sentences or paragrphs are not clear , to be refrased ... Continue reading READ ALL Major Comments: Objective of the study is novel, the manuscript needs a proper english language for better understanding of what the authors are willing to prove by this study. Many sentences or paragrphs are not clear , to be refrased or rewritten. The objective to be clear and same in all parts of the study, whether its abstract,introduction, results, discussion or conclusion of the study. The objectives to be addressed as same. Plz see these >>> Abstract: Rewrite or better to write the sentence.. in the severe stages of periodontitis III and IV….as… In both smokers and nonsmokers stage III and IV periodontitis patients. Moreover, the mean NETosis biomarker‘s mean levels were significantly higher in smoker than in nonsmokers….. Refrase this sentence for better clarity Smoking is one of the main ways in which individuals are exposed to harmful chemicals (cigarette smoke contains at least 4,500 different chemicals), and can cause epigenetic changes that increase inflammation. In addition, smoking generally increases oxidative stress, with excess free oxygen radicals causing cell damage…. Cite a reference for this sentences Several studies have indicated that smokers are more likely to have periodontal disease than nonsmokers, including greater gingival recession, bone attachment , and tooth loss…. What does this mean However, severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful because it maintains the body in an inflammatory state, which destroys tissue….. Lack or defects in neutrophil or its functions… Kindly refrase this sentence and provide a reference Based on the available evidence , this study evaluated the NETosis biomarkers as indicators of inflammation in stage III and stage IV (smokers and nonsmokers) periodontitis groups and compared the findings with those of the controls. Furthermore, we determined the relationship between the NETosis biomarkers and clinical periodontal parameters….. Evidence is already available…. Then what was the idea behind this study … to prove what is already evident…. Better to rewrite the aim or objectives of the study. The type of this study was case control study not clinical trial study… W hat the need to write this here… when already in above para the study design is explained. Study population: Follow the case definition criteria given in the new classification 2017 for periodontitis and implant disease for staging or grading of periodontitis. Inclusion and exclusion criteria to be rewritten… more criteria’s are overlapping among inclusion and exclusion. And the study is done in smokers…. But history of smoking is written as exclusion criteria. Following saliva sampling, the clinical parameters (PLI, BOP, PPD, and CAL) and periodontal status was determined using William’s periodontal probe. Six distinct areas of each tooth (Distobuccal, Buccal, Mesiobuccal, Distolingual, Lingual, and Mesiolingual) were inspected to perform clinical parameters BOP/PPD and CAL only, while the scores of PLI were recorded by examining only four surfaces (mesial, distal, labial/buccal, lingual/palatal). The 3rd molar was excluded from the examination of all parameters, except PLI. PLI was recorded using disclosing agents to detect the presence or absence of dental plaque,29 while the percentage of BOP was noted as 1 present 0 absent.30 The distance from the free gingival margin to the base of the pocket is known as PPD, whereas the distance from the cemento-enamel junction (CEJ) to the base of the pocket in the absence of gingival recession is called CAL. If the margin of the gingiva is at the CEJ, then the CAL was considered to be equal the PPD; when there is gingival recession, CAL was determined by adding the recession depth and PPD; and when the margin was higher than CEJ, in order to determine the CAL, the distance from gingival margin to CEJ was subtracted from the PPD.25 Staging was performed by calculating the CAL plus 2 mm of biological width per root length multiplied by 100 for the most affected tooth in the dentition…. This whole para is confusing… make it simple understandable Pilot study Ten participants from the dental teaching hospital at the Dentistry College/Baghdad University participated in a pilot study to investigate the association between NETosis biomarkers (citH3, ELA, CALPRO, and MPO) as indicators of an inflammatory response in smokers and nonsmokers with periodontitis…… Is it necessary to include these… What is the idea here to include A total of 150 patients with periodontitis were screened to evaluate their eligibility for recruitment, and 75 people were excluded from the study after applying the inclusion and exclusion criteria to patients undergoing periodontal care clinics. Ultimately, only 60 male patients with periodontitis and 25 controls were included in the study (Figure 1)….. Cross check the numbers and figures details. The controls were taken from these 150 or 160 patients? What is control group…. Periodontal healthy individuals? Saliva collection method to be given in details and be clear, Dont repeate the tables or figures or graphs details in the results. Include a para for limitaions of your study and future recommendations. Conclusion part to have some recommendations at the end and some points regarding clinical relevance to the finding of the study. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Javali MA. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r381719 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-381719 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Bouziane A. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r376530 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-376530 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 19 May 2025 Amal Bouziane , Department of Periodontology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco; Laboratory of Biostatistics, Clinical Research and Epidemiology,, Mohammed V University in Rabat, Rabat, Morocco; Research Laboratory in Oral Biology and Biotechnology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.176674.r376530 The manuscript had many methodological and redactional flaws and required improvements recommended at the first submission. Several improvements have not been considered, including the title, Methods, and Results sections. ​​​​​​1. Title: • As NET is a production ... Continue reading READ ALL The manuscript had many methodological and redactional flaws and required improvements recommended at the first submission. Several improvements have not been considered, including the title, Methods, and Results sections. ​​​​​​1. Title: • As NET is a production of NETosis, the abbreviation in the title should be reviewed. • Please specify that periodontitis is a stage III and IV periodontitis. 2. Introduction: ​​​​​​• The sentence “ .. severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful..” still needs clarification. • Text added to abbreviations for the first time is still needed. The list of Abbreviations at the beginning of the manuscript is not mandatory.​​​​​​ ​​​​​​3. Methods: • Please avoid redundancy in the study design. “..not clinical trial study” could be removed. • The case definition of periodontitis, according to EFP and AAP 2017 consensus criteria, should be revised. • “ any periodontal treatment” still need to be clarified. • Smoking was a main exposition in the study. “History of smoking” could not be in exclusion criteria. • In the paragraph “Clinical periodontal parameters, the criteria for staging is not common and need a reference (“ Staging was performed by calculating the CAL plus 2 mm of biological width per root length multiplied by 100 for the most affected tooth in the dentition.”) • Conducting the pilot study is still not justified in the methods. • Please give a clear image of Flow Chart. 4. Results: • It is recommended to avoid redundancy between figures, tables and text for the same results • 0.000 should be replaced by <0.001 for the p value. • Indicate the abbreviations under the tables • The results should be presented as a text narratively (example, the paragraph “Salivary biomarkers levels” ). • The terms used for the same meaning should be unified (example: relationship, correlation, association, on page 8) • The accuracy of a diagnosis test needs a different method to state of Diagnostic accuracy of NETosis biomarkers. • Present the most relevant graphs. • Present the most relevant results ( mean levels, AUC,..) of NETosis biomarkers, such as smokers Vs non-smokers. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Bouziane A. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r376530 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-376530 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Isola G. