Biomedical subjects

S Huber

Publications and source records attributed to S Huber.

At least 37 records · Page 2Linked to original sources

PubMed1998

Analysis of mouse glomerular podocyte mRNA by single-cell reverse transcription-polymerase chain reaction.

Selective investigation of glomerular podocytes is not possible using conventional methods in vivo. Analysis of glomerular epithelium-derived cells in culture yields dubious results because of the rapid dedifferentiation of podocytes. We developed a modification of the polymerase chain reaction (PCR) method previously used to analyze cultured neurons. Podocytes harvested from freshly dissected glomeruli are ideal target cells for this modified, single cell reverse transcription-PCR method to reproducibly identify specific mRNA species from resident intact podocytes in vivo.

Actins↗

PubMed1998

Expression of glucose transporters in human peritoneal mesothelial cells.

Glucose containing solutions, the basis of peritoneal dialysis fluids, affect the proliferation and regeneration of peritoneal mesothelial cells (MsC). The aim of this study was to examine mechanisms of glucose transport into MsC, that is, the expression of facilitative glucose transporters (GLUT) and the Na(+)-dependent glucose transporter (SGLT1) in human primary MsC and a transfected MsC line. Since expression of both transporters is differentiation dependent, we investigated the effects of cell differentiation induced by culturing MsC on membranes or by addition of hexamethylene bisacetamide (HMBA; 6 mM), which enhances SGLT1 expression in LLC-PK1 cells. Levels of mRNA for GLUT1 through GLUT4 and SGLT1 were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of the corresponding proteins was examined by Western blotting and localized by immunofluorescence. Active, Na(+)-dependent glucose transport was assessed by alpha-methyl-D-[14C]glucopyranoside (AMG) with and without the SGLT1-specific inhibitor phlorizin and by patch clamp experiments in NaCl or choline-chloride, For Na(+) dependent glucose uptake choline chloride instead of NaCl served as negative control. Facilitative transport was assessed using 2-fluoro-2-deoxy-[14C]-D-glucose (FDG) with and without the inhibitors cytochalasin B or phloretin. Primary and transfected MsC express GLUT1 and GLUT3 mRNA while no transcripts were found for GLUT2 and GLUT4. No SGLT1 transcript was detectable in subconfluent cells. Semiquantitative RT-PCR analysis documented that the addition of the differentiation inducer HMBA to confluent cultures or growth of MsC on membranes for seven days produced a down-regulation of mRNA for GLUT1, no change for GLUT3, and a substantial increase for SGLT1 mRNA. Under these conditions MsC express SGLT1 protein and possess a Na(+)-dependent glucose uptake as assessed by AMG. Phlorizin (1 mM) inhibits AMG uptake by 30 to 40%. In patch clamp experiments the addition of extracellular glucose depolarized the membrane potential only in the presence of sodium. These results indicate that differentiated MsC express GLUT1, GLUT3, and SGLT1. Further characterization of these transport mechanisms and their regulation may help to understand the cellular effects of glucose on MsC in peritoneal dialysis.

Base Sequence↗

PubMed1998

Chloride channels ClC-2 and ICln mRNA expression differs in renal epithelial ontogeny.

Development-dependent mRNA expression of the chloride channels ClC-2 and ICln was studied by quantitative reverse transcriptase-polymerase chain reaction in rat ureteric bud and cortical collecting duct primary monolayer cultures. Abundance of ClC-2 mRNA increased in ureteric bud cells between embryonic day 15 (E15) and E17, peaked at postnatal day 3 (P3), and was down-regulated at P7 when morphogenesis is complete, suggesting a specific embryonic function. Expression of ICln mRNA, in contrast, up-regulated continuously with development.

Animals↗

PubMed1998

Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes.

Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms' tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease.

Animals↗

PubMed1998

Matrix metalloproteinase-9 (gelatinase B) is selectively elevated in CSF during relapses and stable phases of multiple sclerosis.

