Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, can be induced by immunization with a number of myelin antigens. In particular, myelin oligodendrocyte glycoprotein, a central nervous system (CNS)-specific antigen expressed on the myelin surface, is able to induce a paralytic MS-like disease with extensive CNS inflammation and demyelination in several strains of animals. Although not well understood, the egress of immune cells into the CNS in EAE is governed by a complex interplay between pro and antiinflammatory cytokines and chemokines. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is considered to play a central role in maintaining chronic inflammation. The present study was designed to investigate the previously unexplored role of GM-CSF in autoimmune-mediated demyelination. GM-CSF(-/)- mice are resistant to EAE, display decreased antigen-specific proliferation of splenocytes, and fail to sustain immune cell infiltrates in the CNS, thus revealing key activities for GM-CSF in the development of inflammatory demyelinating lesions and control of migration and/or proliferation of leukocytes within the CNS. These results hold implications for the pathogenesis of inflammatory and demyelinating diseases and may provide the basis for more effective therapies for inflammatory diseases, and more specifically for multiple sclerosis.

H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls.

Following the 2009 H1N1 influenza pandemic, an increased risk of narcolepsy type 1 was observed. Homology between an H1N1 hemagglutinin and two hypocretin sequences has been reported. T cell reactivity to these peptides was assessed in 81 narcolepsy type 1 patients and 19 HLA-DQ6-matched healthy controls. HLA-DQ6-restricted H1N1 hemagglutinin-specific T cell responses were detected in 28.4% of patients and 15.8% of controls. Despite structural homology between HLA-DQ6-hypocretin and -H1N1 peptide complexes, T cell cross-reactivity was not detected. These results indicate that it is unlikely that cross-reactivity between H1N1 hemagglutinin and hypocretin peptides presented by HLA-DQ6 is involved in the development of narcolepsy.

NYK/FLK-1: a putative receptor protein tyrosine kinase isolated from E10 embryonic neuroepithelium is expressed in endothelial cells of the developing embryo.

A protein tyrosine kinase (NYK/FLK-1), bearing all the hallmarks of a growth factor receptor, has been isolated from a cDNA library generated from enriched populations of mouse day 10 embryonic neuroepithelium and from day 18 embryonic colon. Sequence analysis of cDNAs covering the entire coding region of this 5.7 kb mRNA predicted the presence of seven immunoglobulin-like domains in the extracellular region of this molecule. This feature, coupled with the detection of an insert domain bisecting the kinase domain of the predicted protein sequence, places NYK/FLK-1 firmly in the Platelet-derived Growth Factor Receptor-related class of molecules. NYK/FLK-1 is expressed at high levels in adult heart, lung, kidney, brain and skeletal muscle, but is also expressed at lower levels in most other adult tissues. In situ hybridization of day 12.5 embryo sections demonstrated NYK/FLK-1 mRNA expression in endothelial cells lining the dorsal aorta and intervertebral veins. In addition, expression was found in cells lining the capillary plexus which surrounds the developing neuroepithelium, and in the endothelial cells which are found within the embryonic lung, spleen, liver and metanephros.

Expression of two novel eph-related receptor protein tyrosine kinases in mammary gland development and carcinogenesis.

To investigate the involvement of protein tyrosine kinases (PTKs) in the growth control of mammary epithelial cells, we have used PCR based cloning to identify PTKs expressed in a mouse mammary epithelial cell line. This approach led to the isolation of two receptor PTKs of the eph-related subfamily; myk-1, a novel member expressed predominantly in lung, heart and mammary gland and myk-2, a close relative of the human eck gene. Northern blot analysis of RNA from mouse mammary glands at different stages of development revealed that myk-1 and myk-2 expression is induced at puberty and differentially regulated during the estrus cycle. myk-1 and myk-2 expression was down-regulated during the pregnancy induced differentiation of the mammary gland. Over-expression of myk-1 and myk-2 was found in the undifferentiated and invasive mammary tumors of transgenic mice expressing the Ha-ras oncogene. In contrast, no elevated expression of either gene could be detected in the well differentiated and non-metastatic mammary tumors of c-myc expressing transgenic mice. These results indicate that myk-1 and myk-2 expression is induced during the proliferation of the mammary gland and down-regulated by its differentiation.

Baculovirus cDNA libraries for expression cloning of genes encoding cell-surface antigens.

We describe a method for the production of baculovirus-based cDNA libraries. By staining with monoclonal antibodies, single positive cells can be sorted and the virus encoding for the surface epitope can be isolated by limiting dilution. We have used this method to isolate cDNAs encoding several cell-surface antigens.

Expression of two different forms of fibroblast growth factor receptor 1 in different mouse tissues and cell lines.

