Search

Porphyromonas gingivalis (Pg) and its gingipain proteases contribute to Alzheimer's disease (AD) pathogenesis through yet unclear mechanisms. Cellular secretion of small extracellular vesicles or exosomes (EXO) increases with aging as part of the senescence-associated secretory phenotype (SASP). We have shown that EXO isolated from Pg-infected dendritic cells contain gingipains and other Pg antigens and transmit senescence to bystander gingival cells, inducing alveolar bone loss in mice in vivo. Here, EXO were isolated from the gingiva of mice and humans with/without periodontitis (PD) to determine their ability to penetrate the blood–brain barrier (BBB) in vitro and in vivo. PD was induced by Pg oral gavage for 6 weeks in C57B6 mice. EXO isolated from the gingiva or brain of donor Pg-infected (PD EXO) or control animals (Con EXO) were characterized by NTA, Western blot, and TEM. Gingival PD EXO or Con EXO were labeled and injected into the gingiva of uninfected WT mouse model. EXO biodistribution in brains was tracked by an in vivo imaging system (IVIS) and confocal microscopy. The effect of human PD EXO on BBB integrity and permeability was examined using TEER and FITC dextran assays in a human in vitro 3D model of the BBB. Pg antigens (RGP and Mfa-1) were detected in EXO derived from gingival and brain tissues of donor Pg-infected mice. Orally injected PD EXO from donor mice penetrated the brains of recipient uninfected mice and colocalized with hippocampal microglial cells. IL-1β and IL-6 were expressed in human PD EXO and not in Con EXO. Human PD EXO promoted BBB permeability and penetrated the BBB in vitro. This is the first demonstration that microbial-induced EXO in the oral cavity can disseminate, cross the BBB, and may contribute to AD pathogenesis. [ABSTRACT FROM AUTHOR]

Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (Squalus acanthias) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5′ and 3′ RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5′ and 3′ RACE amplifications depend on the presence of a 5′ cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene. [ABSTRACT FROM AUTHOR]

The active form of vitamin D is the hormonally active 1,25(OH)2D3 (Vit D) vitamin, which plays an important role in bone biology and host immunity. The vitamin D receptor (VDR) is a nuclear ligand-dependent transcription factor expressed by many cells. Ligation of VDR by VitD regulates a wide plethora of genes and physiologic functions through the formation of the complex Vit D-VDR signaling cascade. The influence of Vit D-VDR signaling in host immune response to microbial infection has been of interest to many researchers. This is particularly important in oral health and diseases, as oral mucosa is exposed to a complex microbiota, with certain species capable of causing disruption to immune homeostasis. In this review, we focus on the immune modulatory roles of Vit D in the bone degenerative oral disease, periodontitis. [ABSTRACT FROM AUTHOR]

Exosomes (exos) contain molecular cargo of therapeutic and diagnostic value for cancers and other inflammatory diseases, but their therapeutic potential for periodontitis (PD) remains unclear. Dendritic cells (DCs) are the directors of immune response and have been extensively used in immune therapy. We previously reported in a mouse model of PD that custom murine DC-derived exo subtypes could reprogram the immune response toward a bone-sparing or bone-loss phenotype, depending on immune profile. Further advancement of this technology requires the testing of human DC-based exos with human target cells. Our main objective in this study is to test the hypothesis that human monocyte-derived dendritic cell (MoDC)-derived exos constitute a well-tolerated and effective immune therapeutic approach to modulate human target DC and T cell immune responses in vitro. MoDC subtypes were generated with TGFb/IL-10 (regulatory (reg) MoDCs, CD86lowHLA-DRlowPDL1high), E. coli LPS (stimulatory (stim) MoDCs, CD86highHLA-DRhighPDL1low) and buffer (immature (i) MoDCs, CD86lowHLA-DRmedPDL1low). Exosomes were isolated from different MoDC subtypes and characterized. Once released from the secreting cell into the surrounding environment, exosomes protect their prepackaged molecular cargo and deliver it to bystander cells. This modulates the functions of these cells, depending on the cargo content. RegMoDCexos were internalized by recipient MoDCs and induced upregulation of PDL1 and downregulation of costimulatory molecules CD86, HLADR, and CD80, while stimMoDCexos had the opposite influence. RegMoDCexos induced CD25+Foxp3+ Tregs, which expressed CTLA4 and PD1 but not IL-17A. In contrast, T cells treated with stimMoDCexos induced IL-17A+ Th17 T cells, which were negative for immunoregulatory CTLA4 and PD1. T cells and DCs treated with iMoDCexos were immune 'neutral', equivalent to controls. In conclusion, human DC exos present an effective delivery system to modulate human DC and T cell immune responses in vitro. Thus, MoDC exos may present a viable immunotherapeutic agent for modulating immune response in the gingival tissue to inhibit bone loss in periodontal disease. [ABSTRACT FROM AUTHOR]

