Developmentally regulated expression of T cell receptor beta chain variable domains in immature thymocytes.

A minor subset of immature (CD4-,8-) thymocytes that lack expression of the B2A2 antigen was found to express low levels of surface TCR protein as detected by mAbs F23.1 and KJ16 (reacting with protein products of the V beta 8 gene family). Interestingly, F23.1/KJ16 determinants were expressed on a two- to three-fold higher proportion of B2A2- thymocytes than mature lymph node T cells in four independent haplotypes. When expanded in short-term culture with PMA and calcium ionophore, B2A2- thymocytes retained their overexpression of F23.1/KJ16 determinants and showed a fivefold elevated level (relative to lymph node) of V beta 8-specific mRNA. Taken together, these findings suggest that expression of TCR V beta genes, like Ig genes, is developmentally regulated.

Characterization of a novel murine testis-specific serine/threonine kinase.

Using degenerate oligos corresponding to two highly conserved motifs within the protein kinase catalytic domain and a PCR-based cloning strategy, we have isolated a cDNA fragment encoding a new member of the Ser/Thr (serine/threonine) family of protein kinases. Expression analysis revealed that the fragment recognized two transcripts (1.6 and 1.4 kb) exclusively in testis. Using this fragment as a probe, we have cloned a full-length cDNA from a mouse testis cDNA library. The sequence has a 1092-bp open reading frame encoding a protein of 364 amino acids. The N-terminally localized kinase catalytic domain has all the conserved motifs found in other Ser/Thr kinases. Northern blot analysis using the full-length sequence as a probe revealed that the cloned gene corresponds to the 1.6-kb transcript, suggesting the existence of at least two testis-specific novel Ser/Thr kinases. We propose the name testis-specific kinase-1 (TSK-1) for the gene described here. A GenEMBL databank search revealed highest homology to the human gene encoding rac protein kinase-beta and the group of yeast Ser/Thr kinases encoded by SNF-1, nim-1, KIN-1 and KIN-2.

A rapid one-step radiometric assay for hepatitis B surface antigen utilizing monoclonal antibodies.

A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available.

Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain.

Protein tyrosine kinases are pivotal in central nervous tissue development and maintenance. Here we focus on the expression of Ehk-1, a novel Elk-related receptor tyrosine kinase. Ehk-1 gene expression is observed in the developing and adult central nervous system and is highly regulated throughout development at both the messenger RNA and protein levels. Three messenger RNA transcripts of 8.5, 5.9 and 5.1 kb are detectable in the rat brain and a variety of splice possibilities have been identified. However, a major protein species of around M(r) 120,000 predominates throughout development. Ehk-1 messenger RNA and protein levels are highest in the first postnatal week. By in situ messenger RNA hybridization the gene is expressed by all neurons of the adult brain, but mostly in the hippocampus, cerebral cortex and large neurons of the deep cerebellar nuclei, as well as the Purkinje and granular cells of the cerebellum. At earlier stages of development, transcripts are most prominent in the periventricular germinal layers of the brain. Immunohistochemistry reveals a pronounced membrane associated protein expression in immature neurons. In the adult animal, peak reactivity was found in the neuropil with sparing of most perikarya. The spatial and temporal pattern of ehk-1 gene expression suggests a role in both the development and maintenance of differentiated neurons of the central nervous system.

Immunohistochemical localization of EphA5 in the adult human central nervous system.

To better understand the functional role of EphA5 in the adult human central nervous system (CNS), we performed an immunohistochemical mapping study. EphA5, like other members of the Elk/Eph family of receptor tyrosine kinases, was widely distributed in CNS neurons. However, the distribution of the neuronal staining was not uniform. The abundance of stained neurons appeared to increase from the forebrain to the hindbrain and spinal cord. Glial and endothelial tissue was unstained. These findings are consistent with the existence of receptor and ligand gradients in different brain regions. The localization of EphA5 to motor and sensory neurons is consistent with a role of EphA5 in neural plasticity, cell-cell recognition, and topographical orientation of neuronal systems.

Production and characterization of a rat monoclonal antibody against the murine CD3 molecular complex.

We describe the production of a rat monoclonal antibody, 17A2, that detects the T cell receptor-associated CD3 molecular complex. 17A2 cross-competes with a CD3 epsilon-specific reagent and similarly stimulates IL-2 production by T cells. Immunohistological and cell separation applications are shown.

