Expression of Fas (APO-1/CD95) and Fas ligand (FasL) in human neuroblastoma.

BACKGROUND AND PROCEDURE: To determine the possible role of Fas/FasL system in the particularly heterogeneous behaviour of neuroblastoma (NB), we have measured the functional expression of Fas and its ligand, FasL, in primary neuroblastoma samples and cell lines by immunohistochemistry and flow cytometry. RESULTS: Our results reveal that while Fas expression is associated with low stage and more mature tumors, heterogeneous FasL expression was mostly detected in high stage tumors, with our apparent correlation to MYCN amplification. Flow cytometric analysis of cell lines demonstrated a high expression of Fas in epithelial-type, HLA class I positive cell lines, which was lost upon activation with phorbol esters. In contrast, Fas ligand was detected in only a small subset of cell lines. CONCLUSIONS: In some cell lines, cytotoxic assays revealed the ability of NB-associated Fas receptor to transduce an apoptotic signal upon triggering. The pattern of functional Fas/FasL expression in tumours and cell lines suggests that this system may be involved in the evasion of highly malignant neuroblastoma cells to host immune response.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells.

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated whether or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M(r) 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0 degrees C or 37 degrees C. The matrix fraction retained 20-40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37 degrees C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.

Distribution of neuronal and metabolic markers in the human auditory cortex

SUMMARY The human auditory cortex, located on the supratemporal plane of the temporal lobe, is divided in a primary auditory area and several non-primary areas surrounding it. These different areas show anatomical and functional differences. Many studies have focussed on auditory areas in non-human primates, using investigation techniques such as electrophysiological recordings, tracing of neural connections, or immunohistochemical and histochemical staining. Some of these studies have suggested parallel and hierarchical organization of the cortical auditory areas as well as subcortical auditory relays. In humans, only few studies have investigated these regions immunohistochemically, but activation and lesion studies speak in favour of parallel and hierarchical organization, very similar to that of non-human primates. Calcium-binding proteins and metabolic markers were used to investigate possible correlates of hierarchical and parallel organization in man. Calcium-binding proteins, parvalbumin, calretinin and calbindin, modulate the concentration of intracellular free calcium ions and were found in distinct subpopulations of GABAergic neurons in non-human primates species. In our study, their distribution showed several differences between auditory areas: the primary auditory area was darkly stained for both parvalbumin and calbindin, and their expression rapidly decreased while moving away from the primary area. This staining pattern suggests a hierarchical organization of the areas, in which the more darkly stained areas could correspond to an earlier integration level and the areas showing light staining may correspond to higher level integration areas. Parallel organization of primary and non-primary auditory areas was suggested by the complementarity, within a given area, between parvalbumin and calbindin expression across layers. To investigate the possible differences in the energetic metabolism of the cortical auditory areas, several metabolic markers were used: cytochrome oxidase and LDH1 were used as oxidative metabolism markers and LDH5 was used as glycolytic metabolism marker. The results obtained show a difference in the expression of enzymes involved in oxidative metabolism between areas. In the primary auditory area the oxidative metabolism markers were maximally expressed in layer IV. In contrast, higher order areas showed maximal staining in supragranular layers. The expression of LDH5 varied in patches, but did not differ between the different hierarchical auditory areas. The distribution of the two LDH enzymes isoforms also provides information about cellular aspects of metabolic organization, since neurons expressed the LDH1 isoform whereas astrocytes express primarily LDH5, but some astrocytes also contained the LDH1 isoform. This cellular distribution pattern supports the hypothesis of the existence of an astrocyte-neuron lactate shuttle, previously suggested in rodent studies, and in particular of lactate transfer from astrocytes, which produce lactate from the glucose obtained from the circulation, to neurons that use lactate as energy substrate. In conclusion, the hypothesis of parallel and hierarchical organization of the auditory areas can be supported by CaBPs, cytochrome oxidase and LDH1 distribution. Moreover, the two LDHs cellular distribution pattern support the hypothesis of an astrocyte-neuron lactate shuttle in human cortex.

