Biomedical subjects

K Skov

Publications and source records attributed to K Skov.

At least 19 recordsLinked to original sources

PubMed2004

Structure of renal afferent arterioles in the pathogenesis of hypertension.

Renal vascular resistance is increased in essential hypertension, as in genetic models of hypertension. Here we review the evidence that this is at least in part due to structural changes in the afferent arterioles. Rat studies show that the renal afferent arteriole is structurally narrowed in young and adult spontaneously hypertensive rats (SHR). Furthermore, in the second generation of crossbred SHRs/normotensive rats (SHR/WKY F(2)-hybrids), a narrowed afferent arteriole lumen diameter at 7 weeks is a predictor of later development of high blood pressure. The reduced lumen diameter of resistance vessels is accompanied by a decrease in media cross-sectional area in SHR and could therefore be due to inhibited growth. Evidence from a primate model of hypertension has shown a negative correlation between left ventricular hypertrophy and afferent arteriole diameter, but apparently no relation to blood pressure. In SHR, the antihypertensive effect of angiotensin converting enzyme (ACE) inhibitors is mediated through renal vascular mechanisms, while ACE inhibitors (like AT(1) antagonists) have a more persistent effect on blood pressure after treatment withdrawal compared with other antihypertensive drugs. Taken together, the evidence suggests that structural narrowing of the renal afferent arteriole could be an important link in the pathogenesis of primary hypertension, at least in the SHR.

Animals↗

PubMed2003

Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model.

OBJECTIVE: To evaluate the effect of clusterin overexpression on radiation-induced tumour growth rates and apoptosis in human prostate LNCaP cells, as prostate cancer cells are relatively resistant to radiation-induced apoptosis and local recurrences are common, but overexpression of the anti-apoptotic protein clusterin can accelerate progression to androgen-independence and to confer a chemoresistant phenotype in various prostate cancer models. MATERIALS AND METHODS: Western blot analysis and immunohistochemistry were used to compare clusterin expression levels in parental (P) and clusterin-transfected (T) LNCaP cells in vitro and in vivo. The effects of radiation on clusterin-expression in both parental LNCaP/P and clusterin-transfected LNCaP/T tumours were analysed by Northern blot analysis. The cellular response to radiation was determined up to 3 weeks after irradiation using tetrazolium and re-growth assays, and cell-cycle analysis by flow cytometry. RESULTS: Clusterin mRNA expression increased from undetectable to low levels in LNCaP/P tumours after radiation and more than three-fold in LNCaP/T tumours. Clusterin overexpression decreased the radiosensitivity in a time-dependent manner, reducing the extent of growth arrest and apoptosis by up to 54%. Re-growth assays showed that the improved survival rates of LNCaP/T cells after radiation did not change after 3 days, remaining constant over 3 weeks. CONCLUSIONS: These results identify clusterin as a promoter of cell survival that may help mediate resistance to radiation-induced apoptosis. Furthermore, clusterin overexpression seems to provide an extended protection against radiation-induced cell cycle arrest and apoptosis.

Apoptosis↗

PubMed2001

Radiation induced apoptosis in ataxia telangiectasia homozygote, heterozygote and normal cells.

Recent reports suggest that the radiation-induced, p53-dependent, apoptotic response is aberrant in ataxia telangiectasia (AT) cells. We investigated the possibility that an aberrant apoptotic response to ionizing radiation may also be the characteristic of AT heterozygotes and may facilitate in discriminating AT heterozygotes from the general population. Log phase, Epstein Barr virus (EBV) transformed lymphoblastoid cell lines and primary lymphocytes from three AT families were irradiated and the apoptotic response at 30h post radiation was measured by flow cytometry using TUNEL and hypodiploid methods. Our results show that the apoptotic response of AT homozygote (ATM-/-), AT heterozygote (ATM+/-) and normal cells (ATM+/+) to ionizing radiation, measured by the hypodiploid and TUNEL methods using flow cytometry, is dose and time dependent. Furthermore, this response is paradoxical in that ATM (-/-) lymphoblastoid cells were characterized by a reduced post radiation apoptotic response compared to their normal counterparts. Heterozygote (ATM+/-) lymphoblastoid cells displayed an intermediate response to ionizing radiation. In contrast, primary, non-transformed AT cells exhibited the same apoptotic response as their normal counterparts. Our results thus indicate that pre-radiation, EBV-transformed, lymphoblastoid cell lines from individual families may be useful in discriminating ATM status, but patient-derived, primary AT homozygous, heterozygous and normal primary cultured lymphocytes cannot be discriminated by this assay.

