More than 150 novel variants of HLA class I genes detected in German Stem Cell Donor Registry and UCLA International Cell Exchange samples.

High throughput analysis using amplicon-based next-generation sequencing (NGS) of HLA class I genes in samples of registered stem cell donors of the German Stem Cell Donor Registry Düsseldorf revealed 151 novel variants. In addition, four new variants were identified in well-defined samples obtained from the UCLA International Cell Exchange program. New alleles included 37 HLA-A, 57 HLA-B, and 61 HLA-C variant alleles. All variants were confirmed by NGS of HLA-A, HLA-B, and HLA-C genes including the respective 5' and 3' untranslated regions as well as Sanger sequence analysis. Mainly, the variants encompass single nucleotide changes in intronic as well as exonic parts of the genes. We identified intronic variations in 114 new alleles, nonsynonymous nucleotide changes in 25 alleles, synonymous nucleotide changes in nine alleles, and three hybrid alleles. Four alleles carry exonic deletions or insertions resulting in frameshift of peptide translation. Two novel alleles of HLA-C were shown to result in splicing defects of the transcript. Two alleles showed exonic as well as intronic changes. Thirty-four of the new alleles were found in multiple samples.

Prospective, comprehensive, and effective viral monitoring in children undergoing allogeneic hematopoietic stem cell transplantation.

Major advances in the monitoring and treatment of viral infections after hematopoietic stem cell transplantation (HSCT) have been achieved over the last decade. The appropriate extent of viral monitoring and antiviral therapy remains controversial, and reports in pediatric patients receiving allogeneic unmanipulated hematopoietic stem cells (HSCs) are sparse. A total of 40 pediatric patients who underwent HSCT with either peripheral blood stem cells (PBSCs, n = 30) or bone marrow (BM; n = 10) were prospectively monitored every week for viral DNAemia (VDNA) by simultaneous detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), human adenovirus (ADV), and polyoma BK virus (BKV) using real-time TaqMan polymerase chain reaction (PCR). All patients received prophylactic acyclovir and preemptive ganciclovir (GCV) when 500 copies/microg DNA (EBV/HHV6) or >1 copy/microg DNA (CMV) were detected on 2 consecutive measurements. VDNA occurred in 25 of 40 recipients (CMV, 11/40 patients [28%]; EBV, 19/40 [48%]; HHV6, 2/40 [5%]; ADV/BKV, 1/40) and was found exclusively after neutrophil engraftment and in most cases up to day +100. Recurrent VDNA (P = .028) and (readily treatable) viral disease (P = .003) were observed predominantly in patients suffering from nonmalignant diseases, a cohort characterized by delayed lymphocyte engraftment. VDNA occurred more frequently in HLA-mismatched HSCT and in the 24 of 40 patients receiving antithymocyte globulin (ATG). The incidence of EBV, but not that of CMV, was increased in the ATG group. Yet, in these patients, viral loads of both EBV and CMV were higher, but with prompt initiation of preemptive GCV, no posttransplantation lymphoproliferative disorder or other life-threatening morbidities occurred. HHV6 was typically detected at low viral loads (<10(2) copies/microg DNA), with only 5% of HSC recipients fulfilling our HHV6 criteria for triggering GCV treatment. In multivariate analysis, ATG treatment, HLA mismatch, recipient CMV seropositivity, and stem cell source, but not severe acute graft-versus-host disease were identified as independent risk factors for VDNA. This comprehensive viral monitoring program with defined thresholds for initiation of preemptive GCV effectively prevents the development of critical viral disease, even in high-risk patients receiving ATG.

In vitro differentiation of human cord blood-derived unrestricted somatic stem cells towards an endodermal pathway.

