The Oxidative Response of Human Monocytes to Surface Modified Commercially Pure Titanium

Cellular responses to implanted biomaterials are key to understanding osseointegration. The aim of this investigation was to determine the in vitro priming and activation of the respiratory burst activity of monocytes in response to surface-modified titanium. Human peripheral blood monocytes of healthy blood donors were separated, then incubated with surface-modified grade 2 commercially pure titanium (CPT) disks with a range of known surface energies and surface roughness for 30- or 60-min. Secondary stimulation by phorbol 12-myrisate 13-acetate (PMA) following the priming phase, and luminol-enhanced-chemiluminescence (LCL) was used to monitor oxygen-dependent activity. Comparison among groups was made by incubation time using one-way ANOVA. One sample from each group for each phase of the experiment was viewed under scanning electron microscopy (SEM) and qualitative comparisons made. The results indicate that titanium is capable of priming peripheral blood monocytes following 60-min incubation. In contrast, 30 min incubation time lead to reduced LCL on secondary stimulation as compared to cells alone. At both time intervals, the disk with the lowest surface energy produced significantly less LCL compared to other samples. SEM examination revealed differences in surface morphology at different time points but not between differently surface-modified disks. These results are consistent with the hypothesis that the titanium surface characteristics influenced the monocyte activity, which may be important in regulating the healing response to these materials.

Rhinovirus-16 increases ATP release in A549 cells without concomitant increase in production

Human rhinovirus (RV) is the most common cause of upper respiratory tract infection (URTI) and chronic airway disease exacerbation. Cough is present in 50–80% of URTI cases, accompanied by heightened airway hypersensitivity, yet no effective treatment currently exists for this infectious cough. The mechanism by which RV causes cough and airway hypersensitivity in URTI is still unknown despite recent advances in potential therapies for chronic cough.The effect of RV-16 infection (MOI 1) on intracellular ATP stores and ATP release in A549 alveolar epithelial cells was measured.RV-16 infection was found to significantly increase (by 50% from basal at 24 h) followed by decrease (by 50% from basal at 48 and 72 h) intracellular ATP concentrations, while increasing ATP release (from 72 h) independently of secondary stimulation. This effect was mimicked by intercellular adhesion molecule 1 receptor binding alone through ultraviolet-inactivated sham control. In addition, RV-16-infected cells became more sensitive to secondary stimulation with both hypotonic and isotonic solutions, suggestive of a hypersensitive response. These responses were not mediated via increased TRPV4 or pannexin-1 whole-cell expression as determined by Western blotting. Interestingly, the increased ATP release seen was not a result of increased mitochondrial ATP production.Thus, this is the first report demonstrating that RV-16 infection of airway epithelial cells causes hypersensitivity by increasing ATP release. These finding provide a novel insight into the process by which viruses may cause cough and identify a potential target for treatment of viral and post-viral cough.

NK cells acquire epigenetic memory of LPS-induced systemic inflammation

Natural killer cells are unique mediators of innate immunity, and as such, an attractive target for immunotherapy. Following viral infection, NK cells display immune memory properties, defined by heightened responses to re-stimulation, an expansion of specific NK cell sup-populations and a protective role against re-infection. However, similar memory to bacterial infection or systemic inflammation, and the molecular mechanisms behind NK cell memory remain elusive. Here we show that following LPS-induced endotoxemia in mice, NK cells acquire cell-intrinsic memory properties as displayed by an amplified production of IFNγ upon secondary stimulation. NK cell memory is acquired even under the post-endotoxemic suppressive environment and is detectable for at least 9 weeks. Furthermore, we define an epigenetic mechanism essential for NK cell memory, where an H3K4me1-marked latent enhancer is uncovered at the ifng locus. Chemical inhibition of histone methyltransferase activity erased the enhancer and prevented NK cells from acquiring memory. Thus, NK cells develop memory to LPS during endotoxemia, in a histone methylation-dependent manner, which ensures a heightened response to secondary stimulation and confers protection against bacterial infection.

Microglial and Astrocyte priming in the APP/PS1 model of Alzheimer’s Disease: increased vulnerability to acute inflammation and cognitive deficits

Alzheimer’s disease (AD) causes devastating cognitive decline and has no disease-modifying therapies. Neuroinflammation is a significant contributor to disease progression but its precise contribution remains unclear. An emerging literature indicates that secondary inflammatory insults including acute trauma and infection alter the trajectory of chronic neurodegenerative diseases and the roles of microglia and astrocytes require elucidation. The current study, using the APP/PS1 mouse model of AD, demonstrates that microglia are primed by β-amyloid pathology to induce exaggerated IL-1β responses to acute stimulation with LPS or IL-1β. Despite disease-associated NLRP3 inflammasome activation, evidenced by ASC speck formation, APP/PS1 microglial cells show neither IL-1β induction nor NFκB p65 nuclear localisation. Upon secondary stimulation with LPS or IL-1β, NFκB-p65 nuclear localisation and exaggerated pro-IL-1 induction occur. Microglial priming was also unmasked by secondary stimulation with systemic LPS leading to significant cognitive impairment in APP/PS1 mice compared to WT LPS-treated mice. Astrocytes have also recently emerged as displaying significant phenotypic heterogeneity. Here, by-passing microglial priming, and acutely challenging mice with intra-hippocampal IL-1β we demonstrate that astrocytes proximal to Aβ-plaques are also primed to produce exaggerated CCL2, CXCL1 and CXCL10 responses. Many astrocytosis-associated genes in APP/PS1 mice share these exaggerated responses to IL-1β, while others are equally induced in both strains. Collectively the data show that the amyloid-laden brain shows multiple vulnerabilities to secondary inflammatory challenge: both microglia and astrocytes are primed to produce exaggerated secondary inflammation and systemic LPS is sufficient to cause cognitive impairments relevant to delirium, selectively in animals with prior amyloid pathology.

