Biomedical subjects

Peter Wernet

Publications and source records attributed to Peter Wernet.

35 records · Page 2Linked to original sources

PubMed2004

A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential.

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

Adipocytes↗

PubMed2004

Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells.

During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory responses, the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34(+) hematopoietic cells (1) acquire a polarized cell shape upon cultivation, with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement, which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore, polarization of CD34(+) cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together, our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms.

AC133 Antigen↗

PubMed2004

The BCR-ABL1 kinase bypasses selection for the expression of a pre-B cell receptor in pre-B acute lymphoblastic leukemia cells.

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of human pre-B cells. Comparing genome-wide gene expression profiles of BCR-ABL1+ pre-B ALL and normal bone marrow pre-B cells by serial analysis of gene expression, many genes involved in pre-B cell receptor signaling are silenced in the leukemia cells. Although normal pre-B cells are selected for the expression of a functional pre-B cell receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region gene are unresponsive to pre-B cell receptor engagement and exhibit autonomous oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and differentiate into immature B cells expressing immunoglobulin light chains. BCR-ABL1 kinase activity is linked to defective pre-B cell receptor signaling and the expression of a truncated isoform of the pre-B cell receptor-associated linker molecule SLP65. Also in primary leukemia cells, truncated SLP65 is expressed before but not after treatment of the patients with STI571. We conclude that inhibition of BCR-ABL1 reconstitutes selection for leukemia cells expressing a functional (pre-) B cell receptor.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Long-term results of allogeneic penetrating limbo-keratoplasty in total limbal stem cell deficiency.

OBJECTIVE: To determine the prognosis of allogeneic penetrating limbo-keratoplasty in patients with total limbal stem cell deficiency and to find out if donor limbal stem cells survive in the long run. DESIGN: Noncomparative prospective case series. PARTICIPANTS: Forty-eight patients with total limbal stem cell deficiency. INTERVENTION: Allogeneic penetrating limbo-keratoplasty. All patients received systemic cyclosporin A and/or mycophenolate mofetil in the postoperative course. Thirteen patients received grafts with 0 to 1 HLA mismatches in the HLA-A, HLA-B, and HLA-DR loci; 13 patients received grafts with 2 to 6 mismatches; and 22 patients received untyped grafts. MAIN OUTCOME MEASURES: Long-term clear graft survival and survival of donor limbal stem cells. RESULTS: Five years postoperatively, 65% of the grafts with 0 to 1 mismatches, 41% of the grafts with 2 to 6 mismatches, and 14% of the untyped grafts were clear centrally (estimation according to Kaplan-Meier log rank test, P = 0.03). Immunogenetic analysis of epithelial cells from the surface of the graft could be performed successfully in 7 of 9 patients and revealed donor DNA in the epithelium of 5 of these 7 patients up to 56 months postoperatively. CONCLUSIONS: Long-term survival of donor epithelium could be demonstrated immunogenetically in patients undergoing allogeneic penetrating limbo-keratoplasty. Human leukocyte antigen-matched grafts seem to deliver better results than untyped grafts. Progress with matching and immunosuppressive strategies may further improve current results.

Adult↗

PubMed2004

HLA class I/II matching and chronic endothelial cell loss in penetrating normal risk keratoplasty.

