Biomedical subjects

P Meissner

Publications and source records attributed to P Meissner.

At least 19 recordsLinked to original sources

PubMed2003

Validation of a pencil beam model-based treatment planning system for fast neutron therapy.

Treatment planning systems (TPSs) are used to compute dose delivered to the patient. In the case of fast neutron therapy, TPSs are mostly not of general purpose but are dedicated to one facility. This is due to the few fast neutron facilities worldwide and due to the high variation in the neutron energy distributions. Efforts have been undertaken to develop a new TPS that could be applied to all the existing fast neutron facilities. The University Hospital of Essen operates a d (14 MeV) + Be fast neutron beam and the TPS used is based on an empirical model. In a previous study, the empirical model has been evolved to a pencil beam model of 35 monoenergetic neutron beams. Monte Carlo techniques have been utilized to compute distributions of the energy deposition due to primary and scattered neutrons in a simple geometry water phantom. The experimental validation of the method is now presented. Depth dose curves in water of monoenergetic neutrons have been derived from the distributions of energy deposition. The resultant depth dose curves have been utilized in order to determine the depth dose curves of the fast neutron beam of the Essen facility for the 14 radiation field sizes available in this facility. This determination requires the initial neutron spectrum. As this spectrum could not be measured at the Essen facility, the initial neutron spectrum of the Physikalisch Technische Bundesanstalt, Braunschweig, Germany, which operates the same cyclotron, was used. The calculated depth dose curves were compared to experimental depth dose curves that have been obtained in water at the University Hospital of Essen. The comparison between calculated and experimental depth dose curves showed significant deviations in the case of large radiation fields and of depth less than 5 cm. In the case of radiation field areas less than 150 cm2 and depth more than 5 cm (usual clinical situation), the measured and calculated values are in a good agreement. In the case of clinical situation, the dependence on the radiation field size is relatively well taken into account by the model presented here.

Computer Simulation↗

PubMed2002

Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis.

Using transient expression, high amounts (>20 mg/mL) of secreted anti-human Rhesus-D IgG1 were produced in a suspension-adapted HEK293 EBNA cell line (Meissner et al., Biotechnol Bioeng 75: 197-203, 2001). Time of harvest was 3 days after transfection. For the estimation of transfection efficiencies, we routinely co-transfected EGFP reporter DNA. At higher reporter plasmid concentrations, >2% of total transfecting plasmid DNA, a substantial reduction of recombinant antibody synthesis, was observed. This phenomenon was investigated in detail by co-expressing various green fluorescent protein (GFP) reporter constructs, which were targeted at different subcellular locations. Enhanced and humanized GFPs targeted to either the endoplasmic reticulum, the cytosol, or the nucleus reduced recombinant antibody production by 30 to 40% when present at higher concentrations in the transfection solution. The most severe effects were observed when the co-transfected EGFP was targeted to the endoplasmic reticulum, leading to a reduction of up to 80% in the presence of only 5% of reporter DNA. Interestingly, one nuclear-targeted GFP variant that was not codon optimized for expression in human cell lines could be added, to up to almost half of the total amount of transfecting DNA, without adverse effect on antibody production. Although the minimum amount of this reporter DNA needed for fluorescence reading was 10 times higher than for the other variants, it provided a much broader quantity range within which the transfection process could be studied without being negatively affected.

Animals↗

PubMed2002

Empirical description and Monte Carlo simulation of fast neutron pencil beams as basis of a treatment planning system.

The fast neutron beam, used for fast neutron therapy in Essen, is produced by the nuclear reaction of a 14 MeV cyclotron-based deuteron beam on a thick beryllium target. The resulting neutron beam has a continuous energy spectrum with a mean and a maximum energy equal to 5.5 and 18 MeV, respectively. The dose delivered to the patient is computed by a treatment planning system (TPS) based on an empirical model, in which the dose components (neutron and photon) are described by analytical functions. In order to improve the dose calculation, and thus to use the fast neutron beam for other applications (e.g., Boron Neutron Capture Enhancement of Fast Neutron Therapy), in this work we aim to develop a new TPS. For this purpose, a model based on pencil beams of mono-energetic neutrons has been created. The neutron energy ranged from 0.25 MeV up to 17.25 MeV by steps of 0.5 MeV in order to cover the energy range of the Essen facility. The Monte Carlo method was then used to simulate the transport of neutrons within such pencil beams in a homogeneous water phantom. By using Monte Carlo techniques, it is possible to distinguish the energy deposition due to a primary collision in water to that due to scattered neutrons. The energy deposition due to pencil beams of 2.224 MeV photons, coming from hydrogen neutron capture reaction in the phantom or in the collimator, was also determined. In order to complete this work, air filled cylinders have been introduced in the water phantom. It is shown that the resulting depth dose curves for primary neutrons can be easily derived using the homogeneous phantom, and that the description of the effect on scattered neutron dose distribution is more complex. In this work we demonstrate the relevance of Monte Carlo simulations of mono-energetic neutron pencil beams for purposes of neutron treatment planning. Some additional work is still required to describe a clinical situation (continuous energy neutron spectrum) as well as to experimentally validate the method described here.