Apoptosis↗

PubMed2001

Morphology of renal afferent arterioles and glomeruli, heart weight, and blood pressure in primates.

In a Caribbean outbred population of African green monkeys (Cercopithecus aethiops), 5 to 10% of feral adults have elevated blood pressure (BP). We have investigated whether the increased pressure is associated with abnormal renal afferent arteriole structure or glomerular number. In seven young adult (aged 7 to 13 years) male monkeys with consistently high BP (mean BP, 111 mm Hg; ketamine anesthesia) and seven controls (mean BP, 81 mm Hg), the morphology of the renal vasculature has been analyzed in three cortical zones. In each animal, the left kidney vasculature was fixed while relaxed and at known intravascular pressure, and afferent arteriolar diameter and media cross-sectional area were estimated. The right kidney was perfusion-fixed and prepared for unbiased stereologic estimation of glomerular number and size. No difference was found in afferent arteriole lumen diameter or media cross-sectional area, or in glomerular number or size, between the high BP group and controls. There was no difference in heart weight between the two groups, but there was a negative correlation between left ventricle heart weight and afferent arteriole diameter (controls: r = -0.81, P = .025; all animals: r = -0.70, P = .005, slope about 3.5% reduction in lumen diameter for 10% increase in heart weight). The results suggest that cardiac mass and renal afferent arteriole structure may be controlled by a common mechanism unrelated to BP measured in anesthesia. However, the lack of conscious measurements prevents conclusions as to whether this mechanism involves ambulatory BP.

Animals↗

PubMed2001

Quantitative magnetic resonance imaging as marker of synovial membrane regeneration and recurrence of synovitis after arthroscopic knee joint synovectomy: a one year follow up study.

OBJECTIVES: By repeated magnetic resonance imaging (MRI) to study synovial membrane regeneration and recurrence of synovitis after arthroscopic knee joint synovectomy in patients with rheumatoid arthritis (RA) and other (non-RA) causes of persistent knee joint synovitis. METHODS: Contrast enhanced MRI was performed in 15 knees (nine RA, six non-RA) before and one day, seven days, two months, and 12 months after arthroscopic synovectomy. Synovial membrane volumes, joint effusion volumes, and cartilage and bone destruction were assessed on each MRI set. Baseline microscopic and macroscopic assessments of synovitis and baseline and follow up standard clinical and biochemical examinations were available. RESULTS: Synovial membrane and joint fluid volumes were significantly reduced two and 12 months after synovectomy. However, MRI signs of recurrent synovitis were already present in most knees at two months. No significant differences between volumes in RA and non-RA knees were seen. Synovial membrane volumes at two months were significantly inversely correlated with the duration of clinical remission, for all knees considered together (Spearman's correlation r(s)=-0.67; p<0.05), for RA knees (r(s)=-0.76; p<0.05), and for non-RA knees (r(s)=-0.83; p<0.05). Baseline volumes were not significantly correlated with clinical outcome. Only three knees (all RA) showed erosive progression. The rate of erosive progression was not correlated with MRI volumes or with clinical or biochemical parameters. CONCLUSION: The synovial membrane had regenerated two months after arthroscopic knee joint synovectomy and despite significant volume reductions compared with baseline it often showed signs of recurrent synovitis. MRI seems to be valuable as a marker of inflammation, destruction and, perhaps, as a predictor of therapeutic outcome in arthritis.

