• A Comparison of the Levels of Salivary Biomarkers between Conventional Smokers and Electronic Cigarette Users (A Pilot Study)

      Faridoun, Afnan; Meiller, Timothy F. (2019)
      Cigarette smoking is known to alter host response and the release of inflammatory mediators and cytokines in the body. Electronic cigarettes were marketed to provide the same sensation of conventional smoking without detrimental health consequences; however, the safety of their use is an area where more research is needed. Utilizing saliva as a diagnostic medium has been rising, however, there is a lack of studies investigating the effect of e-cigarettes on the salivary biomarker profile. The purpose of this pilot project was to evaluate the salivary cytokines, associated with inflammation, and CRP profile in e-cigarette users, and compare that to cigarette smokers and controls in an attempt to assess the influence of the use of e-cigarettes on salivary biomarkers. Statistically significant elevated levels of IL- 1β accompanied by lower mean levels of IL-1RA in e-cigarette users were detected. TNF- α showed elevated values in e-cigarette users similar to conventional smokers.
    • C-Reactive Protein as a Biomarker of Oral Health and Risk of Cardiovascular Disease in Healthy and CV Disease Subjects

      Alzalzalah, Ahmed; Meiller, Timothy F. (2017)
      Cardiovascular disease (CVD) is a leading global cause of death. Elevated CRP is considered a biomarker for the development of CVD. CRP is elevated in inflammatory periodontal diseases considered by a co-morbidity of CVD. This project contrasted CRP in young healthy, elderly healthy and elderly with CVD subjects relating these to gingival index (GI) and body-mass index (BMI) as parameters of inflammation. The data confirm a correlation between GI, BMI with CRP levels and in turn the risk of CVD. Selected young subjects had CRP levels similar to the elderly CVD subjects. This suggests that young healthy subjects with elevated GI, coupled with high BMI may be at a higher risk to develop CVD, as they age. Future studies should focus on these subjects longitudinally to assess the development of systemic disease, in particular CVD. Modalities to lower risk factors using CRP as a biomarker of efficacy may be developed.
    • Characterization of HSV-1 antigens expressed on human Langerhans cells

      Meiller, Timothy F.; Falkler, William A., Ph.D. (1992)
      Langerhans cells (LC) are immunocompetent antigen presenting cells (APC) which are involved with many infections including those caused by herpes simplex virus (HSV). Viral glycoproteins (g) on the surface of infected cells have been studied in established cell lines. The identification of which viral g participate with LC processing and antigen presentation on APC has not been studied. It is hypothesized that specific HSV-1 viral g species can be identified on the surface of LC very early after viral challenge of LC and are important antigens (Ag) involved in the human immune response to this virus. The purpose of this investigation has been to elucidate the expression of HSV-1 Ag, in vitro, using freshly isolated human LC. These experiments have established techniques for the isolation and enrichment of human LC that retain for up to 18 h post enrichment all of the ultrastructural characteristics and cell markers consistent with those of LC. These LC have been shown to adsorb and replicate HSV-1 virus at a low copy number. Immunofluorescence and ELISA have demonstrated the presence of viral antigens on the surface of membrane fractions of LC 8 h post infection using polyvalent antiserum. Specific viral epitopes related to gD and gE have been identified for the first time on HSV-1 challenged LC at 5 h post infection by two complementary techniques. SDS-PAGE of virally challenged LC membranes demonstrated the emergence of proteins consistent with the electrophoretic mobilities of gD, gE and possibly gH as early as 5 h post infection. Immunoblotting (IB) with monoclonal antibodies (Mab) confirmed the presence of these g in membrane preparations. Dual colloidal gold immunolabelling for transmission electronmicroscopy (TEM), utilizing Mab to LC markers and to epitopes found on viral g demonstrated visual confirmation of the presence of sites reactive with antibodies to gD and gE on the surface of LC 5 h after viral challenge. Although present later in infection larger proteins gB and gC do not appear to be involved in early membrane changes of LC. An epitope of gH was identified by IB, however, visual confirmation by TEM was not accomplished. The 5 h post infection LC, therefore, selectively express epitopes of gD, gE, and gH which may be involved in immunoactivation, particularly in light of the evidence supporting gD as a potent stimulator of neutralizing antibodies. Presentation of such viral antigens by LC would be an early step in this process.
    • Characterization of the response of GM-CSF supplemented THP-1 human monocytes to LPS of oral microorganisms

