Hepatocyte proliferation and gene expression induced by triiodothyronine in vivo and in vitro

Subcutaneous injections of hormone triiodothyronine in rats resulted in peak blood levels at 24 hr with return to baseline by 96 hr. The injections stimulated a liver regeneration response that resembled in timing and in magnitude of DNA synthesis (peak, 24 hr) that induced by 40% hepatic resection. The principal proliferation was of hepatocytes. Although there were some temporal differences from the gene expression of transforming growth factor-alpha, transforming growth factor-beta, and c-Ha-ras that are known to follow partial hepatectomy, the overall profile of these changes was similar to those after partial resection. The effect was liver specific and could be reproduced three times with no diminution in response in the same animal with injections at 10-day intervals. No response was detected in kidney or intestine. This effect in intact animals contrasted with the minimal ability of triiodothyronine to stimulate hepatocytes in culture. However, when the culture medium was enriched with epidermal growth factor, there was a dose-related response to triiodothyronine. The totality of these experiments provides a preliminary basis for the creation with pharmacological techniques of an in vivo hyperplastic hepatic condition permissive of transfection of new genes, as an alternative to partial hepatectomy. Although triiodothyronine was the test agent used, other hepatic growth factors singly or in combination could be candidates for this purpose.     

Inhibition of viral gene expression by human ribonuclease P

External guide sequences (EGSs) are small RNA molecules which consist of a sequence complementary to a target mRNA and render the target RNA susceptible to degradation by ribonuclease P (RNase P). EGSs were designed to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 for degradation. These EGSs were shown to be able to direct human RNase P to cleave the TK mRNA sequence efficiently in vitro. A reduction of about 80% in the expression level of both TK mRNA and protein was observed in human cells that steadily expressed an EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS which carried a single nucleotide mutation that precluded RNase P recognition. Thus, EGSs may represent novel gene-targeting agents for inhibition of gene expression and antiviral activity.     

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.     

Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-L-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-L-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-L-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-L-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a beta-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-L-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-L-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-L-lysine.     

Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro

IPRS is a freely available software system which consists of about 250 library functions in C, and a set of application programs. It is designed to run under UNIX and comes with full source code, system manual pages, and a comprehensive user's and programmer's guide. It is intended for use by researchers in human vision, pattern recognition, image processing, machine vision and machine learning.     

Adenovirus-mediated regulable target gene expression in vivo

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor-ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-kappaB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.     

Laminin chain expression by chick chondrocytes and mouse cartilaginous tissues in vivo and in vitro

We have observed that laminins are expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and adult mouse knee joint by the methods of in situ hybridization, immunohistochemistry, Western blotting, and immunoprecipitation. From in situ hybridization using similar sized RNA probes for different mouse laminin chains, mRNAs for the alpha 1, alpha 2, beta 1, beta 2, and gamma 1 chains were expressed in the chondrocytes of chick embryo sternum, mouse limb bud, and the articular cartilage cap and epiphyseal growth plate of adult mouse knee joint. Through the use of chain-specific antibodies, staining for laminins was observed in the cytoplasm of chondrocytes from chick embryo sternum, mouse limb bud, and adult mouse knee joint. Western blot analysis confirmed the presence of laminin chains in the cells and sternal tissues. Cultured chick embryonic sternal chondrocytes expressed laminin mRNAs in the proliferating stage (2-3 days of culture) but the level increased in the aggregated cells during the maturation stage (5-7 days of culture). Comparable data were also obtained after immunostaining the cells. Thus, laminins are expressed in significant amounts by chondrocytes and may have an important role in cartilage development.     

In vitro and in vivo gene delivery mediated by a synthetic polycationic amino polymer

The r?le of chemotherapy in the treatment of operable soft tissue sarcomas is still debatable. In high-grade tumors, randomized trials using adjuvant chemotherapy have resulted in controversial results. A recent meta-analysis showed a higher median disease-free survival in groups with chemotherapy compared to controls. Doxorubicin, ifosfamide and dacarbazine are the majors drugs. Their combination in adjuvant treatment is currently being investigated. Neoadjuvant chemotherapy has allowed conservative surgery in initially unoperable tumors, but its role in primarily operable tumors remains to be defined.     

Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo

PURPOSE: To determine the potential of somatic gene transfer as a technique for modulating corneal wound healing after superficial keratectomy. METHODS: The transduction of human and rabbit keratocytes with beta-galactosidase and herpes simplex virus thymidine kinase genes was performed. In vitro, human and rabbit keratocytes were transduced with retroviral vectors bearing beta-galactosidase or HStk (herpes simplex virus thymidine kinase) genes. In vivo, rabbit keratocytes were transduced by topical application of vector supernatant after a superficial keratectomy. In vitro and in vivo, expression of the beta-galactosidase gene was examined with histochemical staining. In vitro, ganciclovir cytotoxicity in HStk gene-transduced keratocytes and bystander effect in co-cultures of HStk(+) and HStk(-) keratocytes were measured by determining the degree of confluency of cells in 6-well plates after 10 days of incubation. Corneal haze in rabbits was measured after transduction with Hstk and subsequent treatment with topical ganciclovir. RESULTS: In vitro, both human and rabbit keratocytes were transduced successfully with both beta-galactosidase and HStk genes. Transduction efficiency was greater with human (22%) than with rabbit (16%) cells, and both HStk-transduced cell lines showed dose-dependent ganciclovir cytotoxicity and a significant bystander effect. In vivo, expression of beta-galactosidase within vimentin-positive corneal stromal cells confirmed transduction of keratocytes in the rabbit after superficial stromal keratectomy with an efficiency of 25% to 40%. Postoperative application of topical ganciclovir reduced corneal stromal haze in rabbits. CONCLUSIONS: The ability to genetically transduce stromal keratocytes provides a new strategy for understanding the important cellular and molecular events that influence corneal wound healing, thus offering a potential approach to decrease or prevent corneal haze and scarring after superficial keratectomy.     

H-cadherin expression inhibits in vitro invasiveness and tumor formation in vivo

H-cadherin is a newly characterized cadherin molecule whose expression is decreased in a variety of human carcinoma cells, suggesting that it may play a role in maintaining normal cellular phenotype. To investigate how re-expression of H-cadherin could influence the malignant phenotype of human breast carcinoma cells in vivo, we transfected both control and H-cadherin expression vectors into human breast cancer cells (MDAMB435), which do not express H-cadherin constitutively. We found that invasiveness of these cells could be prevented by transfection with H-cadherin. We also compared the ability of control- and H-cadherin-transfected cells to induce subcutaneous tumors after injection into mammary fat pads of nude mice. Our results show that H-cadherin transfection produced a marked inhibition of tumor growth and modified the morphology of tumor cells: tumors from mice injected with control cells were significantly larger and contained larger cells having a higher degree of pleomorphism than those of tumors generated from carcinoma cells expressing H-cadherin. Altogether, these results indicate that H-cadherin expression antagonizes tumor growth in nude mice, presumably by enhancing cell-cell association in a tissue environment. These findings strongly suggest that H-cadherin could provide a possible target for corrective gene therapy against breast cancer.     

The mouse homeobox gene, Gbx2: genomic organization and expression in pluripotent cells in vitro and in vivo

This paper reviews recent work on a project that uses a computer-aided approach for making 3-D reconstructions of serially sectioned mouse embryos (the digital mouse). The captured images are aligned using a warping program so that almost perfect alignment of adjacent sections is achieved with minimal deformation. The sections that are viewed on the computer screen are in fact computer-generated grey-level images with a resolution of about 10 microm. The reconstructed embryo may then be resectioned in any plane to simulate as near as possible an exact match on the computer screen to the viewer's own material. Individual anatomical domains may then be painted in different colors, and these domains may be selected by querying the textual database containing anatomical and other information. Further, it is now possible to generate 3-D images of individual anatomically-discrete components or related sets of components of a particular system in isolation from the rest of the embryo, or, if required, against a 'ghost-like' image of the intact embryo, or specific parts of an embryo. In the article, examples are given of the use of the system in interpreting the vascular, gut and paraxial mesoderm systems, while both the advantages and disadvantages of this approach are also discussed. The eventual aim will be to provide 3-D reconstructions of mouse embryos from fertilization up to 14 days postcoitum of development. When completed, this project will allow the accurate spatial mapping of gene-expression and cell lineage data onto the digital Atlas of normal mouse embryonic development.     

