Faculty Bibliography

Picoinjection is a promising technique to add reagents into pre-formed emulsion droplets on chip however, it is sensitive to pressure fluctuation, making stable operation of the picoinjector challenging. We present a chip architecture using a simple pressure stabilizer for consistent and highly reproducible picoinjection in multi-step biochemical assays with droplets. Incorporation of the stabilizer immediately upstream of a picoinjector or a combination of injectors greatly reduces pressure fluctuations enabling reproducible and effective picoinjection in systems where the pressure varies actively during operation. We demonstrate the effectiveness of the pressure stabilizer for an integrated platform for on-demand encapsulation of bacterial cells followed by picoinjection of reagents for lysing the encapsulated cells. The pressure stabilizer was also used for picoinjection of multiple displacement amplification (MDA) reagents to achieve genomic DNA amplification of lysed bacterial cells.

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.International Journal of Oral Science advance online publication, 8 August 2014; doi:10.1038/ijos.2014.46.

Limited information is available about the effect of human immunodeficiency virus (HIV) and subsequent antiretroviral treatment on host-microbe interaction. This study aimed to determine the salivary microbial composition in 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that of 10 HIV-seronegative subjects. Both a conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV+ subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, S. mutans, and Candida, in saliva as compared to HIV- subjects. The total cultivable microbial level was significantly correlated with CD4+ T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. Human oral microbe identification microarray (HOMIM), which compared the 16S rRNA genes, showed a clear separation among the three sample groups. Veillonella, Synergistetes, and Streptococcus, were present in all 30 saliva samples. Only minor changes or no changes were observed in the prevalence of Neisseria, Haemophilus, Gemella, Leptotrichia, Solobacterium, Parvimonas and RothiaI. Severn genera were detected only in HIV- samples, including Capnocytophaga, Slackia, Porphyromonas, Kingella, Peptostreptococcaceae, Lactobacillus, and Atopobium. The prevalence of Fusobacterium, Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, and Atopobium was increased after therapy with HAART. In contrast, the prevalence of Aggregatibacter was significantly decreased after HAART. Findings of this study suggest that HIV infection and therapy with HAART could have a significant effect on salivary microbial colonization and composition.

Pancreatic cancer is one of the most lethal cancers worldwide. No effective screening methods exist, and available treatment modalities do not effectively treat the disease. Inflammatory conditions such as pancreatitis represent a well-known risk factor for pancreatic cancer development. Yet only in the past 2 decades has pancreatic cancer been recognized as an inflammation-driven cancer, and the precise mechanisms underlying the pathogenic role of inflammation are beginning to be explored in detail. A substantial amount of preclinical and clinical evidence suggests that bacteria are likely to influence this process by activating immune receptors and perpetuating cancer-associated inflammation. The recent explosion of investigations of the human microbiome have highlighted how perturbations of commensal bacterial populations can promote inflammation and promote disease processes, including carcinogenesis. The elucidation of the interplay between inflammation and microbiome in the context of pancreatic carcinogenesis will provide novel targets for intervention to prevent and treat pancreatic cancer more efficiently. Further studies toward this direction are urgently needed.

We have developed a calcium phosphate glass (CPG) doped with Zn2+ or F- or combined Zn2+ and F- ions, which are naturally found in the human body and play a dual role in bone formation and antibacterial activity. Previously, we have demonstrated that this family of CPGs has superior osteoconductive and resorbable properties in vivo. This study aimed to investigate the antibacterial property of CPGs incorporating Zn2+ and/or F- . We used Streptococcus mutans as a model organism because it is one of the major human oral pathogens and an early colonizer, and it has been associated with several oral infections, such as dental caries, periodontitis, and peri-implantitis. CPGs of 0.01 and 0.05 g were incubated with S. mutans for 0, 2, 4, and 6 h. Serial dilutions were plated in triplicate and colony forming units were determined. The antimicrobial effect of CPG incorporating Zn2+ or F- was greater than CPG incorporating both these ions. CPG without doping produced a moderate antimicrobial effect. This family of CPGs, previously shown to promote new bone formation in vivo, is demonstrated to have superior bactericidal properties. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

