Calcitriol Treatment Decreases Cell Migration, Viability and ß-Catenin Signaling in Oral Dysplasia.

Nearly 90% of oral cancers are characterized as oral squamous cell carcinoma (OSCC), representing the sixth most common type of cancer. OSCC usually evolves from oral potentially malignant disorders that, in some cases, are histologically consistent with a oral dysplasia. The levels of 1α,25 dihydroxyvitamin D3 (1,25-(OH)2D3; calcitriol), the active form of vitamin D3, have been shown to be decreased in patients with oral dysplasia and OSCC. Moreover, treatment with 1,25-(OH)2D3 has been proven beneficial in OSCC by inhibiting the Wnt/ß-catenin pathway, a signaling route that promotes cell migration, proliferation, and viability. However, whether this inhibition mechanism occurs in oral dysplasia is unknown. To approach this question, we used dysplastic oral keratinocyte cultures and oral explants (ex vivo model of oral dysplasia) treated with 1,25-(OH)2D3 for 48 h. Following treatment with 1,25-(OH)2D3, both in vitro and ex vivo models of oral dysplasia showed decreased levels of nuclear ß-catenin by immunofluorescence (IF) and immunohistochemistry (IHC). Consistently, reduced protein and mRNA levels of the Wnt/ß-catenin target gene survivin were observed after treatment with 1,25-(OH)2D3. Moreover, 1,25-(OH)2D3 promoted membranous localization of E-cadherin and nuclear localization of vitamin D receptor (VDR). Functionally, DOK cells treated with 1,25-(OH)2D3 displayed diminished cell migration and viability in vitro.

Reduced C-reactive protein levels after root canal treatment in clinically healthy young apical periodontitis individuals at cardiovascular risk. A prospective study.

AIM: To determine the systemic inflammatory burden, including hsCRP and its monomeric forms, in patients with apical lesions of endodontic origin treated with root canal treatment (RCT). METHODOLOGY: Prospective pre-/post-study. Apical periodontitis (AP) individuals aged 16-40 were included (N = 29). Individuals received RCT and were followed at 1 and 6 months. Fasting blood samples were obtained. Apical lesions of endodontic origin (ALEO) diameter (mm), and periapical index (PAI), were recorded. The serum concentrations of total hsCRP were determined by turbidimetry. Tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-1ß, and soluble (s) E-selectin were assessed by Multiplex assay. Additionally, mCRP forms were determined in the serum of AP patients with a baseline moderate to high cardiovascular risk based on hsCRP stratification (hsCRP ≥1 mg/L) by immunowestern blot (n = 15). Also, CRP isoforms were explored in ALEOs from AP individuals (n = 4). Data were analysed with StataV16. RESULTS: Periapical index and ALEO sizes were reduced at both follow-up visits after RCT (p < .05). Serum levels of TNF-α, IL-6, IL-10, IL-1ß, and sE-selectin did not show significant differences. CRP was borderline reduced at 1 month (p = .04); however, in AP individuals at cardiovascular risk (hsCRP ≥ 1 mg/L), hsCRP and its monomeric isoform significantly decreased at 1 and 6 months (p < .05). CONCLUSIONS: High-sensitivity CRP and mCRP are reduced after RCT in AP individuals at cardiovascular risk.

Inhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis.

PURPOSE: Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/ß-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. EXPERIMENTAL DESIGN: We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. RESULTS: Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear ß-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. CONCLUSIONS: The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.

The salivary peptide histatin-1 enhances bone repair in vivo.

