Brian M. Leo

Collagen and Proteoglycan Abnormalities in the GDF-5-Deficient Mice and Molecular Changes When Treating Disk Cells With Recombinant Growth Factor

Study Design. A magnetic resonance image, histologic, biochemical, and gene expression study was conducted to characterize the effects of growth and development factor-5 (GDF-5) deficiency on the health of the intervertebral disc. Objective. To determine the effect of GDF-5 deficiency on extracellular matrix and gene expression on the intervertebral disc. Summary of Background Data. Developmental and degenerative changes in intervertebral disc are not fully understood. Molecular abnormalities and spontaneous mutations that lead to the deficiency in a normal protein have been useful in understanding the function of certain molecules and the role they play in the structure and health of certain tissues. Although the role of GDF-5 in the disc has not been elucidated, this factor may have an important role in the disc as a result of the well-documented effect of GDF-5 in other chondrogenic tissues. Methods. Intervertebral discs of 20-week-old GDF-5 (−/−) and (+/+) mice were examined radiographically, histologically, biochemically, and with gene expression studies. Cells isolated from GDF-5-deficient mouse discs were treated with recombinant GDF-5 and gene expression was subsequently analyzed. Results. GDF-5 (−/−) mice demonstrated significantly lower T2-weighted signal intensity in the central region of their lumbar discs, and disc histology revealed loss of the normal lamellar architecture of the anulus fibrosus and a shrunken, disorganized nucleus pulposus. Biochemical analysis revealed decreased proteoglycan content but no appreciable change in total collagen content of the discs. Significant downregulation of both aggrecan and type II collagen mRNA, without an appreciable change in type I collagen expression, was noted on gene expression studies. Recombinant GDF-5 treatment of disc cells from the GDF-5-deficient mice resulted in a dose-dependent upregulation of the aggrecan and type II collagen genes. Conclusion. The intervertebral disc is markedly affected by GDF-5 deficiency. This relatively simple (single gene) system with a known molecular defect may be useful in studies designed to define the response of the intervertebral disc to treatment with growth factor in vivo.

In vivo bioluminescent imaging of virus-mediated gene transfer and transduced cell transplantation in the intervertebral disc

Study Design. Work presented here used a small animal model to demonstrate the feasibility and usefulness of in vivo bioluminescent imaging to studying degenerative disc disease. Objectives. To determine the utility of in vivo bioluminescent imaging to monitor the temporal and spatial expression of genetically modified cells within the intervertebral disc of a rodent model. Summary of the Background Data. Noninvasive imaging of genetically engineered cells in the spine has the advantage of allowing events to be tracked without killing the animal and can be used to follow the time course of a particular therapy. Results are presented on the use of Sprague-Dawley rats in experimental studies in which the luciferase reporter gene was delivered to the lumbar intervertebral disc through adenovirus-mediated or cell-based transfer techniques to demonstrate the feasibility to monitor gene expression noninvasively over time. Methods. Tissue culture, disc surgery, and in vivo bioluminescent imaging were used. The intervertebral disc of the rat was either injected in situ with an adenovirus containing the luciferase reporter gene or implanted with fat, bone marrow or intervertebral disc cells transduced ex vivo and contained in a bioresorbable carrier. Results were assessed with in vivo bioluminescent imaging at several time points. Conclusion. Data from 11 animals were obtained with imaging up to 14 days. To our knowledge, this is the first description of in vivo bioluminescence imaging to study spinal conditions. We have characterized the relative expression of three cell types transduced with the Ad-luc virus by ex vivo transfection followed by cell implantation in the rat spine and compared them to one another and to direct infection of Ad-luc adenovirus in situ. Our results demonstrate the feasibility of tracing genetically altered cells in the spine. This technique has the potential to be used to noninvasively track the fate and expression of therapeutic genes within the spine of small animals used in disc research.

Comparison of adherence, cytotoxicity, and Gal/GalNAc lectin gene structure in Entamoeba histolytica and Entamoeba dispar

Abstract The recent development of axenic culture for E. dispar allowed us to examine this amebas ability to bind and lyse target cells and compare it to E. histolytica which has been axenically cultured for years. We found that under axenic conditions, E. dispars adherence to target cells, ligand binding, and cytotoxicity were less than that of E. histolytica . These events were Gal/GalNAc-inhibitable supporting the idea that E. dispar expresses a lectin similar to E. histolytica . Genetic analysis showed that E. dispar had at least two members of the lectin heavy subunit family and four members of the lectin light subunit family that hybridized to ehhgl and ehlgl gene probes. A library screen produced clones which were isolated and sequenced. Derived amino acid sequences showed that the E. dispar heavy and light subunit clones were 86% and 79% identical, respectively, to their E. histolytica counterparts. In particular, the region which contains the epitopes for two adherence-enhancing monoclonal antibodies and a complement-sensitizing monoclonal antibody (amino acids 882–959 of the lectin heavy subunit) were conserved between the species.