Extensive splice variation and localization of the EHK-1 receptor tyrosine kinase in adult human brain and glial tumors.

EHK-1 is a neuronal ELK-related receptor tyrosine kinase which interacts with multiple, membrane-anchored ligands. Recent experiments have suggested a role for some of these ligands in the formation of neuronal pathways. Here, we report the isolation of human EHK-1 cDNAs and the localization of the human EHK-1 gene to chromosome 4q12. Six EHK-1 mRNA splice variants encoding cell-surface receptors with catalytic domains were identified in adult human brain where a 120-kDa EHK-1 protein predominates. Immunohistochemistry for EHK-1 reveals a dendritic staining pattern in cortical neurons and cerebellar Purkinje cells and a marked accumulation of EHK-1 in the somas of pyramidal neurons within the cortex and hippocampus. Interestingly, we have identified lineage aberrant expression of EHK-1 in a number of human gliomas. In addition to functions during development, EHK-1 may be involved in the maintenance of the adult nervous system and contribute to glioma development.

Reciprocal expression of myelin-associated glycoprotein splice variants in the adult human peripheral and central nervous systems.

The L- and S-MAG isoforms differ only at their C-terminus and are believed to be functionally distinct. To obtain information on the relative expression of these alternatively spliced isoforms in humans, we cloned an S-MAG cDNA fragment. The deduced amino-acid sequence of the human S-MAG C-terminus shows fairly conservative substitutions of 4 out of the 10 residues compared to the rodent peptide. Using reverse transcription and a competitive polymerase chain reaction, we show that, in contrast to rodents, the L-MAG splice variant predominates in adult human brain while, like in rodents, S-MAG transcripts are most abundant in peripheral nerve. The results obtained by Western blot analysis and immunohistochemistry are in good agreement with the findings at the mRNA level. Animal experiments may thus be more representative for the role of MAG in human nerve than in brain.

Altered sensitivity of CD81-deficient mice to neurobehavioral effects of cocaine.

CD81, also known as target of the antiproliferative antibody, is known to be expressed in astrocytes and involved in cell adhesion and, recently, we demonstrated its induction exclusively in the accumbens following cocaine. In the present study, the sensitivity of CD81-deficient mice to behavioral effects of cocaine was evaluated. It was found that CD81-deficient mice exhibited altered sensitivity to cocaine as assessed in the place preference conditioning paradigm and locomotor activity. This deficit in place preference conditioning was not accompanied by a deficit in acquisition or retention of water maze behavior. In addition, CD81 knockout mice exhibited higher levels of nucleus accumbens dopamine as compared to their controls. These observations are discussed in the context of the role of CD81 in cocaine-mediated behaviors.

Functional activation of EphA5 receptor does not promote cell proliferation in the aberrant EphA5 expressing human glioblastoma U-118 MG cell line.

Eph receptors are a subfamily of receptor tyrosine kinases (RTKs), that are activated by ephrin ligands and appear to play important roles in axon guidance and cell migration during development of the nervous system. Over-expression or constitutive activation of Eph receptors has been linked with increased proliferation in various tumours. We have recently described lineage aberrant expression of EphA5 in primary human astrocytomas, glioblastomas and in the human glioblastoma U-118 MG cell line. A role for EphA5 expression in these tumours is not apparent, and we have investigated the cellular effects of EphA5 activation using the human glioblastoma U-118 MG cell line as a model. Immunofluorescent staining demonstrated cell surface expression of EphA5. Activation of the EphA5 receptor using an ephrin-A1 recombinant fusion protein resulted in tyrosine phosphorylation of EphA5 in a time-dependent manner. Exposure of U-118 MG glioblastoma cells to ephrin-A1 did not result in significant spontaneous or FCS-stimulated cell proliferation, though a marginal decrease was observed. This is in converse to the effects of Eph activation in other tumour cell lines, and is the first study to investigate EphA5 in glioblastoma cell lines.

Protein kinases expressed in aggregating brain cell cultures during myelination.

