Identification of novel candidate biomarkers related to immune cell infiltration in peri-implantitis.

OBJECTIVE: The present study was performed to identify key biomarkers associated with immune cell infiltration in peri-implantitis through bioinformatic analyses. METHODS: Six peri-implantitis soft tissue samples and six healthy gingiva samples were obtained from GSE106090, and were used to identify immune-associated differentially expressed genes (DEGs) in peri-implantitis. The candidate biomarkers associated with immune cell infiltration were examined by immunohistochemical staining. RESULTS: We identified 2089 upregulated and 2173 downregulated genes. Upregulated DEGs were significantly associated with immune response. Ten key candidate biomarkers were identified in the PPI network, including IL1B, TLR2, TLR4, CCL4, CXCL8, IL10, IL6, CD4, CCL3, and PTPRC. The expression level of the 10 genes increased in peri-implantitis soft tissue samples compared with healthy gingiva samples. The proportion of CD4+ T cells, iTreg, and Tfh in infiltration immune cells increased in peri-implantitis soft tissue samples and were positively correlated with the expression level of candidate biomarkers TLR4, CCL3, CXCL8, and IL1B. Immunohistochemistry showed that there were more lymphocytes in peri-implantitis soft tissue samples, with an increased expression level of TLR4, CCL3, CXCL8, and IL1B. CONCLUSION: Identification of four novel diagnostic biomarkers was helpful for revealing the molecular mechanisms and could serve as a risk predictor for the immune microenvironment in peri-implantitis.

Proteomics and Organoid Culture Reveal the Underlying Pathogenesis of Hashimoto's Thyroiditis.

Hashimoto's thyroiditis (HT) is an autoimmune disease, and its incidence continues to rise. Although scientists have studied this disease for many years and discovered the potential effects of various proteins in it, the specific pathogenesis is still not fully comprehended. To understand HT and translate this knowledge to clinical applications, we took the mass spectrometric analysis on thyroid tissue fine-needle puncture from HT patients and healthy people in an attempt to make a further understanding of the pathogenesis of HT. A total of 44 proteins with differential expression were identified in HT patients, and these proteins play vital roles in cell adhesion, cell metabolism, and thyroxine synthesis. Combining patient clinical trial sample information, we further compared the transient changes of gene expression regulation in HT and papillary thyroid carcinoma (PTC) samples. More importantly, we developed patient-derived HT and PTC organoids as a promising new preclinical model to verify these potential markers. Our data revealed a marked characteristic of HT organoid in upregulating chemokines that include C-C motif chemokine ligand (CCL) 2 and CCL3, which play a key role in the pathogenesis of HT. Overall, our research has enriched everyone's understanding of the pathogenesis of HT and provides a certain reference for the treatment of the disease.

Zoledronic acid impairs oral cancer stem cells by reducing CCL3.

Cancer stem­like cells (CSCs; also referred to as tumor­initiating cells) play crucial roles in tumor progression and aggressiveness. Recent studies have demonstrated the antitumor activity of zoledronic acid (ZA), a third­generation bisphosphonate, in various types of human cancer. However, its effect on oral CSCs and the underlying mechanism remain obscure. The present study demonstrated that ZA suppresses the growth and stemness properties of oral/oropharyngeal squamous cell carcinoma (OSCC) cells. ZA inhibited the malignant characteristics of OSCC cells, such as anchorage­independent growth and epithelial thickening in organotypic raft cultures. Moreover, ZA treatment resulted in suppression of self­renewal capacity, a key feature of CSCs. ZA also inhibited important CSC properties, such as migration and chemo­radioresistance. Mechanistically, ZA exposure significantly decreased chemokine (C­C motif) ligand 3 (CCL3) expression in OSCC cells. It was further demonstrated that CCL3 signaling via its receptor is crucial for supporting the CSC phenotype in OSCC cells. Moreover, an antagonist of the CCL3 receptor reversed the effect of CCL3 on CSC properties, and exogenous CCL3 rescued the suppressaed CSC phenotype in ZA­treated OSCC cells. These results demonstrated that ZA suppresses the CSC phenotype in OSCC cells by reducing CCL3 expression, suggesting that ZA may be an effective therapeutic agent for oral cancer by targeting CSCs.

Elevated CSF concentration of CCL3 and CCL4 in relapsing remitting multiple sclerosis patients.

