[Histological, biochemical and spectroscopic changes of articular cartilage in osteoarthritis: is there a chance for spectroscopic evaluation?]. / Arthrosebedingte histologische, biochemische und spektroskopische Veränderungen am hyalinen Gelenkknorpel: Welchen Stellenwert hat die spektroskopische Evaluation?

BACKGROUND: Non-destructive techniques for the detection and classification of pathological changes of cartilage in the early stages of osteoarthritis are required for arthroscopic and open surgery of joints. Biochemical and histological changes in cartilage with different degrees of destruction were analysed and correlated to changes in the spectroscopic characteristics of cartilage. PATIENTS, MATERIAL AND METHODS: 24 patients (n = 25 knees) with severely destructed knee joints received total knee replacement. The cartilage of the resected joints was classified according to the ICRS system. Defined cartilage specimens were investigated spectroscopically employing NIRS (near-infrared spectroscopy). In the following the cartilage specimens were harvested to determine the content of proteoglycan (GAG) and hydroxyproline (HP) as an essential part of collagen. Histological evaluation of the Mankin score and Otte score was performed using haematoxylin/eosin and safranin-O staining. Spearman's rank correlation coefficient was used to characterise links between the parameters investigated. RESULTS: We found significant correlations between spectroscopic, histological and biochemical characteristics. NIRS corresponded to the content of GAG (ρ = 0.58) and HP (ρ = 0.59), as well as to the Mankin (ρ = 0.55) and Otte (ρ = 0.5) scores. Furthermore, the ICRS classification correlated with histological evaluation (Mankin score ρ = 0.725 and Otte score ρ = 0.736), as to be expected. CONCLUSION: Characteristic cartilage changes in different degrees of osteoarthritis can be detected and evaluated by the spectroscopic method NIRS as a non-destructive technique. However, the quality of this technical evaluation cannot compete with biochemical and histological analysis.

Magnetic spin imaging under ambient conditions with sub-cellular resolution.

The detection of small numbers of magnetic spins is a significant challenge in the life, physical and chemical sciences, especially when room temperature operation is required. Here we show that a proximal nitrogen-vacancy spin ensemble serves as a high precision sensing and imaging array. Monitoring its longitudinal relaxation enables sensing of freely diffusing, unperturbed magnetic ions and molecules in a microfluidic device without applying external magnetic fields. Multiplexed charge-coupled device acquisition and an optimized detection scheme permits direct spin noise imaging of magnetically labelled cellular structures under ambient conditions. Within 20 s we achieve spatial resolutions below 500 nm and experimental sensitivities down to 1,000 statistically polarized spins, of which only 32 ions contribute to a net magnetization. The results mark a major step towards versatile sub-cellular magnetic imaging and real-time spin sensing under physiological conditions providing a minimally invasive tool to monitor ion channels or haemoglobin trafficking inside live cells.

Photoepilation with a diode laser vs. intense pulsed light: a randomized, intrapatient left-to-right trial.

