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Biomedical subjects

G Wolf

Publications and source records attributed to G Wolf.

At least 19 recordsLinked to original sources
PubMed1999

In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits.

In vivo predegeneration of peripheral nerves is presented as a convenient and effective method to obtain activated Schwann cells and an enhanced cell yield following in vitro cultivation. The experiments conducted in rats were aimed at clinical use in gaining Schwann cell suspensions for filling artificial conduits in order to bridge peripheral nerve gaps. The rat sciatic nerve used as a model was transected distally to the spinal ganglia. Predegeneration in vivo was allowed to take place for 1, 2, 3 and 4 days and up to 1, 2 and 3 weeks. The nerve was then resected and prepared for cell cultivation. Schwann cells cultivated from the contralateral untreated nerve served as control. Immunostaining for S100, nerve growth factor receptor and the adhesion molecules N-cadherin and L1 was used to characterize the general state of the cultures. Viability was assessed by fluorescein fluorescence staining, and the proliferation index was determined by bromodeoxyuridine-DNA incorporation. The Schwann cells from predegenerated nerves revealed an increased proliferation rate compared to the control, whereas fibroblast contamination was decreased. Best results were obtained 1 week after predegeneration.

Animals↗
PubMed1999

Tyramide signal amplification in brain immunocytochemistry: adaptation to electron microscopy.

The tyramide signal amplification (TSA) technique is well-established in light microscopic immunohistochemistry and in situ hybridization to improve the signal-to-noise ratio. The present study deals with its adaptation to the electron microscopic level using the pre-embedding technique and a modified protocol. The outcome of immunolabeling of most of the antigens tested in brain tissue, including endothelial and neuronal nitric oxide synthase, glial fibrillary acidic protein, and isolectin B4, was greatly improved. If signal amplification is required, the TSA-technique proved to be reliable with high specificity and good ultrastructural resolution.

Antibody Specificity↗
PubMed1999

Peroxynitrite mediated damage and lowered superoxide tolerance in primary cortical glial cultures after induction of the inducible isoform of NOS.

The effect of the induction of i-NOS in primary glial cultures was studied with respect to the protein levels of reactive oxygen species (ROS) scavenging enzymes and the cytotoxicity of nitric oxide (.NO) formation at different levels of artificially generated superoxide. Stimulation of the cultures by bacterial lipopolysaccharides and gamma-interferon resulted in an induction of i-NOS exclusively in microglial cells. Among the ROS scavenging enzymes superoxide dismutase (Cu/Zn- and Mn-isoform), glutathione peroxidase and catalase only mitochondrial Mn-SOD was found to be upregulated in the course of i-NOS induction (Western blots). Although .NO formation did not affect cell viability at physiological levels of superoxide over a time period of 4 days, it caused an oxidative load particularly in microglial cells as observed by monitoring the oxidation of dichloro-dihydrofluorescein, an indicator for the formation of peroxynitrite and ROS. Elevated levels of superoxide, generated either intracellularly by paraquat or extracellularly via xanthine oxidase and hypoxanthine, resulted dose-dependently in a larger decline of cell viability in the .NO forming cultures compared to controls (release of lactate dehydrogenase, citrate synthase, stainability by propidium iodide, and tetramethylrhodamine). NOS-inhibitors reduced the degree of cell damage to that seen for control cultures, indicating an ONOO--/.NO mediated mechanism of cell damage. Our data support the concept that i-NOS catalyzed .NO-formation leads to an ONOO--mediated increased oxidative load. At physiological levels of superoxide and within a wide range of higher superoxide levels this nitrosative stress is well balanced in cultured glial cells by protective mechanisms.

Animals↗
PubMed1999

Urokinase plasminogen activator receptor (CD87) expression of tumor-associated macrophages in ductal carcinoma in situ, breast cancer, and resident macrophages of normal breast tissue.

