Comparison of efficiency of infection of human gene therapy target cells via four different retroviral receptors.

The relative efficiency of transduction of gene therapy target cells was measured for retroviruses bearing the envelopes of amphotropic murine leukemia virus (MLV-A), xenotropic murine leukemia virus (MLV-X), gibbon ape leukemia virus (GALV), feline leukemia virus subgroup B (FeLV-B), and the feline endogenous virus RD114. These viruses use various cell-surface receptors. Activated peripheral blood lymphocytes (PBL) and primary melanoma cultures were infected relatively poorly by MLV-X pseudotypes. RD114 pseudotypes infected PBL relatively well, whereas bone marrow progenitor cells were efficiently infected by all viruses. Helper-free virus bearing the envelopes of MLV-A, RD114, or GALV was similarly tested. All infected melanoma or bone marrow progenitor cells efficiently, whereas MLV-A was relatively inefficient for infection of PBL. The general utility of RD114 pseudotyped virus for gene delivery coupled with its resistance to inactivation by human serum makes this envelope the most suitable choice for in vivo gene therapy.

Superoxide production by normal and chronic granulomatous disease (CGD) patient-derived EBV-transformed B cell lines measured by chemiluminescence-based assays.

We have compared assays for products of the neutrophil respiratory burst in normal EBV-transformed B cell lines stimulated with agonists of protein kinase C. Those measuring O2- directly or its immediate product, H2O2, were successful. Of these, the most sensitive were the lucigenin- and luminol-based chemiluminescence assays for O2- and H2O2 respectively. Cell lines from CGD patients, with X-linked or autosomal recessive genetic defects in the neutrophil NADPH oxidase, did not respond in these assays, indicative of their inability to produce O2-. The defects in the lines studied encompass both proteins forming the cytochrome b-245 membrane component, and the 47 kDa cytosolic component of the NADPH oxidase. The possession of the disease associated phenotype by these cell lines provides evidence that in the normal situation both neutrophils and B cells produce O2- via the same system.

Abnormal expression of the MOS proto-oncogene in human thyroid medullary carcinoma.

We have been studying the expression of a range of proto-oncogenes in human thyroid tumour tissue by using Northern blot analysis. We have demonstrated the expression of a MOS mRNA of 1 kb in all thyroid samples. Furthermore, in a medullary carcinoma sample we also observed additional mRNA species of 1.7 and 2.2 kb. Southern blot analysis of DNA prepared from the same tumour sample did not reveal a rearrangement of the gene. These findings are the first report of MOS expression in any human tissue, and indicate that MOS oncogene activation might be important in the development of some thyroid tumours.

Flow cytometry analysis of gingival and periodontal ligament cells.

Gingival and periodontal ligament (PDL) fibroblasts are the major cellular components of periodontal soft connective tissues, but the precise differences between these cells are not yet known. In the present study, we have therefore examined the phenotypic and functional features of the cells obtained from gingival and PDL biopsy samples. Spindle-shaped cells characteristic of fibroblasts were the main cell type observed in vitro, although epithelial cells were also present in primary gingival cell cultures. Flow cytometry was used to measure the size and granularity of the cultured cells, and showed that the gingival fibroblasts were smaller and less granular compared with the PDL cells. The expression of certain key extracellular matrix (ECM) proteins, fibronectin, collagen type I, and tenascin was measured by flow cytometry. Analysis of the fluorescence profiles of these cultures showed that the majority of cells expressed fibronectin and that the average fluorescence intensity of this antigen in the PDL cells was higher than that in the gingival fibroblasts. Moreover, the fibronectin-positive PDL cells apparently comprised two subpopulations which expressed fibronectin at different levels, suggesting that the cells in the PDL cultures were functionally heterogeneous. The level of collagen type I was also found to be up-regulated in the PDL compared with the gingival cells and, as with fibronectin, was expressed at two different levels by subsets of the PDL cells. In contrast, tenascin was expressed at very similar levels by both the gingival fibroblasts and PDL cells. In addition, measurement of alkaline phosphatase, a marker enzyme for mineralized tissue-forming cells, showed that the PDL cells had higher activity than the gingival fibroblasts and that the alkaline phosphatase activity in the PDL cells was far more markedly up-regulated by dexamethasone. Our findings demonstrate that, despite their similar spindle-shaped appearance, fibroblasts derived from gingival and PDL tissues appear to display distinct functional activities which are likely to play a vital part in the maintenance of tissue integrity and regenerative processes.

Upregulation of keratinocyte growth factor in cyclosporin A-induced gingival overgrowth.

