Fate of lymphocytes after withdrawal of tofacitinib treatment.

Tofacitinib (Tofa) is an inhibitor of Janus Kinase 3, developed for the treatment of autoimmune diseases and for the prevention of transplant rejection. Due to its selective action on proliferating cells, Tofa can offer a way to block T cell activation, without toxic effects on resting cells. However, few studies have investigated the effects of Tofa on lymphocyte activation in vitro. Our aim was to study the action of Tofa on different lymphocyte subsets after in vitro stimulation and to track the behaviour of treated cells after interruption of the treatment. Peripheral blood lymphocytes were stimulated in vitro with mitogen and treated with two concentrations of Tofa. After a first period in culture, cells were washed and further incubated for an additional time. Lymphocyte subsets, activation phenotype and proliferation were assessed at the different time frames. As expected, Tofa was able to reduce the activation and proliferation of lymphocytes in the first four days of treatment. In addition the drug led to a relative decrease of Natural Killer, B cells and CD8 T cells compared to CD4 T cells. However, treated cells were still viable after the first period in culture and begun to proliferate, strikingly, in a dose dependent manner when the drug was removed from the environment by replacing the culture medium. This novel data does not necessarily predict a similar behaviour in vivo, but can warn about the clinical use of this drug when a discontinuation of treatment with Tofa is considered for any reason.

Differential proteomic analysis of hepatocellular carcinoma.

The principal aim of the present study consisted in the identification of the disregulated proteins associated with the development of hepatocellular carcinoma (HCC). The differences in protein expression between hepatocellular carcinoma (HCC) and the corresponding non-HCC liver tissues were investigated in a cohort of 20 patients using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). The up- and down-regulated protein spots that exhibited 1.5-fold difference signal intensity with statistical significance (p<0.05, t-test, confidence intervals 95%) were excised from the gel and identified by peptide mass fingerprinting using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Thirty-six protein spots corresponding to 29 different disregulated proteins, belonging to heterogeneous metabolic pathways, have been identified. Down-regulated proteins (n=23) were found superior in number than the up-regulated proteins (n=6). Detoxification, carbohydrate metabolism and amino acid biotrasformation represented the main disregulated pathways in HCC. Up-regulation of aldo-keto reductase 1C2, thioredoxin and transketolase, involved in metabolic and regulatory cellular processes including proliferation, differentiation and carcinogenesis were remarkable. These proteins could represent useful biomarkers to provide new insights into global pathophysiologic changes of HCC and for the development of new pharmacological approaches to HCC therapy.

EMILINs interact with anthrax protective antigen and inhibit toxin action in vitro.

The informational spectrum method (ISM) is a virtual spectroscopy method for the fast analysis of potential protein-protein relationships. By applying the ISM approach to the GeneBank protein database the vascular proteins EMILIN1 (Elastin Microfibril Interface Located ProteIN), EMILIN2, MMN1, and MMN2 were identified as additional anthrax PA antigen interacting molecules. This virtual molecular interaction was formally proven by solid phase assays using recombinant proteins. The interaction is independent of the presence of divalent cations and does not involve PA aspartic residue at 683, a critical residue in receptor binding. In fact, the D683A point mutation fully prevented the cell intoxication ability of PA in the presence of Lethal Factor, but it was fully ineffective on the binding of mutated PA to EMILIN1 and EMILIN2. The ISM approach also led to the identification of the potential interaction sites between PA and EMILINs. A PA mutant with a deletion at residue D425 and solid phase protein-protein interaction studies as well as deletion mutant of EMILIN2 confirmed the hypothesized interaction site. Our findings imply that the PA-cell surface receptor interaction is not likely to provide the full explanation for the vascular lesions and prominent hemorrhages that follow Bacillus anthracis infection and spreading and call into play vascular associated proteins such as EMILINs as potential inhibitory proteins.

The relative endogenous expression levels of the IFNAR2 isoforms influence the cytostatic and pro-apoptotic effect of IFNalpha on pleomorphic sarcoma cells.

Based on our previous studies where we found that IFNAR2-1, the short IFNalpha/beta receptor variant, was expressed in pleomorphic sarcoma cells, we decided to determine the relative levels of expression of IFNAR2.1 versus the longer form, named IFNAR2.2, in different pleomorphic sarcoma cells in relation to their response to interferon alpha treatment. When examining a panel of PS cells isolated from surgical specimens, we found that IFNAR2.1 prevailed in 6 out 7 lines analysed and that these generally showed cell cycle arrest and low levels of apoptosis upon IFNalpha treatment. The reverse ratio, i.e. higher constitutive levels of IFNAR2.2 than IFNAR2.1, was associated with an irreversible inhibition of cell growth and pronounced apoptosis. Impairment of tumour growth by low- and high-dose IFNalpha treatment of nude mice inoculated with PS cells expressing predominantly IFNAR2.1 further asserted the effect of the cytokine also in vivo. A proteomic analysis of 120 signalling components in growth arrested, apoptotic PS cells harbouring higher levels of IFNAR2.2 revealed engagement of the canonical Jak/Stat/ISGF3-pathway, the activation of the mitochodrial apoptotic pathway and a potentially novel mechanism of cell cycle blockade unrelated to down-regulation of cyclin A/B and their interacting/regulating kinases. Our results confirm the dominant negative role of IFNAR2.1, but also suggest that the relative endogenous levels of the two IFNalpha/beta receptor isoforms may dictate the signalling pathways triggered by the ligand, such as to cause exclusively cell cycle arrest or induce programmed cell death. This parameter may be of importance for the clinical outcome of IFNalpha treatment of PS.