PubMed Health⌕ Search

Biomedical subjects

Erhard Seifried

Publications and source records attributed to Erhard Seifried.

At least 19 recordsLinked to original sources
PubMed2006

Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells.

To explore the initial steps by which transplanted mesenchymal stem cells (MSCs) interact with the vessel wall in the course of extravasation, we studied binding of human MSCs to endothelial cells (ECs). In a parallel plate flow chamber, MSCs bound to human umbilical vein ECs (HUVECs) similar to peripheral-blood mononuclear cells (PBMCs) or CD34(+) hematopoietic progenitors at shear stresses of up to 2 dynes/cm(2). This involved rapid extension of podia, rolling, and subsequent firm adhesion that was increased when ECs were prestimulated with TNF-alpha. MSC binding was suppressed when ECs were pretreated with function-blocking anti-P-selectin antibody, and rolling of MSCs was induced on immobilized P-selectin, indicating that P-selectin was involved in this process. Preincubation of HUVECs with anti-VCAM-1 or of MSCs with anti-VLA-4 antibodies suppressed binding of MSCs to HUVECs but did not enhance inhibition by anti-P-selectin, indicating that both P-selectin and VCAM-1 are equally required for this process. Intravital microscopy demonstrated the capacity of MSCs to roll and adhere to postcapillary venules in vivo in a mouse model in a P-selectin-dependent manner. Thus, MSCs interact in a coordinated fashion with ECs under shear flow, engaging P-selectin and VCAM-1/VLA-4.

Animals↗
PubMed2006

Role of the monomeric GTPase Rho in hematopoietic progenitor cell migration and transplantation.

To investigate the role of the monomeric guanosine triphosphatase (GTPase) Rho on migration of hematopoietic progenitor cells (HPC), we employed different clostridial toxins which inhibit the Rho family of GTPases. Pretreatment with C2I-C3, a cell-accessible C3 transferase fusion protein that targets Rho, increased chemokinetic migration of the factor-dependent multipotent cell line Factor Dependent Cell Paterson with mixed lineage differentiation potential (FDCP-mix) and of primary lineage marker-depleted HPC in vitro. In contrast, treatment with lethal toxin (LT) from Clostridium sordellii, which predominantly inactivates Rac, and with toxin B from C. difficile, which inactivates Rho, Rac and Cdc42, decreased in vitro migration. When HPC pretreated with LT or toxin B were transplanted into mice, homing to the bone marrow was impaired, whereas C2I-C3 treatment did not alter HPC homing. However, in a competitive hematopoietic repopulation experiment in C57BL/6 mice, pretreatment of bone marrow cells with any of the inhibitors, including the Rho inhibitor C2I-C3, resulted in suppressed donor-type hematopoiesis. Our data indicate that whereas Rac supports HPC cell cycling, migration, short-term homing and hematopoietic regeneration, Rho coordinates down-regulation of HPC migration and is required for hematopoietic regeneration.

ADP Ribose Transferases↗
PubMed2006

Hepatitis B virus genotype G monoinfection and its transmission by blood components.

An acute hepatitis B virus (HBV) infection was diagnosed in a regular apheresis (plasma/platelet) donor by the hepatitis B surface antigen (HBsAg) assay and minipool nucleic acid amplification technology (NAT). The acute infection was confirmed by detection of anti-HBc (IgM) and anti-HBs 2 weeks later. The donor showed no clinical symptoms and had normal alanine aminotransferase levels. He had a history of weekly apheresis plasma or platelet donations. Archived material from the donor and the respective recipients was investigated by sensitive HBV NATs as part of a look-back procedure. HBV DNA was detectable in previous donations as well as in two recipients transfused with platelet concentrates. The rare HBV genotype G was identified in all HBV-DNA-positive samples. Strong evidence of genotype G monoinfection was obtained by clonal sequencing, HBV genotype line probe assay, genotype-specific NATs, and restriction pattern analysis. In contrast to previously described genotype G infections, which all appeared as coinfections with genotype A, neither the hepatitis B e antigen (HBeAg) nor anti-HBe was detectable in any of the samples. This shows that HBeAg is dispensable for viral replication. The delay in detecting HBsAg in both the donor and recipient samples may be explained by either decreased genotype G-specific synthesis of incomplete viral forms in early HBV infection or the lower sensitivity to genotype G of the current HBsAg assays. In conclusion, this reported case of an HBV infection was caused exclusively by genotype G.

