CD28 interaction with filamin-A controls lipid raft accumulation at the T-cell immunological synapse

During physiological T-cell stimulation by antigen presenting cells (APCs), a major T-cell membrane rearrangement is known to occur leading to the organization of 'supramolecular activation clusters' at the immunological synapse. A possible role for the synapse is the generation of membrane compartments where signalling may be organized and propagated. Thus, engagement of the costimulatory molecule CD28 at the immunological synapse promotes the organization of a signalling compartment by inducing cytoskeletal changes and lipid raft accumulation. We identified the actin-binding protein Filamin-A (FLNa) as a novel molecular partner of CD28. We found that, after physiological stimulation, CD28 associated with and recruited FLNa into the immunological synapse, where FLNa organized CD28 signalling. FLNa knockdown by short interfering RNA (siRNA) inhibited CD28-mediated raft accumulation at the immunological synapse and T-cell costimulation. Together, our data indicate that CD28 binding to FLNa is required to induce the T-cell cytoskeletal rearrangements leading to recruitment of lipid microdomains and signalling mediators into the immunological synapse.     

Arachidonic acid suppresses growth of human lung tumor A549 cells through down-regulation of ALDH3A1 expression

Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) in certain normal and tumor cells is associated with protection against the growth inhibitory effect of reactive aldehydes generated during membrane lipid peroxidation. We found that human lung tumor (A549) cells, which express high levels of ALDH3A1 protein, were significantly less susceptible to the antiproliferative effects of 4-hydroxynonenal compared to human hepatoma HepG2 or SK-HEP-1 cells that lack ALDH3A1 expression. However, A549 cells became susceptible to lipid peroxidation products when they were treated with arachidonic acid. The growth suppression of A549 cells induced by arachidonic acid was associated with increased levels of lipid peroxidation and with reduced ALDH3A1 enzymatic activity, protein, and mRNA levels. Furthermore, arachidonic acid treatment of the A549 cells resulted in an increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma), whereas NF-kappaB binding activity was inhibited. Blocking PPARgamma using a selective antagonist, GW9662, prevented the arachidonic acid-mediated reduction of ALDH3A1 expression as well as the growth inhibition of A549 cells, suggesting the central role of PPARgamma in these phenomena. The increase in PPARgamma and the reduction in ALDH3A1 were also prevented by exposing cells to vitamin E concomitant with arachidonic acid treatment. In conclusion, our data show that the arachidonic acid-induced suppression of A549 cell growth is associated with increased lipid peroxidation and decreased ALDH3A1 expression, which may be due to activation of PPARgamma.     

A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees

Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.     

Selective increase in serum IgE following enfuvirtide administration in HIV-1 infected multidrug resistant patients

Enfuvirtide (ENF) is the first HIV-1 entry inhibitor used in clinical practice and is currently administered via the subcutaneous route. We here evaluated whether ENF administration leads to a change in humoral parameters, including IgE, possibly related to ENF-associated injection site reactions (ISRs). A 24-week prospective study was conducted in multidrug resistant patients enrolled in the ENF Early Access Program characterized by CD4 counts < or =100 cells/microlitre and no other active antiretroviral drug. Licensed commercial laboratory assays were used to measure the parameters considered in this study. Results are reported as medians (interquartiles IQR). Statistical analyses were performed using Wilcoxon sign rank, Wilcoxon rank sum and Kruskall Wallis tests. Total IgM, IgA and IgG did not change significantly throughout the study. Conversely, a significant increase in IgE was observed in all patients, in those with normal as well as in those with altered IgE at baseline (BL). ISRs such as induration and number of nodules were more frequent in patients with altered BL IgE. IgE increased significantly in all patients, regardless of the different stratifications in their BL CD4 counts. Of note, the ENF-induced increase in CD4 occurred significantly in all patients, independently of their BL IgE levels. The immunological response associated with ENF treatment is accompanied by a selective increase in IgE levels. Determination of IgE could be used in the monitoring ISRs associated with ENE     

Rapid detection and identification of Mycobacterium tuberculosis by Real Time PCR and Bactec 960 MIGT

We have developed a Real-Time PCR assay to detect M. tuberculosis using the iCycler iQ detection system by TaqMan assay directly on the clinical specimen. A total of 513 clinical samples were taken from patients with suspected tuberculosis and other patients that had an active mycobacterial infection, as well as patients with diagnosed tuberculosis who were receiving antitubercular therapy. The sensitivity and specificity of this assay, 10% and 100%, respectively, were compared to those of conventional microbiological methods.     

A clinical genetic method to identify mechanisms by which pain causes depression and anxiety

Most current methods for assessing pain in animals are based on reflexive measures and require constant interaction between the observer and the animal. Here we explore two new fully automated methods to quantify the impact of pain on the overall behavior of the organism. Both methods take advantage of the animals' natural preference for a dark environment. We used a box divided into two compartments: dark and bright. In the motoric operant task, "AngleTrack", one end of the box was raised so that the animals had to climb uphill to go from the light to the dark compartment. In the thermal operant task, "ThermalTrack", the floor of the dark compartment was heated to a given temperature, while the light compartment remained at 25°C. Rats were individually placed in the light box and their crossing between chambers monitored automatically for 30 minutes. The angle of the box, or the temperature of the dark compartment, was altered to challenge the animals' natural preference. We test the hypothesis that different models of pain (inflammatory or neuropathic) can be differentiated based on performance on these devices. Three groups of rats were tested at five different challenge levels on both tasks: 1) normal, 2) neuropathic injury pain (Spared Nerve Injury), and 3) inflammatory pain (intraplantar injection of Carrageenan). We monitored the position of the animals as well as their rate of switching between compartments. We find significant differences between the three groups and between the challenge levels both in their average position with respect to time, and in their switching rates. This suggests that the angle-track and thermal-track may be useful in assessing automatically the global impact of different types of pain on behavior.     

High sensitivity analysis of oxprenolol in urine by capillary electrophoresis with C18 packed on-line preconcentrator

High sensitivity analysis of oxprenolol in spiked human urine has been performed by capillary zone electrophoresis (CZE) in ammonium formate buffer pH 2.5 using an uncoated capillary with 1cm length C18 on-capillary preconcentrator at the inlet side. The preconcentrator was fabricated in laboratory using the packing method and not encapped C18 5 microm particles as stationary phase material. The packed path was retained into the capillary by sintered stationary phase frits. Before running the CZE analysis, the oxprenolol was eluted from the preconcentrator by injecting a short plug of acetonitrile/water mixtures. With respect to classical CZE, the use of on-line preconcentrator widely increased the method sensitivity allowing the detection of the drug at 0.5 ng/mL (injected concentration). The method showed a linear response in the range of 1-150 ng/mL oxprenolol standard compound. The intra-day repeatability (n = 11) R.S.D. values for migration time, peak area and normalized peak area were 0.72%, 3.96% and 3.66%, respectively, while inter-day repeatability (n = 5 days) R.S.D. values were 2.74%, 9.41% and 9.83%, respectively. The method was successfully applied to the analysis of oxprenolol in extracted urine spiked at 250 pg/mL (oxprenolol LOQ concentration in urine).     

Molecular identification of Tuber magnatum ectomycorrhizae in the field

Tuber ectomycorrhizae in a Tuber magnatum "truffière", located in Central Italy, were studied using molecular methods. Specifically, RFLP-ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the "truffière" under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively).