Biomedical subjects

M Riehle

Publications and source records attributed to M Riehle.

15 recordsLinked to original sources

PubMed2001

Immunogold labelling of fibroblast focal adhesion sites visualised in fixed material using scanning electron microscopy, and living, using internal reflection microscopy.

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell-substrate interface was removed with detergent. The antigen blocking procedure, size of label (5 nm) and duration of silver-enhancement (6 min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided 'sectional' views through the cell. This enabled visualisation of vinculin exclusively within the cell-substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.

Acrylic Resins↗

PubMed2001

Tissue engineering: the biophysical background.

Tissue engineering is the construction, repair or replacement of damaged or missing tissue in humans and other animals. This engineering may take place within the animal body or as tissue constructs to be made in a bioreactor for later grafting into the animal. The minimal set of materials for this are the appropriate types of cell. Usually, however, non-living substrata are used as well. These substrata may be nothing more than materials that bulk up any voids in the damaged tissue and provide the mechanical strength that has been lost when the tissue is damaged or removed. They may serve a similar pair of functions in the bioreactor. They can do much more in terms of pattern formation. The orientations and morphology of the cells, the arrangement of intercellular material as it is laid down and the relationships between different cell types in the repairing or construct tissue are all of importance, for these should resemble the correct normal tissue as closely as possible. Most of these requirements are ones involving pattern formation. This review discusses the various ways in which tissue pattern can be engineered chiefly from a biophysical standpoint. Unpatterned cells are effectively not tissue. This engineering includes the use of topography on the substrata, chemical patterning of adhesive and other cues for the cells, mechanical force application to cause cell orientation and appropriate synthetic responses and electrical fields. The review also discusses the methods used to impart the appropriate cues to and through the materials which are often biodegradable polymers. The article gives particular attention to regions of research and practice where the involvement of the physicist or biophysicist is of importance.

Animals↗

PubMed2000

[C-reactive protein as an independent marker of prognosis in acute coronary syndrome: comparison with troponin T].

BACKGROUND: It has been suggested that inflammatory processes play a role in the pathogenesis of acute coronary syndromes (ACS). C-reactive protein (CRP) is a classic acute phase protein. It is yet unclear whether, in addition to established markers as troponin T (TnT), determination of CRP in patients admitted for ACS contributes significantly to the diagnosis and prognosis of ACS. PATIENTS AND METHODS: We investigated 50 patients with ACS (59.4 SD 13.9 years) in the first hour after admission and 4-24 h later with respect to TnT (Elecsys, Roche Diagnostics) and CRP (biokit, modified Quantex CRP plus, analytical sensitivity 0.02 mg/dL). Fifty percent of the patients were classified as having unstable angina retrospectively. All patients were followed in the 6 weeks post discharge regarding death and recurrent ACS. RESULTS: The cumulative event rate at 6 weeks after discharge was 62.5% for patients being CRP and TnT positive compared to 35.3% in TnT positive and CRP negative patients. In TnT negative patients a positive CRP test predicted 33.3% of events and 28.8% of patients negative for CRP and TnT had events at 42 days post discharge. Logistic regression analysis regarding the primary endpoint including TnT and CRP (4-24 h values), age, gender and diagnosis resulted in independent prediction of ACS or death by TnT (cutoff 0.1 microgram/L, p = 0.048, odds ratio = 7.5) and CRP (cutoff 0.862 mg/dL, p = 0.026, odds ratio = 5.3). Sensitivity/specificity for AMI diagnosis were 69.6%/75% for TnT and 12%/72% for CRP in the first hour and 91.3%/68.2% for TnT and 68%/72% for CRP 4-24 h later. CONCLUSIONS: Besides TnT, high sensitivity CRP determination has no additional value for early AMI diagnosis. The prognosis of these patients during the first 24 hours is significantly and independently predicted by CRP measurements in addition to troponin T.

Acute Disease↗

PubMed1999

Glimepiride (Hoe490) inhibits the rilmakalim induced decrease in intracellular free calcium and contraction of isolated heart muscle cells from guinea pigs to a lesser extent than glibenclamide.

Glibenclamide is a potent inhibitor of the ATP-dependent potassium channel. Opening of the ATP-dependent potassium channel is regarded as a mechanism of ischemic preconditioning. This in vitro study examines the influence of glibenclamide and glimepiride, a new sulfonylurea, on the negative inotropic action of the potassium channel opener rilmakalim in isolated ventricular myocytes. Cardiac myocytes were isolated from adult guinea pig hearts by collagenase perfusion and incubated with rilmakalim (concentration range 0.1-12.0 microM), glibenclamide (concentration range 0.03-3.0 microM) plus rilmakalim (3.0 or 7.5 microM), and glimepiride (0.03-9.0 microM) plus rilmakalim (3.0 or 7.5 microM) and paced by electrical field stimulation. Contractility of the myocytes was evaluated by digital image analysis, intracellular free calcium was determined by means of fura-2 fluorescence measurements, and cell viability was assessed morphologically as well as by measurement of lactate dehydrogenase activity. Rilmakalim reduced the systolic intracellular free calcium and contractility of ventricular myocytes in a concentration dependent manner. This effect was antagonized by glibenclamide at lower concentrations (0.3 microM) than glimepiride (3.0 microM). The smaller antagonistic action of glimepiride on the negative inotropic effect of rilmakalim as compared with glibenclamide most likely reflects a less potent inhibition of ATP-dependent potassium channels by glimepiride.

Adenosine Triphosphate↗

PubMed1999

Bioassay development: the implications of cardiac myocyte motility in vitro.

Cardiac myocytes cultured over microfabricated extracellular recording devices can be used to assay bioactive compounds. However, electrophysiological signals recorded from these devices vary in amplitude with time. Theoretically, changes in signal amplitude arise from myocytes being moved over recording sites by cocultured fibroblasts. To test this, neonatal rat cardiac myocytes were cultured at high densities and low densities on fibronectin-coated glass. After 36.5 h, myocytes were identified by their rhythmic contractions and then time-lapse-recorded for 3.5 h. Length, width, and angle of orientation was then determined every 30 min for five cells in low density and five cells in high-density culture. Low-density cells had mean lengths of 65.3 microm and widths of 35.1 microm, whereas cells in high-density culture had greater mean lengths of 74.2 microm and lower mean widths of 24.3 microm. Length, width, and angle of orientation of cells in low- and high-density culture changed by 4.1%, 11.8%, and 2.7 degrees, and 6.4%, 10%, and 4.6 degrees, respectively, every half hour. We found no evidence of myocyte-fibroblast interactions influencing cell position or shape in low density, but in high density, we found evidence that fibroblast-myocyte interactions could transiently influence cell shape. We conclude that fibroblast-independent changes in cell shape are largely responsible for the changes in signal amplitude recorded from cardiac myocytes cultured on microfabricated extracellular recording devices. However, there is some evidence that myocyte-fibroblast interactions may augment this process in high-density culture. The implications of these findings for bioassay development are discussed.

Animals↗

PubMed1998

A factor preventing melanization of sephadex CM C-25 beads in Plasmodium-susceptible and refractory anopheles gambiae.