The salivary peptide histatin-1 was recently described as a novel osteogenic factor that stimulates cell adhesion, migration, and differentiation in bone-lineage cells. Since these cell responses collectively contribute to bone regeneration, we hypothesized that histatin-1 harbors the capacity to enhance bone tissue repair at the preclinical level. By using a model of monocortical bone defect, we explored the effects of histatin-1 in tibial mineralization and organic matrix formation in vivo. To this end, different amounts of histatin-1 were embedded in one-mm3 collagen sponges and then applied to tibial monocortical defects in C57bl/6 mice. After seven days, mice were euthanized, and samples were processed for subsequent analysis. Micro-computed tomography screening showed that histatin-1 increased intraosseous mineralization, and this phenomenon was accompanied by augmented collagen matrix deposition and closure of cortical defect edges, as determined by Hematoxylin-Eosin and Masson's Trichrome staining. Moreover, immunohistochemical analyses showed that histatin-1 increased the expression of the osteogenic marker alkaline phosphatase, which was accompanied by augmented blood vessel formation. Collectively, our findings show that histatin-1 itself promotes bone regeneration in an orthotopic model, proposing this molecule as a therapeutic candidate for use in bone regenerative medicine.

The GPI-Anchored Protein Thy-1/CD90 Promotes Wound Healing upon Injury to the Skin by Enhancing Skin Perfusion.

Wound healing is a highly regulated multi-step process that involves a plethora of signals. Blood perfusion is crucial in wound healing and abnormalities in the formation of new blood vessels define the outcome of the wound healing process. Thy-1 has been implicated in angiogenesis and silencing of the Thy-1 gene retards the wound healing process. However, the role of Thy-1 in blood perfusion during wound closure remains unclear. We proposed that Thy-1 regulates vascular perfusion, affecting the healing rate in mouse skin. We analyzed the time of recovery, blood perfusion using Laser Speckle Contrast Imaging, and tissue morphology from images acquired with a Nanozoomer tissue scanner. The latter was assessed in a tissue sample taken with a biopsy punch on several days during the wound healing process. Results obtained with the Thy-1 knockout (Thy-1-/-) mice were compared with control mice. Thy-1-/- mice showed at day seven, a delayed re-epithelialization, increased micro- to macro-circulation ratio, and lower blood perfusion in the wound area. In addition, skin morphology displayed a flatter epidermis, fewer ridges, and almost no stratum granulosum or corneum, while the dermis was thicker, showing more fibroblasts and fewer lymphocytes. Our results suggest a critical role for Thy-1 in wound healing, particularly in vascular dynamics.

Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation.

Histatin-1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin-1 has been shown to promote re-epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad-spectrum activity of Histatin-1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin-1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin-1 promoted cell adhesion, spreading, and migration of SAOS-2 cells and MC3T3-E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin-1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin-1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS-2 and MC3T3-E1 cells. Mechanistically, although Histatin-1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of ß-catenin. Strikingly, the effects of Histatin-1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin-1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.

PKD2/polycystin-2 induces autophagy by forming a complex with BECN1.

Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Role of Autophagy in the Microenvironment of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma, the most common type of oral cancer, affects more than 275,000 people per year worldwide. Oral squamous cell carcinoma is very aggressive, as most patients die after 3 to 5 years post-diagnosis. The initiation and progression of oral squamous cell carcinoma are multifactorial: smoking, alcohol consumption, and human papilloma virus infection are among the causes that promote its development. Although oral squamous cell carcinoma involves abnormal growth and migration of oral epithelial cells, other cell types such as fibroblasts and immune cells form the carcinoma niche. An underlying inflammatory state within the oral tissue promotes differential stress-related responses that favor oral squamous cell carcinoma. Autophagy is an intracellular degradation process that allows cancer cells to survive under stress conditions. Autophagy degrades cellular components by sequestering them in vesicles called autophagosomes, which ultimately fuse with lysosomes. Although several autophagy markers have been associated with oral squamous cell carcinoma, it remains unclear whether up- or down-regulation of autophagy favors its progression. Autophagy levels during oral squamous cell carcinoma are both timing- and cell-specific. Here we discuss how autophagy is required to establish a new cellular microenvironment in oral squamous cell carcinoma and how autophagy drives the phenotypic change of oral squamous cell carcinoma cells by promoting crosstalk between carcinoma cells, fibroblasts, and immune cells.

The RabGEF ALS2 is a hypoxia inducible target associated with the acquisition of aggressive traits in tumor cells.