CCL3 and CCL4 are considered as inflammatory cytokines and involved in progression of various neurologic disorders as multiple sclerosis (MS). The aim of this study was to evaluate the association between cerebrospinal fluid (CSF) levels of above mentioned inflammatory cytokines and relapsing remitting multiple sclerosis (RRMS. In this case-control study, 40 unrelated patients (without family relationship) with RRMS and 40 age and sex matched subjects as control group were enrolled. CSF samples obtained from all patients and control group and levels of CCL3 and CCL4 were determined in CSF. The mean CSF level of CCL3 was significantly higher in RRMS patients than healthy controls (29.71±18.56 vs. 10.62±6.85, p<0.01). The CSF levels of CCL4 was also higher in RRMS patients compared with healthy controls (33.62±21.50 vs. 13.74±4.90, p<0.01). We found a positive correlation between CSF levels of CCL3 and disease duration (r=+0.32 and p=0.04) and expanded disability status scale (EDSS) (r=+45, p=0.03). We also found a positive correlation between CSF level of CCL4 and disease duration (r=+0.76 and p<0.01) and EDSS (r=+0.73, p<0.01). Present study showed contribution of CCL3 and CCL4 in MS pathogenesis and suggested them as markers of severity of disease. Investigation  of chemokines responsible for attract  of pathogenic T lymphocyte and macrophage in to the central nerves system(CNS) is crucial for therapeutic targets in MS.

Distinct roles for MDA5 and TLR3 in the acute response to inhaled double-stranded RNA.

The airway epithelial barrier is critical for preventing pathogen invasion and translocation of inhaled particles into the lung. Epithelial cells also serve an important sentinel role after infection and release various pro-inflammatory mediators that recruit and activate immune cells. Airway epithelial barrier disruption has been implicated in a growing number of respiratory diseases including viral infections. It is thought that when a pathogen breaks the barrier and gains access to the host tissue, pro-inflammatory mediators increase, which further disrupts the barrier and initiates a vicious cycle of leak. However, it is difficult to study airway barrier integrity in vivo, and little is known about relationship between epithelial barrier function and airway inflammation. Current assays of pulmonary barrier integrity quantify the leak of macromolecules from the vasculature into the airspaces (or "inside/out" leak). However, it is also important to measure the ease with which inhaled particles, allergens, or pathogens can enter the subepithelial tissues (or "outside/in" leak). We challenged mice with inhaled double stranded RNA (dsRNA) and explored the relationship between inside/out and outside/in barrier function and airway inflammation. Using wild-type and gene-targeted mice, we studied the roles of the dsRNA sensors Toll Like Receptor 3 (TLR3) and Melanoma Differentiation-Associated protein 5 (MDA5). Here we report that after acute challenge with inhaled dsRNA, airway barrier dysfunction occurs in a TLR3-dependent manner, whereas leukocyte accumulation is largely MDA5-dependent. We conclude that airway barrier dysfunction and inflammation are regulated by different mechanisms at early time points after exposure to inhaled dsRNA.

Analysis of Inflammatory Cytokines in Postoperative Fontan Pleural Drainage.

Prolonged pleural drainage is a common complication in patients after Fontan palliation and is associated with short- and long- term morbidities. Among many potential etiologies, prolonged drainage has an inflammatory component, but there are no descriptions of cytokines in Fontan pleural drainage to date. This study aimed to examine the inflammatory make-up of Fontan pleural drainage. This prospective age-range-matched cohort study recruited 25 patients undergoing Fontan procedure and 15 bi-ventricular patients undergoing cardiopulmonary bypass (CPB). Chest tube samples were taken on postoperative day (POD) 1-4, 7, and 10. Cytokines were measured using Bio-Plex Assays. Univariate comparisons were made in patient characteristics and cytokine levels. Median age was 3.7 y (IQR 2.8-3.9) for controls and 2.5 y (IQR 2.1-2.9) in Fontan patients (p = 0.02). Median drainage duration and daily volume was higher in Fontan patients (both p < 0.001). Inflammatory cytokines (IL-17A, IFN-y, MIP-1ß, and TNF-α) were higher in Fontan patients than controls (all p < 0.02). There was an increase in pro-inflammatory cytokines (IL-8, MIP-1ß, and TNF-α) from POD1 to the last chest tube day (LCD) in Fontan patients (all p < 0.0001) and a decrease in the anti-inflammatory cytokine IL-10 (p = 0.001). There was no difference in cytokine concentration from POD1 to LCD among controls. There was a significant association with the cytokine concentration of TNF-α on POD1 and duration of chest tube drainage (p < 0.05). Inflammatory cytokine levels in the pleural fluid of Fontan patients are higher compared to bi-ventricular controls and rise over time where controls do not. This suggests ongoing localized inflammation that is not a result of CPB alone and may be an important contributor to pleural drainage in patients after the Fontan procedure.

