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Vitamin D is the precursor of a hormone (1,25-dihydroxyvitamin D3), which has many biological effects in the skin. The immune modulator properties of vitamin D are mediated in part through effects on regulatory T cells (T-reg). Currently, in psoriasis, the relationship between vitamin D and T-reg has not well elucidated. We assess whether vitamin D status is correlated with circulating T-reg in patients affected by psoriasis and if there is a correlation with the severity of the disease evaluated with Psoriasis Area Severity Index (PASI) score.For each patient we have analyzed, PASI-score, serum levels vitamin D and regulatory T cell percentages. Spearmen's coefficient was used between serum vitamin D levels and the predictors. Subsequently, the independent predictive factors were assessed by Multiple Regression.A total of 26 patients were included in our analysis. Using no parametric Spearman's Coefficient test between serum levels of vitamin D and the single variables, we found an association with T-reg population (p0.001) and with PASI-score (p = 0.04).While vitamin D treatment induces a cytokine profile known to favor the differentiation of T cells with suppressive activity, at the same time, several studies showed how vitamin D can prime for tolerogenic dendritic cells able to favor the differentiation of Treg from T naïve cells. Low levels of vitamin-D may decrease the number of circulatory T-reg, disrupting the immunological homeostasis in psoriatic patients and encouraging the inflammatory activity.

The anaplastic thyroid cancer (ATC) is among the most aggressive human tumors which fail to respond to all the currently available therapeutic approaches. As a consequence most patients die within a few months from diagnosis. In the present preclinical study, the effects of the ZM447439, a functional inhibitor of Aurora kinases, on the growth and tumorigenicity of a panel of ATC derived cell lines (CAL-62, 8305C, 8505C and BHT-101) were evaluated. The treatment of the different ATC cells with ZM447439 inhibited proliferation in a time- and dose-dependent manner, with IC50 comprised between 0.5 mM and 5 mM. Moreover, the drug remarkably impaired the formation of colonies in soft agar of all the cell lines. Consistently with Aurora inhibition, immunofluorescence and immunoblotting experiments demonstrated that Aurora auto-phosphorylation following drug treatment was completely abrogated, and treated cells were characterized by the presence of multiple spindles with short microtubules. In the same experiments we observed the loss of histone H3 phosphorylation on Ser10, specifically due to Aurora-B, after ZM447439 treatment. Time-lapse videomicroscopy and flow cytometric analysis demonstrated that in presence of ZM447439 the cells were able to enter mitosis but not to complete it, becoming polyploid. Almost all the ATC cell lines studied showed increased apoptosis after only 48 h of treatment. In conclusion, our data demonstrate that ZM447439 is effective in reducing cell growth and tumorigenicity of different ATC derived cell lines, and further investigations are needed to exploit its potential therapeutic value for ATC treatment.

Anaplastic thyroid carcinomas (ATC) are highly aggressive tumours unresponsive to any available radio- or chemotherapeutic protocol, with a median survival rate of 4-5 months from the time of diagnosis. We previously demonstrated that ATC are characterized by increased expression of the kinases Aurora-A, -B and -C, involved in the regulation of multiple steps of the mitotic phase. In this study, the in vitro effects of SNS-314 mesylate, a pan-inhibitor of the Aurora kinases, on growth and tumorigenicity of ATC cells were evaluated.The effects of SNS-314 mesylate were assessed on the ATC derived cell lines CAL-62, 8305C, 8505C and BHT-101 by means of cell proliferation assay, immunofluorescence, cytofluorimetry, time lapse microscopy, and colony formation in soft agar.Treatment of the different ATC cells with SNS-314 mesylate inhibited proliferation in a time- and dose-dependent manner, with IC(50) comprised between 2.6 nM and 26.6 nM. CAL-62 cells exposed for 24 h to SNS-314 mesylate 100 nM evidenced a significant augmentation of the apoptotic index. Time-lapse video-microscopy of CAL-62 cells showed that SNS-314 mesylate prevents the completion of mitosis leading to polyploidy. Western blot experiments demonstrated that the auto-phosphorylation of the Aurora kinases as well as histone H3 phosphorylation in CAL-62 treated cells was inhibited. Finally, the drug inhibited colony formation in soft agar of all cell lines.Our results demonstrated that SNS-314 mesylate is capable to efficiently reduce cell growth and tumorigenicity of different ATC derived cell lines suggesting its potential therapeutic value for ATC treatment.