BACKGROUND: Pluripotent unrestricted somatic stem cells (USSC) from UC blood can differentiate into hepatic cells in the in utero sheep model, resulting in 20% human albumin-producing parenchymal hepatic cells without cell fusion or tumor-formation events. Additionally, we have shown in vitro differentiation of USSC by hepatocyte growth factor and oncostatin M induction, causing changes in the gene expression towards the endodermal lineage. Positive glycogen synthase expression and a positive periodic acid-schiff reaction demonstrated a functional production of polysaccharides in the cells. METHODS: We describe the in vitro differentiation of USSC towards an endodermal pathway using different matrices, growth factors and organic substances. Also, co-cultures of USSC with primary cells of endodermal tissue were prepared to mimic the biologic niche. We investigated the effect of direct co-culture of USSC with primary rat hepatocytes or with sheep tissue of endodermal origin. Direct co-cultures were set up to ensure cell-cell contacts. For co-cultures without cell-cell contacts, transwell inlays with 1-microm membranes were used to separate the cells. Furthermore, the effect of endodermally conditioned medium was investigated. Changes in the gene expression patterns were analyzed by RT-PCR. RESULTS: We have shown that USSC can differentiate in vitro into an endodermal-like cell with a phenotype similar to hepatic cells. Differentiation of USSC with growth factors, retinoic acid, matrigel matrix and different co-cultures led to an increased expression of albumin and also to the detection of GSC, SOX 17, Cyp2B6, Cyp3A4, Gys2, HNF4a, ISL-1 and Nkx6.1. In addition, functional albumin secretion was observed. DISCUSSION: Although the differentiation assays demonstrated here produce only an immature hepatocyte-like cell, endodermaly differentiated USSC might be a useful alternative for cell replacement in the future.

High-resolution HLA typing by sequencing for HLA-A, -B, -C, -DR, -DQ in 122 unrelated cord blood/patient pair transplants hardly improves long-term clinical outcome.

To determine the impact of high-resolution (HR) HLA typing with outcomes after UCBT, DNAs of 122 pairs were analysed for HLA class I and class II mismatches (MM) based on HR typing. For HLA-A, -B on low-resolution typing and -DRB1 on HR typing, the following MM situation resulted: no MM (13%), one MM (40%), two MM (36%), three MM (8%), four MM (3%). For A, B, C, DR and DQ based on HR typing the following MM occurred: No MM (4%), one MM (10%), two MM (15%), three MM (22%), four MM (25%), five MM (12%), six MM (6%), seven MM (3%), eight MM (2%). There was no significant association between number of MM (HR) for both HLA-A, -B and -DRB1 and HLA-A, -B, -C, -DRB1 and DQB1 and aGvHD grade II-IV. There was a trend that MM in class I HR were associated with neutrophil recovery; HLA-A locus typing analysed in HvG direction was associated with reduced cumulative incidence of engraftment (P=0.04), the same for C-KIR in HvG direction (P=0.01). No significant correlation was found between numbers of HLA-MM on the HR level with 2-year survival. The analysis shows that the degree of mismatching in UCBT is even higher than expected.

Similar survival following HLA-identical sibling transplantation for standard indication in children with haematologic malignancies: a single center comparison of mobilized peripheral blood stem cell with bone marrow transplantation.

BACKGROUND: Peripheral blood stem cell (PBSC) grafts are increasingly used for autologous and allogeneic haematopoietic stem cell transplantation (alloHSCT) with the aim to hasten neutrophil and platelet engraftment and thereby to reduce transplant-related complications due to infections, bleeding and graft failure. Based on the paucity of data on PBSC transplantation in children we performed a retrospective single-center analysis comparing the outcome of children receiving mobilized PBSC from human leukocyte antigen (HLA)-identical sibling donors to bone marrow (BM) transplant recipients. PATIENTS AND METHODS: Between 1996 and 2004, 16 children with haematologic malignancies and standard indication for alloHSCT underwent PBSC transplantation from HLA-identical sibling donors. The outcome of these children was compared to a historic control group of 19 bone marrow (BM) transplant recipients. Time to neutrophil engraftment, incidence of acute and chronic graft-versus-host disease (GvHD), relapse rate, transplant-related mortality, event-free and overall survival were analyzed. RESULTS: Neutrophil engraftment was achieved significantly faster after PBSC compared to BM transplantation with a median time to neutrophil engraftment of 11 (range: 8-21) and 19 (16-44) days for the PBSC and BM cohort, respectively (p < 0.001). Two of 19 (11 %) BM recipients did not achieve primary neutrophil engraftment and both patients died due to infectious complications. The rate of clinically significant acute GvHD > or = grade II was higher in the PBSC compared to the BM group (75 vs. 39 %; p = 0.045). Incidences of chronic GvHD (PBSC vs. BM: 60 vs. 44 %), death of disease (13 vs. 21 %) and death of complication (13 vs. 16 %) were comparable between both groups (p = ns). With a median follow up of 4.7 years (PBSC) and 10.2 years (BM) overall survival (PBSC vs. BM: 68.6 +/- 13.5 vs. 63.2 +/- 11.1 %; p = 0.65) and event-free survival (67.0 +/- 12.1 vs. 63.2 +/- 11.1 %; p = 0.80) is without demonstrable difference in both groups. CONCLUSIONS: Transplantation of PBSC compared to BM is associated with faster neutrophil engraftment and a higher rate of > or = grade II acute GvHD. As overall survival and event-free survival is similar when using PBSC and BM, PBSC is an alternative stem cell source for HLA-identical sibling transplantation. Further prospective analyses with higher number of patients stratified according to well established risk factors are required to define the precise role of both stem cell sources for children with haematologic malignancies.

