Vaccine immunopotentiators of the future.

Vaccine adjuvants or immunopotentiators comprise a diverse group of molecules or formulations. Despite a wealth of different candidates, there is a need for better vaccine adjuvants in both veterinary and human medicine. For human vaccines, the immunopotentiator choice has been limited to aluminum salts, until recently. By contrast, a whole range of adjuvants is employed for inactivated veterinary vaccines, due to less stringent safety and regulatory requirements and proven superior vaccine performance. This review highlights recent developments and future trends in immunopotentiators.

Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction.

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, facilitates heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues and cells, ALCAM is restricted to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM constructs, revealed that homophilic cell adhesion depended on ligand binding mediated by the membrane-distal amino-terminal immunoglobulin domain and on avidity controlled by ALCAM clustering at the cell surface involving membrane-proximal immunoglobulin domains. Co-expression of a transmembrane ALCAM deletion mutant, which lacks the ligand binding domain, and endogenous wild-type ALCAM inhibited homophilic cell-cell interactions by interference with ALCAM avidity, while homophilic, soluble ligand binding remained unaltered. The extracellular structures of ALCAM thus provide two structurally and functionally distinguishable modules, one involved in ligand binding and the other in avidity. Functionality of both modules is required for stable homophilic ALCAM-ALCAM cell-cell adhesion.

Characterization of recombinant human autoantibody fragments directed toward the autoantigenic U1-70K protein.

The U1-70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of pre-mRNA. All components of the U1 snRNP complex, including the U1-70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization of recombinant human anti-U1-70K single chain autoantibody fragments (anti-hU1-70K scFv) from autoimmune patient-derived phage display antibody libraries. All scFv specifically recognize parts of the hU1-70K protein and its apoptotic 40-kDa cleavage product. In Western blotting assays a number of scFv preferentially recognize the 40-kDa apoptotic cleavage fragment of the U1-70K protein, suggesting a possible involvement of this apoptotic cleavage product in the autoimmune response of patients. The germline gene usage of these recombinant autoantibodies was also determined. Using several U1-70K deletion and point mutants of both human (h) and Drosophila melanogaster (Dm) origin, it was established that the U1-70K epitope that is recognized by the anti-hU1-70K scFv is located within the RNA binding domain.

Caspase-dependent cleavage of nucleic acids.

Autoimmune diseases are frequently characterized by the presence of autoantibodies directed against nucleic acid-protein complexes present in the nucleus of the cell. The mechanisms by which these autoantigenic molecules escape immunological tolerance are largely unknown, although a number of recent observations suggest that modified self-proteins generated during apoptosis may play an important role in the development of autoimmunity. It has been hypothesized that the recognition of these modified self-proteins by the immune system may promote autoantibody production. While apoptosis is specifically characterized by posttranslational modification of proteins, recent findings also show that nucleic acids are modified. This review summarizes the specific cleavages of some of these key nucleic acids, i.e. chromosomal DNA, ribosomal RNA and small structural RNAs (U1 snRNA, Y RNA), in apoptotic cells.

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule.

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.

memA/DRS, a putative mediator of multiprotein complexes, is overexpressed in the metastasizing human melanoma cell lines BLM and MV3.

memA was isolated by subtractive hybridization in which the mRNA repertoire was compared in a panel of human melanoma cell lines with different metastasizing potential. Expression of memA mRNA is elevated in the highly metastasizing human melanoma cell lines and derived xenografts, as compared with the non-metastasizing ones. In a collection of human tumor cell lines and melanoma metastasis lesions, memA mRNA expression could be detected in the A-431 (epidermoid carcinoma), HT-1080 (fibrosarcoma), JEG-3 and JAR (choriocarcinomas) cell lines and in three out of 11 melanoma metastasis lesions. The distribution of memA mRNA in a collection of healthy human organs is also tissue restricted. Sequence analysis revealed that the MEMA protein is identical with a 160 kDa nuclear 'domain rich in serines' (DRS) protein occurring free in the nucleoplasm and in U2-ribonucleoprotein structures. MEMA is also homologous to pinin, a 140 kDa protein associated with the desmosome-intermediate filament complex, and to a 32 kDa porcine neutrophilic protein that was copurified with components of the NADPH-oxidase enzyme complex. The encoded amino acid sequence predicts that the MEMA protein has three coiled-coil domains, one glycine loop domain, is very hydrophilic and contains regions rich in glutamine/proline, glutamic acid and serine residues.

