Measurement of gp130 cytokines oncostatin M and IL-6 in gingival crevicular fluid of patients with chronic periodontitis

Several proinflammatory cytokines can induce periodontal tissue destruction and are thought to be useful indicators or diagnostic markers for periodontitis. Here, we aimed to investigate whether oncostatin M (OSM) was present in gingival crevicular fluid (GCF) and to clarify the correlation of GCF OSM and interleukin-6 (IL-6) levels with the severity of periodontitis. Sixty-two sites in 14 patients were divided into 4 groups based on probing depth (PD) and bleeding on probing (BOP). GCF was collected using paper strips from clinically health sites (PD < or = 3 mm, CAL: 1-3 mm, without BOP, n = 31), mildly diseased sites (PD < or = 3 mm, CAL: 3-5 mm, with BOP, n = 11), moderately diseased sites (PD = 4-6 mm, CAL: 5-8 mm, with BOP, n = 11), and severely diseased sites (PD > 6 mm, CAL: 8-12 mm, with BOP, n = 9). IL-6 and OSM in GCF were quantified by enzyme-linked immunosorbent assay and are expressed as concentrations (pg/ml) and total amounts (pg/site). Correlations of OSM and IL-6 levels with the severity of periodontitis in all groups were determined using Spearman rank correlation (r(s)). Our results showed that OSM and IL-6 were detected in most GCF samples. The total amounts of OSM and IL-6 were significantly positive correlated with severity of diseased sites (OSM: r(s) = 0.526, p < 0.01; IL-6: r(s) = 0.729, p < 0.01). No correlations of OSM or IL-6 concentration in GCF were found with disease severity. OSM and IL-6 levels in GCF were positively correlated to each other when expressed as either concentrations or total amounts (concentrations: r = 0.485, p < 0.01; total amounts r = 0.490, p < 0.01). In conclusion, our findings suggest that IL-6 and OSM may play a role in modulating the inflammatory cascade of chronic periodontitis.     

Group II phospholipase A(2) in human gingiva with periodontal disease

Fourteen kilodalton phospholipase A(2) molecules (PLA(2)) are classified into two groups, I- and II-PLA(2), and only the latter has been considered to play a pathogenetic role in various forms of tissue inflammation. Previously we demonstrated high PLA(2) activity in gingival crevicular fluid (GCF) of patients with periodontal disease, without determining the group of the enzyme involved. In this study, the activity, groups and levels of enzyme in gingiva taken from 13 sites of periodontal disease were determined using both biochemical and radioimmunological methods. A linear correlation between the activity and the level of II-PLA(2) was observed. No I-PLA(2) was found in any of the samples tested. These data suggest that the PLA(2) activity found in the GCF of patients with periodontal disease does not belong to the I-PLA(2) but to the II-PLA(2) group.     

Correlation of MCP-4 and high-sensitivity C-reactive protein as a marker of inflammation in obesity and chronic periodontitis

ObjectivesObesity is increasing in prevalence worldwide and has emerged as a strong risk factor for periodontal disease. Conversely, the remote effects of periodontal disease on various systemic diseases have been proposed. The aim of this study is to determine the presence of MCP-4 and high sensitivity C reactive protein (hsCRP) levels in gingival crevicular fluid (GCF) and serum in obese and non-obese subjects with chronic periodontitis and to find a correlation between MCP-4 and hsCRP in GCF and serum.Materials and methodsForty subjects (20 males and 20 females) were selected and divided into four groups (10 subjects in each group), based on clinical parameters: group NOH (non-obese healthy), group OH (obese healthy), Group NOCP (non-obese with chronic periodontitis) and group OCP (obese with chronic periodontitis). The levels of serum and GCF MCP-4 were determined by ELISA and hsCRP levels were determined by immunoturbidimetry method.ResultsThe mean GCF and serum concentration of MCP-4 was highest for group OCP followed by group NOCP, group OH (in GCF); group OH, group NOCP(in serum) and least in group NOH. The mean hsCRP concentration was highest for group OCP followed by group OH, group NOCP and group NOH. A significant positive correlation was found between serum and GCF MCP-4 and hsCRP levels.ConclusionGCF MCP-4 concentrations increased in periodontal disease compared to health and correlated positively with the severity of disease indicating it as a novel marker of periodontal disease. The serum concentration of MCP-4 was found to be more in obese group as compared to nonobese group indicating it as a marker of obesity. Furthermore, based on the positive correlation of MCP-4 and hsCRP found in this study, it can be proposed that MCP-4 and hsCRP may be the markers linking chronic inflammation in obesity and periodontal disease.     

