Acute environmental temperature variation affects brain protein expression, anxiety and explorative behaviour in adult zebrafish.

This study investigated the effect of 4-d acute thermal treatments at 18 °C, 26 °C (control) and 34 °C on the nervous system of adult zebrafish (Danio rerio) using a multidisciplinary approach based on behavioural tests and brain proteomic analysis. The behavioural variations induced by thermal treatment were investigated using five different tests, the novel tank diving, light and dark preference, social preference, mirror biting, and Y-Maze tests, which are standard paradigms specifically tailored for zebrafish to assess their anxiety-like behaviour, boldness, social preference, aggressiveness, and explorative behaviour, respectively. Proteomic data revealed that several proteins involved in energy metabolism, messenger RNA translation, protein synthesis, folding and degradation, cytoskeleton organisation and synaptic vesiculation are regulated differently at extreme temperatures. The results showed that anxiety-like behaviours increase in zebrafish at 18 °C compared to those at 26 °C or 34 °C, whereas anxiety-related protein signalling pathways are downregulated. Moreover, treatments at both 18 °C and 34 °C affect the exploratory behaviour that appears not to be modulated by past experiences, suggesting the impairment of fish cognitive abilities. This study is the continuation of our previous work on the effect of 21-d chronic treatment at the same constant temperature level and will enable the comparison of acute and chronic treatment effects on the nervous system function in adult zebrafish.

Increase in environmental temperature affects exploratory behaviour, anxiety and social preference in Danio rerio.

The aim of this work is to investigate the effect of a temperature increase on the behaviour of adult zebrafish (Danio rerio) maintained for 21 days at 34 °C (treatment) and 26 °C (control). The temperatures chosen are within the vital range of zebrafish and correspond to temperatures that this species encounters in the natural environment. Previous results showed that the same treatment affects the brain proteome and the behaviour of adult zebrafish by producing alterations in the proteins involved in neurotransmitter release and synaptic function and impairing fish exploratory behaviour. In this study, we have investigated the performance of treated and control zebrafish during environmental exploration by using four behavioural tests (novel tank diving, light and dark preference, social preference and mirror biting) that are paradigms for assessing the state of anxiety, boldness, social preference and aggressive behaviour, respectively. The results showed that heat treatment reduces anxiety and increases the boldness of zebrafish, which spent more time in potentially dangerous areas of the tank such as the top and the uncovered bright area and at a distance from the social group, thus decreasing protection for the zebrafish. These data suggest that the increase in ambient temperature may compromise zebrafish survival rate in the natural environment.

Environmental temperature variation affects brain protein expression and cognitive abilities in adult zebrafish (Danio rerio): A proteomic and behavioural study.

Water temperature is an important environmental parameter influencing the distribution and the health of fishes and it plays a central role in ectothermic animals. The aim of this study is to determine the effects of environmental temperature on the brain proteome and the behavioural responses in zebrafish, a widely used animal model for environmental "omics" studies. Adult specimens of wild-type zebrafish were kept at 18 °C, 34 °C and 26 °C (control) for 21 days. Proteomic data revealed that several proteins involved in cytoskeletal organization, mitochondrial regulation and energy metabolism are differently regulated at the extreme temperatures. In particular, the expression of proteins associated to synapses and neurotransmitter release is down-regulated at 18 °C and 34 °C. In both thermal conditions, fish exhibited a reduced interest for the novel environment and an impairment of cognitive abilities during Y-Maze behavioural tests. The observed pathways of protein expression are possibly associated to functional alterations of the synaptic transmission that may result in cognitive functions impairment at central nervous system level as those revealed by behavioural tests. This study indicates that temperature variations can elicit biochemical changes that may affect fish health and behaviour. This combined approach provides insights into mechanisms supporting thermal acclimation and plasticity in fishes. SIGNIFICANCE: Environmental temperature variation may impact on all levels of biological life. Understanding the impact of thermal variation on the nervous system and animal behaviour is of primary importance since the results obtained can be applied from the ecological to the biomedical fields.

Antisense epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells.