Matrix metalloproteinases (MMPs) are a family of endopeptidases capable of enzymatic digestion of subendothelial basement membrane and other components of the extracellular matrix. Expression of MMP-2, -3, -7 and -9 is increased around multiple sclerosis plaques and in brain tissue in experimental allergic encephalomyelitis. To measure quantitatively the expression of these MMPs and their endogenous inhibitors (TIMP-1 and -2), we analysed samples from 52 patients with relapsing-remitting and primary progressive multiple sclerosis by ELISA (enzyme-linked immunosorbent assay) and substrate-gel electrophoresis (zymography). MMP-9 was increased over controls in 100% of relapsing-remitting multiple sclerosis cases, with similar levels detected in relapses and clinically stable phases of disease. In primary progressive multiple sclerosis, MMP-9 was increased in 57% of CSF samples, but concentrations were below those encountered in the relapsing-remitting form. The selective upregulation of MMP-9 suggests that T-cells and macrophages invading the brain parenchyma and the CSF space are the predominant source of MMP-9 in multiple sclerosis. TIMPs and other MMPs (MMP-2 and -3) were not upregulated or not detectable (MMP-7) in CSF of patients with relapsing-remitting and primary progressive multiple sclerosis. The sustained increase of MMP-9 in clinically stable multiple sclerosis supports the concept that multiple sclerosis is associated with ongoing proteolysis that may result in progressive tissue damage. The selective inhibition of MMP-9 could be a useful approach for the prevention of disease progression in multiple sclerosis.

Cerebrospinal Fluid↗

PubMed1998

Computer assisted retraining of attentional impairments in patients with multiple sclerosis.

OBJECTIVE: To evaluate the efficacy of a computer based retraining of specific impairments of four different attentional domains in patients with multiple sclerosis. METHODS: Twenty two outpatients with multiple sclerosis received consecutively a specific training comprising 12 sessions in each of the two most impaired attention functions. The baseline of attentional deficits, the performance after each training period, and the course of performance in the next nine weeks was assessed by a computerised attention test battery. Additionally, the impact of the training on daily functioning was evaluated with a self rating inventory. RESULTS: Subgroups of patients with multiple sclerosis showing different patterns of attentional impairment could be separated. Significant improvements of performance could almost exclusively be achieved by the specific training programmes. The increase of performance remained stable for at least nine weeks. For quality of life patients reported less attention related problems in everyday situations. CONCLUSIONS: In patients with multiple sclerosis it seems worthwhile to assess attentional functions in detail and to train specific attention impairments selectively.

Activities of Daily Living↗

PubMed1998

Effects of a microbubble contrast agent on breast tumors: computer-assisted quantitative assessment with color Doppler US--early experience.

PURPOSE: To evaluate objectively the effects of a microbubble contrast agent on the color Doppler ultrasound (US) examination of breast lesions. MATERIALS AND METHODS: Forty-seven patients aged 23-71 years underwent color Doppler US before and after intravenous injection of a microbubble contrast agent. A 3-minute computer-assisted assessment of the color pixel density (CPD) was used to evaluate objectively the increase in the number of color Doppler US signals, the transit time of the microbubble bolus, and the potential additional differential diagnostic information. RESULTS: Peak CPD at contrast agent-enhanced color Doppler US was 14.3% +/- 8.1 (mean +/- 1 standard deviation) for carcinomas and 9.3% +/- 4.9 for benign lesions (P = .04). The time to peak enhancement was shorter in carcinomas (38 seconds +/- 20) than in benign tumors (71 seconds +/- 48, P = .02). Final CPD was close or equal to baseline values. With the median of 13% for peak CPD as a threshold, the sensitivity for this parameter was 55%, the specificity was 79%, and the accuracy was 62% (P = .04). For a median time to peak of 50 seconds, the sensitivity was 84%, the specificity was 57%, and the accuracy was 76%. CONCLUSION: After microbubble contrast agent injection, carcinomas and benign lesions behave differently in degree, onset, and duration of Doppler US enhancement. High interindividual variability and temporal variations in the Doppler US signal still limit the value of these criteria for prospective diagnosis.

Adult↗

PubMed1998

Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip.

