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CD56bright and CD56dim NK cell subsets have been proposed to represent either phenotypically and functionally distinct NK cell subsets or, respectively, immature and mature stages of the NK cell lineage. To this regard, using FLT3 ligand, IL-15 and IL-21 cytokine combination, we have recently described the in vitro generation of CD56dim/CD117neg NK cells from CD34+ hematopoietic progenitors, but not from immature CD56bright NK cells (Zamai et al. Cytometry A 2012;81:294-302), suggesting that they represent phenotypically and functionally distinct NK subsets. Based on these observations we have analyzed the NK cells developed in vitro from CD34+ progenitors cultured with FLT3-L plus IL-15 and IL-21, trying to distinguish between the two NK cell subsets. CD56bright /CD117+ NK cells generated after 15 days of culture, early expressed natural cytotoxicity receptors (NCRs), NKG2D, CD161 and CD244, while only a subset expressed granzyme-B, perforin, LFA-1, and CD94- CD159a heterodimer. Differently, virtually all CD56dim/CD117neg NK cells expressed perforin, granzyme B, CD94 and LFA-1, and only a subset of them expressed NCR and CD16 antigens. After 25 days of culture, we observed a significative expansion of the CD56bright/CD117+ compartment, while the “CD56dim NK cell lineage” showed the increase of CD56, CD16 and NCR antigen density, indicating their further maturation and activation. Altogether the data suggest that the two NK subsets originate from two distinct progenitors which, along with their differentiation and activation, generate cells with convergent phenotypes and functions., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Niemann-Pick disease (NPD) type A and B are lysosomal storage disorders (LSD) due to the lack of acid sphingomyelinase (ASM) activity (Schuchman et al., 2001). The enzyme defect results in a pathological accumulation of sphingomyelin (SM) within lysosomes. In many LSD, an accumulation of undegraded substrates in lysosomes due to deficiency of specific lysosomal enzymes impairs the autophagic process (Settembre et al., 2008), but an imbalance of the of autophagic process in NPB cells has never been shown. The purpose of this study is to examine the autophagic response in NPB B lymphocytes by means of flow cytometry, confocal microscopy and western blot techniques. EBV-transformed B Lymphocytes from patients with Niemann-Pick type B were treated with nocodazole (NZ) and wortmannin (WM), two autophagy inhibitors, and rapamicyn (RM), an autophagic inductor. Furthermore we starved cells using a serum-free medium to activate the autophagic process. NPD lymphocytes treated by NZ and RM showed an opposite trend than the expected results for normal cells, in Acridine Orange, Lysotracker Green and CD63 staining, clearly suggesting an impairment of this cellular pathway. Instead, starved cells highlighted a normal behaviour for these markers, indicating a residual ability to enter the process. In conclusion such results suggest the involvement of autophagy and the impairment of lysosomal network before and during NPB cells response to the above-mentioned stimuli. These scenario characterize an imbalance between formation and degradation of autophagic vacuoles (autophagic stress)., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds were extensively studied as anti-gestational agents and some of these molecules were also described as inhibitors of ornithine decarboxylase. Polyamine depletion has been frequently related to the induction of apoptosis and consequently we investigated DL-111-IT and analogs for this effect in myeloid (HL60), neuroblastic (SK-N-MC) and epithelial (BeWo) human tumor cell lines, by means of electron microscopy and DNA electrophoresis. HL60 and SK-N-MC appeared notably sensitive to apoptosis, whereas BeWo responsiveness was variable and frequently associated with necrosis. Our results indicate that the contragestational effect of DL-111-IT and analogs is associated with apoptotic deletion of chorionic tissue and that these molecules, due to their effect on human tumor cell lines, can be considered as antiblastic lead compounds.

Hyperthermia is a known apoptotic inducer and has been recently utilized in combination with chemo-and/or radiotherapy in cancer treatment. In this study we have described its effect on SK-N-MC human neuroblastoma tumor cells, a line which grows as a double adherent and floating population. Considering this particular culture behavior, we also investigated the relationship between hyperthermia and cell adhesiveness by evaluating integrin expression, namely CD11a, which is, as known, closely correlated to cell adhesion properties. By a multiple, ultrastructural and flow cytometrical approach, we have demonstrated that hyperthermia, while triggering apoptosis, also determines a CD11a surface expression decrease in apoptotic and living cells. We thus suggest a further role for this treatment, which, affecting adhesion mechanisms, could down-regulate metastatic diffusion.