Serum-free aggregating rat brain cell cultures provide sufficient cell surface and paracrine interactions between neurons and glial cells for compact myelination. We are interested in the part played in these signalling pathways by protein kinases and have used a PCR cDNA cloning approach to catalogue the protein kinase genes expressed by these cultures. 8 transmembrane protein kinases were identified: IGF1-R, trk B, bFGF-R, c-met, Tyro2, Tyro1, Tyro4 and a novel eck-related gene. The first 4 are receptors for ligands with known trophic functions. Tyro2 is a novel gene related to the EGF-R. The latter 3 belong to the eck gene family of more than 8 highly related putative receptors for, as yet, unknown ligands. 8 cDNAs for intracellular protein kinases were also isolated including 3 novel genes. Ongoing studies are investigating whether these proteins contribute to myelination and/or could be used as therapeutic targets in demyelinating diseases.

Paraproteinaemic neuropathies.

Paraproteinaemia and neuropathy are each relatively frequent and may be associated by chance. However, a number of significant relationships have to be ruled out in the differential diagnosis. Malignant gammopathy should be excluded: multiple myeloma can lead to compression of the spinal cord or cauda equina; primary amyloidosis is occasionally involved; the rare but intriguing POEMS syndrome, consisting of polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes, usually accompanies osteosclerotic myeloma. It can be associated with angio-follicular lymph node hyperplasia and needs to be recognized because radioablative therapy is curative. The 'benign' monoclonal gammopathies of undetermined significance, known as MGUS, are much more frequent. There is an IgM MGUS group with predominantly distal sensorimotor demyelinating polyneuropathy and another rather heterogeneous group with IgG or IgA MGUS and a tendency to a favourable response to plasmapheresis. The role of the monoclonal IgG and IgA antibodies is unclear. This chapter has focused on the pathogenetic mechanisms of neuropathies associated with IgM MGUS. In the majority of cases, monoclonal autoantibodies specific for particular carbohydrate epitopes bind to myelin and are now recognized as the primary cause of the disease manifestations, including widening of the myelin lamellae. While the autoantibodies have been shown to bind complement, the presence of inhibitors is invoked to explain the absence of acute inflammatory changes. The epitopes recognized with the highest affinity by the auto-antibodies are present on the myelin-associated glycoprotein (MAG) and could interfere with cell adhesion and cellular signally processes. In addition, binding to antigenically similar glycoproteins, such as PO, PMP-22 and some acidic glycolipids, may be a contributory factor. It is generally accepted that the anti-MAG autoantibodies are inducing a progressive demyelinating polyneuropathy by modifying axon-Schwann cell interactions.

Recognition of human recombinant myelin associated glycoprotein by anti-carbohydrate antibodies of the L2/HNK-1 family.

The L2 and HNK-1 monoclonal antibodies recognize carbohydrate determinants containing sulfate-3-glucuronate that are prominent on cells of neural crest lineages. In humans these epitopes are most abundant on the Myelin Associated Glycoprotein and it was assumed that they co-localize on the same molecules. Recently, in vitro synthesized carbohydrates have provided a basis for the different recognition requirements of these two antibodies. We now provide in vivo evidence that a human melanoma cell line can produce glycoproteins such as fibronectin, which is recognized by both the L2 and HNK-1 antibodies, and simultaneously a transfected Myelin Associated Glycoprotein carrying only L2-type carbohydrates. Conceivably, the differential expression of L2- and HNK-1 type glycans could have a role in development.

Anti-Thy-1-induced proliferation of immature thymocytes expressing the CD3-associated gamma/delta heterodimer.

A series of rat monoclonal antibodies (mAb) directed against the Thy-1 molecule have been evaluated for their ability to stimulate immature (CD4-8-) murine thymocytes. CD4-8- thymocytes could be induced to proliferate by several anti-Thy-1 mAb provided that exogenous interleukin 1 or interleukin 2 was provided. Apparently non-stimulatory anti-Thy-1 mAb could be rendered stimulatory if cross-linked by a rabbit anti-rat immunoglobulin reagent. Anti-Thy-1-stimulated CD4-8- thymocytes were found to uniformly express CD3 but did not stain positively with F23.1, a mAb reactive with alpha/beta T cell receptors utilizing the V beta 8 gene family. Immunoprecipitation analysis of such stimulated CD4-8- thymocytes indicated that they expressed a CD3-associated heterodimer of 45 kDa/35 kDa most likely corresponding to the product of the gamma and (putative) delta T cell receptor genes. Taken together, these data demonstrate that anti-Thy-1 mAb selectively activate a subset of CD4-8- thymocytes committed to the gamma/delta T cell receptor lineage.