[Long-term results of homologous penetrating limbokeratoplasty in total limbal stem cell insufficiency after chemical/thermal burns]. / Langzeitergebnisse der homologen perforierenden Limbokeratoplastik bei totaler Limbusstammzellinsuffizienz nach Verätzungen/Verbrennungen.

BACKGROUND: Since 1991 homologous penetrating limbokeratoplasty has been performed in 32 patients with severe limbal stem cell insufficiency following chemical/thermal burns. The long-term results considering the effects of HLA matching are presented for the first time. PATIENTS: All patients received systemic cyclosporin A and/or mycophenolate mofetil in the postoperative course. In 9 patients grafts with 0-1 HLA mismatches in the HLA A, B and DR loci, in 6 patients grafts with 2-6 mismatches and in 17 patients untyped grafts were used. Long-term clear graft survival was estimated according to Kaplan and Meier. RESULTS: Five years postoperatively, 50% of the grafts with 0-1 mismatches, 32% of the grafts with 2-6 mismatches and 18% of the untyped grafts were centrally clear (log-rank-test, p>0.05). CONCLUSIONS: Although statistically not significant, HLA matched grafts seem to deliver better results than untyped grafts in penetrating limbokeratoplasty. Improvement of matching strategies and immunosuppression may possibly further improve current results.

Improvement of graft prognosis in penetrating normal-risk keratoplasty by HLA class I and II matching.

BACKGROUND: Owing to contradictory results, HLA matching in penetrating keratoplasty still is equivocal. Different surgical techniques in multicentre studies, missing risk differentiation in high-risk situations, and faulty HLA typing can be identified as main reasons for these contradictory results. In this monocentre study, the value of HLA class I and II matching (A, B, DR loci) was examined in a homogeneous group of 418 normal-risk keratoplasty patients using serological typing techniques for HLA class I and immunogenetic typing techniques for class II. METHODS: Penetrating normal-risk keratoplasty was performed in two groups of patients (group I with 0-2, group II with 3-6 mismatches in the A/B/DR loci). All surgery was carried out by three experienced surgeons according to a standardized scheme. Furthermore, postoperative therapy and controls were standardized. There were no statistically significant differences between the two study groups with regard to the number of ABO or H-Y compatibilities, patient age, patient gender, ratio of previous intraocular surgery, ratio of triple procedures, indication for surgery, follow-up period, donor age, donor gender, post-mortem time of the graft, and endothelial cell density of the graft at the end of organ culture. All HLA typing was performed in a quality-controlled laboratory, serologically for HLA class I (A and B loci) and immunogenetically for HLA class II (DR locus). RESULTS: At 4 years postoperatively, the ratio of clear and rejection-free graft survival was 92% in group I and 66% in group II (Kaplan-Meier estimation, log rank test, P=0.03). Monovariate analysis in the Cox model gave no influence of solitary HLA class I or II matching, but only an influence of combined HLA class I and II matching (P=0.03). CONCLUSIONS: In this monocentre study with proper typing techniques, the beneficial effect of HLA class I plus II matching on clear and rejection-free graft survival could be demonstrated in a homogeneous group of normal-risk keratoplasty patients.

