Juliete A.F. Silva

Signaling pathways associated with the expression of inflammatory mediators activated during the course of two models of experimental periodontitis

AIMS Evaluate the signaling pathways associated with inflammatory mediators activated in two models of experimental periodontitis. MAIN METHODS Two models were used: lipopolysaccharide (LPS) injections and ligature placement. Wistar rats were used and 30 microg LPS from Escherichia coli was injected twice a week into the palatal aspect of the upper molars. Ligatures were placed around lower first molars. A control group received injections of PBS on the palatal gingivae whereas no ligatures were placed on the lower molars. Samples were collected 5, 15 and 30 days and processed for analysis by Western blotting and stereometry. KEY FINDINGS The ligature model was associated with rapid and transient activation of extracellular-regulated kinases (ERK) and p38 mitogen-activated protein kinase (MAPK) as well as of nuclear factor kappa B (NF-kappaB). Activation of these signaling pathways on the LPS model was delayed but sustained throughout the 30-day experimental period. Inflammatory changes induced by both models were similar; however there was a significant reduction on inflammation degree on the ligature model, which paralleled the decrease observed on the activation of the signaling pathways. Activation of signal transducer and activator of transcription (STAT)-3 by phosphorylation of Tyrosine residues and of STAT-5 was observed only on the ligature model. SIGNIFICANCE Regulation of gene expression results from the activation of signaling pathways initiated by receptor-ligand binding of external antigens and also of cytokines produced by the host immune system. Understanding the signaling pathways relevant for a given condition may provide information useful for novel therapeutic approaches.

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes

Molecular mechanisms responsible for periodontal disease (PD) and its worsening in type 1 Diabetes Mellitus (DM1) remain unknown. Cytokine profile and expression levels of collagenases, Mmp14, and tissue inhibitors were determined, as were the numbers of neutrophils and macrophages in combined streptozotocin‐induced DM1 and ligature‐induced PD models. Increased IL‐23 (80‐fold) and Mmp8 expression (25‐fold) was found in DM1. Ligature resulted in an IL‐1β/IL‐6 profile, increased expression of Mmp8, Mmp13, and Mmp14 (but not Mmp1), and transient expression of Timp1 and Reck in non‐diabetics. PD in DM1 involved IL‐1β (but not IL‐6) and IL‐23/IL‐17, reduced IL‐6 and IL‐10, sustained Mmp8 and Mmp14, increased Mmp13 and reduced Reck expression in association with 20‐fold higher counts of neutrophils and macrophages. IL‐23 and Mmp8 expression are hallmarks of DM1. In association with the IL‐1/IL‐6 (Th1) response in PD, one found a secondary IL‐17 (Th17) pathway in non‐diabetic rats. Low IL‐6/TNF‐α suggest that the Th1 response was compromised in DM1, while IL‐17 indicates a prevalence of the Th17 pathway, resulting in high neutrophil recruitment. Mmp8, Mmp13, and Mmp14 expression seems important in the tissue destruction during PD in DM1. PD‐associated IL‐1/IL‐6 (Th1), IL‐10, and Reck expression are associated with the acute‐to‐chronic inflammation transition, which is lost in DM1. In conclusion, IL‐23/IL‐17 are associated with the PD progression in DM1. J. Cell. Physiol. 227: 2441–2450, 2012.

Differential regulation of MMP-13 expression in two models of experimentally induced periodontal disease in rats

OBJECTIVE Evaluate expression of MMP-13 during the course of two models experimentally induced periodontal disease in rats. DESIGN Expression of MMP-13 at mRNA and protein levels was studied, respectively, by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Two experimental models were used: LPS injections and ligature placement. 30mug of LPS from Eschericia coli was injected twice a week into the palatal aspect of upper molars. Ligatures were placed at the gingival margin around lower first molars. Controls received injections of PBS vehicle and no ligatures on lower molars. Samples were collected 5, 15 and 30 days after initiation of periodontal disease and processed for extraction of total RNA, total protein, and routinely processed for histology. RESULTS Both experimental models produced a significant increase on the inflammatory infiltrate that paralleled elevated levels of MMP-13 mRNA and protein at 5 and 15 days. The LPS model was associated with a sustained level of inflammation and increased MMP-13 mRNA throughout the 30 days, whereas the ligature model showed a decrease on the severity of inflammation and MMP-13 mRNA at the 30-day period. Interestingly, MMP-13 protein levels were diametrically contrary to the mRNA levels. CONCLUSION MMP-13 expression during LPS- and ligature-induced experimental periodontal disease follows the increase on severity of inflammation at the earliest periods. At 30 days, there is a decrease on the severity of inflammation on the ligature model associated with decreased MMP-13 mRNA. There is a lack of transcription-translation coupling of MMP-13 gene in both experimental models.