UNLABELLED: Macrophages concentrate urokinase-type plasminogen activator (uPA) at the cell surface by expressing urokinase receptors (uPAR) in order to focus the pericellular space plasminogen-dependent proteolysis important in matrix remodeling and cell movement. This study examines the uPAR levels of tumor-associated macrophages (TAM) of invasive breast carcinomas, of TAMs from ductal carcinoma in situ (DCIS) and of macrophages derived from normal (non-tumor) breast tissue. TAMs from invasive breast carcinomas (n = 30), from DCIS (n = 12), and macrophages from normal breast tissue (n = 30) were cultured and immunocytochemically phenotyped by using a panel of antibodies. Urokinase receptor levels were determined by Western blot analysis and in cell-free supernatants by enzyme-linked immunosorbent assay. Urokinase receptor cell surface fluorescence intensity was determined by FACS and by confocal laser scan microscopy. Urokinase-receptor mRNA was detected by in situ hybridization. TAMs of invasive breast carcinomas and of DCIS possess significantly elevated uPAR levels compared with macrophages derived from normal breast tissue. CONCLUSIONS: activated macrophages with elevated uPAR levels belong to inflammatory areas in close vicinity of infiltrating and non-infiltrating (DCIS) tumor cells. Blood monocytes that possess elevated uPAR-levels may be selectively recruited from the bloodstream to inflammatory sites close to carcinoma cells, and/or breast cancer and precursor lesions may induce elevated uPAR-levels in TAMs by paracrine interactions.

Breast Neoplasms↗
PubMed1999

Angiotensin II stimulates expression of transforming growth factor beta receptor type II in cultured mouse proximal tubular cells.

Tubulointerstitial fibrosis is a common end-point of many chronic renal diseases and contributes to the permanent loss of renal function. There is increasing evidence that the profibrogenic cytokine transforming growth factor TGF-beta plays an essential role in this process by inducing the production of extracellular matrix proteins by tubular cells through an autocrine mechanism. We have previously demonstrated that the vasopeptide angiotensin (ANG) II induces TGF-beta transcription and synthesis in cultured murine proximal tubular cells (MCT cell line). Since the overall effects of TGF-beta on a distinct target cell may also depend on the expression of specific cell surface receptors, the present study was undertaken to test the hypothesis that ANG II modulates expression of TGF-beta receptors in MCT cells. ANG II stimulated protein expression of TGF-beta receptor type II, but not that of type I, in MCT cells as detected by immunofluorescence and western blotting of cell lysates. This stimulated receptor expression was also reflected in an overall increase in specific binding of 125I-labeled TGF-beta1 to intact MCT cells. Coincubation with ANG II and an AT1 receptor antagonist abolished this increase in 125I-labeled TGF-beta1 binding. Furthermore, ANG II also increased steady-state mRNA expression for TGF-beta receptor type II. This stimulation was transduced through AT1 receptors and was independent of TGF-beta released into the culture medium. Transient transfection studies using various length enhancer/promoter elements of the human TGF-beta receptor type II linked to the CAT gene revealed that AP1 sites are a necessary prerequisite for ANG II induced transcriptional activity. ANG II had no effect on TGF-beta receptor types I or II protein or on mRNA expression in syngeneic mesangial cells. Our results provide for the first time convincing evidence that ANG II upregulates TGF-beta receptor type II expression on proximal tubular cells. Since this subtype of receptor is primarily engaged in the initial binding of TGF-beta, an increased receptor expression may result in amplification of the TGF-beta effects on tubular cells. Interference with an activated renin-angiotensin system could therefore counteract the profibrogenic effects of TGF-beta by abolishing ANG II induced expression of TGF-beta receptor type II.

Angiotensin II↗
PubMed1999

[Staging of breast carcinoma. Value of diagnostic imaging].

In women with breast cancer, staging of the tumor plays an important role and often is considered to be the most prognostic and therapeutic factor. The most widely used staging system is based on the pathologic TNM classification. In recent years conservative therapies such as preoperative chemo- or radiotherapy are used more often with limited tissue evaluation resulting in insufficient or even inaccurate staging data. Tumor size as well as nodal involvement is decisive in selecting a treatment method and assessing the response. Thus, more accurate preoperative in vivo staging is necessary. This article therefore gives an overview of the different imaging modalities (mammography, ultrasonography, magnetic resonance imaging, and nuclear medicine) used for breast cancer staging. Furthermore, results of these methods are compared critically with the pathohistologic staging.

Breast↗
PubMed1999

Randomized in vitro and in vivo evaluation of different biopsy needles and devices for breast biopsy.