Adult↗

PubMed1999

Large juxtamedullary glomeruli and afferent arterioles in healthy primates.

BACKGROUND: Differences in the functional demands of superficial and juxtaglomerular nephrons could result in differences in glomerular size between these two types of nephrons. This concept is based on animal models in which the juxtamedullary glomeruli and afferent arteriole diameters are indeed larger than those in the outer cortical zones. However, this difference was not confirmed in human necropsy studies. To obtain further information in living primates, we have made unbiased estimates of glomerular and afferent arteriole dimensions in three cortical zones (superficial, midcortical, and juxtamedullary) in young, adult, normotensive monkeys. METHODS: In each animal (N = 6), the right kidney was perfusion fixed and prepared for unbiased stereological estimation of glomerular number and size. The left kidney vasculature was fixed while relaxed and at a known intravascular pressure. Thereafter, afferent arteriolar dimensions were estimated using light and confocal microscopy. RESULTS: The mean glomerular volume was variable between animals (coefficient of variation, 23%) and was largest in the juxtamedullary zone. Afferent arteriolar lumen diameter varied little between animals (coefficient of variation, 3%), but was also the largest in the juxtamedullary zone. No sclerotic glomeruli were found in any of the animals. CONCLUSION: The findings show that in normal primates, as previously shown in other animals, juxtamedullary glomeruli and afferent arteriolar diameters are larger compared with those in the outer cortical zones.

Animals↗

PubMed1998

Automated fluorescence microscopic measurement of apoptosis frequency following ionizing radiation exposure in cultured mammalian cells.

PURPOSE: To develop and assess an automated image cytometric method of apoptotic cell classification for use under conditions in which apoptosis is a rare event (e.g. fibroblastoid cell lines or low-dose irradiation). METHOD: Image acquisition software was adapted to gather double-stained cell images from slides prepared using cell fixation and staining methods that emphasized apoptotic morphology. Chinese hamster ovary cells (CHO) were classified individually by discriminant analysis of morphological and nuclear texture features calculated for each image. Discriminant functions were constructed from a manually classified set of over 60000 cell images categorized as 'normal', 'apoptotic', 'cell doublets' or 'debris' and all subsequent cell images collected were classified using these functions. RESULTS: Application of this technique resulted in a 99.8% accuracy in classification of the normal cell population, and 81.7% classification accuracy for apoptotic cells. This method was then applied to study the time course of the apoptotic response of CHO cells following X-irradiation. Following irradiation with 5 Gy no increase above control levels of apoptosis was noted until 18 h post-irradiation, which corresponded with the release of the G2 block as determined by DNA-content analysis. Apoptotic frequency increased to a peak level of 12.1 +/- 4.6% at 42 h post-irradiation. CONCLUSIONS: Automated image cytometry provides an efficient and consistent method of apoptosis measurement. This study represents the first detailed characterization of the time course and the role of cell division in CHO cell apoptosis.

Animals↗

PubMed1997

A novel image cytometric method for quantitation of immunohistochemical staining of cytoplasmic antigens.

Evaluation of molecular markers by immunohistochemical labelling of tissue sections has traditionally been performed by qualitative assessment by trained pathologists. For those markers with a staining component present outside of the nucleus, there has been no image histometric method available to reliably and consistently define cell interfaces within the tissue. We present a new method of approximating cellular boundaries to define cellular regions within which quantitative measurements of staining intensity may be made. The method is based upon Voronoi tessellation of a defined region of interest (ROI), and requires only the position of the nuclear centroids within the ROI. Here we describe the VORSTAIN software which has been developed based on the Oncometrics CytoSavant Automated Image Cytometry System. To demonstrate this technique, human breast cancer sections immunohistochemically stained for bcl-2 protein and counter-stained with nuclear methyl green stain were evaluated. Intra-observer variation in the measured values was between 1.5-2.6% and inter-observer variation was between 1.8-4.4%. The primary source of variability was due to difficulties in interpreting the exact position of the nuclear centroids. Analysis of mean staining densities for each slide correlated well with subjective scoring performed by two independent pathologists. Using VORSTAIN, significant variation of staining intensities between regions within the same slide was measured for some sections, indicating a large degree of heterogeneity within the tumours. The ability to accurately quantitate the degree of heterogeneity of molecular marker expression within tumours may be a valuable tool in prognostication.