      Baqui, A. A. M. Abdullahel; Falkler, William A., Ph.D.; Meiller, Timothy F. (1996)
      The effect of granulocyte macrophage colony stimulating factor (GM-CSF) on the differentiation and activation of a human monocyte cell line (THP-1) in the presence of lipopolysaccharide (LPS) from oral organisms has not been investigated. It was hypothesized that GM-CSF treated THP-1 cells are immunologically and functionally hyperactivated in the presence of LPS of oral microorganisms. A study was undertaken to elucidate the immunological expression of activation antigens and production of cytokines by THP-1 cells after treatment with GM-CSF and in response to LPS of the putative periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). LPS of F. nucleatum and P. gingivalis was prepared, characterized by SDS PAGE, standardized by protein concentration and tested for endotoxin content. Morphological changes in THP-1 cells were observed with light, immunofluorescence (IF) and transmission electron microscopy (TEM), following treatment with LPS/PMA (phorbol-12-myristate-13 acetate) and/or GM-CSF at various concentrations and time intervals. The expression of seven different activation antigens namely, CD-11b, CD-11c, CD-14, CD-35, CD-68, CD-71 and HLA-DR, in THP-1 cells was evaluated in this experimental model. Direct labeling of THP-1 cell activation antigens was performed using the Vectastain ABC-AP staining kit with light microscopy. Alternatively, LPS/PMA and/or GM-CSF stimulated and unstimulated cells were stained with FITC labelled antibodies for IF and gold-labelled antibodies were used for TEM. To evaluate chemotaxis as a functional variation in THP-1 cells, an assay was performed using a microchemotaxis chamber and polyvinylpyrolidone free polycarbonate membrane filters. To determine phagocytic activity in the experimental model, an assay was performed using FITC labelled S. cereviseae. Phagocytic uptake of cells was determined with the use of a fluorescence microscope. Production of extracellular cytokines, TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12, by THP-1 cells in the experimental model, was measured by ELISA. Reverse transcription polymerase chain reaction (RT-PCR) of the of TNF-alpha, IL-1 beta and IL-6 cytokines was performed to correlate the cytokine gene transcription with cytokine gene translation (ELISA). Three percent of untreated THP-1 cells expressed HLA-DR; 9%, CD-11b; 8%, CD-11c; 22%, CD-14; 9%, CD-35 and 7%, CD-68 antigens. CD-71 was not expressed in untreated THP-1 cells. Treatment with LPS of F. nucleatum and P. gingivalis and PMA increased the expression of activation antigens. Following treatment with combined GM-CSF and LPS of P. gingivalis and F. nucleatum there was a significant (p<0.05) up-regulation and expression of HLA-DR, CD-11b, CD-11c, CD-35 and CD-71 activation antigens over baseline values. Expression of the LPS receptor, CD-14, was significantly (p<0.05) down-regulated by this treatment for 1-2 d and then up-regulated at 2-4 d. Antigens important in phagocytosis, CD-11b and CD-35, were significantly (p<0.05) up-regulated by GM-CSF. The up-regulation was further demonstrated in phagocytosis functional assays. Stimulation with combined GM-CSF and oral LPS resulted in a significant (p<0.05) escalation in phagocytosis by the THP-1 cells. There was a two-fold increase in chemotactic response with GM-CSF treatment by 4 d, which decreased after 7 d. RT-PCR data indicated that TNF-alpha transcripts were constituitively produced in the THP-1 cell but that translation to a high level of production of functional cytokines required the LPS/GM-CSF stimulus. Gene transcription for IL-6 was detected as early as 5 min post stimulation. (Abstract shortened by UMI.)
    • The Differences in Panoramic Bone Density Between Bisphosphonates (BPs) Patients With, Without, or at Risk for Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ)

