CD21int CD23+ follicular B cells express antigen-specific secretory IgM mRNA as primary and memory responses.

CD21int CD23+ IgM+ mouse follicular B cells comprise the bulk of the mature B-cell compartment, but it is not known whether these cells contribute to the humoral antibody response. We show using a direct RT-PCR method for antigen-specific VH, that FACS-sorted mouse CD21int CD23+ B cells express specific secretory IgM VH transcripts in response to immunization and also exhibit a memory response. The secretory IgM expressed is distinct from the IgG expressed by cells of this phenotype, which we also analyse here, having a distinct broader distribution of CDR-H3 sequences and zero or low levels of somatic mutation in the region analysed. These results imply that cells of the CD21int CD23+ phenotype have distinct IgM+ and IgG+ populations that contribute directly to the humoral antibody and memory responses by expressing antigen-specific secretory immunoglobulin. We also argue that the more diverse CDR-H3 sequences expressed by antigen-experienced IgM+ CD21int CD23+ follicular B cells would place them at the bottom of a recently hypothesized memory B-cell hierarchy.

Recruitment of a distinct but related set of VH sequences into the murine CD21hi/CD23- marginal zone B cell repertoire to that seen in the class-switched antibody response.

Development and maintenance of cells in the murine follicular and marginal zone compartments is thought to involve differing levels of stimulation of the BCR, although it is still not clear which BCR ligands mediate these events. How the delineation between naive and Ag experienced B cell populations relates to cell phenotype and how precise or blurred this delineation is, is also not well understood. In this study, using PCR to analyze the Ab response to phenyl-oxazolone in the mouse, we show that the Ab repertoire of CD21(hi)/CD23(-) marginal zone B cells shows persistent increase in levels of particular IgM after immunization with foreign Ag. Further, we show that these IgMs have different but related VH/CDR3 sequences from those seen in the class-switched response to oxazolone that we have also analyzed. We also detect an effect of Ag on the follicular B cell repertoire that is less persisting. These results provide evidence consistent with the signal-strength model of mature B cell development being extended to include stimulation by foreign Ag, and also further the known zone of influence of foreign Ag on the B cell compartment.

[Study of effect of tongsaimai tablets on experimental diabetic foot model rats].

OBJECTIVE: To observe the effect of Tongsaimai (TSM) tablets in treating foot trauma of diabetic foot (DF) model rats, and discuss its potential mechanism. METHOD: Male SD rats were selected to duplicate the diabetic foot ulcer model and randomly divided into the blank control group, the model group, the metformin treatment group, and TSM 12.44, 6.22, 3.11 g x kg(-1) groups (n = 10). The healing of ulcer wounds were observed on day 1, 4, 8, 13 and 18. After 18 days, a histopathologic examination was conducted for ulcer tissues. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by hydroxylamine and TBA methods. The content of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were determined with the radioimmunoassay. The immunohistochemical method was used to observe the expression of vascular endothelial growth factor (VEGF) in ulcer tissues and the number of capillary vessels. RESULT: TSM could alleviate the pathological changes of diabetic foot rats, accelerate the ulcer healing on 4, 8, 13, 18 d, reduce MDA, IL-6, TNF-alpha, VEGF content in rat serum at 18 d (after the rehabilitation period), and enhance the SOD content. Specifically, the TSM 12.44 g x kg(-1) group showed significant differences compared with the model group (P < 0.05, P < 0.01). At 18 d after the treatment (the late rehabilitation period), the VEGF expression of TSM 12.44, 6.22 g x kg(-1) groups and the number of blood capillaries of the TSM 12.44 g x kg(-1) group were significantly lower than that of the model group (P < 0.05, P < 0.01). CONCLUSION: TSM could promote the foot wound healing of DF model rats, reduce MDA, IL-6 and TNF-alpha levels in serum, increase the SOD content and decrease the VEGF expression and the number of blood capillaries in the late rehabilitation period. Its action mechanism may be related to the inhibition of oxidative stress injury and the inflammatory cell infiltration.