Quantitative Proteomic Analysis of Gingival Crevicular Fluid in Different Periodontal Conditions

Aim

To quantify the proteome composition of the GCF in periodontal health (HH) and in sites with different clinical conditions in chronic periodontitis (CP) subjects.

Material and Methods

5 subjects with HH and 5 with CP were submitted to full-mouth periodontal examination, and GCF sampling. Sites in the CP group were classified and sampled as periodontitis (P, probing depth, PD>4 mm), gingivitis (G, PD≤3mm with bleeding on probing, BOP), and healthy sites (H, PD≤3mm without BOP). GCF proteins were subjected to liquid chromatography electrospray ionization mass spectrometry for identification, characterization and quantification.

Results

230 proteins were identified; 145 proteins were detected in HH, 214 in P, 154 in G, and 133 in H. Four proteins were exclusively detected at HH, 43 proteins at P, 7 proteins at G, and 1 protein at H. Compared to HH group, 35 and 6 proteins were more abundant in P and G (p<0.001), respectively; and 4, 15 and 37 proteins were less abundant in P, G and H (p≤0.01), respectively.

Conclusions

There are marked differences in the GCF proteome according to disease profile. Comprehension of the role of the identified proteins in the etiopathogenesis of periodontal disease may lead to biomarkers definition.     

Application of quantitative proteomic analysis using tandem mass tags for discovery and identification of novel biomarkers in periodontal disease

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, matrix metalloproteinase (MMP)‐9 and neutrophil gelatinase‐associated lipocalin (LCN2), which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP‐9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC‐MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease.     

Effect of a subgingival chlorhexidine chip on the clinical parameters and the levels of alkaline phosphatase activity in gingival crevicular fluid during the non-surgical treatment of periodontitis

The main therapeutic approaches for inflammatory periodontal diseases include the mechanical treatment of root surfaces. Multi-center clinical trials have demonstrated that the adjunctive use of a chlorhexidine (CHX) chip is effective in improving clinical results compared to scaling and root planing (SRP) alone. However, some recent studies failed to confirm these clinical results, nor have any data been reported regarding the capability of the CHX chip in affecting the activity of alkaline phosphatase (ALP) in the gingival crevicular fluid (GCF). This enzyme has been related to a condition of destructive activity of periodontitis. The aim of this study is to provide further data on the clinical and biochemical effects of CHX chips when used as an adjunct to SRP. Eighty-two systemically healthy patients, aged 31-63, with moderate and advanced periodontitis were recruited from the departments of Periodontology of the University of Chieti. In each patient 2 experimental sites, located in two symmetric quadrants, were chosen with a probing depth of > or = 5 mm and bleeding on probing. The 2 sites were selected randomly at the split-mouth level; control sites received SRP alone, and test sites SRP plus 1 CHX chip. Clinical indices, including probing depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), and the ALP activity in GCF were evaluated at baseline and after 6 months. Alkaline phosphatase activity was assayed spectrophotometrically. The PPD and CAL were significantly lower at 6 months as compared to the baseline scores in both treatments (p less than 0.01). The PPD reduction was 2.7 mm in the CHX+SRP group and 1.9 mm in the SRP alone group. The CHX+SRP group showed a significantly greater gain of clinical attachment (mean: 1.4 mm) in comparison with the SRP group (mean: 0.9; p less than 0.05). No differences were observed in the decrease of the percent of BOP-positive sites between the experimental groups. Conversely, the CHX+SRP group underwent a significantly greater decrease (p less than 0.01) of the GCF-ALP activity 6 months after treatment in comparison with the SRP alone group. The adjunctive use of the CHX chip resulted in a significant improvement of pocket reduction and clinical attachment gain as compared with SRP alone. These results were concomitant with a significantly greater reduction of the GCF-ALP activity levels.     

