Ulrich Steidl

Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR

In this study, a quantitative real-time PCR (qPCR) was developed to determine genomic rAAV-2 titers using the Light-Cycler technology. Since the CMV promoter is the most commonly used promoter in gene therapeutic approaches, primers were designed which hybridize with the human CMV promoter sequence. PCR products were detected by the addition of SYBR green. qPCR of a 5 log spanning serial dilution of the vector plasmid containing one CMV promoter per plasmid molecule yielded a high amplification efficiency of 1.99 per cycle. To quantify the copy number of viral genomes, the qPCR curves of adeno-associated virus type 2 (AAV-2) samples were related to a standard curve assessed by the 5 log spanning serial vector plasmid dilution (0.01-100 pg DNA). For validation of the method, rAAV-2 preparations were analyzed by a standard method and qPCR in parallel. As standard method, flow cytometry was used for titration of infectious viral particles on HeLa cells using the Enhanced Green Fluorescent Protein as a marker. A significant correlation was found between the results obtained by flow cytometry and the results from the qPCR over a 5 log range (r=0.85, P<0.0001). The mean ratio between infectious rAAV-2 particles titrated via flow cytometry and genomic copies of rAAV-2 measured by qPCR of the same sample was 1:253. The higher titers found by qPCR might be due to multiple transduction of a single cell or to non-infectious particles generated during rAAV-2 preparation. In conclusion, qPCR is a fast and reliable method for determination of rAAV-2 titers and might be a powerful tool for standardization of rAAV-2 preparations particularly in the context of clinical studies.

Cyclosporine A and Mycophenolate Mofetil vs Cyclosporine A and Methotrexate for graft-versus-host disease prophylaxis after stem cell transplantation from HLA-identical siblings

Summary:The combination of Cyclosporin A (CSA) and Methotrexate (MTX) is considered to be the standard regimen for the prevention of graft-versus-host disease (GVHD) after stem cell transplantation (SCT) from HLA-identical siblings. Mycophenolate Mofetil (MMF) has been widely used for GVHD prophylaxis after nonmyeloablative SCT, but experience following myeloablative therapy is still limited. We retrospectively compared CSA/MTX and CSA/MMF in 93 patients (median age 35 years, range 17–59 years, male subjects 48, female subjects 45) with acute myeloid leukemia (n=33), myelodysplastic syndrome (MDS) (n=3), acute lymphoblastic leukemia (ALL) (n=20) or chronic myeloid leukemia (n=37) who received CSA/MMF (n=26) or CSA/MTX (n=67) as GVHD prophylaxis following high-dose therapy and allogeneic SCT from HLA-identical siblings. No statistically significant differences were found in overall survival, relapse rate, treatment-related mortality and acute or chronic GVHD. Time to myeloid recovery was significantly shorter in patients who received CSA/MMF. We conclude that the combination of CSA/MMF appears equivalent to CSA/MTX for GVHD prophylaxis in patients receiving conventional-intensity SCT from HLA-identical siblings.

Expression of the tyrosine kinase c‐kit is an independent prognostic factor in patients with small cell lung cancer

In a retrospective analysis of 203 patients with small cell lung cancer (SCLC), we examined the prognostic value of c‐kit expression on survival. Expression of c‐kit was examined immunohistochemically in formalin‐fixed, paraffin‐embedded tissue sections. c‐kit was observed in 87.7% of SCLC tumors. Using the Kaplan‐Meier model, we found that lack of c‐kit expression was associated with significantly shorter survival time compared to the presence of c‐kit expression (mean survival 151 ± 27 vs. 358 ± 49 days, p = 0.0084). Moreover, the proportion of c‐kit+ cells within the tumor was also related to survival time. Patients with tumors in which >75% of cells stained positive for c‐kit had a mean overall survival time of 424 (±72) compared to 295 (±67) days for patients with 25–75% c‐kit+ tumor cells. Patients with tumors containing <25% c‐kit+ cells had the worst survival, with 164 (±24) days (p = 0.0033). Further parameters associated with short survival times were low performance status, elevated levels of lactate dehydrogenase and higher stage according to the TNM classification. Multivariate analysis using the Cox regression model showed that the proportion of c‐kit+ cells within the tumor specimen was one of 3 independent prognostic parameters (p = 0.004) for overall survival next to TNM classification (p = 0.001) and performance status (p < 0.001).

