Katerina Chlichlia

Lines of Murine Oligodendroglial Precursor Cells Immortalized by an Activated neu Tyrosine Kinase Show Distinct Degrees of Interaction with Axons In Vitro and In Vivo

Replication‐defective retroviruses expressing the t‐neu oncogene, or a hybrid protein with the neu tyrosine kinase linked to the external region of the human epidermal growth factor receptor (egfr‐neu), were used to establish lines of murine oligodendroglial precursor cells. Differentiation of the t‐neu lines into myelin‐associated glycoprotein (MAG)‐positive oligodendrocytes was induced by dibutyryl cAMP, and the egfr‐neu line showed limited differentiation in vitro upon withdrawal of epidermal growth factor. Cerebellar granule cell neurons expressed mitogens for the cell lines. Upon transplantation into demyelinated lesions, t‐neu line cells engaged with the demyelinated axons whereas the egfr‐neu line cells differentiated further and ensheathed the axons. These cell lines thus interact with neurons in vitro and in vivo and can be used as tools to define the molecules involved in different stages of neuron‐glia interaction.

Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine.

To optimize polynucleotide vaccinations for protective antitumor immunity we used a self-replicating RNA vaccine in which Semliki Forest virus replicase drives RNA expression of the lacZ gene coding for β-galactosidase as model tumor-associated antigen (TAA). This was compared with replicase-deficient control RNA and with lacZ DNA plasmids with respect to gene expression in vitro and in vivo and for vaccination using the mouse ear pinna as an optimal immunization site. In vitro, the highest expression was observed with self-replicating RNA. Gene expression following pinna inoculation of either non-replicating DNA plasmids or self-replicating RNA was similar, lasting for 2–3 weeks. Higher antibody responses were obtained with RNA than with DNA. β-Gal peptide specific CTL memory responses to lacZ DNA or RNA lasted for more than 6 weeks while respective responses induced by lacZ-transfected tumor cells lasted for only 2 weeks. To achieve a protective response against lacZ tumor cells with self-replicating RNA about a 100-fold lower dose of polynucleotide was sufficient in comparison to DNA. The extent of protective antitumor immunity not only depended on the gene dose used for vaccination, but also on the aggressiveness of the lacZ- transfected tumor line used for challenge. In comparison to lacZ-transfected tumor cells as vaccines, polynucleotide vaccination also demonstrated superiority with regard to cross-protection. Protective antitumor immunity could be strongly increased upon co-inoculation of lacZ DNA with IL-2 DNA or IL-12 RNA. IL-2 DNA, but not IL-12 RNA, also augmented the CTL response while IL-12 RNA, but not IL-2 DNA, reduced the antibody response. These results demonstrate efficient protective antitumor immunity after intra-pinna lacZ TAA polynucleotide vaccination and show additional immunomodulatory effects by co-administration of cytokine polynucleotides.

IMMEDIATE EFFECTS OF REVERSIBLE HTLV-1 TAX FUNCTION : T-CELL ACTIVATION AND APOPTOSIS

The tax protein of Human T-cell leukemia virus type 1 (HTLV-1) is important for the transforming properties of this virus in vitro and is considered to be responsible for the early stages of leukemogenesis in infected hosts. To address the early consequences of HTLV-1 tax function, we have constructed fusion proteins containing tax sequence either aminoterminal (taxER) or carboxy-terminal (ERtax) of the hormone binding domain of the human estrogen receptor (ER). Addition of estrogen or the antagonist hydroxytamoxifen to Jurkat T-cells expressing these constructs led to the trans-activation or responsive promoters and upregulation of cell surface markers CD28, CD69 and CD5 but not CD25 (IL2R-alpha subunit) or B7 (ligand for CD28). Additional stimulation of the T-cell receptor CD3 complex, led to the upregulation of CD25. B7 was upregulated by concomittent activation of ERtax and CD3 or CD28 pathways. These events were in part reversible upon withdrawal of hormone and inactivation of ERtax. Severe inhibition of proliferation, and apoptosis was observed with cells which had been subjected to short term (3 days) activation of the tax fusion proteins and the CD3 complex. Induction of ERtax activity for longer than 3 days promoted cell death independently of CD3 stimulation. Co-stimulation through the CD28 cell surface molecule did not suppress induction of apoptosis.