In an experimental study (in vitro and in vivo) we evaluated the efficacy of various biopsy needles/devices for breast biopsy. In vitro, biopsies of five human cadaveric breast specimens were performed using 33 different needles/devices ranging from 14 to 20-gauge. Of these 33 needles/devices, 22 optimally performing needles were selected for the in vivo study. In the clinical part of the study, 44 breast lesions were randomly biopsied with each of the 22 needles/devices under stereotactic guidance. Tissue specimens were analysed quantitatively and qualitatively. Several automatic long-throw guns (Acecut, Asap, Biopty, Magnum) obtained greater tissue areas and had a better histopathologic score than the conventional type of a side-notch needle like Trucut, an aspiration needle like Surecut, or an end-cut needle like Autovac. The automatic long-throw guns performed better than the short-throw Monopty gun. Regardless of needle size (14-20-gauge), breast biopsies should be routinely performed with automated long-throw side-notch guns (Acecut, Asap, Biopty, Magnum).

Adult↗
PubMed1999

Three-dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching.

OBJECTIVE: To develop a new approach to partial zona dissection of oocytes and embryos for facilitating preimplantation genetic sampling and assisted hatching. DESIGN: Controlled clinical study. SETTING: Preimplantation genetic diagnosis and IVF program, Reproductive Genetics Institute/IVF Illinois, Chicago, Illinois. PATIENT(S): Three hundred forty patients undergoing IVF in whom preimplantation genetic diagnosis or assisted hatching was required. INTERVENTION(S): Three-dimensional partial zona dissection and conventional partial zona dissection were performed, with the use of a simple microneedle, on embryos before preimplantation genetic sampling or on day 3 of embryo development before ET. MAIN OUTCOME MEASURE(S): Pregnancy rate, implantation rate, and ease of preimplantation genetic sampling. RESULT(S): A pregnancy rate of 42% and an embryo implantation rate of 17.6% were obtained in the group in which three-dimensional partial zona dissection was performed, compared with a pregnancy rate of 33% and an implantation rate of 14.7% in the control group, which underwent conventional partial zona dissection. Preimplantation genetic sampling can be performed without distortion of the blastomere configuration in the embryo. CONCLUSION(S): Three-dimensional partial zona dissection is a safe and simple mechanical means of creating an opening sufficient for the atraumatic removal of material from oocytes and embryos for preimplantation genetic diagnosis and assisted hatching.

Dissection↗
PubMed1999

The effect of the immunosuppressant FK 506 on peripheral nerve regeneration following nerve grafting.

Nerve allografts are highly antigenic and require the continuous use of immunosuppressive drugs. Neurotoxic complications from immunosuppressant therapy with FK 506 have been noted in the central and peripheral nervous system although an increased rate of axonal regeneration has also been noted. Regeneration of peripheral nerve grafts was assessed in a rat model clinically and morphometrically after treatment for 2 and 6 weeks with two different doses of FK 506. Good regeneration was noted in all groups at 6 weeks. A significantly higher axon count was observed in both the FK 506 groups after 2 weeks regeneration compared with controls. This beneficial effect was not evident after 6 weeks of regeneration. Whether this is related to a pruning mechanism or to a down-regulation of regenerative processes in the nerve due to possible neurotoxic effects of FK 506 remains unknown.

Animals↗
PubMed1999

Hyperuricemia and renal insufficiency associated with malignant disease: urate oxidase as an efficient therapy?

Hyperuricemia is a common finding in patients with malignant diseases. Chemotherapy can induce life-threatening tumor lysis syndrome with severe hyperuricemia, other metabolic abnormalities, and acute renal failure. Intrarenal precipitation of uric acid contributes to renal insufficiency in this situation. Allopurinol, by preventing the conversion of hypoxanthine and xanthine to uric acid, has been long considered the standard pharmacological approach to hyperuricemia and prevention of tumor lysis syndrome. However, allopurinol itself may facilitate precipitation of xanthine crystals and has little influence on already-formed uric acid crystals deposited in the kidney. Urate oxidase further oxidizes uric acid to the highly water-soluble allantoin in mammals, except humans, who lack this enzyme. We report four cases of hyperuricemia (initial serum uric acid concentrations, 14.0 to 25.0 mg/dL) associated with malignant diseases treated with exogenous urate oxidase. Two of the patients showed full-blown tumor lysis syndrome. A single urate oxidase infusion (1,000 U) readily reduced serum uric acid levels in all patients. Furthermore, renal insufficiency, determined by serum creatinine concentrations, improved in three of the four patients. No adverse effects were observed. Currently, a recombinant urate oxidase is undergoing clinical testing and may make this efficient therapy more widely available. We believe that treatment with urate oxidase is a safe and efficient therapy for patients with cancer-associated hyperuricemia and may be effective even in individuals with only moderately elevated serum uric acid concentrations.