One major quantitative trait locus controls melanization of both malaria ookinetes and Sephadex CM beads in a refractory strain of the mosquito, Anopheles gambiae. Hemolymph transferred from a nonmelanizing, Plasmodium-susceptible strain (4arr) to a melanizing, Plasmodium-refractory strain (L35) caused a reduction in the melanization of CM beads. In addition, when beads were first incubated in vivo in susceptible mosquitoes and then recovered, washed, and transferred to refractory mosquitoes, a strong reduction in melanization was observed. No changes in melanization were observed when beads or hemolymph were transferred in the opposite direction or within a strain. Incubation of beads in vitro in refractory or susceptible hemolymph resulted in a reduction of melanization when these beads were subsequently transferred to refractory mosquitoes. This reduction was significantly stronger when susceptible hemolymph was used as the incubating medium. Protection from melanization was observed after 3-, 6-, and 24-h incubations of beads in susceptible mosquitoes with longer incubations resulting in greater protection. Treatment of protected beads with 1 M NaOH resulted in the loss of the protection but treatment with 1% sodium dodecyl sulfate (SDS), 1% SDS/DTT/boiling, or 1 M NaOAc (pH 8.9) did not. These results show that a melanization-preventing factor covalently binds to the surface of CM beads in susceptible mosquitoes and can subsequently prevent melanization in refractory mosquitoes.

Animals↗

PubMed1998

Preliminary study on the suitability of a pharmacological bio-assay based on cardiac myocytes cultured over microfabricated microelectrode arrays.

There are a range of techniques that can be used to assay bioactive compound. One potentially promising technique is a system consisting of microfabricated extracellular recording devices over which electrogenic cells can be grown. To date, research in this area has concentrated on the use of neurons as an electrogenic cell type. However, these cells have limitations. Only small extracellular potentials have been recorded from mammalian neurons cultured over microfabricated electrode arrays. Although such potentials may be of use in assays examining the effects of bio-active compound analogues on firing frequency, they are of little use for more detailed pharmacological studies involving analyses of signal shape. What is required is a system from which much larger extracellular potentials can be recorded. This preliminary study reports on a system based on cardiac myocytes cultured over microfabricated metal microelectrode arrays, from which potentials with a mean amplitude of 16.9 microV can be reliably recorded, which can be reversibly blocked with mumoll-1 concentrations of the sodium ion channel blocker lidocaine. Less common potentials with amplitudes of up to 3.5 mV were also recorded. It is demonstrated that cardiac myocytes cultured over microfabricated micro-electrode arrays can be used in assays of cardioactive compound analogues.

Animals↗

PubMed1996

Synergistic and hierarchical adhesive and topographic guidance of BHK cells.

Guided cell movement is a fundamental process in development and regeneration. We have used microengineered culture substrates to study the interaction between model topographic and adhesive guidance cues in steering BHK cell orientation. Grooves 0.1, 0.5, 1.0, 3.0, and 6.0 microm deep together with pitch-matched aminosilane tracks 5, 12, 25, 50, and 100 microm wide were fabricated on fused silica substrates using photolithographic and dry-etching techniques. The cues were presented to the cells individually, simultaneously in parallel and orthogonally opposed. Cells aligned most strongly to 25-microm-wide adhesive tracks and to 5-microm-wide, 6-microm-deep grooves. Stress fibers and vinculin were found to align with the adhesive tracks and to the grooves and ridges. Cell alignment was profoundly enhanced on all surfaces that presented both cues in parallel. Cells were able to switch alignment from ridges to grooves, and vice versa, depending on the location of superimposed adhesive tracks. Cells aligned preferentially to adhesive tracks superimposed orthogonally over grooves of matched pitch, traversing numerous grooves and ridges. The strength of the cues was more closely matched on narrower 3- and 6-microm-deep gratings with cells showing evidence of alignment to both cues. Confocal fluorescence microscopy revealed two groups of mutually opposed f-actin stress fibers within the same cell, one oriented with the topographic cues and the other with the adhesive cues. However, the adhesive response was consistently dominant. We conclude that cells are able to detect and respond to multiple guidance cues simultaneously. The adhesive and topographic guidance cues modeled here were capable of interacting both synergistically and hierarchically to guide cell orientation.

Animals↗

PubMed1996

Ixodes scapularis (Acari:Ixodidae): status and changes in prevalence and distribution in Wisconsin between 1981 and 1994 measured by deer surveillance.