BACKGROUND: Safe and efficient options for removing unwanted hair are in great demand. Laser devices and intense pulsed light (IPL) sources are the most commonly used treatment modalities. Yet, only a few randomized controlled trials (RCTs) comparing laser and IPL devices are available, and RCTs with long-term results are missing from the literature. OBJECTIVES: To compare the safety and long-term efficacy of diode lasers (DL) and IPL sources for axillary hair removal, we conducted an intrapatient, left-to-right, assessor-blinded and controlled trial. METHODS: IPL (Ellipse Flex PPT; Danish Dermatological Development, Hoersholm, Denmark; λem=600-950 nm) and DL (LightSheer XC system; Lumenis Inc., Santa Clara, CA, U.S.A.; λem=800 nm) treatments were evaluated in 30 study participants (skin type II-III) with unwanted axillary hair growth. Six treatments with each device were carried out at 4-week intervals. Final assessment was conducted 12 months after the last treatment by means of hair counts using close-up photographs. The primary endpoint was reduction in hair growth, analysed on an intention-to-treat and last-observation-carried-forward basis (n=30), and secondary endpoints were patient-rated efficacy, treatment-related pain, adverse effects and treatment duration. RESULTS: Both devices significantly reduced hair counts. Mean reductions from baseline (3 and 12 months after the last treatment) were 59·7% and 69·2% for DL and 42·4% and 52·7% for IPL treatment (P<0·01), respectively. DL treatment induced significantly more pain [3·7±2·1 (DL) vs. 1·6±1·4 (IPL); P<0·01; visual analogue scale] but could be conducted faster [33·1±3·8 s (DL) vs. 40·1±5·0 s (IPL); P<0·01]. No severe side-effects were observed for either therapy. CONCLUSIONS: Both DL and IPL treatments are highly effective, long lasting and safe. DL was found to be more effective than IPL treatment. DL treatment was more painful but less time-consuming than IPL therapy.

Cell cultures in microsystems: biocompatibility aspects.

Bio-Micro-Electro-Mechanical Systems (BioMEMS) are a new tool in life sciences, supporting cell biology research by providing reproducible and miniaturized experimental platforms. In order to cultivate cells in such systems, appropriate microenvironmental conditions are required. Due to the multitude and variety of microbioreactors and cultivated cell types available, standardized cell handling methods and comprehensive biocompatibility data are sparse. The bioreactor developed at Ilmenau University of Technology features BioMEMS consisting of silicon, glass, and polymers, supplied by peripheral components. To verify the system's suitability for cell cultivation, it was necessary to prove whether materials and surfaces are biocompatible. Custom-tailored biocompatibility test procedures along with adequate cell seeding and handling methods had to be developed. According to this, proper positive and negative control samples had to be identified. The cultivation procedures were carried out using osteoblast-like murine fibroblasts (MC3T3-E1) and primary human osteoblasts (hOB). We could provide evidence that cultivation of these cells in our BioMEMS is feasible. In this context the relevant materials and the system's structure can be regarded as to be biocompatible. We could show that cell seeding and handling methods possess a strong impact on growth, development, and cellular activity of cell cultures in BioMEMS. Statistical biocompatibility data for the materials used is given.

High sensitivity magnetic imaging using an array of spins in diamond.

We present a solid state magnetic field imaging technique using a two-dimensional array of spins in diamond. The magnetic sensing spin array is made of nitrogen vacancy (NV) centers created at shallow depths. Their optical response is used for measuring external magnetic fields in close proximity. Optically detected magnetic resonance is read out from a 60 x 60 microm(2) field of view in a multiplexed manner using a charge coupled device camera. We experimentally demonstrate full two-dimensional vector imaging of the magnetic field produced by a pair of current carrying microwires. The presented wide-field NV magnetometer offers, in addition to its high magnetic sensitivity and vector reconstruction, an unprecedented spatiotemporal resolution and functionality at room temperature.

Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.

Transient expression of human telomerase extends the life span of normal human fibroblasts.

We utilized the Cre/lox recombination system to transiently express the catalytic subunit of telomerase (hTERT) in normal diploid foreskin fibroblasts (BJ cells). A retroviral construct containing an hTERT cDNA, flanked by loxP-sites was introduced into near senescent BJ cells (population doubling 85). At population doubling (PD) 92, which exceeds the typical life span of these cells, we excised the gene via Cre-mediated recombination. All clones lost telomerase activity and showed telomere shortening over an additional 50 PDs. Interestingly, the average telomere length in these cells became shorter than in untreated BJ cells at senescence. This may be due to hTERT preferentially elongating the shortest telomeres, leading to greater length uniformity. In summary, transient telomerase expression and only a very small average telomere elongation by hTERT resulted in a 50% increase in life span of human fibroblasts. This suggests a potentially safe use of hTERT in tissue engineering.

