[Show abstract][Hide abstract] ABSTRACT: Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type “1” reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type “2” reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy.
Full-text · Article · Nov 2015 · Frontiers in Genetics
[Show abstract][Hide abstract] ABSTRACT: Introduction:
The pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions.
Periapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti-glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array).
Treg inhibition by anti-glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression.
Because the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.
No preview · Article · Nov 2015 · Journal of endodontics
[Show abstract][Hide abstract] ABSTRACT: In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.
Full-text · Article · Sep 2015 · Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.]
[Show abstract][Hide abstract] ABSTRACT: Bone tissue has a significant potential for healing, which involves a significant the interplay between bone and immune cells. While fracture healing represents a useful model to investigate endochondral bone healing, intramembranous bone healing models are yet to be developed and characterized. In this study, a micro-computed tomography, histomorphometric and molecular (RealTimePCRarray) characterization of post tooth-extraction alveolar bone healing was performed on C57Bl/6 WT mice. After the initial clot dominance (0h), the development of a provisional immature granulation tissue is evident (7d), characterized by marked cell proliferation, angiogenesis and inflammatory cells infiltration; associated with peaks of growth factors (BMP-2-4-7,TGFβ1,VEGFa), cytokines (TNFα, IL-10), chemokines & receptors (CXCL12, CCL25, CCR5, CXCR4), matrix (Col1a1-2, ITGA4, VTN, MMP1a) and MSCs (CD105, CD106, OCT4, NANOG, CD34, CD146) markers expression. Granulation tissue is sequentially replaced by more mature connective tissue (14d), characterized by inflammatory infiltrate reduction along the increased bone formation, marked expression of matrix remodeling enzymes (MMP-2-9), bone formation/maturation (RUNX2, ALP, DMP1, PHEX, SOST) markers, and chemokines & receptors associated with healing (CCL2, CCL17, CCR2). No evidences of cartilage cells or tissue were observed, strengthening the intramembranous nature of bone healing. Bone microarchitecture analysis supports the evolving healing, with total tissue and bone volumes as trabecular number and thickness showing a progressive increase over time. The extraction socket healing process is considered complete (21d) when the dental socket is filled by trabeculae bone with well-defined medullary canals; it being the expression of mature bone markers prevalent at this period. Our data confirms the intramembranous bone healing nature of the model used, revealing parallels between the gene expression profile and the histomorphometric events and the potential participation of MCSs and immune cells in the healing process, supporting the forthcoming application of the model for the better understanding of the bone healing process.
[Show abstract][Hide abstract] ABSTRACT: Th1-polarized host response, mediated by IFN-γ, has been associated with increased severity of periodontal disease as well as control of periodontal infection. The functional polymorphism TBX21-1993T/C (rs4794067) increases the transcriptional activity of the TBX21 gene (essential for Th1 polarization) resulting in a predisposition to a Th-1 biased immune response. Thus, we conducted a case-control study, including a population of healthy controls (H, n=218), chronic periodontitis (CP, n=197), and chronic gingivitis patients (CG, n=193), to investigate if genetic variations in TBX21 could impact the development of Th1 responses, and consequently influence the pattern of bacterial infection and periodontitis outcome. We observed that the polymorphic allele T was significantly enriched in the CP patients compared to CG subjects, while the H controls demonstrated and intermediate genotype. Also, investigating the putative functionality TBX21-1993T/C in the modulation of local response, we observed that the transcripts levels of T-bet, but not of IFN-γ, were upregulated in homozygote and heterozygote polymorphic subjects. In addition, TBX21-1993T/C did not influence the pattern of bacterial infection or the clinical parameters of disease severity, being the presence/absence of red complex bacteria the main factor associated with the disease status and the subrogate variable probing depth (PD) in the logistic regression analysis.
[Show abstract][Hide abstract] ABSTRACT: Introduction
Previous studies describe contrasting molecular profiles of active and inactive periapical granulomas characterized by distinct expression of cytokines, osteoclastogenic factors, and wound healing markers. Although the molecular mechanisms underlying such dichotomy remain unknown, in this study, we investigated the potential involvement of mesenchymal stem cells (MSCs) in determining human and murine periapical lesion activity and outcomes.
Periapical granulomas (n = 83) and control samples (n = 24) were comparatively assessed for the expression levels of 11 mesenchymal stem cell (MSC) markers using real-time polymerase chain reaction. Experimental periapical lesions induced in mice were evaluated for MSC marker expression and the effects of AMD3100 treatment on lesion outcomes.
