| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Carlsson, Björn, et al.
(författare)
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Ex vivo stimulation of cytomegalovirus (CMV)-specific T cells using CMVpp65-modified dendritic cells as stimulators
- 2003
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Ingår i: British Journal of Haematology. - : Wiley. - 0007-1048 .- 1365-2141. ; 121:3, s. 428-38
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Tidskriftsartikel (refereegranskat)abstract
- Cytomegalovirus (CMV) infection is a dangerous complication in immunosuppressed individuals such as allogeneic stem cell transplant patients. CMV disease can be prevented by the early post-transplant transfer of donor-derived, CMV-directed, T cells. Fast and cost efficient methods to generate CMV-specific T cells are, therefore, warranted. The current study utilized peptide-pulsed and adenovirus-transduced dendritic cells (DC) to generate CMV-restricted T cells. After one stimulation with CMV pp65495-503 peptide-pulsed DC and three re-stimulations with peptide-pulsed monocytes, virtually all T cells were CD8+, expressed the relevant T cell receptor and exhibited high peptide-specific lytic activity. After only one stimulation, pp65495-503-restricted T cells could be sorted to a purity of higher than 95% and expanded up to 1000-fold in 2 weeks. This technique may prove useful for the rapid generation of large quantities of specific cytolytic T lymphocytes (CTL) for cell therapy. DC transduced with an adenoviral vector encoding the full-length pp65 protein (Adpp65) were able to simultaneously expand CTL against multiple epitopes of pp65. In addition, they activated CMV-specific CD4+ T-helper cells. This approach would stimulate multiple-epitope populations of pp65-specific T cells and could be made available to patients of any human leucocyte antigen (HLA) haplotype. DC transduced with adenoviral vectors to express full-length antigens may prove to be potent vaccines against viral pathogens and cancer.
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| 3. |
- Cheng, Wing-Shing, et al.
(författare)
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A novel TARP-promoter-based adenovirus against hormone-dependent and hormone-refractory prostate cancer
- 2004
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 10:2, s. 355-364
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Tidskriftsartikel (refereegranskat)abstract
- TARP (T cell receptor gamma-chain alternate reading frame protein) is a protein that in males is uniquely expressed in prostate epithelial cells and prostate cancer cells. We have previously shown that the transcriptional activity of a chimeric sequence comprising the TARP promoter (TARPp) and the PSA enhancer (PSAe) is strictly controlled by testosterone and highly restricted to cells of prostate origin. Here we report that a chimeric sequence comprising TARPp and the PSMA enhancer (PSMAe) is highly active in testosterone-deprived prostate cancer cells, while a regulatory sequence comprising PSAe, PSMAe, and TARPp (PPT) has high prostate-specific activity both in the presence and in the absence of testosterone. Therefore, the PPT sequence may, in a gene therapy setting, be beneficial to prostate cancer patients that have been treated with androgen withdrawal. A recombinant adenovirus vector with the PPT sequence, shielded from interfering adenoviral sequences by the mouse H19 insulator, yields high and prostate-specific transgene expression both in cell cultures and when prostate cancer, PC-346C, tumors were grown orthotopically in nude mice. Intravenous virus administration reveals both higher activity and higher selectivity for the insulator-shielded PPT sequence than for the immediate-early CMV promoter. Therefore, we believe that an adenovirus with therapeutic gene expression controlled by an insulator-shielded PPT sequence is a promising candidate for gene therapy of prostate cancer.
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| 4. |
- Cheng, Wing-Shing, et al.
(författare)
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An oncolytic conditionally replicating adenovirus for hormone-dependent and hormone-independent prostate cancer
- 2006
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Ingår i: Cancer Gene Therapy. - 0929-1903 .- 1476-5500. ; 13:1, s. 13-20
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Tidskriftsartikel (refereegranskat)abstract
- The use of conditionally replicating adenoviruses offers an attractive complementary treatment strategy for localized prostate cancer. We have produced a replicating adenovirus, Ad[I/PPT-E1A], where E1A gene expression is controlled by a recombinant regulatory sequence designated PPT. The PPT sequence comprises a PSA enhancer, a PSMA enhancer and a T-cell receptor gamma-chain alternate reading frame protein promoter, and it is shielded from transcriptional interference from adenoviral backbone sequences by an H19 insulator. Ad[I/PPT-E1A] yields prostate-specific E1A protein expression, viral replication and cytolysis in vitro. Furthermore, Ad[I/PPT-E1A] considerably regresses the growth of subcutaneous LNCaP prostate cancer tumors in nude mice. Importantly, the viral replication and cytolytic effect of Ad[I/PPT-E1A] are independent of the testosterone levels in the prostate cancer cells. This may be beneficial in a clinical setting since many prostate cancer patients are treated with androgen withdrawal. In conclusion, Ad[I/PPT-E1A] may prove to be useful in the treatment of localized prostate cancer.
