Andrew L. Feldhaus

Exogenous PDGF-D Is a Potent Mesangial Cell Mitogen and Causes a Severe Mesangial Proliferative Glomerulopathy

The PDGF family consists of at least four members, PDGF-A, -B, -C, and -D. All of the PDGF isoforms bind and signal through two known receptors, PDGF receptor-alpha and PDGF receptor-beta, which are constitutively expressed in the kidney and are upregulated in specific diseases. It is well established that PDGF-B plays a pivotal role in the mediation of glomerular mesangial cell proliferation. However, little is known of the roles of the recently discovered PDGF-C and -D in mediating renal injury. In this study, adenovirus constructs encoding PDGF-B, -C, and -D were injected into mice. Mice with high circulating levels of PDGF-D developed a severe mesangial proliferative glomerulopathy, characterized by enlarged glomeruli and a striking increase in glomerular cellularity. The PDGF-B-overexpressing mice had a milder proliferative glomerulopathy, whereas the mice overexpressing PDGF-C and those that received adenovirus alone showed no measurable response. Mitogenicity of PDGF-D and -B for mesangial cells was confirmed in vitro. These findings emphasize the importance of engagement of PDGF receptor-beta in transducing mesangial cell proliferation and demonstrate that PDGF-D is a major mediator of mesangial cell proliferation. Finally, this approach has resulted in a unique and potentially valuable model of mesangial proliferative glomerulopathy and its resolution.

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.

Use of Adenovirus-Mediated Gene Transfer to Facilitate Biological Annotation of Novel Genes

As part of a large program of gene annotation, use of adenovirus-mediated gene transfer facilitated rapid progress in the functional evaluation of more than 100 genes. Localized or systemic exposure to gene products expressed by adenovirus-transduced cells led to the discovery of several novel activities through analysis of resulting physiochemical or histological changes. In this summary of the work, we present examples of two studies in which activities of novel growth factors were initially characterized using this approach. In the first example, intravenous delivery of adenovirus encoding different forms of platelet-derived growth factor (PDGF) allowed us to evaluate effects of systemic exposure to two new members of this family, PDGF-C and PDGF-D, and led to specific new hypotheses regarding their roles in diseases of the liver and kidney, respectively. In the second example, localized delivery of adenovirus encoding fibroblast growth factor (FGF)-18 to mouse pinna led to the discovery that this novel FGF is a trophic factor for mature chondrocytes and their progenitors and might be useful for treating cartilage disease. These examples serve to illustrate the potential of in vivo gene delivery approaches to facilitate functional analysis and focus of secondary investigation in a large screening effort.