Inflammatory response under zinc deficiency is exacerbated by dysfunction of the T helper type 2 lymphocyte-M2 macrophage pathway.

Nutritional zinc deficiency leads to immune dysfunction and aggravates inflammation. However, the underlying mechanism remains unknown. In this study, the relationship between macrophage subtypes (M1 and M2) and helper T lymphocytes (Th1 and Th2) was investigated using the spleen from rats fed zinc-deficient or standard diet. In experiment I, 5-week-old male Sprague-Dawley rats were fed a zinc-deficient diet (without zinc additives) or a standard diet (containing 0·01% zinc) for 6 weeks. In experiment II, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; two groups were fed zinc-deficient diets and were injected three times a week with either saline or interleukin-4 (IL-4) (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed a zinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I; GATA-binding protein 3 (GATA-3) protein level, M2 macrophage, CD3+  CD8+ cells, and IL-4/IL-13-positive cells significantly decreased in the spleens of the zinc-deficient group. Additionally, IL-1ß and macrophage inflammatory protein-1α (MIP-1α) mRNA levels significantly increased in the splenic macrophages of the zinc-deficient group. In experiment II; M2 macrophages, CD3+  CD8+ cells, IL-4/IL-13-positive cells, and GATA-3 protein levels significantly increased in the spleens of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Furthermore, IL-1ß and MIP-1α mRNA levels decreased in the splenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by the association with loss of GATA-3, IL-4 and anti-inflammatory M2 macrophages. Importantly, IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune function.

Delay in antibiotic therapy results in fatal disease outcome in murine pneumococcal pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) remains a major cause of death worldwide. Mechanisms underlying the detrimental outcome despite adequate antibiotic therapy and comorbidity management are still not fully understood. METHODS: To model timely versus delayed antibiotic therapy in patients, mice with pneumococcal pneumonia received ampicillin twice a day starting early (24 h) or late (48 h) after infection. Clinical readouts and local and systemic inflammatory mediators after early and late antibiotic intervention were examined. RESULTS: Early antibiotic intervention rescued mice, limited clinical symptoms and restored fitness, whereas delayed therapy resulted in high mortality rates. Recruitment of innate immune cells remained unaffected by antibiotic therapy. However, both early and late antibiotic intervention dampened local levels of inflammatory mediators in the alveolar spaces. Early treatment protected from barrier breakdown, and reduced levels of vascular endothelial growth factor (VEGF) and perivascular and alveolar edema formation. In contrast, at 48 h post infection, increased pulmonary leakage was apparent and not reversed by late antibiotic treatment. Concurrently, levels of VEGF remained high and no beneficial effect on edema formation was evident despite therapy. Moreover, early but not late treatment protected mice from a vast systemic inflammatory response. CONCLUSIONS: Our data show that only early antibiotic therapy, administered prior to breakdown of the alveolar-capillary barrier and systemic inflammation, led to restored fitness and rescued mice from fatal streptococcal pneumonia. The findings highlight the importance of identifying CAP patients prior to lung barrier failure and systemic inflammation and of handling CAP as a medical emergency.

[Effects of recombinant fusion protein interleukin-18 on expression of immune-inflammatory factors in mice infected with Staphylococcus aureus].

OBJECTIVE: To observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo. METHODS: A total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-γ, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2ß mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR. RESULTS: Compared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2ß mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-γ and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-γ in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-γ, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2ß mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-γ. CONCLUSIONS: In the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2ß mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Ellagic acid ameliorates cuprizone-induced acute CNS inflammation via restriction of microgliosis and down-regulation of CCL2 and CCL3 pro-inflammatory chemokines.

Ellagic acid (EA) is a natural phenol antioxidant with various therapeutic activities. However, the efficacy of EA has not been examined in neuro-inflammatory conditions. Microglia making the innate immune system of the central nervous system (CNS) and are imperative cellular mediators of neuro-inflammatory processes. In this study, neuro-protective effects of EA on cuprizone (Cup)-induced acute CNS inflammation evaluated. C57BL/6J mice were fed with chow containing 0.2 % Cup for 3 weeks to induce acute neuro-inflammation predominantly in the corpus callosum (CC). EA was administered at different doses (40 or 80 mg/kg body weight/day/i.p) from the first day of the Cup diet. Microglia activation (microgliosis) and expression of microglia related chemokines during Cup challenge were examined. Results shows that EA significantly decreased the number of activated microglia cells (Iba-1+ cells) and also restricted proliferation of these cell population (Iba-1+/Ki67+ cells) in dose dependent manner. Consequently, concentration of microglial pro-inflammatory chemokines including monocyte chemoattractant protein-1/Chemokine (C-C motif) ligand 2 (MCP-1/CCL2), and macrophage inflammatory protein 1-alpha/Chemokine (C-C motif) ligand 3 (MIP-1-α/CCL3) dramatically reduced in CC after EA treatment. According to this results, we conclude that EA is a suitable therapeutic agent for moderation brain damages in neuro-inflammatory diseases.