Introduction: Fibroblasts are the dominant stromal cells in the gingival lamina propria with a well-established relevance in regulation of inflammation, and in innate immunity. This is exemplified by their hypersecretion of CXCL8, enhancing leukocyte infiltration in chronic and sustained inflammatory conditions. We have previously shown adenosine to be a key metabolic nucleoside that regulates stromal inflammation, but the underlying mechanisms linking adenosine to the metabolic status of fibroblasts and to the resultant inflammatory response are unclear. This study examined, by seahorse real-time cell metabolic analysis, the bioenergetics of the stromal fibroblast response to extracellular adenosine and IL-1β, focusing on CXCL8 secretion by primary human gingival fibroblasts (HGF). Methods: Markers of the glycolytic pathway and mitochondrial biogenesis were tracked through immunoblot. Further, the influence of adenosine on mitochondrial accumulation was measured by uptake of MitoTracker Red fluorescent probe and assessment of the role of FCCP (a mitochondrial uncoupler) in CXCL8 secretion and mitochondrial accumulation. Results: Our results show that the anti-inflammatory response of HGF to extracellular adenosine, typified by reduced CXCL8 secretion, is mediated by mitochondrial oxidative phosphorylation, reflected in higher oxygen consumption rate (OCR). In the presence of IL-1β, adenosine-treated cells induced higher ATP production, basal respiration and proton leak compared to IL-1β without adenosine. Surprisingly, adenosine had no additional effect on the IL-1β-induced higher glycolysis rate demonstrated by the extracellular acidification rate (ECAR). In addition, the higher OCR in adenosine-stimulated cells was not due to the mitochondrial fuel dependency or capacity, but due to an increase in mitochondrial biogenesis and accumulation in the cells with concomitant decrease in mitophagy-required p-PINK1 marker. We detected the accumulation of functional mitochondria with increased activation of the AMPK/SIRT1/PGC-1α pathway. The adenosine-induced uptake of MitoTracker was abrogated by PGC-1α inhibition with SR-12898. In addition, the adenosine effects on reduced CXCL8 were ablated by treatment with FCCP, a potent uncoupler of mitochondrial oxidative phosphorylation. Conclusion: Our findings reveal a key role for mitochondrial bioenergetics in regulation of CXCL8-mediated inflammation by HGF through the adenosine/AMPK/SIRT1/PGC-1α axis. Therapeutically targeting this pathway in gingival fibroblasts might be a promising future strategy to modulate stromal-mediated sustained hyper-inflammatory responses. [ABSTRACT FROM AUTHOR]

