Biomedical subjects

Y Kaneda

Publications and source records attributed to Y Kaneda.

At least 235 records · Page 13Linked to original sources

PubMed1998

HVJ-liposome mediated gene transfer into hepatocytes in vivo.

BACKGROUND/AIMS: The efficient transduction of appropriate target cells will be critical for gene therapy. We evaluated the suitability of hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer for gene therapy of liver diseases. METHODS: The Escherichia coli beta-galactosidase (beta-gal) gene was introduced into rat liver by HVJ-liposome to examine gene transfer efficacy and persistence of expression with or without partial hepatectomy prior to transfection. RESULTS: About 30% of hepatocytes were transduced after portal vein injection. Gene expression was transient, with only 2% of hepatocytes expressing beta-gal after 4 weeks. However, partial hepatectomy performed 24 h prior to injection resulted in persistently high levels of beta-gal for 4 weeks after injection. A 247-bp beta-gal polymerase chain reaction fragment transcript was detected in livers of transfected rats, but not in livers of control rats. The rat livers following gene transfer were histologically normal, and serum glutamic-pyruvic transaminase was not found to be elevated in rats. CONCLUSIONS: Our results demonstrate that HVJ-liposome-mediated gene transfer produced high gene transduction and persistent gene expression in the liver.

Alanine Transaminase↗

PubMed1998

Hemagglutinating virus of Japan-liposome-mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff.

PURPOSE: Late graft failure is a critical problem, particularly in the presence of poor runoff vessels. Intimal hyperplasia is considered to be the main cause of graft failure. We have already reported that intimal thickening of experimental vein grafts in dogs with poor runoff vessels is more pronounced than that in dogs with normal vessels. We and others also have reported that production of nitric oxide (NO) in the endothelium of canine vein grafts is impaired. In the present study, we asked whether in vivo gene transfer of endothelial cell NO synthase (ecNOS) would inhibit intimal hyperplasia of autogenous vein grafts implanted in limbs with poor distal runoff in dogs. METHODS: After exposing femoral veins, the nuclear-targeted lac Zgene, bovine ecNOS cDNA, or control vector plasmid encapsulated in the hemagglutinating virus of Japan-liposomes was infused intraluminally, followed by incubation for 10 minutes at room temperature under a distending pressure of 100 mm Hg. Twenty reversed vein grafts were implanted under normal runoff conditions, and 4 days later these were used to confirm gene transfer to the vein grafts. Twelve reversed vein grafts were implanted under conditions of poor runoff, and 4 weeks after the operation intimal thickening was evident. RESULTS: In vein grafts under normal runoff conditions, lac Z gene transfer exhibited diffuse and frequent X-Gal-positive signals in both medial and adventitial layers 4 days after implantation (n = 3). In case of the ecNOS gene-transferred vein grafts, bovine ecNOS protein was mainly detected in medial smooth muscle cells and adventitial cells 4 days after implantation, determined using immunohistochemical techniques and bovine ecNOS specific antibody (n = 3). In addition, ecNOS-transferred vessels showed intense purple signals by reduced nicotinamide adenine dinucleotide phosphate diaphorase and nitroblue tetrazolium reaction, in both medial and adventitial layers, whereas weak NOS activity was recognized at the adventitial vasa vasorum of the untreated veins or control vector transferred veins (n = 3, respectively). In vein grafts under poor runoff conditions, the intimal thickness at 4 weeks after implantation was significantly reduced by ecNOS gene transfer (n = 4; 90.0 +/- 7.6 microns and 1.18 +/- 0.07 mm2) in comparison with buffer-treated vessels (n = 4; 195.8 +/- 25.7 microns and 2.62 +/- 0.48 mm2) or vector vehicle-treated vessels (n = 4; 193.0 +/- 15.8 microns and 2.65 +/- 0.22 mm2). CONCLUSIONS: Our findings show that gene transfer of ecNOS inhibited intimal hyperplasia of canine vein grafts caused by poor runoff conditions, as a result of an increased local production of NO. Thus ecNOS gene transfer warrants further study as a possible approach to prevent late graft failure.