Detection of Tannerella forsythia and/or Prevotella intermedia might be useful for microbial predictive markers for the outcome of initial periodontal treatment in Koreans

A proportion of diseased sites in periodontal disease do not respond to the initial treatment, which might be due in part to the presence of specific microbial pathogens. The aim of this study was to clarify the value of microbial screening for predicting the outcome of periodontal treatment in Koreans using a polymerase chain reaction (PCR). This study enrolled 32 adults with periodontal disease. Microbial and clinical examinations were performed at the baseline and after the initial treatment (professional toothbrushing, scaling, and root planing). Subgingival plaque samples were taken from four sites in each subject (total 128 samples). PCR was used to detect the four putative pathogenic bacteria. There was an improvement in the average of each clinical measurement after the initial treatment. However, approximately half of the sites exhibiting bleeding upon probing (BOP) at the baseline still exhibited bleeding after treatment. There was a close association between the presence of BOP and the presence of Tannerella forsythia (formerly Bacteroides forsythus) and/or Prevotella intermedia. Furthermore, the sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than the sites where these two species were not detected. Therefore, microbial screening for T. forsythia and P. intermedia might be useful for predicting the treatment outcome in Koreans.     

The efficacy of non-surgical platelet-rich fibrin application on clinical periodontal parameters and periostin level in periodontitis: Clinical trial

Platelet-rich fibrin (PRF) has been widely used in regenerative dentistry due to many growth factors produced. Periostin, a matricellular protein, is a reliable marker for tissue regeneration. Periostin is part of the cellular matrix and regulates bone homeostasis. This study aims to explore the efficacy of PRF in improvement of the clinical periodontal parameters as an adjunct to the scaling and root planing and to evaluate periostin level in gingival crevicular fluid (GCF) at baseline, 1- and 3-month recall visits. Fourteen periodontitis patients who met the inclusion criteria were recruited in this study. Two contralateral periodontal pockets with 4–6 mm in depth in each patient were selected. The sites in every participant were randomly allocated into control sites or test sites. In control sites, only conventional scaling and root planing was carried out. In test sites, however, scaling and root planing method and PRF were applied. Periostin level in GCF and clinical periodontal parameters were measured. The test sites revealed greater relative attachment gain (2.614 ± 0.606 mm and 3.321 ± 0.668 mm) than control sites (1.285 ± 0.671 mm and 1.839 ± 0.632 mm) and a significant pocket reduction (2.535 ± 0.664 mm and 3.321 ± 0.668 mm) than the control sites (1.21 ± 0.508 mm and 1.892 ± 0.655 mm) at 1- and 3-month recall visits respectively. In the test sites, level of periostin (48.83 ± 9.3 ng/μl and 98.90 ± 24.94 ng/μl) were greater than periostin levels in the control sites (42.65 ± 7.03 ng/μl and 49.29 ± 15.14 ng/μl) at 1- and 3-month recall visits respectively. In conclusion, the non-surgical application of PRF as an adjunct to scaling and root planing significantly improved the clinical periodontal parameters through raising periostin level in GCF.     

Quantitation of malondialdehyde in gingival crevicular fluid by a high-performance liquid chromatography-based method

Lipid peroxidation (LPO) has been associated with periodontal disease, and the evaluation of malondialdehyde (MDA) in the gingival crevicular fluid (GCF), an inflammatory exudate from the surrounding tissue of the periodontium, may be useful to clarify the role of LPO in the pathogenesis of periodontal disease. We describe the validation of a method to measure MDA in the GCF using high-performance liquid chromatography. MDA calibration curves were prepared with phosphate-buffered solution spiked with increasing known concentrations of MDA. Healthy and diseased GCF samples were collected from the same patient to avoid interindividual variability. MDA response was linear in the range measured, and excellent agreement was observed between added and detected concentrations of MDA. Samples' intra- and interday coefficients of variation were below 6.3% and 12.4%, respectively. The limit of quantitation (signal/noise=5) was 0.03 μM. When the validated method was applied to the GCF, excellent agreement was observed in the MDA quantitation from healthy and diseased sites, and diseased sites presented more MDA than healthy sites (P<0.05). In this study, a validated method for MDA quantitation in GCF was established with satisfactory sensitivity, precision, and accuracy.     

Anti-HDV IgM as a Marker of Disease Activity in Hepatitis Delta

Background

Hepatitis delta frequently leads to liver cirrhosis and hepatic decompensation. As treatment options are limited, there is a need for biomarkers to determine disease activity and to predict the risk of disease progression. We hypothesized that anti-HDV IgM could represent such a marker.