The influence of the pituitary tumor transforming gene-1 (PTTG-1) on survival of patients with small cell lung cancer and non-small cell lung cancer

Background PTTG-1 (pituitary tumor transforming gene) is a novel oncogene that is overexpressed in tumors, such as pituitary adenoma, breast and gastrointestinal cancers as well as in leukemia. In this study, we examined the role of PTTG-1 expression in lung cancer with regard to histological subtype, the correlation of PTTG-1 to clinical parameters and relation on patients survival. Methods Expression of PTTG-1 was examined immunohistochemically on formalin-fixed, paraffin-embedded tissue sections of 136 patients with small cell lung cancer (SCLC) and 91 patients with non-small cell lung cancer (NSCLC), retrospectively. The intensity of PTTG-1 expression as well as the proportion of PTTG-1 positive cells within a tumor was used for univariate and multivariate analysis. Results PTTG-1 expression was observed in 64% of SCLC tumors and in 97.8% of NSCLC tumors. In patients with SCLC, negative or low PTTG-1 expression was associated with a shorter mean survival time compared with patients with strong PTTG-1 expression (265 ± 18 days vs. 379 ± 66 days; p = 0.0291). Using the Cox regression model for multivariate analysis, PTTG-1 expression was a significant predictor for survival next to performance status, tumor stage, LDH and hemoglobin. In contrast, in patients with NSCLC an inverse correlation between survival and PTTG-1 expression was seen. Strong PTTG-1 expression was associated with a shorter mean survival of 306 ± 58 days compared with 463 ± 55 days for those patients with no or low PTTG-1 intensities (p = 0.0386). Further, PTTG-1 expression was associated with a more aggressive NSCLC phenotype with an advanced pathological stage, extensive lymph node metastases, distant metastases and increased LDH level. Multivariate analysis using Cox regression confirmed the prognostic relevance of PTTG-1 expression next to performance status and tumor stage in patients with NSCLC. Conclusion Lung cancers belong to the group of tumors expressing PTTG-1. Dependent on the histological subtype of lung cancer, PTTG-1 expression was associated with a better outcome in patients with SCLC and a rather unfavourable outcome for patients with NSCLCs. These results may reflect the varying role of PTTG-1 in the pathophysiology of the different histological subtypes of lung cancer.

Inhibition of angiogenesis in vitro by αv integrin–directed antisense oligonucleotides

The integrin αvβ3 plays a central role in angiogenesis. In this study, we used antisense oligodeoxyribonucleotides (ONs) directed against the αv subunit of αvβ3 to inhibit integrin expression. Ten ON sequences, which were selected by systematic alignment of computer-predicted secondary structures of αv mRNA, were transfected into human umbilical vein endothelial cells (HUVECs). Following stimulation by PMA, five antisense ONs significantly inhibited αv mRNA and protein expression in activated HUVEC at a concentration of 0.05 μM with complete prevention of PMA-induced αv up-regulation by the most potent antisense ON. Inhibition of αv expression was associated with significant inhibition of migration of HUVEC by 28% and had no effect on proliferation and apoptosis. Moreover, transfection of antisense ON inhibited the formation of tube-like structures of HUVEC in Matrigel by 44%. In a cell culture model of angiogenesis consisting of a co-culture of endothelial cells with fibroblasts, transfection of antisense ONs resulted in an inhibition of tube formation of 61%. In conclusion, αv antisense ONs are potent inhibitors of angiogenesis in vitro. They might, therefore, be a therapeutic alternative to antagonists, which directly bind to αv integrins, and might be useful for the treatment of malignant tumors and hematological malignancies.

