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Biomedical subjects

T Zander

Publications and source records attributed to T Zander.

At least 19 recordsLinked to original sources
PubMed2006

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA.

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (<10 US dollars) but equally accurate alternative to the Test arrays in determining biotinylated cRNA quality. Forty-one different cRNA samples were hybridized to HG-U133A microarrays from Affymetrix. Ten cRNA samples with a beta-actin 3'/5' ratio >6 were chosen and classified as degraded cRNAs, and 31 samples with a beta-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of beta-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r(2) = 0.6802) between the array and the RT-PCR beta-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean +/- SD, 0.34 +/- 0.09) and degraded (1.71 +/- 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.

Biotinylation↗
PubMed2005

Comparison of the biomechanical effects of posterior and anterior spine-stabilizing implants.

Posteriorly and anteriorly fixed implants for stabilizing unstable spines are available on the market. Differences in the biomechanical behavior of these implant types are not yet fully clear. They were investigated using three-dimensional nonlinear finite element models of the lumbar spine in an intact state, with an anteriorly fixed MACS-TL implant and with posteriorly fixed internal fixators. The bisegmental implants spanned the L3 vertebra, and bone grafts were used with both implant types to replace parts of the two bridged discs. The computer models were loaded with partial body weight and muscle forces simulating standing, flexion, extension and axial rotation. Both implant types have reduced intersegmental rotation for flexion, extension, and axial rotation in the bridged region. The reduction is more pronounced for the MACS-TL implant. The implant type has only a minor effect on intradiscal pressure. Maximum von Mises stresses in the vertebrae are lower for flexion and extension with the MACS-TL implant than with the internal fixator. Very high stresses are predicted for flexion after insertion of internal fixators. For standing and torsion, maximum stresses differ only negligibly between the two implant types. In the period immediately after surgery, patients with osteoporotic vertebrae and who are treated with an internal spinal fixation device should therefore avoid excessive flexion. This study adds new information about the mechanical behavior of the lumbar spine after insertion of posterior and anterior spine-stabilizing implants. This information improves our biomechanical understanding of the spine.

Biomechanical Phenomena↗
PubMed2005

[Effect of vertebral body stiffness before and after vertebroplasty on intradiscal pressure].

Fractures of osteoporotic vertebral bodies are increasingly stabilized with bone cement. The effects of vertebral-body stiffness before and after augmentation with bone cement and of wedge-shaped vertebral body fractures on intradiscal pressure are insufficiently known. In a finite element model of the lumbar spine the elastic modulus of cancellous bone as well as the amount and the elastic modulus of bone cement were varied and the dependency of intradiscal pressure on these parameters was calculated. In addition, a wedge-shaped vertebral-body fracture was simulated. The bulge of the vertebral-body endplate and thus the intradiscal pressure depends strongly on the grade of osteoporosis in the vertebral body. The influence of amount and elastic modulus of bone cement on intradiscal pressure is small. A wedge-shaped vertebral-body fracture causes an anterior shift of upper-body centre of gravity. If this shift is not compensated, it leads to an increased flexion moment that has to be balanced by muscle forces. In addition, this shift leads to a stronger increase of intradiscal pressure than the augmentation of the vertebral body with bone cement.

Bone Cements↗
PubMed2005

Effect of an interspinous implant on loads in the lumbar spine.

Interspinous process implants are increasingly used to treat canal stenoses. Little information exists about the effects of implant height and stiffness on the biomechanical behavior of the lumbar spine. Therefore, a three-dimensional nonlinear finite element model of the osseoligamentous lumbar spine (L1 to L5) was created with a slightly degenerated disc at L3/L4. An interspinous implant was inserted at that segment. Implants with different heights and stiffnesses were studied. The model was loaded with the upper body weight and muscle forces to simulate walking and 25 degrees extension. Implant forces are influenced strongly by the height and negligibly by the elastic modulus of the implant. Intersegmental rotation at implant level is markedly reduced, while intradiscal pressure is slightly increased. Implant size and stiffness have only a minor effect on intradiscal pressure. The maximum von Mises stress in the vertebral arch is strongly increased by the implant.

Elasticity↗
PubMed2005

Effects of the rib cage on thoracic spine flexibility.

Besides protecting the internal organs of the thorax, the rib cage is the site of numerous muscle attachments. It also decreases the overall flexibility of the thoracic spine. This study developed finite element (FE) models of the thoracic spine with and without the rib cage, and the effects of the rib cage on thoracic spine flexibility were determined. The numerical models were validated by comparing the maximum rotation of the models for several loading cases with experimental data in the literature. After adapting the material properties for the discs and ligaments, the calculated maximum rotations differed from the measured median values by less than 1 degrees without the rib cage and by less than 2.5 degrees with it. The rib cage decreased the mean flexibility of the thoracic spine by 23% to 47%, depending on the loading plane. Assuming the ribs to be rigid beams required a corresponding reduction of ligament stiffnesses in order to achieve the same agreement of the maximum rotations with the measured median values. Interconnecting the FE thoracic spine model plus rib cage with the existing detailed FE lumbar spine model improves the simulation of force directions of muscles attached to the rib cage or thoracolumbar spine. In addition, such a model is suitable for determining the effects of lumbar spine implants on spinal balance.