Glinide, but Not Sulfonylurea, Can Evoke Insulin Exocytosis by Repetitive Stimulation: Imaging Analysis of Insulin Exocytosis by Secretagogue-Induced Repetitive Stimulations

To investigate the different effects between sulfonylurea (SU) and glinide drugs in insulin secretion, pancreaticβ-cells were repeatedly stimulated with SU (glimepiride) or glinide (mitiglinide). Total internal reflection fluorescent (TIRF) microscopy revealed that secondary stimulation with glimepiride, but not glucose and mitiglinide, failed to evoke fusions of insulin granules although primary stimulation with glucose, glimepiride, and mitiglinide induced equivalent numbers of exocytotic responses. Glimepiride, but not glucose and mitiglinide, induced abnormally sustained[Ca2+]ielevations and reductions of docked insulin granules on the plasma membrane. Our data suggest that the effect of glinide on insulin secretory mechanisms is similar to that of glucose.

Investigating the Role of Dendritic Cells in Extracorporeal Photopheresis Using an In Vitro Model.

Graft versus Host disease (GvHD) is the major limitation to successful allogeneic hematopoietic stem cell transplantation and contributes significantly to transplant related mortality and morbidity. Steroid refractory or steroid depending GvHD in particular is linked to poor survival or life quality. Conventional immunosuppression has very limited success in these conditions and increases susceptability for infection and relapse. Extracorporeal Photopheresis (ECP) is a promising therapy for acute and chronic GvHD not responding to conventional immunosuppressive therapy. ECP treatment seems not to result in a pan-immunosuppression but has quite selective effects on the pathogenic process in GvHD. The mechanisms of action of ECP in GvHD known so far include lymphocyte apoptosis, cytokine modulation and increased regulatory T cell numbers. It has been suggested that monocytes and dendritic cells (DC) were more resistant to ECP induced apoptosis, so that they might either be directly modulated by ECP or modulated by encounter with apoptotic cells and this way aquiring immunomodulating properties. Here we tested the first hypothesis analysing direct effects of ECP on monocyte derived dendritic cells in vitro. Direct in vitro PUVA treatment leads to activation of monocyte derived immature DCs (upregulation of CD83, CD86, HLA-DR, reduced endocytosis capacity, increased migratory capacity), whereas mature DCs are not affected. However, immature and mature DCs undergo apoptosis later on within 24h-72h. Prestimualtion with either antigen (KLH), IL-12 or co-culture with CD40L expressing cells could not prevent apoptosis. Further DC stimulation through CD40L is abrogated 24h after treatment. DC cytokine analysis (IL-12, TNFα) is pending. After in vitro PUVA primary dermal DCs and Langerhans Cells migrated from skin biopsies undergo apoptosis similarly to monocyte derived DCs. Going along with the early activation, the stimulatory capacity of in vitro PUVA treated immature DCs is enhanced in an MLR on naive T cells. IL-10 levels are decreased as compared to untreated or UVA treated cells. Secondary stimulation through either CD3/CD28 beads or mature DCs leads to reduced proliferation of naive T cells that were primarily stimulated with in vitro PUVA treated as compared to untreated immature DCs. Cytokine analysis after secondary stimulation is pending. We conclude that in our model in vitro PUVA modulates the character especially of immature dendritic cells. However, immunostimulating and immunosuppressive characteristics could be described depending on the assay. Cytokine results after second stimulation (Th1/Th2) are pending. Apoptosis is a major finding and occurs both in monocytes and DCs after 24–72h. Our findings indicate that there might be direct ECP effects on monocytes and DCs going beyond the process of merely depleting antigen presenting cells. Further relevance is currently being investigated.

Downregulation of the DNA-Binding Activity of Nuclear Factor-κB p65 Subunit in Porphyromonas gingivalis Fimbria-Induced Tolerance

ABSTRACT Porphyromonas gingivalis fimbriae induce high levels of nuclear factor-κB (NF-κB)-dependent cytokine release upon primary but not secondary stimulation of monocytic cells (FimA tolerance). In this study, fimbriae induced Toll-like receptor-mediated activation of both p50 and p65 subunits of NF-κB upon primary cellular activation. However, activation of the transactivating p65 subunit (but not of the transcriptionally inactive p50 subunit) was significantly inhibited in fimbria-restimulated cells. Moreover, expression of a NF-κB-dependent reporter gene was inhibited upon secondary stimulation with fimbriae. NF-κB p65 downregulation may thus contribute to induction of FimA tolerance.