Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Antigen-independent immune responses after dendritic cell vaccination

The ability of cultured, antigen-loaded dendritic cells (DCs) to induce antigen-specific T cell immunity in vivo has previously been demonstrated and confirmed. Immune monitoring naturally focuses on immunity against vaccine antigens and may thus ignore other effects of DC vaccination. Here we therefore focused on antigen-independent responses induced by DC vaccination of renal cell carcinoma patients. In addition to the anticipated response against the vaccine antigen KLH, vaccination with CD83⁺ monocyte-derived DCs resulted in a strong increase in the ex vivo proliferative and cytokine responses of PBMCs stimulated with LPS or BCG. In addition, LPS strongly enhanced the KLH-induced proliferative and cytokine response of PBMCs. Moreover, proliferative and cytokine responses of PBMCs stimulated with the homeostatic cytokines IL-7 and IL-15 were also clearly enhanced after DC vaccination. In contrast to LPS induced proliferation, which is well known to depend on monocytes, IL-7 induced proliferation was substantially enhanced after monocyte depletion indicating that monocytes limit IL-7 induced lymphocyte expansion. Our data indicate that DC vaccination leads to an increase in the ex vivo responsiveness of patient PBMCs consistent with a DC vaccination induced enhancement of T cell memory. Our findings also suggest that incorporation of bacterial components and homeostatic cytokines into immunotherapy protocols may be useful in order to enhance the efficacy of DC vaccination and that monocytes may limit DC vaccination induced immunity. Supported by a grant to Martin Thurnher from the kompetenzzentrum medizin tirol (kmt), a center of excellence.     

Bee venom secretory phospholipase A2 and phosphatidylinositol-homologues cooperatively disrupt membrane integrity,abrogate signal transduction and inhibit proliferation of renal cancer cells

Bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P2) act synergistically to induce cell death in tumour cells of various origins with concomitant stimulation of the immune system. Here, we investigated the mechanisms involved in such actions and examined structural requirements of PtdIns-homologues to inhibit tumour cells in combination with bv-sPLA2. Renal cancer cells were treated with bv-sPLA2 alone or in combination with PtdIns-homologues. Inhibitory effects on [³H] thymidine incorporation and intracellular signal transduction pathways were tested. Reaction products generated by bv-sPLA2 interaction with PtdIns(3,4)P2 were identified by mass spectrometry. Among the tested PtdIns-homologues those with a phosphate esterified to position 3 of the inositol head group, were most efficient in cooperating with bv-sPLA2 to block tumour cell proliferation. Growth inhibition induced by the combined action of bv-sPLA2 with either PtdIns(3,4)bisphosphate or PtdIns(3,4,5)trisphosphate were synergistic and accompanied by potent cell lysis. In contrast, PtdIns, which lacked the phosphate group at position 3, failed to promote synergistic growth inhibition. The combined administration of PtdIns(3,4)P2 and bv-sPLA2 abrogated signal transduction mediated by extracellular signal regulated kinase 1 and 2 and prevented transduction of survival signals mediated by protein kinase B. Surface expression of the epidermal growth factor (EGF)-receptor was reduced after PtdIns(3,4)P2-bv-sPLA2 administration and associated with a blockade of EGF-induced signalling. In addition, mass spectroscopy revealed that bv-sPLA2 cleaves PtdIns(3,4)P2 to generate lyso-PtdIns(3,4)P2. In conclusion, we suggest that the cytotoxic activity mediated by PtdIns(3,4)P2 and bv-sPLA2 is due to cell death that results from disruption of membrane integrity, abrogation of signal transduction and the generation of cytotoxic lyso-PtdIns(3,4)P2. This work was supported by a grant to MT of the kompetenzzentrum medizin tirol (kmt), a centre of excellence.     