Tumor hypoxia and the hypoxia inducible factor-1, HIF-1, play critical roles in cancer progression and metastasis. We previously showed that hypoxia activates the endosomal GTPase Rab5, leading to tumor cell migration and invasion, and that these events do not involve changes in Rab protein expression, suggesting the participation of intermediate activators. Here, we identified ALS2, a guanine nucleotide exchange factor that is upregulated in cancer, as responsible for increased Rab5-GTP loading, cell migration and metastasis in hypoxia. Specifically, hypoxia augmented ALS2 mRNA and protein levels, and these events involved HIF-1α-dependent transcription, as shown by RNAi, pharmacological inhibition, chromatin immunoprecipitation and bioinformatics analyses, which identified a functional HIF-1α-binding site in the proximal promoter region of ALS2. Moreover, ALS2 and Rab5 activity were elevated both in a model of endogenous HIF-1α stabilization (renal cell carcinoma) and by following expression of stable non-hydroxylatable HIF-1α. Strikingly, ALS2 upregulation in hypoxia was required for Rab5 activation, tumor cell migration and invasion, as well as experimental metastasis in C57BL/6 mice. Finally, immunohistochemical analyses in patient biopsies with renal cell carcinoma showed that elevated HIF-1α correlates with increased ALS2 expression. Hence, this study identifies ALS2 as a novel hypoxia-inducible gene associated with tumor progression and metastasis.

Effect of Interleukin-17 in the Activation of Monocyte Subsets in Patients with ST-Segment Elevation Myocardial Infarction.

Interleukin- (IL-) 17 is increased in acute myocardial infarction (AMI) and plays a key role in inflammatory diseases through its involvement in the activation of leukocytes. Here, we describe for the first time the effect of IL-17 in the migration and activation of monocyte subsets in patients during ST-segment elevation myocardial infarction (STEMI) and post-STEMI. We analyzed the circulating levels of IL-17 in patient plasma. A gradual increase in IL-17 was found in STEMI and post-STEMI patients. Additionally, IL-17 had a powerful effect on the recruitment of CD14++CD16+/CD14+CD16++ monocytes derived from patients post-STEMI compared with the monocytes from patients with STEMI, suggesting that IL-17 recruits monocytes with inflammatory activity post-STEMI. Furthermore, IL-17 increased the expression of TLR4 on CD14 + CD16 - and CD14++CD16+/CD14+CD16++ monocytes post-STEMI and might enhance the response to danger-associated molecular patterns post-STEMI. Moreover, IL-17 induced secretion of IL-6 from CD14++CD16- and CD14++CD16+/CD14+CD16++ monocytes both in STEMI and in post-STEMI, which indicates that IL-17 has an effect on the secretion of proinflammatory cytokines from monocytes during STEMI and post-STEMI. Overall, we demonstrate that in STEMI and post-STEMI, IL-17 is increased and induces the migration and activation of monocyte subsets, possibly contributing to the inflammatory response through TLR4 and IL-6 secretion.

Wnt/ß-Catenin Signaling in Oral Carcinogenesis.

Oral carcinogenesis is a complex and multifactorial process that involves cumulative genetic and molecular alterations, leading to uncontrolled cell proliferation, impaired DNA repair and defective cell death. At the early stages, the onset of potentially malignant lesions in the oral mucosa, or oral dysplasia, is associated with higher rates of malignant progression towards carcinoma in situ and invasive carcinoma. Efforts have been made to get insights about signaling pathways that are deregulated in oral dysplasia, as these could be translated into novel markers and might represent promising therapeutic targets. In this context, recent evidence underscored the relevance of the Wnt/ß-catenin signaling pathway in oral dysplasia, as this pathway is progressively "switched on" through the different grades of dysplasia (mild, moderate and severe dysplasia), with the consequent nuclear translocation of ß-catenin and expression of target genes associated with the maintenance of representative traits of oral dysplasia, namely cell proliferation and viability. Intriguingly, recent studies provide an unanticipated connection between active ß-catenin signaling and deregulated endosome trafficking in oral dysplasia, highlighting the relevance of endocytic components in oral carcinogenesis. This review summarizes evidence about the role of the Wnt/ß-catenin signaling pathway and the underlying mechanisms that account for its aberrant activation in oral carcinogenesis.