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r377248 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-377248 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 18 Apr 2025 Gaetano Isola , University of Catania, Catania, Italy Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176674.r377248 In the manuscript entitled: "Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients" the authors aimed to assess NETosis biomarkers citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) as indicators of inflammation ... Continue reading READ ALL In the manuscript entitled: "Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients" the authors aimed to assess NETosis biomarkers citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) as indicators of inflammation in the severe stages of periodontitis III and IV in both (smokers and nonsmokers) patients, and to determine the correlation between NETosis biomarkers and clinical periodontal parameters. The authors found that the mean NETosis biomarker‘s mean levels were significantly higher in smoker than in nonsmokers. In addition, the correlations were significant between CALPRO and CitH3 in smokers and between ELA and CitH3 in nonsmokers. The authors concluded that the chosen salivary biomarkers of NETosis revealed elevated clinical accuracy in differentiating the studied periodontitis groups (smokers and nonsmokers) from controls. In addition, cigarette smoking increases the risk of periodontitis, and neutrophils in smokers with periodontitis exhibited more susceptibility to form neutrophil extracellular traps when compared with nonsmokers. Major comments: In general, the idea and innovation of this study regards the analysis of the link between peridontitis and alveolar bone loss mediated by mediators is interesting and novel because the role these aspects in medicine are validated but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this interesting topic with this kind of study design. The study was well conducted by the authors; However, there are some concerns to revise that are described below. The introduction section resumes the existing knowledge regarding the important factor linked with the relationship between periodontitis and systemic diseases. However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, discuss and must cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the relationship between periodontitis peri-implant diseases and systemic diseases and how periodontal treatment could improve alveolar bone loss outcomes: 1) Isola G,et al., 2025 (Ref 1) 2) Isola G,et al., 2025 (Ref 2) The authors should be better specified, at the end of the introduction section, the rationale of the study and the aim of the study. In the central section, should better clarify inclusions and exclusions criteria of the selected sample. Please better state the results obtained in the abstract. The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the relationship regarding the by periodontitis in and risk of oxidative stress evolution in periodontitis patients. The conclusion should reinforce in light of the discussions. In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol. However, this study, in my view does not in its current form satisfy a very high scientific requirement for indexing in this journal and requests a revision before a further re-evaluation of the manuscript. Minor Comments: Abstract: Better formulate the abstract section by better describing the aim of the study Introduction: Please refer to major comments Discussion: Please add a specific sentence that clarifies the results obtained in the first part of the discussion Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No References 1. Isola G, Polizzi A, Serra S, Boato M, et al.: Relationship between periodontitis and systemic diseases: A bibliometric and visual study. Periodontol 2000 . 2025. PubMed Abstract | Publisher Full Text 2. Isola G, Polizzi A, Santagati M, Alibrandi A, et al.: Effect of Nonsurgical Mechanical Debridement With or Without Chlorhexidine Formulations in the Treatment of Peri-Implant Mucositis. A Randomized Placebo-Controlled Clinical Trial. Clin Oral Implants Res . 2025. PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Periodontology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Isola G. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r377248 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-377248 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Silva TA. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r377245 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-377245 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 15 Apr 2025 Tarcília Aparecida Silva , Universidade Federal de Minas Gerais, Belo Horizonte, State of Minas Gerais, Brazil Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176674.r377245 The manuscript entitled “Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis” aims to assess the salivary concentration of citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) in smokers and nonsmokers’ individuals with ... Continue reading READ ALL The manuscript entitled “Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis” aims to assess the salivary concentration of citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) in smokers and nonsmokers’ individuals with periodontitis. The topic is relevant to both dental and medical research. Data from 85 patients were analyzed. The authors conducted a pilot study, sample size calculation and calibration, steps that strengthens the methodology. However, the study presents shortcomings that weaken its clarity and impact, making it unsuitable for indexing in its current form. I have provided criticisms and suggestions for improvement, as follows: Introduction: It is important to clearly state how this study scientifically advance s in relation to previous literature. Introduction: ‘However, severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful because it maintains the body in an inflammatory state, which destroys tissue’. Please rephrase to improve clarity. It would be important to exemplify the conditions in which the lack of neutrophils/Nets results in worse PD e.g. Papillon-Lefévre syndrome. Methods: Considering the potential influence of systemic conditions in the tested salivary biomarkers, it is important to inform how systemic diseases were excluded in this study population. The same concern exists about the medication use, please clearly state how these potential influences were managed. Methods: ‘The patients were advised to clean and floss their teeth to maintain good oral hygiene’. It is not clear if it was a recommendation prior saliva sampling or if patients received instructions (how many sessions) before inclusion in the study. Methods: For how long the saliva samples were stored at -20 o C until ELISA? I advise to report it because it is not the ideal temperature (-80 o C). Methods: Please revise the inclusion/exclusion criteria. Also, include smoking as inclusion criteria for one of groups and detail the criteria e.g. type of cigarettes, etc. Methods: Why only males were included? Vantages and limitations of this choice should be presented in the Discussion section. Methods: Did authors observed differences in the salivary flow between the groups? Please discuss how could this impact the study´s findings considering the effect of smoking in the salivary flow. Results: Data presentation is repetitive. There is no need to represent same results in Tables and Figures. I advise the authors to chose just one presentation for each dataset and focus in the most relevant results. Additional information may be moved to supplementary material. Results: Type and frequency and duration of smoking was evaluated? Maybe authors could use statistical tests to assess the potential influence this variable. Results: Considering differences in the PD parameters in the group’s smoker x non-smoker, variables should be tested in a multivariate model to assess the weight of each one in the target biomarkers. Discussion: The limitation topic should be expanded e.g. one time-point sampling considering the fluctuation of these molecules in saliva, only male included etc. Minor suggestions: Methods: Kits/reagents should be followed by detailed information of product and company (city, state, country). Methods: For ELISA, please cite general details of method, e.g. duplicates were run; data were corrected by protein content or salivary flow, etc. Figure 1: Consider to move it to the supplementary material. Modify ‘polymorph nuclear’ by ‘polymorphonuclear’ Change ‘research groups’ by ‘groups’ Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Dentisty; Oral Pathology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Silva TA. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r377245 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-377245 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 08 Aug 2024 Views 0 Cite How to cite this report: Bouziane A. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.167730.r328722 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v1#referee-response-328722 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 30 Oct 2024 Amal Bouziane , Department of Periodontology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco; Laboratory of Biostatistics, Clinical Research and Epidemiology,, Mohammed V University in Rabat, Rabat, Morocco; Research Laboratory in Oral Biology and Biotechnology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.167730.r328722 The study tries to give some evidence about variations in salivary biomarkers’ (Neutrophil Extracellular Traps) levels in smokers and nonsmokers periodontitis patients. The comparison also concerned periodontally affected patients versus control subjects with a healthy periodontal status. Authors try to ... Continue reading READ ALL The study tries to give some evidence about variations in salivary biomarkers’ (Neutrophil Extracellular Traps) levels in smokers and nonsmokers periodontitis patients. The comparison also concerned periodontally affected patients versus control subjects with a healthy periodontal status. Authors try to bring support to the clinical accuracy of NETosis salivary biomarkers to differentiate periodontitis groups (smokers and nonsmokers) from controls. However, the manuscript has many methodological and redactional flaws and requires improvement. The main issue is related to the design of the study and the presentation of the results. Title : As NET is a production of NETosis, the abbreviation in the title should be reviewed. Please precise that periodontitis is a stage III and IV periodontitis. Introduction : Physiopathology and risk factors of periodontitis need more clarification. Genetics should not be considered as a local factor. The sentence “ .. severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful ..” seems to be contradictory. Idem for “.. The primary defense of periodontal tissues is provided by NETs, and a deficiency in NETs can result in severe early-onset periodontitis. Smoking cigarettes accelerates the production of NETs and PMN-derived proteases, which are responsible for breaking down connective tissue ..”. Please clarify. Please add text to abbreviations at the first time. Methods : The main issue is related to the design of the study. It is not clear if case is Periodontitis group and control is Non periodontitis group, Or case is smokers group and control is Non smokers group. The outcome conditions the sample size and the selection of the study population. Periodontitis cases, including smokers and nonsmokers, should be compared with non-periodontitis controls including smokers and nonsmokers. Please avoid redundancy in the study design. “.. not clinical trial study” could be removed. Please review the case definition of periodontitis according to EFP and AAP 2017 consensus criteria. What do the authors mean by “ any periodontal treatment” in Exclusion criteria? Rarely, a patient did not receive a periodontal treatment in the past, at least a periodontal scaling. Smoking was a main exposition in the study. Smoking could not be an exclusion criterion. Please give a sharp Flow Chart, and add participants with healthy periodontal status. Why the pilot study was conducted? Please explain its part in the methods. Results : The “increase” of parameters implies a prospective design study. Please give an appropriate word. Idem for elevation. The Statistical analysis “ Two qualitative variables, distributional correlations, were tested using the chi-square test. ” Should be clarified. It is more appropriate to focus results to the objectives of the study. Figures 2 and 3 contain repeated information. They could be removed. 0.000 should be replaced by <0.001 for the p value. Indicate the abbreviations under the tables (for example table 5) Please begin the paragraph “Salivary biomarkers levels” with text. Present the most relevant graph in Figure 5. The first paragraph of ROC results should be reformulated. The accuracy of a diagnosis test needs a different method to state of Diagnostic accuracy of NETosis biomarkers. Discussion : How the absence of correlation between CAL and Histon-H3, in stage IV, could be explained comparatively to stage III? (see Table 3) Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Bouziane A. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.167730.r328722 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v1#referee-response-328722 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 07 Apr 2025 Rasha Najim , Master's student, Basic Science Department, University of Baghdad, Baghdad, Iraq 07 Apr 2025 Author Response Greetings Title - I approved - NETosis was mentioned in the title to make the title more influential but we can omit it so I approve the new title ''Evaluation of neutrophil ... Continue reading Greetings Title - I approved - NETosis was mentioned in the title to make the title more influential but we can omit it so I approve the new title ''Evaluation of neutrophil extracellular traps biomarkers as an indication in smokers and nonsmokers biomarkers stage III and IV periodontitis patients'' Introduction - I approved - The clarifying for this sentence is that the balance that occurred in the number neutrophils is crucial for maintaining periodontal homeostasis, Neutrophil equilibrium is important in periodontal homeostasis because periodontitis can develop when there are too many or too few neutrophils in the tissue.as an imbalance can result in defect in NETs production and then periodontitis. Also smoking cigarettes has several potential detrimental effects on periodontal health which impaired defenses against plaque bacteria, and interference with oxidative stress processes. The primary defense of periodontal tissues is provided by NETs, Neutrophils aid in homeostasis and defense by producing neutrophil extracellular taps (NETs). Smoking cigarettes accelerates the production of NETs , which are responsible for breaking down connective tissue. - I approved Methods - The control group is generally healthy persons and also healthy periodontium with BOP less than 10% and also CAL less than 3mm and no periodontal pocket depth less than 4mm and also non smokers any type of smoke. - I approved - The patient didn't do receive any antibiotic or periodontal treatment in the last 3 months before collecting the sample - We mean that we exclude those patients who smoke any other types of smoke like Vape hookah only we select the cigarette's smoke patients - The healthy periodontal status were added in the flow chart - the pilot study was made to obtain the preliminary data for the primary outcome measure, in order to calculate the required sample size Results - Two qualitative variables were tested to calculate the sensitivity and specificity of each salivary biomarker. - I approved - I approved Greetings Title - I approved - NETosis was mentioned in the title to make the title more influential but we can omit it so I approve the new title ''Evaluation of neutrophil extracellular traps biomarkers as an indication in smokers and nonsmokers biomarkers stage III and IV periodontitis patients'' Introduction - I approved - The clarifying for this sentence is that the balance that occurred in the number neutrophils is crucial for maintaining periodontal homeostasis, Neutrophil equilibrium is important in periodontal homeostasis because periodontitis can develop when there are too many or too few neutrophils in the tissue.as an imbalance can result in defect in NETs production and then periodontitis. Also smoking cigarettes has several potential detrimental effects on periodontal health which impaired defenses against plaque bacteria, and interference with oxidative stress processes. The primary defense of periodontal tissues is provided by NETs, Neutrophils aid in homeostasis and defense by producing neutrophil extracellular taps (NETs). Smoking cigarettes accelerates the production of NETs , which are responsible for breaking down connective tissue. - I approved Methods - The control group is generally healthy persons and also healthy periodontium with BOP less than 10% and also CAL less than 3mm and no periodontal pocket depth less than 4mm and also non smokers any type of smoke. - I approved - The patient didn't do receive any antibiotic or periodontal treatment in the last 3 months before collecting the sample - We mean that we exclude those patients who smoke any other types of smoke like Vape hookah only we select the cigarette's smoke patients - The healthy periodontal status were added in the flow chart - the pilot study was made to obtain the preliminary data for the primary outcome measure, in order to calculate the required sample size Results - Two qualitative variables were tested to calculate the sensitivity and specificity of each salivary biomarker. - I