Methods

Samples of 120 HDV-infected patients recruited in an international multicenter treatment trial (HIDIT-2) were studied. Anti-HDV IgM testing was performed using ETI-DELTA-IGMK-2-assay (DiaSorin). In addition, fifty cytokines, chemokines and angiogenetic factors were measured using multiplex technology (Bio-Plex System). A second independent cohort of 78 patients was studied for the development of liver-related clinical endpoints (decompensation, HCC, liver transplantation or death; median follow up of 3.0 years, range 0.6–12).

Results

Anti-HDV IgM serum levels were negative in 18 (15%), low (OD<0.5) in 76 (63%), and high in 26 (22%) patients of the HIDIT-2 cohort. Anti-HDV IgM were significantly associated with histological inflammatory (p<0.01) and biochemical disease activity (ALT, AST p<0.01). HDV replication was independent from anti-HDV IgM, however, low HBV-DNA levels were observed in groups with higher anti-HDV IgM levels (p<0.01). While high IP-10 (CXCL10) levels were seen in greater groups of anti-HDV IgM levels, various other antiviral cytokines were negatively associated with anti-HDV IgM. Associations between anti-HDV IgM and ALT, AST, HBV-DNA were confirmed in the independent cohort. Clinical endpoints occurred in 26 anti-HDV IgM positive patients (39%) but in only one anti-HDV IgM negative individual (9%; p = 0.05).

Conclusions

Serum anti-HDV IgM is a robust, easy-to-apply and relatively cheap marker to determine disease activity in hepatitis delta which has prognostic implications. High anti-HDV IgM levels may indicate an activated interferon system but exhausted antiviral immunity.     

S100A2 level changes are related to human periodontitis

Periodontitis is an inflammatory disease, which, when severe, can result in tooth loss, that affects the quality of life. S100A2 was previously identified as a component of gingival crevicular fluid (GCF) via proteome analysis, but it has not been investigated whether S100A2 plays a role in periodontitis. In this study, we analyzed mRNA expression of S100A2 in gingival tissues from normal and classified periodontal disease patients and compared it to that of S100A8 and S100A9. Quantitative real time-PCR revealed that the mRNA expression levels of S100A2, S100A8, and S100A9 were significantly upregulated in gingival tissues with gingivitis, moderate periodontitis, and severe periodontitis compared to normal tissues. In addition, S100A2 proteins in GCF and the conditioned media of lipopolysaccharide (LPS)-treated Jurkat cells were confirmed by ELISA. S100A2 protein levels were significantly higher in GCF in gingivitis and moderate periodontitis groups than in normal groups. S100A2 mRNA expression and protein secretion were also increased by LPS stimulation. Based on the up-regulation of S100A2 in LPS-stimulated immune cells, gingival tissues and GCF from periodontal disease groups, we conclude that S100A2 is a functional component in the immune response during periodontitis and may serve as a potential biomarker for periodontitis.     

Role of monocyte chemoattractant protein-1 (MCP-1) as an immune-diagnostic biomarker in the pathogenesis of chronic periodontal disease

ObjectiveMonocyte chemoattractant protein-1 (MCP-1) is an important chemokine responsible for the initiation, regulation and mobilization of monocytes to the active sites of severe periodontal inflammation. The present study aims at evaluating the levels of MCP-1 in GCF, saliva and serum and to analyze the changes following phase I periodontal therapy. Assessment of possible correlations between levels of MCP-1 in the three biological fluids was also done.MethodsFifteen healthy and 30 patients of severe chronic periodontitis (diseased) participated in the study. Patients of the diseased group underwent scaling/root planing. Evaluation of PI, GI, PD, CAL and collection of samples of GCF, serum and saliva was done at baseline and 6 weeks following periodontal therapy. MCP-1 levels were quantified in all samples using ELISA.ResultsCompared to healthy controls, MCP-1 levels were statistically significantly higher in GCF (p < 0.001), saliva (p = 0.002) and serum (p < 0.001) in subjects with chronic periodontitis. Levels of MCP-1 in all the three fluids decreased significantly in patients after periodontal therapy (p < 0.001). There was a significant positive correlation between MCP-1 levels in GCF, saliva and serum in patients of chronic periodontitis both pre (r > 0.9) and post-treatment (r > 0.6).ConclusionsThe results suggest that levels of MCP-1 in GCF and saliva can be reliable indicators of severity of periodontal destruction and their serum levels reflect the systemic impact of this local inflammatory disease thereby strengthening the reciprocal oro-systemic association.     