Evaluation of chemokines in gingival crevicular fluid in children with dental caries and stainless steel crowns: A clinico-biochemical study.

AIMS AND OBJECTIVES: The study was conducted to detect the presence of macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß and estimate their levels in gingival crevicular fluid (GCF) in children with dental caries and stainless steel crowns. MATERIALS AND METHODS: A total of 80 children with primary dentition were selected and categorized into four groups with twenty in each group; Group 1 - healthy subjects, Group 2 - dental caries, Group 3 - dental caries involving the pulp, and Group 4 - stainless steel crowns. GCF samples were collected by an extra-crevicular method with microcapillary pipettes. The GCF samples were quantified by ELISA and the levels of MIP-1α and MIP-1ß were determined. RESULTS: MIP-1α and MIP-1ß were detected in all the samples. Highest mean concentration in GCF was obtained for Group 3 followed by Groups 2 and 4 while the lowest concentration was seen in Group 1. This suggests that MIP-1α and MIP-1ß levels in GCF increased proportionately with the inflammation. CONCLUSIONS: GCF serves as a noninvasive diagnostic fluid to measure biomarkers released during dental caries initiation and progression. MIP-1α and MIP-1ß chemokines can be considered as novel biomarkers, in biological mechanism underlying the pathogenesis and inflammation in children with dental caries and stainless steel crowns.

Osteolytic lesions, cytogenetic features and bone marrow levels of cytokines and chemokines in multiple myeloma patients: Role of chemokine (C-C motif) ligand 20.

The relationship between bone marrow (BM) cytokine and chemokine levels, cytogenetic profiles and skeletal involvement in multiple myeloma (MM) patients is not yet defined. This study investigated a cohort of 455 patients including monoclonal gammopathy of uncertain significance (MGUS), smoldering MM and symptomatic MM patients. Skeletal surveys, positron emission tomography (PET)/computerized tomography (CT) and magnetic resonance imaging (MRI) were used to identify myeloma bone disease. Significantly higher median BM levels of both C-C motif Ligand (CCL)3 and CCL20 were found in MM patients with radiographic evidence of osteolytic lesions as compared with those without, and in all MM patients with positive PET/CT scans. BM levels of CCL3, CCL20, Activin-A and Dickkopf-1 (DKK-1) were significantly higher in patients with high bone disease as compared with patients with low bone disease. Moreover, CCL20 BM levels were significant predictors of osteolysis on X-rays by multivariate logistic analysis. On the other hand, DKK-1 levels were related to the presence of MRI lesions independently of the osteolysis at the X-rays. Our data define the relationship between bone disease and the BM cytokine and chemokine patterns highlighting the tight relationship between CCL20 BM levels and osteolysis in MM.

Análise clínica e de marcadores biológicos no fluido do sulco peri-implantar correlacionando-os à mucosa ceratinizada ao redor de implantes dentários / Clinical and biological markers analysis in the peri-implant groove fluid correlating them to the keratinized mucosa around dental implants

