Mark A. Scheper

High mobility group A2 is a target for miRNA-98 in head and neck squamous cell carcinoma

BackgroundHMGA2 expression has been shown to be associated with enhanced selective chemosensitivity towards the topoisomerase (topo) II inhibitor, doxorubicin, in cancer cells. Although the roles of signaling cascades and proteins as regulatory factors in development, neoplasia and adaptation to the environment are becoming well established, evidence for the involvement of regulatory small RNA molecules, such as microRNAs (miRNAs) as important regulators of both transcriptional and posttranscriptional gene silencing is presently mounting.ResultsHere we report that HMGA2 expression in head and neck squamous cell carcinoma (HNSCC) cells is regulated in part by miRNA-98 (miR-98). Albeit HMGA2 is associated with enhanced selective chemosensitivity towards topoisomerase (topo) II inhibitor, doxorubicin in HNSCC, the expression of HMGA2 is thwarted by hypoxia. This is accompanied by enhanced expression of miRNA-98 and other miRNAs, which predictably target HMGA2. Moreover, we show that transfection of pre-miR-98™ during normoxia diminishes HMGA2 and potentiates resistance to doxorubicin and cisplatin. These findings implicate the role of a miRNA as a key element in modulating tumors in variable microenvironments.ConclusionThese studies validate the observation that HMGA2 plays a prominent role in governing genotoxic responses. However, this may only represent cells growing under normal oxygen tensions. The demonstration that miRNA profiles are altered during hypoxia and repress a genotoxic response indicates that changes in microenvironment in eukaryotes mimic those of lower species and plants, where, for example, abiotic stresses regulate the expression of thousands of genes in plants at both transcriptional and posttranscriptional levels through a number of miRNAs and other small regulatory RNAs.

Farnesol-Induced Apoptosis in Candida albicans

ABSTRACT Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.

Microbial interactions and differential protein expression in Staphylococcus aureus–Candida albicans dual-species biofilms

The fungal species Candida albicans and the bacterial species Staphylococcus aureus are responsible for a majority of hospital-acquired infections and often coinfect critically ill patients as complicating polymicrobial biofilms. To investigate biofilm structure during polymicrobial growth, dual-species biofilms were imaged with confocal scanning laser microscopy. Analyses revealed a unique biofilm architecture where S. aureus commonly associated with the hyphal elements of C. albicans. This physical interaction may provide staphylococci with an invasion strategy because candidal hyphae can penetrate through epithelial layers. To further understand the molecular mechanisms possibly responsible for previously demonstrated amplified virulence during coinfection, protein expression studies were undertaken. Differential in-gel electrophoresis identified a total of 27 proteins to be significantly differentially produced by these organisms during coculture biofilm growth. Among the upregulated staphylococcal proteins was l-lactate dehydrogenase 1, which confers resistance to host-derived oxidative stressors. Among the downregulated proteins was the global transcriptional repressor of virulence factors, CodY. These findings demonstrate that the hyphae-mediated enhanced pathogenesis of S. aureus may not only be due to physical interactions but can also be attributed to the differential regulation of specific virulence factors induced during polymicrobial growth. Further characterization of the intricate interaction between these pathogens at the molecular level is warranted, as it may aid in the design of novel therapeutic strategies aimed at combating fungal–bacterial polymicrobial infection.

A Novel Immune Evasion Strategy of Candida albicans : Proteolytic Cleavage of a Salivary Antimicrobial Peptide

Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps), involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap) family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the first defined mechanism behind the enhanced susceptibility of HIV+ individuals to oral candidiasis since the emergence of HIV.

Oral health status of 207 head and neck cancer patients before, during and after radiotherapy

