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The Coast Guard is an organization committed to safeguarding the marine environment, that requires specialized personnel. Therefore, it is necessary to integrate the "basic" preparation, with "specialist" training in compliance with the high standards of a Q.M.S. of training, which uses teaching methods advanced, classroom and lessons, case studies, but also practice in the field. The latter methodology plays an important role since learning by doing, even more than theory, guarantees the effectiveness of learning, emotional involvement and the formation of experiential memory of learners.

Marginal zone (MZ) B cells, identified as surface (s)IgM(high)sIgD(low)CD23(low/-)CD21(+)CD38(-) B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27(+) and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared ("stereotyped") sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli.

The mechanisms by which adenomatous polyposis coli (APC) gene mutations contribute to colorectal tumourigenesis and progression are still not fully understood. Using in vitro mouse embryonic stem cells, APC mutations have been proposed to dysregulate the interactions between kinetochores and microtubules during mitosis, leading to chromosomal instability (CIN) and aneuploidy. A link between APC mutations and aneuploidy in vivo among human sporadic colorectal adenomas has not been reported previously and was therefore investigated in the present series of 61 adenomas. Multi-parameter flow cytometry, based on scattering and fluorescence from the DNA-specific 4,6-diamidino-2-phenylindole-2-hydrochloride (DAPI) dye, which separates epithelial from stromal lymphocyte nuclei, was used to evaluate the DNA index (DI) and to sort epithelial nuclei. Additionally, DNA extracted from these sorted nuclei was used to analyse APC mutations by DNA sequencing. Aneuploidy was present in 20 of 61 adenomas (33%), with 15 of these 20 cases (75%) having a near-diploid DI (DI different from 1 and less than 1.3). APC mutations were detected in 19 adenomas (31%): 12 were within or downstream of the mutation cluster region (MCR), roughly defined by codons 1200-1500, and seven were upstream of the MCR. Overall, the prevalence of aneuploidy in APC wild-type and mutated adenomas was 26% and 47%, respectively, and no statistically significant association was found between APC status and DI (p = 0.142). However, when APC mutations were subdivided into two groups, ie occurring within/downstream of the MCR and upstream of the MCR, the association of APC mutations within and downstream of the MCR with aneuploidy was statistically significant (p = 0.017). In conclusion, the present data suggest that the type of APC mutation may play a role in the origin of CIN in vivo in human sporadic colorectal adenomas and that APC mutations within and downstream of the MCR, and large-scale chromosomal alterations, may co-operate in the progression of a subgroup of adenomas.

Interaction of NK cells with autologous immature dendritic cells (iDCs) results in reciprocal activation. We have previously reported that NK cells trigger iDC to polarize and secrete IL-18; in turn, DC-activated NK cells secrete the nuclear protein/proinflammatory cytokine high mobility group box protein 1 (HMGB1), which induces DC maturation and prevents DC from lysis. However, activated NK cells can also kill iDC. To investigate whether effector and maturative properties may coexist or segregate in different NK subsets, human NK cell clones were generated and analyzed for their effects on iDC. We found that the ability of different NK cell clones to induce iDC maturation is unlinked to their phenotypic and cytolytic features but correlates with the relocation of HMGB1 from nucleus to cytoplasm. “Maturative” NK cell clones secrete HMGB1 spontaneously. It is interesting that secretion is strongly enhanced by engagement of the surface molecule NKp30 but only slightly induced by triggering of the activating NK receptor CD16. However, culturing freshly isolated NK cells for 1 week with low doses of anti-CD16 triggers the relocation of HMGB1 from nucleus to cytoplasm and its spontaneous secretion, resulting in a stronger maturation potential of the NK cells. Together, our data indicate that NK cells comprise functionally different subsets, endowed with different capacities to secrete HMGB1 and to induce maturation of autologous iDC. Nonetheless, maturation properties can be modulated by different stimuli. This suggests that depending on the environmental stimuli, NK/iDC interaction can lead to different outcomes, thus influencing immune response.

Whether in vivo specific gene mutations lead to chromosomal instability (CIN) and aneuploidy or viceversa is so far not proven. We hypothesized that aneuploidy among human sporadic colorectal adenomas and KRAS2 and APC mutations were not independent. Additionally, we investigated if 1p34–36 deletions by dual target FISH were associated with aneuploidy. Among 116 adenomas, 29 were DNA aneuploid by flow cytometry (25%) and 29 were KRAS2 mutated (25%). KRAS2 mutations were associated with aneuploidy (P=0.02). However, while G–C and G–T transversions were strongly associated with DNA aneuploidy (P=0.007), G–A transitions were not. Within a second series of 61 adenomas, we found, instead, that APC mutational status and aneuploidy by flow cytometry were not associated. However, a statistically significant association was found with specific APC mutations, i.e., occurring in the mutation cluster region (MCR, codons 1200–1500) or downstream (P=0.016). Finally, the correlation of 1p34–36 deletions with flow cytometric and FISH detected aneuploidy was also significant (P=0.01). Specific KRAS2 and APC mutations and loss of genes in the 1p34–36 region appear associated with aneuploidy suggesting that these events are not independent and may cooperate in inducing human sporadic colorectal adenomas. A cause effect relationship between gene mutations and aneuploidy remains, however, to be demonstrated.