Computer Simulation↗
PubMed2004

Comparison of different isolation techniques prior gene expression profiling of blood derived cells: impact on physiological responses, on overall expression and the role of different cell types.

Owing to its clinical accessibility, peripheral blood is probably the best source for the assessment of differences or changes in gene expression associated with disease or drug response and therapy. Gene expression patterns in peripheral blood cells greatly depend on temporal and interindividual variations. However, technical aspects of blood sampling, isolation of cellular components, RNA isolation techniques and clinical aspects such as time to analysis and temperature during processing have been suggested to affect gene expression patterns. We therefore assessed gene expression patterns in peripheral blood from 29 healthy individuals by using Affymetrix microarrays. When RNA isolation was delayed for 20-24 h-a typical situation in clinical studies-gene signatures related to hypoxia were observed, and downregulation of genes associated with metabolism, cell cycle or apoptosis became dominant preventing the assessment of gene signatures of interindividual variation. Similarly, gene expression patterns were strongly dependent on choice of cell and RNA isolation and preparation techniques. We conclude that for large clinical studies, it is crucial to reduce maximally the time to RNA isolation. Furthermore, prior to study initiation, the cell type of interest should already be defined. Our data therefore will help to optimize clinical studies applying gene expression analysis of peripheral blood to exploit drug responses and to better understand changes associated with disease.

Adult↗
PubMed2003

Influence of graded facetectomy and laminectomy on spinal biomechanics.

Facetectomy and laminectomy are techniques for decompressing lumbosacral spinal stenosis. Resections of posterior bony or ligamentous parts normally lead to a decrease in stability. The degree of instability depends on the extent of resection, the loading situation and the condition of the intervertebral discs. The correlation between these parameters is not well understood. In order to investigate how these parameters relate to one another, a three-dimensional, non-linear finite element model of the lumbosacral spine was created. Intersegmental rotations, intradiscal pressures, stresses, strains and forces in the facet joints were calculated while simulating an intact spine as well as different extents of resection (left and bilateral hemifacetectomy, hemilaminectomy and bilateral laminectomy, two-level laminectomy), disc conditions (intact and degenerated) and loading situations (pure moment loads, standing and forward bending). The results of the modelling showed that a unilateral hemifacetectomy increases intersegmental rotation for the loading situation of axial rotation. Expanding the resection to bilateral hemifacetectomy increases intersegmental rotation even more, while further resection up to a bilateral laminectomy has only a minor additional effect. Hemilaminectomy and laminectomy only differ in their effect for ventriflexion and muscle-supported forward bending. Two-level laminectomy increases the intersegmental rotation only for standing. Degenerated discs result in smaller intersegmental rotations and higher disc stresses at the respective levels. Decompression procedures affect the examined biomechanical parameters less markedly in degenerated than in intact discs. Resection of posterior bony or ligamentous elements has a stronger influence on the amount than on the distribution of stresses and deformations in a disc. It has only a minor effect on the biomechanical behaviour of the adjacent region. Spinal stability is decreased after a laminectomy for forward bending, and after a two-level laminectomy for standing. For axial rotation, spinal stability is decreased even after a hemifacetectomy. Patients should therefore avoid excessive axial rotation after such a treatment.

Biomechanical Phenomena↗
PubMed2002

[Effect of implants for replacement of a vertebral body on the mechanical function of the lumbar spine].

A three-dimensional, nonlinear finite element model of the lumbar spine was created. A bisegmental internal spinal fixation device and successively two kinds of vertebral body replacements were integrated into the computer model. The model was loaded with pure moments in the three anatomical main planes as well as with forces that are expected during standing. A vertebral body replacement drastically reduces the mobility in the implant region. An implant has only a minor influence on the mobility and stresses in the adjacent regions for the loading cases chosen. A preload, for example, caused by additional distraction from the precisely fitted ventral implant, exerts a very strong effect on the stresses in the end plates that are in contact with the implant. Different sizes of the contact area between implant and vertebral body have only a local effect on stress distribution.

Computer Simulation↗
PubMed2002

Genetic instability in Hodgkin's lymphoma.