The aim of this retrospective study was to describe the oral health status of patients before, during, and after radiotherapy (RT) for the treatment of head and neck cancer (HNC). Before RT, the following data was collected: presence of unrecoverable teeth, residual roots, unerupted teeth, use of dentures, periodontal alterations, caries, candidiasis, and xerostomia. Mucositis, candidiasis, and xerostomia were evaluated during RT. Patients continued to be followed after RT for evaluation of mucositis, candidiasis, xerostomia, radiation caries, and osteoradionecrosis. For statistical analysis, 95% confidence intervals (CI) were determined using sample size, population, and percentages. Before RT, 120 (57.9%) patients presented with alterations in the oral cavity namely, 85 (41.0%) with periodontal disease, 44 (21.2%) with residual roots, 25 (12.0%) with caries, 15 (7.2%) with candidiasis, and 12 (5.8%) had an unerupted tooth present. Xerostomia was a complaint of 19 patients (9.1%). Restorations were indicated for 33 patients (15.9%), whereas extraction was indicated for 104 (50.2%) patients. During RT, mucositis was found in 80 (61.7%) patients, candidiasis in 60 (45.8%), and xerostomia was a complaint of 82 patients (62.6%). After RT, mucositis persisted in 21 patients (19.2%), candidiasis was identified in 23 patients (21.1%), and xerostomia was reported by 58 patients (53.2%). Radiation caries developed in 12 patients (11.0%), whereas six patients (5.5%) developed osteoradionecrosis. The demographic profile herein presented will be useful as baseline data to provide additional epidemiological information and to determine future measures for prevention and treatment of RT-induced complications and sequelae.

Granulomatous Foreign-Body Reaction Involving Oral and Perioral Tissues After Injection of Biomaterials: A Series of 7 Cases and Review of the Literature

PURPOSE Injectable implants used for soft-tissue augmentation may lead to a granulomatous foreign-body reaction. The aim of this report is to present 7 new cases of foreign-body granulomas involving the oral and perioral tissues, after injection of biomaterials to achieve soft-tissue augmentation. In addition, the clinical and epidemiological profile of this condition is summarized, based on a review of the English-language literature of all previously described cases. PATIENTS AND METHODS We report on 7 new cases of granulomatous foreign-body reaction involving the oral and perioral tissues after the injection of biomaterials. A comprehensive literature review is also presented. RESULTS The literature search revealed 49 cases of this condition affecting the oral and perioral tissues. Our 7 patients were female, with a mean age of 52.8 years (range, 34 to 70 years). The lower lip was affected in 4 cases, 1 case was located in the upper lip, 1 case in the buccal mucosa, while 1 case involved 2 different sites (upper lip and buccal mucosa). Histopathologic examination revealed numerous cells with clear, often multiple, cytoplasmic vacuoles, bearing a resemblance to lipoblasts. Immunohistochemistry revealed diffuse positivity for the histiocytic marker CD68. CONCLUSIONS The diagnosis of granulomatous foreign-body reactions may be challenging because of their microscopic resemblance to liposarcoma, and because of the occasional reluctance of patients to report the previously performed esthetic procedure. A clinical history, histopathologic examination, and immunohistochemical analysis (as needed) are essential in achieving an accurate diagnosis.

Protection of the oral mucosa by salivary histatin‐5 against Candida albicans in an ex vivo murine model of oral infection

The oral cavity is a primary target for opportunistic infections, particularly oral candidiasis caused by Candida albicans. A commensal fungus commonly colonizing mucosal surfaces, under conditions of immune dysfunction, C. albicans can become a pathogen causing recurrent infections. Yet, the role of host oral innate immunity in the development of candidiasis is not fully elucidated. Specifically, the host salivary antimicrobial peptide histatin-5 (Hst-5) has been proposed to play a protective role in the oral cavity against C. albicans. However, investigations demonstrating its efficacy on oral tissue have been lacking. To this end, in this study, an ex vivo murine model of oral infection was developed. Viable C. albicans counts and histopathological analyses demonstrated a significant protective effect for Hst-5 on mouse oral tissue against C. albicans. More importantly, host saliva exerted a comparable anticandidal effect. However, this effect was neutralized upon treatment of saliva with proteases and C. albicans, previously shown to degrade Hst-5, indicating that Hst-5 is likely the salivary component responsible for the observed protection. Combined, the findings from this study demonstrate for the first time the efficacy of salivary Hst-5 in protecting host oral tissue against C. albicans infection, thereby affirming the therapeutic potential of this natural host peptide.

Histopathologic findings in bone from edentulous alveolar ridges: a role in osteonecrosis of the jaws?

