La Sindrome Antifosfolipidi: revisione critica degli attuali aspetti diagnostici, patogenetici e terapeutici

La sindrome antifosfolipidi (aPS) identifica una condizione ad aumentato rischio di occlusione vascolare e/o complicanze gravidiche, la cui definizione è stata stabilita nel 2005 sulla base di un consenso internazionale. I pazienti sono definiti affetti da aPS se presentano almeno un criterio clinico (occlusione vascolare e/o complicanza gravidica) e un criterio di laboratorio, in un definito periodo di tempo; i criteri di laboratorio che definiscono la aPS sono la positività ripetuta (confermata dopo 12 settimane) per la ricerca degli anticorpi del tipo lupus anticoagulante (LA) e/o la positività per gli anticorpi in fase solida anti-cardiolipina (aCL) o anti- β2glicoproteina I (β2GPI). Nonostante i progressi degli ultimi 20 anni dovuti all’interesse di molti specialisti, immunologi, ematologi, reumatologi o ginecologi, la aPS rimane non completamente conosciuta in molti aspetti, tuttora controversi e discussi. Scopo della presente tesi è di presentare un aggiornamento sui criteri di laboratorio attualmente applicati, sulle ipotesi patogenetiche in discussione e sulle proposte terapeutiche nelle varie e differenti manifestazioni cliniche, con particolare riferimento alla terapia anticoagulante

Cornelia de Lange syndrome and cancer: An open question

[No abstract

PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages.

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location

A Mutant-p53/Smad Complex Opposes p63 to Empower TGFβ-Induced Metastasis

SummaryTGFβ ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFβ-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFβ acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFβ-dependent inhibition of p63 function

Comparison by qRT-PCR of Stemness and Differentiation Marker Expression in Cultured Colon Cells and Microdissected Colon Crypt Populations.

<p>mRNA levels of Lgr5, Msi-1 and CK20 genes were evaluated by qRT-PCR on FACS-sorted PKH^high, PKH^low and PKH^neg cells at the 3^rd week of culture (Panel A) and on Differentiated (Diff), TA and CBC cells (Panel C) isolated by laser microdissection from colon crypts (as shown by the picture reported in Panel B). Data are shown as mean values ± SD calculated as described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043379#s2" target="_blank"><i>Materials and Methods</i></a>, normalized to the housekeeping gene β₂-microglobulin and relative to the reference sample (PKH^low for sorted cultured cells, and TA for cells isolated by microdissection). * p<0.05.</p

<i>In vitro</i> Differentiation of Cultured PKH^pos Cells.

<p>(A) After 3 weeks of culture the spheroids obtained from PKH-stained cells maintained in serum-free medium in non-adherent plates (upper panel) were dissociated, and PKH^pos cells were FACS-sorted and cultured in the presence of 10% FCS in adherent plates or included into Matrigel. In both conditions, the cells changed their morphology (lower panel), and in the presence of Matrigel they formed branched structures surrounding a “hole”. (B) Cytofluorimetric analysis of Msi-1 and Lgr5 expression in PKH^pos cells cultured for 3 weeks in serum-free conditions (Spheroids) and after 10 days in differentiating conditions (Differentiated). (C) WB analysis of Msi-1 and Muc-1 proteins in Spheroids and Differentiated cell lysates. α-tubulin was used as a control for protein contents. (D) qRT-PCR analysis of CK20, Muc-1 and Muc-2 expression in Spheroids and Differentiated cells. Data are calculated as mean values ± SD as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043379#s2" target="_blank"><i>Materials and Methods</i></a>, normalized to the housekeeping gene β₂-microglobulin and relative to the reference sample (spheroid cells). *p<0.05. (E) Confocal microscopy analysis of differentiated cells. In the presence of serum and adhesion conditions, the cells presented a fragmented PKH26 dye pattern along the cellular membrane, completely loosing the expression of Msi-1 while acquiring Muc-1 (sample # 1) and CK20 (samples #2) expression.</p

Expression of Msi-1 and CK20 in Cultured Colon Cells.

<p>(A) Immunohistochemical analysis of CK20 showed labeling of the epithelial cells along the crypt wall and at the mucosal surface, but not at the crypt bottom. (B) Cytofluorimetric analysis of Msi-1 and CK20 expression by PKH^neg, PKH^low and PKH^high populations at the 3^rd week of culture; a small subset of PKH^high cells co-expressed both markers. One representative experiment out of 4 consecutive is shown. (C) Confocal microscopy analysis of CK20 and Msi-1 expression in PKH^pos cells. The cells co-expressing both markers are indicated by an arrow. Magnification 40×. (D) Immunofluorescence analysis of Msi-1 and CK20 expression in frozen colon sections. Double-positive cells are evident at the very crypt base. (E) qRT-PCR analysis of Muc-1 and Muc-2 expression on FACS-sorted Msi-1⁺/CK20⁻ and Msi-1⁺/CK20⁺ subsets at the 3^rd week of culture. Data are calculated as mean values ± SD as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043379#s2" target="_blank"><i>Materials and Methods</i></a>, normalized to the housekeeping gene β₂-microglobulin and relative to the reference sample (Msi-1⁺/CK20⁻ cells). * p<0.05.</p

Sequences of the primers used in qRT-PCR experiments.

<p>Sequences of the primers used in qRT-PCR experiments.</p