OBJECTIVE: Chronic endothelial cell loss of the graft is very common after penetrating keratoplasty. The aetiology of this is unknown. Clinically, non-identifiable immune reactions have been suspected. Recently, we were able to demonstrate that proper human leucocyte antigen (HLA) matching is a suitable means to reduce classical immune reactions in normal risk keratoplasty patients. In this study, we therefore investigated whether HLA-matched grafts also experience less chronic endothelial cell loss. METHODS: A homogenous group of 223 normal risk keratoplasty patients was divided into six groups with different degrees of HLA matching (group 1 with unknown HLA data, group 2 with up to two mismatches, group 3 with three mismatches, group 4 with four mismatches, group 5 with five mismatches and group 6 with six mismatches on the HLA A, B, DR loci). All serological HLA A, B, C and all moleculargenetic HLA DRB, DRQB typings of donors and recipients were performed in a single laboratory accredited by the American Society for Histocompatibility and Immunogenetics. Only patients with at least three postoperative endothelial cell density values were included in the study. The slopes of the regression lines for each individual scatterplot of endothelial cell density values plotted against postoperative time (linear regression, lost cells/mm2/day), and after logarithmic transformation (exponential regression, annual relative loss of cells) were evaluated, respectively. RESULTS: There were no statistically significant differences between the six groups. CONCLUSION: Whereas proper HLA matching at present standards is already a suitable means to reduce identifiable immune reactions and to prolong graft survival even in normal risk keratoplasty patients, the same HLA matching procedures are not effective in reducing the extent of chronic endothelial cell loss. For several reasons this does not yet exclude, however, the possibility that the underlying cause of chronic endothelial cell loss is immunological.

Cell Count↗

PubMed2003

[Stem cell therapy in acute myocardial infarction].

Remodeling of the left ventricle after myocardial infarction represents a major cause of infarct-related heart failure and death. Experimental data suggest that bone marrow-derived cells may contribute to the healing of myocardial infarction. The selective intracoronary transplantation of autologous, mononuclear bone marrow cells is safe and seems to be effective under clinical conditions concerning heart function, myocardial perfusion, and metabolism. The therapeutic effect may be attributed to bone marrow cell-associated myocardial regeneration and neovascularization.

Angioplasty, Balloon, Coronary↗

PubMed2003

T lymphoid differentiation in human bone marrow.

The unique role of the thymus in the development of T cells was established >4 decades ago. To elucidate how uncommitted lymphoid progenitor cells are instructed to migrate from bone marrow to the thymus to undergo T lymphoid differentiation, we generated and analyzed a genome-wide gene expression profile of CD7+ CD10+ human bone marrow T cell lineage precursors (TLPs) by using the serial analysis of gene expression technique. Unexpectedly, the serial analysis of gene expression profile identified a high number of (pre-) T cell receptor antigen (TCR)-related transcripts in bone marrow TLPs. To determine the configuration of the TCRbeta locus in these cells at a quantitative level, we sorted and analyzed bone marrow TLPs from five donors by single-cell PCR. Similar proportions of TLPs harbored TCRbeta germ-line alleles, D-J, or V-DJ gene rearrangements. Thus, bone marrow TLPs are heterogenous with respect to TCRbeta rearrangement status, suggesting an active recombination machinery that is consistent with the expression of RAG1, RAG2, and TdT in this population. As a hallmark of ongoing TCRbeta V-DJ rearrangement, we could amplify broken-ended recombination-signal sequence DNA intermediates from bone marrow TLPs, but not from mature T cells by ligation-mediated PCR. Approximately half of the TCRbeta rearrangements were compatible with the expression of a functional pre-TCR, which is in agreement with surface expression of pre-Talpha on bone marrow TLPs as shown by confocal laser microscopy and flow cytometry. At a frequency <0.5% of mononucleated cells in human bone marrow, this population is rare, yet it exemplifies T lymphoid differentiation in the human already before immigration into the thymus.

Base Sequence↗

PubMed2003

Rapid and highly efficient gene transfer into natural killer cells by nucleofection.

Natural killer (NK) cells are important mediators of virus- and tumor-specific immune responses. The transfection of genes into NK cells has been proven difficult and so far requires infection with virus-based vectors. Here, the application of a novel nonviral, electroporation-based gene transfer method is described for the rapid and highly efficient transient transfection of NK cell lines as well as freshly isolated NK cells. In contrast to conventional methods, this technique, termed nucleofection, leads to direct transfer of DNA into the nucleus. Using reporter proteins H-2K(k), luciferase+, and enhanced yellow green fluorescent protein (EYFP) as independent read-out systems, transfection efficiencies of well over 50% were achieved in transient transfection assays. The highest luciferase activity could be measured only 4 h after transfection, whereas EYFP, when analyzed by flow cytometry, showed expression peaks after 28 h. Interestingly, best transfection efficiencies were achieved with non-dividing NK cells. The novel nuclear gene transfer method presented here is highly useful for the analysis of NK cell-specific gene regulation and should facilitate the development of NK cell-based gene therapy approaches.