Differential gene expression underlying the functional distinctions of primary human CD34+ hematopoietic stem and progenitor cells from peripheral blood and bone marrow

Abstract: The restorative capacity of human CD34+ hematopoietic cells is clinically used in the autologous and allogeneic transplant setting to support cytotoxic therapy. We examined gene expression patterns of highly enriched bone marrow CD34+ (BM‐CD34+) or G‐CSF‐mobilized peripheral blood CD34+ (PB‐CD34+) cells by cDNA array technology, quantitative real‐time RT‐PCR, and flow cytometry, to identify molecular causes underlying the functional differences between circulating and sedentary hematopoietic stem and progenitor cells. The greater cell cycle and DNA synthesis activity of BM‐CD34+ compared to PB‐CD34+ cells was reflected by the 2‐ to 5‐fold higher expression of 9 genes involved in cell cycle, 11 genes regulating DNA synthesis, and the cell cycle‐initiating transcription factor E2F‐1. The 2‐ to 3‐fold greater expression of 5 pro‐apoptotic genes in PB‐CD34+ cells indicated a higher apoptotic activity, which could functionally be corroborated by apoptosis assays. Thrombin receptor (PAR1), known to play a role in trafficking of malignant cells, was 3.6‐fold higher expressed in circulating CD34+ cells than in BM‐CD34+ cells. Guidance via thrombin receptor might molecularly mediate stem cell migration. In summary, our study provides gene expression profiles of primary human CD34+ hematopoietic cells of blood and marrow. Our data molecularly confirm and explain the finding that CD34+ cells residing in the bone marrow are cycling more rapidly, whereas circulating CD34+ cells consist of a higher number of quiescent stem and progenitor cells. Moreover, our data give novel molecular insights into stem cell migration and differentiation.

Molecular Biology of Hematopoietic Stem Cells

Human CD34+ hematopoietic stem and progenitor cells are capable of maintaining a life-long supply of the entire spectrum of blood cells dependent on systemic needs. Recent studies suggest that hematopoietic stem cells are, beyond their hematopoietic potential, able to differentiate into nonhematopoietic cell types, which could open novel avenues in the field of cellular therapy. Here, we concentrate on the molecular biology underlying basic features of hematopoietic stem cells. Immunofluorescence analyses, culture assays, and transplantation models permit an extensive immunological as well as functional characterization of human hematopoietic stem and progenitor cells. New methods such as cDNA array technology have demonstrated that distinct gene expression patterns of transcription factors and cell cycle genes molecularly control self-renewal, differentiation, and proliferation. Furthermore, several adhesion molecules have been shown to play an important role in the regulation of hematopoiesis and stem cell trafficking. Progress has also been made in elucidating molecular mechanisms of stem cell aging that limit replicative potential. Finally, more recent data provide the first molecular basis for a better understanding of transdifferentiation and developmental plasticity of hematopoietic stem cells. These findings could be helpful for non-hematopoietic cell therapeutic approaches.

Non-small lung cancer cells are prime targets for p53 gene transfer mediated by a recombinant adeno-associated virus type-2 vector

In this study, we elucidated the potential of recombinant adeno-associated virus type-2 (rAAV-2) vectors for lung cancer gene therapy. Cell lines of the three major histological subtypes of non-small cell lung cancer (NSCLC) were highly susceptible for rAAV-2 showing transduction rates between 63.4 and 98.9%. In contrast, cell lines of small cell carcinomas were resistant to rAAV-2 infection. For restoration of p53 function in p53 deficient NSCLC, a rAAV-2 vector was constructed containing wt p53 cDNA. Following transduction with rAAV-p53, cell growth of all NSCLC cell lines was significantly reduced in a dose-dependent manner between 44 and 71.7% in comparison with rAAV-GFP transduced cells. The reduction of tumor cell growth was associated with increased apoptosis. Adding cisplatin to rAAV-p53-infected cells led to a significant growth inhibition between 81 and 91% indicating a synergistic effect between cisplatin and rAAV-p53. Interestingly, the tumor cells surviving cisplatin and rAAV-p53 treatment were inhibited in their ability to form colonies as reflected by a reduction of colony growth between 57 and 90.4%. In conclusion, rAAV-2 vectors exhibit a strong tropism for NSCLC. Successful inhibition of tumor cell growth following transduction with a rAAV-p53 vector underlines the potential role of rAAV-2 in cancer gene therapy.

Transplantation of allogeneic CD34+-selected cells followed by early T-cell add-backs: favorable results in acute and chronic myeloid leukemia.