Acute Disease↗
PubMed2006

Founder mutation Arg485Pro led to recurrent compound heterozygous GGCX genotypes in two German patients with VKCFD type 1.

Congenital combined deficiency of the vitamin-K-dependent coagulation factors (VKCFD) represents a rare autosomal recessive inherited bleeding disorder caused by mutations in either the gamma-glutamyl carboxylase gene (VKCFD type 1) or the vitamin K epoxide reductase gene (VKCFD type 2). Four different mutations of the gamma-glutamyl carboxylase gene (GGCX) have so far been reported in three unrelated patients with VKCFD type 1. Here we report on a fourth patient who presented with two compound heterozygous missense mutations of the GGCX gene, His404Pro and Arg485Pro. The His404Pro mutation has not been described previously, while the Arg485Pro mutation has been reported in another compound heterozygous VKCFD type 1 patient from Germany. Most interestingly, haplotype analysis revealed that Arg485Pro is due to a founder mutation, suggesting that this mutation is present in the German population at some low frequency. The founder mutation explains that the only two compound heterozygous VKCFD type 1 patients known today originated from Germany.

Adult↗
PubMed2006

FACS technology used in a new rapid bacterial detection method.

Culture methods for bacterial detection (BacT/ALERT or Pall eBDS) are currently implemented in blood donor screening procedures in many countries. Experience in the first years after implementation of these detection assays showed that although the analytical sensitivity was extremely high (about 1 CFU mL(-1)), the majority of these platelets were still transfused before a positive screening result was attained. Rapid technologies were developed to more effectively prevent transfusion-transmitted bacterial infection. In this study, a new rapid bacterial detection method based on fluorescence-activated cell sorting (FACS) technology was developed. Bacteria were stained with thiazole orange dye for 5 min and measurement was taken immediately after staining. The entire process took only 30 min. Six transfusion-relevant bacteria strains were tested in a spiking study. Without pre-incubation in a special bacteria growth medium, analytical sensitivity ranged between 10(5) CFU mL(-1) (Klebsiella oxytoca and Serratia marcesens) and 10(3) CFU mL(-1) (Escherichia coli). Sensitivity could be improved to 10(1) CFU mL(-1) for all tested bacteria by adding a pre-incubation step (6 h at 37 degrees C). Although preliminary in nature, results of our study suggest that bacterial detection by FACS technology in conjunction with a pre-incubation step offers a sensitive alternative technology to culture methods. Additionally, it provides the benefit of a rapid test time and the opportunity of preventing bacterial transmitted infections more effectively.

Bacteriological Techniques↗
PubMed2006

A comparison of three rapid bacterial detection methods under simulated real-life conditions.

BACKGROUND: Bacterial screening of all produced platelet concentrates (PCs) is implemented in many countries to reduce the risk of transfusion-transmitted sepsis. This study compares three rapid bacterial detection methods by imitating real-life conditions. STUDY DESIGN AND METHODS: The sensitivity of a solid-phase scanning cytometer (optimized Scansystem, Hemosystem), fluorescence-activated cell sorting (FACS) analysis, and 16S RNA in-house nucleic acid testing (NAT) was evaluated by spiking PCs with four transfusion relevant bacteria (Staphylococcus aureus, Bacillus cereus, Klebsiella pneumoniae, and Escherichia coli ). Two different inocula (10 colony-forming units [CFUs]/mL and 10 CFUs/bag) were used to simulate real-life conditions. Samples were taken at 12, 16, 20, and 24 hours after spiking. RESULTS: With the high inoculum, NAT had a 100 percent rate of positive testing for all four types of bacteria (10/10 replicates) at each time point. With the exception of E. coli, the sensitivity of FACS and optimized Scansystem was comparable for the high inoculum. With the low inoculum, 60 percent of E. coli, 80 percent of B. cereus, 90 percent of K. pneumoniae, and 100 percent of S. aureus were NAT-positive 12 hours after spiking. In contrast, only 20 percent of E. coli, 10 percent of B. cereus, and 70 percent of K. pneumoniae were FACS-positive with the low inoculum 12 hours after spiking. CONCLUSIONS: In summary, the preliminary data revealed a higher sensitivity for NAT in comparison to FACS and optimized Scansystem under the defined study conditions. To imitate real-life conditions, further spiking studies with a low inoculum (10 CFUs/bag) and slower growing organisms should be conducted to examine the sensitivity of available detection systems.

