In vitro and in silico processes to identify differentially expressed proteins

We present an integrated proteomics platform designed for performing differential analyses. Since reproducible results are essential for comparative studies, we explain how we improved reproducibility at every step of our laboratory processes, e.g. by taking advantage of the powerful laboratory information management system we developed. The differential capacity of our platform is validated by detecting known markers in a real sample and by a spiking experiment. We introduce an innovative two-dimensional (2-D) plot for displaying identification results combined with chromatographic data. This 2-D plot is very convenient for detecting differential proteins. We also adapt standard multivariate statistical techniques to show that peptide identification scores can be used for reliable and sensitive differential studies. The interest of the protein separation approach we generally apply is justified by numerous statistics, complemented by a comparison with a simple shotgun analysis performed on a small volume sample. By introducing an automatic integration step after mass spectrometry data identification, we are able to search numerous databases systematically, including the human genome and expressed sequence tags. Finally, we explain how rigorous data processing can be combined with the work of human experts to set high quality standards, and hence obtain reliable (false positive < 0.35%) and nonredundant protein identifications.     

HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation

Objective

CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3).

Methods and Results

CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL₃ compared to non-lipidated apoA-I. In vivo, in an apoE^-/- mouse “flow cessation model,” in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively.

Conclusions

These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE^-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both in vitro and in vivo. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression.     

Use of Formalin-Fixed Paraffin-Embedded Samples for Gene Expression Studies in Breast Cancer Patients

To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.     

Stereotactic radiation therapy for skull base recurrences: Is a salvage approach still possible?

AimA literature review was performed to analyse the role of stereotactic radiotherapy given in a single shot or in a fractionated fashion for recurrent skull base tumours in order to ascertain if it can be a real salvage approach.BackgroundThe management of recurrent skull base tumours can have a curative or palliative intent and mainly includes surgery and RT.Materials and methodsOne-thousand-ninety-one articles were found in the search databases and the most relevant of them were analysed and briefly described.ResultsData on recurrences of meningioma, pituitary adenoma, craniopharyngioma, chordoma and chondrosarcoma, vestibular schwannoma, glomus jugulare tumours, olfactory neuroblastoma and recurrences from head and neck tumours invading the base of skull are reported highlighting the most relevant results in terms of local control, survival, side effects and complications.ConclusionsIn conclusion, it emerges that SRS and FSRT are effective and safe radiation modalities of realize real salvage treatment for recurrent skull base tumours.     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

Taking EPR "Snapshots" of the Oxidative Stress Status in Human Blood

Assessment of oxidative stress status (OSS) in human tissues is still troublesome. Using an innovative EPR-radical-probe we successfully measured the instantaneous concentration of ROS directly in peripheral blood of athletes and normally active workers during 60 min controlled exercise. The probe employed was bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate, which quantitatively and instantaneously reacts with oxygen-centered radicals (including superoxide) to yield the parent nitroxide, which is sufficiently persistent to be measured by EPR. Our measurements suggest that while at rest normally active individuals may benefit more from antioxidant supplementation than athletes; conversely, during exercise athletes may benefit more from supplementation. Our method allows reliable, quick, and non-invasive quantitative determination of OSS in human peripheral blood.     

Similar Trends in Serum VEGF-D Levels and Kidney Angiomyolipoma Responses with Longer Duration Sirolimus Treatment in Adults with Tuberous Sclerosis

Context

We have previously shown that serum VEGF-D is elevated at baseline, correlates with kidney angiomyolipoma size at baseline and 12 months, and decreases with sirolimus treatment in adults with tuberous sclerosis complex (TSC). To further investigate the utility of serum VEGF-D for longer term monitoring of TSC kidney disease, we present VEGF-D level results with 24 month follow-up.

Objective

To compare 24 month VEGF-D levels in two subgroups of sirolimus treated patients (OFF SIROLIMUS AFTER 12 MONTHS or ON SIROLIMUS AFTER 12 MONTHS).

Design and Intervention(s)

Serum VEGF-D was measured in samples collected from subjects enrolled in a phase 2 multicenter trial evaluating sirolimus for the treatment of kidney angiomyolipomas associated with TSC or TSC/LAM. All participants were treated with sirolimus from 0–12 months. During months 12–24, sirolimus was discontinued in one subgroup. The other subgroup was treated with additional sirolimus.