Animals↗

PubMed1998

Impaired thyroid function in murine toxoplasmosis.

A decline in serum thyroxine (T4) occurs in Nya: NYLAR female mice infected with Toxoplasma gondii. To ascertain whether the hypothyroxinaemia might be the result of primary thyroid dysfunction, 2 parameters of thyrofollicular cell function were monitored to determine (a) if the cell surface membrane receptors for thyroid-stimulating hormone (TSH) were operative, and (b) whether the cyclic adenosine monophosphate (cAMP)-dependent cascade of intracellular events leading to the release of T4 was responsive to exogenous cAMP. Our results indicated that both parameters were intact and functional in the infected-mouse thyrocytes. However, the elicited T4 responses were distinctly diminished in magnitude, reflecting a lack of readily available thyroidal T4 reserves. Because the continuing synthesis, storage, and release of T4 is dependent on the pulsatile stimulation of the thyroid by TSH, we suggest that the depletion of T4 reserves is likely due to perturbation of the pulsatile release of TSH from the pituitary, rather than to primary thyroid malfunction.

Animals↗

PubMed1998

Aetiology of thyroidal dysfunction in murine toxoplasmosis.

Mice infected with Toxoplasma gondii manifest a rapid decline in serum thyroxine (T4) levels. To locate the locus of the hypothyroxinaemia, the integrity of the pituitary-thyroid axis of infected mice was assessed by a thyrotropin-releasing hormone (TRH) assay. A rise in serum T4 after inoculation of TRH implies the release of thyrotropin (thyroid-stimulating hormone) from a functionally intact pituitary. Administration of a single, large-dose (1 microgram) bolus of TRH to infected mice induced a positive, although subnormal, T4 response. In contrast, when infected mice were pre-treated with a series of low-dose (5 ng) pulses of TRH prior to the bolus challenge, the T4 response was markedly enhanced. We suggest that the multiple inoculations of low-dose pulses of TRH 'primed' the pituitary (and secondarily the thyroid) and led to replenishment of their readily available hormone reserves and the heightened response to stimulation. These observations indicate that the locus of thyroid dysfunction is in the hypothalamus, not the pituitary or thyroid, and apparently involves impairment of the pulsatile release of TRH.

Animals↗

PubMed1998

The induction of apoptosis in HeLa cells by the loss of LBP-p40.

To analyze the function of the laminin-binding protein precursor p40 (LBP-p40) in higher eukaryotic cells, plasmid DNA expressing antisense or sense cDNA for p40 under the control of the LacSwitch system was introduced into HeLa cells. Stable transformants were isolated, and the expression of p40 was assayed by Western and Northern blotting. The expression level of p40 was not affected in HeLa cell transformants cultured in 10% serum-supplemented media with the induction of antisense (AS)-p40 with 5 mM IPTG. However, both the protein and message for endogenous p40 in serum-depleted media with 5 mM IPTG were reduced to about 30 - 10% of the expression level in serum-free media without 5 mM IPTG. Colony formation was inhibited with the suppression of p40. AS-p40 clones died in 7 days when cultured in serum-depleted media with 5 mM IPTG, while clones without 5 mM IPTG AS-p40 clones never died, even in serum-depleted media. Additionally, sense (S)-p40 clones and control CAT clones survived more than 2 weeks in serum-free media with 5 mM IPTG. DNA fragmentation assay revealed that cell death induced by the reduction of AS-p40 resulted from apoptosis. Both the inhibition of cell growth and apoptotic cell death were partially rescued by the transfer of the p40 cDNA expression vector to AS-p40 clones. Moreover, the introduction of a synthetic hammerhead ribozyme for LBP-p40 using a fusigenic viral liposome suppressed the message for LBP-p40 even in the presence of 10% serum, and it also induced apoptosis.

Apoptosis↗

PubMed1998

Persistent gene expression in rat liver in vivo by repetitive transfections using HVJ-liposome.