Human rhabdomyosarcoma cells express membrane epidermal growth factor receptor (ECF-R), which could confer responsiveness to EGF and transforming growth factor-alpha (TGF-alpha) of autocrine or paracrine origin. To study the role played by this growth factor circuit in the proliferation and differentiation of myogenic neoplastic cells, human rhabdomyosarcoma EGF-R-expressing cells (RD/18 clone) have been transfected with a plasmid containing a fragment of the EGF-R cDNA in the antisense orientation. In vitro growth and differentiative ability were studied on six antisense-transfected clones (AS) in comparison to parental RD/18 cells and to cells transfected with the plasmid containing only the neomycin resistance gene (NEO). A reduced EGF-R membrane expression was found in AS clones by decreased immunofluorescence with an anti-EGF-R monoclonal antibody. All AS transfectants had a greatly impaired proliferative ability, even when cultured in fetal bovine serum-containing medium. Proliferation of AS clones was completely blocked in medium supplemented with 2% horse serum. The differentiation ability of AS clones was heterogeneous, ranging from clones with a percentage of myosin-positive cells higher than controls to clones with a negligible myosin expression. Therefore, the growth impairment determined by the loop interruption is not sufficient to switch on the differentiation program. The role played by EGF-R in the proliferation of human rhabdomyosarcoma cells suggests that this receptor could constitute a target for a therapeutic approach.

Expression of transduced carcinoembryonic antigen gene in human rhabdomyosarcoma inhibits metastasis.

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface glycoprotein belonging to the immunoglobulin superfamily. CEA has been involved in vitro in adhesion mechanisms, but little is known about the function of this glycoprotein in vivo in normal tissue differentiation and malignancy. With regard to the relationship between CEA expression and tissue differentiation, it has been reported that transfection of the CEA gene in rat L6 myoblasts results in a complete block of myogenic differentiation. To extend investigations to the transformed myogenic counterpart and examine CEA effects on differentiation and malignancy outside the colon system, we have transfected the human CEA gene in human rhabdomyosarcoma cells. Human rhabdomyosarcoma cells transfected with the CEA gene correctly expressed membrane CEA anchored via glycosylphosphatidylinositol and secreted CEA in the medium. CEA gene transfer in human rhabdomyosarcoma cells, which display a limited differentiation ability, does not further inhibit myogenic differentiation or alter in vitro proliferation or natural killer sensitivity. CEA transfection does not affect s.c. growth in nude mice, but the ectopic expression of CEA in human rhabdomyosarcoma cells can strongly inhibit their metastatic ability to lungs and adrenals after i.v. injection. The impairment of metastatic potential correlates with a reduction in the homotypic adhesion properties of the cells. These data suggest that CEA, in some systems, can interfere with intercellular adhesion and, at least for cells not metastatic to the liver, can act as an anti-metastatic molecule.

Expression of functional CD40 on human osteosarcoma and Ewing's sarcoma cells.

CD40, a membrane glycoprotein of the tumor necrosis factor receptor family, is expressed by several tumor types, including B-cell lymphomas, carcinomas, and melanoma, but little is known concerning its expression by sarcoma. We used flow cytometry to analyze the expression of CD40 in human cell lines derived from 12 osteosarcomas, 6 Ewing's sarcomas, and 5 rhabdomyosarcomas. Detectable CD40 levels ranging from low to very high were found in one-third of osteosarcomas, whereas five of six Ewing's sarcomas expressed intermediate levels of CD40; all rhabdomyosarcomas were CD40-negative. At the tissue level, two of eight primary high-grade osteosarcomas showed CD40-positive immunostaining. Osteosarcoma cells and Ewing's sarcoma cells expressing CD40 were treated with recombinant soluble CD40 ligand to analyze CD40 function. Treatment with soluble CD40 ligand increased the level of apoptotic cells and stimulated the transcription of matrix metalloproteinase 9 gene, enhancing matrix metalloproteinase 9 enzyme secretion. The results indicate that in human osteosarcoma and Ewing's sarcoma, CD40 is a functional receptor whose engagement can have opposite effects on tumor cell survival and malignancy.

Transduction of genes coding for a histocompatibility (MHC) antigen and for its physiological inducer interferon-gamma in the same cell: efficient MHC expression and inhibition of tumor and metastasis growth.

