Biomedical subjects

M Goerner

Publications and source records attributed to M Goerner.

9 recordsLinked to original sources

PubMed2006

The initial phase of graft-versus-host disease is associated with a decrease of CD4+CD25+ regulatory T cells in the peripheral blood of patients after allogeneic stem cell transplantation.

The mechanisms that induce and control the alloimmune inflammation of graft-versus-host disease (GvHD) after allogeneic stem cell transplantation (allo-SCT) are still incompletely understood. In the murine system, GvHD can be suppressed by CD4(+)CD25(+) regulatory T cells (TREG), which are generally involved in the suppression of inflammatory reactions. A disruption of the homeostasis between TREG and conventional T cells might therefore be associated with the inflammatory reactions of GvHD. We repetitively measured the frequency of TREG in the peripheral blood of 29 patients within the first 71-373 days after allo-SCT and correlated the results with the clinical course. We demonstrate that the initial phase of GvHD is associated with a significant reduction of TREG in the peripheral blood, while at later stages and during intensified immunosuppressive therapy, increased numbers of TREG appear. These results might indicate a pathogenic role for reduced numbers of TREG in the induction of human GvHD.

Adult↗

PubMed2005

Molecular monitoring of tumour load kinetics predicts disease progression after non-myeloablative allogeneic stem cell transplantation in multiple myeloma.

BACKGROUND: Non-myeloablative allogeneic stem cell transplantation followed by immunomodulatory therapies is considered a potentially curative approach in the treatment of multiple myeloma and most effective in a minimal residual disease setting. PATIENTS AND METHODS: The aim of this study was to find the most sensitive real-time PCR assay (TaqMan), based on the IGH rearrangement, to quantify the tumour load of 11 patients with multiple myeloma after non-myeloablative allogeneic transplantation. Patient-allele specific primers (ASO) and the TaqMan probe were derived from CDR2 and CDR3 hypervariable regions of IGH, while consensus primers were located within the FR3 and FR4/JH regions. Four different approaches of primer combinations were tested. RESULTS: ASO-forward and -reverse primers together with the clone-specific TaqMan probe were the most sensitive approach compared with the JH (P=0.071) or the FR3 consensus primer (P <0.001). The detection limit amounted to 1/10(4)-1/10(5) cells. Consecutively, 120 samples from 11 patients prior and post allogeneic transplantation were analysed. Three patients reached complete clinical remission accompanied by molecular remission. Disease progression or relapse was seen in six patients. In five, molecular progressive disease was detected prior to the clinical diagnosis of progression or relapse. CONCLUSION: Patient-specific real-time IGH-PCR provides the opportunity for earlier treatment intervention.

Adult↗

PubMed2005

Long-term outcome of nonmyeloablative allogeneic transplantation in patients with high-risk multiple myeloma.

Conventional treatment or autologous transplantation has not been able to achieve long-term remission in patients with multiple myeloma (MM). Nonmyeloablative allogeneic transplantation might offer an option for cure without the high mortality associated with conventional conditioning. Here we present a retrospective analysis of patients with high-risk MM treated with nonmyeloablative allogeneic transplantation. In all, 52 patients with relapsed MM or high-risk features at diagnosis received 2 Gy TBI alone (n=3) or with fludarabine (n=49) as conditioning. Patients were heavily pretreated with a median of eight cycles of conventional chemotherapy and one or more autologous transplants for all but one patient. Regimen-related toxicity was low. Acute graft-versus-host disease II-IV occurred in 37% of patients, and 70% experienced chronic graft-versus-host disease (cGvHD). Median follow-up was 567 days, and transplant-related mortality was 17% in total. Estimated progression-free and overall survival at 18 months was 29.4 and 41.1%, respectively. Patients with cGvHD had a significantly higher progression-free survival, as did patients with up to eight cycles of pretreatment chemotherapy vs those with nine or more. In this highly pretreated patient group, disease control was unsatisfactory and our results suggest that a potential strategy might be to perform allogeneic transplant earlier in the course of the disease.

Adult↗

PubMed2001

Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.