Three-dimensional reconstructions of normal and aneurysmatic left ventricles in vivo using transesophageal echocardiography.

OBJECTIVE: To perform three-dimensional surface reconstructions to provide spatial delineations of a normal and an aneurysmatic left ventricle, using transesophageal echocardiography. DESIGN: Prospective study. SETTING: University hospital. PARTICIPANTS: Eight patients in cardiogenic shock admitted to the intensive care unit and two patients undergoing surgery with general anesthesia. INTERVENTIONS: Using a multiplane transesophageal echocardiography probe, nine echocardiographic cross-sectional images of the heart at approximately 20 degrees angular increments were obtained from midesophageal level in each patient for three-dimensional surface reconstructions. Multiple determinations of cardiac output using the thermodilution principle were also made in each patient to verify the accuracy of three-dimensional data sets. MEASUREMENTS AND MAIN RESULTS: End-diastolic and end-systolic volumes were determined from three-dimensional data sets using the disc-summation method. Stroke volume was derived as the difference between end-diastolic and end-systolic volumes. Stroke volume was also calculated from thermodilution cardiac output measurements and heart rate. Correlation and limits of agreement between stroke volumes derived by the two methods were determined. Three-dimensional wire-frame models of a normal and an aneurysmatic left ventricle at end-systole were constructed from the nine echocardiographic cross-sectional images. Correlation coefficient between stroke volume derived from three-dimensional data sets using the disc-summation method and that measured by the thermodilution method was 0.91 (p < 0.001). Wire-frame models reveal a normal symmetric cavity and an aneurysmal cavity in sharp relief. CONCLUSIONS: Three-dimensional surface reconstruction can be performed from multiple cross-sectional images obtained using an unmodified commercially available multiplane transesophageal echocardiography probe, to reveal the left ventricular cavity in sharp relief. High correlation between stroke volume calculated from three-dimensional data sets and that measured by the thermodilution method attests to the accuracy of the three-dimensional data sets.

DNA strand breaks (comet assay) and embryo development effects in grass shrimp (Palaemonetes pugio) embryos after exposure to genotoxicants.

Grass shrimp embryos develop in egg sacs (stages 1-10) attached to the female for 14-20 days after which they 'hatch' from the egg sacs into a swimming zoea stage (stage 11). Until they emerge from the egg sacs, embryos depend on lipids and lipovitellin stored within the egg. The percent of embryos which hatch after exposure to toxicants relative to controls was the basis of an embryo development assay. Exposure of embryos to chromium(III) chloride, sodium chromate, mercuric chloride, and 2-methyl-1,2-naphthoquinone (MNQ) resulted in a reduced hatching rate. In addition to effects on embryo development, DNA strand damage tests were carried out on contaminant-exposed embryos, using the single-cell electrophoresis method often referred to as comet assay. Development of stage 4 embryos was more affected by MNQ exposure than stage 7 embryos. The hatching rates of stages 4 and 7 embryos exposed to MNQ (172 micrograms/l) were 0 and 90%, respectively. DNA strand damage, measured as DNA tail moments, were 3.4 and 4.4, respectively. Thus, exposure of an early embryo stage to MNQ prevented full embryo development while development of later embryo stages was not affected. It may be that the DNA repair systems are more efficient in later embryo stages than in early stages and thus DNA damaged in the early stages affects development.

DNA damage as a bivalve biomarker.