Adipose Tissue↗

PubMed2001

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells.

Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

Bioreactors↗

PubMed2001

Mesenchymal differentiation and organ distribution of established human stromal cell lines in NOD/SCID mice.

Two human stromal cell lines were established previously from bone marrow-derived primary long-term cultures by immortalization using the SV40 large T antigen and cellular cloning. After irradiation, the fibroblast-like cell lines L87/4 and L88/5 support hematopoietic differentiation of allogeneic cord blood cells in vitro. The stromal cells do not express CD34 and CD50, but some adhesion molecules and integrins, such as CD44, CD54 and CD58. Their expression profiles on RNA and protein levels are suggestive of their osteogenic potency. The quality and quantity of osteocalcin and osteopontin protein expression depended on the culture conditions. Expression of the osteogenic markers increased over time in culture, especially in cells growing in clusters. The stromal cells also expressed collagens I and V, but did not show any expression of collagens II and III. The potentially osteoblastic stromal cells were transplanted into NOD/ SCID recipient mice by intravenous injection and were found in various mesenchymal organs up to 10 weeks after transplantation. Osteocalcin-positive human stromal cells could be detected in the bone marrow, thymus, liver, brain and gut of the recipient animals. In summary, there is evidence that human bone-marrow-derived stromal cells have to be considered mesenchymal progenitors, persistently expressing osteogenic markers in vitro and in vivo.

Animals↗

PubMed1997

Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10.

Virally infected cells degrade intracellular viral proteins proteolytically and present the resulting peptides in association with major histocompatibility complex (MHC) class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). These cells are normally prone to CTL-mediated elimination. However, several viruses have evolved strategies to avoid detection by the immune system that interfere with the pathway of antigen presentation. Epstein-Barr virus (EBV) expresses a predominantly late protein, the BCRF1 gene product vIL-10, that is similar in sequence to the human interleukin-10 (hIL-10). We show here that vIL-10 affects the expression of one of the two transporter proteins (TAPs) associated with antigen presentation. Similarly, hIL-10 showed the same activity. Expression of the LMP2 and TAP1 genes but not expression of TAP2 or LMP7 is efficiently downregulated, indicating a specific IL-10 effect on the two divergently transcribed TAP1 and LMP2 genes. Downregulation of TAP1 by IL-10 hampers the transport of peptide antigens into the endoplasmatic reticulum, as shown in the TAP-specific peptide transporter assay, their loading onto empty MHC I molecules, and the subsequent translocation to the cell surface. As a consequence, IL-10 causes a general reduction of surface MHC I molecules on B lymphocytes that might also affect the recognition of EBV-infected cells by cytotoxic T cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

[Change in bronchial hyperreactivity with nCPAP respiration in patients with sleep related respiratory disorders].

Own results of 23,174 patients suffering from sleep apnoea syndrome showed that about 4 per cent of these patients have an increase of bronchial reactivity. In 60 patients with assured bronchial hyperreactivity we performed an inhalative bronchial provocation test using carbachol. Tests were performed before and after two days of treatment with nCPAP in the early morning. 23 of our 60 patients showed a decrease of PD20 FEV1 after nCPAP. The other patients did not show any significant differences. Our results demonstrate that in patients with increased bronchial reactivity, nCPAP therapy may aggravate bronchial hyperreactivity.

Adult↗

PubMed1997

[Effect of n-CPAP therapy on outcome of cold provocation].