Hematopoietic stem cell donor registry strategies for assigning search determinants and matching relationships.

Registries and cord blood banks around the world collect and store the HLA types of volunteers in order to identify matched unrelated donors for patients requiring hematopoietic stem cell transplantation. This task is complicated by the many formats in which HLA types are provided by the testing laboratories (types obtained by serology vs by DNA-based methods; high vs intermediate vs low resolution) and by the need to identify which of these diverse types are most likely to match the HLA assignments of a searching patient as closely as possible. Conversion of the assignments to 'search determinants' may be included within the algorithm used to select and prioritize a list of potentially suitable donors, either as an aid to matching or as a tool to optimize the performance of comparisons within large data files. The strategies used by registries to create search determinants are described. A set of search determinants, utilized by the National Marrow Donor Program, is provided as an example and is intended to initiate further discussion aimed at understanding the process used by each registry with the possibility of developing a standard process among registries worldwide.

Individual analysis of expected time on the waiting list for HLA-matched corneal grafts.

OBJECTIVE: Recent studies report the beneficial effect of HLA matching for long-term prognosis of penetrating keratoplasty (kp). This improvement of prognosis, however, has to be weighed against the additional time on the waiting list due to the search for a HLA-compatible graft. Reliable estimation of this additional waiting period is a prerequisite for informed consent on the waiting policy. METHODS: A mathematical model based on survival analysis and HLA haplotype frequencies was used to estimate time on the waiting list for each of 1,400 HLA-typed patients registered at the Lions Cornea Bank NRW. Additionally, the waiting period of each patient was retrospectively determined. Both values were tested for correlation. This analysis was performed for acceptance of up to two mismatches on HLA-A, -B and -DR. RESULTS: When accepting two, one and zero mismatches, median predicted waiting period was 1 +/- 6, 7 +/- 49 and 17 +/- 159 months respectively. Median waiting period in retrospective simulation was 1 +/- 3, 5 +/- 9 and 15 +/- 14 months. Correlation of values from the predictive formula and simulation was statistically significant (p < 0.0001). CONCLUSION: Predicted time on the waiting list is a valuable tool for management of HLA matching in kp.

Predicting time on the waiting list for HLA matched corneal grafts.

Recent studies report the beneficial effect of HLA matching for the long-term prognosis of penetrating keratoplasty (KP). This improvement in prognosis, however, has to be weighed against the additional waiting time while a HLA compatible graft is found. Precise estimation of this additional waiting period is a prerequisite for informed consent on the waiting policy. A mathematical model based on survival analysis and HLA haplotype frequencies was used to estimate time on the waiting list for each of 1400 HLA typed patients registered at the Lions Cornea Bank North Rhine Westfalia (NRW). Additionally, the waiting period for each patient was retrospectively determined. Both values were tested for correlation. This analysis was performed for acceptance of up to two mismatches on HLA-A, -B and -DR. The median predicted waiting period was compatible with the median waiting period in retrospective simulation. Correlation of both entities was statistically significant also. Predicted time on the waiting list as derived from the patient's HLA genotype and a comprehensive database of HLA haplotype frequencies is thus a valuable tool for management of HLA-matching in KP.

[DNA PCR typing of non-human primates]. / DNA-PCR-Typisierung an nicht-menschlichen Primaten.

Human beings and non human primates show similarities in the non coding DNA range too, but up to now there are only a few data. This paper presents first results of a study dealing with a larger spectrum of species and individuals, considering the genetic marker HLA-DQA1, LDLR, GYPA, HBGG, D7S8, GC (partionally coding) and VWA, FES, F13B, TH01, CD4, FGA (not coding). The results show that not only the apes can be typed but also Macaca sylvanus as a member of the Cercopithecoidea. In contrast to earlier publications there is an unexpected larger similarity between the allele ranges of the apes studied and those of human beings.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Novel mutations of the von hippel-lindau tumor-suppressor gene and rare DNA hypermethylation in renal-cell carcinoma cell lines of the clear-cell type.