Journal Article↗
PubMed1999

Effect of arginine on basal and high potassium-induced efflux of [3H]D-aspartate from rat striatal slices.

There are conflicting reports in the literature regarding the effects of nitric oxide as well as the involvement of the cyclic GMP pathway on the transmitter release. To study the influence of the availability of the nitric oxide precursor arginine on the glutamate transmission process, rat striatal slices preloaded with the tritiated glutamate analogue D-aspartate were used. L-Arginine stimulated in a concentration-dependent way (0.01-10.0 mM) the high potassium-induced efflux of [3H]D-aspartate. The basal release was increased only by 10 mM L-arginine. Neither the basal nor the depolarization-induced efflux of [3H]D-aspartate was affected by D-arginine. The L-arginine effect was abolished by the nitric oxide synthase inhibitor L-arginine methyl ester and was not modified by cyclic GMP. Only at high concentrations of L-arginine (10 mM) could an elevation of cyclic GMP level be demonstrated. The results are discussed in terms of direct presynaptic action of nitric oxide on [3H]D-aspartate efflux and a possible modulation of glutamate release by the availability of arginine.

Animals↗
PubMed1999

Selective targeting of somatostatin receptor 3 to neuronal cilia.

Recently, five members of the somatostatin receptor family have been cloned. However, little is known about their cellular and subcellular localization in the central nervous system. Using specific anti-peptide antisera, we observed somatostatin receptor 3-like immunoreactivity in many brain regions, including the cerebral cortex, hippocampus, hypothalamus, amygdala and cerebellum. In all of these regions (except for the cerebellar cortex), somatostatin receptor 3-like immunoreactivity was selectively targeted to 4-8-microm-long rod-shaped profiles which did not co-localize with axonal or dendritic markers. One immunoreactive profile was always associated with one neuronal cell body. This staining pattern was resistant to colchicine treatment and showed a closely overlapping distribution with somatostatin receptor 3 messenger RNA, suggesting that the receptor protein is not transported over long distances. Electron microscopic analysis revealed that somatostatin receptor 3-like immunoreactivity is localized to the plasma membrane of neuronal cilia which extended into an intercellular pocket and showed a 9+0 filament pattern in their basal body and proximal segments. Thus, somatostatin receptor 3 demonstrates a unique example of a G-protein-coupled receptor not localized to "classical" pre- or postsynaptic sites, but selectively targeted to neuronal cilia. The presence of the somatostatin receptor 3 receptor on neuronal cilia suggests that these presumably non-motile cilia may not merely represent developmental remnants, but rather function as chemical sensors of the immediate milieu.

Amino Acid Sequence↗
PubMed1999

Photodynamic effects induced by meso-tetrakis[4-(carboxymethyleneoxy)phenyl] porphyrin on isolated Sarcoma 180 ascites mitochondria.

Using mitochondria isolated from Sarcoma 180 ascites tumour in Swiss mice as a model system, we have evaluated the ability of a novel porphyrin, meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (H2T4CPP), to induce damage on photosensitization. Oxidative damage to mitochondria, one of the primary and crucial targets of the photodynamic effect, is assessed by measuring products of lipid peroxidation such as thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH), besides the loss of activity of the mitochondrial marker enzyme succinate dehydrogenase (SDH). Analysis of product formation, the effect of deuteration and selective inhibition by scavengers of reactive oxygen species (ROS) show that the damage observed is due mainly to singlet oxygen (1O2) and to a minor extent to hydroxyl radicals (.OH). The 1O2 generation and triplet lifetime of this porphyrin have also been estimated. Fluorescence spectroscopy, used to ascertain the binding of this porphyrin to the mitochondrial proteins, shows a rapid association within 0-2 h and a decline thereafter. Confocal microscopy reveals intracellular localisation of this porphyrin in cells in vitro. Our overall results suggest that the porphyrin H2T4CPP, due to its ability to bind to mitochondrial protein components and to generate ROS upon photoexcitation, may have potential applications in photodynamic therapy.

Animals↗
PubMed1999

Induction of permeability transition in pancreatic mitochondria by cerulein in rats.