Integrin expression and function is largely modulated by cell activation. Here we provide evidence that long term activation of human NK cells results in a marked modulation of beta 1-integrin expression and adhesive functions. By flow cytometry and immunochemical analysis we have detected induction of alpha 1 beta 1 and alpha 2 beta 1, increased expression of alpha 4 beta 1 and alpha 5 beta 1, and decline of alpha 6 beta 1 on CD3-CD56+ NK cells generated from 10-day coculture of nonadherent PBMC with irradiated RPMI 8866 EBV+ lymphoblastoid B cell line. Adhesion assays performed on extracellular matrix-coated plates showed that, unlike fresh NK cells, long term-activated NK cells bind to native collagen I via alpha 2 beta 1 and to heat-denatured collagen I in an RGD-dependent manner, although they lose the ability to bind to laminin. In regard to the adhesion to FN, no major quantitative changes are observed after long term NK cell activation. However, whereas alpha 4 beta 1 and alpha 5 beta 1 completely mediate the adhesion of fresh NK cells to fibronectin, binding of activated NK cells is only partially beta 1-dependent and seems to involve also non-beta 1-integrin(s) recognizing and RGD sequence. The modulation of beta 1-integrin expression and the acquisition of new adhesive properties on long term-activated NK cells may be relevant for their traffic and tissue localization during inflammation and immune response.

We used Fourier Transibrm Infrared Spectroscopy (FTIR) combined with flow cytometry to study the apoptosis and necrosis processes in Jurkat, a lymphocyte cell lìne. The apoptosis was induced in the cells by a chemical agent, the actinomycin D, while the necrosis was induced lowering the pH value to 4.2. The apoptotic events were analysed by flow cytometry (using annexin V and propidium iodide) and contemporary monitored by FTIR spectroscopy at diflèrent times after the treatment. This comparison allowed us to find in the IR spectrum, between 3000 cm 1 and 2800 c--1, a "marker band" of the apoptosis corresponding to the exposure of phosphatidylserine on the outer leaflet of the membrane. A marker of a specific cellular process obtained by using a non-destructive technique such as FTIR spectroscopy, has a great signifìcance in the diagnostic medicine providing a tool for detecting pathologies in vivo.

In order to define a cellular model suitable for studying, in vitro, the molecular properties and functions of neurotrophin receptors in human lymphocytes, TrkA, TrkB, TrkC and p75(NTR) expression was investigated in a panel of EBV immortalized lymphoblastoid (LCL) and Burkitt lymphoma-derived cell lines (BLs) compared to primary B lymphocytes by RT-PCR and flow cytometric analysis. Our data show that trkA and trkB are transcribed in most B cell lines of normal and malignant origin. For several of them, we also gained first evidence of trkC expression in B cells. All cell lines and primary B cells lack p75(NTR) expression. These data suggest that neurotrophin receptors expression in the B cell lines correlates to some extent with the phenotypic maturation stage and endogenous viral activity levels. Our data suggest that TrkA and TrkB, once activated, provide a partial rescue from apoptosis, whereas TrkC stimulates the progression through the cell cycle without affecting cell survival. Finally, the identification of a number of cell lines showing single expression of one of the Trk receptors has disclosed the availability of a cellular tool for further studies on their function, and mechanisms of signal transduction in the B cell moiety in the absence of p75(NTR).

