Faculty Bibliography

More than 37 million people are living with human immunodeficiency virus 1 (HIV), and more people than ever received lifesaving antiretroviral therapy worldwide. HIV-1 infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation. We investigated the impact of HIV-1 infection on the GI microbiome and its association with host immune activation. The data indicated that the microbiome was different in HIV-positive and HIV-negative individuals. The initial sequence analysis of saliva indicated that there were major differences in the phyla of Bacteroidetes, Firmicutes, Proteobacteria, and TM7. Phylum Tenericutes was only seen in HIV-positive saliva. At the family level, we identified differences in Streptococcacea, Prevotellaceae, Porphyromonadaceae, and Neisseriaceae, whereas data from various sites in GI tract indicated that Prevotella melaninigencia, Fusobacterium necrophorum, Burkholderia, Bradyrhizobium, Ralstonia, and Eubacterium biforme were predominant but differentially present at various sites. Furthermore, there was a decrease in seven proteins associated with the alternative complement pathway and an increase in 6 proteins associated with the lectin and classical complement pathways. The correlation with a shift in complement pathways suggests that compromised immunity could be responsible for the observed dysbiosis in the GI microbiome.

Recent years have seen a massive expansion in our understanding of how we interact with our microbial colonists. The development of new, rapid sequencing techniques such as pyrosequencing and other next-generation sequencing systems have enabled us to begin to characterise the constituents of our diverse microbial communities, revealing the astonishing genetic richness that is our microbiome. Despite this, our ignorance of how these communities change over the course of an HIV infection is profound. Whilst some steps have been made to characterise the HIV microbiome at selected sites, these reports are still limited and much remains to be done. It has become apparent, however, that host-microbiota interactions are perturbed during HIV infections, with microbial translocation of potential pathogens linked to a variety of different HIV complications, including more rapid progression of disease. The use of probiotics and prebiotics has been investigated as treatments to alleviate symptoms for a variety of conditions, and is now being proposed for the treatment of symptoms associated with HIV. However, this is a new area of investigations and many questions remain unanswered. What we know about both of these topics is a drop in the ocean compared with what we need to know. In this article, we report on a workshop where these two major under-investigated research areas were presented, and future directions explored and discussed.

BACKGROUND: Subantimicrobial dose doxycycline (SDD) has been used as an adjunct in periodontal treatment due to its matrix metalloproteinase inhibition properties. Although the benefits of SDD therapy, such as improvement in the parameters of periodontal probing depth and clinical attachment level, have been proven in multiple clinical studies, the comprehension of other biological mechanisms of action on periodontitis remains poorly investigated. Therefore, this animal model study evaluated the effects of SDD monotherapy on the expressions of the following key pro-inflammatory genes: Proteinase-Actived Receptor-2 (PAR2), Tumor Necrosis Factor alpha (TNF-alpha), Interleukin-17 (IL-17), and. IL-1beta. METHODS: Male Wistar rats were randomly assigned to: A) Control group: no ligature-induced periodontitis and no treatment; B) Ligature group: ligature-induced periodontitis and placebo treatment and C) Ligature + doxycycline group: ligature-induced periodontitis and SDD treatment. After the experimental time, animals were sacrificed and Reverse Transcriptase-Polymerase Chain Reaction was performed to analyze the mRNA expression of IL-1beta, IL-17, TNF-alpha, and PAR2 in gingival tissue samples. Histological analyses were performed on the furcation region and mesial gingiva of mandibular first molars to measure periodontal bone loss and collagen content. RESULTS: SDD administration significantly downregulated PAR2, IL-17, TNF-alpha, and IL-1beta mRNA expressions (p<0.05). In addition, SDD treatment was accompanied by lower rates of alveolar bone loss (p<0.05) and by maintenance of the amount of gingival collagen fibers. CONCLUSIONS: These findings reveal new perspectives regarding SDD efficacy since it can be partially related to pro-inflammatory gene expression modulation, even considering PAR2 and IL-17, which has not been investigated thus far.

Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis.

The accumulation of amyloid-beta (Abeta) plaques is a central feature of Alzheimer's disease (AD). First reported in animal models, it remains uncertain if peripheral inflammatory and/or infectious conditions in humans can promote Abeta brain accumulation. Periodontal disease, a common chronic infection, has been previously reported to be associated with AD. Thirty-eight cognitively normal, healthy, and community-residing elderly (mean age, 61 and 68% female) were examined in an Alzheimer's Disease Research Center and a University-Based Dental School. Linear regression models (adjusted for age, apolipoprotein E, and smoking) were used to test the hypothesis that periodontal disease assessed by clinical attachment loss was associated with brain Abeta load using 11C-Pittsburgh compound B (PIB) positron emission tomography imaging. After adjusting for confounders, clinical attachment loss (>/=3 mm), representing a history of periodontal inflammatory/infectious burden, was associated with increased PIB uptake in Abeta vulnerable brain regions (p = 0.002). We show for the first time in humans an association between periodontal disease and brain Abeta load. These data are consistent with the previous animal studies showing that peripheral inflammation/infections are sufficient to produce brain Abeta accumulations.

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.

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.

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.