Expression of the B cell-associated tyrosine kinase gene Lyn in primary neuroblastoma tumours and its modulation during the differentiation of neuroblastoma cell lines.

The src-related intracellular protein tyrosine kinase Lyn is a signal transducing molecule for surface immunoglobulin M and is expressed predominantly in hemopoietic cells. We report here the expression of the lyn gene in human neuroblastoma. In surgical tumour samples lyn transcripts were found preferentially at early stages whereas they were barely detectable in highly malignant tumours. In a cloned human neuroblastoma cell line, Be(2)C, lyn mRNA levels increased during neuronal differentiation induced by retinoic acid. Lyn mRNA levels were undetectable and did not respond to retinoic acid in a glial-type neuroblastoma clone, SH-EP. Retinoic acid-induced glial differentiation was associated with a reduction of lyn transcripts in a clonal I-type neuroblastoma cell line, SH-IN, which shares properties of both neuronal- and glial-type clones. Like pp60c-src Lyn may be involved in a signalling pathway of neuroblasts committed to neuronal differentiation.

Abnormal expression of T cell receptor genes in Lyt-2- L3T4- lymphocytes of lpr mice: comparison with normal immature thymocytes.

The autoimmunity and lymphoproliferation characteristic of lpr mice are age and thymus dependent. The accumulating Lyt-2- L3T4- T cells express only minimal cell-surface antigen receptor. After stimulation with phorbol myristic acetate (PMA) and interleukin 2, essentially normal levels of surface antigen receptor were expressed by the lpr Lyt-2- L3T4- subpopulation but remained undetectable in the corresponding normal immature thymocyte population. We have found near normal T cell receptor (TCR)-alpha and -beta mRNA expression by these lpr cells. The phenotypically comparable Lyt-2- L3T4- subset of normal thymocytes expressed approximately 10-fold less TCR-alpha mRNA than normal lymph node cells and somewhat higher TCR-beta mRNA. After cultivation of this immature thymocyte subpopulation with PMA and interleukin 2, TCR-alpha mRNA levels remained low, and TCR-beta transcripts were found to be subnormal. By using the same culture conditions, the corresponding mRNA levels of the lpr subset tended to resemble those of Lyt-2- L3T4- thymocytes. Fresh and cultured Lyt-2- L3T4- normal thymocytes showed comparably high levels of full-length TCR-gamma transcripts. In contrast, the fresh lpr subset had barely detectable levels of TCR-gamma mRNA. Upon cultivation, however, these levels increased over 200-fold to within the range observed in the Lyt-2- L3T4- thymocyte subset. No induction of the gamma gene was observed in similarly cultured normal lymph node T cells. The lpr cells may therefore correspond to an as yet undefined stage of normal T cell differentiation.

T cell lineages in the thymus of lpr/lpr mice. Evidence for parallel pathways of normal and abnormal T cell development.

Autoimmune mice homozygous for the lpr/lpr (lpr) gene develop a profound lymphadenopathy resulting from the accumulation of immature Thy-1+ Lyt-2- L3T4- cells in peripheral lymphoid tissues. The source of these cells is not known although the presence of a thymus is necessary to manifest both the lymph node enlargement and the autoimmunity. For this reason and the fact that the abnormal lpr cell phenotypically resembles immature thymocytes, we studied the thymus in lpr mice. Adult lpr thymuses were found to contain an immature population phenotypically identical to the peripherally accumulating cells, including the expression of B220 and Pgp-1 antigens as well as the presence of surface T cell receptor molecules as defined by the antibody KJ16-133. Evidence is presented that some of these lpr precursor T cells are capable of intrathymic differentiation, whereas the vast majority are exported unchanged to the lymph nodes where a portion differentiate further into mature T cells. This lpr-specific lineage could be distinguished from a normal component of the lpr thymus by surface phenotype and immunohistology. The results suggest that the massive accumulation of cells in lpr lymph nodes is not so much the result of abnormal proliferation of T cells as abnormal intrathymic differentiation. In addition, a minor subpopulation of normal Lyt-2- L3T4- thymocytes was identified that resembles the phenotype of the lpr cell and similarly expresses surface T cell receptor molecules. The presence of two parallel lineages in the lpr thymus thus also provides insight into normal T cell development.