Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array.

Monoclonal antibody F77 was previously raised against human prostate cancer cells and has been shown to recognize a carbohydrate antigen, but the carbohydrate sequence of the antigen was elusive. Here, we make multifaceted approaches to characterize F77 antigen, including binding analyses with the glycolipid extract of the prostate cancer cell line PC3, microarrays with sequence-defined glycan probes, and designer arrays from the O-glycome of an antigen-positive mucin, in conjunction with mass spectrometry. Our results reveal F77 antigen to be expressed on blood group H on a 6-linked branch of a poly-N-acetyllactosamine backbone. We show that mAb F77 can also bind to blood group A and B analogs but with lower intensities. We propose that the close association of F77 antigen with prostate cancers is a consequence of increased blood group H expression together with up-regulated branching enzymes. This is in contrast to other epithelial cancers that have up-regulated branching enzymes but diminished expression of H antigen. With knowledge of the structure and prevalence of F77 antigen in prostate cancer, the way is open to explore rationally its application as a biomarker to detect F77-positive circulating prostate cancer-derived glycoproteins and tumor cells.

A novel internally controlled real-time reverse transcription-PCR assay for HIV-1 RNA targeting the pol integrase genomic region.

Given the worldwide increasing spread of HIV-1 genetic variants, it is mandatory that assays used for nucleic acid testing for HIV-1 detect all existing groups and subtypes of HIV-1. In this report the development and evaluation of a quantitative real-time HIV-1 RT-PCR assay that targets a conserved region within the pol integrase domain is described. As an internal control reaction, endogenous glyceraldehyde-3-phosphate-dehydrogenase transcripts were detected in a multiplex configuration. The detection limit (95% cut-off value) was determined by probit analysis and calculated as 281 IU/ml of HIV-1 RNA. Within-run and between-run coefficients of variation were below 15 and 27%, respectively, indicating high reproducibility. The described assay detected all tested HIV-1 isolates representing groups M, O and N. Within group M, quantitative test results correlated well with viral loads as determined by the automated Abbott RealTime HIV-1 assay. Based on the testing of 1206 confirmed HIV-1 RNA negative blood donor samples, assay specificity was found to be 100%. The rate of inhibition was 0.37%. The described HIV-1 real-time RT-PCR was validated according to regulatory guidelines and is applicable to the screening of blood donors as well as the determination of HIV-1 viral load.

Lack of F8 mRNA: a novel mechanism leading to hemophilia A.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8 gene was detected. Using 2 common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription-polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARA locus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, which points to a novel mechanism leading to HA.

Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA.

Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.

Perioperative monitoring of primary and secondary hemostasis in coronary artery bypass grafting.