Periodontal Disease-Associated Compensatory Expression of Osteoprotegerin Is Lost in Type 1 Diabetes Mellitus and Correlates with Alveolar Bone Destruction by Regulating Osteoclastogenesis

Alveolar bone resorption results from the inflammatory response to periodontal pathogens. Systemic diseases that affect the host response, such as type 1 diabetes mellitus (DM1), can potentiate the severity of periodontal disease (PD) and accelerate bone resorption. However, the biological mechanisms by which DM1 modulates PD are not fully understood. The aim of this study was to determine the influence of DM1 on alveolar bone resorption and to evaluate the role of receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) in osteoclastogenesis in rats. PD was induced by means of ligature in nondiabetic and in streptozotocyn-induced DM1 rats. Morphological and morphometric analyses, stereology and osteoclast counting were performed. RANKL and OPG mRNA levels, protein content, and location were determined. PD caused alveolar bone resorption, increased the number of osteoclasts in the alveolar bone crest and also promoted changes in RANKL/OPG mRNA expression. DM1 alone showed alveolar bone destruction and an increased number of osteoclasts at the periapical and furcal regions. DM1 exacerbated these characteristics, with a greater impact on bone structure, resulting in a low OPG content and a higher RANKL/OPG ratio, which correlated with prominent osteoclastogenesis. This work demonstrates that the effects of PD and DM1 enhance bone destruction, confirms the importance of the RANKL signaling pathway in bone destruction in DM1 in animal models and suggests the existence of alternative mechanisms potentiating bone degradation in PD.

A new paradigm in the periodontal disease progression: gingival connective tissue remodeling with simultaneous collagen degradation and fibers thickening.

Bacterial dental plaque is considered to be the main cause of periodontal diseases, but progression of the disease is also related to the host inflammatory response. The earliest affected tissue is the gingiva, but the specific mechanisms involved in the onset of this condition remain unclear. Frequently, collagen degradation is pointed as the main marker of periodontal disease progression, but the organization of the fibers in the gingival tissue is still unknown. The aim of the present study was to investigate the gingival extracellular matrix in a model of ligature-induced periodontal disease. Analysis of the microbiota indicated a progressive increase in the ratio of Gram-negative/Gram-positive microorganisms. There was no difference in the organization of reticulin fibers next to the epithelial basement membrane, whereas the arrangement of collagen fibers in the gingival connective tissue was significantly affected. Animals with inflammation presented a reduction of 35% in the total area occupied by collagen fibers. However, these fibers were thicker and more densely packed. These alterations involve type I, type III and type VI collagens as determined by immunohistochemistry. The results demonstrated the occurrence of marked reorganization of the gingival extracellular matrix in response to the inflammatory process, indicating a new paradigm in the periodontal disease progression: collagen degradation and fibers thickening, simultaneously.

Macrophage roles in the clearance of apoptotic cells and control of inflammation in the prostate gland after castration

Androgen deprivation results in massive apoptosis in the prostate gland. Macrophages are actively engaged in phagocytosing epithelial cell corpses. However, it is unknown whether microtubule‐associated protein 1 light chain 3 alpha (LC3)‐associated phagocytosis (LAP) is involved and contribute to prevent inflammation.

Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment

Twenty horses were used in the experiment, for composed control group, (Cg) instrumented group, (Ig;without intestinal obstruction), treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment) and non-treated group (Ntg;submitted to intestinal obstruction without treatment). Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.

Lipocalina associada à gelatinase de neutrófilos (NGAL) e calprotectina no tecido laminar de equinos após obstrução jejunal, tratados ou não com hidrocortisona

Laminitis is a severe hoof condition in horses that may cause intense suffering. In this study, leukocyte infiltration in hoof laminar tissue was investigated in horses subject to intestinal obstruction using immunohistochemistry to detect calprotectin, and zymography to detect neutrophil gelatinase associated lipocalin (NGAL). There were four groups: the Control Group (Gc), with seven horses, without surgical procedures; the Sham-operated Group (Gi), with five horses that were subjected to surgical procedure without intestinal obstruction; the No Treat group (Gnt), with four horses subjected to intestinal obstruction (jejunal distention using an intraluminal balloon) without treatment; and Treated group (Gt), with four horses subjected to intestinal obstruction and treated with hydrocortisone. Positive calprotectin imunostaining was detected in all experimental groups, with increase cell counts in horses of the distended group compared with the control group. NGAL expression was increased in Gd compared with Gc e Gi. The Gt did not differ from the others. In conclusion, small intestine distension can promote leukocyte infiltration in equine hoof laminar tissue, and NGAL zymography was considered a useful method for leukocyte tissue detection in horses. New studies will be conducted to verify the possible beneficial anti-inflammatory effects of hydrocortisone in hoof of horses with intestinal obstruction.

The origin of prostate gland-secreted IgA and IgG

The prostate secretes immunoglobulin (Ig) A (IgA) and IgG; however, how immunoglobulins reach the secretion, where the plasma cells are located, whether immunoglobulins are antigen-specific and where activation of the adaptive response occurs are still unknown. Immune cells, including CD45RA+ cells, were scattered in the stroma and not organized mucosae-associated lymphoid-tissue. IgA (but not IgG) immunostaining identified stromal plasma cells and epithelial cells in non-immunized rats. Injected tetramethylrhodamine-IgA transcytosed the epithelium along with polymeric immunoglobulin receptor. Oral immunization with ovalbumin/mesopourous SBA-15 silica adjuvant resulted in more stromal CD45RA+/IgA+ cells, increased content of ovalbumin-specific IgA and IgG, and the appearance of intraepithelial CD45RA+/IgG+ cells. An increased number of dendritic cells that cooperate in other sites with transient immunocompetent lymphocytes, and the higher levels of interleukin-1β, interferon-γ and transforming growth factor-β, explain the levels of specific antibodies. Nasal immunization produced similar results except for the increase in dendritic cells. This immunomodulatory strategy seems useful to boost immunity against genitourinary infections and, perhaps, cancer.