Alisol B 23-acetate adjusts bile acid metabolisim via hepatic FXR-BSEP signaling activation to alleviate atherosclerosis.

BACKGROUND: Postmenopausal women have a high incidence of atherosclerosis. Phytosterols have been shown to have cholesterol-lowering properties. Alisa B 23-acetate (AB23A) is a biologically active plant sterol isolated from Chinese herbal medicine Alisma. However, the atherosclerosis effect of AB23A after menopause and its possible mechanism have not been reported yet. PURPOSE: To explore whether AB23A can prevent atherosclerosis by regulating farnesoid X receptor and subsequently increasing fecal bile acid and cholesterol excretion to reduce plasma cholesterol levels. METHODS: Aortic samples from premenopausal and postmenopausal women with ascending aortic arteriosclerosis were analyzed, and bilateral ovariectomized (OVX) female LDLR-/- mice and free fatty acid (FFA)-treated L02 cells were used to analyze the effect of AB23A supplementation therapy. RESULTS: AB23A increased fecal cholesterol and bile acids (BAs) excretion dependent on activation of hepatic farnesoid X receptor (FXR) in ovariectomized mice. AB23A inhibited hepatic cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) via inducing small heterodimer partner (SHP) expression. On the other hand, AB23A increased the level of hepatic chenodeoxycholic acid (CDCA), and activated the hepatic BSEP signaling. The activation of hepatic FXR-BSEP signaling by AB23A in ovariectomized mice was accompanied by the reduction of liver cholesterol, hepatic lipolysis, and bile acids efflux, and reduced the damage of atherosclerosis. In vitro, AB23A fixed abnormal lipid metabolism in L02 cells and increased the expression of FXR, BSEP and SHP. Moreover, the inhibition and silencing of FXR canceled the regulation of BSEP by AB23A in L02 cells. CONCLUSION: Our results shed light into the mechanisms behind the cholesterol-lowering of AB23A, and increasing FXR-BSEP signaling by AB23A may be a potential postmenopausal atherosclerosis therapy.

Salidroside ameliorates orthopedic surgery-induced cognitive dysfunction by activating adenosine 5'-monophosphate-activated protein kinase signaling in mice.

Perioperative neurocognitive disorders (PND) are the most common postoperative complications with few therapeutic options. Salidroside, a plant-derived compound, has gained increased attention as a treatment for various neurological diseases and particularly as a modifier of microglia-mediated neuroinflammation. However, the effect of salidroside on orthopedic surgery-induced cognitive dysfunction and the underlying mechanisms are largely unknown. Here, we found that salidroside greatly attenuated cognitive impairment in mice after orthopedic surgery. Neuroinflammation in the mouse hippocampus was also attenuated by salidroside. Meanwhile, salidroside treatment induced a switch in microglial polarization to the anti-inflammatory phenotype. In vitro, salidroside suppressed the expression of proinflammatory cytokines and induced a switch in microglial phenotype to the anti-inflammatory phenotype. Mechanistically, molecular docking studies revealed the potential AMPK activation activity of salidroside. And salidroside did up-regulated the AMPK pathway proteins. Moreover, AMPK antagonist abolished the effects of salidroside in vivo and in vitro. Taken together, our results demonstrated that salidroside effectively suppressed PND by suppressing microglia-mediated neuroinflammation through activating AMPK pathway, and it might be a novel therapeutic approach for PND.

Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers.