Early clinical, histological, and immunohistochemical findings in suspected acute graft-versus-host disease and their association with patient outcomes.

BACKGROUND/OBJECTIVES: Acute graft-versus-host disease (aGVHD) is a serious condition after allogeneic hematopoietic stem cell transplantation (HSCT), frequently involving skin, gut, and liver. It can be difficult to diagnose early, yet this is vital for adequate management. We sought to identify initial clinical and histopathological features in children with suspected GVHD and the association with clinical course and outcomes. METHODS: Retrospective study of patients with skin biopsies for suspected aGVHD from 2006 to 2016. We collected demographic and clinical information, histologic, and immunohistochemical (IHC) findings, and outcomes during follow-up. Bivariate and multivariate analyses were done to identify risk factors associated with remission, development of severe/life-threatening aGVHD, and mortality. RESULTS: We included 42 patients, 15 females. Skin manifestations occurred 51 days (median) after HSCT. On biopsy, 76.2% had mild (stage 1-2) skin aGVHD; during the course of the disease, severity and systemic involvement increased to global grade III/IV in 66.6%. All patients received treatment; 15 are in remission from aGVHD and 23 have died. Histologic features were diagnostic in 83.3%. On bivariate and multivariate analysis, we identified initial clinical and histologic findings that were associated with the measured outcomes: odds of remission from aGVHD were increased when focal vacuolar changes were found on skin biopsy (OR 6.028; 95%CI:1.253-28.992) but decreased by initial hepatic aGVHD (OR 0.112; 95%CI: 0.017-0.748); severe/life-threatening aGVHD was associated with initial gastrointestinal aGVHD (OR 6.054; 95%CI:1.257-29.159); and odds of mortality were decreased with male donor (OR 0.056; 95%CI:0.004-0.804), nulliparous female donor (OR 0.076; 95%CI:0.009-0.669), and focal vacuolar changes on skin biopsy (OR 0.113; 95%CI:0.017-0.770). CONCLUSIONS: We found novel indicators predictive of remission, severity, and mortality in children with aGVHD. Further studies of this condition in children are needed.

Nuclear accumulation of ß-catenin is associated with endosomal sequestration of the destruction complex and increased activation of Rab5 in oral dysplasia.

Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of ß-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of ß-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the ß-catenin destruction complex allows nuclear accumulation of ß-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of ß-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3ß, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3ß, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the ß-catenin destruction complex leads to stabilization and nuclear accumulation of ß-catenin in oral dysplasia.

Nuclear localization of ß-catenin and expression of target genes are associated with increased Wnt secretion in oral dysplasia.

OBJECTIVES: To evaluate the localization of ß-catenin in oral dysplastic cells, the expression of target genes upregulated in oral dysplasia, and the role of Wnt ligands in these events. MATERIALS AND METHODS: Subcellular localization of total and non-phosphorylated (transcriptionally active) ß-catenin was evaluated by immunofluorescence and biochemical fractionation in dysplastic oral keratinocytes (DOK), non-dysplastic oral keratinocytes (OKF6), oral squamous carcinoma cells (CAL27) and primary oral keratinocytes. Tcf/Lef-dependent transcription was measured by luciferase reporter assays. Expression of target genes, survivin and cyclin D1, was evaluated by RT-qPCR and Western blotting. Wnt secretion was inhibited with the inhibitor of porcupine, C59. Wnt3a and ß-catenin were evaluated in biopsies by tissue immunofluorescence. RESULTS: Immunofluorescence and fractionation experiments showed augmented nuclear ß-catenin (total and transcriptionally active) in DOK, when compared with OKF6 and CAL27 cells. Intriguingly, conditioned medium from DOK promoted nuclear accumulation of ß-catenin and Tcf/Lef-dependent transcription in OKF6 and primary oral keratinocytes, suggesting the participation of secreted factors. Treatment of DOK with C59 decreased Wnt3a secretion, nuclear ß-catenin and the expression of survivin and cyclin D1 at both mRNA and protein levels. Accordingly, DOK secreted higher Wnt3a levels than OKF6, and inhibition of Wnt3a secretion prevented DOK-induced Tcf/Lef-dependent transcription in OKF6. These observations were confirmed in clinical samples, since tissue immunofluorescence analysis showed simultaneous expression of Wnt3a and nuclear ß-catenin in oral dysplasia, but not in healthy mucosa biopsies. CONCLUSION: These data indicate that secretion of Wnt ligands is critical for ß-catenin nuclear localization and expression of target genes in oral dysplasia.

