Killer immunoglobulin-like receptors (KIR) and their HLA-ligands in Italian paroxysmal nocturnal haemoglobinuria (PNH) patients.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic disorder characterized by expansion of phosphatidylinositol glycan-A-defective progenitor(s). Immune-dependent mechanisms, likely involving a deranged T cell-dependent autoimmune response, have been consistently associated with the selection/dominance of PNH precursors. Natural killer (NK) lymphocytes might participate in PNH pathogenesis, but their role is still controversial. NK activity is dependent on the balance between activating and inhibiting signals. Key component in such regulatory network is represented by killer immunoglobulin-like receptors (KIR). KIR are also involved in the regulation of adaptive cytotoxic T cell response and associated with autoimmunity. This study investigated on the frequency of KIR genes and their known human leukocyte antigen (HLA) ligands in 53 PNH Italian patients. We observed increased frequency of genotypes characterized by ≤2 activating KIR as well as by the presence of an inhibitory/activating gene ratio ≥3.5. In addition, an increased matching between KIR-3DL1 and its ligand HLA-Bw4 was found. These genotypes might be associated with lower NK-dependent recognition of stress-related self molecules; this is conceivable with the hypothesis that an increased availability of specific T cell targets, not cleared by NK cells, could be involved in PNH pathogenesis. These data may provide new insights into autoimmune PNH pathogenesis.

TGF-ß1 exposure induces epithelial to mesenchymal transition both in CSCs and non-CSCs of the A549 cell line, leading to an increase of migration ability in the CD133+ A549 cell fraction.

Metastasis is the leading cause of death by cancer. Non-small-cell lung cancer (NSCLC) represents nearly 85% of primary malignant lung tumours. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Transforming growth factor-ß1 (TGF-ß1) is the major inductor of EMT. The aim of this study is to investigate TGF-ß1's effect on cancer stem cells (CSCs) identified as cells positive for CD133, side population (SP) and non-cancer stem cells (non-CSCs) identified as cells negative for CD133, and SP in the A549 cell line. We demonstrate that TGF-ß1 induces EMT in both CSC and non-CSC A549 sublines, upregulating the expression of mesenchymal markers such as vimentin and Slug, and downregulating levels of epithelial markers such as e-cadherin and cytokeratins. CSC and non-CSC A549 sublines undergoing EMT show a strong migration and strong levels of MMP9 except for the CD133(-) cell fraction. OCT4 levels are strongly upregulated in all cell fractions except CD133(-) cells. On the contrary, wound size reveals that TGF-ß1 enhances motility in wild-type A549 as well as CD133(+) and SP(+) cells. For CD133(-) and SP(-) cells, TGF-ß1 exposure does not change the motility. Finally, assessment of growth kinetics reveals major colony-forming efficiency in CD133(+) A549 cells. In particular, SP(+) and SP(-) A549 cells show more efficiency to form colonies than untreated corresponding cells, while for CD133(-) cells no change in colony number was observable after TGF-ß1 exposure. We conclude that it is possible to highlight different cell subpopulations with different grades of stemness. Each population seems to be involved in different biological mechanisms such as stemness maintenance, tumorigenicity, invasion and migration.

Prognostic value of cancer stem cells, epithelial-mesenchymal transition and circulating tumor cells in lung cancer.

The epithelial-mesenchymal transition (EMT) is a program involved in embryonic development that is often activated during cancer invasion and metastasis. CD133 is the main marker identifying cancer stem cells (CSCs) in lung cancer. Circulating tumor cells (CTCs) are demonstrated to be useful as a biomarker for the diagnosis and treatment of cancer. The aim of this study was to correlate EMT, CSCs and CTCs with patient prognosis to verify whether they can contribute to better stratification of lung cancer patients at risk for recurrent and metastatic disease. Pulmonary venous blood was drawn after major pulmonary surgery in 45 patients with resectable non-small cell lung cancer (NSCLC) in order to identify CTCs. For the same patients, we also constructed prognostic lung tissue microarrays (TMA) for CD133 and c-kit and evaluated CSC and EMT markers using flow cytometry. Cytokeratin-positive cells were detectable in 11 (23.9%) cases. c-kit expression was heterogeneous in prognostic TMAs while CD133 expression was low or absent which was also confirmed by flow cytometry and RT-PCR. Flow cytometric analysis showed that the mean percentage of cells with CD133 expression was 1.6%. CD90 and CD326 markers were co-expressed with a mean percentage of 10.41%. When CD133 and CD90/CD326 expression was correlated with follow-up, CD133 showed a higher correlation with deceased patients when compared with CD90/CD326 co-expression (32.5 vs. 9.5%). CD133 expression demonstrated a strong significant association with patients exhibiting progressive disease when compared to CD90/CD326 expression (15 vs. 7.1%). CD133 may be significantly associated with invasion and metastatic spread of NSCLC. The co-expression of CD90, CD326 and CD133 has definite prognostic value in patients with NSCLC.

Triphasic waves and cerebral tumors.

Three patients with cerebral tumor involving diencephalic midline structures (2 malignant gliomas, 1 craniopharyngioma) and no metabolic abnormalities developed disorientation in time and place or coma with triphasic waves (TW) seen on electroencephalograms. Serial EEG recordings showed persistence or disappearance of TW depending on poor or good outcome of the antiedema treatment. TW have been described with disorders affecting the brain diffusely, as metabolic encephalopathies, dementing processes, cerebral carcinomatosis and baclofen intoxication. The findings described here demonstrate that TW may occur in patients with brain tumor involving subcortical midline structures.