As the aging population grows, chronic age-related bone degenerative diseases become more prevalent and severe. One such disease, periodontitis (PD), rises to 70.1% prevalence in Americans 65 years and older. PD has been linked to increased risk of other age-related diseases with more serious mortality and morbidity profiles such as Alzheimer's disease and cardiovascular disease, but the cellular and biological mechanisms remain unclear. Recent in vitro studies from our group indicate that murine dendritic cells (DCs) and T cells are vulnerable to immune senescence. This occurs through a distinct process involving invasion of DCs by dysbiotic pathogen Porphyromonas gingivalis (Pg) activating the senescence associated secretory phenotype (SASP). Exosomes of the Pg-induced SASP transmit senescence to normal bystander DC and T cells, ablating antigen presentation. The biological significance of these findings in vivo and the mechanisms involved were examined in the present study using young (4-5mo) or old (22-24mo) mice subjected to ligature-induced PD, with or without dysbiotic oral pathogen and injection of Pg-induced DC exosomes. Senescence profiling of gingiva and draining lymph nodes (LN) corroborates role of advanced age and PD in elevation of senescence biomarkers beta galactosidase (SA-ß-Gal), p16 INK4A p21Waf1/Clip1, IL6, TNF?, and IL1ß, with attendant increase in alveolar bone loss, reversed by senolytic agent rapamycin. Immunophenotyping of gingiva and LN revealed that myeloid CD11c+ DCs and T cells are particularly vulnerable to senescence in vivo under these conditions. Moreover, Pg-induced DC exosomes were the most potent inducers of alveolar bone loss and immune senescence, and capable of overcoming senescence resistance of LN T cells in young mice. We conclude that immune senescence, compounded by advanced age, and accelerated by oral dysbiosis and its induced SASP exosomes, plays a pivotal role in the pathophysiology of experimental periodontitis. [ABSTRACT FROM AUTHOR]

Chronic subdural hematoma or nonacute subdural hematoma (NASH) remains a common neurosurgical disease, with an incidence of 1.7–20.6:100,000 individuals. Surgical evacuation of chronic subdural hematoma can be complicated by inadequate drainage and recurrence rates up to 20%–30% in some series. We examine the safety and efficacy of endoscope-assisted NASH evacuation and review the literature on the technique. A consecutive, single-center series of endoscope-assisted NASH evacuations was reviewed to assess patient factors, imaging, and recurrence. A systematic literature review was performed using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A total of 9 patients who underwent endoscope-assisted NASH evacuation were included. This technique showed some improved evacuation of hematoma, removal of membranes, coagulation of at-risk blood vessels, directed placement of surgical drains, visualization of embolization efficacy, coagulation of friable membranes, and removal of acute clot. Only 1 episode of NASH recurrence (11.1%) was seen at the last follow-up. A systematic review of 14 studies with 1353 patients identified the use of membranectomy in 57% of cases and a recurrence rate of 0%–8.8%. Endoscopic visualization can be a useful adjunct in the modern treatment of NASH. Reduced risk of recurrence was seen compared with those of historical surgical drainage methods including burr holes (20–30%). The inclusion of endoscopic visualization in the modern era with middle meningeal artery embolization may potentially combine methods that can dramatically reduce the recurrence of NASH. [ABSTRACT FROM AUTHOR]

We recently reported that the oral mucosal pathogen Porphyromonas gingivalis, through its 67-kDa Mfal (minor) fimbria, targets the C-type lectin receptor DC-SIGN for invasion and persistence within human monocyte-derived dendritic cells (DCs). The DCs respond by inducing an immunosuppressive and Th2-biased [CD4.sup.+] T-cell response. We have now purified the native minor fimbria by ion-exchange chromatography and sequenced the fimbria by tandem mass spectrometry (MS/MS), confirming its identity and revealing two putative N-glycosylation motifs as well as numerous putative O-glycosylation sites. We further show that the minor fimbria is glycosylated by ProQ staining and that glycosylation is partially removed by treatment with [beta](1-4)-galactosidase, but not by classic N- and O-linked deglycosidases. Further monosaccharide analysis by gas chromatography-mass spectrometry (GC-MS) confirmed that the minor fimbria contains the DC-SIGN-targeting carbohydrates fucose (1.35 nmol/mg), mannose (2.68 nmol/mg), N-acetylglucosamine (2.27 nmol/mg), and N-acetylgalactosamine (0.652 nmol/mg). Analysis by transmission electron microscopy revealed that the minor fimbria forms fibers approximately 200 nm in length that could be involved in targeting or cross-linking DC-SIGN. These findings shed further light on molecular mechanisms of invasion and immunosuppression by this unique mucosal pathogen. doi: 10.1128/JB.00275-10