Human papillomavirus infection in oral squamous cell carcinomas from Chilean patients.

Human papillomavirus (HPV) is the causal agent of cervical, anogenital and a subset of oropharyngeal carcinomas. In addition, the role of HPV in oral carcinogenesis has been suggested, although the findings are inconclusive. In this study, using conventional polymerase chain reaction (PCR) and genotyping by specific PCR and DNA sequencing, we analyzed the HPV presence in 80 oral squamous cell carcinomas (OSCCs) from Chilean subjects. In addition, we determined the expression of p16, p53, pRb and Ki-67 using immunohistochemistry (IHC). The CDKN2A (p16) promoter methylation was evaluated using methylation-specific PCR (MSP). HPV sequences were found in 9/80 (11%) OSCCs. Non-statistically significant association with p53, pRb, Ki-67 and p16 levels were found (p=0.77; 0.29; 0.83; 0.21, respectively). HPV-16 and 18 were the most prevalent HPV genotypes in 8/9 (89%) OSCCs. In addition, CDKN2A (p16) was methylated in 39% of OSCCs. No association with HPV presence (p=0.917) was found. These results suggest that HPV positive OSCCs are entities that do not resemble the molecular alterations of HPV-associated tumors in a Chilean population. More studies are warranted to determine the role of HPV in OSCCs.

Sarcomatoid (spindle cell) carcinoma of tongue: a report of two cases.

Sarcomatoid Carcinoma (SC) is an unusual and aggressive variant of squamous cell carcinoma, which frequently recurs and metastasizes; for this reason, the right diagnosis is very important. It is considered to be a biphasic tumor made up of cells from squamous and spindle cells carcinoma with a sarcomatous aspect, but of epithelial origin. The diagnosis often represents a clinical-pathological challenge where the study with immunohistochemical technique (IHC) is key to the histopathological diagnosis. The reported cases related to oral mucosa are limited. In this work we present two SC cases where the use of IHC allowed us to achieve a conclusive diagnosis.

C-reactive protein expression is up-regulated in apical lesions of endodontic origin in association with interleukin-6.

INTRODUCTION: C-reactive protein (CRP) is the prototype component of acute-phase proteins induced ultimately by interleukin (IL)-6 in the liver, but it is unknown whether periradicular tissues locally express CRP. The present study aimed to identify whether CRP messenger RNA synthesis occurs in situ within apical lesions of endodontic origin (ALEOs) and healthy periodontal ligament and its association with IL-6 and to determine their protein levels and tissue localization. METHODS: Patients with asymptomatic apical periodontitis and healthy volunteers presenting at the School of Dentistry, University of Chile, Santiago, Chile, were enrolled. ALEOs and healthy teeth were obtained and processed for either immunohistochemistry and double immunofluorescence to assess IL-6 and CRP tissue localization, whereas healthy periodontal ligaments were processed as controls for real-time reverse-transcription polymerase chain reaction for their RNA expression levels and multiplex assay to determine their protein levels. Statistic analysis was performed using the unpaired t test or Mann-Whitney test according to data distribution and Pearson correlation. RESULTS: IL-6 and CRP were synthesized in ALEOs, whereas their RNA expression and protein levels were significantly higher when compared with healthy periodontal ligament. IL-6 and CRP immunolocalized to the inflammatory cells, vascular endothelial cells, and mesenchymal cells. Both, IL-6 and CRP colocalized in ALEOs, and a positive correlation was found between their expression levels (P < .05). CONCLUSIONS: IL-6 and CRP messenger RNA are constitutively expressed in periodontal ligament and up-regulated in ALEOs along with higher protein levels. Given their pleiotropic effects, IL-6 and CRP protein levels in apical tissues might partially explain the development and progression of ALEOs as well as potentially asymptomatic apical periodontitis-associated systemic low-grade inflammation.