Previous studies have shown that the choice of envelope protein (pseudotype) can have a significant effect on the efficiency of retroviral gene transfer into hematopoietic stem cells. This study used a competitive repopulation assay in the dog model to evaluate oncoretroviral vectors carrying the envelope protein of the endogenous feline virus, RD114. CD34-enriched marrow cells were divided into equal aliquots and transduced with vectors produced by the RD114-pseudotype packaging cells FLYRD (LgGLSN and LNX) or by the gibbon ape leukemia virus (GALV)-pseudotype packaging cells PG13 (LNY). A total of 5 dogs were studied. One dog died because of infection before sustained engraftment could be achieved, and monitoring was discontinued after 9 months in another animal that had very low overall gene-marking levels. The 3 remaining animals are alive with follow-ups at 11, 22, and 23 months. Analyses of gene marking frequencies in peripheral blood and marrow by polymerase chain reaction revealed no significant differences between the RD114 and GALV-pseudotype vectors. The LgGLSN vector also contained the enhanced green fluorescent protein (GFP), enabling us to monitor proviral expression by flow cytometry. Up to 10% of peripheral blood cells expressed GFP shortly after transplantation and approximately 6% after the longest follow-up of 23 months. Flow cytometric analysis of hematopoietic subpopulations showed that most of the GFP-expressing cells were granulocytes, although GFP-positive lymphocytes and monocytes were also detected. In summary, these results show that RD114-pseudotype oncoretroviral vectors are able to transduce hematopoietic long-term repopulating cells and, thus, may be useful for human stem cell gene therapy.

Amino Acid Transport System ASC↗

PubMed2001

Fast confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine by LC/MS/MS using negative atmospheric-pressure chemical ionisation (APCI).

A fast method using automated solid-phase extraction (SPE) and short-column liquid-chromatography coupled to tandem mass-spectrometry (LC/MS/MS) with negative atmospheric-pressure chemical ionisation (APCI) has been developed for the confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine samples. This highly specific method which combines chromatographic separation and MS/MS-analysis can be used for the confirmation of positive immunoassay results with a NIDA cut-off of 15ng/ml. The conjugates of THC-COOH were hydrolysed prior to SPE, and a standard SPE was performed using C18-SPE columns. No derivatisation of the extracts was needed as in GC/MS analysis, and the LC run-time was 6.5min by gradient elution with a retention time of 2.4min. Linearity of calibration was obtained in the range between 0 and 500ng/ml (correlation coefficient R(2)=0.998). Using linear regression (0-50ng/ml) the limit of detection (LOD) was 2.0ng/ml and the limit of quantitation (LOQ) was 5.1ng/ml; day-to-day reproducibility and precision were tested at 15 and 250ng/ml and were 13.4ng/ml+/-3.3% and 255.8ng/ml+/-4.5%, respectively.

Chromatography, Liquid↗

PubMed2000

Expansion and transduction of nonenriched human cord blood cells using HS-5 conditioned medium and FLT3-L.

Cord blood (CB) stem cell transplantations have been associated with delayed hematopoietic engraftment. This has most likely been due to the limited numbers of hematopoietic short-term repopulating cells in CB. Ex vivo expansion of CB has been attempted, and expansion of CD34-enriched CB has been successful; however, CD34 enrichment procedures are in general associated with substantial cell loss. Thus, we have studied culture conditions for expansion of nonenriched CB. Nonenriched CB cells were cultured for 21 days in the presence of conditioned medium from the HS-5 stromal cell line and FLT3-L or alternatively in the presence of FLT3-L, stem cell factor (SCF), megakaryocite growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF) (FSMG), either on fibronectin fragment CH-296-coated dishes or on uncoated dishes. With all four culture conditions, the number of mononuclear cells initially decreased until day 7 and then increased until the end of the expansion cultures. Overall expansion using HS-5 and FLT3-L resulted in superior expansion of MNC and CFU-C (44-/34-fold) for both cultures with and without CH-296 compared to FSMG (18-/17-fold). Expansion on CH-296 was less efficient than expansion on tissue culture-treated wells without CH-296 for both conditions. We then studied the best time for transduction on nonenriched CB. In contrast to enriched CD34 cells, we found for both conditions, HS-5/FLT3-L and growth factor cocktail, higher transduction efficiencies when cells were transduced on day 7 as compared to day 2. Gene transfer rates up to 45% were achieved with both conditions, which corresponded with the increased number of cells in S phase on day 7 compared to day 2. We conclude that HS-5 and FLT-3L allow efficient expansion and transduction of nonenriched CB.

Cell Culture Techniques↗

PubMed1999

The use of granulocyte colony-stimulating factor during retroviral transduction on fibronectin fragment CH-296 enhances gene transfer into hematopoietic repopulating cells in dogs.

