The regulation of miRNAs by reconstituted high-density lipoproteins in diabetes-impaired angiogenesis

Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. We recently found that reconstituted high-density lipoproteins (rHDL) rescue diabetes-impaired angiogenesis. microRNAs (miRNAs) regulate angiogenesis and are transported within HDL to sites of injury/repair. The role of miRNAs in the rescue of diabetes-impaired angiogenesis by rHDL is unknown. Using a miRNA array, we found that rHDL inhibits hsa-miR-181c-5p expression in vitro and using a hsa-miR-181c-5p mimic and antimiR identify a novel anti-angiogenic role for miR-181c-5p. miRNA expression was tracked over time post-hindlimb ischaemic induction in diabetic mice. Early post-ischaemia when angiogenesis is important, rHDL suppressed hindlimb mmu-miR-181c-5p. mmu-miR-181c-5p was not detected in the plasma or within HDL, suggesting rHDL specifically targets mmu-miR-181c-5p at the ischaemic site. Three known angiogenic miRNAs (mmu-miR-223-3p, mmu-miR-27b-3p, mmu-miR-92a-3p) were elevated in the HDL fraction of diabetic rHDL-infused mice early post-ischaemia. This was accompanied by a decrease in plasma levels. Only mmu-miR-223-3p levels were elevated in the hindlimb 3 days post-ischaemia, indicating that rHDL regulates mmu-miR-223-3p in a time-dependent and site-specific manner. The early regulation of miRNAs, particularly miR-181c-5p, may underpin the rescue of diabetes-impaired angiogenesis by rHDL and has implications for the treatment of diabetes-related vascular complications

Varicella Zoster Virus infection of human matured monocyte derived dendritic cells & its impact on their effector functions

Varicella Zoster Virus (VZV) is a species specific alpha human herpes virus that causes both Varicella (chickenpox) and Herpes Zoster (shingles) in human hosts. It has been hypothesized that host dendritic cells (DC) play a pivotal role in the pathogenesis of VZV during the early stages of infection. DC have previously been shown to be permissive to VZV infection and capable of transmitting the virus to human T lymphocytes. VZV has also been shown to employ a variety of mechanisms to evade detection and clearance by the host immune system. The primary aim of this study was to identify the underlying mechanisms beneath the virus induced modulation of mature DC effector functions. Observation of the kinetics of VZV mediated downregulation of the DC immune molecules CD80, CD83 and CD86 in addition to the block of Late viral gene products demonstrated that de novo viral protein synthesis of the immediate early or early class, were required to mediate modulation of CD80, CD83 and CD86. Blocking of the host cell proteasome had a restorative effect on the cell surface expression of CD86 in VZV infected mature DC. Analysis of a panel of gene transcripts involved in the effector functions of human DC in VZV infected mature DC identified significant changes in cytokine and chemokine expression (IL-8 & MIF), cytokine and chemokine receptor expression (CXCR-4 & FLT-3) and other functionally important immune molecule expression (CD44, CD80 & HLA-DPA1). Some of these changes suggest that VZV actively induces changes to host DC transcripts in order to maintain an immature state and thus negate the effector function of mature DC. The migratory capabilities of VZV infected mature DC was shown to be compromised. Furthermore, it was shown that the few still capable of migration had significantly reduced levels of cell surface CD83. Taken together, the results presented identify a wide range of mechanisms by which VZV modulates mature DC effector functions

Varicella-Zoster Virus Inhibition of the NF-κB Pathway during Infection of Human Dendritic Cells: Role for Open Reading Frame 61 as a Modulator of NF-κB Activity

Dendritic cells (DC) are antigen-presenting cells essential for initiating primary immune responses and therefore an ideal target for viral immune evasion. Varicella-zoster virus (VZV) can productively infect immature human DCs and impair their function as immune effectors by inhibiting their maturation, as evidenced by the expression modulation of functionally important cell surface immune molecules CD80, CD86, CD83, and major histocompatibility complex I. The NF-κB pathway largely regulates the expression of these immune molecules, and therefore we sought to determine whether VZV infection of DCs modulates the NF-κB pathway. Nuclear localization of NF-κB p50 and p65 indicates pathway activation; however, immunofluorescence studies revealed cytoplasmic retention of these NF-κB subunits in VZV-infected DCs. Western blotting revealed phosphorylation of the inhibitor of κBα (IκBα) in VZV-infected DCs, indicating that the pathway is active at this point. We conclude that VZV infection of DC inhibits the NF-κB pathway following protein phosphorylation but before the translocation of NF-κB subunits into the nucleus. An NF-κB reporter assay identified VZV open reading frame 61 (ORF61) as an inhibitor of tumor necrosis factor alpha-induced NF-κB reporter activity. Mutational analysis of ORF61 identified the E3 ubiquitin ligase domain as a region required for NF-κB pathway inhibition. In summary, we provide evidence that VZV inhibits the NF-κB signaling pathway in human DCs and that the E3 ubiquitin ligase domain of ORF61 is required to modulate this pathway. Thus, this work identifies a mechanism by which VZV modulates host immune function

Characterization of B-cell subpopulations in patients with chronic rhinosinusitis

Background: Recent research suggest that B and plasma cells may play an important role in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). The purpose of this study was to subcharacterize the B cell response in the sinus mucosa of control and CRS patients. Methods: Representative tissue samples and peripheral blood samples were obtained from controls, CRS without nasal polyps (CRSsNP) and CRSwNP. Using single-cell suspension flow cytometry these samples were analyzed for overall and stage-specific B and plasma cell percentages. Results: Both atopic and nonatopic CRSwNP patients showed an increase in local numbers of naive, active, and memory B cells compared to controls. CRSsNP patients only showed local elevations of naive B cells. Plasma cells were only significantly elevated in the sinus tissue of atopic CRSwNP patients. These local tissue increases did not correlate with increased numbers of circulating B cells. Conclusion: This study provides further evidence of an important role of B cells in CRSwNP patients. The local increase appears to be independent of a systemic response. (C) 2013 ARS-AAOA, LLC