Cell Division↗

PubMed2003

HLA class I and II matching improves prognosis in penetrating normal-risk keratoplasty.

BACKGROUND: HLA matching in penetrating keratoplasty is still neglected in most eye clinics. This is due to contradictory results of studies performed in the past. Different surgical techniques in multicenter studies, missing risk differentiation in high-risk situations and faulty HLA typing can be identified as the main reasons for these contradictory results. In this monocenter study, the value of HLA class I and II matching (A, B, DR loci) was examined in a homogenous group of 398 normal-risk keratoplasty patients using modern typing techniques. 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). Surgery was done by 3 experienced surgeons according to a standardized scheme. Also, postoperative therapy and controls were standardized. There were no statistically significant differences between the two study groups as regards number of AB0 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. HLA typing was performed in a quality-controlled laboratory, serologically for HLA class I (A and B loci) and moleculargenetically for HLA class II (DR locus). RESULTS: Four years postoperatively, the ratio of clear and rejection-free graft survival was 91% in group I and 67% 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 monocenter 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 homogenous group of normal-risk keratoplasty patients.

Cornea↗

PubMed2002

Recipient cytokine genotypes for TNF-alpha and IL-10 and the minor histocompatibility antigens HY and CD31 codon 125 are not associated with occurrence or severity of acute GVHD in unrelated cord blood transplantation: a retrospective analysis.

BACKGROUND: In HLA-identical sibling bone marrow transplantation, certain recipient cytokine gene polymorphism genotypes and minor histocompatibility differences influence the occurrence and severity of acute graft-versus-host disease (aGvHD). The present study investigated the role of cytokine tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 gene polymorphisms HY, HA-1, and CD31 minor histocompatibility antigen (mHag) mismatch in the development of aGvHD after unrelated cord blood (CB) transplant (CBT). METHODS: DNA samples of 115 CB recipients and their unrelated CB grafts were analyzed for genotype associated with TNF-alpha (TNFd3/d3) and IL-10 (IL-10(-1064), 11-16) and for disparities in major and three minor histocompatibility antigens, HY, HA-1, and CD31 codon 125. Results were correlated with the incidence of aGvHD grades II to IV. RESULTS: Neither the donor nor the recipient GvHD risk alleles TNFd3/d3 and IL-10(-1064) (11-16) were associated with the development of aGvHD grades II to IV and I to IV. Because of the heterogeneity of CBTs, the data were reanalyzed separately for patients with malignancies (n=83) or with inborn errors (n=24). No significant association was observed between the severity of aGvHD and the possession of either TNFd3/d3 or IL-10 (11-16) genotypes. Mismatches for the mHags HY, HA-1, and CD31 exon 125 between donor and recipient did not associate with aGvHD grades II to IV. CONCLUSIONS: In contrast to HLA-identical sibling bone marrow transplantation, in mismatched unrelated CBT, neither the cytokine genotypes TNFd3/d3 alone or in combination with IL-10(-1064) alleles nor the minor histocompatibility antigens HY, HA-1, and CD31 exon 125 were associated with aGvHD grades II to IV. Further determination of the cytokine gene polymorphism genotypes in CBTs compared with bone marrow transplants may identify those polymorphisms that could be potential predictive markers for the occurrence of aGvHD.

Acute Disease↗

PubMed2002

Nucleolin as activator of human papillomavirus type 18 oncogene transcription in cervical cancer.

High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18(+) cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16(+) SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18(+) precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18(+) carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis.

Base Sequence↗

PubMed2002

Crucial role of DNA methylation in determination of clonally distributed killer cell Ig-like receptor expression patterns in NK cells.