Synthetic nanoparticle formulations have the potential for tumor-targeted gene delivery. Receptor-targeted nanocomplex (RTN) formulations comprise mixtures of cationic liposomes and targeting peptides that self-assemble on mixing with nucleic acids. RTN formulations were prepared containing different polyethylene glycol (PEG)ylated lipids with esterase-cleavable linkers (e.g., ME42) to promote intracellular PEG detachment and nanoparticle disassembly. In addition, integrin-targeting peptides (peptide ME27) were tested with endosomal furin- and cathepsin B-cleavable peptide linkers located between the integrin-binding ligand and the K(16) nucleic acid-binding domain to promote intracellular disengagement from the receptor. ME42/ME27 RTNs formed stable particles of <200 nm in isotonic salt buffers, compared with 4-microm particles formed by un-PEGylated RTNs. Transfection efficiency by PEG-modified, cleavable RTNs improved approximately 2-fold in 4 different cell lines, with 80% efficiency in murine neuroblastoma cells. In an in vivo model of neuroblastoma, ME42/ME27 RTNs delivering luciferase genes were tumor specific, with little expression in other organs tested. PEGylation of the RTNs enhanced luciferase transfection 5-fold over non-PEG formulations, whereas the cleavability of the peptide ME27 enhanced transfection 4-fold over that of RTNs with noncleavable peptides. Cleavability of the lipid for in vivo transfections had no effect. PEGylated, cleavable RTN formulations offer prospects for tumor-specific therapeutic gene transfer.

Alisol B 23-acetate activates ABCG5/G8 in the jejunum via the LXRα/ACAT2 pathway to relieve atherosclerosis in ovariectomized ApoE-/- mice.

Phytosterols have been shown to improve blood lipid levels and treat atherosclerosis. This research investigated the effects of phytosterol Alisol B 23-acetate (AB23A) on jejunum lipid metabolism and atherosclerosis. The results show that intragastric administration of AB23A can significantly reduce atherosclerotic plaque area and lipid accumulation in the jejunum of ovariectomized ApoE-/- mice fed a high-fat diet and can also improve the lipid mass spectra of the plasma and jejunum. In vitro studies have shown that AB23A can increase cholesterol outflow in Caco-2 cells exposed to high fat concentrations and increase the expression of ATP-binding cassette transfer proteins G5/G8 (ABCG5/G8), the liver X receptor α (LXRα). Furthermore, inhibition of LXRα can significantly eliminate the active effect of AB23A on decreasing intracellular lipid accumulation. We also confirmed that AB23A has a negative effect on Acyl-CoA cholesterol acyltransferase 2 (ACAT2) in Caco-2 cells cultured in the high concentrations of fat, and we found that AB23A further reduces ACAT2 expression in cells treated with the ACAT2 inhibitor pyripyropene or transfected with ACAT2 siRNA. In conclusion, we confirmed that AB23A can reduce the absorption of dietary lipids in the jejunum by affecting the LXRα-ACAT2-ABCG5/G8 pathway and ultimately exert an anti-atherosclerotic effect.

Receptor-targeted nanocomplexes optimized for gene transfer to primary vascular cells and explant cultures of rabbit aorta.

We have developed new, synthetic vector formulations that display high efficiency of gene transfer to vascular cells and tissues. The formulations comprise cationic liposomes and cationic, receptor-targeting peptides that self assemble on mixing with plasmid DNA into receptor-targeted nanocomplexes (RTNs). One such RTN formulation was optimal for transfection of primary smooth muscle cells (LYD-1), while a second was optimal for transfection of rabbit aortic explants (LYD-2). In both RTNs, the peptide was a 16-lysine motif linked to the targeting sequence CYGLPHKFCG via a short spacer sequence. The major difference between LYD-1 and LYD-2 lay in the cationic lipid component, where LYD-1 contained ditetradecyl trimethyl ammonium (DTDTMA), an unsaturated, cationic lipid with a 14-carbon alkyl tail, whereas LYD-2 contained 2,3-dioleyloxypropyl-1-trimethyl ammonium chloride (DOTMA), a cationic lipid with an 18-carbon unsaturated alkyl tail. LYD-2 transfections of aortic explants were effective with incubations performed at room temperature for as little as 30 minutes, with either saline or glucose-based solutions. Transgene expression in the explants peaked at 5 days and persisted for 14 days. The kinetics of transfected gene expression, along with the efficacy of transfection with short incubation times, indicate that these new formulations may be useful tools in the development of molecular therapies for cardiovascular diseases.