Genetic instability is a characteristic feature of the malignant Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin's lymphoma and the lymphocytic and histiocytic (L&H) cells in lymphocyte predominant Hodgkin's lymphoma. Genetic instability can be classified into four major categories: distinct DNA mutations (microsatellite instability); numerical aberrations (chromosomal instability); structural aberrations (translocation instability); and gains and losses of chromosomal regions. In Hodgkin's lymphoma (HL), HRS cells and L&H cells show somatically mutated clonally rearranged immunoglobulin genes, thus characterizing these cells genetically as germinal center B cells. These cells furthermore show mutations of oncogenes and tumor suppressor genes in some cases (p53, IkappaBalpha, CD95/Fas). They do not, however, display microsatellite instability, as they have a proficient mismatch repair machinery. In contrast, HRS and L&H cells frequently harbor recurrent but not specific numerical and structural aberrations as detected by classical cytogenetics and fluorescence in situ hybridization analysis. Results from molecular genetic studies using comparative genomic hybridization and allelotyping (LOH) indicate typical genetic patterns in HL with gains and losses of distinct chromosomal regions. In some instances, candidate genes possibly involved in the malignant transformation of HRS cells and L&H cells have been characterized (JAK2, c-REL, MDM2). In summary, using molecular genetics it might be possible in the near future to elucidate some of the complex genetic instabilities observed in HL.

Chromosome Aberrations↗
PubMed2002

Prognostic factors in Hodgkin's lymphoma.

Risk-adapted treatment strategies have constituted a major issue since the beginning of clinical research into Hodgkin's disease (HD). Various prognostic factors have been identified and several of those considered for staging procedures, resulting in strictly stage-dependent treatment recommendations for patients suffering from HD. These factors may be subdivided in host-related (e.g. age, sex) and tumour-related (e.g. number of tumour cells, growth characteristics, spread of tumour cells, resistance to apoptosis) factors. Owing to the striking improvement of the overall prognosis in HD patients it may be difficult to identify novel prognostic factors analysing the minority of patients with a fatal outcome. However, especially in advanced-stage disease, improved treatment results were achieved by the introduction of more aggressive treatment regimens, resulting in an increased toxicity rate. Thus, partially in contrast to earlier work in this field, future prognostic factors are needed for identification of those patients that have a good prognosis and might be susceptible to overtreatment. During the Fifth International Symposium on Hodgkin's Lymphoma, promising results on several new prognostic markers were presented. Furthermore, a joint effort to design new studies on large, well characterised patient groups has been initiated.

Biomarkers, Tumor↗
PubMed2001

Loss of heterozygosity in the Hodgkin-Reed Sternberg cell line L1236.

Hodgkin-Reed Sternberg cells are derived from germinal centre B-cells in most cases. Somatic mutations affecting their rearranged immunoglobulin genes were detected, rendering potential functional rearrangements non-functional. Under physiological conditions such cells would be designated to undergo apoptosis within the germinal centre. In search for the specific transforming event that prevents Hodgkin-Reed Sternberg cells from programmed cell death, cytogenetic analyses were broadly performed but did not reveal specific chromosomal aberrations. Analysis of these cells on the molecular level is difficult to perform due to the scarcity of the cells in the lymphoma tissue and the given limitations of in situ studies. To overcome these limitations, the cell line L1236, known to be derived from Hodgkin-Reed Sternberg cells in situ, was chosen for allelotype analysis. Using a panel of microsatellite loci assigned to nearly all chromosomal arms, regions of loss of heterozygosity were detected on chromosomal arms 6p, 9q and 17p. The size of lost segments was estimated by amplification of additional microsatellite loci mapped to the respective regions. Further analyses of single Hodgkin-Reed Sternberg cells will reveal whether LOH affecting these regions is a recurrent event in HD and to which extent the smallest commonly affected region can be estimated.

Alleles↗
PubMed2001

Estimation of muscle forces in the lumbar spine during upper-body inclination.

OBJECTIVE: To estimate the muscle forces during upper-body inclination and to determine their influence on stress distribution in the annulus fibrosus of the lumbar spine discs. DESIGN: The muscle forces and stresses were calculated using a non-linear finite element model of the lumbar spine. BACKGROUND: Little is known about the influence of muscle forces on the deformation of, and stresses in, the lumbar spine. In most studies, muscle forces are neglected. METHODS: Three-dimensional non-linear finite element models of the ligamentous lumbar spine, with and without internal spinal fixators, were created. They were validated by use of experimental data from in vitro measurements on cadaver specimens. In a second step, the influence of muscle forces on stresses in the annulus fibrosus of the lumbar spine discs was investigated in a parameter study. This was done for different inclination angles of the upper-body. RESULTS: Good agreement between analytical and experimental results proved achievable when loading with pure moments in the three main planes of the lumbar spine. For inclination of the upper-body, the flexion angle clearly has a strong influence on the stresses in the lumbar spine while the influence of local muscles was small. The stress distribution in the discs differed considerably when the muscle forces are neglected and only a pure moment is applied. CONCLUSIONS: This study confirmed earlier ones that have shown that muscle forces should not be neglected when studying the stresses in the lumbar spine. The local dorsal muscles, however, have only a small influence on the stresses in the discs. RELEVANCE: For investigations of the biomechanical effects of spinal implants and surgical procedures, experimental or analytical methods are used. Due to the complexity involved, as well as to a lack of information, muscle forces are often neglected. Our study showed that muscles do in fact have a major influence on the mechanical behaviour of the spine and should always be taken into account.