On-pump cardiac surgery is accompanied by complex alterations of hemostasis. The excessive postoperative bleeding has been attributed to acquired platelet dysfunction, impaired plasmatic coagulation, and increased fibrinolysis. The characterization of the hemostatic defects responsible for bleeding is crucial for specific treatment and optimal clinical management of the patient. For rapid determination of platelet-dependent primary hemostatic capacity (PHC), the Platelet Function Analyzer PFA-100 system is available. To evaluate the PFA performance in perioperative monitoring, a study was performed in 49 patients selected for low bleeding risk undergoing selective primary coronary artery bypass grafting (CABG). We compared PHC with Simplate bleeding time (BT) and platelet aggregometry. Furthermore, we analyzed global hemostasis by thromboelastography (TEG) and plasmatic coagulation by standard clotting tests prothrombin time (PT, Quick), activated partial thromboplastin time (aPTT), thrombin time (TT) and clotting factors and fibrinolysis by batroxobin (reptilase) time (RT). In all patients BT was postoperatively increased by 1.5- to 2-fold irrespective of perioperative complications and decreased to mildly prolonged values on the first postoperative day (1st day). In patients without complications, PHC in both collagen-adenosine diphosphate closure time (CADP-CT: 83 seconds preop, 78 seconds postop, and 74 seconds 1st day) and collagen-epinephrine closure time (CEPI-CT: 98 seconds preop, 95 seconds postop, 85 seconds 1st day) remained nearly stable. Apart from a patient with postoperative moderate thrombocytopenia, in bleeding patients no other significant defect of postoperative platelet hemostatic capacity was observed. However, on 1st day, the PHC of those patients was significantly reduced compared with non-bleeding patients. In patients with postoperative myocardial ischemia, increased PHC was identified by significantly shorter postoperative CADP-CT (66 seconds vs. 83 seconds) than in uncomplicated patients. By aggregometry, partial platelet dysfunction was observed in some patients without correlation to bleeding complications. In seven of 9 patients the postoperative bleeding complication was attributed to prolonged heparin anticoagulation and/or mildly enhanced fibrinogenolysis/fibrinolysis by TEG and standard plasmatic coagulation tests (TEG: k time 18 minutes vs. 8 minutes; aPTT: 47 seconds vs. 32 seconds; TT: 18.0 seconds vs. 12.3 seconds) and (RT: 19.5 seconds vs. 17.7 seconds). The impairment of PHC, platelet aggregation, and clotting factors observed on the 1st day in bleeding and in intra-aortic balloon pump (IABP) patients are most likely secondary effects, for example, loss of active platelets and clotting factors, to the primary postoperative bleeding or implantation of the IABP. In conclusion, our data indicate that in standard CABG procedures highly variable alterations of the hemostatic system occur after cardiopulmonary bypass (CPB) even in patients with assumed low operative risks. For identification of post-CPB bleeding complications, thromboelastography, aPTT, and TT and heparin and batroxobin (reptilase) time as fibrinolysis-sensitive assays are useful. Platelet function appears to be rapidly restored in uncomplicated CABG. PHC determination by PFA-100 demonstrates a high specificity for adequate platelet function and, therefore, could be beneficial in improved transfusion of platelet concentrates. PHC testing by PFA-100 may help identify postoperative platelet hyper-reactivity associated with myocardial lesion.

gammaAla82Gly represents a common fibrinogen gamma-chain variant in Caucasians.

Screening of 200 blood donors for the presence of polymorphisms in three fibrinogen genes (FGA, FGB, FGG), revealed two individuals with a heterozygous missense mutation (c.323C > G, gammaAla82Gly) in the FGG gene. This mutation has been reported previously to cause mild hypofibrinogenaemia. Analysis of an additional 416 blood donors showed two more heterozygous gammaAla82Gly mutations, resulting in an overall gammaAla82Gly allele frequency of 0.0032. Haplotype analysis demonstrated that the gammaAla82Gly mutation originated from a common founder. From these data we estimated that homozygous individuals for gammaAla82Gly should occur at a frequency of 1: 95 000, suggesting that hypofibrinogenaemia represents a more frequent condition in the population than so far believed.

Gene expression analysis in platelets from a single donor: evaluation of a PCR-based amplification technique.

BACKGROUND: Genetic analysis of platelet mRNA may facilitate the diagnosis of disorders affecting the megakaryocytic-platelet lineage. Its use, however, is limited by the exceptionally small yield of platelet mRNA and the risk of leukocyte contamination during platelet preparation. METHODS: We depleted platelet suspensions of leukocytes by filtration and used a PCR-based RNA amplification step [switching mechanism at the 5' end of RNA templates (SMART)]. We tested the reliability and precision of the RNA amplification procedure by use of real-time PCR to measure quantities of specific transcripts: von Willebrand factor (vWF), A-subunit of coagulation factor XIII (F13A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Microarray analysis was performed on platelet RNA with and without amplification. RESULTS: Microgram quantities of platelet-specific cDNAs were produced from as little as 50 ng of total platelet RNA or 40 mL of whole blood. At cycle numbers <16, amplification of all transcripts tested was exponential with slightly more efficient amplification of low-abundance transcripts. Expression profiling of 9850 genes gave identical results for 9815 genes (1576 positive/8239 negative). Eight transcripts failed to be amplified by the SMART procedure. Expression of vWF, F13A, and GAPDH transcripts showed only minor day-to-day variations in three healthy individuals. CONCLUSION: The proposed protocol makes extremely small amounts of platelet RNA available for gene expression analysis in single patients.