[Recombinant AAV1 mediated vascular endothelial growth factor gene expression promotes angiogenesis and improves neural function: experiment with rats].

OBJECTIVE: To investigate the therapeutic effect of vascular endothelial growth factor (VEGF) gene expression mediated by recombinant AAV1 (rAAV1) vector in brain ischemia and the mechanism thereof. METHODS: Sixty-four SD rats were randomly divided into 2 equal groups and received intra-ventricular injection with rAAV1-VEGF or rAAV1-lacZ as controls. 21 days later the rats underwent transient middle cerebral artery occlusion (MCAO). Neurological severity score (NSS) was recorded 1, 2, 3, 7, 14, and 21 days after MCAO. 48 rats were sacrificed 21 days after MCAO and brains were taken out from 48 rats. Immune quantitative analysis was used to identify the quantity of VEGF expression. Immunohistochemistry was used to identify the site of VEGF expression. Immunofluorescence double labeling of von Willebrand factor (vWF) and 5-bromodeoxy-uridine (BrdU) was performed to detect the proliferation of endothelial cells. Fluorescein isothiocyanate (FITC)-dextran was infused into the caudal vein of 8 rats from each group and then the rats were killed with their brains taken out to evaluate the cerebral microvessel perfusion and microvessel density. RESULTS: The NSSs of the VEGF group 7, 14, and 21 days after MCAO were all significantly lower than those of the control group (all P < 0.05), and the VEGF165 protein expression quantity was 27 times as that of the control group (P < 0.05). Immunohistochemistry demonstrated that VEGF expression was distributed mainly in the caudate putamen, corpus callosum, choroid plexus, and hippocampus in the VEGF group, while no expression was detected in the control group. The microvessel density of the VEGF group was 157 +/- 13, significantly higher than that of the control group [(89 +/- 9), P < 0.05]. BrdU +/vWF + endothelial cells were detected in the area adjacent to the MCAO. The density of microvessel infused with FITC-dextran was (152,617 +/- 13,076) microm2/mm2 in the VEGF group, significantly higher than that of the control group [(91,658 +/- 6577) microm2/mm2 P < 0.05]. CONCLUSION: rAAV1 mediates the VEGF gene expression in multiple structures in the brain and attenuates the neurological deficit of MCAO. VEGF gene transfer may stimulate angiogenesis and improves blood supply in brain. Neovascularization may be a therapeutic strategy for brain ischemia.

Application to vascular adventitia of a nonviral vector for TIMP-1 gene therapy to prevent intimal hyperplasia.

Somatic gene transfer continues to have potential for the study and therapy of cardiovascular disease. We have developed a modular, self-assembling, nonviral system consisting of Lipofectin, integrin-targeting peptides, and plasmid DNA (LID) and we have applied this to a model of vascular injury, rat carotid angioplasty. Marker gene studies identified transfection of adventitial cells after vector delivery to that layer. Human tissue inhibitor of metalloproteinase-1 (hTIMP-1) was tested as a therapeutic gene product after direct application to the exposed adventitial layer. Vascular LID.hTIMP-1 transfection was confirmed by polymerase chain reaction and gene expression by immunohistochemistry at 7 days. Neointimal areas were 0.160 +/- 0.078 and 0.225 +/- 0.052 mm(2) for LID.hTIMP-1-transfected (n = 14) and LID.pCI-transfected (n = 12) vessels, respectively, at 14 days, and 0.116 +/- 0.068 mm(2) (n = 14) and 0.194 +/- 0.095 mm(2) (n = 14), respectively, at 28 days, representing a 29 and 40% reduction in neointimal hyperplasia at 14 and 28 days, respectively, after balloon dilatation. Neointima-to-media ratios were similarly reduced. In addition, expansile remodeling after balloon injury was inhibited at 14 days, the area within the external elastic lamina being 0.50 +/- 0.02 and 0.61 +/- 0.02 mm(2) in LID.hTIMP-1- and LID.pCI-transfected arteries, respectively (p < 0.0005). We have demonstrated an effective system of therapeutic gene transfer, particularly targeting the arterial adventitia, where transfer of genes involved in matrix remodeling and cell migration may be useful.