Uma faixa adequada de mucosa ceratinizada (MC) é importante para garantir condições mínimas necessárias para o estabelecimento da homeostasia do periodonto de proteção. Frente à infecção bacteriana, os tecidos periodontais e peri-implantares desenvolvem uma resposta imune inflamatória local, resultando na produção e liberação de diversos mediadores inflamatórios que podem ser encontrados no fluido do sulco gengival e peri-implantar. Entretanto, é escassa a literatura acerca dos níveis desses mediadores em sítios peri-implantares considerando a faixa de MC. Assim, o objetivo deste trabalho foi avaliar a associação entre a quantidade e qualidade da MC peri-implantar e parâmetros clínicos e a qualidade da resposta imune através da análise da concentração de mediadores inflamatórios (IL-1, IL-4, IL-6, IL-8, MIP-1, TNF- e VEGF) presentes no fluido peri-implantar humano antes (T1) e depois (T2) da raspagem subgengival, através de imunoensaio. Parâmetros clínicos avaliaram índice de placa (IP), supuração a sondagem (S), profundidade de sondagem (mesial-PSM, centro-PSC e distal-PSD), índice de sangramento (mesial-ISM, centro-ISC e distal-ISD), nível de inserção relativo (NIR), largura (LMC) e espessura (EMC) da MC na face vestibular. Amostras de fluido sulcular foram coletadas e analisadas. Os implantes foram divididos em grupos de acordo com a faixa de MC (G12mm e G2>2mm) e espessura de MC (GA11mm e GB1>1mm; GA21,5mm e GB2>1,5mm). Foram avaliados 20 pacientes (11 homens e 9 mulheres) com idade entre 40 e 80 anos (53,45±10,32), que apresentaram 42 implantes (G1=25 e G2=17). Os resultados clínicos demonstraram diferença estatística significativa apenas entre T1 e T2 dentro do G1 para IP (T1=56% e T2=16%) e ISM (T1=68% e T2=40%). Foi observada diferença estatística entre G1 e G2 apenas para IL-1 em T2 (G1=9,77pg/ml±12,44 e G2=30,13pg/ml±32,29). Intra-grupos, todas as citocinas aumentaram significativamente, mas apenas no G2, demonstrando diferença de reatividade entre grupos. Quanto à espessura da MC (GA1=6 e GB1=36), resultados clínicos revelaram diferença inter-grupos para ISC em T2 (GA1=16,67% e GB1=61,11%) e intra-grupos para IP no GB1 (T1=52,78% e T2=27,78%). Houve aumento significativo no GB1 para todas as citocinas, exceto VEGF, assim como para IL-1 no GA1. Quando a amostra foi redistribuída em GA2=24 e GB2=18, os resultados clínicos indicaram diferença estatística inter-grupos para PSC em T2 (GA2=2,58mm±1,06 e GB2=3,11mm±1,02) e intra-grupos para IP (T1=62,5% e T2=20,83%) e PSC (T1=2,92mm±1,18 e T2=2,58mm±1,06) no GA2 e para ISM (T1=55,56% e T2=27,78%) no GB2. Intra-grupos observou-se aumento significativo para todas as citocinas no GA2 exceto VEGF, assim como IL-8 no GB2. Conclui-se que as diferenças clínicas apresentadas tenderam a evidenciar a importância da MC principalmente após o preparo inicial e, além disso, uma faixa de MC maior que 2mm influenciou os níveis dos mediadores inflamatórios avaliados após a raspagem subgengival. Adicionalmente, a falta de diferença estatística significativa na comparação entre grupos com diferentes espessuras de MC, bem como tal diferença ora no grupo espesso ora no grupo fino quando se adotam diferentes valores de corte (1mm ou 1,5mm respectivamente), demonstra resultados inconclusivos, ressaltando a importância de novas pesquisas para responder esta questão.(AU)

An adequate keratinized mucosa (KM) width is important to ensure minimal conditions necessary to establish protect periodontium homeostasis. When a bacterial infection occurs, periodontal and peri-implant tissues develop a local inflammatory immune response that results in production and release of several inflammatory mediators that may be found in gingival crevicular and in peri-implant fluids. However, there is a lack of literature concerning about the levels of these mediators in peri-implant sites considering KM width. The aim of this study was to evaluate the association between KM peri-implant quantity and quality and clinical parameters and immune response quality by analyzing the inflammatory mediators concentration (IL-1, IL-4, IL-6, IL-8, MIP-1, TNF- and VEGF) present in human peri-implant fluid before (T1) and after (T2) subgingival scaling, by immunoassay. Clinical parameters evaluated plate index (PI), probing suppuration (S), probing depth (mesial-PDM, center-PDC and distal-PDD), bleeding index (mesial-BIM, center-BIC and distal-BID), relative attachment level (RAL), keratinized mucosa width (KMW) and thickness (KMT) on the buccal face. Sulcular fluid samples were collected and analyzed. The implants were divided in groups according KM width (G12mm and G2>2mm) and KM thickness (GA11mm and GB1>1mm, GA21,5mm and GB2>1,5mm). Twenty patients (11 men and 9 women) aged 40 to 80 years (53,45±10,32) were evaluated, with 42 implants (G1=25 and G2=17). Clinical results showed a significant statistical difference only between T1 and T2 within G1 for PI (T1=56% and T2=16%) and BIM (T1=68% and T2=40%). Statistical difference was observed between G1 and G2 only for IL-1 in T2 (G1=9,77pg/ml±12,44 and G2=30,13pg/ml±32,29). Intra-groups, all cytokines increased significantly, but only in G2, showing reactivity difference between groups. As to KM thickness (GA1=6 and GB1=36), clinical results revealed intergroup differences for BIC in T2 (GA1=16,67% and GB1=61,11%) and intra-groups for PI in GB1 (T1=52,78% and T2=27,78%). There was a significant increase in GB1 for all cytokines except VEGF, as well as for IL-1 in GA1. When the sample was redistributed in GA2=24 and GB2=18, clinical results indicated statistical inter-group differences for PDC in T2 (GA2=2,58mm±1,06 and GB2=3,11mm±1,02) and intra-groups for PI (T1=62,5% and T2=20,83%) and PDC (T1=2,92mm±1,18 and T2=2,58mm±1,06) in GA2 and for BIM (T1=55,56% and T2=27,78%) in GB2. Intra-groups were observed significantly increase for all cytokines in GA2 except VEGF, as well as IL-8 in GB2. Concluded that clinical differences presented tended to show the KM importance principally after the initial preparation and, in addition, KM width greater than 2mm influenced the inflammatory mediators levels evaluated after subgingival scaling. Additionally, the absence of significant statistic difference between groups when comparing the keratinized mucosa thickness, as well as this difference sometimes in the thick group or in the thin group when different court values was adopted (1mm or 1,5mm respectively), show inconclusive results, emphasizing the importance of new research that may answer this question.(AU)