Effects of retinoic acid and estrogens on oxytocin gene expression in the rat uterus: in vitro and in vivo studies

We and others have previously identified functional estrogen (E) and retinoic acid (RA) response elements in the human and rat oxytocin (OT) gene promoters. Whereas there is no direct evidence for a significant role of E or RA in the regulation of rat hypothalamic OT gene expression, we have recently demonstrated that in vivo administration of E strongly stimulates uterine OT gene expression. Here, we show that in vivo administration of RA similarly induces a significant increase in uterine OT gene expression. Moreover, we report that the E and RA effects are reproducible in vitro. Using short-term uterine organ explant cultures derived from 18-day pregnant rats, we found that E (50 nM) and RA (0.4 nM) increased OT mRNA levels 5.2- and 3-fold, respectively, suggesting a direct action of these agents on uterine OT gene expression. Finally, we analyzed uterine E and RA receptor gene expression during pregnancy. Using semi-quantitative Northern blot analysis, we found that mRNAs encoding the E receptor, the RA receptor alpha and RA receptor beta are present in rat uterus and that their levels rise by 3.7-, 3.6- and 5.8-fold, respectively, between day 14 of gestation and term. Taken together, the data suggest that, at term, the rat uterus has an increased capacity to respond to E and RA, and that both agents may be involved in mediating the dramatic increase of OT mRNA accumulation observed in the uterus at term.     

In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA

We used electromyographic analysis to determine the muscle activity of the shoulder muscles during the lift-off test and during resisted internal rotation. The activity in the upper and lower subscapularis muscle during a lift-off test from the region of the midlumbar spine was approximately 70% of maximal voluntary contraction. This level was significantly higher than for all the other muscles tested (P < 0.05). The lift-off test with the hand placed in the region of the midlumbar spine resulted in one-third more electromyographic activity in the subscapularis muscle than when the test was modified and performed with the hand at the buttocks region. A resisted lift-off test resulted in higher activities in all the muscles, but only a small increase in the pectoralis major muscle. The pectoralis major muscle was significantly more active during resisted internal rotation with the arm in front of the body. Comparison of activity in the upper subscapularis with that in the lower subscapularis muscle showed no significant differences during any of the tests. This study documents the importance of the subscapularis muscle during the lift-off test and suggests that other potential internal rotators of the humerus have a limited role in maintaining internal rotation when the arm is placed behind the back.     

A Wnt signaling pathway controls hox gene expression and neuroblast migration in C. elegans

Immunization regimens that induce a broadly reactive cytolytic T lymphocyte (CTL) response specific for lentiviral antigens have emerged as the leading candidates in efficacy trials conducted in both animal modelshumans. To date, lentivirus vaccination strategies have overlooked one such immunization strategy, namely the use of particulate antigens. To evaluate the efficacy of targeting antigen into the phagocytic pathway to elicit a cell-mediated immune response to lentiviral antigens, we initiated the first study of a particulate-based vaccination protocol using a large animal model system. Gradient-purified equine infectious anemia virus (EIAV) was covalently coupled to glutaraldehyde-activated iron oxide beads. In vitro studies demonstrated the effectiveness of the inactivated whole virus particulate to prime antigen presenting cells for the activationexpansion of virus-specific CD8(+) CTL. The in vivo effectiveness of the particulate antigen was evaluated by experimental immunization of ponies. Ponies receiving the viral particulate vaccinechallenged with infectious EIAV had a delayed progression to diseasea reduced viral load compared with infected ponies that had not been vaccinated. Interestingly, in vitro virus-specific CTL activity was detected in only one of four immunized animals at the day of challenge. The beneficial effects of the particulate vaccine regimen were not clearly associated with any in vitro measurable parameters of the virus-specific cellular or humoral immune responses elicited by the vaccine at the day of challenge. However, within 3 weeks after virus challenge, anamnestic humoral responses characterized by a rapid emergence of neutralizing activity in the seruma predominance of conformationally dependent epitopes recognized by virus-specific antibodies were observed in the vaccinates. Taken together, further studies are clearly warranted in large animal model systems using a particulate-based vaccine regimen considering the beneficial effects of this regimen in our studythe protective effects of particulate antigen delivery in the murine model.