A statewide survey of blacklegged ticks, Ixodes scapularis Say, on white-tailed deer was conducted in 1994 to examine the status and changes in the prevalence and geographic distribution of this tick in Wisconsin. I. scapularis adults were collected at 17 of 26 deer registration stations, including stations in eastern (Poy Sippi) and southern Wisconsin (Monroe) without previously reported populations. Nearly all of the stations where 1 or no ticks were collected were in the eastern 1/3 of the state. The prevalence of I. scapularis in 1994 was compared with prevalence estimates taken in similar surveys during 1981 and 1989. A significant increase in tick prevalence occurred at stations along the Wisconsin River Valley, which previously defined the edge of the eastern range of I. scapularis populations and in southern Wisconsin.

Animals↗

PubMed1995

Effect of mosquito age and reproductive status on melanization of sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae.

Malaria-refractory and -susceptible strains of the mosquito vector, Anopheles gambiae, differ in their response to negatively-charged Sephadex CM-25 beads. CM-25 beads elicit a much stronger melanization reaction in refractory mosquitoes than in susceptible mosquitoes. Light microscopic and scanning electron microscopic studies documented a progression from early stages with small spots of melanin adhering to CM-25 beads to late stages where spots had grown and coalesced to form a dark dense capsule. This reaction occurred maximally during the first 18 hr after inoculation; female mosquitoes aged 3-5 days showed 48% of beads heavily melanized by 18 hr postinoculation in the refractory strain and 0-10% in the susceptible strain. Female mosquito age and reproductive status strongly affected the ability to melanize beads. All of the beads were completely melanized in the refractory strain on the day immediately following eclosion (Day 0); thereafter these levels decreased steadily until the last time point on Day 7, when only 23% of beads were melanized in this strain. In the susceptible strain, 53% of beads were heavily melanized on Day 0 and 0-10% were melanized at all other times. At Day 1 and 2 after blood feeding, 85 and 88% of beads, respectively, were heavily melanized in refractory females in comparison with control mosquitoes of the same age which heavily melanize 23-58% of the beads. Blood feeding had little effect on the ability to melanize beads in the susceptible strain.

Aging↗

PubMed1994

Subtraction scanning acoustic microscopy reveals motility domains in cells in vitro.

Scanning acoustic microscopy (SAM) observes all mechanical properties of living cells. Subtraction of the SAM images (SubSAM) of live cells was developed as a method for investigating minimal changes in cellular topography and elasticity. The image formation in the SubSAM takes into account the motion of cell mass as well as the changes of tension. High spatial and temporal resolution of the SubSAM revealed the structure of motile processes that develops at increasing time intervals, thus allowing the arising complexity of motion to be registered and investigated. Independent spots of activity emerge on a quiescent background as motility domains; they may change position, divide, merge, or disappear after a long time interval. In addition, zones of quiescence were identified over central parts of cytoplasmic lamellae. Nonmalignant (Ep: tadpole epidermal cells, XTH2: endothelial cells from tadpole hearts, 3T3 cells) and neoplastic cells (K2 cells of rat fibrosarcoma, A870N cells selected from K2) were investigated with the SubSAM. Three types of domains of subcellular cytoplasmic motility were identified in time series of two-dimensional SubSAM iamges in normal and neoplastic cells. Of them only the wave-like domain is self-evident, being derived from ruffling and protruding activity at the cell margin. Two other domains wait for detailed analysis. The oscillating domain is a visualization of tension within the cell(s), and the nucleating domain indicates intracellular processes possibly preceding locomotion. Differences in motile domains were found between low K2 and high A870N metastatic cells. The dynamics of motility domains of the A870N cells resembled that of the highly motile Ep cells. Cell morphotype and motile activity of the A870N cells are significantly influenced by the pH of the medium. It became evident that identification of the otherwise invisible motile domains in living cells by SubSAM opens a new approach to a characterization of cell motility in vitro and to an understanding of early cellular reactions to various stimuli.

Animals↗

PubMed1994

Ouabain and digitoxin as modulators of chick embryo cardiomyocyte energy metabolism.

