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.

Methods for studying primary cilia in heart tissue after ischemia-reperfusion injury.

Cardiovascular diseases are the leading cause of death and disability worldwide. After heart injury triggered by myocardial ischemia or myocardial infarction, extensive zones of tissue are damaged and some of the tissue dies by necrosis and/or apoptosis. The loss of contractile mass activates a series of biochemical mechanisms that allow, through cardiac remodeling, the replacement of the dysfunctional heart tissue by fibrotic material. Our previous studies have shown that primary cilia, non-motile antenna-like structures at the cell surface required for the activation of specific signaling pathways, are present in cardiac fibroblasts and required for cardiac fibrosis induced by ischemia/reperfusion (I/R) in mice. I/R-induced myocardial fibrosis promotes the enrichment of ciliated cardiac fibroblasts where the myocardial injury occurs. Given discussions about the existence of cilia in specific cardiac cell types, as well as the functional relevance of studying cilia-dependent signaling in cardiac fibrosis after I/R, here we describe our methods to evaluate the presence and roles of primary cilia in cardiac fibrosis after I/R in mice.

Unexpected Findings in Hereditary Breast and Ovarian Cancer Syndrome: Low-Level Constitutional Mosaicism in BRCA2.

Hereditary breast and ovarian cancer syndrome (HBOC) is a clinical entity characterized by an increased risk of developing breast and ovarian cancer. The genetic diagnosis is based on the identification of heterozygous germinal variants in HBOC susceptibility genes. However, it has recently been described that constitutional mosaic variants can contribute to the aetiology of HBOC. In constitutional mosaicism, individuals have at least two genotypically distinct populations of cells that arise from an early post-zygote event. The mutational event occurs early enough in development to affect several tissues. It is detected in germinal genetic studies as low variant allele frequency (VAF) variants (<30%) that are generally overlooked during the prioritization process. Constitutional mosaic variants can affect both somatic and germinal cells, and thus can be passed to the offspring and have important consequences for genetic counselling. In this work, we report the c.9648+1G>A mosaic variant in the BRCA2 gene and propose a diagnostic algorithm to deal with potential mosaic findings identified by Next Generation Sequencing (NGS).

Appropriateness of drug prescriptions in patients with chronic kidney disease in primary care: a double-center retrospective study.

BACKGROUND: Chronic kidney disease (CKD) is a highly prevalent disease worldwide. A basic pillar for the management of a patient with CKD is the safe use of drugs. Inadequate dosing of medication or contraindicated drugs in renal impairment can lead to negative outcomes. The primary objective was to analyse the drug prescriptions of patients with CKD from two primary care centres to see if they were optimally adapted to the patient's estimated glomerular filtration rate (eGFR). METHODS: A retrospective observational study was conducted in two urban primary care centres. The study period was between September-October 2019. Patients over 18 years of age, with established CKD and with an eGFR less than 60 mL/min/1.73m2 for at least three months were included. Their demographic data (age and sex) and clinical variables such as associated comorbidities, eGFR value were retrospectively registered. Finally, their medication plans were reviewed in order to detect: inappropriate prescribing (IP), defined as an incorrect dose/frequency or contraindicated drug according to the renal function of the patient; nephrotoxic drugs and drugs with a high sodium content. RESULTS: A total of 273 patients were included. The most common patient profile was an elderly woman, polymedicated, with other concomitant diseases and with mild CKD. Two hundred and one IPs were detected, 13.9% of which were contraindicated drugs. Of all patients, 49.1% had been prescribed at least one IP on their medication plan, 93.8% had some potentially nephrotoxic drug and 8.4% had drugs with a high sodium content prescribed. CONCLUSIONS: Patients with CKD are at increased risk of medication-related problems. It is necessary to implement measures to improve the safety in the prescription of drugs in patients with CKD.

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.

Stability of 5% vancomycin ophthalmic solution prepared using balanced salt solution after freezing for 90 days.