Biomarkers, Tumor↗

PubMed1996

Effects of an angiotensin-converting enzyme inhibitor, a calcium antagonist, and an endothelin receptor antagonist on renal afferent arteriolar structure.

Narrowed afferent arteriolar diameter in young, spontaneously hypertensive rats (SHR) may be a contributor to later development of high blood pressure. Thus, treatment that causes dilation of the afferent arterioles in SHR may inhibit the redevelopment of high blood pressure when treatment is withdrawn. We treated SHR with an ACE inhibitor (cilazapril, 5 to 10 mg/kg per day, high; 1 mg/kg per day, low), a calcium antagonist (mibefradil, 20 to 30 mg/kg per day), and an endothelin receptor antagonist (bosentan, 100 mg/kg per day) from age 4 to 20 weeks. Untreated SHR and Wistar-Kyoto rats were also investigated. At 20 weeks, the rats were killed, and morphology of the afferent arterioles was studied. Other SHR (untreated, high cilazapril, low cilazapril, mibefradil) were treated in exactly the same way and then followed to 32 weeks without treatment. The morphometric studies showed that cilazapril increased the lumen diameter in the afferent arterioles and decreased the media-lumen ratio in a dose-dependent manner. On withdrawal of cilazapril treatment, the reduction in blood pressure persisted. Mibefradil tended to increase afferent arteriolar diameter, whereas it did not alter media-lumen ratio. The persistent effect on blood pressure was only moderate after withdrawal of mibefradil. Bosentan had no effect on renal afferent arteriolar structure or blood pressure. In conclusion, cilazapril was more effective than mibefradil in altering afferent arteriolar structure and caused the most persistent effect on blood pressure after treatment withdrawal. The association of increased afferent arteriolar diameter and lower blood pressure level after withdrawal of treatment may suggest a pathogenic role for afferent arteriolar diameter in the development of high blood pressure in SHR.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1996

Multi-organ dysfunction associated with high-dose carboplatin therapy prior to autologous transplantation.

The specific contribution of high-dose carboplatin to regimen-related toxicity after autologous transplantation has been difficult to determine, particularly in patients receiving cyclophosphamide or ifosfamide. We report five cases of severe multi-system failure in patients receiving high-dose carboplatin in addition to other cytotoxics prior to autologous transplantation. Two of these patients did not receive ifosfamide or cyclophosphamide as part of their chemotherapy. The clinical picture consisted of early onset acute renal failure, arrhythmias and/or myocardial hypokinesia, mental obtundation and other neurological deficits and, in two patients, an acute myopathy. In the two patients in whom serum platinum was measured, levels were very high at the time of marrow infusion. All patients had received prior cisplatin therapy but showed either normal or only modestly impaired creatinine clearance before the transplant. These observations are consistent with a specific clinical syndrome associated with carboplatin toxicity and suggest that creatinine clearance may be inadequate as an indicator of potential renal failure when high-dose carboplatin is used.

Adult↗

PubMed1996

Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5.

The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles. Platination significantly decreased immunofluorescence levels (approximately 4-fold less than EF5) after 3 h in hypoxia, but also increased levels after exposure in air (approximately 1.5 x) such that the hypoxic ratio decreased from approximately 50 to approximately 13. Platinated EF5 also showed significantly greater cytotoxicity than its parent in both aerobic and hypoxic cells. These results are consistent with targeting of EF5 to DNA, which was confirmed qualitatively by confocal microscopy.

Animals↗

PubMed1994

Number and size of renal glomeruli in spontaneously hypertensive rats.