Bisphosphonate-related osteonecrosis of the jaws (BONJ) is characterized by a breach in the oral mucosa with exposure of necrotic bone. Although bisphosphonates impact multiple biologic processes, including bone turnover and vascularity, factors contributing to the pathogenesis of BONJ remain poorly understood. In this retrospective analysis, the histopathologic findings from 154 alveolar bone specimens obtained during osteotomy preparation for dental implant placement were reviewed from 147 consecutively treated patients [male (79); female (68); Caucasian (141); African-American (6)]. The alveolar ridge sites had been edentulous for 1 year or longer. None of the patients in this study had a history of bisphosphonate therapy or clinical evidence of BONJ. Two pathologists, masked, using predetermined criteria, reviewed and substantiated the pathology reports provided by the licensed pathology service. In selected cases, special stains had been conducted to help establish the presence of bacteria. The histopathologic findings for the core specimens were as follows: 76 viable bone (49.4%); 54 nonviable bone (35.0%); and 24 osteomyelitis (15.6%). These histopathologic findings indicate that the edentulous jaw can contain regions of nonviable bone and microbial biofilm formation for 1 year or more after tooth extraction and mucosal healing. Regions of necrotic bone and subclinical infection may contribute to the development of untoward clinical events, such BONJ and early implant failure.

Expression and alterations of the PTEN / AKT / mTOR pathway in ameloblastomas

OBJECTIVES Recently, an allelic loss of phosphatase and tensin homologue (PTEN) was shown to occur in ameloblastomas. In carcinogenesis, loss of PTEN allows for overactivity of the phosphatidylinositol-3-kinase/protein kinase B (PI3K / AKT) pathway inducing an upregulation of mammalian-target of rapamycin (mTOR) and its downstream effector ribosomal-subunit-6 kinase (S6K); allowing for uncontrolled cell proliferation, apoptosis inhibition and cell cycle deregulation. METHODS Thirty ameloblastomas and five dental follicles were studied, looking at the immunohistochemical expression of total PTEN and AKT, as well as their phosphorylated (p) active forms, and the downstream effector and indicator of mTOR activity p70 ribosomal-subunit-6 kinase (pS6K). Also assessed was the expression of extracellular-signal-regulated kinase (ERK), which cross talks with AKT. RESULTS Total PTEN was absent in 33.3% of ameloblastomas, while its stabilized, phosphorylated(ser380 / thr382 / thr383) form was absent in 83.3% of tumors. In contrast, AKT was expressed in 83.3% of ameloblastomas, showing high expression of the p-thr(308)AKT and p-ser(473) AKT forms in 93.3% and 56.6% of cases, respectively. Further, the mTOR activated pS6K(ser240 / 244) was detected in 86.7% of ameloblastomas, while ERK was overexpressed in 70.0% of the cases. CONCLUSION Immunohistochemical analysis of aberrant signaling in the PI3K/AKT/mTOR pathway in ameloblastomas may represent a valuable tool for elucidating pathogenesis, aggressiveness and selecting optimal therapeutics.

Are we on the brink of nonsurgical treatment for ameloblastoma

OBJECTIVE Recent identification of altered molecular signaling pathways in neoplasia has begun to elucidate mechanisms of oncogenesis, differentiation, and tumor progression, and to suggest plausible nonsurgical considerations for treatment. Here we review the sonic hedgehog (SHH) and PI3K/Akt/mTOR signaling pathways, their role in ameloblastoma, a locally aggressive odontogenic tumor, and evidence for consideration of therapeutic approaches that target these molecular pathways. In so doing, some of the gaps will be revealed that may impel investigations and translate to patient care, helping to minimize or eliminate the need for extensive surgery. STUDY DESIGN This is a comprehensive review of the literature regarding alterations in signaling mechanisms associated with ameloblastomas. In addition, this review attempts to explore and discuss possible inhibitors to these pathways that may have utility in treating ameloblastoma. RESULTS The expression of SHH signaling molecules in ameloblastomas at the mRNA and protein levels has intimated that these molecules may play a role in cell proliferation of these tumors. Immunohistochemical analysis has revealed aberrant signaling in the PI3K/Akt/mTOR pathway in ameloblastomas and appears to be a valuable tool for elucidating pathogenesis and aggressiveness, and selecting optimal therapeutics. CONCLUSION The understanding of altered pathways in ameloblastoma may soon provide nonsurgical options for the treatment of this condition. The demonstration of cross talk in SHH signal transduction with PI3K signaling through Akt has shown that these pathways converge to control the Gli transcription factors. Thus, tumors that entirely depend on active SHH signaling for survival/growth and maintenance may well be susceptible targets for combined chemotherapy with SHH-specific inhibitors together with PI3K, Akt, or mTOR blocking agents. Some of these inhibitors could be used locally, thereby minimizing major systemic effects.