Mapping the structural core of human cerebral cortex.

Structurally segregated and functionally specialized regions of the human cerebral cortex are interconnected by a dense network of cortico-cortical axonal pathways. By using diffusion spectrum imaging, we noninvasively mapped these pathways within and across cortical hemispheres in individual human participants. An analysis of the resulting large-scale structural brain networks reveals a structural core within posterior medial and parietal cerebral cortex, as well as several distinct temporal and frontal modules. Brain regions within the structural core share high degree, strength, and betweenness centrality, and they constitute connector hubs that link all major structural modules. The structural core contains brain regions that form the posterior components of the human default network. Looking both within and outside of core regions, we observed a substantial correspondence between structural connectivity and resting-state functional connectivity measured in the same participants. The spatial and topological centrality of the core within cortex suggests an important role in functional integration.

Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice.

This study demonstrates that endogenously produced interferon gamma (IFN-gamma) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-gamma (i.e., IFN-gamma receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-gamma-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-gamma-insensitive p53(-/-) mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-gamma-insensitive mice, we found IFN-gamma's actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-gamma. Thus, IFN-gamma forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.

Efficiency of recombinant human TNF in human cancer therapy.

Recombinant human tumour necrosis factor (TNF) has a selective effect on angiogenic vessels in tumours. Given that it induces vasoplegia, its clinical use has been limited to administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs. When combined with the alkylating agent melphalan, a single ILP produces a very high objective response rate. In melanoma, the complete response (CR) rate is around 80% and the overall objective response rate greater than 90%. In soft tissue sarcomas that are inextirpable, ILP is a neoadjuvant treatment resulting in limb salvage in 80% of the cases. The CR rate averages 20% and the objective response rate is around 80%. The mode of action of TNF-based ILP involves two distinct and successive effects on the tumour-associated vasculature: first, an increase in endothelium permeability leading to improved chemotherapy penetration within the tumour tissue, and second, a selective killing of angiogenic endothelial cells resulting in tumour vessel destruction. The mechanism whereby these events occur involves rapid (of the order of minutes) perturbation of cell-cell adhesive junctions and inhibition of alphavbeta3 integrin signalling in tumour-associated vessels, followed by massive death of endothelial cells and tumour vascular collapse 24 hours later. New, promising approaches for the systemic use of TNF in cancer therapy include TNF targeting by means of single chain antibodies or endothelial cell ligands, or combined administration with drugs perturbing integrin-dependent signalling and sensitizing angiogenic endothelial cells to TNF-induced death.

Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically regulated in cortical astrocytes following sleep deprivation in mice.

STUDY OBJECTIVES: There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called "astrocyte-neuron lactate shuttle" (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifically in astrocytes following sleep deprivation. Astrocytes were purified by fluorescence-activated cell sorting from transgenic mice expressing the green fluorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. DESIGN: 6-hour instrumental sleep deprivation (TSD). SETTING: Animal sleep research laboratory. PARTICIPANTS: Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. INTERVENTIONS: Basal sleep recordings and sleep deprivation achieved using a modified cage where animals were gently forced to move. MEASUREMENTS AND RESULTS: Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, α-2-Na/K pump, Glt1, and Ldha mRNAs were significantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not significant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. CONCLUSIONS: This study shows that TSD induces the expression of genes associated with ANLS specifically in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle.

Guidelines for imaging retinoblastoma: imaging principles and MRI standardization.

Retinoblastoma is the most common intraocular tumor in children. The diagnosis is usually established by the ophthalmologist on the basis of fundoscopy and US. Together with US, high-resolution MRI has emerged as an important imaging modality for pretreatment assessment, i.e. for diagnostic confirmation, detection of local tumor extent, detection of associated developmental malformation of the brain and detection of associated intracranial primitive neuroectodermal tumor (trilateral retinoblastoma). Minimum requirements for pretreatment diagnostic evaluation of retinoblastoma or mimicking lesions are presented, based on consensus among members of the European Retinoblastoma Imaging Collaboration (ERIC). The most appropriate techniques for imaging in a child with leukocoria are reviewed. CT is no longer recommended. Implementation of a standardized MRI protocol for retinoblastoma in clinical practice may benefit children worldwide, especially those with hereditary retinoblastoma, since a decreased use of CT reduces the exposure to ionizing radiation.