Renal-cell carcinoma (RCC) is the most common neoplasm of the kidney, accounting for about 3% of all adult malignancies. Histopathologically, 80% of all cases can be classified as clear-cell RCC. Of these, approximately 55% to 70% are associated with mutations in the von Hippel-Lindau (VHL) tumor-suppressor gene. Here, new mutations of the VHL gene were defined by the use of temperature gradient gel electrophoresis and subsequent sequencing. In addition, DNA hypermethylation, an alternative mechanism of VHL gene silencing, was evaluated by methylation-specific PCR. Twenty-six clear-cell, 3 chromophilic, and 2 chromophobic RCC cell lines were analyzed. Among the clear-cell RCC cell lines tested, 12 (47%) contained 13 mutations overall: 8 (62%) in exon 1, 3 (23%) in exon 2, and 2 (15%) in exon 3. Ten of these mutations have thus far not been described. All single base pair changes were transversions. Six mutations led to alteration of a single amino acid. Seven mutations generated a frameshift or a stop codon. One cell line contained a complex duplication of 36 bp. All cell lines with mutations showed loss of heterozygosity in the VHL gene. No mutations could be detected in the chromophilic or chromophobic RCC samples. Significant hypermethylation was not observed in any of the cell lines. These data provide further evidence that distinct mutations in the VHL gene are a characteristic feature of clear-cell RCC. In contrast, hypermethylation of the gene is probably a rare event. The high frequency of transversion mutations suggests a role for exogenous carcinogens in the etiology of clear-cell RCCs.

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German caucasoid patients and their corresponding 821 potential unrelated stem cell transplants.

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.

Donor selection process for allogeneic hematopoietic stem cell transplantation at the university hospital of Düsseldorf (1997-1998).

BACKGROUND: Transplantation of hematopoietic stem cells (HSC) is an effective treatment for a number of patients with life-threatening hematologic diseases. HSC donors can be found in the family of the patient or in registries of unrelated donors. In the present study, the search procedure within the last two years for an allogeneic HSC donor at the University of Düsseldorf is analyzed. PATIENTS AND METHODS: During 1997 and 1998, an early search for a related HSC donor in the family was performed for 70 high risk pediatric patients. During the same period, the search for an unrelated HSC donor for 116 adult and pediatric patients was performed. Low resolution HLA-A and -B typing was performed by serology in combination with DNA-typing. High resolution typing of HLA-A, -B and -C was carried out by DNA-sequencing. Low resolution HLA-DRB- und HLA-DQB1-typing was done solely by DNA-typing and high resolution typing of these genes was performed by DNA-sequencing. MAIN RESULTS: For 51 of 70 high risk pediatric patients (73%), no family donor could be defined, 16 of 70 patients (23%) had a genotypically identical sibling and for three of 70 patients (4%) an HLA-acceptable donor in the extended family could be identified. The search for an unrelated HSC donor was successful in 74% of the adult and pediatric patients lacking such a family donor. Most noteworthy, nearly all of the HLA-acceptable donors were identified from that group of donors in the registries, which were not only HLA-A and HLA-B, but also HLA-DR pretyped. CONCLUSION: These data show, that a growing number of pediatric patients with high risk leukemia need an unrelated HSC donor and that HLA-ABDR-pretyped registries present the optimal prerequisite to identify an HSC donor for most of the patients. In addition, 25% of the patients with no family or unrelated HSC donor require HSC transplants from alternative donors like unrelated Cord Blood (CB) from high quality cord blood banks.

Establishment of experimental conditions for the rapid detection of mutations in the von Hippel-Lindau gene by parallel temperature gradient gel electrophoresis.

Renal cell carcinoma (RCC) is the most common adult kidney neoplasm and is present in approximately 3% of all adult malignancies. Mutations of the von Hippel-Lindau (VHL) gene had been found in 55-70% of sporadic RCC and appear to be a critical event in the pathogenesis of clear-cell RCC. Several screening procedures for the detection of mutations in the VHL gene such as single-strand conformation polymorphism analysis, Southern blot and polymerase chain reaction (PCR)-employing methods had been used. Recently, the perpendicular temperature gradient gel electrophoresis (TGGE) was successfully applied to detect genetic alterations in the VHL gene. Here, the experimental conditions for parallel TGGE were established. The results on eleven samples with mutations in different exons demonstrate that parallel TGGE can be used as a simple and rapid method to detect mutations in the VHL gene. Several samples can be run at the same time, making parallel TGGE ideal for the screening of a large number of samples.