approved - I approved Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 07 Apr 2025 Rasha Najim , Master's student, Basic Science Department, University of Baghdad, Baghdad, Iraq 07 Apr 2025 Author Response Greetings Title - I approved - NETosis was mentioned in the title to make the title more influential but we can omit it so I approve the new title ''Evaluation of neutrophil ... Continue reading Greetings Title - I approved - NETosis was mentioned in the title to make the title more influential but we can omit it so I approve the new title ''Evaluation of neutrophil extracellular traps biomarkers as an indication in smokers and nonsmokers biomarkers stage III and IV periodontitis patients'' Introduction - I approved - The clarifying for this sentence is that the balance that occurred in the number neutrophils is crucial for maintaining periodontal homeostasis, Neutrophil equilibrium is important in periodontal homeostasis because periodontitis can develop when there are too many or too few neutrophils in the tissue.as an imbalance can result in defect in NETs production and then periodontitis. Also smoking cigarettes has several potential detrimental effects on periodontal health which impaired defenses against plaque bacteria, and interference with oxidative stress processes. The primary defense of periodontal tissues is provided by NETs, Neutrophils aid in homeostasis and defense by producing neutrophil extracellular taps (NETs). Smoking cigarettes accelerates the production of NETs , which are responsible for breaking down connective tissue. - I approved Methods - The control group is generally healthy persons and also healthy periodontium with BOP less than 10% and also CAL less than 3mm and no periodontal pocket depth less than 4mm and also non smokers any type of smoke. - I approved - The patient didn't do receive any antibiotic or periodontal treatment in the last 3 months before collecting the sample - We mean that we exclude those patients who smoke any other types of smoke like Vape hookah only we select the cigarette's smoke patients - The healthy periodontal status were added in the flow chart - the pilot study was made to obtain the preliminary data for the primary outcome measure, in order to calculate the required sample size Results - Two qualitative variables were tested to calculate the sensitivity and specificity of each salivary biomarker. - I approved - I approved Greetings Title - I approved - NETosis was mentioned in the title to make the title more influential but we can omit it so I approve the new title ''Evaluation of neutrophil extracellular traps biomarkers as an indication in smokers and nonsmokers biomarkers stage III and IV periodontitis patients'' Introduction - I approved - The clarifying for this sentence is that the balance that occurred in the number neutrophils is crucial for maintaining periodontal homeostasis, Neutrophil equilibrium is important in periodontal homeostasis because periodontitis can develop when there are too many or too few neutrophils in the tissue.as an imbalance can result in defect in NETs production and then periodontitis. Also smoking cigarettes has several potential detrimental effects on periodontal health which impaired defenses against plaque bacteria, and interference with oxidative stress processes. The primary defense of periodontal tissues is provided by NETs, Neutrophils aid in homeostasis and defense by producing neutrophil extracellular taps (NETs). Smoking cigarettes accelerates the production of NETs , which are responsible for breaking down connective tissue. - I approved Methods - The control group is generally healthy persons and also healthy periodontium with BOP less than 10% and also CAL less than 3mm and no periodontal pocket depth less than 4mm and also non smokers any type of smoke. - I approved - The patient didn't do receive any antibiotic or periodontal treatment in the last 3 months before collecting the sample - We mean that we exclude those patients who smoke any other types of smoke like Vape hookah only we select the cigarette's smoke patients - The healthy periodontal status were added in the flow chart - the pilot study was made to obtain the preliminary data for the primary outcome measure, in order to calculate the required sample size Results - Two qualitative variables were tested to calculate the sensitivity and specificity of each salivary biomarker. - I approved - I approved Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Hadzic Z. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.167730.r315884 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v1#referee-response-315884 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 09 Sep 2024 Zerina Hadzic , Sarajevo School of Science and Technology, Sarajevo, Federation of Bosnia and Herzegovina, Bosnia and Herzegovina Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.167730.r315884 The text of the research article titled: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients requires major correction. The abstract is not clear and it does not give all the important details for understanding the ... Continue reading READ ALL The text of the research article titled: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients requires major correction. The abstract is not clear and it does not give all the important details for understanding the research article, especially in the methodology section. From the title, abstract, but also Introduction it is not clear that the authors aimed to determine the salivary parameters only in male patients. It is also not elaborated clearly why the female patients were excluded. The introduction does not give any inside in the other similar studies and the background for their examination in patients with periodontitis. The classification of the patients in the stage III and IV periodontitis groups is not correct. Stage III and stage IV in the segment of bone loss are radiologicaly with bone loss to mid-third of root or beyond, but are separated by the number of teeth lost, and with complexity of stage III being applicable to stage IV and additional complexity including masticatory disfunction, bite collapse, drifting, flaring and less then remaining 20 teeth (10 opposing pairs). [ ref 1 ] Also it is not clear if the patients consumed and antioxidants and if this was an exclusion criteria applied. In the saliva sampling section it is not clear: did the patients brush before the sampling? how were samples defrosted? Also the time frame of saliva sampling is not clear. The authors state: Salivary samples were conducted between 9 am and 12 pm. Is the 12 pm: noon or 00:00, since by definition noon is neither AM or PM. Also, study included PLI in 3rd molars but excluded the 3rd molar for other clinical periodontal indices. Were the 3rd molars included in the determination of periodontitis stage? Conclusion is not written clearly and does not correspond to the precise results and study groups. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No References 1. Tonetti MS, Greenwell H, Kornman KS: Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J Periodontol . 2018; 89 Suppl 1 : S159-S172 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Periodontal disease, Salivary biomarkers, Oxidative stress, Inflammatory biomarkers, Biofilm. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Hadzic Z. Reviewer Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.167730.r315884 ) The direct URL for this report is: https://f1000research.com/articles/13-907/v1#referee-response-315884 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 07 Apr 2025 Rasha Najim , Master's student, Basic Science Department, University of Baghdad, Baghdad, Iraq 07 Apr 2025 Author Response This research was approved for its title and content after submitting a protocol to the Council of the College of Dentistry at the university of Baghdad. the abstract of the ... Continue reading This research was approved for its title and content after submitting a protocol to the Council of the College of Dentistry at the university of Baghdad. the abstract of the research sheds light on the research in general but all the details are mentioned in the research in detail. All salivary samples in this research were taken from males exclusively and females were excluded from the beginning before starting the research to provide standardization of the salivary samples on this study and also due to that hormonal fluctuations during female's lifetime may provide an exaggerated inflammatory response to the clinical periodontal parameters and produce significant changes in the periodontium according to previous studies. All the periodontitis patients taken in this study were sever stages stage III and stage IV and we depend on the clinical periodontal parameters according to the mentioned ref. (46) that depend on calculating the CAL and presence of PPD