Fibroblast growth factor-18 is a trophic factor for mature chondrocytes and their progenitors.

OBJECTIVE: The aim of this study was to examine the effects of recombinant human Fgf18 on chondrocyte proliferation and matrix production in vivo and in vitro. In addition, the expressions of Fgf18 and Fgf receptors (Fgfr) in adult human articular cartilage were examined. METHODS: Adenovirus-mediated transfer of Fgf18 into murine pinnae and addition of FGF18 to primary cultures of adult articular chondrocytes were used to assess the effects of FGF18 on chondrocytes. In situ hybridization was used to examine the expression of Fgf18 and Fgfr s in adult human articular cartilage. RESULTS: Expression of Fgf18 by adenovirus-mediated gene transfer in murine pinnae resulted in a significant increase in chondrocyte number. Chondrocytes were identified by staining with toluidine blue and a monoclonal antibody directed against type II collagen. Fgf18, Fgfr 2-(IIIc), Fgfr 3-(IIIc), and Fgfr 4 mRNAs were detected within these cells by in situ hybridization. The nuclei of the chondrocytes stained with antibodies to PCNA and FGF receptor (FGFR) 2. Addition of FGF18 to the culture media of primary articular chondrocytes increased the proliferation of these cells and increased their production of extracellular matrix. To assess the receptor selectivity of FGF18, BaF3 cells stably expressing the genes for the major splice variants of Fgfr1-3 were used. Proliferation of cells expressing Fgfr 3-(IIIc) or Fgfr 2-(IIIc) was increased by incubation with FGF18. Using FGFR-Fc fusion proteins and BaF3 cells expressing Fgfr 3-(IIIc), only FGFR 3-(IIIc)-Fc, FGFR 2-(IIIc)-Fc or FGFR 4-Fc reduced FGF18-mediated cell proliferation. Expression of Fgf18, Fgfr 3-(IIIc) and Fgfr 2-(IIIc) mRNAs was localized to chondrocytes of human articular cartilage by in situ hybridization. CONCLUSION: These data demonstrate that Fgf18 can act as a trophic factor for elastic chondrocytes and their progenitors in vivo and articular chondrocytes cultured in vitro. Expression of Fgf18 and the genes for two of its receptors in chondrocytes suggests that Fgf18 may play an autocrine role in the biology of normal articular cartilage.

Platelet-derived growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor.

We have characterized platelet-derived growth factor (PDGF) C, a novel growth factor belonging to the PDGF family. PDGF-C is a multidomain protein with the N-terminal region homologous to the extracellular CUB domain of neuropilin-1, and the C-terminal region consists of a growth factor domain (GFD) with homology to vascular endothelial growth factor (25%) and PDGF A-chain (23%). A serum-sensitive cleavage site between the two domains allows release of the GFD from the CUB domain. Competition binding and immunoprecipitation studies on cells bearing both PDGF alpha and beta receptors reveal a high affinity binding of recombinant GFD (PDGF-CC) to PDGF receptor-alpha homodimers and PDGF receptor-alpha/beta heterodimers. PDGF-CC exhibits greater mitogenic potency than PDGF-AA and comparable or greater mitogenic activity than PDGF-AB and PDGF-BB on several mesenchymal cell types. Analysis of PDGF-CC in vivo in a diabetic mouse model of delayed wound healing showed that PDGF-CC significantly enhanced repair of a full-thickness skin excision. Together, these studies describe a third member of the PDGF family (PDGF-C) as a potent mitogen for cells of mesenchymal origin in in vitro and in vivo systems with a binding pattern similar to PDGF-AB.

Molecular cloning, chromosome mapping and characterization of UBQLN3 a testis-specific gene that contains an ubiquitin-like domain.