Bacterial Infections↗
PubMed2006

Detection of heterozygous large deletions in the antithrombin gene using multiplex polymerase chain reaction and denatured high performance liquid chromatography.

The present study reports a method for the easy, rapid and cost effective detection of heterozygous large deletions. As a model gene all exons of the antithrombin gene were amplified in a one tube multiplex polymerase chain reaction (PCR) and the products separated according to their size by reverse-phase ion-pair high performance liquid chromatography. A significant reduction in the height of a peak in the probandOs sample compared to in the control indicates the presence of a large deletion of the corresponding allele. Using this approach we identified heterozygous deletions in four patients: the deletions affected exons 1 and 2, exon 7 and the whole antithrombin gene.

Antithrombin III↗
PubMed2005

Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblastic leukemia.

OBJECTIVE: Natural killer (NK) cell-mediated cytotoxicity contributes to the innate immune response against numerous malignancies, including leukemias. Acute lymphoblastic leukemias (ALL) often display a high degree of resistance, the mechanisms of which have not been elucidated. METHODS: We used the well-characterized NK cell line NK-92 as a model to investigate whether mechanisms commonly implicated in tumor escape from NK cell killing are relevant for ALL. RESULTS: We demonstrate selective resistance of B-precursor ALL to NK-92 cytotoxicity even in the absence of inhibitory killer cell immunoglobulin-like receptors (KIR), except for KIR2DL4. We also show that human leukocyte antigen-G, a ligand of KIR2DL4, expressed on a subset of ALL, does not mediate resistance of NK-cell mediated lysis. Similarly, intracellular adhesion molecule/lymphocyte function-associated antigen-1 interaction did not contribute significantly to resistance. In contrast the NK-sensitive T-ALL (MOLT-4) expressed moderate amounts of MHC class I chain-related gene AB (MICA/B) a ligand for the NK cell activating receptor NKG2D, while expression of MICA/B was absent in resistant B-ALL cell lines. CONCLUSIONS: The NK cell-resistance of B-lineage ALLs does not appear to involve inhibitory mechanisms, but suggests deficient NK cell activation. Thus, immunostrategies designed to enhance ALL sensitivity toward NK cell-mediated cytotoxicity should focus on mechanisms of NK cell activation.

B-Lymphocytes↗
PubMed2005

Transplantation of human acute myeloid leukemia (AML) cells in immunodeficient mice reveals altered cell surface phenotypes and expression of human endothelial markers.

To better characterize acute myeloid leukemia (AML) development in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we transplanted samples from patients with AML or KG-1 and EOL-1 cell lines. We found 9/12 primary AML samples and both cell lines to engraft within 2-8 weeks, with 5-80% human cells in bone marrow. Compared with freshly isolated AML cells, percentages of human CD33+, CD38+, CD31+ CD13+ or CD15+ subpopulations increased after transplantation, whereas percentages of CD34+ cells decreased. Engrafted mice frequently showed expression of human endothelial cell markers. Thus, transplantation of human AML into NOD/SCID mice reveals expression of hematopoietic and endothelial differentiation markers.

Acute Disease↗
PubMed2005

Induction and detection of human mesenchymal stem cell migration in the 48-well reusable transwell assay.