PURPOSE: Osteonecrosis of the jaw (ONJ) has been reported to be associated with patients receiving bisphosphonate (BP) therapy. There are many reports that suggest that the time of exposure to BPs is a significant risk factor for ONJ and that the greatest risk occurs after dentoalveolar surgery. The aim of this study was to retrospectively investigate the duration of BP therapy and related events before the onset of ONJ based on an intravenous (IV) or oral route of administration. MATERIALS AND METHODS: We conducted a retrospective cohort study of patients referred to our institution to identify the onset of ONJ based on the exposure to BP therapy and associated triggers (ie, dentoalveolar surgery or spontaneous occurrence) based on the route of BP administration. Demographic data (ie, age, gender, and race), medical diagnosis related to BP therapy, and information as to whether the BP therapy was continued at the time of ONJ diagnosis were also collected. RESULTS: We reviewed the records for 114 patients with a history of ONJ. We divided patient cohorts by route of BP administration, with 76 patients having a history of IV BP therapy and 38 patients having a history of oral BP therapy. The overall onset of ONJ was earlier in the IV BP group (median, 3 years) compared with the oral BP group (median, 5 years). There was no statistical difference in the duration to occurrence of ONJ associated with dental extraction compared with spontaneous occurrence for both the IV and oral BP groups. CONCLUSIONS: The median onset of ONJ for patients undergoing IV BP therapy occurs earlier than the median onset for patients undergoing oral BP therapy, and there was no difference in onset occurring spontaneously and after dental extraction. The significance of these findings suggests that patients who receive IV BP therapy should be closely evaluated after the initiation of BP therapy. The lack of evidence suggesting greater onset after dental extraction may provide clinical support for dentoalveolar surgery that is indicated for patients with a history of BP therapy. Research focusing on the clinical circumstances and physiologic events during early antiresorptive therapy may provide insight as to the critical risk factors.

Oral Diseases (2012) Objective: Infection has been hypothesized as a contributing factor to bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ). The objective of this study was to determine the bacterial colonization of jawbone and identify the bacterial phylotypes associated with BRONJ. Materials and methods: Culture-independent 16S rRNA gene-based molecular techniques were used to determine and compare the total bacterial diversity in bone samples collected from 12 patients with cancer (six, BRONJ with history of BP; six, controls without BRONJ, no history of BP but have infection). Results: Denaturing gradient gel electrophoresis profile and Dice coefficient displayed a statistically significant clustering of profiles, indicating different bacterial population in BRONJ subjects and control. The top three genera ranked among the BRONJ group were Streptococcus (29%), Eubacterium (9%), and Pseudoramibacter (8%), while in the control group were Parvimonas (17%), Streptococcus (15%), and Fusobacterium (15%). H&E sections of BRONJ bone revealed layers of bacteria along the surfaces and often are packed into the scalloped edges of the bone. Conclusion: This study using limited sample size indicated that the jawbone associated with BRONJ was heavily colonized by specific oral bacteria and there were apparent differences between the microbiota of BRONJ and controls.

We report a clinical study that examines whether HIV infection affects Streptococcus mutans colonization in the oral cavity. Whole stimulated saliva samples were collected from 46 HIV-seropositive individuals and 69 HIV-seronegative control individuals. The level of S. mutans colonization was determined by conventional culture methods. The genotype of S. mutans was compared between 10 HIV-positive individuals before and after highly active antiretroviral therapy (HAART) and 10 non-HIV-infected control individuals. The results were analyzed against viral load, CD4+ and CD8+ T-cell counts, salivary flow rate, and caries status. We observed that S. mutans levels were higher in HIV-infected individuals than in the non-HIV-infected control individuals (p = 0.013). No significant differences in S. mutans genotypes were found between the two groups over the six-month study period, even after HAART. There was a bivariate linear relationship between S. mutans levels and CD8+ counts (r = 0.412; p = 0.007), but not between S. mutans levels and either CD4+ counts or viral load. Furthermore, compared with non-HIV-infected control individuals, HIV-infected individuals experienced lower salivary secretion (p = 0.009) and a positive trend toward more decayed tooth surfaces (p = 0.027). These findings suggest that HIV infection can have a significant effect on the level of S. mutans, but not genotypes.

ABSTRACT: BACKGROUND: Bacterial infections have been linked to malignancies due to their ability to induce chronic inflammation. We investigated the association of oral bacteria in oral squamous cell carcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5 cm distant from the same patient (n = 10). By using culture-independent 16S rRNA approaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, we assessed the total bacterial diversity in these clinical samples. RESULTS: DGGE fingerprints showed variations in the band intensity profiles within non-tumor and tumor tissues of the same patient and among the two groups. The clonal analysis indicated that from a total of 1200 sequences characterized, 80 bacterial species/phylotypes were detected representing six phyla, Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library, 12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species, Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I were highly associated with tumor site where as Granulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius was present in 70% of both non-tumor and tumor sites. CONCLUSIONS: The underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumor and tumor sites of OSCC subjects indicated a shift in bacterial colonization. These most prevalent or unique bacterial species/phylotypes present in tumor tissues may be associated with OSCC and needs to be further investigated with a larger sample size.