and all the parameters were assessed by inter-examiner as well as intra-examiner calibration. All the salivary samples as mentioned in this research were collected from specialized governmental dental educational centers and their working hours are necessarily from morning until 3 pm therefore the samples were taken in the morning period and before any periodontal treatment for the purpose of accuracy in collecting salivary samples. This research was approved for its title and content after submitting a protocol to the Council of the College of Dentistry at the university of Baghdad. the abstract of the research sheds light on the research in general but all the details are mentioned in the research in detail. All salivary samples in this research were taken from males exclusively and females were excluded from the beginning before starting the research to provide standardization of the salivary samples on this study and also due to that hormonal fluctuations during female's lifetime may provide an exaggerated inflammatory response to the clinical periodontal parameters and produce significant changes in the periodontium according to previous studies. All the periodontitis patients taken in this study were sever stages stage III and stage IV and we depend on the clinical periodontal parameters according to the mentioned ref. (46) that depend on calculating the CAL and presence of PPD and all the parameters were assessed by inter-examiner as well as intra-examiner calibration. All the salivary samples as mentioned in this research were collected from specialized governmental dental educational centers and their working hours are necessarily from morning until 3 pm therefore the samples were taken in the morning period and before any periodontal treatment for the purpose of accuracy in collecting salivary samples. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 07 Apr 2025 Rasha Najim , Master's student, Basic Science Department, University of Baghdad, Baghdad, Iraq 07 Apr 2025 Author Response This research was approved for its title and content after submitting a protocol to the Council of the College of Dentistry at the university of Baghdad. the abstract of the ... Continue reading This research was approved for its title and content after submitting a protocol to the Council of the College of Dentistry at the university of Baghdad. the abstract of the research sheds light on the research in general but all the details are mentioned in the research in detail. All salivary samples in this research were taken from males exclusively and females were excluded from the beginning before starting the research to provide standardization of the salivary samples on this study and also due to that hormonal fluctuations during female's lifetime may provide an exaggerated inflammatory response to the clinical periodontal parameters and produce significant changes in the periodontium according to previous studies. All the periodontitis patients taken in this study were sever stages stage III and stage IV and we depend on the clinical periodontal parameters according to the mentioned ref. (46) that depend on calculating the CAL and presence of PPD and all the parameters were assessed by inter-examiner as well as intra-examiner calibration. All the salivary samples as mentioned in this research were collected from specialized governmental dental educational centers and their working hours are necessarily from morning until 3 pm therefore the samples were taken in the morning period and before any periodontal treatment for the purpose of accuracy in collecting salivary samples. This research was approved for its title and content after submitting a protocol to the Council of the College of Dentistry at the university of Baghdad. the abstract of the research sheds light on the research in general but all the details are mentioned in the research in detail. All salivary samples in this research were taken from males exclusively and females were excluded from the beginning before starting the research to provide standardization of the salivary samples on this study and also due to that hormonal fluctuations during female's lifetime may provide an exaggerated inflammatory response to the clinical periodontal parameters and produce significant changes in the periodontium according to previous studies. All the periodontitis patients taken in this study were sever stages stage III and stage IV and we depend on the clinical periodontal parameters according to the mentioned ref. (46) that depend on calculating the CAL and presence of PPD and all the parameters were assessed by inter-examiner as well as intra-examiner calibration. All the salivary samples as mentioned in this research were collected from specialized governmental dental educational centers and their working hours are necessarily from morning until 3 pm therefore the samples were taken in the morning period and before any periodontal treatment for the purpose of accuracy in collecting salivary samples. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 08 Aug 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 4 5 6 Version 2 (revision) 07 Apr 25 read read read read read Version 1 08 Aug 24 read read Zerina Hadzic , Sarajevo School of Science and Technology, Sarajevo, Bosnia and Herzegovina Amal Bouziane , Mohammed V University in Rabat, Rabat, Morocco; Mohammed V University in Rabat, Rabat, Morocco; Mohammed V University in Rabat, Rabat, Morocco Tarcília Aparecida Silva , Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Gaetano Isola , University of Catania, Catania, Italy Mukhatar Ahmed Javali , King Khalid University,, Abha, Saudi Arabia Zina Ali Daily , University of Al-Ameed,Karbala, Karbala, Iraq Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Daily Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 02 Dec 2025 | for Version 2 Zina Ali Daily , University of Al-Ameed,Karbala, Karbala, Iraq 0 Views copyright © 2025 Daily Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The work of this study is clear and given an information to the severity of periodontitis in smokers and nonsmokers. However, it is better to add a few words about the statistical method within the methods of the abstract and add the value of p in the results of the abstract for further clarification. Regarding the use manual ELISA technology to determine the levels of citH3, ELA, CALPRO, and MPO biomarkers in saliva but it was not clarifying how to obtain these levels, so it would be preferable to explain the processes of ELISA. It is also preferable to add a source to an existing paragraph within the article and explain the reason for that (The calibrated periodontal evaluations were performed on five patients which recorded every two hours for each of the six sites per tooth). Who did the examinations? Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Immunology, genetics, periodontics, oncology, endocrine disorders, cardiovascular diseases and dental implants I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Daily ZA. Peer Review Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r434266) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-434266 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Javali M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 28 May 2025 | for Version 2 Mukhatar Ahmed Javali , King Khalid University,, Abha, Saudi Arabia 0 Views copyright © 2025 Javali M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Major Comments: Objective of the study is novel, the manuscript needs a proper english language for better understanding of what the authors are willing to prove by this study. Many sentences or paragrphs are not clear , to be refrased or rewritten. The objective to be clear and same in all parts of the study, whether its abstract,introduction, results, discussion or conclusion of the study. The objectives to be addressed as same. Plz see these >>> Abstract: Rewrite or better to write the sentence.. in the severe stages of periodontitis III and IV….as… In both smokers and nonsmokers stage III and IV periodontitis patients. Moreover, the mean NETosis biomarker‘s mean levels were significantly higher in smoker than in nonsmokers….. Refrase this sentence for better clarity Smoking is one of the main ways in which individuals are exposed to harmful chemicals (cigarette smoke contains at least 4,500 different chemicals), and can cause epigenetic changes that increase inflammation. In addition, smoking generally increases oxidative stress, with excess free oxygen radicals causing cell damage…. Cite a reference for this sentences Several studies have indicated that smokers are more likely to have periodontal disease than nonsmokers, including greater gingival recession, bone attachment , and tooth loss…. What does this mean However, severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful because it maintains the body in an inflammatory