Histopathological Grading of Oral Mucosal Chronic Graft-versus-Host Disease: Large Cohort Analysis.

Graft-versus-host disease (GVHD) can manifest as acute or chronic complications in patients after hematopoietic cell transplantation (HCT). Oral chronic GVHD (cGVHD) occurs in approximately 70% of HCT recipients and includes lichenoid-like mucosal reactions, restricted mouth opening, and salivary gland dysfunction. However, the underlying histopathological presentation remains to be validated in large cohorts. We characterized the histopathological features of oral mucosal cGVHD and devised a scoring model in a large patient cohort (n = 112). Oral mucosal biopsy sections (n = 303) with and without oral cGVHD were identified from archived and current HCT recipients with additional healthy controls. Histological screening was performed on hematoxylin and eosin-stained and periodic acid-Schiff-stained sections. A points-based grading tool (0 to 19, grade 0 to IV) was established based on intraepithelial lymphocytes and band-like inflammatory infiltrate, atrophic epithelium with basal cell liquefaction degeneration, including apoptosis, as well as separation of epithelium and pseudo-rete ridges. Validation involved 62 biopsy specimens, including post-HCT (n = 47) and healthy (n = 15) specimens. Remaining biopsy specimens (n = 199) were blindly graded by 3 observers. Histological severity was correlated with clinical diagnostic and distinctive features, demonstrating a spectrum of individual patient severity, including frequent signs of subclinical GVHD in healthy mucosa. However, oral cGVHD presented with significantly higher (P < .001) scores compared with HCT controls, with moderate to high positive likelihood ratios for inflammatory infiltrate, exocytosis, and basal membrane alterations. The grade II-IV biopsy specimens demonstrated a histopathological diagnosis of active mucosal lichenoid-like cGVHD, highlighting the importance of correlating clinical presentation with the dynamic histopathological processes for improved patient stratification. In addition, this tool could be used for assessing treatments, pathological processes, and immune cellular content to provide further insight into this debilitating disease.

Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

OBJECTIVE: Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. MATERIALS AND METHODS: Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E2, interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1ß stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. RESULTS: PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p < .001). Short-term exposure to 0.005% PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p < .001). IL-1ß stimulation induced an inflammatory response with a significant increase in the secretion of PGE2, IL-6, IL-8 and MMP-1. Treatment of IL-1ß stimulated fibroblasts in combination with PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE2, IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE2, IL-6, IL-8 or MMP-1 by gingival fibroblasts. CONCLUSIONS: Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1ß-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

Perspectives on oral chronic graft-versus-host disease from immunobiology to morbid diagnoses.

Chronic Graft-versus-Host Disease (cGVHD) is a major long-term complication, associated with morbidity and mortality in patients following allogenic hematopoietic cell transplantation (HCT) for immune hematopoietic disorders. The mouth is one of the most frequently affected organs after HCT (45-83%) and oral cGVHD, which may appear as the first visible sign. Manifestations present with mucosal lichenoid lesions, salivary gland dysfunction and limited oral aperture. Diagnosis of oral cGVHD severity is based on mucosal lesions with symptoms of sensitivity and pain and reduced oral intake. However, diagnostic difficulties arise due to subjective definitions and low specificity to cover the spectrum of oral cGVHD. In recent years there have been significant improvements in our understanding of the underlying oral cGVHD disease mechanisms. Drawing upon the current knowledge on the pathophysiology and biological phases of oral cGVHD, we address oral mucosa lichenoid and Sjogren's Syndrome-like sicca syndromes. We consider the response of alloreactive T-cells and macrophages to recipient tissues to drive the pathophysiological reactions and biological phases of acute inflammation (phase 1), chronic inflammation and dysregulated immunity (phase 2), and subsequent aberrant fibrotic healing (phase 3), which in time may be associated with an increased malignant transformation rate. When formulating treatment strategies, the pathophysiological spectrum of cGVHD is patient dependent and not every patient may progress chronologically through the biological stages. As such there remains a need to address and clarify personalized diagnostics and management to improve treatment descriptions. Within this review, we highlight the current state of the art knowledge on oral cGVHD pathophysiology and biological phases. We address knowledge gaps of oral cGVHD, with a view to facilitate clinical management and improve research quality on lichenoid biology and morbid forms of oral cGVHD.

Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation.

Endochondral bone formation is orchestrated by mesenchymal cell condensation to form cartilage anlagen, which act as a template for bone formation and eventual mineralization. The current study performed gene expression analysis to examine pre- and post-mineralization stages (E15 and E19) of endochondral bone formation, using fetal metatarsal long bones as a model. An extensive number of genes were differentially expressed, with 543 transcripts found to have at least 2-fold up-regulation and 742 with a greater than 2-fold down-regulation. A bioinformatics approach was adopted based on gene ontology groups, and this identified genes associated with the regulation of signaling and skeletal development, cartilage replacement by bone, and matrix degradation and turnover. Transcripts linked to skeletal patterning, including Hoxd genes 10-12, Gli2 and Noggin were considerably down-regulated at E19. Whereas genes associated with bone matrix formation and turnover, ACP5, MMP-13, bone sialoprotein, osteopontin, dentin matrix protein-1 and MMP-9 all were distinctly up-regulated at this later time point. This approach to studying the formation of the primary ossification center provides a unique picture of the developmental dynamics involved in the molecular and biochemical processes during this intricately regulated process.