A competitive repopulation assay in the dog was used to develop improved gene transfer protocols for hematopoietic stem cell gene therapy. Using this assay, we previously showed improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched marrow cells were cocultivated on gibbon ape leukemia virus (GALV)-based retrovirus vector-producing cells. In the present study, we have investigated the use of fibronectin fragment CH-296 and 2 growth factor combinations to further improve gene transfer efficiency. CD34-enriched marrow cells from each dog were prestimulated for 24 hours and then divided into 3 equal fractions. Two fractions were placed into flasks coated with either CH-296 or bovine serum albumin (BSA) and virus-containing medium supplemented with growth factors, and protamine sulfate was replaced 4 times over a 48-hour period. One fraction was cocultivated on irradiated PG13 (GALV-pseudotype) packaging cells for 48 hours. In 2 animals, cells of the different fractions were transduced in the presence of human FLT-3 ligand (FLT3L), canine stem cell factor (cSCF), and human megakaryocyte growth and development factor (MGDF), and in 2 other dogs, transduction was performed in the presence of FLT3L, cSCF, and canine granulocyte-colony stimulating factor (cG-CSF). The vectors used contained small sequence differences, allowing differentiation of cells genetically marked by the different vectors. After transduction, nonadherent and adherent cells from all 3 fractions were pooled and infused into lethally irradiated dogs. Polymerase chain reaction and Southern blot analysis were used to determine the persistence of the transferred vectors in the peripheral blood and marrow cells after transplantation. The highest levels of gene transfer were obtained when cells were transduced in the presence of FLT3L, cSCF, and cG-CSF (gene transfer levels of more than 10% for more than 8 months so far). Compared with the 2 animals that received cells transduced with FLT3L, cSCF, and MGDF, gene transfer levels were significantly higher when dogs received cells that were transduced in the presence of cG-CSF. Transduction on CH-296 resulted in gene transfer levels that were at least as high as transduction by cocultivation. In summary, the overall levels of gene transfer obtained with these conditions should be sufficiently high to allow stem cell gene therapy studies aimed at correcting genetic diseases in dogs as a model for human gene therapy.

Animals↗

PubMed1999

Improved gene transfer into canine hematopoietic repopulating cells using CD34-enriched marrow cells in combination with a gibbon ape leukemia virus-pseudotype retroviral vector.

We have used dogs to study gene transfer into hematopoietic stem cells, because of the applicability of results in dogs to human transplantation and the availability of canine disease models that mimic human diseases. Previously we reported successful gene transfer into canine marrow repopulating cells, however, gene transfer efficiency was low, usually below 0.1% (Kiem et al, Hum Gene Ther 1996; 7: 89). In this study we have used CD34-enriched marrow cells to study different retroviral pseudotypes for their ability to transduce canine hematopoietic repopulating cells. Cells were divided into two equal fractions that were cocultivated for 72 h with irradiated packaging cells producing vector with different retroviral pseudotypes (GALV, amphotropic or 10A1). The vectors used contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into four dogs after a myeloablative dose of 920 cGy total body irradiation. Polymerase chain reaction (PCR) analysis of DNA from peripheral blood and marrow after transplant showed that the highest gene transfer rates (up to 10%) were obtained with the GALV-pseudotype vector. Gene transfer levels have remained stable now for more than 18 months. Southern blot analysis confirmed the high gene transfer rate. Interference studies on canine D17 cells revealed that 10A1 virus behaved like an amphotropic virus and was not able to use the GALV receptor. In summary, our results show improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched cells are transduced by cocultivation on a GALV-pseudotype packaging cell line in combination with a GALV-pseudotype vector. Furthermore, these results demonstrate that the monoclonal antibody to canine CD34 used in this study is able to enrich for hematopoietic repopulating cells.

Animals↗

PubMed1999

The use of CD34+-selected PBPC after high-dose chemotherapy in breast cancer patients is associated with prolonged recovery time and increased infectious complications.

High-dose chemotherapy with autologous stem cell rescue can result in autotransplantation of tumor cells. A possible approach to reduce tumor cell contamination is the positive selection of CD34+ PBPC, but this might be associated with a prolonged recovery time as well as an increased risk of infectious complications because of the loss of committed progenitor cells. To investigate this aspect, we compared two sequentially treated cohorts of high-risk breast cancer patients. Both groups received the same high-dose chemotherapy regimen followed by autologous peripheral stem cell transplantation. Group I received CD34+-selected blood progenitor cells, and group II received nonselected blood progenitor cells. We compared these two identically treated groups with regard to recovery time, need for blood products, infectious complications, need for antibiotic treatment, and length of the transplantation-related hospital stay. We found a prolonged recovery time for neutrophils up to 0.5 x 10(9)/L (14 days in the selected group/10 days in the nonselected group) and platelets up to 30 x 10(9)/L (29/12 days), associated with an increased requirement for RBC transfusions (5/3 U) and platelet transfusions (10/2 U). The rate of severe infectious complications (2/0), the need for nonprophylactic antibiotic treatment (15/10), and the length of the hospital stay (25/21 days) in group I were also increased. We conclude that positive selection of PBPC should not be used routinely until randomized studies show a clear long-term benefit of using CD34+-selected stem cell products in breast cancer patients.

Antigens, CD34↗