BACKGROUNDnThe aim of this study was to investigate preservation of anti-leukemic activity and protection from opportunistic infections after transplantation of allogeneic + cells in patients with hematologic malignancies and bad prognosis. Methods Thirty-three patients [median age 42 years, range 23-55 years, diagnosis AML/myelodysplastic syndrome (MDS) 14, ALL nine, CML seven and multiple myeloma (MM) three] received myeloablative conditioning followed by infusion of selected CD34+ cells from matched unrelated donors (31) or HLA-identical siblings (two). Early donor lymphocyte infusions (DLI; 0.5 and 1.0 x 10(6) CD3+ cells/kg) were given while patients were on immunosuppressive therapy.nnnRESULTSnNinety-seven per cent of patients engrafted and 24 of 29 patients surviving more than 30 days received at least one pre-emptive DLI. Three patients (10%) developed acute (a)GvHD (two grade I-II, one grade III-IV) spontaneously, and 16 patients (67%) developed aGvHD after DLI (12 grade I-II, four grade III-IV). Eight of 24 evaluable patients developed chronic (c)GvHD (33%, six limited, two extensive). After a median follow-up of 590 days (range 138-1610 days) 18 patients were alive (55%), 16 in complete remission (CR), one in hematologic and one in molecular relapse. Seven patients died after relapse (21%) and eight died from transplantation-related causes (24%). Patients with myeloid malignancies had a significantly better survival than patients with ALL or MM (74%+/-10 vs. 30%+/-13, P<0.05).nnnDISCUSSIONnEarly pre-emptive low-dose DLI following transplantation of selected CD34+ cells from unrelated donors after myeloablative conditioning is feasible and effective without undue toxicity, especially in patients with myeloid malignancies.

Keloide. Eine dermale fibroproliferative erkrankung unbekannter ursache

ZusammenfassungEs wird ein Überblick über die neuesten Forschungsergebnisse der Pathogenese der Keloide gegeben, und bestehende Behandlungsmäglichkeiten werden diskutiert. Keloide treten bevorzugt bei jüngeren Patienten nach Traumata der Haut auf. Sie wachsen infiltrativ in die umgebende gesunde Haut und neigen nur selten zu spontanen Remissionen. Ein Großteil der Patienten klagt über Juckreiz im Bereich der Keloide und Einschränkung der Lebensqualität. Das klinische Bild der Keloide ist seit langem bekannt, dennoch gibt es nur wenige Hinweise auf ihre Pathogenese. Neue Untersuchungen zeigen, dass Stärungen in der Signaltransduktion des Wachstumsfaktors TGFβ eine Rolle spielen kännten. Außerdem weisen klinische Beobachtungen auf eine familiäre Häufung der Keloide hin, sodass auch genetische Ursachen in Betracht gezogen werden müssen. Die bestehenden Behandlungsmäglichkeiten sind unbefriedigend. Neben der Kontaktkryotherapie und dem Unterspritzen der Läsionen mit Kortikoid-Kristallsuspension werden neue Therapieverfahren wie der gepulste Farbstofflaser eingesetzt. Auch Externa wie Zwiebelextrakte und Okklusivpflaster kommen neben Druckbandagen zur Anwendung.AbstractWe review recent advances in keloid research and treatment. Keloids are benign tumours of the skin. They tend to occur in younger patients after different kinds of injuries, infections or they may develop spontaneously. In contrast to hypertrophic scars, keloids are not confined to the original wound, but grow into the corresponding healthy skin. They rarely recede within time. Most patients complain of itching and suffer from impairment of their quality of life. Only little is known about the pathogenesis of keloids, although they have been clinically well characterized for a long time. New results point towards a disruption in the signaling pathway of TGFβ. In addition there seems to be evidence of an increased familiar incidence and therefore the genetic background should be screened. Successful treatment of keloids is difficult. As contact cryotherapy and intralesional corticosteroid injections are in most cases not successful, the pulsed dye-laser seems to be a promising alternative. In addition topical treatment with extracts of onions, occlusive silicone sheets and pressure improves the clinical appearance of keloids.