Local levels of biomarkers after surgical and nonsurgical debridement of residual pockets and nonresidual sites in diabetic patients: a 12-month follow-up.

There is scarce evidence on suitable approaches for the treatment of unresponsive or residual periodontal sites in diabetic patients. This study assessed the effects of surgical debridement (SD) and nonsurgical debridement (NSD), associated with amoxicillin and metronidazole, on clinical and immunological outcomes of residual pockets and adjacent healthy sites in patients with type 2 diabetes. A split-mouth, randomized controlled trial was conducted in 21 patients presenting at least 2 residual pockets in contralateral quadrants 12 months after basic nonsurgical periodontal therapy. Patients received systemic antibiotics, and contralateral quadrants were assigned to receive SD or NSD. The changes in clinical parameters were evaluated from baseline to 12 months. Local levels of 14 cytokines and chemokines were measured with multiplex bead immunoassays at baseline and 3 and 12 months after therapy. There were no statistically significant differences between SD and NSD for changes in clinical parameters from baseline to 12 months (P > 0.05). There was a significantly greater increase in the levels of granulocyte-macrophage colony-stimulating factor and interleukin 6 from baseline to 3 months in the healthy sites adjacent to residual pockets receiving SD (P < 0.05). A significant decrease in the levels of monocyte chemoattractant protein-1 and macrophage inflammatory protein 1α occurred from baseline to 12 months in the residual pockets treated by SD (P < 0.05). In conclusion, SD and NSD resulted in similar clinical benefits at 12 months. The short-term increase in the levels of proinflammatory biomarkers in SD sites probably can be attributed to tissue trauma and healing, and the long-term decrease in the levels of chemotactic factors in residual pockets treated by surgery may reflect remission of infection and stable wound healing in these sites at 12 months.

Influence of type 2 diabetes on local production of inflammatory molecules in adults with and without chronic periodontitis: a cross-sectional study.

BACKGROUND: Pathological changes in periodontal tissues are mediated by the interaction between microorganisms and the host immune-inflammatory response. Hyperglycemia may interfere with this process. The aim of this study was to compare the levels of 27 inflammatory molecules in the gingival crevicular fluid (GCF) of patients with type 2 diabetes, with and without chronic periodontitis, and of chronic periodontitis subjects without diabetes. A putative correlation between glycated haemoglobin (HbA1c) and levels of the inflammatory molecules was also investigated. METHODS: The study population comprised a total of 108 individuals, stratified into: 54 with type 2 diabetes and chronic periodontitis (DM + CP), 30 with chronic periodontitis (CP) and 24 with type 2 diabetes (DM). Participants were interviewed with the aid of structured questionnaire. Periodontal parameters (dental plaque, bleeding on probing and periodontal pocket depth) were recorded. The GCF levels of the 27 inflammatory molecules were measured using multiplex micro-bead immunoassay. A glycated haemoglobin (HbA1c) test was performed for patients with diabetes by boronate affinity chromatography. RESULTS: After adjustment for potential confounders, the DM + CP group had higher levels of IL-8 and MIP-1ß, and lower levels of TNF-α, IL-4, INF-γ, RANTES and IL-7 compared to the CP group. Moreover, the DM + CP group had lower levels of IL-6, IL-7 and G-CSF compared to the DM group. The DM group had higher levels of IL-10, VEGF, and G-CSF compared to the CP group. The levels of MIP-1α and FGF were lower in diabetes patients (regardless of their periodontal status) than in chronic periodontitis subjects without diabetes. Diabetes patients (DM + CP and DM) had higher Th-2/Th-1 ratio compared to the CP group. HbA1c correlated positively with the pro-inflammatory cytokines (Pearson correlation coefficient = 0.27, P value: 0.02). CONCLUSION: Type 2 diabetes and chronic periodontitis may influence the GCF levels of inflammatory molecules synergistically as well as independently. Type 2 diabetes was associated with high Th-2/Th-1 ratio, and modulated the local expression of molecules involved in the anti-inflammatory and healing processes.

