Short exposure of albumin to high concentrations of malondialdehyde does not mimic physiological conditions

Malondialdehyde (MDA), a major lipid peroxidation product, spontaneously binds to, and modifies proteins. In vivo, proteins are physiologically exposed to micromolar MDA concentrations for long periods. In order to mimic this process in vitro, protein modification is often performed by short exposure to millimolar MDA concentrations, also in order to generate antigenic structures for antibody production.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.     

Loss of miR‐101 expression promotes Wnt/β‐catenin signalling pathway activation and malignancy in colon cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer‐related mortality in Western countries. Although the aberrant expression of several microRNAs (oncomiRs) is associated with CRC progression, the molecular mechanisms of this phenomenon are still under investigation. Here we show that miR‐101 expression is differentially impaired in CRC specimens, depending on tumour grade. miR‐101 re‐expression suppresses cell growth in 3D, hypoxic survival and invasive potential in CRC cells showing low levels of miR‐101. Additionally, we provide molecular evidence of a bidirectional regulatory mechanism between miR‐101 expression and important CRC pro‐malignant features, such as inflammation, activation of the Wnt/β‐catenin signalling pathway and epithelial–mesenchymal transition (EMT). We then propose that up‐regulated miR‐101 may function as a tumour suppressor in CRC and that its pharmacological restoration might hamper the aggressive behaviour of CRC in vivo. MiR‐101 expression may also represent a cancer biomarker for CRC diagnosis and prognosis. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Comparison of two nucleic acid extraction and testing systems for HCMV-DNA detection and quantitation on whole blood specimens from transplant patients

Quantitative detection of human cytomegalovirus (HCMV) DNA on whole blood is currently the primary choice for virological monitoring in transplant patients and for determining the appropriate antiviral strategy, however specific issues of variability remain in terms of extraction methods, amplification efficiency, and variability. This study compared the performance characteristics of two nucleic acid extraction and testing systems for HCMV-DNA quantitation, the artus^® CMV QS-RGQ kit, associated with a fully automated DNA extraction and assay set up by Qiagen (system 1) and the Q-CMV Real Time Complete kit by Nanogen, associated with a semiautomated nucleic acid extraction system by Biomérieux (system 2) in 189 specimens from transplant patients and 10 from 2012 HCMV Quality Control for Molecular Diagnostics (QCMD). The two systems exhibited a 80.4% concordance. Differences between the two systems were within ±1 log₁₀ copies/ml of the averaged log₁₀ results for 88.9% of the tested specimens. For all qualitatively discordant specimens, mean viral load was ≤3 log₁₀ copies/ml. Considering viral load measurement, system 1 gave earlier positives that system 2, with a 14.8% of specimens resulted positive at low viral loads with system 1 and negative with system 2. In QCMD specimens, difference was below 0.7 log₁₀ copies/ml for both the systems.     

Detection of human cytomegalovirus in transbronchial biopsies from lung transplant recipients

The role of human cytomegalovirus (HCMV) in lung transplantation (LT) and drawbacks related to viral quantification in bronchoalveolar lavage (BAL) underline the potential usefulness of investigating other specimens. Thirty-three LT recipients were prospectively studied by HCMV quantitative real time PCR on matched transbronchial biopsy (TBB), BAL, and whole blood specimens. Overall, 27/33 patients turned out HCMV-positive in at least one specimen: 7.1 %, 37.1 %, and 13.5 % of TBB, BAL, and blood samples, respectively. No significant association between HCMV on all types of specimens and acute rejection, lymphocytic bronchiolitis, bronchiolitis obliterans and bronchiolitis obliterans syndrome was found. HCMV pneumonia was associated to HCMV detection on TBB (p = 0.003) and whole blood (p = 0.008), not on BAL (p = 0.47). The highest mean viral load was detected in TBB from cases with HCMV pneumonia in comparison to all other cases, suggesting the potential use of HCMV investigation in TBB for evaluating posttransplant complications.     

Inhibition of aquaporin-1 dependent angiogenesis impairs tumour growth in a mouse model of melanoma

Prohibiting angiogenesis is an important therapeutic approach for fighting cancer and other angiogenic related diseases. Research focused on proteins that regulate abnormal angiogenesis has attracted intense interest in both academia and industry. Such proteins are able to target several angiogenic factors concurrently, thereby increasing the possibility of therapeutic success. Aquaporin-1 (AQP1) is a water channel membrane protein that promotes tumour angiogenesis by allowing faster endothelial cell migration. In this study we test the hypothesis that AQP1 inhibition impairs tumour growth in a mouse model of melanoma. After validating the inhibitor efficacy of two different AQP1 specific siRNAs in cell cultures, RNA interference experiments were performed by intratumoural injections of AQP1 siRNAs in mice. After 6 days of treatment, AQP1 siRNA treated tumours showed a 75 % reduction in volume when compared to controls. AQP1 protein level, in AQP1 knockdown tumours, was around 75 % that of the controls and was associated with a significant 40 % reduced expression of the endothelial marker, Factor VIII. Immunofluorescence analysis of AQP1 siRNA treated tumours showed a significantly lower microvessel density. Time course experiments demontrated that repeated injections of AQP1 siRNA over time are effective in sustaining the inhibition of tumour growth. Finally, we have confirmed the role of AQP1 in sustaining an active endothelium during angiogenesis and we have shown that AQP1 reduction causes an increase in VEGF levels. In conclusion, this study validates AQP1 as a pro-angiogenic protein, relevant for the therapy of cancer and other angiogenic-related diseases such as psoriasis, endometriosis, arthritis and atherosclerosis.