Osteoadherin accumulates in the predentin towards the mineralization front in the developing tooth.

BACKGROUND: Proteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following, matrix maturation, and finally mineral deposition. Alizarin Red S staining for calcium deposition showed clear bone-like nodules, which support matrix maturation and mineralization. CONCLUSIONS: These studies provide new evidence for the role of OSAD in the mineralization process and its specific localization in the predentin layer accumulating at the mineralization front highlighting its role in tooth development.

Substitution of bovine dentine sialoprotein with chondroitin sulfate glycosaminoglycan chains.

Dentine sialoprotein (DSP) represents 5-8% of all non-collagenous proteins present in the tooth, but, together with dentine phosphoprotein, has been shown to be vital for correct tooth formation. Recently, the existence of a highly glycosylated form of porcine DSP has been reported and it was shown to possess glycosaminoglycan (GAG) chains. The current investigation confirms that this is also the case for bovine DSP and has further characterized these carbohydrates. Dentine sialoprotein was purified from bovine dentine extracts by anion exchange chromatography and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and mass spectroscopy. An increase in molecular mass was observed, from 120 kDa to greater than 250 kDa, with a corresponding rise in anionic strength. Cellulose acetate electrophoresis and western blotting indicated the presence of chondroitin sulfate GAG chains within these dentine fractions. Further examination using sequential digestion with chondroitinase AC and N-glycosidase cleaved the samples first to 95 kDa and then to 80 kDa, respectively, confirming a high level of glycosylation. These results support the classification of bovine DSP as a proteoglycan, and that the carbohydrate substitutions may contribute to the functional properties of DSP.

Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen.

Dentine phosphoprotein (DPP) has been proposed to both promote and inhibit mineral deposition during dentinogenesis. The present study aimed to investigate the molecular interactions of DPP and dephosphorylated DPP (DPP-p) with hydroxyapatite (HAP). Bovine DPP was purified and dephosphorylated by alkaline phosphatase to obtain DPP-p. DPP and DPP-p adsorption to HAP was determined along with their ability, when free in solution or bound to collagen, to influence HAP-induced crystal growth. Absorption isotherms suggested that lower DPP concentrations (1.5-6.25 microg ml(-1)) demonstrated a reduced affinity for HAP compared with higher protein concentrations (12.5-50.0 microg ml(-1)). Dephosphorylated DPP had a much reduced affinity for HAP compared with DPP. Dentine phosphoprotein inhibited seeded HAP crystal growth, in a dose-dependent manner, whilst removal of the phosphate groups reduced this inhibition. When bound to collagen fibrils, DPP significantly promoted the rate of HAP crystal growth over 0-8 min. Conversely, DPP-p and collagen significantly decreased the rate of crystal growth over 0-18 min. These results indicate a major role for the phosphate groups present on DPP in HAP crystal growth. In addition, concentration-dependent conformational changes to DPP, and the interaction with other matrix components, such as collagen, are important in predicting its dual role in the mineralization of dentine.

Modulation of collagen fibrillogenesis by dentinal proteoglycans.

Studies have identified different pools of proteoglycan (PG) species present within the unmineralized matrix of the predentine, the transitional phase at the predentine-dentine interface and the mineralized dentine. These PGs alter with respect to the chemical nature of their glycosaminoglycan (GAG) chains and as a result of extracellular processing of the macromolecule in the matrix. The present study has examined the influence of the PGs isolated from these phases and the influence of the attached GAG chains, upon their ability to control collagen fibrillogenesis. PGs isolated from the three phases were characterized and determined to contain a mixture of decorin and biglycan. Results have indicated that predentine PGs, which are substituted with a higher proportion of dermatan sulfate, significantly delayed fibril formation while ultimately promoting the formation of thicker fibrils. Removal of the GAG chains further delayed fibrillogenesis, leading to the formation of thinner fibrils, compared with the collagen-only control. PGs isolated from predentine-dentine, which contained a higher proportion of chondroitin sulfate, also significantly delayed fibrillogenesis, resulting in thicker collagen fibrils. GAG chains attached to the predentine-dentine interface PGs played a role in the timing of fibrillogenesis with fibril formation initiated at the same time as the collagen control, but yielding thicker fibrils. Dentine PGs significantly inhibited fibrillogenesis and fibril thickness over concentrations of 50-25 microg/mL protein. In conclusion, the PGs isolated from the distinct phases have indicated differing roles in the orchestrated organization of the extracellular matrix during dentinogenesis, with roles for both the core protein and attached GAG chains indicated.