High-dose cyclophosphamide induces specific tumor immunity with concomitant recruitment of LAMP1/CD107a-expressing CD4-positive T cells into tumor sites.

Cancer chemotherapy regimens, particularly those employing high-dose cytotoxic drugs such as cyclophosphamide (CTX), have been considered to be immune suppressive. However, we observed that a single administration of high-dose CTX abolished tumors arising from subcutaneous injection of a mouse hepatoma cell line and subsequently induced specific tumor immunity. Depletion of T cells, specifically CD4(+) T cells, abrogated the CTX-mediated tumor regression. CTX treatment induced the rapid recruitment of CD4(+) T cells into the tumors, and these recruited cells initiated expression of LAMP1/CD107a, a cytotoxic granule molecule, and granzyme B in the absence of antigen presentation at draining lymph nodes and proliferation in the tumor tissues. Moreover, CTX enhanced the expression of a CC chemokine, CCL3, in tumor tissues, and CTX-mediated tumor regression was attenuated in mice deficient in CCR5, the receptor for this chemokine. Consistently, less CTX-induced accumulation of intratumoral LAMP1/CD107a-expressing CD4(+) T cells was observed in mice receiving splenocytes derived from CCR5-deficient mice than in those receiving splenocytes derived from WT mice. Thus, CTX induces the expression of CCL3, which induces the intratumoral migration of CD4(+) T cells expressing cytotoxic molecules, leading to tumor eradication and subsequent specific tumor immunity.

The alteration of inflammatory markers and apoptosis on chronic prostatitis induced by estrogen and androgen.

PURPOSE: The age-related decline of the testosterone-to-estrogen (T-to-E2) ratio in serum is associated with the increased prevalence of prostatic inflammation. The goal of the study was to induce prostatic inflammation with E2 and androgen treatment and to explore the inflammatory markers and apoptosis on prostatitis. METHODS: Castrated SD rats were treated with E2 and different doses of androgens to achieve an elevated concentration of E2 and a wide range of the androgen-to-E2 ratio in serum. Inflammatory markers TNF-α, COX-2 and MIP-1α were immunohistochemically stained. Apoptosis detection was evaluated by TUNEL staining. E2, T and DHT concentrations in serum were measured, and the relative weight of the prostate and seminal vesicles were determined. RESULTS: T was anti-inflammatory at the doses which normalized or over stimulated the growth of the prostate and seminal vesicles. Experimentally, prostatitis induced by E2 alone increased the prostatic levels of the inflammatory markers TNF-a, COX-2 and MIP-1a. As signs of anti-estrogenic actions, androgens dose-dependently decreased the expression of TNF-α, COX-2 and MIP-1α. Prostatitis induced by E2 alone caused extensive apoptosis in the castrate-resistant cells and E2-induced apoptosis occurred dependently of T manipulation. CONCLUSIONS: Estrogen-alone-induced inflammatory response could promote the expression of inflammatory markers; however, T supplementation reduces the expression of inflammatory markers and E2-induced apoptosis occurs dependently on T manipulation in prostatitis.

Increased expression of CCL4/MIP-1ß in CD8+ cells and CD4+ cells in sarcoidosis.