Severe postoperative complications adversely affect long-term survival after r1 resection for pancreatic head adenocarcinoma.

BACKGROUND: Survival after pancreatic head adenocarcinoma surgery is determined by tumor characteristics, resection margins, and adjuvant chemotherapy. Few studies have analyzed the long-term impact of postoperative morbidity. The aim of the present study was to assess the impact of postoperative complications on long-term survival after pancreaticoduodenectomy for cancer. METHODS: Of 294 consecutive pancreatectomies performed between January 2000 and July 2011, a total of 101 pancreatic head resections for pancreatic ductal adenocarcinoma were retrospectively analyzed. Postoperative complications were classified on a five-grade validated scale and were correlated with long-term survival. Grade IIIb to IVb complications were defined as severe. RESULTS: Postoperative mortality and morbidity were 5 and 57 %, respectively. Severe postoperative complications occurred in 16 patients (16 %). Median overall survival was 1.4 years. Significant prognostic factors of survival were the N-stage of the tumor (median survival 3.4 years for N0 vs. 1.3 years for N1, p = 0.018) and R status of the resection (median survival 1.6 years for R0 vs. 1.2 years for R1, p = 0.038). Median survival after severe postoperative complications was decreased from 1.9 to 1.2 years (p = 0.06). Median survival for N0 or N1 tumor or after R0 resection was not influenced by the occurrence and severity of complications, but patients with a R1 resection and severe complications showed a worsened median survival of 0.6 vs. 2.0 years without severe complications (p = 0.0005). CONCLUSIONS: Postoperative severe morbidity per se had no impact on long-term survival except in patients with R1 tumor resection. These results suggest that severe complications after R1 resection predict poor outcome.

Charge dependent substrate activity of C3' and N3 functionalized, organometallic technetium and rhenium-labeled thymidine derivatives toward human thymidine kinase 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).

Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome.

Cancer/testis (CT) genes are normally expressed in germ cells only, yet are reactivated and expressed in some tumors. Of the approximately 40 CT genes or gene families identified to date, 20 are on the X chromosome and are present as multigene families, many with highly conserved members. This indicates that novel CT gene families may be identified by detecting duplicated expressed genes on chromosome X. By searching for transcript clusters that map to multiple locations on the chromosome, followed by in silico analysis of their gene expression profiles, we identified five novel gene families with testis-specific expression and >98% sequence identity among family members. The expression of these genes in normal tissues and various tumor cell lines and specimens was evaluated by qualitative and quantitative RT-PCR, and a novel CT gene family with at least 13 copies was identified on Xq24, designated as CT47. mRNA expression of CT47 was found mainly in the testes, with weak expression in the placenta. Brain tissue was the only positive somatic tissue tested, with an estimated CT47 transcript level 0.09% of that found in testis. Among the tumor specimens tested, CT47 expression was found in approximately 15% of lung cancer and esophageal cancer specimens, but not in colorectal cancer or breast cancer. The putative CT47 protein consists of 288 amino acid residues, with a C-terminus rich in alanine and glutamic acid. The only species other than human in which a gene homologous to CT47 has been detected is the chimpanzee, with the predicted protein showing approximately 80% identity in its carboxy terminal region.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NPâeuro0/00>âeuro0/00chitosan DNA NPâeuro0/00=âeuro0/00DNA unloaded chitosan NPâeuro0/00>âeuro0/00control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Selective accumulation of mature DC-Lamp+ dendritic cells in tumor sites is associated with efficient T-cell-mediated antitumor response and control of metastatic dissemination in melanoma.

The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.