      Al-Rasheed, Laila Mahmoud; Meiller, Timothy F. (2014)
      Background: Since late 2003, there have been numerous reports in the literature illustrating the association between bisphosphonate use and the appearance of avascular necrosis of the jaws. Bisphosphonate-related osteonecrosis of the jaw (BRONJ) represents a growing concern for oral- and maxillofacial practice. Although many studies have been conducted in recent years evaluating the early manifestations of BRONJ, a clearer understanding of the significance of early radiographic alterations could improve the identification of patients at increased risk of this disease and may help to guide interventions. The purpose of this study is to compare the bone density of the posterior mandible in the panoramic radiographs of patients with BRONJ, patients who received bisphosphonate therapy and did not develop BRONJ, patients who received bisphosphonate therapy and eventually developed BRONJ, and controls. Materials and Methods: A convenience sample, consisted of 54 digital panoramic radiographs was divided in to four groups. Group 1 represented controls i.e. patients that have never been exposed to bisphosphonates of any kind and Group 2 represented the "at risk patients" i.e. received bisphosphonates but never developed BRONJ. Group 3A consists of early panoramic radiographs of patients who are on bisphosphonate therapy but at that time did not develop BRONJ. Group 3B is the BRONJ group, including those of 3A that went on to develop BRONJ and subjects that presented with BRONJ. The radiographs were evaluated using Planmeca Romexis software fully supported for radiographs were evaluated using Planmeca Romexis software fully supported for Windows operating system. The minimum and maximum density, averages and SD of the bone at the angle of the mandible on the right and the left sides were compared using ttest. Results: There was no significant difference in bone density of the digital panoramic radiographs between patients at risk of BRONJ, having BRONJ, and normal patients. Although the data is not significant, the findings indicate a clear trend whereas the minimum and maximum densities are highest in individuals taking BPs and these values decline as BRONJ develops, as compared with the controls. Conclusion: More studies are required to definitively determine the diagnostic and prognostic value of simple radiographic imaging as relates to the problem of BRONJ.
    • Immunohistochemistry Analysis of Ki-67 and P53 Proteins in Oral Lichen Planus Compared to Other Oral Epithelial Lesions

      Akbar, Aqdar Abdullah; Meiller, Timothy F. (2014)
      Aim: The goal of this project was to determine if selected biomarkers of oral cancer are expressed in lichen planus and to identify the relationship, if any, of these biomarkers to malignant transformation. Methods: Six each of archived specimens with confirmed histopathological diagnosis of oral benign fibroma, oral reticular lichen planus, oral erosive lichen planus, oral mild to moderate dysplasia, oral severe dysplasia, and oral squamous cell carcinoma were selected. A total of 35 acceptable specimens were stained using an immunohistochemistry method for Ki67 and P53 proteins. Specimens were scanned with Aperio Scanscope CS and slides were analyzed visually, for expression patterns, and also using Aperio software. Results: The results of the present study demonstrated that the expression of P53 in oral lichen planus was 40.3 %, compared to 31.6% in oral benign fibroma, 46.3% in oral erosive lichen planus, 43.9% in oral mild to moderate dysplasia, 46.4% in oral severe dysplasia, and 47.2% in oral squamous cell carcinoma. On the other hand, Ki67 expression for oral lichen planus was 32.3% compared to 19.1% in oral benign fibroma, 40.3% in oral erosive lichen planus, 36% in oral mild to moderate dysplasia, 44.3% in oral severe dysplasia, and 48.9% in oral squamous cell carcinoma. Conclusion: Expression of the P53 and Ki67 biomarkers do not consistently correlate with malignant transformation of oral lichen planus appear to be associated with premalignant lesions of dysplasia. P53 and Ki67 are not a definitive diagnostic tool and further investigation to other biomarkers is needed.
    • Panoramic Bone Density in the Posterior Mandible in Patients with, without, or at risk of developing Medication-Related Osteonecrosis of the Jaw (MRONJ)

      ALFARHAN, ISRA; Meiller, Timothy F. (2017)
      Early radiographic changes of MRONJ could help in identifying patients at increased risk of developing the disease. This study compared and contrasted bone density in the posterior mandible of patients with, without, or at risk of developing MRONJ. This was a retrospective study of 46 patients (18 with MRONJ, 20 at risk but without MRONJ, and 8 controls with no exposure to MRONJ associated drugs). ImageJ software was used for bone density evaluation of the radiographs. Bone density was significantly higher in MRONJ patients when compared to at risk patients and controls. In a sub-cohort of patients with MRONJ where we had pre- and post-MRONJ images, the density was significantly higher in the pre-MRONJ radiographs when compared to after MRONJ radiographs and also higher than the at risk radiographs (p-value = 0.03). ImageJ analysis of the panoramic radiographs was successful in detecting significant differences in bone density in our sample.
    • The Role of miRNAs in Early Detection of Oral Cancer

