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Biomedical subjects

R I Garver

Publications and source records attributed to R I Garver.

29 records · Page 2Linked to original sources
PubMed1991

Modulation of bFGF in lung fibroblasts by TGF-beta and PDGF.

Growth factors produced by alveolar macrophages are thought to promote the fibroblast proliferation within interstitial spaces of fibrotic lungs. This study investigated the possibility that the macrophage-produced growth factors might modulate the expression of basic fibroblast growth factor (bFGF) by lung fibroblasts. To evaluate this question, bFGF gene expression and protein production were evaluated in normal adult human lung fibroblast cell lines. Under normal culture conditions, the fibroblasts expressed the bFGF gene as two major transcripts (7.1, 3.7 kb). The addition of fetal calf serum (FCS) to serum-starved fibroblasts caused a 5- to 10-fold increase in bFGF expression. Steady-state bFGF expression was increased 108% by platelet-derived growth factor (PDGF) and 602% by transforming growth factor-beta (TGF-beta). Insulin-like growth factor-1 had no significant effect on bFGF expression. Nuclear runoff studies demonstrated that both PDGF and TGF-beta increased the relative rates of bFGF transcription in the fibroblasts. Western blot analysis of lysates from fibroblasts treated with either PDGF or TGF-beta had no detectable increase in bFGF protein above unstimulated controls. However, the simultaneous addition of PDGF and TGF-beta, or FCS, produced a marked increase in bFGF. These experiments show that two growth factors present in the alveolar airspace compartment of fibrotic lungs can promote the expression of bFGF within lung fibroblasts.

Adult↗
PubMed1991

Serum response heterogeneity among nonsmall cell lung cancer cell lines.

This study examined the morphology, in vitro growth, and two genetic responses to serum stimulation in the nonsmall cell lung cancer (NSCLC) cell lines SK-Lu-1, SK-MES-1, A427, and A549. Morphologically, all four were NSCLC: SK-Lu-1 was undifferentiated, the remainder were adenocarcinoma variants. SK-Lu-1 and SK-MES-1 were slow growing with low-anchorage independent growth capacity; the A427 and A549 lines were fast growing with high-anchorage independent growth capacity. All of the lines expressed basic fibroblast growth factor (bFGF) as a dominant 7.1 kb transcript at amounts significantly lower than in control human lung fibroblasts. bFGF expression could be upregulated by serum exposure in several nontransformed human cell lines, but only the SK-Lu-1 NSCLC cells increased bFGF after serum exposure (482%) compared with a peak increase of 1222% in the fibroblast controls. All of the NSCLC cell lines increased c-fos in response to the same serum stimulations. These results show that growth-factor gene expression can be modulated in NSCLC, and that significant differences exist among NSCLC cell lines commonly used as laboratory correlates of human disease.

Blood Proteins↗
PubMed1988

Emphysema associated with complete absence of alpha 1- antitrypsin in serum and the homozygous inheritance [corrected] of a stop codon in an alpha 1-antitrypsin-coding exon.

Homozygous inheritance of the null bellingham alpha 1-antitrypsin (alpha 1AT) gene is associated with early-onset emphysema, resulting from the lack of alpha 1AT to protect the lung from neutrophil elastase. Cloning and sequencing of the null bellingham gene demonstrated that the promoter region, coding exons, and all exon-intron junctions were normal except for a single base substitution in exon III, causing the normal lys217 (AAG) to become a stop codon (TAG). Evaluation of genomic DNA of family members by using oligonucleotides directed toward this region demonstrated that the index case had inherited this mutation in a homozygous fashion. Although the consequences to the individual (i.e., emphysema) are identical to those associated with the common homozygous Z mutation, the homozygous null bellingham form of alpha 1AT deficiency has a very different genetic basis.

Adult↗
PubMed1987

Clonal gene therapy: transplanted mouse fibroblast clones express human alpha 1-antitrypsin gene in vivo.

A retroviral vector was used to insert human alpha 1-antitrypsin (alpha 1AT) complementary DNA into the genome of mouse fibroblasts to create a clonal population of mouse fibroblasts secreting human alpha 1AT. After demonstrating that this clone of fibroblasts produced alpha 1AT after more than 100 population doublings in the absence of selection pressure, the clone was transplanted into the peritoneal cavities of nude mice. When the animals were evaluated 4 weeks later, human alpha 1AT was detected in both sera and the epithelial surface of the lungs. The transplanted clone of fibroblasts could be recovered from the peritoneal cavities of those mice and demonstrated to still be producing human alpha 1AT. Thus, even after removal of selective pressure, a single clone of retroviral vector-infected cells that expressed an exogenous gene in vitro, continued to do so in vivo, and when recovered, continued to produce the product of the exogenous gene.

Animals↗
PubMed1987

Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT.

Animals↗
PubMed1978

Characterization of the anti-stem cell activity of anti-mouse brain serum.