BACKGROUND: Drug-induced gingival overgrowth (GO) is a frequent and adverse side-effect associated principally with the administration of the immunosuppressive drug cyclosporin A (CsA) and also certain anti-epileptic and anti-hypertensive drugs. It is characterized by a marked increase in the thickness of the epithelial layer and the accumulation of excessive amounts of connective tissue. Although the mechanism by which the drugs cause GO is not yet understood, keratinocyte growth factor (KGF), which is a potent epithelial cell mitogen, has been implicated in other hyperplastic conditions, including mammary and prostatic hyperplasia, and could also be involved in the molecular pathology of GO. METHODS: Immunohistochemistry was used to examine the expression of KGF in normal gingiva (NG) and GO tissue sections. The relative level of KGF mRNA in GO tissue and cells was compared with that of NG tissue and fibroblast cells using the semi-quantitative reverse transcribed-polymerase chain reaction (RT-PCR) and DNA sequencing was carried out to confirm the identity of the PCR product. RESULTS: KGF antigen and mRNA were readily detected in the GO tissue immunohistochemically and by RT-PCR, respectively, but were not expressed in the NG tissue. Moreover, KGF transcripts were found to be approximately 2 times higher in the GO than in the NG fibroblasts in vitro, although the difference was not statistically significant. CONCLUSIONS: This study has shown, for the first time, that the level of KGF is elevated in GO and suggests that KGF may have an important role in the enhanced epithelial proliferation associated with GO.

Flow cytometry analysis of guided tissue regeneration-associated human periodontal cells.

BACKGROUND: Expanded polytetrafluoroethylene (ePTFE) barrier membranes have been widely used for guided tissue regeneration (GTR) of the human periodontal ligament (PL). However, the precise cellular and molecular events involved in the re-growth of the new tissue are still unclear. METHODS: Retrieved membranes and the newly-regenerated soft tissue (RT) underlying the membranes were used to examine the cells associated with GTR compared with normal human PL and gingival cells. Flow cytometry (FCM) was used, for the first time, to analyze the spindle-shaped fibroblast-like cells which were adherent to these membranes and the cells which grew out of the RT. RESULTS: The results showed that the membrane-associated (M) cells had the lowest rate of proliferation and appeared to be larger and more granular than the other types of cell. Moreover, both the M- and RT-derived cells were found to express higher levels of the extracellular matrix (ECM) proteins collagen type 1, fibronectin, tenascin, and decorin. In addition, evidence based on FCM profiles identified distinct sub-populations of GTR cells in which fibronectin expression was markedly up-regulated compared with normal PL cells and which also differed in size and granularity. CONCLUSIONS: The results of this study show that cells associated with GTR barrier membranes and with the underlying tissue appear to have distinct phenotypic and functional activities consistent with the production of new periodontal connective tissue and periodontal regeneration.

Polymerase chain reaction analysis of oestrogen and androgen receptor expression in human gingival and periodontal tissue.

Oestrogen and androgen receptors mediate the effects of their respective hormones by acting as ligand-activated transcription factors that control a wide range of biological processes. In order to determine whether periodontal and gingival tissues could respond to oestrogen and androgen, the specific and highly sensitive reverse-transcribed polymerase chain reaction (PT-PCR) was used to examine the presence of the mRNAs corresponding to these receptors. Expression of the androgen receptor was readily detected in periodontal and gingival tissue and in fibroblasts derived from these tissues, but transcripts for the oestrogen receptor were not detected in these samples. Moreover, treatment of the cultured fibroblasts with the oestrogen diethylstilbesterol (DES) or the androgen dihydrotestosterone (DHT) did not induce the expression of the oestrogen receptor mRNA; nor did the hormones modify the activity of the androgen receptor gene. These results suggest that, while periodontal and gingival tissues are not able to respond directly to oestrogen, they may nevertheless be highly sensitive to the anabolic effects of androgens.

Expression of growth-factor receptors in normal and regenerating human periodontal cells.

Growth factors are biologically active mediators that bind to specific receptors on target cells and regulate genes involved in cell growth, wound healing and regeneration. The expression of these receptors is thus of fundamental importance for the response of the cells to the factors. The aim here was to examine, using immunohistochemistry and flow cytometry, the expression of growth factor receptors in normal gingiva, periodontal ligament and in cells derived from these tissues, and also in regenerated tissues following guided tissue regeneration (GTR). By immunocytochemistry platelet-derived growth factor receptor-alpha (PDGF-Ralpha) was not detected in any of the tissues, whereas the PDGF-Rbeta and transforming growth factor-beta receptor types I and II (TGF-beta RI, RII) appeared to be upregulated in regenerated tissues compared with gingival and periodontal ligament tissues. Epidermal growth factor receptor (EGF-R) was also notably elevated in the regenerated tissue and was strongly expressed in the gingival epithelium but not in the periodontal ligament. Neither were fibroblast growth factor receptor-I (FGF-RI) or insulin-like growth factor receptor (IGF-R) detected in the periodontal ligament, nor in the gingiva, but they sometimes stained weakly in the regenerated tissues. Flow cytometry (FCM) showed that all the cells derived from the normal gingiva and the periodontal ligament expressed the PDGF-Rbeta, whereas the TGF-beta RI and RII, FGF-RI and IGF-R were detected in only a proportion of the total cells. In contrast, none of the cells expressed the PDGF-Ralpha or the EGF-R. These observations show that the growth factor receptors are differentially expressed by the periodontal tissues and cells and suggest that the corresponding factors may also be differentially involved in periodontal wound healing and regeneration.