The mouse mammary carcinoma TS/A, of BALB/c (H-2d) origin, was transfected with the murine interferon-gamma (IFN-gamma) gene (Int. J. Cancer 55: 320, 1993). We used IFN-gamma transfectants as recipients for a second round of transfections with murine allogeneic class I histocompatibility (H-2b) genes that are modulated by IFN. Transfectants with either gene alone, as well as parent TS/A cells (TS/A-pc), were used as controls. Only double transfectants expressed high levels of the allogeneic H-2b genes, while in H-2b single transfectants the expression was very low (but was induced by treatment with exogenous IFN-gamma). The tumorigenic potential of IFN-gamma or H-2b single transfectants was reduced in comparison to TS/A-pc. IFN-gamma+H-2Kb double transfectants were almost nontumorigenic, while IFN-gamma+H-2Db clones gave rise to tumors in about one-half of mice. The experimental metastatic ability of all IFN-gamma+H-2b double transfectants was very low. IFN-gamma single transfectants were known to induce a strong macrophage response in the host. The expression of allogeneic H-2 antigens added a T-lymphocyte-mediated response that accounted for the lower tumorigenicity of double transfectants. These results show that it is possible to steer the immune response evoked by tumor cells for therapeutic purposes. Moreover, the high H-2 expression obtained in IFN-gamma+H-2b double transfectants suggests that single IFN-gamma transfectants are ideal recipients for all IFN-sensitive genes. This approach can be used also for other general-purpose inducers of gene expression.

The immune response elicited by mammary adenocarcinoma cells transduced with interferon-gamma and cytosine deaminase genes cures lung metastases by parental cells.

The parental cells of the TSA murine mammary adenocarcinoma (TSA-pc) were transfected with both the interferon-gamma (IFN-y) gene and the cytosine deaminase (CD) suicide gene to obtain a therapeutic vaccine active against TSA-pc lung metastases. Even in the absence of treatment with the prodrug 5-fluorocytosine (5-FC), the local growth of double transfectants (CD-y clones) was inhibited by a marked recruitment of granulocytes and macrophages. In mice harboring TSA-pc micrometastases, therapeutic vaccination with either IFN-gamma or CD single transfectants reduced the number of lung nodules, whereas CD-gamma double transfectants abrogated metastasis growth in up to 80% of mice. Treatment of mice with 5-FC did not alter the curative efficacy of CD-gamma double-transfectant cells. By contrast, in mice vaccinated with CD single-transfectant cells, 5-FC treatment caused a significant loss of their curative activity. Host T cells played an active role in the cure of lung metastases, because vaccination of nude mice with CD-gamma cells was uneffective.

Inhibition of lung colonisation of a mouse mammary carcinoma by therapeutic vaccination with interferon-alpha gene-transduced tumor cells.

A spontaneously metastatic murine mammary adenocarcinoma, TSA, has been transduced with the gene for interferon alpha1 (IFN-alpha). Transfectants were used for the immunotherapy of mice bearing lung colonies induced by the intravenous inoculation of non-transduced parental cells. A significant reduction in the number of tumor colonies was obtained when repeated subcutaneous administrations of mitomycin C-blocked transfectant cells were given, commencing 3 days after an intravenous challenge with TSA cells. Intraperitoneal vaccination induced a stronger anti-tumor response than subcutaneous vaccination, and the proportion of tumor-free mice reached 50%. The potency of IFN-alpha transfectants was similar to that of IFN-gamma transfectants previously obtained from TSA. Admixture of IFN-alpha and IFN-gamma transfectant cells in the same vaccine did not increase the curative effect over that of single vaccines. In nude mice vaccination with IFN-alpha or IFN-gamma transfectants did not lead to a reduction in the number of lung colonies, indicating that an intact T cell response was required for the therapeutic effect observed in immunocompetent mice.

Inhibition of poly(ADP-ribose) polymerization preserves the glutathione pool and reverses cytotoxicity in hydrogen peroxide-treated lymphocytes.

DNA damage caused by oxygen radicals activates poly(ADP-ribosyl) polymerase (pADPRP), a nuclear enzyme that utilizes NAD+ as substrate. It has been demonstrated that pharmacological inactivation of pADPRP rescues human lymphocytes damaged by oxygen radicals, but not those damaged by equitoxic doses of ionizing radiation. In the present paper we demonstrate that the NAD+ pool decreases after both damaging treatments and is preserved in a similar fashion by pADPRP inhibition. On the contrary, the ATP pool, cell energy charge and reduced thiols are decreased only by the administration of oxygen radicals, and are preserved if poly(ADP)ribosylation is inhibited. In fact, treatment with oxidant agents depletes the cell energy pools owing to the simultaneous demands of the glutathione (GSH)/NADPH cycle and pADPRP-driven NAD+ consumption, while in irradiated cells only the latter mechanism operates. We suggest that, when pADPRP is inhibited, enough energy is available for the preservation of cell thiols, thereby allowing oxidant-treated cells to survive and undergo mitosis. Thus, GSH and energy shortage appear to be the main cause of cell death in oxidant-injured cells.