Bivalves have been used in numerous environmental assessment studies, chiefly because they are sessile deposit or suspension feeding organisms found in or near sites of environmental concern, and they can be easily collected, sorted and deployed at sites of interest. Monitoring studies utilizing bivalves currently rely on the comparison of growth, survival, and contaminant bioaccumulation. Data gathered from 'reference' sites are compared with those of populations at assessment sites. These studies require extended periods of exposure, lasting weeks to months, and the use of a well defined population of test organisms of similar size, age, and condition. In many cases time and resources require researchers to restrict their sampling to the organisms on-hand at a particular site without the benefit of any reference data. Therefore more versatile and sensitive assessment methods are needed. Because effects at higher levels of organization such as growth, development, and survival are initiated at the molecular and cellular levels attempts have been made to identify useful biomarkers at these levels. The proposed advantages of molecular/cellular biomarkers are that they will respond to stress predictably and more rapidly, and will be indicative of the mechanisms of toxicity thereby yielding a rudimentary characterization of the contaminant(s) influencing them. In the following communication we will report on past and current developments in the monitoring of DNAdamage as an environmental biomarker.

DNA damage in mussels at sites in San Diego Bay.

Identification and assessment of introduced and other toxicants is crucial to any comprehensive study of contaminants within the marine environment. The relationship between DNA single-strand breaks and the exposure of marine organisms to environmental contaminants was examined at sites in San Diego Bay, CA. A comprehensive assessment of the extent and consequences of marine environmental contamination in the area of Naval Station San Diego was conducted in the summer of 1995. The study addressed contamination sources, distributions, concentrations, transport, sediment-water exchange, biological effects, and degradation. The biological effects portion of the study (this paper) included contaminant bioaccumulation, organismal growth, and the determination of DNA single-strand breaks using the Comet assay. DNA damage was determined in hemocytes collected from deployed and resident mussels, Mytilus edulis, at six stations in and around the Naval Station San Diego. Deployed mussels were exposed on station for approximately 30 days in plastic mesh bags, placed 1 m above the bottom. Hemocyte samples were collected on days 0, 12, and 32. It was found that stations exhibiting the extremes of contaminant exposure, both highest and lowest concentrations, were easily identified using growth and DNA damage measurements. Sediment chemistry and bioaccumulation data indicated, Hg, Cu, and Zn, to be the most notable contaminants. The Comet assay, and in particular germ cell DNA damage determinations, were found to respond rapidly to station contaminants. Results from this study and an earlier 1993 study suggest that the non-sediment associated effects observed at one station may have been the result of the photoactivation of accumulated PAHs.

Gene transfer into hepatocytes and human liver tissue by baculovirus vectors.

Gene therapy of liver diseases requires the development of efficient vectors for gene transfer in vivo. Retroviral and adenoviral vectors have been shown to deliver genes efficiently into hepatocytes in vitro and in vivo. However, these vectors do not allow for exclusive infection of the liver which would be highly advantageous for in vivo gene therapy strategies. We have recently demonstrated that genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) efficiently deliver genes into cultured cells and have a strong preference for hepatocytes of different origin. Baculoviral gene transduction efficiency into human hepatocytes was determined to approach 100% and expression levels are high, provided that gene expression is controlled by mammalian promoters. In this report, we present further properties of baculoviruses regarding their use for hepatocyte gene transfer. Baculovirus-mediated gene expression declines rapidly in the hepatocellular carcinoma cell line Huh7 and more slowly in primary cultures of mouse hepatocytes. Direct application of baculoviruses for gene delivery to the liver in vivo is hampered by serum components, presumably by complement. However, we demonstrate here that baculoviral gene transfer is feasible in ex vivo perfused human liver tissue. This result suggests the development of a strategy using baculoviral vectors for liver-directed gene therapy.

Left ventricular volume calculations using a multiplanar transoesophageal echoprobe; in vitro validation and comparison with biplane angiography.