MCS marker expression was prevalent in periapical granulomas compared with controls, whereas CD29, CD73, CD90, CD146, CD166, NANOG, Stro-1, and CXCR4 expressions were higher in inactive than active lesions. Experimental periapical lesion inactivity was also associated with an increased expression of MSC markers. The inhibition of MSC mobilization to the periapex by AMD3100 resulted in increased lesion sizes; decreased expression of MSCs and wound healing markers; and increased expression of interleukin 1 beta (IL-17β), interleukin 17 (IL-17), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and nuclear factor kappa-B ligand (RANKL).
Our results show that MSC markers are overexpressed in inactive human and experimental periapical lesions and that MSC mobilization results in the attenuation of experimental lesion progression associated with immunosuppressive and prohealing mechanisms.
Full-text · Article · Oct 2014 · Journal of Endodontics
[Show abstract][Hide abstract] ABSTRACT: Previous studies demonstrate that the balance between pro- and anti-inflammatory
mediators determines the stable or progressive nature of periapical granulomas by
modulating the balance of the osteoclastogenic factor RANKL and its antagonist OPG.
However, the cytokine networks operating in the development of periapical lesions are
quite more complex than what the simple pro- versus anti-inflammatory mediators'
paradigm suggests. Here we simultaneously investigated the patterns of Th1, Th2, Th9,
Th17, Th22, Thf, Tr1 and Tregs cytokines/markers expression in human periapical
Full-text · Article · Jul 2014 · Journal of applied oral science: revista FOB
[Show abstract][Hide abstract] ABSTRACT: Objective: While RANK/RANKL/OPG comprises the major osteoclastogenic system involved in inflammatory osteolysis, osteoclast costimulatory molecules (OCMs, i.e. DAP-12/TREM-2/SIRPb1; FcRgamma/OSCAR/PIR-A) also play important roles in osteoclastogenesis. We investigated the expression of such molecules in human and experimental periodontitis (PD), with special attention to the FcRgamma-related complex.
Method: and Results: Using RealTimePCR and FACS, we detected a higher expression of OCMs in human PD samples than in controls, and that DAP-12/TREM-2/SIRPb1 and FcRgamma/OSCAR/PIR-A complexes increased along experimental PD in C57Bl/6 in mice; especially in CCR2+ and CCR5+ osteoclast precursors. In vitro activation of CCR2+CCR5+ osteoclast precursors via TLR and TNF-alpha resulted in a marked increase of OCMs expression; a finding correlated with decreased bone loss presented in vivo by CCR2KO, CCR5KO, TLR4KO and TNFp55KO mice strains after PD induction. Focused in the FcRgamma/OSCAR/PIR-A, our data shows that CCR2+CCR5+RAW cells in vitro FcRgamma stimulation (with IgG) increase osteoclastogenesis. Accordingly, while absence of FcRgamma ligands (in BKO mice) lead to decreased bone loss in vivo along ePD, Ig transfer to BKO mice recovers the bone loss phenotype in a Ig dose dependent way.
Conclusion: We demonstrated an increased expression OCMs in human and experimental PD and that Ig/FcRgamma axis contribute to osteoclastogenesis and bone loss in vivo and vitro.
[Show abstract][Hide abstract] ABSTRACT: Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae
in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.
Preview · Article · Mar 2014 · Journal of Visualized Experiments
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis and lymphangiogenesis are the processes of neovascularization that evolve from preexisting blood and lymphatic vessels. There are few studies on angiogenesis and none on lymphangiogenesis in leprosy. Thus, the role of neovascularization in the pathophysiological mechanisms of the disease was studied across the spectrum of leprosy, its reactional states and its residual lesions.
Seventy-six biopsies of leprosy skin lesions and seven healthy controls were selected. Fifty-five serum samples were used for the detection of CD105 by ELISA. Histological sections were stained with antibodies against CD31 (blood and lymphatic vessels), D2-40/podoplanin (lymphatic vessels), and CD105/endoglin (neovessels). Microvessels were counted in 100 high-power fields (400x) and the number of vessels was evaluated in relation to the extension of the inflammatory infiltrate (0-3), to the bacillary index (0-6) and to the clinical forms. Angiogenesis, as marked by CD31 and CD105, was observed across the leprosy spectrum, compared with the controls. Additionally, there was a positive correlation between these markers with extension of the infiltrate (p <0.0001). For D2/40, lymphangiogenesis was observed in the tuberculoid form (p <0.0001). There was no statistical significance for values of CD105 detected in plasma by ELISA.