The effects of 0.1 nmol/l ouabain (CAS 630-60-4) and digitoxin (CAS 71-63-6) on oxygen consumption rate (OCR) and the contractility of cultured chick embryo cardiomyocytes were investigated using a perfusion system which allowed microscopic observation during the experiment. contractility was assessed by a novel image analysis procedure. During substrate free perfusion the OCR was increased on application of 0.1 nmol/l ouabain or digitoxin for 60 min, whilst contractility was not affected. Digitoxin (0.1 nmol/l) elicited no change in the OCR in the presence of glucose, pyruvic acid, lactic acid and caprylic acid. Ouabain (0.1 nmol/l) did not affect the OCR or contractility in the presence of glucose, pyruvic acid and lactic acid, however together with caprylic acid the OCR was increased significantly. These results demonstrate a hitherto unknown stimulation of fatty acid utilization by low concentrations of ouabain.

Animals↗

PubMed1993

In vitro approach to 'uremic cardiomyopathy'.

Cardiovascular complications determine the prognosis of patients with chronic renal failure. The contribution of compounds retained during uremia to specific myocardial lesions is controversial. We investigated the contractility of spontaneously beating mouse cardiac myocytes in culture under perfusion with sera derived from patients on maintenance hemodialysis and test solutions containing possible toxins. Cellular contractility under defined environmental conditions is determined by a computer-assisted digital image analysis. 'Uremic sera', creatinine, urea, and combinations of these compounds reduce inotropy of the cultured heart cells, induce arrhythmias or asynchronies in a concentration-dependent manner. We propose the myocyte perfusion technique as an in vitro approach to identify cardiotoxins in the body fluids of chronically uremic patients.

Animals↗

PubMed1991

Effects of ouabain and digitoxin on the respiration of chick embryo cardiomyocytes in culture.

The effect of digitoxin (CAS 71-63-6) and ouabain (g-strophantin, CAS 630-60-4) on respiration, morphology and beating activity of cardiomyocytes in culture derived from embryonic chick hearts has been investigated. The drugs were applied in a perfusion system using a protein- and substrate-free perfusion medium (BSS) at two concentration of K+ (5.4 and 4 mmol/l). In either K+ concentration oxygen consumption was 0.13 +/- 0.05 nmol O2 h-1 per 1000 cells. During 3 h of perfusion with BSS oxygen consumption declines only slightly to 79 +/- 15% of the initial value. No relation was found between beating frequency and oxygen consumption. Increase in respiration ranged from 5 to 45% and lasted between 5 and 120 min. At concentrations being inhibitory to the Na+/K(+)-ATPase (greater than or equal to 1 mumol/l) ouabain stimulated respiration by about 20% at 4 mmol/l K+ and 10% at 5.4 mmol/l K+ while digitoxin was effective at 5.4 mmol/l only (a transient increase of 20%). At 0.1 nmol/l, (a concentration below the KD of the high affinity binding site of the Na+/K(+)-ATPase) ouabain caused a long lasting activation of respiration by about 30%, digitoxin induced a transient rise of up to 20% at 5.4 mmol/l K+. At 4 mmol/l K+ digitoxin did not affect respiration while ouabain caused a transient increase. The lowest concentration of ouabain inducing a reproducible activation of oxygen consumption was 0.1 pmol/l. At this concentration digitoxin was no longer effective. At 1 fmol/l respiration was stimulated only occasionally.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1982

Renal tubular acidosis: effects of etacrynic acid on renal acid and calcium excretion.

The fundamental disorder in renal tubular acidosis--the impaired excretions of hydrogen ions--can favourably be influenced by etacrynic acid (Hydromedin). Net acid and calcium excretion was measured in eight patients with incomplete RTA I and five controls before and during treatment with the diuretic: urinary pH was lowered and net acid excretion increased (p less than 0.05) with only slight rise in urinary calcium. Etacrynic acid appears to be particularly suitable for the long term treatment of patients with incomplete RTA I and calculous disease.

Acidosis, Renal Tubular↗