Our own investigations comprising 23,174 patients suffering from sleep apnoea showed that about 4 per cent of these patients suffer from a hyperreactive bronchial system. In some of these patients treatment with nCPAP causes coughing or mild dyspnoea even after having been previously asymptomatic. Loss of water and heat on the surface of bronchial mucosa may induce reversible bronchoconstriction. We examined in 60 patients suffering from obstructive sleep apnoea whether mechanical treatment with nCPAP would cause a change in bronchial reactivity. Cold air hyperventilation was used in provocation testing. Provocation tests were performed before and after a 3-day treatment with nCPAP in the early morning. In some patients with previously positive reaction, application of nCPAP alone decreased the lung function. Cold air hyperventilation challenge may be helpful to detect possible risks in patients using nCPAP, and to minimise such risks.

Adult↗

PubMed1996

Rapid proliferation of B cells from adenoids in response to Epstein-Barr virus infection.

EBV is a human tumor virus that is associated with different types of tumors. A unique feature of EBV is its capability to infect and immortalize human B cells both in vivo and in vitro. In cell culture, this progress is termed immortalization and infected B cells grow out to permanent, so-called lymphoblastoid cell lines. During our experiments, we observed that B lymphocytes derived from adenoids are infected efficiently by EBV and proliferate much more rapidly than any other known type of B cell. High concentrations of adhesion molecules and of CD21, the EBV receptor, present on these cells may account for this phenomenon. Adenoid B cells may therefore represent a particular subpopulation of preactivated B lymphocytes that can greatly simplify and enhance the production of lymphoblastoid cell lines for, e.g., antigen-presenting cells for gene therapeutic approaches and similar applications.

Adenoids↗

PubMed1996

Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.

We present a detailed analysis of cytokine expression patterns of the two permanent human bone marrow stromal cell lines, L87/4 and L88/5. These cell lines, previously established in our laboratory, are highly radiotolerant without cell detachment and support long-term cultures of CD(34+)-enriched human cord blood cells. RT-PCR analysis of 22 different cytokines or cytokine receptor mRNAs showed an almost identical expression pattern in the two stromal cell lines compared to primary human Dexter-type stroma. Since stromal feeder lines employed in long-term cultures usually are irradiated and grown in media containing corticosteroids, we analyzed the impact of irradiation and dexamethasone on cytokine production in the two cell lines by RT-PCR, Northern blot analysis, bioassays, and RIAs. By RT-PCR analysis, constitutive mRNA expression of c-kit, G-CSF, GM-CSF, IL-1 beta, IL-6, IL-7, IL-8, IL-11, Kit ligand (KL), LIF, M-CSF, MIP-1 alpha, TGF-beta, and TNF-alpha was demonstrated in both cell lines, with L87/4 a more potent cytokine producer than L88/5. Northern blot data showed an increase in mRNA levels for GM-CSF, IL-1 beta, and LIF by irradiation and IL-1 alpha treatment in both cell lines. IL-1 alpha-induced GM-CSF, IL-1 beta, IL-6, IL-11, and LIF mRNA levels were reduced by the addition of dexamethasone, whereas dexamethasone had no influence on the amounts of IL-1 alpha-induced G-CSF mRNA. L87/4 and, to a lower extent, L88/5 cells showed dexamethasone-dependent increases in KL mRNA, while KL mRNA levels were not stimulated by IL-1 alpha.

Base Sequence↗

PubMed1995

Cloning, sequence, and expression of mouse protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin. A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria. We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase. The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation of Escherichia coli SASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library. The sequence of this cDNA along with 5' untranslated sequence obtained by 5' rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp. This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870. The protein as expressed in E. coli is sensitive to inhibition by the diphenyl ether herbicide acifluorfen. Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb.

Amino Acid Sequence↗

PubMed1995

Identification of two new mutations in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. Six missense mutations in the UROIIIS gene, a deletion and an insertion have already been described in CEP. This work brings further evidence for the heterogeneity in the genetic defect found in CEP. Two new mutations are described, a point mutation (V99A) and a frame-shift mutation (633insA) in the same patient who had a mild to moderate form of Günther's disease. The mutation (V99A) had a detectable residual activity when expressed in Escherichia coli while the insertion (633insA), which introduced a premature stop, had no activity. In the patients studied in our laboratory, the mutation C73R, associated with a severe phenotype, remains the most frequently seen.

Base Sequence↗

PubMed1995

[The effect of nCPAP respiration on hyperreactivity in patients with sleep related respiratory disorder].

Of the 8,973 patients with sleep related breathing disorders examined in our department, about 4% were found to have a hyperreagible bronchial system. Provocation tests were performed before and after a 3-day CPAP therapy in 8 male patients with confirmed bronchial hyperreagibility. Two of these 8 patients revealed a marked increase of the respiratory path resistance and a decrease in FEV1.0 of 30% in the provocation test after 3 days. This did not occur with the other patients. From these findings, it is concluded that CPAP therapy can lead to an increase in the hyperreagibility in some patients with hyperreagible bronchial systems. However, further investigations are needed to identify the underlying causal relationships.