Oxygen radicals induce stress proteins and tolerance to oxidative stress in human lymphocytes.

A set of eight proteins is induced in peripheral blood lymphocytes from normal donors by exposure to hydrogen peroxide or to xanthine oxidase plus hypoxanthine. Four of them (hsp90, hsp72 and proteins 65 and 50 kDa) are also expressed after heat shock, together with proteins 110, 100 and 38 kDa. Among proteins induced after oxidative stress is a 32 kDa protein-probably corresponding to heme oxygenase-1 (HO-1)- and a 27 kDa protein, both known to be induced by reactive oxygen species. Although ionizing radiation is known to generate a number of pro-oxidant intermediates, using our one-dimensional electrophoresis system we can detect no differences in the proteins synthesized after exposure to gamma-ray doses between 5 and 20 Gy as compared with control cells. Pre-exposure to a mild hyperthermia or to moderate oxidative stress significantly increases survival of lymphocytes challenged with high doses of reactive oxygen species, in conditions compatible with a protective rôle exerted by stress proteins. The increase in survival is accompanied by the maintenance of the proliferative capacity of the cells. The physiological rôle played by stress proteins in prevention and repair of damage and the relationships between stress protein induction, oxidative state, proliferation and mode of cell death are discussed.

Differential effect of L-histidine in human lymphocytes damaged by different oxygen radical producing systems.

The effect of histidine on damage induced by oxygen radicals was studied in peripheral blood lymphocytes treated with free oxygen radical-inducing agents: hydrogen peroxide, xanthine oxidase plus hypoxanthine, bleomycin and gamma-rays. L-Histidine, at a concentration of 1 mM, was found to potentiate both cell killing and inhibition of PHA-stimulated cell division brought about by hydrogen peroxide or xanthine oxidase plus hypoxanthine. In contrast, L-histidine did not affect gamma-ray- or bleomycin-induced cell killing and inhibition of PHA-stimulated cell division. We suggest that L-histidine potentiation of cell damage is mainly mediated by interaction of the amino acid with hydrogen peroxide and/or iron rather than with other reactive oxygen species. In addition, these results also indicate that hydrogen peroxide produced by gamma-radiation- or bleomycin-treated cells plays no role in the toxic effects elicited by these agents.

[Urologically silent Grawitz tumor. General framework of paraneoplastic syndromes and personal experiences]. / Tumore di Grawitz urologicamente silente. Inquadramento generale delle sindromi paraneoplastiche ed esperienze personali.

The symptomatological triad of Grawitz tumour (haematuria, renal mass, lumbar pain) is rarely observed, whereas clinical signs and symptoms indicative of an internist pathology. An account is given of the more common paraneoplastic syndromes that may be associated with renal adenocarcinoma. If correctly evaluated, these permit early diagnosis and resolutive surgery. Reference is made to personal experience in emphasising that there are extremely few diagnostic features in cases of urologically silent Grawitz tumour.

Spectrophotometric determination of thiols in human lymphocytes.

A spectroscopic method for thiol analysis, based on the complexation reaction with Pd(II), is described. The proposed method is simple and sensitive and can be used for a rapid analysis of thiols in human lymphocytes.

Modulation of caspase-3 activity by zinc ions and by the cell redox state.

It is known that DNA fragmentation during apoptosis is controlled by a number of factors, a crucial step being the caspase-operated cleavage of ICAD, the DNase inhibitor. We have previously demonstrated that hydrogen peroxide-treated lymphocytes undergo apoptosis without formation of a DNA ladder; however, the use of micromolar amounts of a Zn(2+) chelator allowed DNA cleavage at internucleosomal sites. Such results were extended in the present work, thus allowing their framing into the events related to alterations in the redox state of the cell. Apoptosis in hydrogen peroxide-treated lymphocytes was found to occur with caspase-3 activation, but the enzyme activity was found to be impaired, thus affecting internucleosomal fragmentation as well as nuclear morphology. Caspase-3 activity was found to resume upon mild Zn(2+) chelation. These results provide as well an experimental model from which apoptotic events upstream and downstream of caspase-3 activity can be examined.