Most viral vectors are highly immunogenic and are of limited use for somatic gene therapy that requires repetitive administrations. We have developed a highly efficient gene transduction procedure useful for repetitive transfections using liposome containing hemagglutinating virus of Japan (HVJ-liposome). The Escherichia coli beta-galactosidase (beta-gal) gene was embodied in HVJ-liposome, and introduced directly into the caudal lobe of rat liver that was transiently isolated from a systemic circulation. A 116 kDa beta-gal protein was detected in transfected rat liver tissues by Western blot analysis and it was expressed in more than two-thirds of the liver by histological staining. It was found that the transfection efficiency was not affected by repetitive transfections. In support of these findings, antibody response to HVJ-liposome detected in the rat sera was weak and transient. Furthermore, cytotoxic T lymphocytes were not elicited against autologous rat hepatocytes that were transfected in vivo using HVJ-liposome. Thus, our results demonstrate that the isolation of a target liver from systemic circulation and the direct administration of foreign genes using HVJ-liposomes are useful for high gene transduction and persistent gene expression in the liver.

Animals↗

PubMed1998

Inhibition of intimal hyperplasia after vein grafting by in vivo transfer of human senescent cell-derived inhibitor-1 gene.

The senescent cell-derived inhibitor (sdi)-1 (p21) protein has been identified as a downstream mediator of the tumor suppressor p53 in the cell cycle regulation. In this study, we focused on the function of sdi-1 gene in inhibiting vascular smooth muscle cell (VSMC) proliferation after vein grafting in a rabbit model. To test the hypothesis, we transfected human sdi-1 gene by an intra-operative approach. Accompanied by markedly increased sdi-1 protein, the significant increase in PCNA-stained VSMCs in vein grafts was inhibited by transfection of sdi-1 gene. Moreover, at 2 weeks after transfection, transfer of sdi-1 gene resulted in a significant inhibition in neointimal formation, compared with control vector. Of importance, immunohistological studies determining the expression pattern of myosin heavy isoforms, adult type specific SM2 and embryonic specific SMemb/NMHC-B, demonstrated expression of the adult phenotype of VSMCs in the neointima of sdi-1 gene-transfected vein grafts at 2 weeks after the operation, while the neointima was predominantly composed of embryonic phenotype of VSMCs in the control grafts. Overall, these results demonstrate that a single intraluminal incubation of human sdi-1 gene can result in a significant inhibition of neointimal formation after vein grafting, associated with phenotypic change of VSMCs from neonatal to adult type in a rabbit model. Inhibition of hyperplasia in a graft model by transfection of sdi-1 gene may be due to the change in VSMC phenotype from neonatal to adult, in addition to the inhibition of VSMC growth.

Actins↗

PubMed1998

Sustained transgene expression in vitro and in vivo using an Epstein-Barr virus replicon vector system combined with HVJ liposomes.

For long-term gene expression in tissues, we constructed an Epstein-Barr virus (EBV) replicon-based plasmid, pEB, containing the latent viral DNA replication origin (oriP) and EBV nuclear antigen-1 (EBNA-1). When pEB was transferred to human cells (HeLa-S3, HEK 293 and FS 3) and rodent cells (BHK-21) using HVJ-cationic liposomes, luciferase expression was observed in those cells for at least 10 days. Luciferase activity was two to 10 times higher in those cell lines on and after day 3 post-transfection of pEBActLuc compared with plasmids without the EBV replicon sequence. Southern blot analysis showed that the pEB vector luciferase gene was maintained extrachromosomally in BHK-21 cells. In human cells, transformation was five to 20 times more efficient with pEBc than with pcDNA3, and 18-35% of the introduced EBV replicon plasmid was replicated autonomously. The luciferase gene or lacZ gene was introduced into mouse liver using HVJ-AVE liposomes. Luciferase gene expression was observed for at least 35 days in cells transfected with pEBActLuc, whereas it was not detected on day 14 in cells transfected with pActLuc, which lacks the EBV sequence. By the transfer of pEBActNlacF, the lacZ gene expression rate in hepatocytes was approximately 35 and 12% on days 7 and 35, respectively.

