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Natural killer (NK) cells are critical players against tumors. The outcome of anti-tumor vaccination protocols depends on the efficiency of NK-cell activation, and efforts are constantly made to manipulate them for immunotherapeutic approaches. Thus, a better understanding of NK-cell activation dynamics is needed. NK-cell interactions with accessory cells and trafficking between secondary lymphoid organs and tumoral tissues remain poorly characterized. Here, we show that upon triggering innate immunity with lipopolysaccharide (LPS), NK cells are transiently activated, leave the lymph node, and infiltrate the tumor, delaying its growth. Interestingly, NK cells are not actively recruited at the draining lymph node early after LPS administration, but continue their regular homeostatic turnover. Therefore, NK cells resident in the lymph node at the time of LPS administration become activated and exert anti-tumor functions. NK-cell activation correlates with the establishment of prolonged interactions with dendritic cells (DCs) in lymph nodes, as observed by two-photon microscopy. Close DC and NK-cell contacts are essential for the localized delivery of DC-derived IL-18 to NK cells, a strict requirement in NK-cell activation.

Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells (NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen associated molecular pattern (PAMP) activated-DCs in inducing NK cell-mediated anti-tumor responses. Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4 dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory conditions are also evaluated. Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs. © 2010 Copyright SPIE - The International Society for Optical Engineering.

P53 is a tumor suppressor used as marker for early cancer diagnosis and prognosis. We have studied constructs based on gold nanoparticles (NPs) decorated with specific anti-p53 antibodies and with a fluoresceine derivative, FITC. The interaction of gold surface plasmons with fluorophores bound within few nanometers from the surface, likely induces changes in the fluorophore excited state lifetime. Indeed we found previously that this parameter follows linearly the p53 concentration in solutions (in vitro conditions) up to 200-400 pM, depending on the size of the NP, with a 5 pM uncertainty. We have evaluated here the nanosensor specificity for p53 by testing it in-vitro against bovine serum albumine, beta-lactolglobulin and lysozyme. Moreover, the titration of total cell extracts from p53+/+ or p53-/- cells with the p53antibody decorated gold NPs, indicates that this construct can also be used to detect the presence of p53 in total cell extracts and it will be therefore a valuable tool also for in vivo screening.

The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

P53 is a tumor suppressor used as marker for early cancer diagnosis and prognosis. We have studied constructs based on gold nanoparticles (NPs) decorated with specific anti-p53 antibodies and with a fluoresceine derivative, FITC. The interaction of gold surface plasmons with fluorophores bound within few nanometers from the surface, likely induces changes in the fluorophore excited state lifetime. Indeed we find that this parameter follows linearly the p53 concentration in solutions (in vitro conditions) up to 200-400 pM, depending on the size of the NP, with a 5 pM uncertainty. We have then evaluated the nanosensor specificity for p53 by testing it in-vitro against a number of globular proteins: bovine serum albumin (BSA), beta-lactoglobulin and lysozyme. The experiments indicate that, apart from BSA, which is notoriously a sticky protein, the other globular proteins do not compete for the p53 recognition by the p53 antibody. The titration of total cell extracts from p53+/+ or p53-/- cells with the p53antibody decorated gold NPs, indicates that the fluorophore lifetime is highly sensitive to the presence of p53 in the cell extracts. The nanocostrcut discussed here can then be used to detect the presence of p53 in total cell extracts and it will be therefore a valuable tool also for in vivo screening.

Pediatric acute lymphoblastic leukemia (ALL) comprises genetically distinct subtypes. However, 25% of cases still lack defined genetic hallmarks. To identify genomic aberrancies in childhood ALL patients nonclassifiable by conventional methods, we performed a single nucleotide polymorphisms (SNP) array-based genomic analysis of leukemic cells from 29 cases. The vast majority of cases analyzed (19/24, 79%) showed genomic abnormalities; at least one of them affected either genes involved in cell cycle regulation or in B-cell development. The most relevant abnormalities were CDKN2A/9p21 deletions (7/24, 29%), ETV6 (TEL)/12p13 deletions (3/24, 12%), and intrachromosomal amplifications of chromosome 21 (iAMP21) (3/24, 12%). To identify variation in expression of genes directly or indirectly affected by recurrent genomic alterations, we integrated genomic and gene expression data generated by microarray analyses of the same samples. SMAD1 emerged as a down-regulated gene in CDKN2A homozygous deleted cases compared with nondeleted. The JAG1 gene, encoding the Jagged 1 ligand of the Notch receptor, was among a list of differentially expressed (up-regulated) genes in ETV6-deleted cases. Our findings demonstrate that integration of genomic analysis and gene expression profiling can identify genetic lesions undetected by routine methods and potential novel pathways involved in B-progenitor ALL pathogenesis.