The induction and detection of chemotactic migration in mesenchymal stem cells (MSC) are complicated by their adherent nature. We describe here the experimental details for inducing the transmigration of MSC seeded in microchemotaxis chambers and reliably identifying the translocated cells. A combination of 8-microm pore-sized membranes and a two-step staining procedure resulted in the detection of MSC migration against a gradient of human plasma which plateaued after 4 h. Microscopic enumeration of the transmigrated cells clearly distinguished contaminating nonmigrated cells from transmigrated cells. Chemokinetic and chemotactic effects could be separated and were influenced by precoated fibronectin or vitronectin. Thus, the technique described allows rapid and reliable induction and determination of migration in adherent MSC.

Bone Marrow Cells↗
PubMed2005

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.

Amino Acid Substitution↗
PubMed2005

Optimized Scansystem platelet kit for bacterial detection with enhanced sensitivity: detection within 24 h after spiking.

BACKGROUND AND OBJECTIVES: The prevention and detection of bacterial contamination of platelet concentrates remains a major challenge for transfusion medicine. To be suitable for blood-transfusion services, the contamination detection method must be highly sensitive, easy to perform and preferably of low cost. In this spiking study, we evaluated the new optimized Scansystem Platelet Kit detection method for use on apheresis platelets. STUDY DESIGN AND METHODS: Apheresis platelet concentrates (APCs) were individually spiked with 10 colony-forming units (CFU)/ml of one of 10 different strains of bacteria. The spiked APCs were analysed at specific time-points during incubation by using the optimized Scansystem Platelet Kit. Bacterial enumeration was performed by plating onto blood agar. RESULTS: All the bacterial strains tested were detected by using the optimized Scansystem Platelet Kit when sampled 24 h after spiking. Compared to the Scansystem standard kit, sensitivity was increased to < 50 CFU/ml. The identity of the spiked bacteria was confirmed by Gram staining and DNA fingerprinting. CONCLUSION: The optimized Scansystem Platelet Kit was able to reliably detect, within 70 min, 10 transfusion-relevant bacterial species in APCs when a sample volume was taken 24 h after spiking. This is the first study carried out by using the optimized Scansystem bacterial detection that was found to have an enhanced sensitivity compared to the standard kit.

Bacteria↗
PubMed2005

Nucleic acid test screening of blood donors for orthopoxviruses can potentially prevent dispersion of viral agents in case of bioterrorism.

BACKGROUND: Microbiologic agents such as variola virus (VAR) are very attractive for terrorism. As a result of international collaboration under the WHO eradication campaign, smallpox was declared eradicated in 1980. Therefore, the immunization programs were discontinued worldwide. Because most people are now immunologically naive, VAR is considered to be a potential threat agent or bioterrorist weapon. Real-time polymerase chain reaction (PCR) followed by melting analysis was developed for fast and safe analysis and allows differentiation of VAR from other orthopoxviruses (OPVs) like vaccinia or camelpox virus. STUDY DESIGN AND METHODS: A RealArt Orthopox LC PCR kit (Artus GmbH) was used to amplify OPV sequences from blood donor samples. A total of 31,500 blood donor samples were tested in minipools of up to 96 samples. To evaluate the sensitivity of the assay, routine donor minipools (90 +/- 6 samples per pool) were spiked with vaccinia virus used as positive control. RESULTS: Specificity was 100 percent because none of 31,500 blood donors was positive for the presence OPV. The detection limit of the assay was 10.6 copies per PCR procedure. Therefore, a sensitivity of 1590 copies per mL was calculated. Overall, 0.28 percent of test results had to be considered invalid owing to negative internal controls. CONCLUSION: The RealArt Orthopox LC PCR kit enables reliable detection of OPV DNA in viremic blood donor samples, even at the beginning of the disease when patients present minor clinical symptoms, and could be implemented in our routine screening procedure immediately. Thus, the assay could potentially help to prevent dispersion of viral agents by blood transfusion in case of bioterrorism.