The sequence of the ubiquitin protein is highly conserved between species and has facilitated the cloning of numerous ubiquitin-like proteins. In the present study, we report the cloning of the cDNA for human ubiquilin 3 (UBQLN3). The deduced amino acid sequence of UBQLN3 contains a UBQ domain (ubiquitin-like) in the amino terminus as well as two highly conserved domains found in several recently cloned ubiquitin-like proteins. One of these domains, termed the NP domain, is a highly conserved 93 amino acid region present in UBQLN3 and several ubiquitin-like proteins. The last conserved domain is the UBA domain (ubiquitin-associated) found in a variety of proteins of the ubiquination pathway. The human UBQLN3 gene was mapped to the 11p15 region of chromosome 11. Northern blot analysis of multiple human and mouse tissues demonstrated UBQLN3 mRNA expression specifically in testis.

Molecular cloning, chromosome mapping and characterization of a testis-specific cystatin-like cDNA, cystatin T.

The cystatin superfamily of cysteine proteinase inhibitors consists of three major families. In the present study, we report the cloning of the cDNA for mouse cystatin T, which is related to family 2 cystatins. The deduced amino acid sequence of cystatin T contains regions of significant sequence homology including the four highly conserved cysteine residues in exact alignment with all cystatin family 2 members. However, cystatin T lacks some of the conserved motifs believed to be important for inhibition of cysteine proteinase activity. These characteristics are seen in two other recently cloned genes, CRES and Testatin. Thus, cystatin T appears to be the third member of the CRES/Testatin subgroup of family 2 cystatins. The mouse cystatin T gene was mapped on a region of chromosome 2 that contains a cluster of cystatin genes, including cystatin C and CRES. Northern blot analysis demonstrated that expression of mouse cystatin T is highly restricted to the mouse testis. Thus, a shared characteristic of the cystatin family 2 subgroup members is an expression pattern limited primarily to the male reproductive tract.

Expression of chimeric granulocyte-macrophage colony-stimulating factor/interleukin 2 receptors in human cytotoxic T lymphocyte clones results in granulocyte-macrophage colony-stimulating factor-dependent growth.

Adoptive immunotherapy with ex vivo-expanded antigen-specific cytotoxic T lymphocytes (CTLs) has been shown to clear viral infections and eliminate tumors in murine models. Clinical trials have also reported promising data for the use of adoptive immunotherapy to treat cytomegalovirus (CMV) and Epstein-Barr viral (EBV) infections in bone marrow transplant recipients. For these indications, the need for ex vivo-expanded CTLs is often short lived, until the immune system is reconstituted by the donor transplant. In chronic disease settings, increased longevity of adoptively transferred CTLs and generation of memory will be necessary. The additional administration of helper functions normally supplied by antigen-specific T helper (Th) cells will probably be essential for long-term survival of adoptively transferred CTLs. Toward this goal of supplying helper functions, we transduced human CTLs with chimeric GM-CSFR/IL-2R receptors that deliver an IL-2 signal on binding GM-CSF. Clones expressing the chimeric receptors proliferated in response to GM-CSF. Stimulation with antigen induced GM-CSF production and resulted in an autocrine growth loop such that the CTL clones proliferated in the absence of exogenous cytokines. This type of genetic modification has potential for increasing the circulating half-life and, by extension, the efficacy of ex vivo-expanded CTLs.

A CD2/CD28 chimeric receptor triggers the CD28 signaling pathway in CTLL.2 cells.

We are evaluating strategies to enhance the in vivo proliferation and function of adoptively transferred antigen-specific T cells. Although the CD28 costimulatory pathway is important for T cell activation and proliferation, the expression of the ligands for CD28 is highly restricted. We have generated a chimeric receptor composed of the signaling domains of CD28 and the extracellular domain of CD2 which binds the widely expressed ligand CD58. The CD2/CD28 chimeric receptor was introduced into CTLL.2 cells via retrovirus infection and was shown to be expressed on the cell surface. By monitoring early and late components of the CD28 signaling pathway, the chimeric receptor was demonstrated to trigger the CD28 pathway in response to CD2 cross-linking. The possible utility of the CD2/CD28 chimeric receptor for adoptive immunotherapy is discussed.

Manipulation of T cell costimulatory and inhibitory signals for immunotherapy of prostate cancer.