Animals↗

PubMed1998

Early biological effect of in vivo gene transfer of platelet-derived growth factor (PDGF)-B into healing patellar ligament.

To define the early biological effect of in vivo introduction of the PDGF-B gene on the healing of ligaments, a HVJ-liposome suspension containing platelet-derived growth factor (PDGF)-B cDNA was injected directly into the injured patellar ligament of 14-week-old male Wistar rats. Rats were killed at 1, 4 and 8 weeks for the morphological analysis of angiogenesis by laminin immunohistochemistry and of collagen deposition by Masson's Trichrome staining and collagen I immunohistochemistry. PDGF-B gene transfer caused the enhanced expression of PDGF in healing ligament up to 4 weeks after transfection, leading to an initial promotion of angiogenesis and subsequent enhanced collagen deposition in the wound. Enhanced and accelerated matrix synthesis in the PDGF-B gene introduced healing ligament suggests that this gene transfer technique may be a potentially useful tool for improving soft tissue repair.

Animals↗

PubMed1998

Efficient transfer of oligonucleotides and plasmid DNA into the whole heart through the coronary artery.

Several of the current techniques for transfer of both oligonucleotide and plasmid DNA into the myocardium are impaired by low efficiency and toxicity. To improve gene transfer techniques, especially into the whole heart, a gene transfer method involving liposome in conjunction with a viral envelope (HVJ-liposome) was essayed as an alternative. FITC-labeled oligonucleotide (F-ODN) and the cDNA of beta-galactosidase (beta-gal) were introduced into the myocardium by coronary infusion of HVJ-liposome during cardioplegic arrest of adult Sprague-Dawley rat hearts. Then, transfected heart was ectopically transplanted into another rat abdomen of the same strain to maintain the transfected heart long enough to allow for protein synthesis. After 3 days of transfection, transfected heart was excised and the efficiency of gene transfection was evaluated. FITC was detected in the nuclei of more than 70% of the myocytes and endothelial cells both in the epicardium and endocardium. beta-Gal was expressed in the cytosol of more than 50% of the myocytes. beta-Gal expression was demonstrated by Western blotting analysis at day 3 after transfection and continued for at least 14 days. No significant histological damage of the myocardium or leakage of CPK were detected in the rats transfected by the HVJ-liposome method. These results clearly demonstrate that the hearts were efficiently transfected both by oligonucleotide and plasmid DNA as a result of coronary infusion of HVJ-liposome during cardioplegic arrest. This thus appears to be an efficient method for gene transfer into the whole heart, providing a new tool for research and therapy for heart diseases.

Animals↗

PubMed1998

A comparison of in vivo gene delivery methods for antisense therapy in ligament healing.

To determine the most efficient in vivo delivery method of oligonucleotides for antisense therapy in ligament healing, fluorescence-labelled phosphorothioate oligodeoxynuleotides (ODN) were introduced into 12 rabbit ligament scars 2 weeks after injury using haemagglutinating virus of Japan (Sendai virus: HVJ)-conjugated liposomes. We compared the efficiency of cellular uptake of fluorescence as a percentage of all cells in each scar using three delivery procedures: (1) direct free-hand injection into the ligament scar using a conventional syringe; (2) systematic direct sca injection using a repeating 10 microliters dispenser and a square mesh grid system; and (3) injection into the feeding (femoral) artery. Results showed that there was a significant difference in fluorescence uptake by scar cells on day 1 after injection between the three delivery methods: (1) direct free-hand, 9.7 +/- 7.6% (average +/- s.d.); (2) systematic direct, 58.4 +/- 15.9%; and (3) intra-arterial, 0.2 +/- 0.1%. Systematic direct injection was most efficient and it resulted in 25.9 +/- 13.0% of scar cells being labeled at 7 days after transfection. We then introduced antisense ODN for the rabbit proteoglycan, decorin, into ligament scars with this delivery method and confirmed a significant inhibition of decorin mRNA expression in antisense-treated scar tissues in vivo both at 2 days (42.3 +/- 14.7% of sense control +/- s.d.; P < 0.0025) and 3 weeks (60.5 +/- 28.2% of sense control +/- s.d.; P < 0.024) after treatment, compared with sense ODN-treated scars. Decorin was significantly suppressed also at protein level in antisense-treated scars at 4 weeks (66.6 +/- 35.7% of sense control +/- s.d.; P < 0.045) after treatment. These results demonstrate that in vivo transfection efficiency in ligament scars is 'delivery system dependent' and that introduction of antisense ODN for the small proteoglycan, decorin, with this delivery method can lead to significant suppression of its expression over 3 weeks both at mRNA and protein levels. Thus, an effective model for the potential manipulation of scar composition and quality in ligament healing has been established.