Biomechanical Phenomena↗
PubMed2000

[Immunohistochemical detection of complement factors: a reliable method for the diagnosis of myasthenia gravis].

A 57-year-old woman suffered from generalized muscular weakness without diplopia or dysphagia for about 2 years. An abnormal decremental response on low frequency nerve stimulation and improvement upon administration of edrophonium chloride suggested a diagnosis of myasthenia gravis. However, this diagnosis remained uncertain, since repeated tests for antiacetylcholine receptor antibodies were negative. In addition, the patient reported that she was never able to keep up with peers in prolonged physical activities since childhood. For this reason, a congenital myasthenic syndrome was suspected and an intercostal muscle biopsy performed for special end-plate studies. Immunohistochemistry of the muscle biopsy revealed membrane attack complex deposits at the end-plates. This finding definitely confirmed the diagnosis of autoimmune myasthenia gravis. In conclusion, immunohistochemistry for complement deposits at the end-plates is a simple and reliable method of confirming the diagnosis of myasthenia gravis in uncertain cases.

Biopsy↗
PubMed1999

Somatic mutations within the untranslated regions of rearranged Ig genes in a case of classical Hodgkin's disease as a potential cause for the absence of Ig in the lymphoma cells.

Hodgkin-Reed-Sternberg (H-RS) cells are clonal B cells carrying Ig gene rearrangements. However, in situ hybridization methods failed to demonstrate Ig gene expression in H-RS cells of classical Hodgkin's disease (HD). Because somatic mutations rendering potentially functional Ig gene rearrangements nonfunctional were detected in some cases of the disease, it was speculated that H-RS cells in classical HD may have lost the ability to express antigen receptor as a rule. Recently, we established a novel cell line (L1236) from H-RS cells of a patient with mixed cellularity subtype of HD. L1236 cells harbor a potentially functional VH1 and a potentially functional Vkappa3 gene rearrangement. However, no antibody expression was detected. To show potential reasons for this lack of Ig expression, we analyzed the genomic organization of the Ig genes and their transcription in the primary and cultivated H-RS cells of this patient. The H-RS cells were found to have switched their isotype to IgG4, confirming their mature B-cell nature. By amplifying cDNA from L1236 cells as well as from frozen biopsy material transcripts of the Vkappa3 and the VH1 gene rearrangement were detected for both sources of cDNA. However, Northern blot hybridization of L1236 RNA failed to demonstrate VH1 and Vkappa3 transcripts, indicating only a low level of transcription. Sequence analysis of the promoter and leader regions of the VH1 gene rearrangement from L1236 cells as well as from lymphoma-affected tissue showed a somatic mutation in the conserved octamer motif of the promoter region. Somatic mutations were also detected within the 3' splice site of the leader intron and adjacent nucleotides in the rearranged Vkappa light chain gene, leading to aberrant splicing. These mutations might prevent the generation of adequate amounts of functional Ig gene transcripts as template for translation into protein. Thus, mutations in H-RS cells that prevent Ig gene expression might also be located outside the coding region of the Ig genes.

Base Sequence↗
PubMed1999

A new heat shock protein that binds nucleic acids.

We describe the isolation of Hsp15, a new, very abundant heat shock protein that binds to DNA and RNA. Hsp15 is well conserved and related to a number of RNA-binding proteins, including ribosomal protein S4, RNA pseudouridine synthase, and tyrosyl-tRNA synthetase. The region shared between these proteins appears to represent a common, but previously unrecognized, RNA binding motif. Filter binding studies showed that Hsp15 binds to a 17-mer single-stranded RNA with a dissociation constant of 9 microM in 22.5 mM Hepes, pH 7. 0, 5 mM MgCl2. A role of Hsp15 in binding nucleic acids puts this protein into a different functional category from that of many other heat shock proteins that act as molecular chaperones or proteases on protein substrates.

Amino Acid Sequence↗
PubMed1998

Reconstitution of a protein disulfide catalytic system.

Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA. Here we demonstrate the direct reoxidation of DsbA by DsbB. We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion. The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction. Membranes from a dsbB null mutant display no DsbA reoxidation activity. The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a Km = 10 microM. The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.

Catalysis↗