      Almubarak, Hussain Mohammed; Meiller, Timothy F.; Mao, Li, M.D. (2012)
      MicroRNAs, commonly noted as miRNAs (miRs), are evolutionary conserved small non-coding RNAs that negatively regulate gene expression post-transcriptionally. Recently, miRNAs have been identified as potentially important biomarkers in various cancers including oral squamous cell carcinoma. The hypothesis of this study was that the expression of miRNAs is different in squamous cell carcinoma in African Americans as compared to Caucasians; as well as when compared to Age, Sex, Location, Recurrence, Grade of cancer, Stage of cancer, Neural invasion, Muscular invasion, and HPV 16/18 or HPV 31/33/51 status. Fifty cases of oral squamous cell carcinoma were matched to the Maryland Cancer Registry (MCR) database and were selected for one Caucasian and one African American in matched sets for age, sex and location. To detect potentially significant miRNAs, two different epithelial cell lines were selected to represent the normal epithelium (HaCaT and NOK) and compared to the dysplastic epithelium (Leuk-1) and the oral cancer (SCC-9 and SCC-25). The miRNAs profiling was performed for the cell lines and several dysregulated miRNAs were identified. The microarray data were validated by the real-time PCR. The expression of miR-708, miR-193a-5p, miR-155, miR-584, miR-663, and miR-886-5p suggested their significant potential at the early stages of tumorigenesis. However, miR-30d, miR-34a, miR-34c-3p, miR-138, and miR-486-5p were dysregulated only in cancer cell lines reflecting their potential at the late stages. The prominently dysregulated miR-708 and miR-193a-5p were selected for further analysis. The lentiviral transduction targeting miR-708 and miR-193a-5p was used to reverse their originally detected expression levels. Immunoblotting was performed on Survivin, a miR-708's direct target in Renal Cell Carcinoma, and on previously studied biomarkers including Hexokinase II, Beclin 1, and LC3. The expression patterns of the detected miRNAs were matched with microarrays results of the 50 paraffin-embedded tissue samples. Significant difference was found based on race (miR-34c-3p), sex (miR-193a-5p), location (miR-193a-5p & miR-34a), grade (miR-486-5p), and HPV status (miR-584). The survival analysis showed a significant difference based on age, location, and grade. The bioinformatics analysis supported our findings and showed consistent results with other miRNAs studies in the literature. The high expressions of miR-584 and miR-486-5p deserve further investigation.
    • The Role of pH in Patients Receiving Long Term Bisphosphonate Therapy

      Alhouli, Munawer; Meiller, Timothy F. (2013)
      Purpose: Bisphosphonate (BP)-associated osteonecrosis of the jaw (BON) lacks a defined pathophysiologic mechanism and treatment regimen. This current study was designed to be part of a parent BON pathogenesis series of protocols to investigate the contributing factors of causation. The following hypothesis was tested: Subjects receiving long term bisphosphonate therapy that have developed BON have a more acidic oral environment i.e. as measured by salivary pH than normal (no BP exposure) subjects and those receiving long term bisphosphonate therapy that have not developed BON. This study assessed the pH of saliva in subjects receiving long term bisphosphonate therapy so that possible oral buffers could eventually be developed and investigated as possible treatments i.e. to reduce the acidic effects BPs such as zoledronic acid (ZA) have in BON wound healing. Methods: Using the fully IRB approved HIPAA partial waiver for recruitment, subjects were selected based on history and physical examination in order to document previous and/or current use of bisphosphonates. Standard of care physical examination was used to determine the presence or absence of BON lesions intraorally. As part of a fully IRB approved protocol the informed consent process was used and for those willing to participate and having signed the consent document, we collected the single sample of saliva. We employed these ex-vivo studies using pH measurement on ~5 ml of fresh unstimulated whole saliva collected over a 5 minute time period at this single visit. Results: Ex-vivo, we have shown in this study that patients with active lesions of BON have more acidic saliva as compared to those without BON or in those not taking a BP. Conclusion: These findings suggest that acidic salivary pH is directly associated with BON, may play a role in the initiation and prolongation of oral BON and may eventually support the potential role of using salivary buffers to offset acidic saliva as an adjunct therapeutic treatment for BON, specifically to speed wound healing.
    • Role of Survivin and Hexokinase II in the regulation of Autophagy and Apoptosis in Oral Squamous Cell Carcinoma