Animals↗

PubMed1998

Inhibition of growth of human vascular smooth muscle cells by overexpression of p21 gene through induction of apoptosis.

The senescent cell-derived inhibitor (sdi)-1 protein (p21 product) has been identified as a downstream mediator of the tumor suppressor p53 in the regulation of cell cycle progression through a G1 phase checkpoint. Given the importance of cell cycle inhibition for the treatment of restenosis, in this study we focused on the function of p21 gene in inhibiting proliferation of vascular smooth muscle cells (VSMC). To test the hypothesis, we transfected human p21 gene into human aortic VSMC using hemagglutinating virus of Japan-liposome-mediated transfer. Initially, we examined the successful transfection of human p21 gene into VSMC. p21 protein was increased in VSMC transfected with p21 vector as compared with control vector. Accompanied by increased p21 protein, transfection of p21 vector resulted in a significant decrease in number of VSMC induced by 2% serum (P<.01). Although p21 has been reported to play an important role in the regulation of apoptosis in some cells, apoptosis mediated by p21 is still controversial. Therefore, we hypothesized that overexpression of p21 mediates apoptosis in human VSMC, in addition to the blockade of cell cycle progression. First, we assessed the concordance between morphologic analysis and apoptosis as determined by nuclear staining with Hoechst 33342. Cells transfected with p21 gene exhibited the characteristic features of cell shrinkage, membrane blebbing, and rounding that are typical of apoptotic death. Of greater interest, a significant increase in apoptotic cells was observed in VSMC transfected with p21 vector as compared with control vector (P<.01). These results were confirmed by the measurement of DNA fragmentation. Consistent with nuclear staining, DNA fragmentation in VSMC transfected with human p21 gene was significantly increased as compared with that in VSMC transfected with control vector (P<.05). To study the molecular mechanisms of apoptosis mediated by overexpression of p21 gene, the protein levels of bax, a promoter of apoptosis, and bcl-2, an inhibitor of apoptosis, were also measured by Western blotting. Overexpression of p21 gene significantly increased protein of bax (P<.05), whereas transfection of p21 gene did not alter bcl-2 protein. Importantly, the ratio of bax to bcl-2 was significantly increased in VSMC transfected with human p21 vector as compared with control vector (P<.05). Overall, these results demonstrated that inhibition of VSMC growth by overexpression of human p21 gene was accompanied by induction of apoptosis through an inappropriate increase in bax protein. These results suggest that regulation of cell cycle by p21 may be closely linked to programmed cell death/apoptosis in human VSMC.

Aorta↗

PubMed1998

Conditioned medium from HepG2 cells transfected with human apolipoprotein(a) gene stimulates growth of human vascular smooth muscle cells: effects of overexpression of human apolipoprotein(a) gene.