Bioterrorism↗
PubMed2005

VKORC1 haplotypes and their impact on the inter-individual and inter-ethnical variability of oral anticoagulation.

In order to elucidate the role of VCORC1 sequence variants in warfarin sensitivity, we established a complete SNP map of the VKORC1 gene locus in 200 blood donors from Western Germany. Nearly all of the genetic variability of the VKORC1 gene in Europeans is reflected by three main haplotypes. Recently described polymorphisms associated with low warfarin dose requirement (dbSNP:rs9934438; dbSNP:rs17878363) were found in complete linkage disequilibrium with the VKORC1*2 haplotype. In two patient cohorts of European origin with either increased coumarin sensitivity (n= 14) or partial coumarin resistance (n=36) the VKORC1*2 frequency varied highly significant between the two groups and also when compared to 200 blood donor controls (coumarin sensitive 96%, coumarin resistant 7%, controls 42%) thus demonstrating a strong association between these two phenotypes and the VKORC1 haplotype (p = 1.6 x 10(-8) for coumarin sensitive and p = 1.9 x 10(-8) for coumarin resistant). Analysis of database derived VKORC1 genotypes of African Americans and Chinese revealed that haplotype frequencies in these populations differ significantly from the European sample (for VKORC1*2: Europeans 42%, Chinese 95%, African Americans 14%). These observations suggest VKORC1 as principal genetic modulator of the ethnic differences in warfarin response. Since hereditary pharmacodynamic (VKORC1) and pharmacokinetic (CYP2C9) factors account for up to 50% of the inter-individual variability of the warfarin response, these genetic markers may serve as clinically relevant predictors of warfarin dosing in future studies.

Administration, Oral↗
PubMed2005

Prolonged preterm rupture of fetal membranes, a consequence of an increased maternal anti-fetal T cell responsiveness.

A fetus, although semi-allogeneic, is usually accepted by the maternal immune system. However, complications, including alloresponsive mechanisms, are thought to be potentially detrimental for a successful pregnancy. Therefore, we compared allogeneic T cell responses of nonpregnant women with the response of healthy pregnant women and pregnant women who have various gestation-associated diseases. Peripheral blood mononuclear cells (PBMCs) of all three groups were stimulated with PBMCs from unrelated volunteers. Pregnant women had significantly reduced stimulation indices (SIs) compared with nonpregnant women. Exposing PBMCs from pregnant women to PBMCs of their own fetus led to a further significant decrease of SIs. Among the two groups of pregnant individuals, SIs of women with prolonged preterm rupture of fetal membranes (PPROM) were significantly higher when the maternal PBMCs were stimulated with PBMCs of their own fetus. This phenomenon could not be observed after stimulation with PBMCs from unrelated volunteers. In addition, an increased humoral immune response was assessed for women with PPROM in comparison with women with uncontrollable preterm labor. Our results revealed a strongly reduced allogeneic T cell response of PBMCs from pregnant women that was further down-regulated when PBMCs from their own fetus were used as stimulators. By contrast, data from women with PPROM suggest an increased maternal T cell response specifically toward the fetal HLA antigens.

Adult↗
PubMed2005

High-throughput purification of viral RNA based on novel aqueous chemistry for nucleic acid isolation.

BACKGROUND: Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive. METHODS: We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch. RESULTS: The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated. CONCLUSIONS: Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.

Autoanalysis↗
PubMed2005

Identification of 32 novel mutations in the factor VIII gene in Indian patients with hemophilia A.

Seventy-five unrelated hemophilia A patients from India were analyzed for factor VIII gene defects. Intron 22 inversion was identified in 22 patients and intron 1 inversion in 2 patients. In the remaining 51 patients without inversions screening the FVIII gene by denaturing high performance liquid chromatography (DHPLC) revealed 42 different mutations in 44 unrelated subjects. These included 14 missense, 7 nonsense, 9 splice site, 8 deletional, 3 insertional mutations and one indel mutation. Of these, 32 were novel gene alterations. The hotspots included intron 22 inversion, CpG and adenine runs.

Binding Sites↗