The identification of potentially useful immune-based treatments for prostate cancer has been severely constrained by the scarcity of relevant animal research models for this disease. Moreover, some of the most critical mechanisms involved in complete and proper antitumoral T cell activation have only recently been identified for experimental manipulation, namely, components involved in the costimulatory pathway for T cell activation. Thus, we have established a novel syngeneic murine prostate cancer model that permits us to examine two distinct manipulations intended to elicit an antiprostate cancer response through enhanced T cell costimulation: (i) provision of direct costimulation by prostate cancer cells transduced to express the B7.1 ligand and (ii) in vivo antibody-mediated blockade of the T cell CTLA-4, which prevents T cell down-regulation. In the present study we found that a tumorigenic prostate cancer cell line, TRAMPC1 (pTC1), derived from transgenic mice, is rejected by syngeneic C57BL/6 mice, but not athymic mice, after this cell line is transduced to express the costimulatory ligand B7.1. Also, we demonstrated that in vivo antibody-mediated blockade of CTLA-4 enhances antiprostate cancer immune responses. The response raised by anti-CTLA-4 administration ranges from marked reductions in wild-type pTC1 growth to complete rejection of these cells. Collectively, these experiments suggest that appropriate manipulation of T cell costimulatory and inhibitory signals may provide a fundamental and highly adaptable basis for prostate cancer immunotherapy. Additionally, the syngeneic murine model that we introduce provides a comprehensive system for further testing of immune-based treatments for prostate cancer.

The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex.

CD69 is a rapidly induced T cell activation Ag that is also expressed in an inducible fashion on cells of most, if not all, hematopoietic lineages. Molecular cloning has shown that CD69 is a type II membrane glycoprotein that is a member of the C-type lectin family. In this report we have shown that induction of CD69 mRNA in activated murine thymocytes and T cells is very rapid, peaking between 30 and 60 min poststimulation, and transient, dropping to nearly resting levels by 8 h. An analysis of the mouse CD69 gene structure showed the gene to consist of 5 exons and have a phorbol ester-inducible promoter element within the first 700 bp upstream of the start of transcription. Chromosomal mapping placed the mouse CD69 gene on the long arm of chromosome 6 near the NK gene complex that contains the related NKR-P1 and Ly-49 gene families. The human CD69 gene mapped to chromosome 12p13 near the related NKG2 gene cluster and in a region associated with rearrangements in approximately 10% of cases of childhood acute lymphocytic leukemia.

Targeted disruption of the Oct-2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element-mediated gene activation.

The Oct-2 protein is a tissue-specific POU-homeodomain transcription factor. It has been considered to represent a developmental regulator of immunoglobulin gene expression by virtue of its interaction with a functionally essential octamer element found in immunoglobulin gene promoters. This proposal has been most strongly challenged by several in vitro transcription analyses which have shown that the related ubiquitous factor Oct-1 can activate transcription from immunoglobulin gene promoters as efficiently as Oct-2. We have genetically analyzed Oct-2 function by using gene targeting to disrupt both alleles of the locus in the murine B cell line WEHI-231. This cell line expresses productively rearranged immunoglobulin genes as well as the Oct-2 gene at high levels which are comparable to those observed in activated murine splenic B cells. In spite of a drastic reduction in Oct-2 levels (20-fold), no effect was observed on the expression of endogenous immunoglobulin genes or on the activity of a transfected immunoglobulin promoter or a heterologous promoter with a single octamer element. In contrast, expression of a reporter construct containing multiple octamer motifs upstream of a heterologous promoter was severely reduced in the double-disruptant cells. The differential responses of the single- and multiple-octamer motif reporter constructs in the mutant B cells are unlikely to be a consequence of differing concentration requirements for activation by Oct-2. The two constructs are activated equivalently over the same range of Oct-2 concentration in a non-B cell. These results provide genetic support for the existence of an Oct-2-independent, but octamer element-dependent, B cell-specific pathway for immunoglobulin gene transcription. They also genetically reveal a distinct Oct-2-dependent pathway of octamer-mediated gene activation. This study demonstrates the feasibility of targeting a diploid locus in a somatic mammalian cell line. Extension of this approach to genes encoding other transcription factors will allow a genetic dissection of their functions within the context of cell lines representing various differentiation states.