Lipoprotein(a) [Lp(a)] is well known to stimulate growth of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. Its mechanism is postulated to be decreased in active transforming growth factor (TGF)-beta. However, the exact mechanisms and cellular processing from apolipoprotein(a) [apo(a)] to Lp(a) have not yet been clarified because no cultured cells producing apo(a) are available. Therefore, it is necessary to establish apo(a)-producing cells to study the role of apo(a). We evaluated the effects of overexpression of human apo(a) gene on human aortic VSMC growth. First, we tested whether transfection of apo(a) gene into human hepatoma cells, HepG2 cells, producing human apoB resulted in the formation of Lp(a). Transfection of apo(a) gene into HepG2 cells resulted in detectable levels of Lp(a) in the medium, as assessed by ELISA and Western blot, whereas no Lp(a) was detected in the medium of HepG2 cells transfected with control vector and untransfected HepG2 cells. Expression of apo(a) mRNA was also confirmed by reverse transcription-polymerase chain reaction. In contrast, Western blotting showed a single band detected by specific anti-apo(a) antibody, but not anti-apoB antibody, in the medium of apo(a)-transfected VSMCs. These results demonstrate that Lp(a) can be formed from apo(a) on HepG2 cells, whereas transfection of apo(a) gene into VSMCs resulted in the production of apo(a) alone but not Lp(a). Next, we examined the biological effects of overexpression of apo(a) gene on growth of VSMCs and endothelial cells. Incubation of cultured medium of HepG2 cells transfected with apo(a) gene with human VSMCs or endothelial cells resulted in a significant increase in cell number compared with the conditioned medium of HepG2 cells transfected with control vector. In contrast, transfection of apo(a) gene directly into VSMCs caused no significant effect on VSMC growth. Therefore, we measured TGF-beta concentration in the conditioned medium of VSMCs. However, using ELISA, only latent but not active TGF-beta was detected in the medium of VSMCs. Moreover, addition of neutralizing anti-TGF-beta antibody did not alter VSMC growth. These results suggest that Lp(a) could stimulate growth of VSMCs via the independent mechanisms from the inhibition of TGF-beta activation. Overall, these data demonstrate that overexpression of apo(a) gene in cells producing apoB results in formation of Lp(a), resulting in a mitogenic action on human endothelial cells and VSMCs. These results provide new information to understand the mechanisms of the mitogenic action of Lp(a) and suggest the role of Lp(a) in the pathogenesis of atherosclerosis.

Apolipoproteins↗

PubMed1998

Amino acids and peptides. XXXI. Preparation of analogs of the laminin-related peptide YIGSR and their inhibitory effect on experimental metastasis.

Analogs of a partial sequence peptide of laminin, i.e., Tyr-Ile-Gly-Ser-Arg (YIGSR) analogs and Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg (CDPGYIGSR) analogs, were prepared by the solid-phase method and their inhibitory effects on experimental metastasis of B16-BL6 melanoma cells were examined. YIGSR analogs in which Ile was replaced by other hydrophobic amino acids (Met, Leu, Phe) were inhibitory. Cys-containing analogs of YIGSR were also prepared, but were less active than the parent peptide, YIGSR. Among them, CYIGSR was easily oxidized to form a disulfide bond. A Cys-containing YIGSR analog cyclized through a disulfide bond, cyclo(CYIGSRC)G, was prepared. The disulfide bond formation was performed on the resin by the silyl chloride-sulfoxide method and by the iodine oxidation method. The yield of the silyl chloride-sulfoxide method was much better than that of the iodine oxidation method.

Animals↗

PubMed1998

Heterogeneous induction of apoptosis in colon cancer cells by wild-type p53 gene transfection.

To examine the effects of wild-type (wt)-p53 gene transfer on cancer cell growth and apoptosis induction, we transduced human wt-p53 cDNA into three colon cancer cell lines either with or without a mutation of the p53 gene using the HVJ-cationic liposome method. Wt-p53 gene transfer, thus, induced an apparent growth arrest in all cell lines, but its enhancement of the apoptotic rate varied (from about 4 to 70 times). The simultaneous doxorubicin treatment was able to enhance growth arrest and the apoptosis induction rate. These findings suggest that wt-p53 gene transfer using HVJ-cationic liposomes seems to be a potentially effective therapeutic strategy, however wt-p53 gene transfer still appears to be more effective in combination with other cytotoxic treatments.