BACKGROUND: Biplane angiographic and transthoracic echocardiographic volume calculations have shown to be sufficiently reliable in symmetric hearts; however, they are unreliable in the presence of aneurysmatic distortions. Multiplane transoesophageal echocardiography offers unobstructed cross-sectional views of the heart from one stable transducer position with the potential of imaging irregular cavity forms more accurately. It was the purpose of this in vitro study to compare the precision of multiplanar transoesophageal echocardiography to that of biplane angiography in determining left ventricular volumes, especially in aneurysmatic models. METHODS: Seven silicon rubber models of the left ventricle from post-mortem specimens (four with aneurysms) were filled with 30 different volumes (range 153-256 ml, 197 +/- 30 ml). Echocardiographic cross-sections (20 degree rotational steps) were obtained from different transducer positions, utilizing a multiplanar probe with a central rotational axis. Volumes were calculated using the disc-summation method. For comparison the same volumes were determined by standard biplane angiography. The minimum number of echo cross-section necessary to optimize precision was analysed by calculating volumes for each increasing equidistant rotational step. RESULTS: Linear correlation between measured volume using a multiplanar transoesophageal echoprobe and true volume was high (r = 0.97) and significantly better than for biplane angiography (r = 0.88; P < 0.001). Measurement bias and imprecision were also significantly lower with multiplanar echocardiography than with biplane angiography (3.9 +/- 7.1% vs 11.1 +/- 15.4%, and 2.0 +/- 3.7% vs 5.9 +/- 8.3%; P < 0.001). Precision of biplane angiographic volume measurements was significantly influenced by the presence of aneurysmatic distortions. Multiplanar echo volumes, however, were not influenced by left ventricular geometry and transducer positions. Nine echo cross-sections provided optimal precision. CONCLUSIONS: Three-dimensional echocardiographic volume calculations using a multiplanar transoesophageal echoprobe and the disc-summation method provide precise measurements unaffected by left ventricular geometry and transducer position in an in vitro setting. Standard biplane angiography is significantly less precise.

[Gender, work load and list profile in general practice. Experiences from a patient listing project]. / Kjønn, arbeidsbelastning og listeprofil i allmennpraksis. Erfaringer fra fastlegeforsøket.

A list patient project was carried out in four Norwegian municipalities in 1993. Female general practitioners from one of the involved communities soon showed frustration about the their experience that the new system had increased their work load. Although the new system was intended primarily to serve the needs of the population, the problems experienced by the female doctors were taken as warnings about future problems for the servants of the system. We therefore wanted to study relationships between experienced work load, list profiles and procedures for establishment of the patient lists among male and female doctors in Trondheim and Tromsø, the two largest project municipalities. Our data confirm the impression of increased work load and dissatisfaction with the list profiles. We find that female doctors have shorter lists and a higher proportion of female patients, and are less accessible. We discuss elements which might influence these problems in the future, especially as regards list profiles, the establishment of lists, and priorities related to people's free choice of doctor.

Patients with sexually transmitted disease: a well-defined HIV risk group in general practice?

Since patients with sexually transmitted disease (STD) form the most apparent risk group for HIV infection in general practice, differences in sexual behaviour in patients with and without STD were studied. Patients fulfilling at least one of four clinical criteria for suspicion of STD were offered four microbiological tests and a serological HIV test and were asked to complete a questionnaire concerning possible HIV exposure. Control patients were selected from a sample of ordinary consulting patients in general practice, without clinical suspicion of STD. The answers to the questionnaires given by six male and 52 female patients with a positive microbiological test for STD were compared with answers given by control patients matched for sex, age, education and geographical region. Females with STD had multiple male partners more often than controls. STD patients were more uncertain whether they had had sex with a HIV-positive person, and they reported more previous gonorrhea than the control patients. The proportion of patients who reported practicing oral sex was the same in both groups, while data for anal sex were insufficient. There were no significant differences in the use of condoms with a new partner, although there was a higher proportion of never-users of condoms among the STD patients. Identifying STD patients on clinical grounds is difficult in general practice. Clinical criteria have limited sensitivity because of asymptomatic infections, and the specificity is low. Patients with microbiologically confirmed STD should receive HIV-related attention, but attention to STD patients alone is not enough. Many people without STD have sexual behaviour which may exposure them to the HIV virus. The only means to a selective approach is increased attention to an appropriate sexual medical history.