BLyF, a novel cell-type- and stage-specific regulator of the B-lymphocyte gene mb-1.

The mb-1 gene encodes an integral membrane protein that appears to be required for the surface expression and signalling function(s) of the immunoglobulin receptor on B lymphocytes. The gene is expressed in a lineage-restricted manner. It is activated early in B-cell ontogeny, continues to be expressed in mature B cells, but is turned off in terminally differentiated plasma cells. We have identified the mb-1 promoter and functionally tested its activity by transient transfections. A 737-bp promoter fragment preferentially stimulates accurately initiated transcription in mb-1-expressing B cells. Deletion analysis of the promoter suggests the presence of two functional domains, proximal and distal. Both domains independently activate transcription from a heterologous promoter. The distal domain functions in a cell-type- and stage-specific manner, activating transcription in B cells but not in T cells or plasma cells. A 25-bp element within this domain is necessary and sufficient for activity. This element is recognized by a novel cell-type- and stage-specific transcription factor termed BLyF. The binding of BLyF completely correlates with the ability of the regulatory element to stimulate transcription. Thus, BLyF appears to positively regulate transcription of the mb-1 gene. Our results also suggest that the inactivity of the mb-1 locus in plasma cells is not simply due to the loss of BLyF activity.

Molecular analysis of the Corynebacterium glutamicum lysl gene involved in lysine uptake.

Two Corynebacterium glutamicum mutants defective in lysine uptake were identified by analysing mutants resistant to S-(2-aminoethyl)-cysteine (AEC). A 5.6 kb genomic DNA fragment restoring AEC sensitivity and lysine uptake was isolated. A 4.2 kb subfragment was sequenced and three open reading frames were identified. Subcloning and gene disruption experiments showed that only the first open reading frame, termed lysl, is involved in lysine uptake. Lysl consists of 501 amino acids with a Mr of 53600. The hydrophobicity profile suggests that the lysl gene product is an integral membrane protein with 13 transmembrane segments. The amino acid sequence of lysl displays strong homology to that of the arcD gene product of Pseudomonas aeruginosa, which is proposed to act as an arginine-ornithine antiporter. Investigation of the influence of the lysl gene on lysine secretion suggests the existence of a separate lysine efflux system in C. glutamicum.

Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria.

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.

Nucleotide sequence of the L gene of vesicular stomatitis virus (New Jersey): identification of conserved domains in the New Jersey and Indiana L proteins.

The nucleotide sequence of the L gene of vesicular stomatitis virus, New Jersey serotype (Hazelhurst subtype), was determined. Primer extension dideoxy sequencing of genomic RNA using reverse transcriptase initiated within the adjacent G gene provided a consensus sequence of 6522 nucleotides. The G/L intergenic junction spanned 21 nucleotides and contained a pseudo transcription start signal as well as two sequences (10 and 6 nucleotides in length) which are reiterated within the L coding region. The predicted L mRNA was 6398 nucleotides long and contained a single open reading frame corresponding to an L protein encompassing 2109 amino acids with a MW of 241,546. Comparison of the amino acid sequence of this New Jersey serotype L protein to that previously reported for the L protein of the serologically and genetically distinct Indiana serotype (M. Schubert, G. G. Harmison, and E. Meier (1984). J. Virol. 51, 505-514.) revealed a high degree of functional homology. In addition, six regions (43 to 103 amino acids in length) which displayed a high percentage of identical amino acids (85 to 96%) were identified. Five of these regions were clustered within the amino-terminal half of the L protein. Two of these regions contained sequences, 41 amino acids in length, which were significantly similar to corresponding regions of the L proteins of the paramyxoviruses Sendai and Newcastle disease virus. These structurally conserved regions may correspond to functional domains of the multifunctional L protein.