Apoptosis↗

PubMed1998

HVJ (Sendai virus) liposome-mediated gene transfer: current status and future perspectives (review).

Haemagglutinating virus of Japan (HVJ; Sendai virus), a member of the mouse paramyxovirus family, has been combined with liposomes to produce a novel gene transfer system, namely HVJ liposomes. This vector system is defined as a , constructed with inactivated viral particles and non-viral (artificial) multi- or unilamellar liposomes containing gene expression cassettes and has several advantages in comparison with other viral or non-viral systems. Many studies have shown that this vector system can, not only produce efficient gene transfer using reporter genes, but also with resulting in vivo functional changes in several animal models of diseases. Despite these results, it is likely that the construct will need to be modified to improve gene transfer and expression efficiency and also to extend the potential disease targets. We review the present status of this hybrid vector system and also discuss possible modifications for future application to either in vivo analysis of specific gene expression or human gene therapy strategies for congenital or acquired diseases.

Forecasting↗

PubMed1998

Cerebral anomalies in congenital murine toxoplasmosis: a preliminary report.

Gravid Nya: NYLAR strain mice were infected with Toxoplasma gondii on embryonic day 7. During the remaining 12 days of gestation, considerable fetal wastage occurred. At birth, pups were sickly and growth-stunted, and postnatal mortality was high. After 1 month, surviving weanlings were killed and autopsied. Coronal sections of the cerebrum revealed: numerous cortical and periventricular cystic cavitations, some undergoing calcification; ventricular enlargement and marked periventricular edema; subependymal nodules bulging into the ventricles; loss of subependymal germinal cells; and meningeal and perivascular inflammation. We suggest, as a working hypothesis, that hematogenously transported parasites invaded the vascular endothelium of the fetal cerebrum, inducing endothelial cell activation, recruitment of inflammatory cells, and focal inflammatory lesions in the endothelium. These inflammatory lesions triggered an intravascular coagulopathy, leading to the formation and deposition of microthrombi in cerebral capillaries and the development of numerous infarcts. Hypoxic-ischemic necrosis of the infarcted tissues led to the creation of the cystic cavitations.

Animals↗

PubMed1998

Introduction of DNA into the rat and primate trabecular meshwork by fusogenic liposomes.

PURPOSE: To evaluate the feasibility of introducing exogenous genes and phosphorothioate oligonucleotides into the anterior chamber tissues of rats and monkeys using the authors' fusogenic liposomes. METHODS: Hemagglutinating virus of Japan liposomes containing LacZ DNA-high-mobility group 1 complexes or fluorescein isothiocyanate (FITC)-labeled phosphorothioate oligonucleotides were prepared and injected into the anterior chambers of rats (3 microliters) and rhesus monkeys (30 microliters). The expression of LacZ DNA was visualized histochemically by beta-Galactosidase assay and was followed for as long as 60 days in rats and 30 days in monkeys. FITC-labeled phosphorothioate oligonucleotides were observed by fluorescence microscopy for as long as 14 days in rats and 7 days in monkeys. RESULTS: Injection of LacZ DNA-high-mobility group 1 complexes encapsulated in hemagglutinating virus of Japan liposomes resulted in blue staining in the trabecular meshwork and iris-ciliary body of rats and selectively in the trabecular meshwork of monkeys at the concentrations used. This LacZ expression lasted for as long as 14 days after injection in both animals. Phosphorothioate oligonucleotides (3 microM) also were introduced into the rat trabecular meshwork and iris-ciliary body and into the primate trabecular meshwork when encapsulated in hemagglutinating virus of Japan liposomes, although the injection of naked FITC-labeled phosphorothioate oligonucleotides at the same concentration resulted in little fluorescence in any anterior chamber tissue. CONCLUSIONS: This study shows that the use of hemagglutinating virus of Japan liposomes can transfer LacZ DNA and phosphorothioate oligonucleotides to adult rat and primate trabecular meshwork. This system may enable progress in glaucoma research and in the development of nonviral somatic gene therapy of the trabecular meshwork to treat glaucoma.

Animals↗