Intra-ventricular infusion of rAAV1-EGFP resulted in transduction in multiple regions of adult rat brain: a comparative study with rAAV2 and rAAV5 vectors.

Most gene transfer studies conducted in the central nervous system (CNS) with recombinant adeno-associated virus (rAAV) vectors have been carried out by direct intra-parenchymal injection. However, this delivery method usually results in transduction of cells in only a limited region and is quite invasive, which may hamper its potential clinical application. Injection of viral vectors into the cerebrospinal fluid (CSF) may provide an alternative strategy for widespread gene delivery to the CNS via the subarachnoid space. In this study we compared the transduction abilities of rAAV types 1, 2, and 5 when infused directly into the right lateral cerebral ventricle of adult rats. Multiple structures in the vicinity of the lateral ventricle were transduced by rAAV1, but not by rAAV2 or rAAV5 vectors. Double immunolabeling showed that the transduced cells included not only neurons, but also glia. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments demonstrated that rAAV1-mediated EGFP mRNA expression was significantly higher than that induced by either rAAV2 or 5. Our data suggest that intra-ventricular infusion of rAAV1 vectors provides a useful method for broad gene delivery to cells in the adult rat CNS.

Inhibition of LN-308 glioma cell proliferation and migration by retinoic acid amide through activation of Akt pathway.

The present study was performed to investigate the effect of retinoic acid amide (RAA) on the expression of integrin α3ß1, rate of cell proliferation and migration in p53-deficient glioma cell line, LN-308. The results revealed promotion of integrin α3 expression, reduction in proliferation and migration in RAA treated cells compared to the control LN-308 glioma cells. Promotion of RAA induced integrin α3ß1 expression led to the enhancement in cyclin-dependent kinase nuclear localization and activation of Akt pathway. In addition, RAA treatment inhibited the expression of nuclear factor-κB, Bcl-2 and epidermal growth factor receptor (EGFR). These factors are responsible for promoting the rate of cell proliferation and survival in the carcinoma cells. Thus RAA treatment inhibits rate of LN-308 glioma cell proliferation and migration through increase in integrin α3ß1 expression and activation of Akt pathway. Therefore, RAA can be of therapeutic importance for the treatment of glioma.

Diversification of specificity after maturation of the antibody response to the HIV gp41 epitope ELDKWA.

During maturing antibody responses the increase in affinity for target antigens is achieved by genetic diversification of antibody genes followed by selection for improved binding. The effect this process has on the specificity of antibody for variants of the antigen is not well-defined, despite the potential role of antibody diversification in generating enhanced protection against pathogen escape mutants, or novel specificities after vaccination. To investigate this, a library of single amino-acid substitution epitope variants has been screened with serum obtained at different time-points after immunization of mice with the HIV gp41 peptide epitope ELDKWA. The serum IgG response is shown to mature and increase affinity for ELDKWA, and the titre and affinity of IgG against most epitope variants tested increases. Furthermore there is a bias towards high affinity serum IgG binding to variant epitopes with conservative substitutions, although underlying this trend there is also significant binding to many epitopes with non-conservative substitutions. Thus, maturation of the antibody response to a single epitope results in a broadening of the high-affinity response toward variant epitopes. This implies that many pathogen epitope escape variants that could manifest as single amino-acid substitutions would not emerge by escaping immune surveillance.

Integrin-targeted nanocomplexes for tumour specific delivery and therapy by systemic administration.