Hyperstimulation with cholecystokinin analogue cerulein induces a mild edematous pancreatitis in rats. There is evidence for a diminished energy metabolism of acinar cells in this experimental model. The aim of this study was to demonstrate permeability transition of the mitochondrial inner membrane as an early change in mitochondrial function and morphology. As functional parameters, the respiration and membrane potential of mitochondria isolated from control and cerulein-treated animals were measured, and changes in volume and morphology were investigated by swelling experiments and electron microscopy. Five hours after the first injection of cerulein, the leak respiration was nearly doubled and the resting membrane potential was decreased by about 17 mV. These alterations were reversed by extramitochondrial ADP or did not occur when cyclosporin A was added to the mitochondrial incubation. A considerable portion of the mitochondria isolated from cerulein-treated animals was swollen and showed dramatic changes in morphology such as a wrinkled outer membrane and the loss of a distinct cristae structure. These data provide evidence for the opening of the mitochondrial permeability transition pore at an early stage of cerulein induced pancreatitis. This suggests that the permeability transition is an initiating event for lysis of individual mitochondria and the initiation of apoptosis and/or necrosis, as had been shown to occur in this experimental model.

Animals↗
PubMed1999

Prevention of age-related aneuploidies by polar body testing of oocytes.

PURPOSE: We previously demonstrated that aneuploidy-free oocytes may be preselected by testing the first and second polar bodies removed from oocytes following their maturation and fertilization. The present paper describes the results of the application of the method in 659 in vitro fertilization cycles from patients of advanced maternal age. METHODS: Using micromanipulation techniques, 3943 oocytes were tested by polar body sampling and fluorescent on situ hybridization analysis using specific probes for chromosomes 13, 18, and 21. RESULTS: Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of the aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. Most errors in the first meiotic division were represented by chromatid malsegregation. The transfer of embryos deriving from 1558 of 1829 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies and 88 healthy children born after confirmation of the polar body diagnosis. CONCLUSIONS: Polar body testing of oocytes provides an accurate and reliable approach for prevention of age-related aneuploidies in in vitro fertilization patients of advanced maternal age.

Adult↗
PubMed1999

Birth of healthy children after preimplantation diagnosis of thalassemias.

BACKGROUND: Preimplantation genetic diagnosis (PGD) allows couples at risk of having children with thalassemia to ensure the healthy outcome of their pregnancy. METHODS: Seventeen PGD clinical cycles were initiated for Cypriot couples at risk of having children with different thalassemia mutations, including IVSI-110, IVSI-6, and IVS II-745. Unaffected embryos for transfer were selected by testing oocytes, using first and second polar body (PB) removal and nested polymerase chain reaction analysis followed by restriction digestion. RESULTS: Unaffected embryos were selected in 16 of 17 PGD cycles. Of 166 oocytes studied from these cycles, 110 were analyzed by sequential analysis of both the first and the second PB, resulting in preselection and transfer of 45 unaffected embryos. This resulted in seven pregnancies and in the birth of five healthy thalassemia-free children. The embryos predicted to have inherited the affected allele were not transferred. Analysis of these embryos confirmed the PB diagnosis. CONCLUSIONS: Sequential first and second PB testing of oocytes is reliable for PGD of thalassemia and is a feasible alternative to prenatal diagnosis in high-risk populations.

Alleles↗
PubMed1999

Interaction of nitric oxide donors and ascorbic acid on D-[3H] aspartate efflux from rat striatal slices.

There are conflicting reports in the literature concerning the neuroprotective effect of ascorbic acid on excitotoxic processes in which excessive glutamate release and nitric oxide are supposed to be major factors. To study the influence of ascorbate on the nitric oxide modulated glutamate release rat striatal slices, preloaded with the tritiated glutamate analog D-aspartate, were used. The high potassium-induced efflux of D-[3H]aspartate was concentration dependently stimulated by the nitric oxide donors sodium nitroprusside, S-nitroso-N-acetylpenicillamine (SNAP) or 5-amino 3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1), as well as by solutions of gaseous nitric oxide and, interestingly, by cyanide. Only the stimulation of D-[3H]aspartate release by SNAP and nitroprusside was affected by ascorbate in terms of a highly significant potentiation. Ascorbate was shown to exert its effect primarily by influencing the decomposition of these nitric oxide donors rather than by a direct interaction of ascorbate with nitric monoxide on glutamate release.

Animals↗