Faculty Bibliography

Background: Ageing occurs as a complex interaction of cell autonomous and non-cell autonomous mechanisms. Functional decline is a hallmark of aging in multiple tissues, including the intestine, and this process is thought to be driven in part by deterioration in resident stem cell function. The intestine has a rapid turnover of every 3-5 days, owing this to the presence of Lgr5+ intestinal stem cells (ISCs), that resides at the bottom of the crypt along with their niche providing Paneth cells. Therefore, we investigated how this compartment is affected with aging.
Method(s): Four and 22 months old C57BL/6 male mice were used for aging phenotype studies. Young and old mice received rapamycin (4 mg/kg) or salicylate (2 mg/mL) for 28 days. Neutralizing antibodies to IL-1beta, TNF-alpha, IFN-gamma or IgG1 were i.p. injected at a 300 mug dose for 3 weeks. Isolated crypts from Lgr5-EGFP mice were re-suspended in TrypLE Express with Rock inhibitor and DNAse I, filtered and centrifuged. Cells were then re-suspended in FACS buffer containing PE-conjugated anti-CD24 antibody and APC-conjugated anti-Epcam antibody for 15 min at 4degreeC, and analyzed by MoFlo. ISC proliferation was assessed using an ex vivo organoid assay. Briefly, isolated crypts were re-suspended in matrigel, transferred to a 48-well plate to solidify at 37degreeC, and 250 muL crypt culture medium (ADF, Pen/Strep, HEPES, Glutamax, N2, B27, N-acetyl-L-cysteine, Noggin, EGF and R-Spondin) was added to each well and maintained at 37degreeC. The organoid formation was quantified on day 9. Parabiosis surgery was carried out by the Einstein Chronobiosis core.
Result(s): As compared to young mice, old mice harbor declines in ISC proliferation, impaired mucosal barrier integrity, and shifts in the gut immune cell composition, including increased CD8⁺ T cells and dendritic cells (P<0.05). We next generated isochronic (Y-Y, O-O) and heterochronic (Y-O) C57BL/6 male parabionts and observed impaired ISC function in young parabionts exposed to old blood (P<0.05). Rapamycin or salicylate treatment restored ISC function in old mice (P<0.05), without further suppressing mTOR signaling. However, rapamycin reduced plasma cytokines to more youthful levels (P<0.05), suggesting a potential role for inflammation in mediating the stem cell dysfunction with aging. Ex vivo screening assays confirmed that TNFalpha and IFNgamma potently disrupt ISC proliferation (P<0.05), while in vivo treatment with a TNFalpha-or IFNgamma-neutralizing-antibody restored ISC function in old mice (P<0.05). Upon single cell ablation in the crypts, the motion of neighboring cells was dramatically impaired, and cell debris 'lingered' in the crypts of old animals (P<0.05).
Conclusion(s): We propose that intestinal aging happens as a complex interplay of cell autonomous and non-cell autonomous pathways. Moreover, IFN-gamma and TNF-alpha can act as lead progeronic factors to drive a decline in intestinal stem cell function with aging

Various surface modifications have been tried for enhancing osseointegration of the dental implants like mechanical and/or chemical treatments and deposition of calcium phosphate coatings. The objective of this research was to develop calcium-phosphate based thin coatings with antibacterial and bioactive properties for potential application in dental implants. Titanium (Ti) discs were immersed in different calcifying solutions: CaP (positive control), F-CaP, Zn-CaP and FZn-CaP and incubated for 24 h. Negative control was uncoated Ti discs. Coated surfaces were characterized using X-ray diffraction, scanning electron microscopy and energy dispersive spectroscopy. Antibacterial properties were tested using Porphyromonas gingivalis because of its strong association with periodontal and peri-implant infections. Bacterial adhesion and colonization were studied at different timepoints. The coated surfaces had compositional characteristics similar to that of bone mineral and they inhibited the growth, colonization and adherence of P. gingivalis, resulted in reduced thickness of biofilms and bacterial inhibition in the culture medium as compared to the positive and negative controls (p < 0.05). There was no significant difference between the experimental groups (p > 0.05). All CaP coatings demonstrated in vitro bioactivity (formed carbonate hydroxyapatite when immersed in SBF). Such coatings can enhance osseointegration and prevent infection in implants, thereby improving the success rates of implants

The mechanism underlying bone impairment in patients with diabetes mellitus, a metabolic disorder characterized by chronic hyperglycaemia and dysregulation in metabolism, is unclear. Here we show the difference in the metabolomics of bone marrow stromal cells (BMSCs) derived from hyperglycaemic (type 2 diabetes mellitus, T2D) and normoglycaemic mice. One hundred and forty-two metabolites are substantially regulated in BMSCs from T2D mice, with the tricarboxylic acid (TCA) cycle being one of the primary metabolic pathways impaired by hyperglycaemia. Importantly, succinate, an intermediate metabolite in the TCA cycle, is increased by 24-fold in BMSCs from T2D mice. Succinate functions as an extracellular ligand through binding to its specific receptor on osteoclastic lineage cells and stimulates osteoclastogenesis in vitro and in vivo. Strategies targeting the receptor activation inhibit osteoclastogenesis. This study reveals a metabolite-mediated mechanism of osteoclastogenesis modulation that contributes to bone dysregulation in metabolic disorders.

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.

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.

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.

People with Down syndrome (DS) are at an increased risk for Alzheimer's disease (AD). After 60 years of age, >50% of DS subjects acquire dementia. Nevertheless, the age of onset is highly variable possibly because of both genetic and environmental factors. Genetics cannot be modified, but environmental risk factors present a potentially relevant intervention for DS persons at risk for AD. Among them, inflammation, important in AD of DS type, is potential target. Consistent with this hypothesis, chronic peripheral inflammation and infections may contribute to AD pathogenesis in DS. People with DS have an aggressive form of periodontitis characterized by rapid progression, significant bacterial and inflammatory burden, and an onset as early as 6 years of age. This review offers a hypothetical mechanistic link between periodontitis and AD in the DS population. Because periodontitis is a treatable condition, it may be a readily modifiable risk factor for AD.

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.