Adult↗

PubMed1995

[Effect of n-CPAP therapy on the 24-hour blood pressure profile in severe obstructive sleep apnea syndrome].

We examined circadian blood pressure rhythm before and two days after onset of n-CPAP therapy in 50 patients, suffering from polysomnograhpically ensured severe obstructive sleep-apnea-syndrome. Measurements were performed using a non-invasive blood pressure registration. Patients with normal circadian blood pressure conditions and those with disturbed circadian rhythm could be differentiated. N-CPAP therapy may normalize circadian blood pressure conditions in some patients.

Adult↗

PubMed1995

[Polysomnographic results before and after uvulopalatopharyngoplasty (UPPP)].

Uvulo-palato-pharyngo-plastic is one possibility for treatment of obstructive sleep-apnea. We examined in a prospective study design 36 patients, suffering from polysomnographically ensured obstructive sleep-apnea-syndrome, who were undergone uvulo-palato-pharyngo-plastic. We analysed pre- und postoperative polysomnographic data. Most of patients demonstrated a decrease of amount of apneas, duration of apneas and an increase of minimal oxygen-desaturation. Patients who had high amounts of apneas (AI > 40/h) or who had a Broca-index > 140 p.c. gained not as much from surgical treatment as other patients did.

Adult↗

PubMed1995

[Theophylline acceptance in long-term therapy of patients with obstructive sleep related respiratory disorder].

In 1,150 patients with sleep apnea syndrome, the apnea number, the morning theophyllin level, the symptom of morning exhaustion were recorded for the first night of theophyllin therapy and in the follow-up period of up to 5 years; in addition, side effects were noted at the end of the observation period. Prior to start of the therapy, the average apnea number was 97 per night; this decreased to 25 per night in the initial therapy. In the observation period of between 3 and 28 months, the number of apnea phases increased slightly on average. The symptoms of morning exhaustion initially decreased to 60% but increased again by about 20% over the next five years. In responders, theophyllin reduces the number of apnea in the long term course; however, frequent therapy controls are needed to determine the optimal dosage. Longitudinally, patients with an apnea index < 20/profited most from this drug therapy.

Adult↗

PubMed1994

Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity.

We describe the establishment of two permanent Simian virus 40-transformed human stromal cell lines, designated L87/4 and L88/5, derived from the bone marrow of a hematologically normal male patient. Both cell lines show a fibroblastoid morphology and do not express hematopoietic cell markers. L87/4 but not L88/5 expresses the macrophage marker CD68. The most remarkable feature of these new stromal cell lines is their ability to persist as growth-arrested adherent feeder cells after ionizing-irradiation at doses up to, and exceeding 20 Gy (L87/4). This renders them particularly useful for studying aspects of feeder dependence of hematopoietic cell development in long-term culture. Both cell lines are able to function as feeder cells, supporting the long-term proliferation of CD34+ human cord blood cells as well as the clonogenic growth of the human Burkitt lymphoma B-cell line BL70.

Bone Marrow Cells↗

PubMed1994

Expression of a cloned protoporphyrinogen oxidase.

The previously cloned hem Y gene of Bacillus subtilis (Hansson, M., and Hederstedt, L. (1992) J. Bacteriol. 174, 8081-8093) has been expressed in Escherichia coli. The expressed protein has been shown to be the penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4) and, thus, the gene designation should be hem G. This represents the first report of the expression of a cloned protoporphyrinogen oxidase from any source. The enzyme is present in the soluble cytoplasmic fraction and is, thus, unlike all previously reported eukaryotic or prokaryotic protoporphyrinogen oxidases, which are membrane-bound. It utilizes molecular oxygen as a terminal electron acceptor, and protoporphyrinogen IX, mesoporphyrinogen IX, and coproporphyrinogen III serve as substrates. The diphenyl ether herbicide acifluorfen, which is a strong inhibitor of the eukaryotic enzyme, is only weakly inhibitory. The enzyme has a predicted molecular weight of 51,200, which corresponds well with molecular weight determination via high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis. In addition the enzyme contains a putative dinucleotide binding region at the amino terminus, which is consistent with the previously demonstrated presence of a flavin moiety in the characterized mammalian enzymes.

Amino Acid Sequence↗