Relationships of anti-PAc (361-386) peptide salivary IgA antibody, eosinophils and basophils with periodontal status in the elderly

The amino acid residues 361-386 of Streptococcus mutans PAc includes an important region associated with the interaction between S. mutans and salivary components. We investigated the relationships between levels of the anti-PAc (361-386) peptide antibody (PPA) in saliva and periodontal status in 281 elderly subjects (mean age 77 years; 118 females, 163 males) by assessing dental calculus (CA), attachment loss (AL), pocket depth (PD), bleeding on probing (BOP) and various blood parameters. Enzyme-linked immunosorbent assay results revealed that subjects with a PPA level of greater than 0.1 (PPA detected group) showed a lower average value for number of sites with more than 6 mm of AL/6 points x 100/tooth (rAL6) than those with a PPA level of less than 0.1 (PPA not detected group). Furthermore, average values for rAL6 were significantly lower in the PPA detected group, and BOP, AL and rAL6 correlated positively and significantly with the percentage of eosinophils present in leukocytes in female subjects in both groups. PPA level had a negative correlation with percentages of basophils and eosinophils. The results indicate that systemic increases in numbers of eosinophils and basophils are associated with the development of periodontal diseases, while PPA level may be a useful indicator of periodontal status.     

Tumor necrosis factor-α converting enzyme (TACE) increases RANKL expression in osteoblasts and serves as a potential biomarker of periodontitis

Periodontitis is a very prevalent disease. Therefore, biomarkers for the early and standard diagnoses of periodontitis are urgently needed. TACE is a membrane-bound metalloprotease. Although a recent study suggested that TACE levels in GCF are elevated during periodontal disease, the levels of TACE in GCF at different stages of chronic periodontitis have not been determined. Here, we analyzed the protein levels of TACE in GCF from periodontal disease subjects and confirmed that the protein levels of TACE were higher in the moderate periodontitis groups. TACE is known to be a NF-κB ligand that stimulates RANKL secretion in osteoblasts. To understand the effects of TACE on RANKL and OPG in osteoblasts, we treated MG63 cells with TACE. We observed an increase in RANKL protein expression but a decrease in OPG protein expression. Our data suggest that TACE can induce RANKL expression and promote osteoclastogenesis, thus worsening the outcome of periodontitis.     

Brain α-Ketoglutarate Dehydrotenase Complex Activity in Alzheimer's Disease

We measured the activity of the a-ketoglutarate dehydrogenase complex (α-KGDHC), a rate-limiting Krebs cycle enzyme, in postmortem brain samples from 38 controls and 30 neuropathologically confirmed Alzheimer's disease (AD) cases, in both the presence and absence of thiamine pyrophosphate (TPP), the enzyme's cofactor. Statistically significant correlations between brain pH and lactate levels and α-KGDHC activity in the controls were observed, suggesting an influence of agonal status on the activity of α-KGDHC. As compared with the controls, mean α-KGDHC activity, with added TPP, was significantly (p < 0.005) reduced in AD brain in frontal (-56%), temporal (-60%), and parietal (-68%) cortices, with the reductions (-25 to -53%) in the occipital cortex, hippocampus, amygdala, and caudate failing to reach statistical significance. In the absence of exogenously administered TPP, mean a-KGDHC activity was reduced to a slightly greater extent in all seven AD brain areas (-39 to -83%), with the reductions now reaching statistical significance in the four cerebral cortical areas and hippocampus. A statistically significant negative correlation was observed between α-KGDHC activity and neurofibrillary tangle count in AD parietal cortex, the brain area exhibiting the most marked reduction in enzyme activity; this suggests that the enzyme activity reduction in AD brain may be related to the disease process and severity. In each brain area examined, TPP produced a greater stimulatory effect on α-KGDHC activity in the AD group (23–280% mean stimulation) as compared with the controls (-4 to ±50%); this TPP effect could be explained by reduced endogenous TPP levels in AD brain. Reduced brain α-KGDHC activity could be consequent to loss of neurons preferentially enriched in α-KGDHC, a premortem reduction in TPP levels (which may have affected enzyme stability), elevated brain levels of the α-KGDHC inhibitor ammonia, or an actual failure in the expression of the gene encoding the enzyme. We suggest that a defect in this key Krebs cycle enzyme could contribute to an impairment of cerebral energy metabolism and the brain dysfunction in AD.     