Galactosyltransferase and sialyltransferase are located in different subcellular compartments in HeLa cells

Galactosyl- and sialyltransferase have been localized by double immunofluorescence labeling in HeLa cells. Galactosyltransferase was found in a compact juxtanuclear structure previously shown to represent the Golgi apparatus (J. Roth and E.G. Berger (1982) J. Cell Biol. 93, 223-9), whereas sialyltransferase was localized to vesicles spread over the whole cytoplasm. These findings indicate different compartments for both transferases and support a model of subcompartmentation of glycosylation steps along the secretory pathway.     

The primary structure of human ribonuclease/angiogenin inhibitor (RAI) discloses a novel highly diversified protein superfamily with a common repetitive module.

Immunological screening of a lambda gt11 library, constructed from HeLa mRNA, yielded several ribonuclease/angiogenin inhibitor (RAI) cDNA clones containing 900-bp inserts. Northern blot analysis revealed that the length of the RAI mRNA is approximately 1.9 kb. Construction and screening of a eukaryotic cDNA expression library (HeLa) containing preferentially complete cDNA inserts led to the isolation of a full length clone. The complete nucleotide sequence was determined. The C-terminal amino acid sequence deduced from the cDNA is identical to the peptide sequence obtained from a CNBr fragment of RAI, confirming the identity of the clone. The deduced primary structure of RAI consists of eight homologous tandem repeats with remarkable periodicity of leucine and cysteine residues. Each repeat is derived from the duplication of a leucine-rich 28-amino-acid module. This prototype module is closely related to a repetitive 24-amino-acid motif of unclear function, previously found in proteins involved in important biological processes such as blood coagulation, embryonic development, cell morphogenesis and signal transduction. Although homologous, the RAI modules show distinct differences in length and amino acid composition to the modules of this group of proteins, demonstrating their high potential of variability, necessary for adaptation to very diverse roles. Based on our results we propose that these repetitive modules are a common structural feature of a novel protein superfamily whose members exert their function by highly specific protein-protein interactions.     

Mycobacterial lipoarabinomannans: modulators of dendritic cell function and the apoptotic response

The molecular bases of Mycobacterium tuberculosis pathogenicity remain unclear. We report here how M. tuberculosis mannosylated lipoarabinomannans contribute to the survival of bacilli in the human reservoir by (i) inhibiting IL-12 production by macrophages and dendritic cells and (ii) modulating M. tuberculosis-induced macrophage apoptosis.     

Serum antibodies against Saccharomyces cerevisiae: a new prognostic indicator in metastatic renal-cell carcinoma

PURPOSE: A recent study reported that a diet rich in bread and refined cereals might have an unfavorable role in the development of renal cell carcinoma (RCC). To test whether an underlying intolerance of bread ingredients is responsible for the unfavorable influence of bread on RCC, we examined patient sera for the presence of food-specific IgG. EXPERIMENTAL DESIGN: A commercial test was used to detect food-specific IgG directed against a panel of 113 food antigens in sera of 54 patients with metastatic RCC. Kaplan-Meier estimates were used for univariate survival analysis, and differences in survival curves were assessed with the log-rank test. Multivariate survival analysis was done using a Cox regression model. RESULTS: We found that RCC patients with elevated serum levels of IgG antibodies against S. cerevisiae, commonly known as baker's yeast and yet another bread component, have an unfavorable clinical course. Median survival of patients with high levels of S. cerevisiae IgG was only 17.8 months, whereas median survival of patients with low S. cerevisiae IgG was 43.8 months (P = 0.0022; log-rank). Multivariate survival analysis identified high levels of S. cerevisiae IgG as a strong and independent prognostic risk factor (risk ratio 4.6, P = 0.001; 95% CI 1.61-13.08). CONCLUSIONS: Our findings indicate that serum levels of IgG against S. cerevisiae may predict survival in patients with metastatic RCC. The data suggest not cereals but baker's yeast being the critical component of bread that may cause immune deviation and impaired immunosurveillance in predisposed RCC patients.