OBJECTIVE: A reduced number of nephrons, whether acquired or congenital, as has been observed in certain inbred rat strains, may lead to systemic hypertension. The present study estimated the total number of glomeruli and mean glomerular size in kidneys of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. METHODS: Unbiased stereological methods were used on immersion-fixed kidneys from 12-week-old male SHR (n = 9) and age-matched WKY rats (n = 11). RESULTS: The total number of glomeruli was significantly reduced in the SHR compared with in the WKY rats. Sclerotic glomeruli were not found in either group. No significant difference in mean glomerular volume was observed, but the total glomerular volume was reduced by 25% in the SHR compared with in the WKY rats. CONCLUSION: SHR have fewer glomeruli than WKY rats, but of similar size, resulting in a reduced total glomerular volume in adult SHR. These findings are consistent with the hypothesis that the kidney plays an important role in the pathogenesis of genetic hypertension.

Animals↗

PubMed1994

The effect of two topoisomerase inhibitors on low-dose hypersensitivity and increased radioresistance in Chinese hamster V79 cells.

A preliminary investigation of the effect of topoisomerase inhibitors on the structure of the survival curve at low doses has been carried out in Chinese hamster V79 cells, where there is a deviation from the predicted response to radiation (Marples and Joiner, Radiat. Res. 113, 41-51, 1993). Cells were treated with one representative drug for each enzyme (topoisomerase I or II) prior to X irradiation in air and assessed for cell survival using automated microscopic location of cells. VP-16 causes little or no effect, while camptothecin has a measurable effect up to 2 Gy. The results are discussed in terms of the role of DNA damage and cell cycle in increased radioresistance, which encourage further investigation of the effects of this class of drugs at low doses.

Animals↗

PubMed1994

A preliminary investigation into the extent of increased radioresistance or hyper-radiosensitivity in cells of hamster cell lines known to be deficient in DNA repair.

The response to low doses of X rays was assessed in cells of three hamster cell lines which are defective in DNA repair and was compared with their parental lines. Cells of the V79-derived double-strand break repair-deficient line XR-V15B showed no radioresistance in the 0.5-Gy range compared with the V79B wild type, but instead showed an exponential response. Cells of the single-strand break repair-deficient line EM9 showed hyper-radiosensitivity and exhibited increased radioresistance. Most interestingly, cells of the UV-20 cell line appeared to respond exponentially, as a continuation of the hyper-radiosensitive portion of the curve, with no evidence of increased radioresistance. This line is defective in an incision step of excision repair and is sensitive to crosslinking agents. Further studies are warranted to address the possible role of single- and double-strand break repair and excision repair in hyper-radiosensitivity and increased radioresistance.

Animals↗

PubMed1994

Can colony size be used to detect low-dose effects?

Many insults at low doses are defined as nontoxic, even though obvious effects do occur. Among these are changes in colony size when clonogenic survival is assessed. Early attempts to quantify radiation effects on colony size have been reported, but the time-consuming nature of these experiments did not encourage the use of this parameter as an end point. Recently, however, developments in image cytometry technology have provided alternative, less labor-intensive means of measuring colony size. These techniques have been used in our ongoing investigations of radiation effects at low doses. Data accumulated to date show a measurable dose dependence of colony size in clones classed as survivors. This dose dependence is characterized by fluctuations in the 0-1.5-Gy dose range, followed by a gradual decrease in colony size at higher doses. The fluctuations at low doses correspond qualitatively to the concavity, thought to be indicative of inducible repair phenomena, that has been observed in cell survival curves in the same dose range. This concavity was also seen in the current study, but its detection appeared to be dependent on the method used to score survivors.

Animals↗

PubMed1992

Morphology of renal afferent arterioles in spontaneously hypertensive rats.

