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41.
da Silva FH Pereira VG Yasumura EG Tenório LZ de Carvalho LP Lisboa BC Matsumoto PK Stilhano RS Samoto VY Calegare BF Brandão Lde C D'Almeida V Filippo TR Porcionatto M Toma L Nader HB Valero VB Camassola M Nardi NB Han SW 《Genetic vaccines and therapy》2012,10(1):2-11
Background
Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity.Methods
MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses.Results
After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months.Conclusions
These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice. 相似文献42.
Sara D'Angelo Jacob Glanville Fortunato Ferrara Leslie Naranjo Cheryl D Gleasner Xiaohong Shen Andrew RM Bradbury Csaba Kiss 《MABS-AUSTIN》2014,6(1):160-172
In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries. 相似文献
43.
Fortunato Ferrara Sara D’Angelo Tiziano Gaiotto Leslie Naranjo Hongzhao Tian Susanne Gr?slund Elena Dobrovetsky Peter Hraber Fridtjof Lund-Johansen Silvia Saragozza Daniele Sblattero Csaba Kiss Andrew RM Bradbury 《MABS-AUSTIN》2015,7(1):32-41
Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. 相似文献
44.
Lúcia de Paula Célio L Silva Daniela Carlos Camila Matias-Peres Carlos A Sorgi Edson G Soares Patrícia RM Souza Carlos RZ Bladés Fábio CS Galleti Vânia LD Bonato Eduardo DC Gonçalves Érika VG Silva Lúcia H Faccioli 《Genetic vaccines and therapy》2007,5(1):1-7
The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs. 相似文献
45.
We have purified and characterized a phenobarbital (PB)-inducible hepatic mitochondrial cytochrome P-450 (P-450), termed P-450mt4, which is distinctly different from the previously characterized mitochondrial isoforms. The level of induction of P-450mt4 by PB in the male livers is nearly 20-fold, as against a marginal induction in the female livers, suggesting that it may be a male predominant isoform. P-450mt4 shows a close resemblance to microsomal P-450b (the major PB-inducible form) with respect to electrophoretic migration (apparent molecular mass of 50 kDa) and immunological cross-reactivity, although it exhibits a distinct isoelectric pH (pI 6.9 vs 6.5 for P-450b), peptide fingerprint pattern, and amino acid composition. Further, the N-terminal sequence analysis shows over 90% positional identity (39 out of 42) between P-450mt4 and P-450b, suggesting that it is a close relative of the P-450 IIB gene family. In vitro reconstitution experiments show that P-450mt4 can metabolize a wide range of substrates such as benzphetamine, (dimethylamino)antipyrine, aflatoxin B1, and vitamin D3, exclusively in the presence of mitochondrial-specific ferredoxin and ferredoxin reductase as electron carriers. P-450mt4 is translated as a 53-kDa precursor, which is transported into mitochondria under in vitro conditions and processed into a mature 50-kDa protein. These results provide conclusive evidence for the occurrence of a male-specific P-450 belonging to the IIB gene family in rat liver mitochondria. 相似文献
46.
Aileen?G?RowanEmail author Koichiro?Suemori Hiroshi?Fujiwara Masaki?Yasukawa Yuetsu?Tanaka Graham?P?Taylor Charles?RM?Bangham 《Retrovirology》2014,11(1):116
Background
Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4+ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.Results
Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.Conclusions
HTLV-1 gene expression in primary CD4+ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.47.
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49.
B G Niranjan H Raza R M Shayiq C R Jefcoate N G Avadhani 《The Journal of biological chemistry》1988,263(1):575-580
Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria. 相似文献
50.
Characterization of a female-specific hepatic mitochondrial cytochrome P-450 whose steady-state level is modulated by testosterone 总被引:1,自引:0,他引:1
Polyclonal antibody to mitochondrial P-450c27/25 reacted with two proteins of apparent molecular masses of 52 kilodaltons (kDa) and 50 kDa from the female rat liver mitochondrial proteins bound to an omega-octylaminoagarose column. The two proteins were purified to greater than 85% homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography, and both were found to be P-450 as judged by dithionite-reduced CO difference spectra. Both of the P-450 forms required mitochondrial-specific ferredoxin and ferredoxin reductase for in vitro reconstitution of enzyme activities, suggesting that they are mitochondrial forms. The 52-kDa P-450 exhibited the properties of mitochondrial 27/25-hydroxylase with respect to high vitamin D3 25-hydroxylase activity [1.4 nmol (nmol of P-450)-1 min-1] and N-terminal amino acid sequence. The 50-kDa P-450, on the other hand, lacked significant vitamin D3 25-hydroxylase activity, but showed 17 beta-reductase [0.380-0.400 nmol (nmol of P-450)-1 min-1] and 17 beta-oxidase [0.1-0.16 nmol (nmol of P-450)-1 min-1] activities with both androgens and estrogens as substrates. Immunoblot analysis of proteins using a monoclonal antibody specific for P-450c27/25 showed a 2-3-fold higher level of this enzyme in the female liver mitochondria than in the males. Similarly, use of a polyclonal antibody in the immunoblot analysis showed that the 50-kDa P-450 is female-specific. The relative level of P-450c27/25 was reduced significantly in castrated females, while the level of the female-specific 50-kDa P-450 was increased. However, the levels of both enzymes were increased in castrated males.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献