ICE-proteases mediate HTLV-I tax-induced apoptotic T-cell death

The Tax protein of Human T-cell leukemia virus type 1 (HTLV-1) is important for the T-cell immortalizing properties of this virus in vitro and is considered to be responsible for the early stages of leukemogenesis in infected hosts. Tax can upregulate expression of TNF-α and TNF-β, as well as potentiate apoptosis in activated T-cells and in serum starved murine fibroblasts. To examine the role of CD95 (APO-1/Fas) and ICE-proteases in Tax-mediated active T-cell death, Jurkat T cells expressing (APOS) or lacking (APOR) cell surface expression of CD95 (APO-1/Fas) were genetically modified to express hormone-inducible HTLV-1 Tax constructs. Hormone-inducible action of Tax alone was sufficient to promote programmed cell death in CD95-expressing Jurkat T-cell clones. In contrast, clones lacking CD95 surface expression were resistant to the antiproliferative action of Tax. Both APOS and APOR clones exhibited Tax-dependent upregulation of CD95 ligand and TNF-α. Blocking experiments suggested that while the apoptotic action of Tax critically required ICE-protease function it was largely independent of cell surface interaction of CD95 ligand or TNF-α with their corresponding receptors. These observations strongly implicate ICE-proteases in Tax-induced T-cell death, and suggest a possible involvement of CD95 in this process.

HTLV-1 Tax: Linking transformation, DNA damage and apoptotic T-cell death

The human T-cell leukemia virus type I (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4-positive T-cell neoplasia. The HTLV-1 proto-oncogene Tax, a potent transcriptional activator of cellular and viral genes, is thought to play a pivotal role in the transforming properties of the virus by deregulating intracellular signaling pathways. During the course of HTLV-1 infection, the dysregulation of cell-cycle checkpoints and the suppression of DNA damage repair is tightly linked to the activity of the viral oncoprotein Tax. Tax activity is associated with production of reactive oxygen intermediates (ROS), chromosomal instability and DNA damage, apoptotic cell death and cellular transformation. Changes in the intracellular redox status induced by Tax promote DNA damage. Tax-mediated DNA damage is believed to be essential in initiating the transformation process by subjecting infected T cells to genetic changes that eventually promote the neoplastic state. Apoptosis and immune surveillance would then exert the necessary selection pressure for eliminating the majority of virally infected cells, while escape variants acquiring a mutator phenotype would constitute a subpopulation of genetically altered cells prone to neoplasia. While the potency of Tax-activity seems to be a determining factor for the observed effects, the cooperation of Tax with other viral proteins determines the fate and progression of HTLV-1-infected cells through DNA damage, apoptosis, survival and transformation.

DNA vaccination with asparaginyl endopeptidase (Sm32) from the parasite Schistosoma mansoni: anti-fecundity effect induced in mice.

DNA-based vaccine technology was used to induce an immune response in mice against a schistosome cysteine proteinase, asparaginyl endopeptidase (Sm32). The cDNA coding for Sm32 was cloned in a mammalian expression vector under control of the CMV promoter/enhancer and expressed for the first time in transfected mammalian cells as well as in mice immunized with the Sm32-encoding DNA construct. These mice developed antibodies which recognized the native protein not only in homogenates of Schistosoma mansoni worms but also in the gut on cryostat sections of the parasites. This DNA vaccine led to an anti-fecundity effect: female worms of a challenge infection produced 37% less eggs than those growing in naïve mice. The results suggest that Sm32 may be a candidate antigen for the generation of an anti-pathology vaccine against schistosomes.