The anti-stem cell activity of a high-titer rabbit anti-mouse brain serum preparation has been further characterized. Following absorption with bone marrow and erythrocytes the antiserum had dose-dependent cytotoxicity against pluripotent stem cells. Rigorous absorptions with bone marrow, spleen, liver, erythrocytes, and thymus failed to remove the anti-stem cell activity of the serum. Adult brain, the immunogen, but not neonatal brain, removed a substantial amount of the activity against stem cells. Maximal cytotoxicity occurred both with and without complement and was maximal following only a 4 degrees C incubation of cells with serum. The anti-stem cell activity was present in the serum globulin fraction. No increase in the frequency of microcolonies or, with longer growth periods, in splenic macrocolonies was observed, suggesting that CFU-s were completely inactivated by exposure to the antiserum. Injected antiserum also reduced CFU-s in vivo.

Animals↗
PubMedDate not supplied

Coacervate microspheres as carriers of recombinant adenoviruses.

The therapeutic utility of recombinant adenoviruses (rAds) is limited in part by difficulties in directing the viruses to specific sites and by the requirement for bolus administration, both of which limit the efficiency of target tissue infection. As a first step toward overcoming these limitations, rAds were encapsulated in coacervate microspheres comprised of gelatin and alginate followed by stabilization with calcium ions. Ultrastructural evaluation showed that the microspheres formed in this manner were 0.8-10 microM in diameter, with viruses evenly distributed. The microspheres achieved a sustained release of adenovirus with a nominal loss of bioactivity. The pattern of release and the total amount of virus released was modified by changes in microsphere formulation. Administration of the adenovirus-containing microspheres to human tumor nodules engrafted in mice showed that the viral transgene was transferred to the tumor cells. It is concluded that coacervate microspheres can be used to encapsulate bioactive rAd and release it in a time-dependent manner.

Adenoviridae↗
PubMedDate not supplied

Quantitative and in vivo activity of adenoviral-producing cells made by cotransduction of a replication-defective adenovirus and a replication-enabling plasmid.

Achieving limited recombinant viral replication may provide a means of amplifying viral-mediated gene transfer in vivo. We have previously shown that cotransduction of an E1-defective adenovirus with a plasmid containing the deleted E1 functions into prostate carcinoma cells resulted in E1-defective virus production by those cells. The studies described here have extended these findings to more firmly establish the capacity of the trans complementation approach to achieve amplification of recombinant viral transgene expression. The recombinant virus used for all the studies was AdCMV-luc which contained a luciferase expression cassette; the replication-enabling plasmid, pE1, encoded the E1 functions deleted from AdCMV-luc. Quantitative in vitro studies with the HeLa cell line showed that for each plaque forming unit of AdCMV-luc originally exposed to the cells, 0.54 x 10(3) new replication-defective viruses were detected in supernatants and lysates over the following 4 days. Multiple cell lines were shown to support new virus production following cotransduction of AdCMV-luc and pE1. Small numbers of replication-competent viruses were detected in the lysates and supernatants from the cotransduced cells such that for every 10(5) replication-defective viruses approximately two replication-competent viruses were produced. Tumor nodules produced by engrafting a mixture of AdCMV-luc/pE1-cotransduced HeLa cells with uninfected HeLa cells yielded much higher levels of luciferase expression than control tumors containing mixtures of cells infected with AdCMV-luc alone. In total, these results demonstrate new virus production by cells receiving a replication-defective adenovirus and a replication-enabling plasmid are capable of amplifying recombinant viral transgene expression in vivo.

Adenoviridae↗
PubMedDate not supplied

Use of a tissue-specific promoter for targeted expression of the herpes simplex virus thymidine kinase gene in cervical carcinoma cells.

Molecular chemotherapy strategies have been developed for a number of epithelial malignancies based on selective delivery and expression of a toxin-encoding gene into the cancer cells. To date, these strategies have not been explored in the context of carcinoma of the cervix, despite the fact that a variety of factors suggest this as an appropriate disease for this gene therapy approach. One limitation in this respect is that appropriate tissue-specific promoters for selective toxin gene expression have not been defined for cervical carcinoma. In this regard, the secretory leukoprotease inhibitor (SLPI) gene has been shown to be constitutively expressed in many epithelial carcinoma cells including the uterine cervix. Thus, we investigated the utility of the SLPI gene as a tissue-specific promoter for regulatory control of the herpes simplex virus thymidine kinase gene for in vitro treatment of cervical carcinoma cells. For this analysis, a gene construct was derived with the herpes simplex virus thymidine kinase gene under regulatory control of the 5' upstream regions of the SLPI gene. Transient transduction of three human cervical carcinoma cell lines with the SLPI-thymidine kinase (TK) construct was followed by treatment with the prodrug ganciclovir. Crystal violet staining was subsequently used to assess cell viability. In this analysis, it was shown that the SLPI-TK construct directed TK-mediated killing in two of three tested cervical cell lines, with the two cell lines being positive for SLPI. In addition, mixing experiments established that cervical carcinoma cells could exhibit a bystander effect which potentially augments the efficacy of molecular chemotherapy approaches. These findings may allow for the development of efficacious, target-specific, toxin gene therapy strategies for cervical carcinoma in human patients.

Carcinoma↗