Prognosis for Sixth Nerve Palsy Arising from Paranasal Sinus Disease

Background: The abducens nerve, cranial nerve VI (CNVI), is the medial-most nerve in the cavernous sinus. Its close proximity to the sphenoid sinus makes it susceptible to injury, invasion, or compression from a sphenoid pathology leading to horizontal gaze diplopia. A wide range of literature describes myriad causes for CNVI palsy, but there is a lack of references that point to paranasal sinus pathology as an etiology, as well as the prognosis and timeline for resolution. Here, we describe a series of patients that presented with CNVI palsy, their management, and prognosis for recovery. This study was designed to evaluate and understand prognostic factors predicting disease course and likelihood of resolution in patients with abducens nerve palsy. Methods: A multi-institutional retrospective review was performed of all patients presenting with CNVI palsy between 2009 and 2012. The demographic data, radiological features, treatment regimens, and disease courses were analyzed. Results: Fifteen patients at four institutions were identified. Seven patients had neoplasms originating from the paranasal sinuses, three suffered from allergic fungal sinusitis, three patients had invasive fungal sinusitis, one patient had fibrous skull base dysplasia, and one had chronic bacterial sinusitis. The average follow-up time from presentation was 9 months (range, 1-16 months). Thirteen patients underwent surgery, three received chemotherapy, and four had radiation therapy. CNVI palsy resolved in 50% of the cases, with an average time to resolution of 6 weeks (range, 2-12 weeks). Conclusion: Paranasal sinus pathology is a rare cause of CNVI palsy. A number of factors may help to predict prognosis in these patients. Masses compressing, but not destroying or invading, the cavernous sinus had optimal posttreatment outcomes with full resolution occurring as early as 2 weeks. Destructive lesions that invaded CNVI and its vasculature, i.e., invasive fungus, were negative indicators for recovery. Knowledge of factors that affect recovery can help clinicians predict disease course and prognosis for resolution of the defect

Summary schematic comparing effects of broad-spectrum inhibition by M3 on atherosclerosis.

<p>The effects of broad-spectrum chemokine inhibition by M3 was demonstrated via two models of atherosclerosis—‘rapid promotion’ and ‘slow progression’. In the rapid promotion model M3 inhibits chemokine activity, causing suppression of inflammatory monocytes, reducing adherence to the endothelium so they accumulate in the circulation rather than enter the plaque. This leads to a reduction in plaque macrophages and a suppression in lipid deposition in the descending thoracic aorta, which develops later, but not in the aortic sinus that would contain plaque of a more advanced stage. In the ‘rapid promotion’ model, aortic phosphorylated p65 was lower which may also have contributed to the reduction in plaque macrophages. In the more gradual slower progressive model, we saw an increase in plaque SMCs, a marker of improved plaque stability, with an overall reduction in atherosclerotic lesion area in the aortic sinus and descending thoracic aorta. Despite the decrease in lesion area, there was no effect on circulating monocytes or plaque macrophage content. This may be explained by the decline in M3 protein and activity at the later time points of this study and the overall lower levels of inflammation in this chow-fed model.</p

M3 reduces plaque macrophage content and p65 activation when the rate of plaque development is more rapid.

<p>Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (<i>n</i> = 10–12/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” for details. Upper panels are representative images of Mac-3⁺ macrophages (brown staining) in aortic sinus sections. Quantification of macrophage staining within plaques (μm²) for <b>A.</b> the ‘rapid promotion’ model and <b>B.</b> the ‘slow progression’ model. Phosphorylated p65 levels were measured in aortic arch samples for <b>C.</b> the ‘rapid promotion’ model and <b>D.</b> the ‘slow progression’ model. p65 mRNA levels were determined in aortic arch samples for <b>E.</b> the ‘rapid promotion’ model and <b>F.</b> the ‘slow progression’ model. Data expressed as mean±SEM. *<i>p</i><0.05, **<i>p</i><0.01, ****<i>p</i><0.0001.</p

M3 inhibits chemokine activity <i>in vitro</i>.

<p>The Boyden chamber migration assay was used to assess <b>A.</b> CCR2-, <b>B.</b> CCR5- and <b>C.</b> CX₃CR1-directed cell migration <i>in vitro</i>. 293T cells were transfected with plasmids encoding CCR2, CCR5 or CX₃CR1. <b>D.</b> Migration of human primary monocytes towards CCL2, CCL5, CX₃CL1 and CXCL12 in Boyden chamber migration assay. Representative images of Calcein AM labelled monocytes migrated to the underside of transwell membranes with and without purified M3 protein (100 ng/ mL) are shown. Data are mean±SEM. *<i>p</i><0.05, **<i>p</i><0.01.</p

M3 reduces lipid deposition in descending aortas.

<p>Two models of ‘rapid promotion’ or ‘slow progression’ of atherosclerosis were established in which a HFD or regular chow were fed and AdM3 or AdGFP were infused (<i>n</i> = 10–12/group). See “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173224#sec002" target="_blank">Materials and Methods</a>” for details. Images are representative sections of Oil Red O stained descending thoracic aortas. Oil Red O staining was quantified as a percentage of total thoracic aorta area for the ‘rapid promotion’ model and the ‘slow progression’ model. Data are mean±SEM. *<i>p</i><0.05.</p