Systemic effects of cytokines released by gene-transduced tumor cells: marked hyperplasia induced in small bowel by gamma-interferon transfectants through host lymphocytes.

Cells transduced with cytokine genes are currently used to enhance the anti-tumor and immunomodulatory effects of these molecules in cancer therapy. The sustained release of cytokine thus obtained can perturb many homeostatic systems of the host. We have previously shown that the murine mammary adenocarcinoma TS/A transfected with the murine gamma-interferon (IFN-gamma) gene stimulates a strong immune response that impairs tumor growth. Mice bearing tiny tumors have serum IFN-gamma levels constantly exceeding 100 IU/ml. Therefore, we asked which systemic effects can be triggered in mice by such transfectants. BALB/c mice bearing tumors produced by clone 16.6000 cells (which release 6,000 IU/ml of IFN-gamma in culture) were compared to normal mice and to mice with tumors produced by parent cells transfected with the neomycin resistance gene (NEO cells, no IFN-gamma release). Histological studies revealed a marked hyperplasia of small bowel in mice bearing 16.6000 tumors; the villi and crypts of these mice were > 1.5 times longer than those of normal mice and of mice bearing NEO tumors. In vivo administration of bromodeoxyuridine evidenced a 2.5-3 times increase in the proliferative score of the intestinal crypts of mice bearing 16.6000 tumors compared to control mice. No intestinal alterations were observed in nude mice bearing 16.6000 tumors. T lymphocytes thus appear to play a causal role in this phenomenon.

Gene expression profile analysis in human T lymphocytes from patients with Down Syndrome.

Down Syndrome (DS) is caused by the presence of three copies of the whole human chromosome 21 (HC21) or of a HC21 restricted region; the phenotype is likely to have originated from the altered expression of genes in the HC21. We apply the cDNA microarray method to the study of gene expression in human T lymphocytes with trisomy 21 in comparison to normal cells. Two patients with DS were investigated, along with two normal subjects as a control, all being tested in independent, duplicated cell culture experiments. The most consistent finding was the overexpression of the superoxide dismutase gene (SOD1), located on 21q, and of MHC DR beta 3 (HLA-DRB3), GABA receptor A gamma 2 (GABRG2), acetyltransferase Coenzyme, A 2 (ACAT2) and ras suppressor protein 1 (RSU1) genes. When the data were clustered according to chromosome localization, the HC21 gene set showed, on average, the highest expression in DS cells in all the experiments. Moreover, separate clustering of patients and controls was obtained when analysis was restricted to HC21 gene expression values. These findings reinforce the specific gene dosage theory for the pathogenesis of the DS phenotype, and show a consistent overexpression of the SOD1 gene on 21q.

White cell apoptosis in packed red cells.

BACKGROUND: After the removal of the buffy coat, packed red cell (RBC) transfusion units still contain white cells that may undergo apoptosis as a result of storage conditions (1-6 degrees C). The aim of the present study was the evaluation of this phenomenon in view of the possible influence it may have on febrile nonhemolytic transfusion reactions. STUDY DESIGN AND METHODS: Three independent methods (microscopy, DNA electrophoresis, and cytometry) were used to evaluate apoptosis in white cells present in 13 RBC units. Of these units, 10 had been collected into CPD/saline-adenine-glucose-mannitol and 3 into CPDA-1; each bag was split in two parts, one of which was irradiated. RBCs were stored at 1 to 6 degrees C, and samples were periodically withdrawn for study. The proliferative capacity of stored lymphocytes was evaluated after phytohemagglutinin stimulation and tritiated thymidine incorporation. RESULTS: Apoptosis was found to occur in both granulocytes and lymphocytes, starting from the first 48 to 72 hours of storage. The choice of the anticoagulant-preservative solution and the effect of irradiation did not influence the amount and the timing of the apoptotic phenomenon. Lymphocyte proliferative capacity was found to decrease sharply with storage time. CONCLUSION: Conditions of storage in RBCs induce consistent apoptosis in residual white cells. The possible clinical implications of the relationships between apoptosis and the induction of biologic response modifiers (that may cause interleukin-mediated febrile non-hemolytic transfusion reactions) and between apoptosis and immune reactions remain to be elucidated.