state, which destroys tissue….. Lack or defects in neutrophil or its functions… Kindly refrase this sentence and provide a reference Based on the available evidence , this study evaluated the NETosis biomarkers as indicators of inflammation in stage III and stage IV (smokers and nonsmokers) periodontitis groups and compared the findings with those of the controls. Furthermore, we determined the relationship between the NETosis biomarkers and clinical periodontal parameters….. Evidence is already available…. Then what was the idea behind this study … to prove what is already evident…. Better to rewrite the aim or objectives of the study. The type of this study was case control study not clinical trial study… W hat the need to write this here… when already in above para the study design is explained. Study population: Follow the case definition criteria given in the new classification 2017 for periodontitis and implant disease for staging or grading of periodontitis. Inclusion and exclusion criteria to be rewritten… more criteria’s are overlapping among inclusion and exclusion. And the study is done in smokers…. But history of smoking is written as exclusion criteria. Following saliva sampling, the clinical parameters (PLI, BOP, PPD, and CAL) and periodontal status was determined using William’s periodontal probe. Six distinct areas of each tooth (Distobuccal, Buccal, Mesiobuccal, Distolingual, Lingual, and Mesiolingual) were inspected to perform clinical parameters BOP/PPD and CAL only, while the scores of PLI were recorded by examining only four surfaces (mesial, distal, labial/buccal, lingual/palatal). The 3rd molar was excluded from the examination of all parameters, except PLI. PLI was recorded using disclosing agents to detect the presence or absence of dental plaque,29 while the percentage of BOP was noted as 1 present 0 absent.30 The distance from the free gingival margin to the base of the pocket is known as PPD, whereas the distance from the cemento-enamel junction (CEJ) to the base of the pocket in the absence of gingival recession is called CAL. If the margin of the gingiva is at the CEJ, then the CAL was considered to be equal the PPD; when there is gingival recession, CAL was determined by adding the recession depth and PPD; and when the margin was higher than CEJ, in order to determine the CAL, the distance from gingival margin to CEJ was subtracted from the PPD.25 Staging was performed by calculating the CAL plus 2 mm of biological width per root length multiplied by 100 for the most affected tooth in the dentition…. This whole para is confusing… make it simple understandable Pilot study Ten participants from the dental teaching hospital at the Dentistry College/Baghdad University participated in a pilot study to investigate the association between NETosis biomarkers (citH3, ELA, CALPRO, and MPO) as indicators of an inflammatory response in smokers and nonsmokers with periodontitis…… Is it necessary to include these… What is the idea here to include A total of 150 patients with periodontitis were screened to evaluate their eligibility for recruitment, and 75 people were excluded from the study after applying the inclusion and exclusion criteria to patients undergoing periodontal care clinics. Ultimately, only 60 male patients with periodontitis and 25 controls were included in the study (Figure 1)….. Cross check the numbers and figures details. The controls were taken from these 150 or 160 patients? What is control group…. Periodontal healthy individuals? Saliva collection method to be given in details and be clear, Dont repeate the tables or figures or graphs details in the results. Include a para for limitaions of your study and future recommendations. Conclusion part to have some recommendations at the end and some points regarding clinical relevance to the finding of the study. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Javali MA. Peer Review Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r381719) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-381719 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Bouziane A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 19 May 2025 | for Version 2 Amal Bouziane , Department of Periodontology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco; Laboratory of Biostatistics, Clinical Research and Epidemiology,, Mohammed V University in Rabat, Rabat, Morocco; Research Laboratory in Oral Biology and Biotechnology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco 0 Views copyright © 2025 Bouziane A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript had many methodological and redactional flaws and required improvements recommended at the first submission. Several improvements have not been considered, including the title, Methods, and Results sections. ​​​​​​1. Title: • As NET is a production of NETosis, the abbreviation in the title should be reviewed. • Please specify that periodontitis is a stage III and IV periodontitis. 2. Introduction: ​​​​​​• The sentence “ .. severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful..” still needs clarification. • Text added to abbreviations for the first time is still needed. The list of Abbreviations at the beginning of the manuscript is not mandatory.​​​​​​ ​​​​​​3. Methods: • Please avoid redundancy in the study design. “..not clinical trial study” could be removed. • The case definition of periodontitis, according to EFP and AAP 2017 consensus criteria, should be revised. • “ any periodontal treatment” still need to be clarified. • Smoking was a main exposition in the study. “History of smoking” could not be in exclusion criteria. • In the paragraph “Clinical periodontal parameters, the criteria for staging is not common and need a reference (“ Staging was performed by calculating the CAL plus 2 mm of biological width per root length multiplied by 100 for the most affected tooth in the dentition.”) • Conducting the pilot study is still not justified in the methods. • Please give a clear image of Flow Chart. 4. Results: • It is recommended to avoid redundancy between figures, tables and text for the same results • 0.000 should be replaced by <0.001 for the p value. • Indicate the abbreviations under the tables • The results should be presented as a text narratively (example, the paragraph “Salivary biomarkers levels” ). • The terms used for the same meaning should be unified (example: relationship, correlation, association, on page 8) • The accuracy of a diagnosis test needs a different method to state of Diagnostic accuracy of NETosis biomarkers. • Present the most relevant graphs. • Present the most relevant results ( mean levels, AUC,..) of NETosis biomarkers, such as smokers Vs non-smokers. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Bouziane A. Peer Review Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r376530) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-376530 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 18 Apr 2025 | for Version 2 Gaetano Isola , University of Catania, Catania, Italy 0 Views copyright © 2025 Isola G. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In the manuscript entitled: "Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients" the authors aimed to assess NETosis biomarkers citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) as indicators of inflammation in the severe stages of periodontitis III and IV in both (smokers and nonsmokers) patients, and to determine the correlation between NETosis biomarkers and clinical periodontal parameters. The authors found that the mean NETosis biomarker‘s mean levels were significantly higher in smoker than in nonsmokers. In addition, the correlations were significant between CALPRO and CitH3 in smokers and between ELA and CitH3 in nonsmokers. The authors concluded that the chosen salivary biomarkers of NETosis revealed elevated clinical accuracy in differentiating the studied periodontitis groups (smokers and nonsmokers) from controls. In addition, cigarette smoking increases the risk of periodontitis, and neutrophils in smokers with periodontitis exhibited more susceptibility to form neutrophil extracellular traps when compared with nonsmokers. Major comments: In general, the idea and innovation of this study regards the analysis of the link between peridontitis and alveolar bone loss mediated by mediators is interesting and novel because the role these aspects in medicine are validated but further studies on this topic could be an innovative issue in this field could be open a creative matter of debate in literature by adding new information. Moreover, there are few reports in the literature that studied this interesting topic with this kind of study design. The study was well conducted by the authors; However, there are some concerns to revise that are described below. The introduction section resumes the existing knowledge regarding the important