Embryonic epithelia at the tip of the ureteric bud (UB) face the interspace between epithelial and mesenchymal cells and are fundamentally involved in reciprocal signaling during early nephrogenesis. To characterize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17 (day E17)] microdissected from the outer cortex. Cells at the UB tip had a high whole cell conductance (14 +/- 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in nonepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective current fraction, by contrast, activated slowly during strong hyperpolarization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipette, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 (out of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two different lengths were obtained, which might be due to two alternatively spliced ClC-2 mRNA isoforms. This first and combined approach by patch-clamp and single cell RT-PCR techniques to embryonic epithelia indicates that 1) cells at the UB tip express a phenotype remarkably different from that of postembryonic collecting duct principal cells and that 2) ClC-2 is likely to have a key role in early nephrogenesis.

Animals↗

PubMed1998

Activated protein C resistance assay performance: improvement by sample dilution with factor V-deficient plasma.

OBJECTIVE: To evaluate a modification of a commercially available reagent kit (COATEST APC Resistance Kit) for functional activated protein C (APC) resistance testing, and to determine the ability of the modified assay to demonstrate APC resistance in patients receiving warfarin. DESIGN: Functional APC resistance testing was performed using both the first-generation COATEST APC Resistance Kit and a modified, or second-generation, version of the COATEST assay that uses predilution of the patient sample with factor V-deficient plasma. Factor V genotyping for APC resistance (FV R506Q) was performed using a well-characterized polymerase chain reaction-restriction fragment length polymorphism method. SETTING: University medical center. PATIENTS: Seventy-three individuals referred for hypercoagulability testing who were not receiving warfarin therapy and 29 patients with a history of venous thrombosis who were receiving warfarin therapy. MAIN OUTCOME MEASURE: Sensitivity and specificity as determined by comparing functional APC resistance to the FV R506Q genotype. RESULTS: In 73 patients referred for hypercoagulability testing, but not receiving warfarin therapy, a sensitivity of 0.86 and a specificity of 0.75 were obtained with the first-generation COATEST assay. In contrast, a sensitivity and specificity of 1.0 were obtained when the second-generation COATEST assay was employed. In 29 patients receiving warfarin, the first-generation assay exhibited a sensitivity and specificity of 0.88 and 0.95, respectively, whereas the sensitivity and specificity for the second-generation assay was 1.0. CONCLUSIONS: Predilution of patient plasma with factor V-deficient plasma results in improved sensitivity and specificity of the COATEST APC Resistance Kit, thus offering a simple modification to enhance APC resistance determination in the routine clinical laboratory setting.

Blood Coagulation Disorders↗

PubMed1997

Reciprocal expression of myelin-associated glycoprotein splice variants in the adult human peripheral and central nervous systems.

The L- and S-MAG isoforms differ only at their C-terminus and are believed to be functionally distinct. To obtain information on the relative expression of these alternatively spliced isoforms in humans, we cloned an S-MAG cDNA fragment. The deduced amino-acid sequence of the human S-MAG C-terminus shows fairly conservative substitutions of 4 out of the 10 residues compared to the rodent peptide. Using reverse transcription and a competitive polymerase chain reaction, we show that, in contrast to rodents, the L-MAG splice variant predominates in adult human brain while, like in rodents, S-MAG transcripts are most abundant in peripheral nerve. The results obtained by Western blot analysis and immunohistochemistry are in good agreement with the findings at the mRNA level. Animal experiments may thus be more representative for the role of MAG in human nerve than in brain.

Adult↗

PubMed1997

[Diagnosis and differential diagnosis in multiple sclerosis].

The diagnosis of multiple sclerosis (MS) relies on the occurrence of characteristic symptoms, on the patient's history and on the correct interpretation of nowadays very sensitive but not very specific auxillary examinations. This paper reviews the diagnostic criteria and typical signs and symptoms of the disease. The significance of cerebrospinal fluid (CSF) analysis, magnetic resonance imaging (MRI) and evoked potentials is discussed, with emphasis on their predictive value concerning the development of MS after a first episode with symptoms suggestive of MS. A wide range of other diseases mimicking MS, like infectious, autoimmune, granulomatous, metabolic and hereditary diseases is reviewed. A checklist for important points in patient history and further investigations is proposed.