Extensive research in humans and animal models has begun to unravel the complex mechanisms that drive the immunopathogenesis of periodontitis. Neutrophils mount an early and rapid response to the subgingival oral microbiome, producing destructive enzymes to kill microbes. Chemokines and cytokines are released that attract macrophages, dendritic cells, and T cells to the site. Dendritic cells, the focus of this review, are professional antigen-presenting cells on the front line of immune surveillance. Dendritic cells consist of multiple subsets that reside in the epithelium, connective tissues, and major organs. Our work in humans and mice established that myeloid dendritic cells are mobilized in periodontitis. This occurs in lymphoid and nonlymphoid oral tissues, in the bloodstream, and in response to Porphyromonas gingivalis. Moreover, the dendritic cells mature in situ in gingival lamina propria, forming immune conjugates with cluster of differentiation (CD) 4+ T cells, called oral lymphoid foci. At such foci, the decisions are made as to whether to promote bone destructive T helper 17 or bone-sparing regulatory T cell responses. Interestingly, dendritic cells lack potent enzymes and reactive oxygen species needed to kill and degrade endocytosed microbes. The keystone pathogen P. gingivalis exploits this vulnerability by invading dendritic cells in the tissues and peripheral blood using its distinct fimbrial adhesins. This promotes pathogen dissemination and inflammatory disease at distant sites, such as atherosclerotic plaques. Interestingly, our recent studies indicate that such P. gingivalis-infected dendritic cells release nanosized extracellular vesicles called exosomes, in higher numbers than uninfected dendritic cells do. Secreted exosomes and inflammasome-related cytokines are a key feature of the senescence-associated secretory phenotype. Exosomes communicate in paracrine with neighboring stromal cells and immune cells to promote and amplify cellular senescence. We have shown that dendritic cell-derived exosomes can be custom tailored to target and reprogram specific immune cells responsible for inflammatory bone loss in mice. The long-term goal of these immunotherapeutic approaches, ongoing in our laboratory and others, is to promote human health and longevity. [ABSTRACT FROM AUTHOR]

Complementary DNAs encoding homologs of the mammalian aqua-glyceroporins (termed AQPe) and aquaporin-1 isoforms (termed AQP1) were isolated from the European eel. The AQP amino acid sequences share 35-54% identity with other known human AQPs. Although AQPe mRNA expression was approximately equivalent along the entire length of the gut, AQP1 expression was the highest in the posterior/rectal segment. Seawater (SW) acclimation increased AQP1 mRNA abundance by 5- and 17-fold in the anterior, 14- and 23-fold in the mid-, and 9- and 7-fold in the posterior/rectal gut regions of yellow and silver eels, respectively. SW acclimation had an effect on AQPe mRNA expression only in the midintestine of silver eels, where a small but significant 1.7-fold increase in abundance was measured. Western blots using an eel AQP1-specific antibody identified the presence of a major immunoreactive 28-kDa protein, primarily within the posterior/rectal segment. A 3-wk SW transfer induced an increase in AQP1 protein abundance in all intestinal segments, with the posterior/rectal region still expressing protein levels ~40- and 8-fold higher than the anterior and mid-segments, respectively. Strong AQP1 immunofluorescence was detected within the vascular endothelium in both freshwater (FW)- and SW-acclimated eels and in the epithelial apical brush border in the posterior/ rectal gut regions of SW-acclimated eels. Cortisol infusion into FW eels had no effect on intestinal AQPe mRNA expression but induced increases in AQP1 mRNA and protein levels. These results provide evidence for the presence of a SW-induced and steroid-regulated AQP water channel pathway within the intestine of the European eel. Anguilla anguilla; gastrointestinal tract; AQP1; AQPe; corticosteroid