Retroviral transduction of human periodontal cells with a temperature-sensitive SV40 large T antigen.

The periodontal ligament (PDL) is considered to contain subpopulations of cells responsible for the development, repair and regeneration of the periodontium. Cell cultures have been used as model systems in order to understand the complex cellular and biochemical events underlying these processes. In order to obtain long-term cultures of these cells that can be cloned and characterized, primary cultures of PDL and gingival cells were infected with an amphotropic retroviral construct encoding a temperature-sensitive SV40 large T antigen (tsT). After selection for drug resistance, the cells expressed the T antigen and proliferated at 34 degrees C for more than 40 passages. However, when the T antigen was inactivated by incubation at 39 degrees C, the cultures became growth-arrested and the granularity of the cells increased, possibly as a result of differentiation. Reverse transcribed-polymerase chain reaction and flow cytometry showed that the tsT-transduced cells expressed a number of soft and hard connective-tissue antigens, including osteocalcin, osteonectin, osteopontin, collagen type I and alkaline phosphatase. Moreover, incubation of the transduced PDL cells at 39 degrees C was found to upregulate the expression of osteocalcin, osteopontin and collagen type I, but downregulate osteonectin. At this temperature, the presence of the dexamethasone downregulated type I collagen, while vitamin D3 had no effect on the expression of any of the antigens examined. Under all culture conditions, antigen expression was far higher in the transduced PDL cells than the gingival cells. The findings thus show that growth of the tsT-transduced PDL and gingival cells is temperature-dependent and that the presence of the T antigen increases their lifespan but does not ablate the expression of certain of their characteristic phenotypic and functional features.

Expression and splicing of the fibronectin gene in healthy and diseased periodontal tissue.

Fibronectin is a major component of the extracellular matrix and is considered to have an important role in chronic inflammatory periodontal disease. The fibronectin gene product has been shown to be subject to alternative splicing in 3 regions, each generating different mRNA transcripts associated specifically with normal adult tissue, embryogenesis, tissue regeneration, and wound healing. In the present study, using the reverse-transcribed polymerase chain reaction to examine splicing profiles of the primary transcript, we found that healthy periodontal tissue expressed all alternatively spliced embryonic isoforms, indicative of the extensive and ongoing rebuilding processes which occur in these tissues. In marked contrast, only the exon-skipped transcripts were generated in tissue from chronic inflammatory periodontal disease patients. The loss of the high molecular weight isoforms in lesional tissues may be due to the excess production of inflammatory mediators in this disease, since we observed that high concentrations of the cytokine IL-1beta caused down-regulation of these transcripts in normal periodontal cells in tissue culture. Moreover, we also demonstrated that growth factors likely to be involved in periodontal regeneration and repair, such as PDGF, IGF-1 and TGF-beta, elicited pronounced upregulation of the embryonic isoforms of fibronectin in these cells. Although the functional activities of the antigens corresponding to the alternatively spliced variants of fibronectin are not yet known, our finding that they are selectively expressed suggests that they have highly specific roles in both periodontal breakdown and repair.

X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X-linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.

Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis.

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

Efficient retroviral transduction of human bone marrow progenitor and long-term culture-initiating cells: partial reconstitution of cells from patients with X-linked chronic granulomatous disease by gp91-phox expression.

The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X-CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X-CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase.

p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox.

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91-phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91-phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.

Restriction fragment length polymorphisms in the ETS-1 proto-oncogene. Comparison of Saudi and Western populations.

We have cloned part of the ETS 1 proto-oncogene and demonstrated the presence of two polymorphic Sst I restriction sites. A probe derived from one of our clones revealed the presence of 8.3 kb, 9.5 kb and/or 11.5 kb fragments on Southern blots of human DNA samples. The relative frequencies of these alleles appear to be significantly different between Saudi and Western populations, but there are no apparent differences in these frequencies between Saudi non-leukemic and leukemic individuals.

Detection of the H-RAS oncogene in human thyroid anaplastic carcinomas.

We have transfected high-molecular-weight DNA from human thyroid carcinomas into murine 3T3 cells. As a result we identified several foci of morphologically distinct transformed cells in each of the tumour DNA transfected cultures. After a total of three rounds of transfection, the transformed cells were shown to form tumours in nude mice. Southern blot analysis of DNA prepared from third-round transfectants demonstrated the presence of human Alu repetitive sequences and, after hybridization with probes for known oncogenes, indicated the presence of the human H-RAS oncogene in 3T3 cells transfected with three out of four anaplastic carcinoma DNA samples. It appears therefore that activation of RAS genes may be an important event in the development of the anaplastic thyroid tumours.