Regulation of human factor IX expression using doxycycline-inducible gene expression system.

Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovectors: a response vector expressing hFIX (Ad5.TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5.CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5.TRE.hFIX in all cell lines in combination with Ad5.CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5.CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5.CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.

Allogeneic dendritic cells fused with tumor cells: preclinical results and outcome of a clinical phase I/II trial in patients with metastatic renal cell carcinoma.

Therapeutic vaccination with dendritic cells (DC) can lead to tumor regression in animal models and has shown promising results in the first clinical trials of metastatic renal cell carcinoma and malignant melanoma. In vitro data and results of a clinical phase I/II trial using DC tumor fusions in patients with progressive metastatic renal cell carcinoma are presented here. In addition to toxicity and feasibility, complex immune monitoring was a point of interest. DC precursor cells were obtained from the peripheral blood mononuclear cells (PBMCs) of healthy donors and were fused with either allogeneic (8 patients) or autologous (4 patients) renal tumor cells. In total, 12 patients with progressive metastatic renal cell carcinoma were treated with an average of 2.8 x 10(7) tumor cells fused with 1.8 x 10(7) DC each administered on days 0, 28, and 56 intradermally. Fusion efficacy for the tumor cells used was 14.3% +/- 7.8%. Cell viability was 59.8% +/- 6.8% after fusion and irradiation. We observed no adverse effects and no difference in clinical outcome between the allogeneic and the autologous treatment. Eight patients remained in a progressive disease state and four patients in a stable disease state. T-cell immunity was carefully monitored before, during, and after treatment. Delayed-type hypersensitivity (DTH) reaction using tumor cells was positive after treatment in 7 of 12 patients, 2 of whom were found to have stable disease. An increase in the reactivity against recall antigens was seen in most patients. Interestingly, cytotoxicity of peripheral blood lymphocytes (PBLs) against renal cell carcinoma cells increased during treatment as well as the percentage of interferon-gamma-secreting cells. This effect was significantly enhanced within the group that had stable disease. The lack of adverse effects together with positive immunologic signs justifies further investigation of this novel therapeutic approach. Further studies are necessary to test for clinical effectiveness in patients with tumors, especially those with less advanced disease.

Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcome of a first clinical phase I/II trial.

In this study we have presented in vitro data and results of a preliminary clinical trial using dendritic cells (DC) in patients with progressive metastatic renal cell carcinoma. DC precursor cells were obtained from peripheral blood mononuclear cells (PBMC). DC were pulsed with autologous tumor cell lysate if available. In total, 15 patients were treated with a median of 3.95 x 10(6) DC administered and ultrasound-guided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease (PD), 7 patients showed stable disease (SD), and one patient displayed a partial response (PR). Most interestingly, the patient who was treated with the highest number of DC (14.4 x 10(6) DC/vaccine) displayed a PR. Delayed-type hypersensitivity (DTH) reaction using autologous tumor lysate was positive in 3 out of 13 patients, including the patient with PR. Two out of 3 patients receiving additional treatment with keyhole limpet hemocyanin (KLH) showed reactivity to KLH after vaccination. CD3+CD4+ and CD3+CD28+ cells as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy. In conclusion, our observations confirm the capability of tumor-lysate pulsed autologous DC vaccines to stimulate an immune response in patients with metastatic renal cell carcinoma even in the presence of a large tumor burden. The lack of adverse effects together with immunologic effects support further investigation of this novel therapeutic approach. Further studies are necessary to demonstrate clinical effectiveness in cancer patients, in particular in patients with less advanced disease.