We present a new perfusion technique that allows arteries down to the level of capillaries to be fixed while relaxed and under a known intravascular pressure. Through a catheter inserted into the right renal artery of 12-week-old male spontaneously hypertensive rats (n = 9) and control Wistar-Kyoto rats (n = 11), the kidney vessels were rinsed with human plasma, relaxed by papaverine, and perfused with a casting resin containing microspheres. The microspheres (12 microns) became trapped in the glomeruli of the kidney and, together with a closing of the venous outflow, they caused the flow through the kidney to stop, so that the intravascular pressure was raised to the level of the input perfusion pressure (100 mm Hg). The resin material was allowed to harden, and the kidney was immersion-fixed and prepared for histomorphometrical investigations. This technique made it possible to measure both the structurally determined lumen diameter and the corresponding media thickness under clearly defined conditions. The lumen diameter of afferent arterioles close to the glomeruli showed a 17% reduction in spontaneously hypertensive rats (15.4 +/- 0.6 microns; mean +/- SEM) compared with Wistar-Kyoto rat arterioles (18.5 +/- 0.3 microns, p < 0.001). However, this was not due to media hypertrophy, because media cross-sectional area was smaller (p < 0.001) in spontaneously hypertensive rats (210 +/- 6 microns 2) compared with Wistar-Kyoto rats (274 +/- 16 microns 2). We conclude that the lumen reduction in renal afferent arterioles in spontaneously hypertensive rats is not the result of an encroachment on the lumen by a hypertrophic media.

Analysis of Variance↗

PubMed1991

Interaction of platinum drugs with clinically relevant x-ray doses in mammalian cells: a comparison of cisplatin, carboplatin, iproplatin, and tetraplatin.

Whereas the interaction between radiation and platinum complexes has never been pronounced in radiobiological experiments (to 30 Gy in mammalian cells), there have been reports of interest in this combination in the clinic, where fractionated doses of approximately 2 Gy are used. Our studies on the marked interaction in hypoxia at the 80% survival level (1-2.5 Gy) with cisplatin have been extended to second generation platinum drugs of clinical interest. The studies in the lower radiation dose region have been facilitated by the use of the cell analyzer DMIPS to identify individual cells and follow them microscopically to assess for clonogenic ability. Chinese hamster V79 cells were used, which were exposed to drug for 1 hr prior to irradiation in hypoxia (or air). None of the drugs give an enhancement ratio (ER) greater than 1.3 in the high radiation dose region, whereas all can produce ER80% (ER calculated at iso-survival of 80%) of 2 or higher at low doses in hypoxic cells. The enhancement of radiation kill in oxic V79 cells (ER's to 1.1 at 1-2% S) disappears at low doses (ER80% = 1.0) except for tetraplatin, where a moderate ER80% (to 1.64) was measured. Comparison of the hypoxic interaction on a concentration basis suggests that cisplatin is the best drug at low x-ray doses and low concentrations, but the interaction reaches a plateau at ER80% approximately 2.0. Tetraplatin continues to give better interaction with increasing concentration (up to ER80% = 3.7 at 25 microM). Interaction of radiation with the less toxic drugs, iproplatin and carboplatin, used at around 100 microM can be improved by longer exposure times prior to irradiation. Comparison on the basis of toxicity, for which the plating efficiency was used, suggests that cisplatin gives a better interaction than the three newer drugs for a given level of toxicity in hypoxic V79 cells.

Animals↗

PubMed1978

In-111 transferrin labeling studied by perturbed angular correlations.

The technique of perturbed angular correlations (P.A.C.) was used to study transferrin labeled with radioactive indium. Three forms of the labeled system were studied: a) the In-111 was bound to transferrin with no iron present at either of the binding sites; b) the In-111 probe was used to label transferrin that was 33% saturated with iron; and c) the label was attached to saturated Fe-transferrin. The measured perturbation factors, G22(t), indicate that the three binding states are clearly differentiated. Cases (a) and (b) exhibit characteristic features of time-independent quadrupole interactions and the average quadrupole interaction frequencies, omegaq, were determined in these cases.

Binding Sites↗