Sarcoidosis is a granulomatous disease with an increased accumulation of T cells in lungs as a result of on-site proliferation and chemotaxis induced by chemokines. It has already been demonstrated that CCL3-5 levels were increased in BAL fluid of sarcoidosis patients. To analyze the expression of CCL3-5 chemokines by T-cell subtypes (CD4+, CD8+, Th1, Th2, Tc1 or Tc2) in the lungs of sarcoidosis patients, fifteen untreated sarcoidosis patients and eighteen control subjects were enrolled in this study. CD4+ and CD8+ cells were isolated from BAL fluid by positive magnetic selection. The expression of CCL3-5 and other cytokines in CD4+ and CD8+ cells were measured by flow cytometry. The percentage of CD4+ or CD8+ cells expressing CCL4 were significantly higher in sarcoidosis patients (22.3% and 58.1%) compared to those seen in healthy subjects (11.1% and 16.5%, P = 0.04 and P = 0.02, respectively). In addition, the expression of CCL3, CCL4 and CCL5 was significantly elevated in CD8+ cells (8.9%, 58.1% and 2.1%) compared to CD4+ cells (2.1%, 22.3% and 0.7%; P = 0.04, P = 0.009 and P = 0.04, respectively), whereas CCL4 was expressed by significantly more Tc1 than Th1 cells in sarcoidosis patients (P = 0.006). Our study shows the possible role of CD8+ cells and CD4+ cells in recruiting T cells to the site of inflammation in sarcoidosis through the release of CCL4, either alone or together with Th1/Tc1-associated cytokines.

Bone marrow plasma macrophage inflammatory protein protein-1 alpha(MIP-1 alpha) and sclerostin in multiple myeloma: relationship with bone disease and clinical characteristics.

The aim of the study was to investigate the expression of MIP-1 alpha and sclerostin in bone marrow of patients with multiple myeloma (MM), the possible association of the sclerostin and MIP-1 alpha with MBD and the clinical characteristics. 53 patients (29 M, 24 F), median age 64 years was studied. MIP-1 alpha, sclerostin and bone-specific alkaline phosphatase (bALP) levels were quantified using an enzyme-linked immunosorbent assay (ELISA). Sclerostin and MIP-1 alpha mRNA expression was determined by RT-PCR. PTH and 1,25(OH) 2D3 levels were measured with an electrochemiluminescence immunoassay. The sclerostin and MIP-1 alpha concentrations in patients with MM were higher than those in the controls. RT-PCR analysis verified that the bone marrow mononuclear cells (BMMNCs) of most patients showed sclerostin and MIP-1 alpha mRNA expression. The sclerostin and MIP-1 alpha levels in patients with ISS stage III disease were significantly higher than those in patients with ISS stage II disease (p=0.01 and 0.06). The sclerostin and MIP-1 alpha levels in patients with BMD in group C were significantly higher than those in group A+B. There was positive correlation between sclerostin levels and MIP-1 alpha, beta2-microglobulin and aCa levels. A negative association was seen between sclerostin levels and bALP, HB and ALB levels. The MM patients with high sclerostin levels (>0.72 ng/ml) had significantly shorter median survival than those with low sclerostin levels (≤0.72 ng/ml) (χ(2)=7.574, p=0.006). Our findings support the positive relationship between sclerostin levels and MIP-1alpha levels deserve further detailed research.

Salivary biomarkers associated with gingivitis and response to therapy.

BACKGROUND: Salivary biomarkers are potentially important for determining the presence, risk, and progression of periodontal disease. However, clinical translation of biomarker technology from lab to chairside requires studies that identify biomarkers associated with the transitional phase between health and periodontal disease (i.e., gingivitis). METHODS: Eighty participants (40 with gingivitis, 40 healthy) provided saliva at baseline and 7 to 30 days later. An additional sample was collected from gingivitis participants 10 to 30 days after dental prophylaxis. Clinical parameters of gingival disease were recorded at baseline and the final visit. Salivary concentrations of interleukin (IL)-1ß, IL-6, matrix metalloproteinase (MMP)-8, macrophage inflammatory protein (MIP)-1α, and prostaglandin E2 (PGE2) were measured. RESULTS: Clinical features of health and gingivitis were stable at both baseline visits. Participants with gingivitis demonstrated significantly higher bleeding on probing (BOP), plaque index (PI), and gingival index (GI) (P ≤0.002) and a significant drop in BOP, PI, and GI post-treatment (P ≤0.001). Concentrations of MIP-1α and PGE2 were significantly higher (2.8 times) in the gingivitis group than the healthy group (P ≤0.02). After dental prophylaxis, mean biomarker concentrations did not decrease significantly from baseline in the gingivitis group, although concentrations of IL-1ß, IL-6, and MMP-8 approached healthy levels, whereas MIP-1α and PGE2 concentrations remained significantly higher than in the healthy group (P ≤0.04). Odds ratio analyses showed that PGE2 concentrations, alone and in combination with MIP-1α, readily discriminated gingivitis from health. CONCLUSIONS: Salivary PGE2 and MIP-1α discriminate gingivitis from health, and patients with gingivitis who return to clinical health continue to produce inflammatory mediators for weeks after dental prophylaxis.