factor linked with the relationship between periodontitis and systemic diseases. However, as the importance of the topic, the reviewer strongly recommends, before a further re-evaluation of the manuscript, to update the literature through read, discuss and must cites in the references with great attention all of those recent interesting articles, that helps the authors to better introduce and discuss the relationship between periodontitis peri-implant diseases and systemic diseases and how periodontal treatment could improve alveolar bone loss outcomes: 1) Isola G,et al., 2025 (Ref 1) 2) Isola G,et al., 2025 (Ref 2) The authors should be better specified, at the end of the introduction section, the rationale of the study and the aim of the study. In the central section, should better clarify inclusions and exclusions criteria of the selected sample. Please better state the results obtained in the abstract. The discussion section appears well organized with the relevant paper that support the conclusions, even if the authors should better discuss the relationship regarding the by periodontitis in and risk of oxidative stress evolution in periodontitis patients. The conclusion should reinforce in light of the discussions. In conclusion, I am sure that the authors are fine clinicians who achieve very nice results with their adopted protocol. However, this study, in my view does not in its current form satisfy a very high scientific requirement for indexing in this journal and requests a revision before a further re-evaluation of the manuscript. Minor Comments: Abstract: Better formulate the abstract section by better describing the aim of the study Introduction: Please refer to major comments Discussion: Please add a specific sentence that clarifies the results obtained in the first part of the discussion Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No References 1. Isola G, Polizzi A, Serra S, Boato M, et al.: Relationship between periodontitis and systemic diseases: A bibliometric and visual study. Periodontol 2000 . 2025. PubMed Abstract | Publisher Full Text 2. Isola G, Polizzi A, Santagati M, Alibrandi A, et al.: Effect of Nonsurgical Mechanical Debridement With or Without Chlorhexidine Formulations in the Treatment of Peri-Implant Mucositis. A Randomized Placebo-Controlled Clinical Trial. Clin Oral Implants Res . 2025. PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Periodontology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Isola G. Peer Review Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r377248) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-377248 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Silva T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 15 Apr 2025 | for Version 2 Tarcília Aparecida Silva , Universidade Federal de Minas Gerais, Belo Horizonte, State of Minas Gerais, Brazil 0 Views copyright © 2025 Silva T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript entitled “Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis” aims to assess the salivary concentration of citrullinated histone H3 (citH3), neutrophil elastase (ELA), calprotectin (CALPRO), and myeloperoxidase (MPO) in smokers and nonsmokers’ individuals with periodontitis. The topic is relevant to both dental and medical research. Data from 85 patients were analyzed. The authors conducted a pilot study, sample size calculation and calibration, steps that strengthens the methodology. However, the study presents shortcomings that weaken its clarity and impact, making it unsuitable for indexing in its current form. I have provided criticisms and suggestions for improvement, as follows: Introduction: It is important to clearly state how this study scientifically advance s in relation to previous literature. Introduction: ‘However, severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful because it maintains the body in an inflammatory state, which destroys tissue’. Please rephrase to improve clarity. It would be important to exemplify the conditions in which the lack of neutrophils/Nets results in worse PD e.g. Papillon-Lefévre syndrome. Methods: Considering the potential influence of systemic conditions in the tested salivary biomarkers, it is important to inform how systemic diseases were excluded in this study population. The same concern exists about the medication use, please clearly state how these potential influences were managed. Methods: ‘The patients were advised to clean and floss their teeth to maintain good oral hygiene’. It is not clear if it was a recommendation prior saliva sampling or if patients received instructions (how many sessions) before inclusion in the study. Methods: For how long the saliva samples were stored at -20 o C until ELISA? I advise to report it because it is not the ideal temperature (-80 o C). Methods: Please revise the inclusion/exclusion criteria. Also, include smoking as inclusion criteria for one of groups and detail the criteria e.g. type of cigarettes, etc. Methods: Why only males were included? Vantages and limitations of this choice should be presented in the Discussion section. Methods: Did authors observed differences in the salivary flow between the groups? Please discuss how could this impact the study´s findings considering the effect of smoking in the salivary flow. Results: Data presentation is repetitive. There is no need to represent same results in Tables and Figures. I advise the authors to chose just one presentation for each dataset and focus in the most relevant results. Additional information may be moved to supplementary material. Results: Type and frequency and duration of smoking was evaluated? Maybe authors could use statistical tests to assess the potential influence this variable. Results: Considering differences in the PD parameters in the group’s smoker x non-smoker, variables should be tested in a multivariate model to assess the weight of each one in the target biomarkers. Discussion: The limitation topic should be expanded e.g. one time-point sampling considering the fluctuation of these molecules in saliva, only male included etc. Minor suggestions: Methods: Kits/reagents should be followed by detailed information of product and company (city, state, country). Methods: For ELISA, please cite general details of method, e.g. duplicates were run; data were corrected by protein content or salivary flow, etc. Figure 1: Consider to move it to the supplementary material. Modify ‘polymorph nuclear’ by ‘polymorphonuclear’ Change ‘research groups’ by ‘groups’ Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Dentisty; Oral Pathology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Silva TA. Peer Review Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.176674.r377245) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-907/v2#referee-response-377245 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Bouziane A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 30 Oct 2024 | for Version 1 Amal Bouziane , Department of Periodontology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco; Laboratory of Biostatistics, Clinical Research and Epidemiology,, Mohammed V University in Rabat, Rabat, Morocco; Research Laboratory in Oral Biology and Biotechnology, Faculty of Dental Medicine,, Mohammed V University in Rabat, Rabat, Morocco 0 Views copyright © 2024 Bouziane A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The study tries to give some evidence about variations in salivary biomarkers’ (Neutrophil Extracellular Traps) levels in smokers and nonsmokers periodontitis patients. The comparison also concerned periodontally affected patients versus control subjects with a healthy periodontal status. Authors try to bring support to the clinical accuracy of NETosis salivary biomarkers to differentiate periodontitis groups (smokers and nonsmokers) from controls. However, the manuscript has many methodological and redactional flaws and requires improvement. The main issue is related to the design of the study and the presentation of the results. Title : As NET is a production of NETosis, the abbreviation in the title should be reviewed. Please precise that periodontitis is a stage III and IV periodontitis. Introduction : Physiopathology and risk factors of periodontitis need more clarification. Genetics should not be considered as a local factor. The sentence “ .. severe periodontitis is caused by a lack of neutrophils, and an excess of neutrophils appears to be harmful ..” seems to be contradictory. Idem for “.. The primary defense of periodontal tissues is provided by NETs, and a deficiency in NETs can result in severe early-onset periodontitis. Smoking cigarettes accelerates the production of NETs and PMN-derived proteases, which are responsible for breaking down connective tissue ..”