Adult↗

PubMed1997

Epithelial nephrogenesis.

The metanephric, or definitive, kidney forms as a result of inductive processes between tissues of two distinct embryologic origins, the metanephric mesenchymal blastema and the ureteric bud. After inductive signalling between these primordial tissues, mesenchymal cells aggregate next to the branching ureteric bud tip, convert to epithelial cells and differentiate into the diverse cell populations of the nephron. The ureteric bud enters branching morphogenesis and gives rise to the collecting duct system. Nephrogenesis has become a target system by which to study developmental processes including embryonic inductive interactions, mesenchyme-to-epithelium conversion, cell lineage pathways, epithelial cell polarization, branching morphogenesis and spatio-temporal expression of transcription factors. This review summarizes data on cellular and extracellular events during epithelial metanephrogenesis.

Animals↗

PubMed1997

Reduced glucose metabolism in the frontal cortex and basal ganglia of multiple sclerosis patients with fatigue: a 18F-fluorodeoxyglucose positron emission tomography study.

To investigate the pathophysiology of fatigue in MS, we assessed cerebral glucose metabolism (CMR-Glu) in 47 MS patients using PET and 18F-fluorodeoxyglucose. Applying the Fatigue Severity Scale (FSS), we first compared MS patients with severe fatigue (MS-FAT, n = 19, FSS > 4.9) and MS patients without fatigue (MS-NOF, n = 16, FSS < 3.7) on a pixel-by-pixel basis using Statistical Parametric Mapping (SPM95). Second, we compared FSS values of all 47 patients covering the whole range of this scale with CMRGlu using an analysis of covariance (SPM95). In addition, we determined global CMRGlu by region-of-interest analysis. Sixteen healthy subjects served as control subjects (CON). Global CMRGlu was significantly lower in both MS groups compared with CON (CON 43.3 +/- 6.9 mumol/100 mL/min, MS-FAT 34.7 +/- 4.4, MS-NOF 35.4 +/- 4.5) but was not related to fatigue severity. Comparing the two MS groups, SPM95 analysis revealed predominant CMRGlu reductions bilaterally in a prefrontal area involving the lateral and medial prefrontal cortex and adjacent white matter, in the premotor cortex, putamen, and in the right supplementary motor area of MS-FAT. In addition, there were CMRGlu reductions in the white matter extending from the rostral putamen toward the lateral head of the caudate nucleus. FSS values were inversely related to CMRGlu in the right prefrontal cortex. CMRGlu in the cerebellar vermis and anterior cingulate was relatively higher in MS-FAT than in MS-NOF patients. CMRGlu of both regions showed positive correlations with FSS values. Our data suggest that fatigue in MS is associated with frontal cortex and basal ganglia dysfunction that could result from demyelination of the frontal white matter.

Adult↗

PubMed1996

[Multiple sclerosis: therapy with recombinant beta-1b interferon: initial results with 30 multiple sclerosis patients in northwest Switzerland].

Recombinant interferon beta-1b has been registered with the Swiss health authorities since August 1995. Due to a special arrangement with health insurances it has been possible to prescribe this medication since spring 1995. We report on our experience with the first 30 consecutively treated multiple sclerosis patients. Indication, adverse event profile and clinical response to treatment are described. The most common side effects were local injection site reactions (63%), influenza-like symptoms (50%) and fatigue (33%). As compared to the prestudy period we observed a 49% reduction in the exacerbation rate. Compliance was excellent, possibly due to strict selection and extensive information about possible effects and side effects.

Adjuvants, Immunologic↗

PubMed1996

Differential expression of perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis.

Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8+ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3+CD8+ and CD3+ CD4+ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8+ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest (a) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and (b) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin- and secretion-dependent mechanism of muscle fiber injury.

Base Sequence↗

PubMed1996

Resistance to Activated Protein C: Comparison of Three Different PCR Methods for Detection FV R506Q.