Human NK cells are characterized by the expression of surface receptors of the killer cell Ig-like receptor (KIR) family, which are involved in the specific recognition of pathogenic target cells. Each NK cell expresses and maintains an individual subset of inhibitory and stimulatory KIR and in this way contributes to a diversified NK cell repertoire. To date, the molecular basis for generation of clonally distributed KIR expression patterns has been elusive. Here, analyses of DNA methylation patterns of KIR genes in NK cell lines as well as in NK cells, freshly isolated from peripheral blood, demonstrated that a small CpG island surrounding the transcriptional start site of each KIR gene is consistently demethylated in expressed KIR and methylated in unexpressed KIR. DNA-demethylating treatment resulted in a rapid and stable induction of transcription and cell surface expression of all formerly unexpressed KIR in NK cell lines, NK cell clones, and freshly isolated NK cells, but not in other cell types. In vitro methylation of KIR CpG islands repressed reporter gene expression in NK cells. We conclude that clonal patterns of KIR expression are mainly epigenetically determined and maintained through DNA methylation.

5' Untranslated Regions↗

PubMed2002

Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans.

BACKGROUND: Experimental data suggest that bone marrow-derived cells may contribute to the healing of myocardial infarction (MI). For this reason, we analyzed 10 patients who were treated by intracoronary transplantation of autologous, mononuclear bone marrow cells (BMCs) in addition to standard therapy after MI. METHODS AND RESULTS: After standard therapy for acute MI, 10 patients were transplanted with autologous mononuclear BMCs via a balloon catheter placed into the infarct-related artery during balloon dilatation (percutaneous transluminal coronary angioplasty). Another 10 patients with acute MI were treated by standard therapy alone. After 3 months of follow-up, the infarct region (determined by left ventriculography) had decreased significantly within the cell therapy group (from 30+/-13 to 12+/-7%, P=0.005) and was also significantly smaller compared with the standard therapy group (P=0.04). Likewise, infarction wall movement velocity increased significantly only in the cell therapy group (from 2.0+/-1.1 to 4.0+/-2.6 cm/s, P=0.028). Further cardiac examinations (dobutamine stress echocardiography, radionuclide ventriculography, and catheterization of the right heart) were performed for the cell therapy group and showed significant improvement in stroke volume index, left ventricular end-systolic volume and contractility (ratio of systolic pressure and end-systolic volume), and myocardial perfusion of the infarct region. CONCLUSIONS: These results demonstrate for the first time that selective intracoronary transplantation of autologous, mononuclear BMCs is safe and seems to be effective under clinical conditions. The marked therapeutic effect may be attributed to BMC-associated myocardial regeneration and neovascularization.

Angioplasty, Balloon, Coronary↗

PubMed2002

Placental/umbilical cord blood for unrelated-donor bone marrow reconstitution: relevance of nucleated red blood cells.

Placental/umbilical cord blood (PCB) is a source of hematopoietic stem cells for bone marrow reconstitution. Engraftment speed and survival are related to the total nucleated cell (TNC) dose of the graft. This study explored the possible influence on engraftment of nucleated red blood cells (NRBCs) in the graft. Automated hematology analyzers were used to enumerate TNCs. NRBCs were counted by visual examination or by using an automated analyzer. Hematopoietic progenitor cells were enumerated as either colony-forming cells or CD34(+) cells. Transplant centers reported on transplant outcome in 1112 patients given PCB grafts through September 2001. NRBCs correlated with progenitor cell numbers. Both white blood cell and NRBC dose were independently predictive of myeloid engraftment speed. Because NRBC dose predicted engraftment speed, inclusion of NRBCs in the TNC count does not reduce the effectiveness of the prefreezing TNC count as an index of the quality of a PCB unit as a graft. The correlation between the number of NRBCs and the number of hematopoietic progenitor cells probably reflects the involvement of early stem cells in erythroid responses.

Bone Marrow Cells↗

PubMed2002

Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes.