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| 5. |
- Cheng, Wing-Shing, et al.
(författare)
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Characterization of the androgen-regulated prostate-specific T cellreceptor gamma-chain alternate reading frame protein (TARP) promoter.
- 2003
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Ingår i: Endocrinology. ; 144:8, s. 3433-40
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Tidskriftsartikel (refereegranskat)abstract
- TARP (T cell receptor gamma-chain alternate reading frame protein) is uniquely expressed in males in prostate epithelial cells and prostate cancer cells. Here we demonstrate that TARP expression is regulated by testosterone at the transcriptional level through specific binding of androgen receptor to an androgen response element in the proximal TARP promoter. We further demonstrate that the promoter specifically initiates reporter gene expression in TARP-positive prostate cancer cell lines. To develop a regulatory sequence for prostate-specific gene expression, we constructed a chimeric sequence consisting of the TARP promoter and the prostate-specific antigen (PSA) enhancer. We found that in the prostatic adenocarcinoma cell line LNCaP, the transcriptional activity of the regulatory sequence consisting of a TARP promoter and PSA enhancer is 20 times higher than the activity of a regulatory sequence consisting of the PSA promoter and PSA enhancer. Thus, our studies define a regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer cells and may therefore play a role in prostate cancer gene therapy.
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| 6. |
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| 7. |
- Cheng, Wing-Shing, 1974-
(författare)
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TARP Promoter-Based Prostate Cancer Gene Therapy : From Development to Application
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Prostate cancer is one leading cause of cancer-related death among men in Western countries. The standard therapies for localized prostate cancer include radical prostatectomy and radiation therapy. Such measures are relatively effective in the short term, but many patients ultimately relapse. These patients may benefit from a combination of standard therapy and oncolytic virus therapy. My work aimed to develop viruses for this purpose.TARP is a protein that in males is specifically expressed in prostate epithelial and cancer cells. In my thesis, I characterized the TARP promoter and showed that TARP expression is regulated at the transcriptional level by testosterone through binding of the androgen receptor in the proximal TARP promoter. I further developed TARP promoter-based regulatory sequences for prostate-specific gene expression. A sequence comprising a PSA enhancer, a PSMA enhancer and the TARP promoter was constructed and designated PPT. An adenoviral vector containing the PPT sequence shielded from transcriptional interference by an H19 insulator showed high prostate-specific transcriptional activity in human cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer the PPT sequence is not active. Therefore, a two-step transcriptional amplification (TSTA) system was used together with the PPT sequence to develop an adenovirus that confers prostate-specific transgene expression also in murine cells.I constructed a conditionally replicating adenovirus where the E1A gene expression is controlled by an H19 insulator-shielded PPT regulatory sequence, Ad[I/PPT-E1A]. This virus exhibited absolute prostate specificity in terms of E1A expression, viral replication and cytolysis in vitro and in vivo. Importantly, our virus is active both in the presence and absence of testosterone, which may prove beneficial for patients treated by hormonal withdrawal. Hopefully, my work will improve existing gene therapy strategies for prostate cancer and in the long term improve the prognosis for patients with prostate cancer.
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| 8. |
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| 9. |
- Danielsson, Angelika, 1981-, et al.
(författare)
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The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication
- 2011
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:2, s. e14700-
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Tidskriftsartikel (refereegranskat)abstract
- The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR) has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction demonstrating that the main effect by which FK228 enhances transgene expression is transduction independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2, was transduced with Ad[CD40L]. One unexpected finding was that the transgene expression of an adenoviral vector with the prostate-specific PPT promoter decreased in the human prostate cancer cell line LNCaP when FK228 was administered. This is probably a consequence of phenotypic alteration of LNCaP towards a neuroendocrine phenotype after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may alter the phenotype of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected.
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| 10. |
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