Rapid method for successful HLA class I and II typing from cadaveric blood for direct matching in cornea transplantation.

BACKGROUND: The aim of the study was to establish fast methods for postmortem HLA class I and II typing of cornea donors using cadaveric blood. METHODS: The commercially available reagents Lymphokwik MN and Dynabeads were evaluated here to provide an enriched living mononuclear cell (MNC) population and B-cell population for HLA class I and II typing of cadaveric blood by serology. Cadaveric blood was obtained 1-80 h post mortem. After isolation of living B-cells and B-cell-depleted living MNC's, cells were serologically typed by double-fluorescence cytotoxicity assay for HLA class I and II antigens. RESULTS: In 373 (81%) of 461 cadaveric blood samples HLA class I typing, and in 36 (62%) of 56 cadaveric blood samples HLA-class II typing, by serology was successful and accomplished within 5 h. Results from the serological HLA class I typing were confirmed by the results of HLA class I typing by RNA-based sequencing in seven cases. To improve the HLA class II typing, DNA typing using PCR with sequence-specific primers was performed in 148 samples and reverse hybridization of PCR-amplified DNA to immobilized HLA class II specific primers in 270 samples. These data were confirmed by DNA-based sequencing in five cases and by sequence-specific oligonucleotide hybridization in all cases. CONCLUSIONS: These results lead to the following typing strategy: HLA class I typing should be performed by serology. HLA class II typing should be performed by DNA technology because of its relative independence of the quality of the blood sample. The strategy we have developed is very successful and fast for tissue typing post mortem, thus expanding the time available for ideal HLA matching, increasing the number of available HLA-matched corneas and therefore reducing the number of graft rejections.

German consensus on immunogenetic donor search for transplantation of allogeneic bone marrow and peripheral blood stem cells.

In Germany allotransplantation of bone marrow or peripheral blood stem cells is presently performed by 34 different teams operating more or less independently. Thus, strategies of immunogenetic donor search, use of the various tissue typing techniques and policy on acceptable HLA mismatches in related and unrelated settings may vary considerably from one transplant centre to another. This paper summarises the results of the first German consensus meeting on immunogenetic donor search for bone marrow/peripheral blood stem cell grafting. The main goal of the participating transplant physicians and immunogeneticists was to define national standards for the above issues.

Differential transfection efficiency rates of the GM-CSF gene into human renal cell carcinoma lines by lipofection.

One of the major questions in any gene therapy approach is the selection of the appropriate vector system. Here, the optimization of a gene transfer protocol for renal cell carcinoma using lipofection as a nonviral gene transduction system was evaluated. To select the promoter which gives the highest expression, different plasmids which are able to express Escherichia coli beta-galactosidase gene as a reporter gene under the control of different promoters were tested: human cytomegalovirus promoter (pCMVbeta), simian virus 40 promoter (pSVbeta), adenovirus promoter (ADbeta), and herpes simplex virus thymidine kinase promoter (TKbeta). The pCMVbeta revealed the highest expression of the beta-gal gene in the renal cell carcinoma (RCC) lines. Thus this CMV promoter was selected for the expression of the granulocyte-macrophage colony stimulator factor (GM-CSF) gene. Three different lipids (LipofectAmine, LipofectAce, and Lipofectin) were compared for their transduction efficiency, and the optimal conditions for quantitatively high lipofection rates were established. The consistently best results regarding gene expression as well as viability of the RCC lines were obtained when Lipofectin was used. Gene expression was monitored by a specific enzyme-linked immunosorbent assay and functionally validated by a cell proliferation test. The GM-CSF expression profile showed a peak at 48 hours after transfection and was still detectable after 5 days. Here the feasibility of efficient lipofection of the GM-CSF gene into RCC lines is demonstrated. Most importantly, considerable differences in the relative quantity of GM-CSF gene transfer into the different RCC lines was observed here. This may be of critical relevance for the design of any clinical gene transduction protocol in tumor cell vaccination attempts.