Background: Resistance to activated protein C(APC) is the most prevalent identifiable risk factor for inherited thrombophilia. Over 90% of APC resistance results from a single point mutation in the Factor V gene. The mutation, termed FV R506Q of FV Leiden, predicts an abnormal Factor Va protein in which arginine, at amino acid position 506, is replaced by glutamine, rendering Factor Va resistant to proteolytic inactivation by APC, thus establishing a life long hypercoagulable state. The current study compared three different polymerase chain reaction (PCR)-based approaches for the detection of FV R506Q. Methods and Results: Sixty-seven patient blood samples were genotyped by (1) analyzing for loss of a Mnl I recognition site, an acquired restriction-fragment-length polymorphism (RFLP) due to FV R506Q; (2) primer-engineered RFLP wherein the presence of FV R506Q results in generation of a novel Nla III recognition site; and (3) allele-specific PCR. Sixty-five of 67 patient samples yielded concordant genotype results by all three PCR methods. Of the remaining 2 of the 67 patients, a "nondiagnostic" result was obtained for either allele-specific PCR or primer-engineered RFLP. Conclusions: A comparative analysis of 67 patient samples demonstrated that primer engineered RFLP and allele-specific PCR offer feasible alternative or confirmatory testing approaches to Mnl I RFLP for the detection of FV R506Q. A high degree of diagnostic concordance was observed for all three methods, and no false positive or negative results were observed with the Mnl I RFLP technique.

Journal Article↗

PubMed1996

Factor V R506Q gene mutation analysis by PCR-RFLP: optimization, comparison with functional testing for resistance to activated protein C, and establishment of cell line controls.

Resistance to activated protein C (APC) has been recently identified as a highly prevalent risk factor for the development of venous thrombosis. In the majority of cases, APC resistance correlates with the presence of a single point mutation in the factor V gene (FV R506Q). The mutation is present in 3% to 5% of the general population and in up to 50% of patients with a personal and family history of venous thrombosis. In the current study, the authors have optimized and implemented for clinical diagnosis a method for detection of FV R506Q using the polymerase chain reaction coupled with restriction fragment length polymorphism analysis (PCR-RFLP). Forty-one healthy adults and 139 patients referred for hypercoagulability testing were genotyped and their APC resistance ratios determined using commercially available reagents (COATEST APC Resistance Kit). Comparative analysis indicated that if functional APC resistance was defined as per manufacturer's guidelines, a significant number of individuals with a normal factor V genotype were categorized as APC resistant and conversely, a significant number of individuals heterozygous for FV R506Q were categorized as non-APC resistant. These results indicate that comparative functional and genotypic analyses in the individual clinical laboratory setting are critical for establishing normal ranges and cut-off values for functional APC resistance due to FV R506Q. To facilitate molecular evaluation of APC resistance, Epstein-Barr virus (EBV) immortalized B-lymphocyte cell lines were established from individuals heterozygous and homozygous for FV R506Q.

Adult↗

PubMed1996

Detection of clonal immunoglobulin heavy-chain gene rearrangements in cases of suspected lymphoproliferative disorders: comparison of polymerase chain reaction and Southern blot analysis.

Demonstration of clonality is supportive of a diagnosis of malignancy in cases of lymphoproliferative disorders. Determination of clonality at the molecular level is currently accomplished by Southern analysis; however, the polymerase chain reaction offers a potential alternative that is rapid, simple, and less expensive. To test its feasibility as a diagnostic test, we amplified the DNA from 121 suspected lymphoproliferative disorders submitted for gene rearrangement studies. In comparison to Southern analyses, a sensitivity of 70% and specificity of 96% were obtained. To test the effect of primer variability in the joining region of the heavy-chain gene, we substituted a more degenerate primer but found no changes in sensitivity or specificity. We conclude that the polymerase chain reaction has current application with minute or fixed specimens and may generally serve as a rapid, initial evaluation for B-cell clonality, followed by Southern analysis in negative cases. However, higher overall sensitivity must be achieved before this technique can replace Southern analysis as the method of choice in determining clonal gene rearrangements.

Base Sequence↗