Systemic lupus erythematosus (SLE)-prone female MRL-lpr / lpr (MRL-lpr) mice were treated with mouse or rat IFN-gamma under different experimental conditions, both prophylactically in 6- to 8 week-old animals and therapeutically in 12- to 18-week-old SLE-affected mice. It was found that IFN-gamma heterogeneously modulated the course of the disease in MRL-lpr mice. When administered prophylactically, IFN-gamma favorably modulated the histological, serological and clinical signs of the disease. Relative to untreated or PBS-treated control animals, the MRL-lpr mice which received IFN gamma were virtually free of inflammatory infiltration of the kidneys and the lungs, had lower levels of azotemia with reduction of both circulating IgG1, IgG2a and IgG3 and anti-double strand (ds) and single strand (ss) DNA antibodies, milder skin vasculitis, significantly reduced enlargement of their lymph nodes and lower weight of the spleens. IFN-gamma also lowered the rate of mortality of MRL-lpr mice. In contrast to these findings, therapeutically administered IFN-gamma worsened the course of the disease in MRL-lpr mice, which exhibited increased proteinuria, higher levels of IgG2a and IgG3 and anti-ds and -ss DNA antibodies, more aggressive nephritis and died at an earlier age than PBS-treated control mice. The dichotomic effect of IFN-gamma on disease manifestation in MRL-lpr mice offers new insights into the complex role of this cytokine in the regulation of systemic autoimmunity such as SLE.

The human T cell leukemia/lymphoma virus (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL). CD4+ lymphocytes are the preferential targets of infection, even though other cell types can be infected in vitro by the virus. Although ATL cells show CD3 and CD4 surface markers, some ATL-derived cell lines were reported to express also myeloid antigens. In order to analyze possible phenotypic changes induced by HTLV-I after infection of human lymphocytes, CD4+ cells were isolated from peripheral blood of three healthy donors, by separation through immunomagnetic beads. CD4+ lymphocytes were then infected by coculture with irradiated HTLV-I producing MT-2 cells. The phenotypic profile of infected cells was studied by flow cytometric analysis using monoclonal antibodies against lymphoid (CD3, CD4, TCR alpha/beta) and myelomonocitic markers (CD13, CD14, CD15, CD33, CD34). The results show that HTLV-I immortalized cell lines coexpressed CD13, CD33 and lymphoid markers. No expression of CD14, CD15 and CD34 was observed. These data suggest that the presence of both myeloid and lymphoid phenotype in HTLV-I infected T cells is the results of an induction rather than a selection mechanism.

Recent data indicate that integrin-generated signals can modulate different receptor-stimulated cell functions in both a positive (costimulation) and a negative (inhibition) fashion. Here we investigated the ability of beta 1 integrins, namely alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors, to modulate CD16-triggered phospholipase activation in human NK cells. beta 1 integrin simultaneous cross-linking selectively inhibited CD16-induced phospholipase D (PLD) activation, without affecting either phosphatidylinositol-phospholipase C or cytosolic phospholipase A2 (PLA2) enzymatic activity. CD16-induced secretory PLA2 (sPLA2) protein release as well as its enzymatic activity in both cell-associated and soluble forms were also found to be inhibited upon beta 1 integrin coengagement. The similar effects exerted by specific PLD pharmacological inhibitors (2,3-diphosphoglycerate, ethanol) suggest that in our experimental system, sPLA2 secretion and activation are under the control of a PLD-dependent pathway. By using pharmacological inhibitors (2,3-diphosphoglycerate, wortmannin, ethanol) we also demonstrated that PLD activation is an important step in the CD16-triggered signaling cascade that leads to NK cytotoxic granule exocytosis. Consistent with these findings, fibronectin receptor engagement, by either mAbs or natural ligands, resulted in a selective inhibition of CD16-triggered, but not of PMA/ionomycin-induced, degranulation that was reversed by the exogenous addition of purified PLD from Streptomyces chromofuscus.