Leakiness of the swimbladder wall of teleost fishes must be prevented to avoid diffusional loss of gases out of the swimbladder. Guanine incrustation as well as high concentrations of cholesterol in swimbladder membranes in midwater and deep‐sea fish has been connected to a reduced gas permeability of the swimbladder wall. On the contrary, the swimbladder is filled by diffusion of gases, mainly oxygen and CO2, from the blood and the gas gland cells into the swimbladder lumen. In swimbladder tissue of the zebrafish and the Japanese eel, aquaporin mRNA has been detected, and the aquaporin protein has been considered important for the diffusion of water, which may accidentally be gulped by physostome fish when taking an air breath. In the present study, the expression of two aquaporin 1 genes (Aqp1aa and Aqp1ab) in the swimbladder tissue of the European eel, a functional physoclist fish, was assessed using immunohistochemistry, and the expression of both genes was detected in endothelial cells of swimbladder capillaries as well as in basolateral membranes of gas gland cells. In addition, Aqp1ab was present in apical membranes of swimbladder gas gland cells. The authors also found high concentrations of cholesterol in these membranes, which were several fold higher than in muscle tissue membranes. In yellow eels the cholesterol concentration exceeded the concentration detected in silver eel swimbladder membranes. The authors suggest that aquaporin 1 in swimbladder gas gland cells and endothelial cells facilitates CO2 diffusion into the blood, enhancing the switch‐on of the Root effect, which is essential for the secretion of oxygen into the swimbladder. It may also facilitate CO2 diffusion into the swimbladder lumen along the partial gradient established by CO2 production in gas gland cells. Cholesterol has been shown to reduce the gas permeability of membranes and thus could contribute to the gas tightness of swimbladder membranes, which is essential to avoid diffusional loss of gas out of the swimbladder. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis (P. gingivalis) is a unique pathogen implicated in severe forms of periodontitis (PD), a disease that affects around 50% of the US population. P. gingivalis is equipped with a plethora of virulence factors that it uses to exploit its environment and survive. These include distinct fimbrial adhesins that enable it to bind to other microbes, colonize inflamed tissues, acquire nutrients, and invade cells of the stroma and immune system. Most notable for this review is its ability to invade dendritic cells (DCs), which bridge the innate and adaptive immune systems. This invasion process is tightly linked to the bridging functions of resultant DCs, in that it can disable (or stimulate) the maturation function of DCs and cytokines that are secreted. Maturation molecules (e.g., MHCII, CD80/CD86, CD40) and inflammatory cytokines (e.g., IL-1b, TNFa, IL-6) are essential signals for antigen presentation and for proliferation of effector T-cells such as Th17 cells. In this regard, the ability of P. gingivalis to coordinately regulate its expression of major (fimA) and minor (mfa-1) fimbriae under different environmental influences becomes highly relevant. This review will, therefore, focus on the immunoregulatory role of P. gingivalis fimbriae in the invasion of DCs, intracellular signaling, and functional outcomes such as alveolar bone loss and immune senescence. [ABSTRACT FROM AUTHOR]

Background: The macrophage is an innate immune defense cell involved in pathogen recognition and clearance. Aim: In view of the diversity of the macrophage phenotype and function, the present study investigated how Enterococcus faecalis infection affects the differentiation, phenotype and cytokine profile of macrophages. Methods: Murine bone marrow-derived stem cells were co-cultured with E. faecalis before and after differentiation. Macrophage M0 polarization towards M1 or M2 was initiated at day 6 by addition of LPS and INF-γ, or IL-4 and IL-13, respectively. Results: E. faecalis did not inhibit macrophage differentiation and were identified within macrophages. Viability of the macrophages infected with E. faecalis prior to differentiation was enhanced, evidenced by apoptosis inhibition, as was expression of CD38 and IRF5 proteins, indicators of M1-like polarization. These M1-like macrophages expressed an aberrant cytokine mRNA profile, with reduction in inflammatory cytokines IL-1β and IL-12 and increase in regulatory cytokine IL-10. No changes in TNF-α or TGF-β1 were detected, compared with the control groups. This atypical M1-like phenotype was retained even upon stimulation with growth factors that normally trigger their development into M2 macrophages. Conclusions: These findings suggested that E. faecalis infection of bone marrow-derived stem cells during differentiation into macrophages induces an atypical M1-like phenotype associated with intracellular bacterial survival. [ABSTRACT FROM AUTHOR]