Hermann Herbst

Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein.

Human endogenous retrovirus sequences (HERVs) reside in the genomes of primates and humans for several million years. The majority of HERVs is non-coding but a limited set is intact and can express proteins. We have recently identified an almost intact HERV-K(HML-2) provirus on chromosome 7 and have documented that most patients with germ cell tumors (GCTs) display antibodies directed against proteins of HERV-K(HML-2). To address whether these proteins merely represent tumor markers or contribute to neoplastic transformation, we examined the transforming potential of various HERV sequences and studied physical interactions between HERV and cellular proteins by yeast two-hybrid and biochemical assays. cORF, a protein encoded by the C-terminal open reading frame within the env gene, supports tumor growth in nude mice and associates with the promyelocytic leukemia zinc finger protein (PLZF). The interaction domains map between amino acid residues 21 and 87 of cORF, and between residues 245 and 543 of PLZF. PLZF is critical for spermatogenesis in mice. Abnormal spermatogenesis or maturation of gonocytes is thought to predispose humans to the development of germ cell tumors. Thus, cORF of human endogenous retroviruses may contribute to tumor development by interfering with processes during spermatogenesis that involve PLZF.

Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors

Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model.

Glycolipids as markers of murine T and B lymphoblastoid tumour cell lines

Cell surface glycosphingolipids are thought to be implicated in processes of cell-cell recognition and growth regulation, receptor function, and malignant transformation [l-3]. The identification and the structural analysis of these membrane components are also expected to afford an insight into the process of thymusderived (T) and bone marrow-derived (B) lymphocyte differentiation, since activated T and B lymphocytes express different sets of glycosphingolipids f4,5]. It is, therefore, not unre~onable to assume that tumour celi lines of T and of B cell origin will each yield characteristic glycosphingolipid distribution patterns relating either to the expression of tumour-associated moieties orto components typical for certain functional states during differentiation and/or maturation. Here, spontaneous or chemically induced, transplantable murine T and B lymphoblastoid tumours, adapted to in vitro conditions of growth [6,7] were used in a comparative qualitative and quantitative study of ganglioside and neutral glycolipid distribution patterns revealed by hip-performance thin-layer chromatography (HPTLC) after biosynthetic sugar labelhng [ 51. We document here differences not only between T and B cell lines but also among T and 3 cell lines that may be used as biochemical markers.

Grouping of haemolytic streptococci by monoclonal antibodies: Determinant specificity, cross-reactivity and affinity

Streptococcal group polysaccharide (CHO), A-, A-variant-, B-, C-, D- and G-specific monoclonal antibodies were prepared by the hybridoma technique employing spleen cells of several inbred mouse strains which are either high or low responders to the group A-CHO. The isotypes of these reagents were restricted to the class mu and IgG subclasses gamma 3 and--in small numbers--gamma 1. Two distinct categories of antibodies were identified for all but group D specificity: those which agglutinate suspended bacteria but do not precipitate purified soluble antigen, and those which show both agglutinating and precipitating properties. The group D antibodies described here were only of the latter category. The reactions were inhibitable by haptens in as far as these were known. Cross-reactions were observed in group-A-specific antibodies with E and L polysaccharides. Most G-CHO-specific antibodies cross-reacted with B-CHO. Association constants determined by fluorescence quenching measurements were for binding of complete A and C polysaccharides in the range of 10(6) to greater than 10(8) M-1, and for hapten binding by A-, Av- and C-CHO-specific antibodies in the range of 10(3) to 10(4) M-1. These results support a model of steric arrangements of antigenic determinants on A-variant bacteria and solubilized antigen [42] and allow its extension to streptococcal groups A, B, C and G. This model explains the observed functional differences by postulating single, terminal determinants which interact with the prevailing non-precipitating antibodies and internal repeating determinants which react with precipitins, respectively. No significant differences were found in the reactivity patterns to these streptococcal group antigens between strains of mice in terms of their ability to respond with high or low serum antibody titres to group A-CHO. On the other hand, within high and low responder strains, different kinetics of the optimal timing of fusion after initiation of the secondary immune reaction by boosting was observed. Low responders were most efficiently used for fusion 1.5 days later than high-responder spleen cells. This feature is interpreted to indicate an earlier proliferation of B lymphocytes in high responders, due to either an improved responsiveness to T-lymphocyte help or a reduced reactivity with T suppressor cells in comparison to low-responder B lymphocytes.