The fibroblast growth factor receptors (FGFRs) form a multigene family of at least four members, all having extracellular regions consisting of either two or three immunoglobin-like (Ig-like) domains. By RNase protection analysis we have analyzed the expression of FGFR-1 mRNA in various tissues and cell lines and demonstrated that all of the cell lines studied expressed at least two different forms of the FGFR-1 at similar levels. Although muscle and heart express forms having either two [FGFR-1 short (FGFR-1S)] or three [FGFR-1 long (FGFR-1L)] Ig-like domains, the developing brain and adult brain express only mRNA encoding the longer form. The two forms of the receptor were characterized further by stably introducing expression vectors expressing them into Rat-2 fibroblasts and FDC-P1 myeloid cells. Treatment of the transfected Rat-2 cells with acidic FGF (aFGF) or basic FGF (bFGF) resulted in focus formation. The transformed phenotype was observed even without addition of ligand after growth in culture for greater than 2 months. Cross-linking of 125I-labeled bFGF (125I-bFGF) to Rat-2 cells expressing either FGFR-1L or FGFR-1S yielded two similar complexes of 150 and 110 kDa. Although Rat-2 cells expressing FGFR-1L yielded similar complexes with 125I-labeled aFGF (125I-aFGF), only the 150-kDa complex was formed with cells expressing FGFR-1S. The 150-kDa complex was also observed when 125I-aFGF or 125I-bFGF was cross-linked to FDC-P1 cells expressing FGFR-1L. Significantly, these complexes were only observed when heparin was present in the cross-linking reaction. FDC-P1 cells expressing FGFR-1 bound aFGF and bFGF with high affinity but only in the presence of heparin. The factor dependence of these cells could be switched from interleukin 3 to FGF in the presence of heparin.

Role of the c-myc and the N-myc proto-oncogenes in the immortalization of neural precursors.

To study the role of c-myc and N-myc in the immortalization of neural precursors, we infected neuroepithelial cells isolated from 10-day-old mouse embryos with a new retrovirus vector, pzen, harboring either the c-myc or the N-myc oncogene. The immortalized cell lines contain high levels of the virally expressed myc protein. The amount of myc proteins correlated with the capacity of the cells to differentiate spontaneously in vitro into neurons and glia; cell lines expressing high levels of myc protein can differentiate spontaneously while cell lines expressing low levels of the myc protein resemble epithelial cells. Addition of acidic or basic fibroblast growth factor enhanced differentiation of most cell lines. Some of the cell lines produced neurotrophic growth factors capable of supporting the growth of other cell lines at low density. There was no significant difference between cell lines immortalized with c-myc or with N-myc. Most of the immortalized cells lines generated from bipotential precursors are capable of differentiating into neurons and glia.

Two forms of the basic fibroblast growth factor receptor-like mRNA are expressed in the developing mouse brain.

The embryonic neuroepithelium gives rise to the components of the central nervous system in the mature animal. To study the early development of the murine central nervous system we have sought to isolate growth factor receptors from the neuroepithelium of the neural tube of 10-day-old mouse embryos. Because many growth factor receptors are members of the protein-tyrosine kinase family, we have used the polymerase chain reaction to amplify mRNA sequences from 10-day-old mouse embryo neuroepithelium; these sequences lie between the nucleotide sequences of two highly conserved amino acid motifs from the catalytic domain of protein-tyrosine kinases. By using this technique we have isolated a clone encoding the murine basic fibroblast growth factor receptor (bFGF-R), as well as a shorter form of this mRNA. This latter cDNA comprised 75% of the bFGF-R cDNA clones isolated from the immortalized neuroepithelial cell lines. This variant mRNA, designated here as N-bFGF-R, appears to be expressed at higher levels in neuronal cells in early stages of development. The bFGF-R is a member of a multigene family, as demonstrated by Southern blot analysis and the cloning of two other members of this family.

Immortalization of mouse neural precursor cells by the c-myc oncogene.

Immortalized cell lines have been generated from embryonic mouse neuroepithelium by infection with a retrovirus containing the c-myc oncogene. The morphology and the antigenic phenotype of the cloned cell lines are characteristic of normal neuroepithelium. Although the cell lines are stable and do not spontaneously differentiate, morphological changes can be induced with both acidic and basic fibroblast growth factor. Fibroblast growth factor at 5 ng/ml stimulates differentiation of the neuroepithelial cells, and it has been shown that the cloned cell line 2.3D can differentiate into astrocytes, containing glial fibrillary acidic protein, and neurons, expressing the A2B5 marker and neurofilaments. This indicates that some cells in the neuroepithelium at embryonic day 10 are multipotent and are not restricted to either the glial or neuronal cell lineage. The cell lines also can be induced with interferon gamma to express class I and class II histocompatibility antigens. The response of the c-myc-immortalized cell lines to these two factors is similar to that observed with freshly isolated neuroepithelium and suggests that such immortalized precursor populations are representative of the cells found in the developing neuroepithelium.

Tyrosine kinases: from viral oncogenes to developmental regulators.

Protein tyrosine kinases (PTKs) play a central role in cellular regulation by virtue of their participation in, and control of, signal transduction pathways; they act as a molecular interface between the cell's environment and intercellular metabolism. The mammary gland, unlike most organs, undergoes most of its morphogenesis in juvenile and adult life. The epithelium goes through hormonally controlled cycles of proliferation and regression, the fully differentiated state only being reached at the end of pregnancy. These features make the mammary gland an amenable tissue to study the involvement of PTKs in epithelial cell development and differentiation. We have used a PCR-based molecular cloning strategy to identify PTKs from murine mammary gland cells. Amongst 70 kinase clones characterized we found 3 PTKs previously undescribed in mouse, 4 known PTKs and 5 serine threonine kinases. Expression studies revealed differential tissue specificity and developmental regulation of the 3 previously undescribed PTKs. These results substantiate the view that PTKs are involved in the regulation of cellular differentiation.