Angiogenesis is present across the spectrum of leprosy and in its reactional forms. The increase in the number of vessels, as detected by CD31 and CD105 staining, is related to the extension of the inflammatory infiltrate. Samples from reactional lesions have a higher number of CD31+ and CD105+ stained vessels, which indicates their involvement in the pathophysiological mechanisms of the reactional states. The regression of lesions is accompanied by the regression of neovascularization. Drugs inhibiting angiogenesis may be relevant in the treatment of leprosy, in addition to multidrugtherapy, and in the prevention of the development of reactions.
[Show abstract][Hide abstract] ABSTRACT: The development of regulated immune responses is essential in the balance between tolerance and immunity to antigens. Immune-related disorders are the result of inappropriate destruction of normal tissue by unbalanced immune inflammatory reactions, and comprise autoimmune (a failure of immune self-tolerance) and inflammatory disorders (an excessive inflammatory reaction to exogenous antigens). Among highly prevalent chronic inflammatory diseases, periodontal diseases (PD) are characterized by the inflammatory bone resorption of the teeth supporting structures. PD is the most prevalent form of bone pathology in humans and a modifying factor of the systemic health of patients. PD presents a complex etiology, where microbial factors trigger a chronic inflammatory immune response, whose nature and strength are related to the lesions stability or progression. Such response, and consequently the PD outcome, also can be modulated by local, systemic and environmental factors, and by the host genetic background, but the extent of each contributing factor to the pathogenesis of PD remains unknown. In fact, over the years the contribution of microbial, genetic and modifying factors (a series of co-morbidities including diabetes and rheumatoid arthritis) have been investigated in PD. However, in the view of the potential overlapping or additive/synergistic effects of such factors in alveolar bone loss, its individual investigation does not allow the determination of its individual and exact role in the determination of bone loss severity that typify PD. Therefore, this review article will discuss the individual and associated risks that single nucletotide polymorphisms (SNPs), classic pathogens (such as the red complex and A. actinomycetemcomitans) and modifying factors (such as diabetes and rheumatoid arthritis) confer to the PD-associated bone loss. Interestingly, the genetic-environmental interaction is quite diverse, ranging from additive effects to dominant scenarios, where genetic predisposition and the intensity and frequency of microbial challenge can result in distinct outcomes. Also, the modulator effect of co-morbidities is characterized beyond a simple exacerbation of inflammatory reaction and bone loss process, comprising a significant modulation of host responsiveness, which even involves the participation of commensal flora.
[Show abstract][Hide abstract] ABSTRACT: The development of periapical granulomas is dependent on the host response and involves Th1, Th2, Th17, and Treg-related cytokines. The discovery of new Th9 and Th22 subsets, with important immunomodulatory roles mediated by interleukin (IL)-9 and IL-22, respectively, emphasizes the need for reevaluation of current cytokine paradigms in context of periapical lesions. We investigated the expression of IL-9 and IL-22 in active and stable human granulomas and throughout experimental lesion development in mice.
Periapical granulomas (N = 83) and control specimens (N = 24) were evaluated regarding the expression of IL-9 and IL-22 via real-time polymerase chain reaction. Experimental periapical lesions were induced in mice (pulp exposure and bacterial inoculation) and the lesions evolution correlation with IL-9 and IL-22 expression kinetics was evaluated.
IL-9 and IL-22 mRNA expression was higher in periapical lesions than in control samples; higher levels of IL-9 and IL-22 were observed in inactive than in active lesions. In the experimental lesions model, increasing levels of IL-9 and IL-22 mRNA were detected in the lesions, and inverse correlations were found between IL-9 and IL-22 and the increase of lesion area in the different time point intervals.
Our results suggest that Th9 and Th22 pathways may contribute to human and experimental periapical lesion stability.
No preview · Article · Jan 2013 · Journal of endodontics
[Show abstract][Hide abstract] ABSTRACT: In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.
Full-text · Article · Sep 2012 · Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.]
[Show abstract][Hide abstract] ABSTRACT: Objective: While cytokines have been implicated in bone loss process, its role in bone repair process remains unknown. The objective of this study was to characterize the role of TNF-α and IL-10 in regulating the outcome of alveolar bone healing process after tooth extraction and the underlying gene expression changes along the repair in IL-10KO and TNFp55KO strains compared to C57Bl/6(WT) mice.
Method: Maxillas were collected for molecular (RealTimePCR) and histomorphometric analysis 0, 7, 14, 21, 28 and 42 days after extraction of right upper incisor.