Setting

Adult TSC participants were recruited from six clinical sites in the United States (comprehensive TSC clinics, 5; urology clinic, 1).

Patients

There were 28 TSC patients who completed all 24 months of the study and serum samples were available at 24 months from 18/28 patients.

Main Outcome Measure(s)

We compared the percent change in VEGF-D levels (baseline to 24 months) in patients from the two treatment subgroups.

Results

At 24 months, VEGF-D levels decreased by 67% compared with baseline (to 787±426 pg/ml) in the ON SIROLIMUS AFTER 12 MONTHS group versus a 13% decrease (to 2971±4014 pg/ml) in the OFF SIROLIMUS AFTER 12 MONTHS group (p = 0.013, Mann-Whitney test). A similar trend was observed in kidney angiomyolipoma size but not in pulmonary function tests. Conclusions Serum VEGF-D may be useful for monitoring response to treatment with sirolimus and kidney angiomyolipoma size in patients with TSC, but confirmation is needed.

Trial Registration

Clinical trials.gov {"type":"clinical-trial","attrs":{"text":"NCT00126672","term_id":"NCT00126672"}}NCT00126672.     

The habitat functional response links seasonal third‐order selection to second‐order landscape characteristics

Determining how animals respond to differences in resource availabilities across spatiotemporal extents is critical to our understanding of organism distributions. Variations in resource distribution leading to changes in spatial arrangements across landscapes are indicative of a habitat functional response. Our goal was to assess how resource availabilities influenced both second‐order (i.e., home ranging behavior) and third‐order (i.e., habitat or resource selection) selection by feral pigs (Sus scrofa) in an agricultural landscape. We defined agriculturally based seasons to estimate home range characteristics using autocorrelated kernel density estimation within each season. We then modeled home range size as a function of resource availability (i.e., resource selection analyses) to determine whether individual behaviors were predicted by shifts in home ranging behavior. Both home range analyses and resource selection analyses indicated seasonal differences in selection for agricultural resources as availabilities changed, suggesting second‐ and third‐order selection is mechanistically linked through a habitat functional response.     

Interleukin-7-aggravated joint inflammation and tissue destruction in collagen-induced arthritis is associated with T-cell and B-cell activation

Introduction

We sought to investigate the capacity of interleukin (IL)-7 to enhance collagen-induced arthritis and to study by what mechanisms this is achieved.

Methods

Mice received multiple injections with IL-7 or phosphate-buffered saline (PBS) as a control. Arthritis severity and incidence were determined by visual examination of the paws. Joint destruction was determined by assessing radiographs and immunohistochemistry of the ankle joints. Total cellularity and numbers of T-cell and B-cell subsets were assessed, as well as ex vivo production of interferon-γ (IFN-γ), IL-17, and IL-4. Proinflammatory mediators were measured in serum with multianalyte profiling.

Results

IL-7 increased arthritis severity and radiology-assessed joint destruction. This was consistent with IL-7-increased intensity of cell infiltrates, bone erosions, and cartilage damage. Splenic CD19⁺ B cells and CD19⁺/GL7⁺ germinal center B cells, as well as CD4 and CD8 numbers, were increased by IL-7. IL-7 expanded memory T cells, associated with increased percentages of IFN-γ-, IL-4-, and IL-17-producing CD4⁺ T cells. On antigen restimulation of draining lymph node cells in vitro IL-7 treatment was found to increase IFN-γ and IL-17 production, whereas IL-4 was reduced. IL-7 also increased concentrations of proinflammatory mediators, indicative of T-cell activation (sCD40L), vascular activation (VCAM-1, VEGF), tissue destruction (fibroblast growth factor-basic (FGF-b), LIF), and chemotaxis (MIP-1γ, MIP-3β, lymphotactin, MDC, and MCP-5).

Conclusions

In arthritic mice, IL-7 causes expansion of T and B cells, associated with increased levels of proinflammatory mediators. IL-7 intensifies arthritis severity and joint destruction, accompanied by increased Th1 and Th17 activity. These data indicate that IL-7 could be an important mediator in arthritic conditions and that targeting IL-7 or its receptor represent novel therapeutic strategies.