Murine Vκ25 and Vκ27 Amino-Acid Sequences of C57B1/6 Origin: Monoclonal Antibodies 17S29.1 and 22S25.1 Specific for the Group A-Streptococcal Polysaccharide

Antibodies 17S29.1 and 22S25.1 are monoclonal, hybridoma-derived gamma 3 kappa murine immunoglobulins with specificity for N-acetyl-glucosamine beta 1----3-linked to the L-rhamnose backbone structure, the immunodeterminant of the streptococcal Group A polysaccharide. The VL 17S29.1 amino-acid sequence is the third complete one reported from an antibody with this specificity, the second fully determined V kappa 25 structure and the first complete V kappa sequence of C57B1/6 origin derived from a carbohydrate-specific antibody. VL22S25.1 is a member of the V kappa 27 isotype of murine immunoglobulin VL regions. V kappa 17S29.1 and the determined part of the V kappa 22S25.1 sequence are compared to the previously described V kappa regions of streptococcal Group A polysaccharide-specific antibodies and to 12 selected partial and complete V kappa regions of antibodies with other specificities, predominantly to carbohydrate antigens. Both V kappa 17S29.1 and V kappa 22S25.1 increase the variability of known murine V kappa regions. They are the most homologous to the other V kappa regions derived from antibodies with streptococcal Group A polysaccharide specificity and share with them the amino-acid residue Arg74, so far characteristic for V kappa regions from antibodies with this specificity. The analysis of groups of independently expressed, highly homologous V kappa regions, namely V kappa 17S29.1 and V kappa 2S1.3 as one and V kappa 7S34.1 and V kappa 22S25.1 as a second group, offers the possibility of estimating the minimal number of V kappa germline genes involved in the immune response to the structurally defined streptococcal Group A polysaccharide antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal Antibodies Detecting Epstein-Barr Virus Strain and Group Specific Antigenic Determinants

Abstract Monoclonal antibodies were prepared against the four main surface and envelope polypeptides (p80, p140, p240, p340) of the QIMR-WIL transforming Epstein-Barr-virus (EBV) strain and several clones were obtained with reactivity to solely p80, p140, p340, and to p240/p340 simultaneously. The immunofluorescence reaction on viable EBV transformed cells suggests that p80, p240, and p340 may be part of the membrane-antigen (MA) complex defined by immunofluorescence techniques. Differences in the reactivity with the corresponding proteins of B95-8 (transforming) and P3HR-1 (non-transforming) EBV strains demonstrate that these reagents are able to detect strain- and group-specific determinants. Thus, monoclonal antibodies now provide a serological basis for EBV typing.

A Set of Monoclonal Antibodies to Streptococcal Group Polysaccharide Antigens

Abstract Group antigens of hemolytic streptococci are conventionally defined by agglutination and precipitation reactions with rabbit antisera. A set of monoclonal hybridoma-derived mouse antibodies reported here confirms and extends the serology of Groups A, A-variant, B, C, D and G streptococci. These monoclonal reagents identify in all Group polysaccharides two determinants, one terminal and one linear along the repeating units. Terminal determinants are recognized in the agglutination of vaccines and in binding reactions; linear determinants of the repeating sugar moieties are recognized both by binding (including agglutination) and precipitation of the isolated polysaccharide. Fine specificity analyses of the Groups B, D, and G reveal in comparison to conventional rabbit antisera distinct specificities of monoclonal antibodies.

Monoclonal Antibody Secreting Mouse Hybridoma Clones Express Different Sets of Membrane Glycosphingolipids in Different Antigenic Systems

Abstract B-cell hybridomas developed for the secretion of monoclonal antibodis are also a rich source of membrane material for biochemical analyses of other than Ig,H-2 and Ia-antigens. Here, the glycosphingolipid labelling patterns of hybridomas are reported, which produced antibodies against streptococcal group A polysaccharides (ACHO), azobenzene arsonate (ABA) or phosphorylcholine (PC). While the Sp2/O tumour cell line revealed by high performance thin layer chromatography (HPTLC) only 3 ganglioside bands, hybridomas exhibited up to 50 additional GSL. In the streptococcal system at least three different GSL-patterns could be distinguished. These were independent of H-2, Ig-allotype, -class, and -subclass. Distinct GSL patterns were found for hybridomas of the ABA and PC systems without correlation to the major idiotypes. However, there is the indication that IgM and IgG antibody producing hybridomas show as a result of differentiation unique but distinct GSL patterns.

Monoclonal Antibodies Against Membrane Glycolipids of an Anti-Streptococcal Group a b-Cell Hybridoma

Abstract A B-cell hybridoma producing monoclonal antibody specific for Streptococcal Group A polysaccharide was used as a source of murine lymphocyte membrane glycolipids. Mouse monoclonal antibodies were raised against purified gangliosides and neutral glycosphingolipids and were detected using an ELISA assay. We report the preliminary characterization of the neutral glycosphingolipid antigens recognized by two hybridomas.