The segregation of killer cell immunoglobulin-like receptor ( KIR) genes was determined for a panel of 21 Caucasoid families: 23 different KIR gene patterns were found and could be assigned to combinations of 16 different haplotypes. Four loci were held in common by all haplotypes: KIR2DL4, KIR3DL2, the putative pseudogene KIR3DL3 and KIR2DL2/KIR2DL3, the latter likely being alleles of one gene. Group A haplotypes, which have a unique combination of seven KIR genes, were found at 80% frequency in the family panel, the polygenic group B haplotypes at 65% frequency. KIR gene segregation was fully determined for the nine group B haplotypes, which occurred at highest frequencies in both the family panel and a panel of unrelated individuals. The group B haplotypes carried between seven and 11 KIR genes and encoded inhibitory KIR for one, two, or all three major HLA class I epitopes. Analysis of human leucocyte antigen (HLA) class I genotypes revealed that most, but not all, individuals possess an inhibitory KIR for a self HLA class I epitope. The number of stimulatory KIR genes in group B haplotypes varied considerably between one and five. The data show that group B haplotypes possess a broad spectrum of KIR gene patterns, which is largely complementary to the KIR gene set of group A haplotypes. The results suggest that rapid diversification of group B haplotypes is the result of pathogen-mediated selection for KIR genotypes that have more than the set of KIR genes provided by the group A haplotype.

Female↗

PubMed2002

Sp1 as G1 cell cycle phase specific transcription factor in epithelial cells.

Sp1 binding sites have been identified in enhancer/promoter regions of several growth and cell cycle regulated genes, and it has been shown that Sp1 is increasingly phosphorylated in G1 phase of the cell cycle. Interactions of Sp1 with proteins involved in control of cell cycle and tumor formation have been reported. Here we show that expression of Sp1 protein predominates in the G1 phase of the cell cycle in epithelial cells. This is achieved by proteasome-dependent degradation. Inhibition of endogeneous Sp1 activity by a dominant-negative Sp1 mutant was associated with a cell cycle arrest in G1 phase, a strongly reduced expression of cyclin D1, the EGF-receptor and increased levels of p27Kip1. We have thus identified Sp1 as an important regulator of the cell cycle in G1 phase.

Cell Cycle Proteins↗

PubMedDate not supplied

Clinical-scale generation of dendritic cells in a closed system.

Immunotherapy of malignant diseases based on dendritic cells (DCs) pulsed with tumor antigens is a promising approach. Therefore, there is a demand for large-scale, clinical-grade ex vivo generation of DCs. Here, a procedure is presented that combines monocyte selection and tissue culture in closed systems under current good manufacturing practice conditions. Leukocytes from three patients with urologic cancers were collected by leukapheresis and subjected to immunomagnetic enrichment. From leukapheresis products containing 1.6 +/- 0.2 x 1010 (mean +/- SEM) leukocytes with a frequency of CD14+ monocytes of 18.7 +/- 2.3%, monocytes were enriched to 94.3 +/- 2.2%. CD14+ cell recovery was 67.0 +/- 4.7%. After 6 days of culture in Teflon bags in X-Vivo 15 medium supplemented with autologous plasma, GM-CSF, and IL-4, cells showed an immature DC phenotype and efficient antigen uptake. Following an additional 3 days of culture in the presence of GM-CSF, IL-4, IL-1beta, IL-6, TNFalpha, and PGE(2), cells (82.0 +/- 5.8% CD83+) displayed a mature DC morphology and phenotype, including expression of CD11b, CD11c, CD18, CD25, CD40, CD54, CD58, CD80, CD86, HLA class I, and HLA-DR as well as expression of CCR7 but not CCR5. The mature DC phenotype remained stable for at least 5 days in the absence of cytokines. Yield of DC was 14.0 +/- 4.7% and viability was 91.9 +/- 3.5%. Mature DCs effectively clustered with naive T cells and potently induced allogeneic T-cell proliferation and IL-2 and IFNgamma but not IL-4 production. Thus, this procedure allows large-scale generation of stably mature, Th1 responses inducing DCs under cGMP conditions in a closed system from cancer patients and is therefore well suited for immunotherapy.

Antibodies, Monoclonal↗