      Desai, Prapti; Scheper, Mark; Meiller, Timothy F. (2012)
      Survivin, an inhibitor of apoptosis and hexokinase II, a key enzyme in glycolysis are under-expressed in normal oral tissue; while they are over-expressed in oral squamous cell carcinoma (OSCC). Previous studies in our lab have shown a direct correlation between survivin and hexokinase II, using bromo-pyruvic acid, a known hexokinase II inhibitor. Additionally, it has also been shown that there is an increase in the expression of these two biomarkers with the increase in severity of premalignant lesions. These data were obtained from our patient database at Oral Pathology Consultants (OPC) which was compared to the Maryland Cancer Registry (MCR) for a cancer match. Based on this previous work, we decided to study two of the cancer hallmarks in OSCCs; dysregulation of apoptosis and altered cellular metabolism, and molecules associated with them; survivin and hexokinase II. Interestingly, survivin was found to be over-expressed in 80% of OSCCs. Hence, our hypothesis was that in cells during survivin over-expression, treatments with DNA damaging agents will lead to induction of autophagy in OSCCs. To prove this hypothesis, two well known DNA damaging drugs, cisplatin and paclitaxel were used. The basic aim of the study was to down-regulate survivin and study the effect of this down-regulation on autophagy proteins, beclin-1 and mammalian light chain protein kinase, LC3 (autophagy marker). Using a premalignant cell line (Leuk-1) and malignant cell lines (SCC 9 and SCC 25), we have shown down-regulation of survivin using cisplatin and paclitaxel. Survivin is known to inhibit caspases, hence its down-regulate is likely to activate the process of apoptosis. Our western blot results represent down-regulation of beclin-1 as well as the active form of LC3, LC3-II. However, beclin-1 forms a complex with anti-apoptotic proteins, Bcl-2/xL and its down-regulation with chemotherapeutic drugs disturbs the complex, increasing the loss of mitochondrial membrane integrity and ultimately apoptosis. Also, LC3 is a high turn-over protein which can be incorrectly reflected on a western blot. However, further validation of the processes activated due to beclin-1 and LC3 down-regulation will raises a strong possibility of cross-talk between autophagy and apoptosis. Further, literature studies have shown that activation of one signaling pathway regulates key players at transcription and translational levels, which in turn activate or down-regulate other signaling pathways. Our findings support the novel role of the survivin/hexokinase II axis in the regulation of autophagy and apoptosis, in both dysplastic oral lesions and in OSCC.  
    • A soft-tissue in vitro model of bisphosphonate induced localized non-traditional calciphylaxis

      Alfouzan, Weam; Meiller, Timothy F. (2013)
      Background: Bisphosphonates (BPs) such as zoledronic acid (ZA) are widely used to treat complications of bony metastases in cancer patients. One of the serious adverse effects of ZA treatment is Bisphosphonates osteonecrosis (BON). The pathophysiologic mechanisms of BON are not fully elucidated. Scheper et al (2012) suggest that BON is correlated with a localized non-traditional calciphylaxis. In this project, we studied the effect of ZA on soft tissue by calcium deposition. Also, we hypothesized that albumin will reverse that effect. Materials and Methods: Normal Oral Keratinocytes (NOK) and Human Gingival Fibroblats (HGF) were treated by different ZA concentration and Bovine Serum Albumin (BSA). Immunoflurosence and Western Blot were used to study the treatments effects. Results: There is an increase in calcium concentration when cells were treated with ZA. However, there is a decrease when cells were treated with albumin. Conclusion: Albumin can bind to the calcium released following ZA treatment. Our finding might suggest a method of BON prevention. Further studies are needed to determine if such effects are clinically relevant for in vivo models of BON.
    • The Diagnostic Utility of Cancer Stem Cell Marker CD44 in Early Detection of Oral Cancer

      Alsalem, Bader; Meiller, Timothy F. (2019)
      The incidence of Oral Squamous Cell Carcinoma (OSCC) is expected to increase in the coming decades. In recent years, cancer biomarkers have emerged as a promising diagnostic and prognostic tool for various cancers. The cluster differentiation antigen (CD44) is among the most frequently identified cancer stem cell markers in solid tumors. Using immunohistochemistry labeling in previously diagnosed specimens, we aimed to analyze the utility of CD44 in early diagnosis of OSCC. Biopsy specimen subgroups of lateroventral tongue revealed an increased proportion of cells staining positive for CD44 in the epithelial samples of OSCC compared with erosive lichen planus and oral dysplastic lesions. CD44, in combination with other oral biomarkers, therefore, has the potential to assist in the early diagnosis of OSCC. Future studies with larger sample sizes and multiple biomarkers should be carried out, utilizing a pre-determined IHC staining and interpretation strategy to promote reproducibility of evidence.