PURPOSE: To evaluate the stability of 5% vancomycin ophthalmic solution prepared using balanced salt solution (BSS) and stored at -20°C in polypropylene containers. METHODS: Six batches of vancomycin 50 mg/mL eyedrops were aseptically prepared. One bottle of each batch was analyzed immediately after preparation, and the rest were stored at -20°C and analyzed using high-performance liquid chromatography (HPLC) at 30, 60, and 90 days to test their physicochemical stability and sterility. Thereafter, bottles were removed from the freezer and stored at 5°C for 30 days, with HPLC and other analyses repeated 105 and 120 days after preparation. All samples were analyzed in triplicate. Stability was defined as the absence of particles, color variation, or changes in pH and a remaining vancomycin concentration of 90% to 110% of the initial concentration. The sterility of the ophthalmic solution was evaluated by using soybean-casein digest broth with resins; samples were incubated for 7 days and checked daily for signs of microbial growth. RESULTS: There was no particle formation or sign of precipitation in any of the solutions throughout the duration of the study, regardless of the storage conditions. No change in color or turbidity was observed. The pH and osmolarity remained unchanged during storage at -20°C and after thawing. The vancomycin concentration remained within 10% of the initial concentration during the 90-day period of storage at -20°C and the subsequent 30 days after thawing. Sterility was preserved in all samples. CONCLUSION: A 5% solution of vancomycin prepared using BSS was physicochemically and microbiologically stable when stored at -20°C for 90 days. After thawing, this extemporaneous formulation remained stable when refrigerated at 5°C for 30 days.

Dysregulated healing response participates in the pathophysiology of temporomandibular joint ankylosis.

This study aimed to characterize samples from patients diagnosed with TMJ ankylosis, using both clinical and histological data. Both clinical and histological analyses of retrieved tissue samples from patients with primary TMJ ankyloses were performed retrospectively (1980-2012). All patients had been subjected to primary arthroplasty. Our study analyzed connective tissue differentiation, ossification patterns, and bone resorption, using histology and immunohistochemistry. Fifteen case records, with a sex ratio of 4:1 (men:woman) and a median age of 8 years, were collected. Six patient samples reported a previous inflammatory event. Histologically, 15 samples exhibited fibrous tissue. Among these, 13 displayed bone at different stages of maturity (fibrous/bony ankylosis). Eleven samples showed aberrant cartilage, characterized by hypertrophic chondrocyte-like cells at the bone/cartilage interface. Four samples revealed inflammatory infiltrate; in one case, this was organized as a lymphoid follicle. Eleven samples showed bone resorption by attached osteoclasts. Interestingly, non-attached osteoclasts were detected, suggesting locally impaired bone remodeling. An association between the presence of mature/lamellar bone and the presence of osteoclasts was observed (p = 0.03). No association was found between previous history of either trauma or infection and the histological type of ankylosis (p = 0.74). There was no association between the histological presence of inflammation or infection and the type of ankylosis (p = 0.63 and p = 0.87, respectively). Retrieved TMJ ankylosis tissues displayed both aberrant ossification and reduced focal bone resorption, suggesting a dysregulated healing response.

Dose banding of intravenous ganciclovir: Banding scheme proposal and audit of toxicity and efficiency.

WHAT IS KNOWN AND OBJECTIVE: Dose banding is a strategy to optimize processing without reducing patient safety. Prescribed doses are rounded up or down to predetermined standard doses. Although it has been mostly used in chemotherapy, other drugs are suitable for this strategy, such as the antiviral ganciclovir. The aim of this work is to assess the safety and efficiency of a dose-rounding system for intravenous ganciclovir. METHODS: Dose bands were established for a maximum of 10% variation from the individualized dose. The number of annual preparations that expired before use and the number of ganciclovir vials saved were documented as indicators of efficiency. Toxicity was assessed comparing haematological parameters before and after ganciclovir treatment in a sample of patients who received doses above the theoretical dose (n = 121) and in the rest of the cohort (n = 129). RESULTS AND DISCUSSION: Five ganciclovir standard doses were established. It was shown that the bulk of the preparations (83.7%) had a maximum variation between the exact dose prescribed and the adjusted dose of ±10%. Three years after its implementation, a mean of 2848 annual preparations were compounded. The average percentage of annual expired preparations was lower than 1% of the total compounded doses, and the dose-rounding system allowed for saving 699 manufactured ganciclovir vials annually. There was no significant difference between haemoglobin and leucocyte levels measured before and after ganciclovir treatment in both groups. WHAT IS NEW AND CONCLUSION: Ganciclovir dose banding allows for efficient management of preparations without an increased risk of acute haematological side effects.

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.

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.

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.

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.