Result: In C57Bl/6WT mice the initial formation of clot (0 hours) was followed by the transient appearance of foci of inflammatory infiltrate (7 days) and gradual (7-42 days) formation of connective tissue, vessels and bone. TNFp55KO strain presented increased counts of inflammatory cells, in spite of a slight decrease in the KC/CXCL1, MCP-1/CCL-2, MIP1α/CCL3 and IL-10 expression, and a delayed transition of granulation to bone tissue, associated with increased COL-I and decreased CBFA-1, ALP, OCN and PHEX expression, despite the higher proportion of osteoclasts in the late periods. IL10KO strain showed an increased density of inflammatory cells associated with higher expression of pro-inflammatory cytokines and chemokines (IL-1β, TNF-α, KC/CXCL1, MCP-1/CCL-2 and MIP-1α/CCL3) in the initial periods, followed by a significant delay in bone repair, evidenced by lower density of bone matrix associated with lower expression of CBFA-1, ALP, OCN and PHEX and higher density of osteoclasts.
Conclusion: Therefore both cytokines interferes in alveolar bone repair through mechanisms involving the control of inflammatory cell migration and modulation of osteogenic markers expression, since that the absence of IL-10 is associated with higher inflammatory activity and bone resorption concomitant with lower bone formation, while the deficiency of TNF-α affect the recruitment of inflammatory infiltrates and the kinetics of alveolar bone healing.
[Show abstract][Hide abstract] ABSTRACT: Periodontitis comprises a group of multifactorial diseases in which periodontopathogens accumulate in dental plaque and trigger host chronic inflammatory and immune responses against periodontal structures, which are determinant to the disease outcome. Although unusual cases of non-inflammatory destructive periodontal disease (NIDPD) are described, their pathogenesis remains unknown. A unique NIDPD case was investigated by clinical, microbiological, immunological and genetic tools. The patient, a non-smoking dental surgeon with excessive oral hygiene practice, presented a generalized bone resorption and tooth mobility, but not gingival inflammation or occlusion problems. No hematological, immunological or endocrine alterations were found. No periodontopathogens (A. actinomycetemcomitans, P. gingivalis, F. nucleatum and T. denticola) or viruses (HCMV, EBV-1 and HSV-1) were detected, along with levels of IL-1β and TNF-a in GCF compatible with healthy tissues. Conversely ALP, ACP and RANKL GCF levels were similar to diseased periodontal sites. Genetic investigation demonstrated that the patient carried some SNPs, as well HLA-DR4 (*0404) and HLA-B27 alleles, considered risk factors for bone loss. Then, a less vigorous and diminished frequency of toothbrushing was recommended to the patient, resulting in the arrest of alveolar bone loss, associated with the return of ALP, ACP and RANKL in GCF to normality levels. In conclusion, the unusual case presented here is compatible with the previous description of NIDPD, and the results that a possible combination of excessive force and frequency of mechanical stimulation with a potentially bone loss prone genotype could result in the alveolar bone loss seen in NIDPD.
Full-text · Article · Feb 2012 · Journal of applied oral science: revista FOB
[Show abstract][Hide abstract] ABSTRACT: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process at the periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions.
Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio).
We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001).
The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success.
No preview · Article · Feb 2012 · Journal of endodontics
[Show abstract][Hide abstract] ABSTRACT: Current literature on chronic periodontitis genetics encompasses numerous single nucleotide polymorphisms-focused case-control studies with inconsistent and controversial results, which typically disregards the exposure concept embraced by case-control definition. Herein, we propose a case-control design reappraisal by clear phenotype selection, where chronic gingivitis represents a genetically resistant phenotype/genotype opposing the susceptible cohort.
The hypothesis was tested in healthy, chronic periodontitis and gingivitis groups through Real-time PCR-based allelic discrimination of classic variants IL1B-3954, IL6-174, TNFA-308, IL10-592 and TLR4-299.
Observed allele/genotype frequencies characterize the healthy group with an intermediate genetic profile between periodontitis and gingivitis cohorts. When comparing genotype/allele frequencies in periodontitis versus healthy and periodontitis versus gingivitis scenarios, the number of positive associations (2-4) and the degree of association (p and odds ratio values) were significantly increased by the new approach proposed (periodontitis versus gingivitis), suggesting the association of IL1B-3954, TNFA-308, IL10-592 and TLR4-299 with periodontitis risk. Power study was also significantly improved by the new study design proposed when compared to the traditional approach.
The data presented herein support the use of new case-control study design based on the case-control definition and clear resistance/susceptibility phenotypes selection, which can significantly impact the study power and odds of identification of genetic factors involved in PD.
Full-text · Article · Jan 2012 · Journal Of Clinical Periodontology