Tocilizumab in the treatment of patients with rheumatoid arthritis in real clinical practice: results of an Italian observational study

To describe the effectiveness and safety of tocilizumab (TCZ), an interleukin-6 receptor inhibitor, in a cohort of patients with rheumatoid arthritis (RA) recruited in clinical practice

Molecular evidence for a thymus-independent partial T cell development in a FOXN1-/- athymic human fetus.

The thymus is the primary organ able to support T cell ontogeny, abrogated in FOXN1(-/-) human athymia. Although evidence indicates that in animal models T lymphocytes may differentiate at extrathymic sites, whether this process is really thymus-independent has still to be clarified. In an athymic FOXN1(-/-) fetus, in which we previously described a total blockage of CD4(+) and partial blockage of CD8(+) cell development, we investigated whether intestine could play a role as extrathymic site of T-lymphopoiesis in humans. We document the presence of few extrathymically developed T lymphocytes and the presence in the intestine of CD3(+) and CD8(+), but not of CD4(+) cells, a few of them exhibiting a CD45RA(+) naïve phenotype. The expression of CD3εεpTα, RAG1 and RAG2 transcripts in the intestine and TCR gene rearrangement was also documented, thus indicating that in humans the partial T cell ontogeny occurring at extrathymic sites is a thymus- and FOXN1-independent process

Differential effect of 2-hydroxyoleic acid enantiomers on protein (sphingomyelin synthase) and lipid (membrane) targets

The complex dual mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent anti-tumor compound used in membrane lipid therapy (MLT), has yet to be fully elucidated. It has been demonstrated that 2OHOA increases the sphingomyelin (SM) cell content via SM synthase (SGMS) activation. Its presence in membranes provokes changes in the membrane lipid structure that induce the translocation of PKC to the membrane and the subsequent overexpression of CDK inhibitor proteins (e.g., p21Cip1). In addition, 2OHOA also induces the translocation of Ras to the cytoplasm, provoking the silencing of MAPK and its related pathways. These two differential modes of action are triggered by the interactions of 2OHOA with either lipids or proteins. To investigate the molecular basis of the different interactions of 2OHOA with membrane lipids and proteins, we synthesized the R and S enantiomers of this compound. A molecular dynamics study indicated that both enantiomers interact similarly with lipid bilayers, which was further confirmed by X-ray diffraction studies. By contrast, only the S enantiomer was able to activate SMS in human glioma U118 cells. Moreover, the anti-tumor efficacy of the S enantiomer was greater than that of the R enantiomer, as the former can act through both MLT mechanisms. The present study provides additional information on this novel therapeutic approach and on the magnitude of the therapeutic effects of type-1 and type-2 MLT approaches. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy

Identification of lymphocytes at extrathymic sites of differentiation in a Nude/SCID human fetus.

<p>(A, B) Immunohistochemical detection of lymphocytes at extrathymic sites of differentiation in FOXN1^−/− human fetus (16 weeks of gestation). (A) Stem cells, B cells and NK cells were detected by immunohistochemical stain for CD34 (brown), CD20 (brown) and CD56 (brown) in intestinal and liver sections obtained from a FOXN1^−/− human fetus (16 weeks of gestation) or an aged-matched control fetus. In CD34 and CD20 stained intestine sections from WT and FOXN1^−/− fetuses original magnification was 100x. (B) As negative control, intestinal sections from FOXN1^−/− fetus were counterstained with hematoxylin and with the isotype control (primary antibody omitted) by DAB. As positive control, hyperplastic lymph node sections were stained for CD3 (brown) by DAB. Intestinal or liver sections of FOXN1^−/− human fetus were counterstained with hematoxylin and eosin (H&E). Immunohistochemical analysis of intestinal or liver sections of a FOXN1^−/− human fetus and an aged-matched control fetus using anti-CD3 staining to mark T cells, anti-CD4 staining to mark T helper cells and anti-CD8 staining to mark cytotoxic T cells. DAB, 200x. Representative results from two independent experiments with two distinct samples are shown. (C, D) Confocal microscopy of FOXN1^−/− intestinal sections. (C) Labeling with anti-human CD4 (red) and anti-human CD8 (green). (D) Labeling with anti-human CD7 (green) and anti-human CD3 (red). Representative results from three independent experiments are shown. (E) RT-PCR analysis of CD3ε intestinal expression. CD3ε transcript expression in human skin fibroblasts (negative control), intestinal lymphocytes from WT or FOXN1^−/− fetuses is shown. Blanck, no cDNA. β-actin was used as loading control. Representative results from three independent experiments are shown. (F) Quantitative real-time PCR showing the expression of mRNAs encoding CD3ε (relative to β-actin) in skin fibroblasts (negative control), thymus (positive control) and intestinal tissue of control and FOXN1^−/− fetuses (16 weeks of gestation). Representative results from two independent experiments are shown.</p

Local production of T cells in the FOXN1^−/− human intestine.

<p>(A) Quantitative real-time PCR showing the expression of mRNAs encoding RAG1, RAG2 and pTα (relative to β-actin) in skin fibroblasts (negative control), thymus (positive control) and intestinal tissue of control and FOXN1^−/− fetuses (16 weeks of gestation). (B) Comparison of TCR Vβ-region usage between intestinal lymphocytes (black bars) and CBMCs (white bars) from the FOXN1^−/− fetus. Experiments were repeated three times in (A) and two times in (B).</p

Lymphocytes with naive phenotype in cord blood and intestine.

<p>Flow cytometry of CBMCs from normal and FOXN1^−/− fetuses matched for gestational age (16 weeks of gestation). Dot plots show the expression pattern of the naïve cell markers. (A) Frequencies of CBMCs expressing both CD45RA and CD3 markers. (B) Frequencies of CBMCs expressing both CD62L and CD3 markers. (C) Frequencies of CBMCs coexpressing CD45RA and CD62L markers. (D) Frequencies of CBMCs coexpressing CD45RA and CD27 markers. Experiment in (A), (B), (C) and (D) was repeated two times. Data were obtained by gating first on viable cells and later on CD45⁺ cells (A and B) or finally also on CD3⁺ (C and D). (E) Confocal microscopy of fetal intestinal sections labeled with anti-human CD3 (red) and anti-human CD45RA (green). Representative results from three independent experiments with two samples are shown.</p

Detection of extrathymically derived T lymphocytes in the cord blood of FOXN1^−/− human fetus.

<p>(A) Flow cytometry analysis of CBMCs from WT (left dot plots) or FOXN1^−/− (right dot plots) fetuses (16 weeks of gestation). CD7 and CD2 together with the CD8α and CD8β expression patterns for the gated CD45⁺CD3⁻ cells are shown. CD8α and CD4 expression is shown for the gated CD45⁺CD3⁺ cells. Numbers indicate the frequency of the cells within the gate. Experiment was repeated two times. Data were obtained by gating first on viable cells and later on CD45⁺ cells. (B) RT-PCR analysis of CD3ε expression in CBMCs. The expression of CD3ε transcript in human skin fibroblasts (negative control), human thymus (positive control), CBMCs from WT or FOXN1^−/− fetuses is shown. Blanck, no cDNA. β-actin was used as loading control. Representative results from three independent experiments are shown. (C) Quantitative real-time PCR showing the expression of mRNAs encoding CD3ε (relative to β-actin) in skin fibroblasts (negative control), thymus (positive control) and CBMCs from WT or FOXN1^−/− fetuses (16 weeks of gestation). Representative results from two independent experiments are shown.</p