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells.

We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas.

The role of TLR2, TLR4 and CD36 in macrophage activation and foam cell formation in response to oxLDL in humans.

UNLABELLED: Toll-like receptor (TLR)2, TLR4 and CD36 are central in inflammation and the development of atherosclerosis. Oxidized low-density lipoprotein (oxLDL) plays a critical role in this disease through its involvement in the formation of foam cells and the activation of leukocytes. The aim of this research was to analyze the role of TLR2, TLR4 and CD36 in foam cell differentiation and macrophage activation. METHODS: Human macrophages were incubated with monoclonal antibodies specific for TLR2, TLR4 and CD36 prior to stimulation with oxLDL. Subsequently, we analyzed foam cell formation, cytokine secretion, histocompatibility complex (MHC) class II molecules and CD86 expression and T cell proliferation. RESULTS: The stimulation of macrophages with oxLDL induced foam cell formation, cytokine secretion, HLA-DR and CD86 expression and T cell proliferation. The blockage of TLR2, TLR4 and CD36 reduced the secretion of IL-1ß, IL-6 and IL-8, the expression of HLA-DR and CD86, T cell proliferation and foam cell formation. However, the blockage of TLR2 did not affect the formation of foam cells. CONCLUSION: Our study demonstrates that TLR2, TLR4 and CD36 participate in the immune response to oxLDL by inducing an increase in pro-inflammatory cytokines, the expression HLA-DR and CD86 and the proliferation of T cells. However, TLR2 does not participate in the formation of foam cells, while TLR4 and CD36 play a relevant role in this process. These findings suggest that the activation of these receptors by oxLDL contributes to the pathogenesis of atherosclerosis.

High levels of CXC ligand 12/stromal cell-derived factor 1 in apical lesions of endodontic origin associated with mast cell infiltration.

INTRODUCTION: CXC ligand 12/stromal-derived factor-1 (CXCL12/SDF-1) is a pleiotropic chemokine that regulates the influx of a wide range of leukocytes. The aim of this study was to characterize CXCL12/SDF-1 in apical lesions (ALs) of endodontic origin, with special emphasis in associated immune cell populations. METHODS: In this case-control study, 29 individuals with chronic apical periodontitis and 21 healthy volunteers were enrolled. ALs and healthy periodontal ligament samples were obtained for tissue homogenization, immune Western blotting, and enzyme-linked immunosorbent assay to determine CXCL12/SDF-1 forms and levels. Anatomopathologic diagnosis, immunostaining for CXCL12/SDF-1, CD117-CXCL12/SDF-1, and toluidine blue were also performed to identify tissue and cell localization. Finally, a set of tissue samples were digested and analyzed by flow cytometry to identify CXCL12/SDF-1 in different immune cell populations. Data were analyzed with Stata v11 and WinDi 2.9 software, and significance was considered if P < .05. RESULTS: CXCL12/SDF-1 was predominantly identified as monomers; levels of CXCL12/SDF-1 were significantly higher in ALs compared with controls, and it was primarily localized to inflammatory infiltrates. Expression of CXCL12/SDF-1 was colocalized to mast cells in tissue sections. Furthermore, CD117(+) mast cells were the second most frequent infiltrating cells and the main CXCL12/SDF-1 expressing cells, followed by CD4(+) lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells. CONCLUSIONS: ALs of endodontic origin demonstrated higher levels of CXCL12/SDF-1 compared with controls. CXCL12/SDF-1 was identified in immune cell populations, whereas mast cells represented the major CXCL12/SDF-1 expressing cells, suggesting that this chemokine might play a central role in apical tissue destruction, most probably inducing persistent recruitment of immune cells, particularly of mast cells.