Nanoparticle formulations offer opportunities for tumour delivery of therapeutic reagents. The Receptor-Targeted Nanocomplex (RTN) formulation consists of a PEGylated, endosomally-cleavable lipid and an RGD integrin-targeting, endosomally-cleavable peptide. Nancomplexes self-assemble on mixing with plasmid DNA to produce nanoparticles of about 100 nm. The environmentally-sensitive linkers promote intracellular disassembly and release of the DNA. RTNs carrying luciferase genes were administered intravenously to mice carrying subcutaneous neuroblastoma tumours. Luciferase expression was much higher in tumours than in liver, spleen and lungs while plasmid biodistribution studies supported the expression data. Transfection in tumours was enhanced two-fold by integrin-targeting peptides compared to non-targeted nanocomplexes. RTNs containing the interleukin-2 (IL-2) and IL-12 genes were administered intravenously with seven doses at 48 h intervals and tumour growth monitored. Tumours from treated animals were approximately 75% smaller on day 11 compared with RTNs containing control plasmids with one third of treated mice surviving long-term. Extensive leukocyte infiltration, decreased vascularization and increased necrotic areas were observed in the tumours from IL2/IL12 treated animals. Splenocytes from re-challenged mice displayed enhanced IL-2 production following Neuro-2A co-culture, which, combined with infiltration studies, suggested a cytotoxic T cell-mediated9 tumour-rejection process. The integrin-targeted RTN formulation may have broader applications in the further development of cancer therapeutics.

Recombinant adeno-associated virus serotype 1-vascular endothelial growth factor promotes neurogenesis and neuromigration in the subventricular zone and rescues neuronal function in ischemic rats.

OBJECTIVE: Vascular endothelial growth factor (VEGF) enhances neurogenesis in ischemic brains. However, in most circumstances, endogenous VEGF expression is limited and insufficient to prevent brain damage. We transferred the VEGF gene into brain tissue with recombinant adeno-associated virus serotype 1 (rAAV1) vectors and determined the effect of VEGF expression on neurogenesis and recovery of neurological function after brain ischemia. METHODS: Two groups (n = 32) of Sprague Dawley rats received intraventricular injection of AAV1-VEGF or AAV1-lacZ. Twenty-one days after gene transfer, rats underwent transient middle cerebral artery occlusion, and neurological severity score was measured 1, 2, 3, 7, 14, and 21 days later. Immunostaining was used to identify the quantity and distribution of VEGF expression. Double-immunofluorescence for doublecortin and bromodeoxyuridine or neuronal nuclei was performed to detect neurogenesis and the migration of neural progenitor cells. RESULTS: VEGF expression reduced the size of cerebral infarction and improved neurological function. It also enhanced the proliferation of neural progenitor cells in the subventricular zone and promoted their migration to the ischemic lesion. Neural precursors in the subgranular zone of the dentate gyrus were also increased; however, most of these cells did not move to the ischemic lesion and integrated with their region of origin. CONCLUSION: rAAV1-mediated expression of VEGF in the rat brain reduces the size of the infarcted lesion and promotes recovery of neurological function, likely by enhancing neurogenesis in the subventricular zone and promoting neural precursor migration to brain tissue around the core of the ischemic lesion.

Targeting lipopolyplexes using bifunctional peptides incorporating hydrophobic spacer amino acids: synthesis, transfection, and biophysical studies.

We have developed efficient synthetic routes to two hydrophobic amino acids, suitably protected for solid-phase peptide synthesis, and have successfully synthesized peptides containing these or other hydrophobic amino acids as spacers between a Lys16 moiety and an integrin-targeting motif. These peptides have in turn been used to formulate a range of lipopolyplex vectors with Lipofectin and plasmid DNA. The transfection efficiencies of these vectors and their aggregation behavior in buffers and in serum have been studied. We have shown that vectors containing peptides incorporating long linkers that are entirely hydrophobic are less efficient transfection agents. However, linkers of equivalent length that are in part hydrophobic show improved transfection properties, which is probably due to the improved accessibility of the integrin-binding motif.

Efficient transfection of non-proliferating human airway epithelial cells with a synthetic vector system.