. Please clarify. Please add text to abbreviations at the first time. Methods : The main issue is related to the design of the study. It is not clear if case is Periodontitis group and control is Non periodontitis group, Or case is smokers group and control is Non smokers group. The outcome conditions the sample size and the selection of the study population. Periodontitis cases, including smokers and nonsmokers, should be compared with non-periodontitis controls including smokers and nonsmokers. Please avoid redundancy in the study design. “.. not clinical trial study” could be removed. Please review the case definition of periodontitis according to EFP and AAP 2017 consensus criteria. What do the authors mean by “ any periodontal treatment” in Exclusion criteria? Rarely, a patient did not receive a periodontal treatment in the past, at least a periodontal scaling. Smoking was a main exposition in the study. Smoking could not be an exclusion criterion. Please give a sharp Flow Chart, and add participants with healthy periodontal status. Why the pilot study was conducted? Please explain its part in the methods. Results : The “increase” of parameters implies a prospective design study. Please give an appropriate word. Idem for elevation. The Statistical analysis “ Two qualitative variables, distributional correlations, were tested using the chi-square test. ” Should be clarified. It is more appropriate to focus results to the objectives of the study. Figures 2 and 3 contain repeated information. They could be removed. 0.000 should be replaced by <0.001 for the p value. Indicate the abbreviations under the tables (for example table 5) Please begin the paragraph “Salivary biomarkers levels” with text. Present the most relevant graph in Figure 5. The first paragraph of ROC results should be reformulated. The accuracy of a diagnosis test needs a different method to state of Diagnostic accuracy of NETosis biomarkers. Discussion : How the absence of correlation between CAL and Histon-H3, in stage IV, could be explained comparatively to stage III? (see Table 3) Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 07 Apr 2025 Rasha Najim, Master's student, Basic Science Department, University of Baghdad, Baghdad, Iraq Greetings Title - I approved - NETosis was mentioned in the title to make the title more influential but we can omit it so I approve the new title ''Evaluation of neutrophil extracellular traps biomarkers as an indication in smokers and nonsmokers biomarkers stage III and IV periodontitis patients'' Introduction - I approved - The clarifying for this sentence is that the balance that occurred in the number neutrophils is crucial for maintaining periodontal homeostasis, Neutrophil equilibrium is important in periodontal homeostasis because periodontitis can develop when there are too many or too few neutrophils in the tissue.as an imbalance can result in defect in NETs production and then periodontitis. Also smoking cigarettes has several potential detrimental effects on periodontal health which impaired defenses against plaque bacteria, and interference with oxidative stress processes. The primary defense of periodontal tissues is provided by NETs, Neutrophils aid in homeostasis and defense by producing neutrophil extracellular taps (NETs). Smoking cigarettes accelerates the production of NETs , which are responsible for breaking down connective tissue. - I approved Methods - The control group is generally healthy persons and also healthy periodontium with BOP less than 10% and also CAL less than 3mm and no periodontal pocket depth less than 4mm and also non smokers any type of smoke. - I approved - The patient didn't do receive any antibiotic or periodontal treatment in the last 3 months before collecting the sample - We mean that we exclude those patients who smoke any other types of smoke like Vape hookah only we select the cigarette's smoke patients - The healthy periodontal status were added in the flow chart - the pilot study was made to obtain the preliminary data for the primary outcome measure, in order to calculate the required sample size Results - Two qualitative variables were tested to calculate the sensitivity and specificity of each salivary biomarker. - I approved - I approved View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Bouziane A. Peer Review Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.167730.r328722) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-907/v1#referee-response-328722 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Hadzic Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 09 Sep 2024 | for Version 1 Zerina Hadzic , Sarajevo School of Science and Technology, Sarajevo, Federation of Bosnia and Herzegovina, Bosnia and Herzegovina 0 Views copyright © 2024 Hadzic Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The text of the research article titled: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients requires major correction. The abstract is not clear and it does not give all the important details for understanding the research article, especially in the methodology section. From the title, abstract, but also Introduction it is not clear that the authors aimed to determine the salivary parameters only in male patients. It is also not elaborated clearly why the female patients were excluded. The introduction does not give any inside in the other similar studies and the background for their examination in patients with periodontitis. The classification of the patients in the stage III and IV periodontitis groups is not correct. Stage III and stage IV in the segment of bone loss are radiologicaly with bone loss to mid-third of root or beyond, but are separated by the number of teeth lost, and with complexity of stage III being applicable to stage IV and additional complexity including masticatory disfunction, bite collapse, drifting, flaring and less then remaining 20 teeth (10 opposing pairs). [ ref 1 ] Also it is not clear if the patients consumed and antioxidants and if this was an exclusion criteria applied. In the saliva sampling section it is not clear: did the patients brush before the sampling? how were samples defrosted? Also the time frame of saliva sampling is not clear. The authors state: Salivary samples were conducted between 9 am and 12 pm. Is the 12 pm: noon or 00:00, since by definition noon is neither AM or PM. Also, study included PLI in 3rd molars but excluded the 3rd molar for other clinical periodontal indices. Were the 3rd molars included in the determination of periodontitis stage? Conclusion is not written clearly and does not correspond to the precise results and study groups. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No References 1. Tonetti MS, Greenwell H, Kornman KS: Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J Periodontol . 2018; 89 Suppl 1 : S159-S172 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Periodontal disease, Salivary biomarkers, Oxidative stress, Inflammatory biomarkers, Biofilm. I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 07 Apr 2025 Rasha Najim, Master's student, Basic Science Department, University of Baghdad, Baghdad, Iraq This research was approved for its title and content after submitting a protocol to the Council of the College of Dentistry at the university of Baghdad. the abstract of the research sheds light on the research in general but all the details are mentioned in the research in detail. All salivary samples in this research were taken from males exclusively and females were excluded from the beginning before starting the research to provide standardization of the salivary samples on this study and also due to that hormonal fluctuations during female's lifetime may provide an exaggerated inflammatory response to the clinical periodontal parameters and produce significant changes in the periodontium according to previous studies. All the periodontitis patients taken in this study were sever stages stage III and stage IV and we depend on the clinical periodontal parameters according to the mentioned ref. (46) that depend on calculating the CAL and presence of PPD and all the parameters were assessed by inter-examiner as well as intra-examiner calibration. All the salivary samples as mentioned in this research were collected from specialized governmental dental educational centers and their working hours are necessarily from morning until 3 pm therefore the samples were taken in the morning period and before any periodontal treatment for the purpose of accuracy in collecting salivary samples. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Hadzic Z. Peer Review Report For: Evaluation of NETosis (Neutrophil Extracellular Traps) biomarkers in smokers and nonsmokers periodontitis patients [version 2; peer review: 1 approved, 3 approved with reservations, 2 not approved] . F1000Research 2025, 13 :907 ( https://doi.org/10.5256/f1000research.167730.r315884) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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