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Mol Ther. 2002 Aug;6(2):272-8.
Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks.
Veldwijk MR, Topaly J, Laufs S, Hengge UR, Wenz F, Zeller WJ, Fruehauf S.
German Cancer Research Center, Im Neuenheimer Feld 280 D-69120, Heidelberg, Germany.
Despite the clinical application of adeno-associated virus (AAV) gene therapy, the titration of viral stocks has not yet been standardized. This complicates the comparison of viral stocks between laboratories. Functional titering of AAV is time-consuming, requires the manipulation of hazardous material, and often has a high degree of variability. We established an optimized real-time quantitative polymerase chain reaction (RQ-PCR) titration assay to determine viral titers and compared it with a functional green fluorescent protein (GFP)-based titration method. With a combination of improved lysis procedures and RQ-PCR protocols we could decrease the intraexperimental coefficient of variation (CV) from 0.24 +/- 0.03 to 0.042 +/- 0.004 and the interexperimental CV from 0.34 +/- 0.06 to 0.093 +/- 0.028 following functional and RQPCR-based titration, respectively. This low variability conforms to even the strictest quality standards required, for example, in clinical laboratories. The highly standardized titration by RQPCR described here will be especially advantageous for groups working on AAV-based gene therapy in a good manufacturing practice setting.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12349826&dopt=Abstract
Virology. 2002 Sep 1;300(2):205-16.
Coreceptor Switch of [MLV(SIVagm)] pseudotype vectors by V3-loop exchange.
Steidl S, Stitz J, Schmitt I, Konig R, Flory E, Schweizer M, Cichutek K.
Department of Medical Biotechnology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany.
Retroviral vectors derived from murine leukemia virus (MLV) have been pseudotyped with a variant of the envelope glycoprotein (Env) of nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to result in [MLV(SIVagm-wt)] vector particles. The variant env gene encodes a full-length surface envelope glycoprotein (SU) and a C-terminally truncated transmembrane protein (TM). To change the coreceptor usage of this vector from CCR5 to CXCR4, which is predominant on human CD4-positive lymphocytes, the putative V3-loop of SIVagm SU was replaced by that of the T cell tropic HIV-1 variant BH10. The resulting [MLV(SIVagm-X4)] vectors were shown to specifically transduce CD4/CXCR4-positive cell lines, demonstrating the equivalent function in cell entry and choice of coreceptor usage of the V3-loops of SIVagm and HIV-1. These modified vectors were able to transduce primary human lymphocytes and were resistant to neutralization by sera from HIV-1-infected individuals. The [MLV(SIVagm-X4)] pseudotype vector generated is thus a promising candidate vector, e.g., for in vivo gene therapy of HIV-1 infection.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12350351&dopt=Abstract
Urology. 2002 Sep;60(3):531-6.
Integrin alpha(v) and coxsackie adenovirus receptor expression in clinical bladder cancer.
Sachs MD, Rauen KA, Ramamurthy M, Dodson JL, De Marzo AM, Putzi MJ, Schoenberg MP, Rodriguez R.
Brady Urological Institute, Johns Hopkins Hospital, Baltimore, Maryland 21287, USA.
OBJECTIVES: To evaluate the expression of the coxsackie and adenovirus receptor (CAR) and alpha(v) integrins in clinical specimens of bladder cancer to determine the susceptibility to adenoviral gene therapy. Efficient adenovirus-based gene therapy requires binding of the virus to CAR and involves the alpha(v) integrins. Studies on bladder cancer cell lines have shown that low adenoviral transduction rates were associated with low-level expression of CAR. Integrin alpha(v) expression increases in various tumors suggest its importance in differentiation, proliferation, and migration. CAR is structurally a member of the Ig-type superfamily of cell-cell adhesion molecules, suggesting that its expression may also be related to the state of tumor differentiation. METHODS: We performed immunohistochemistry for CAR and integrin alpha(v) expression in bladder cancer specimens in 50 paraffin-embedded tumor-normal pairs and confirmed the results by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of 11 separate bladder tumors and 4 separate normal bladder controls. RESULTS: Immunochemistry demonstrated a stage and grade-dependent decrease in CAR expression (90.0%, 83.3%, and 31.3% of normal urothelium and superficial and invasive transitional cell carcinoma [TCC] and 83.3% and 39.5% of low and high-grade TCC, respectively). Furthermore, we found a stage and grade-dependent increase in alpha(v) integrin expression (13.3%, 46.0%, and 56.3% of normal urothelium, superficial TCC, and invasive TCC and 25% and 52.6% of low and high-grade TCC, respectively). Quantitative RT-PCR analysis confirmed a downregulation at the CAR gene expression level. CONCLUSIONS: This down-regulation may have a major impact on developing adenoviral-based gene therapy modalities. In addition, we propose that loss of CAR expression decreases rigid cell adhesion, possibly increasing the migratory potential. Loss of CAR expression correlates with the invasive phenotype in our analysis of bladder cancer. Simultaneously, the finding of increased alpha(v) expression in invasive cancer suggests a pathogenesis that involves heterophilic adhesion and migration of these cells on various extracellular ligands.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12350512&dopt=Abstract
Blood. 2002 Oct 15;100(8):2724-31.
Gene transfer improves erythroid development in ribosomal protein S19-deficient Diamond-Blackfan anemia.
Hamaguchi I, Ooka A, Brun A, Richter J, Dahl N, Karlsson S.
Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University Hospital, Sweden.
Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Forty percent of the patients are blood transfusion-dependent. Recent reports show that the ribosomal protein S19 (RPS19) gene is mutated in 25% of all patients with DBA. We constructed oncoretroviral vectors containing the RPS19 gene to develop gene therapy for RPS19-deficient DBA. These vectors were used to introduce the RPS19 gene into CD34(+) bone marrow (BM) cells from 4 patients with DBA with RPS19 gene mutations. Overexpression of the RPS19 transgene increased the number of erythroid colonies by almost 3-fold. High expression levels of the RPS19 transgene improved erythroid colony-forming ability substantially whereas low expression levels had no effect. Overexpression of RPS19 had no detrimental effect on granulocyte-macrophage colony formation. Therefore, these findings suggest that gene therapy for RPS19-deficient patients with DBA using viral vectors that express the RPS19 gene is feasible.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12351378&dopt=Abstract
Natural Herbal Supplement: Hair Million
for increasing number of folks in aging society. Loss of hair is a visible problem, and affects the appearance and changes identity of a person.
The phenomenon of hair thinning and hair loss is most commonly associated with natural aging, although there are many other causes of hair loss, which include inherited or genetic conditions, illnesses, malnutrition, stress, hormonal problems, chemotherapy, and use of some drugs.
Hair Million is an alternative solution to hair loss problems. Anecdotally, it shows prositive results and improvement for age-related hair thinning and hair loss for a fraction of people who take it. We do not know the mechanisms of action as to how Hair Million works to help stop hair loss, and promote hair growth. We only know by anecdotal observations. There has been no clinical trials nor placebo controlled statistical analysis on the efficacy of Hair Million on hair loss and hair growth. However, there are two merits in this hair restoration herbal formula:
Firstly, Hair Million is rather inexpensive, and secondly, it is made of well known herbs that are safe when consumed in regular quantities.
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