Periodontal Disease in Type 1 Diabetes Mellitus: Influence of Pubertal Stage and Glycemic Control

ObjectiveThough gingivitis is common in children with type 1 diabetes mellitus (T1DM), the overall periodontal health in T1DM during the pubertal stage is less well-characterized. The study was undertaken to explore the possible influence of puberty and metabolic derangement on periodontal health in T1DM.MethodsIn this cross-sectional study, 110 subjects between 10-18 years with T1DM and 52 healthy siblings of similar age were evaluated for pubertal stage, glycosylated hemoglobin (HbA1c), and periodontal health. Simplified oral hygiene index (OHIS), gingival index (GI), plaque index (PI), bleeding on probing (BOP), and probing depth (PPD) were evaluated at 4 sites per tooth as per 6 Ramfjord index teeth used to assess periodontal disease (PD).ResultsPD not merely gingivitis was significantly higher in T1DM (84/110, 76.36%) than the control group (28/52, 53.8%) (P = .004). Irrespective of pubertal status, children with T1DM had worse GI, PI, BOP, and PPD than nondiabetic subjects, although OHIS was better in diabetes. In both T1DM and nondiabetic subjects, pubertal subjects showed significantly worse OHIS, PPD, BOP, and GI than prepubertal subjects. PD was correlated with pubertal stage, age, and HbA1c, although less strongly with the duration of diabetes. In logistic regression, pubertal stage was a stronger predictor of PD (OR = 14.26) than age (OR = 2.22), and HbA1c (OR = 1.5) rather than the presence of diabetes and its duration.ConclusionsThough pubertal status, age, and poor glycemic control rather than the presence of diabetes and its duration are associated with gingivitis and other forms of PD, puberty had a more profound effect in the pathogenesis of PD in T1DM.     

Influence of storage conditions on MALDI‐TOF MS profiling of gingival crevicular fluid: Implications on the role of S100A8 and S100A9 for clinical and proteomic based diagnostic investigations

Gingival crevicular fluid (GCF) may be a source of diagnostic biomarkers of periodontitis/gingivitis. However, peptide fingerprints may change, depending on GCF collection, handling and storage. We evaluated how storage conditions affect the quality and the reproducibility of MALDI‐TOF profiles of this fluid. GCF was collected on paper strips from four subjects with healthy gingiva. Our findings demonstrated that sample storage conditions significantly affect GCF peptide pattern over time. Specifically, the storage of GCF immediately extracted from paper strips generates less variations in molecular profiles compared to the extraction performed after the storage. Significant spectral changes were detected for GCF samples stored at –20°C directly on the paper strips and extracted after three months, in comparison to the freshly extracted control. Noteworthy, a significant decrease in the peak area of HNP‐3, S100A8, full‐length S100A9 and its truncated form were detected after 3 months at –80°C. The alterations found in the “stored GCF” profile not only may affect the pattern‐based biomarker discovery but also make its use not adequate for in vitro diagnostic test targeting S100A8, S100A9 proposed as potential diagnostic biomarkers for periodontal disease. In summary, this study shows that the best preserved signatures were obtained for the GCF samples eluted in trifluoroacetic acid and then immediately stored at –80°C for 1 month. The wealth of information gained from our data on protein/patterns stability after storage might be helpful in defining new protocols which enable optimal preservation of GCF specimen.     

Incidence of Prevotella intermedia and Prevotella nigrescens in Periodontal Health and Disease

The incidence of black-pigmented rods (BPRs), especially Prevotella intermedia and Prevotella nigrescens, in periodontal health and disease were examined. Furthermore, the degradative enzyme activities of P. intermedia were compared among the strains from periodontal health and disease. Microbiological specimens were collected from subgingival crevice or periodontal pocket by paper point. The BPRs were found in 71.1% of periodontally healthy subjects (n = 45), and in 47.1% of healthy sites (n = 34) and 87.8% of active sites (n = 41) among periodontally diseased patients. Porphyromonas gingivalis was detected only in active sites of periodontally diseased patients (17.8% of 180 strains). P. intermedia was the predominant BPR in both healthy and active sites (37.3 and 41.7%, respectively) of the patients. However, P. nigrescens was the predominant BPR (70.5% of 173 strains) in periodontally healthy subjects. The enzyme activities of esterase, esterase-lipase, acid-phosphatase and α-fucosidase of P. intermedia strains isolated from active sites in patients were significantly higher (P < 0.05) than those of healthy subjects. The results suggest that P. intermedia might increase the activity of degradative enzymes under a certain condition and support the progression of periodontitis.     