Differential microRNA Profiles and Their Functional Implications in Different Immunogenetic Subsets of Chronic Lymphocytic Leukemia

Critical processes of B-cell physiology, including immune signaling through the B-cell receptor (BcR) and/or Toll-like receptors (TLRs), are targeted by microRNAs. With this in mind and also given the important role of BcR and TLR signaling and microRNAs in chronic lymphocytic leukemia (CLL), we investigated whether microRNAs could be implicated in shaping the behavior of CLL clones with distinct BcR and TLR molecular and functional profiles. To this end, we examined 79 CLL cases for the expression of 33 microRNAs, selected on the following criteria: (a) deregulated in CLL versus normal B-cells; (b) differentially expressed in CLL subgroups with distinct clinicobiological features; and, (c) if meeting (a) + (b), having predicted targets in the immune signaling pathways. Significant upregulation of miR-150, miR-29c, miR-143 and miR-223 and downregulation of miR-15a was found in mutated versus unmutated CLL, with miR-15a showing the highest fold difference. Comparison of two major subsets with distinct stereotyped BcRs and signaling signatures, namely subset 1 [IGHV1/5/7-IGKV1(D)-39, unmutated, bad prognosis] versus subset 4 [IGHV4-34/IGKV2-30, mutated, good prognosis] revealed differences in the expression of miR-150, miR-29b, miR-29c and miR-101, all down-regulated in subset 1. We were also able to link these distinct microRNA profiles with cellular phenotypes, importantly showing that, in subset 1, miR-101 downregulation is associated with overexpression of the enhancer of zeste homolog 2 (EZH2) protein, which has been associated with clinical aggressiveness in other B-cell lymphomas. In conclusion, specific miRNAs differentially expressed among CLL subgroups with distinct BcR and/or TLR signaling may modulate the biological and clinical behavior of the CLL clones.

Preliminary study on sex-related inflammatory reactions in mice infected with Schistosoma mansoni

The aim of this study was to investigate the contribution of the sex of both the parasite and the host to the inflammatory response induced in unisexual infections of Schistosoma mansoni in mice. Organ weight, cell count and the delayed type hypersensitivity reaction were used as tools in this comparative study. The inflammatory reactions differed as a function of the sex of both the host and the parasite. Female mice showed a stronger inflammatory reaction to schistosome infection than males, while male schistosomes induced a stronger inflammatory response compared to females. The host-related differences in the inflammatory reaction may reflect differences in the factors affecting the immune defence of male and female mice. The differences in the inflammatory response induced by the parasite are discussed in terms of the quantity and quality of antigens among male and female worms.

Multiple-checkpoint inhibition of thymic stromal lymphopoietin–induced TH2 response by TH17-related cytokines

BACKGROUND The interplay between allergy and autoimmunity has been a matter of long debate. Epidemiologic studies point to a decreased frequency of allergy in patients with autoimmune diseases. However, recent studies suggest that IL-17 and related cytokines, which play a central role in autoimmunity, might also promote allergy. OBJECTIVE To address this controversy, we systematically studied the interactions between T(H)17-related cytokines and the thymic stromal lymphopoietin (TSLP)-mediated proallergic pathway. METHODS We used human primary dendritic cells (DCs), T cells, and skin explants. A novel geometric representation and multivariate ANOVA were used to analyze the T(H) cytokine profile. RESULTS We show that IL-17A specifically inhibits TSLP production but increases proinflammatory IL-8 production in human skin explants exposed to TNF-α and IL-4. This inhibitory activity was confirmed in cultured skin explants of atopic dermatitis lesions. At the T-cell level, T(H)17-polarizing cytokines (IL-1β, IL-6, TGF-β, and IL-23) inhibited T(H)2 differentiation induced by TSLP-activated DCs. This led to a global dominance of a T(H)17-polarizing environment over TSLP-activated DCs, as revealed by clustering and computational analysis. CONCLUSIONS Our data indicate that T(H)17-related cytokines are negative regulators of the TSLP immune pathway. This might explain the decreased frequency of allergy in patients with autoimmunity and suggests new means of manipulating proallergic responses.

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 109 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 109 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.