MEMD, a new cell adhesion molecule in metastasizing human melanoma cell lines, is identical to ALCAM (activated leukocyte cell adhesion molecule).

From a differential mRNA display comparing a non- and a highly metastasizing human melanoma cell line, we isolated and characterized memD. memD is preferentially expressed in the highly metastasizing melanoma cell lines of a larger panel. The encoded protein, MEMD, is identical to activated leukocyte cell adhesion molecule (ALCAM), a recently identified ligand of CD6. ALCAM is involved in homophylic (ALCAM-ALCAM) and heterophylic (ALCAM-CD6) cell adhesion interactions. We have studied MEMD/ALCAM cell-cell interactions between human melanoma cells. The expression of this cell adhesion molecule not only correlates with enhanced metastatic properties and with aggregational behavior of human melanoma cells as tested by FACS analysis, but transfection experiments also make clear that MEMD/ALCAM expression is essential for cell-cell interaction of the investigated human melanoma cells. As the melanoma cell lines analyzed are all CD6 negative, these results strongly suggest that MEMD/ALCAM is an adhesion molecule mediating homophylic clustering of melanoma cells. MEMD/ALCAM expression is not restricted to subsets of leukocytes and melanoma cells, it can also be found in healthy organs and in several other malignant tumor cell lines. Besides, MEMD/ALCAM is also expressed in cultured endothelial cells, pericytes and melanocytes, in xenografts derived from the radial and vertical growth phase and in 4 of 13 melanoma metastasis lesions. The potential role is discussed of MEMD/ALCAM mediated cell-cell interactions in migration of mobile cells (ie, activated leukocytes, metastasizing tumor cells) through tissues.

Expression of nma, a novel gene, inversely correlates with the metastatic potential of human melanoma cell lines and xenografts.

nma, a novel gene, was isolated by using a subtractive hybridization technique in which the gene expression was compared in a panel of human melanoma cell lines with different metastatic potential. nma mRNA expression (1.5 kb) is high in poorly metastatic human melanoma cell lines and xenografts and completely absent in highly metastatic human melanoma cell lines. Fluorescence in situ hybridization combined with the analysis of a panel of human-rodent somatic cell hybrids indicated that the nma gene is located on human chromosome 10, in the region p11.2-p12.3. Sequence analysis of nma showed no homologies with other known genes or proteins, except for several partially sequenced cDNAs. The predicted amino acid sequence suggests that the protein encoded by nma contains a transmembrane domain. Expression of nma is high in human kidney medulla, placenta and spleen, low in kidney cortex, liver, prostate and gut and absent in lung and muscle. Whereas nma is not expressed in normal skin tissue, expression is high in melanocytes and in 3 out of 11 melanoma metastases tested.

nmb, a novel gene, is expressed in low-metastatic human melanoma cell lines and xenografts.

From a subtractive cDNA library, we isolated several cDNA clones which showed differential expression between highly and lowly metastatic human melanoma cell lines. One clone, designated nmb, showed preferential expression in the low-metastatic cell lines and was chosen for further characterization. Sequence analysis revealed that this clone represents a novel gene, encoding a putative transmembrane glycoprotein which showed the highest homology to the precursor of pMEL17, a melanocyte-specific protein. nmb RNA expression was absent in most tumor-cell lines tested and not restricted to the melanocytic lineage. Transfection of a partial nmb cDNA into a highly metastatic melanoma cell line (BLM) resulted, in 2 of 3 transfectants, in slower subcutaneous tumor growth and, in 1 of 3 transfectants, in reduction of the potential for spontaneous metastasis in nude mice.