Interleukin-12 and interleukin-16 in periodontal disease

The immune system plays an important role in the pathological process of periodontitis. Interleukin-12 (IL-12) is produced by monocytes, macrophages and neutrophils. These cells are proinflammatory infiltrates in periodontitis tissues. High IL-12 will contribute to the immune reaction to Th1 type. IL-12 is an inducer of INF-r production. IFN-gamma itself can also activate IL-12 production. Lipopolysaccharides (LPS) of periodontopathogens are also activators of IL-12. Interleukin-16 (IL-16) can cause the high affinity of IL-2 receptors on CD4+ cells and is chemotaxis to Th1 cells and CD4+ T cells. IL-16 can stimulate monocytes to produce proinflammatory cytokines and is highly associated with inflammation including arthritis, enteritis and allergic rhinitis. However, the information on IL-12 and IL-16 in periodontitis is not clear. In this study, 105 GCF samples were collected from 19 periodontal disease patients and 6 healthy ones. The clinical periodontal indices, the habits of cigarette smoking and alcohol drinking were recorded. ELISA was used to determine the levels of IL-12 and IL16 in the GCF. In the non-smoking/non-alcohol-drinking individuals: (1) the total amount of IL-12 (but not IL-16) was significantly higher in chronic periodontitis (CP) sites than gingivitis (G) or healthy (H) sites; (2) the diseased sites (CP + G) had a significantly higher total amount of IL-12 (but not IL-16) than the H sites. Among CP sites, both the concentration and total amount of IL-16 (but not IL-12) were significantly higher in alcohol drinkers/cigarette smokers as compared to the non-drinkers/non-smokers. CP sites of the drinkers/smokers also had significantly deeper probing pocket depth than sites of those without these two habits. IL-12 and IL-16 may be related to the pathogenesis of periodontal disease, but within the periodontitis sites, IL-16 may be related to disease severity in alcohol drinkers/smokers.     

Effect of Nadir CD4+ T Cell Count on Clinical Measures of Periodontal Disease in HIV+ Adults before and during Immune Reconstitution on HAART

Background

The contribution of HIV-infection to periodontal disease (PD) is poorly understood.  We proposed that immunological markers would be associated with improved clinical measures of PD.

Methods

We performed a longitudinal cohort study of HIV-infected adults who had started highly active antiretroviral therapy (HAART) <2 years. PD was characterized clinically as the percent of teeth with ≥1 site with periodontal probing depth (PPD) ≥5.0mm, recession (REC) >0mm, clinical attachment level (CAL) ≥4.0mm, and bleeding on probing (BOP) at ≥4 sites/tooth and microbiologically as specific periodontopathogen concentration. Linear mixed-effects models were used to assess the associations between immune function and PD.

Results

Forty (40) subjects with median 2.7 months on HAART and median nadir CD4+ T-cell count of 212 cells/μl completed a median 3 visits. Over 24 months, CD4+ T-cell count increased by a mean 173 cells/µl (p<0.001) and HIV RNA decreased by 0.5 log₁₀ copies/ml (p<0.001); concurrently, PPD, CAL and BOP decreased by a mean 11.7%, 12.1%, and 14.7% respectively (all p<0.001). Lower nadir CD4+ T-cell count was associated with worse baseline REC (-6.72%; p=0.04) and CAL (9.06%; p<0.001). Further, lower nadir CD4+ T-cell count was associated with a greater relative longitudinal improvement in PPD in subjects with higher baseline levels of Porphyromonas gingivalis (p=0.027), and BOP in subjects with higher baseline levels of Porphyromonas gingivalis or Treponema denticola (p=0.001 and p=0.006 respectively). Longitudinal changes from baseline in CD4+ T-cell count and level of HIV RNA were not independently associated with longitudinal changes in any clinical markers of PD.

Conclusion

Degree of immunosuppression was associated with baseline gingival recession. After HAART initiation, measures of active PD improved most in those with lower nadir CD4+ T-cell counts and higher baseline levels of specific periodontopathogens. Nadir CD4+ T-cell count differentially influences periodontal disease both before and after HAART in HIV-infected adults.