BACKGROUND: The poor transfection efficiency of most synthetic vectors in non-dividing, airway epithelial cells is limiting the development of gene therapy for respiratory diseases such as cystic fibrosis. METHODS: Cultured airway epithelial cells, quiescent or replicating, were transfected with a lipid/peptide synthetic vector system and transfection efficiency assessed by luciferase assays or flow cytometry for green fluorescent protein expression. Tight junctions were disrupted by treatment with ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Cell division was inhibited by aphidicolin and assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and assay by fluorescence microscopy or flow cytometry. In vivo transfection was performed by intratracheal instillation. RESULTS: Initial studies showed that this synthetic vector transfected 8% of non-dividing epithelial cells in culture compared with 45% of subconfluent, dividing cells. However, formulation of the vector with EGTA, which disrupts cellular tight junctions, induced a four-fold increase in the transfection efficiency of confluent, non-mitotic cells. The EGTA enhancement of transfection also correlated with a four-fold increase in vector binding to the cells, presumably via exposed basolateral receptors. Flow cytometry and double immunofluorescent staining for reporter gene expression and BrdU incorporation indicated efficient transfection of non-proliferating cells in a confluent monolayer by this vector. Furthermore, in vivo transfection of murine lung by intratracheal administration showed a six-fold enhancement of luciferase expression when lungs were pre-treated with EGTA. CONCLUSIONS: This study confirms the hypothesis that a lipid/peptide vector transfects non-dividing cells with high efficiency which contributes to its previously reported efficiency in transfecting bronchial epithelium, making it a good choice for gene transfer studies in the lung.

High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system.

BACKGROUND: Gene therapy strategies for the treatment of vascular disease such as the prevention of post-angioplasty restenosis require efficient, non-toxic transfection of vascular cells. In vitro studies in these cells contribute to vector development for in vivo use and for the evaluation of genes with therapeutic potential. The aim of this project was to evaluate a novel synthetic vector consisting of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), which combine to form the LID vector complex. METHODS: Cultures of porcine smooth muscle cells and endothelial cells were established and then transfected with the LID vector, using the reporter genes luciferase and green fluorescent protein and the metalloprotease inhibitor TIMP-1. RESULTS: The LID vector system transfected primary porcine vascular smooth muscle cells and porcine aortic endothelial cells with efficiency levels of 40% and 35%, respectively. By increasing the relative DNA concentration four-fold, incubation periods as short as 30 min achieved the same levels of luciferase transgene expression as 4 h incubations at lower DNA concentrations. The transfection did not affect cell viability as measured by their proliferative potential. Serum levels of up to 20% in the transfection medium had no adverse affect on the efficiency of transfer and gene expression in either cell type. Transfections with the cDNA for TIMP-1 produced protein levels that peaked at 130 ng/ml per 24 h and persisted for 14 days at 10 ng/ml per 24 h. CONCLUSION: This novel vector system has potential for studies involving gene transfer to cardiovascular cells in vitro and in vivo.

Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector.

BACKGROUND: The pig lung, given its gross anatomical, histological and physiological similarities to the human lung, may be useful as a large animal model, in addition to rodents, in which to assess the potential of vectors for pulmonary airway gene transfer. The aim of this study was to assess the utility of the pig lung as a model of gene transfer to the human lung with a synthetic vector system. METHODS: The LID vector system consists of a complex of lipofectin (L), integrin-binding peptide (I) and plasmid DNA (D). LID complexes containing a beta-galactosidase reporter gene under a CMV promoter or a control plasmid at1 mg/3 ml PBS, or 3 ml buffer, was administered to the right lower lobe of the pig lung through a bronchoscope. Pigs were culled at 48 h and lung sections prepared for immunohistochemical and histological analysis. Bronchoalveolar lavage fluid was collected and analysed for TNF-alpha by ELISA. RESULTS: Immunohistochemical staining for the beta-galactosidase reporter gene indicated high efficiency of gene transfer by the LID vector to pig bronchial epithelium with 46% of large bronchi staining positively. There was no evidence for vector-specific inflammation assessed by leukocytosis and cytokine production. CONCLUSIONS: This study demonstrates the use of the pig for studies of gene transfer in the lung and confirms in a second species the potential of the LID vector for gene therapy of pulmonary diseases such as cystic fibrosis.