Breast deformation during the course of radiotherapy: The need for an additional outer margin.

PURPOSE: The aim of this retrospective study was to investigate and quantify the extent of breast deformation during the course of breast cancer (BC) radiotherapy (RT). The magnitude of breast deformation determines the additional outer margin needed for treatment planning to deliver a full dose to the target volume. This is especially important when using inverse planning techniques. METHODS: A total of 93 BC patients treated with RT and with daily CBCT image guidance were selected for this study. Patients underwent either only breast-conserving surgery (BCS) (n = 5), BCS with sentinel node biopsy (n = 57) or BCS with radical axillary node dissection (n = 31). The treatment area included the whole breast and chest wall (54%) or also the axillary lymph nodes (46%). 3D-registration was conducted between 1731 CBCT images and the respective planning CT images to assess the difference in breast surface. RESULTS: The largest maximum breast surface expansion (MBSE) was 15 mm; the average was 2.4 ±â€¯2.1 mm. In 294 fractions (17%), the MBSE was ≥5 mm. An outer margin of 8 mm would have been required to cover the whole breast in 95% of the treated fractions. There was a statistically significant correlation between the MBSE and body mass index (r = 0.38, p = 0.001). CONCLUSIONS: Significant changes in the breast surface occur during the course of BC RT which should be considered in treatment planning. An additional margin outside the breast surface of at least 8 mm is required to take into account the anatomical changes occurring during BC RT.

Transport of Iodine Is Different in Cartilage and Meniscus.

Contrast enhanced computed tomography (CECT) has been proposed for diagnostics of cartilage and meniscus injuries and degeneration. As both tissues may be imaged simultaneously, CECT could provide a method for comprehensive evaluation of knee joint health. Since the composition and structure of cartilage and meniscus are different, we hypothesize that transport characteristics of anionic contrast agents also differ between the tissues. This would affect interpretation of CECT images and warrants investigation. To clarify this, we aimed to determine the transport kinematics of anionic iodine (q = -1, M = 126.9 g/mol), assumed to not be significantly affected by the steric hindrance, thus providing faster transport than large molecule contrast agents (e.g., ioxaglate). Cylindrical samples (d = 6 mm, h = 2 mm) were prepared from healthy bovine (n = 10) patella and meniscus, immersed in isotonic phosphate-buffered NaI solution (20 mgI/mL), and subsequently imaged with a micro-CT at 20 time points up to 23 h. Subsequently, normalized attenuation and contrast agent flux, as well as water, collagen, and proteoglycan (PG) contents in the tissues were determined. Normalized attenuation at equilibrium was higher (p = 0.005) in meniscus. Contrast agent flux was lower (p = 0.005) in the meniscus at 10 min, but higher (p < 0.05) between 30 and 120 min. In both tissues, contrast agent distribution at equilibrium suggested an inverse agreement with the depth-wise PG distribution. In conclusion, iodine transport into cartilage and meniscus was different, especially between the first 2 hours after the immersion. This is an important finding which should be considered during simultaneous CECT of cartilage and meniscus.

Bioconcentration, biotransformation and elimination of pyrene in the arctic crustacean Gammarus setosus (Amphipoda) at two temperatures.

The influence of temperature on the bioaccumulation, toxicokinetics, biotransformation and depuration of pyrene was studied in the arctic marine amphipod Gammarus setosus. A two-compartment model was used to fit experimental values of total body burden, total metabolites and parent pyrene concentrations and to calculate toxicokinetic variables derived for two experimental treatments (2 and 8 °C). No statistically significant differences were observed with temperature for these toxicokinetic variables or bioconcentration factors. Contrarily, the Q10 values suggested that the toxicokinetic variables ke and km were temperature-dependent. This may be explained by the high standard deviation of the Q10 values. Q10 is the variation in the rate of a metabolic reaction with a 10 °C increase in temperature. Depuration rate constants were calculated from linear best fit equations applied to measured pyrene concentrations over time during the depuration phase of the experiment. During depuration, the parent pyrene was eliminated in two stages with faster elimination observed at 8 °C compared to 2 °C. This finding was also indicated by the Q10. No changes in total body burdens of metabolite concentrations were observed during the monitoring of depuration over a period of 96 h. The biotransformation pathway of pyrene in G. setosus was also investigated in this study with two main phase II biotransformation products discovered by liquid chromatography. These products are conditionally identified as the sulphate and glucose conjugates of 1-hydroxy-pyrene. Overall, the study contributes new knowledge to the understanding of the fate of PAHs in arctic biota. In particular, the study provides valuable insight into the bioaccumulation and biotransformation of an important PAH and its metabolites in a species that serves as both a predator and prey in the arctic ecosystem.

Contrast enhanced imaging of human meniscus using cone beam CT.

OBJECTIVE: Meniscal injuries can lead to mechanical overloading of articular cartilage and eventually to knee osteoarthritis. The objective was to evaluate the potential of contrast enhanced computed tomography (CECT) to image contrast agent (CA) diffusion in human menisci with a clinical cone beam CT scanner. DESIGN: Isolated human menisci (n = 26) were imaged using magnetic resonance imaging (MRI) and CECT in situ. Diffusion of anionic CA into the meniscus was imaged for up to 30 h. The results of CECT were compared with water, collagen and proteoglycan (PG) contents, biomechanical properties, age and histological and MR images of the samples. RESULTS: Diffusion of CA required over 25 h to reach equilibrium. The CA partition (the CA concentration in the tissue divided by that in the bath) at the 40 min time point correlated significantly with that at the 30 h time point in both lateral (r = 0.706, P = 0.007) and medial (r = 0.669, P = 0.012) menisci. Furthermore, CA partition in meniscus after 30 h of diffusion agreed qualitatively with the distribution of PGs. CONCLUSION: The cross-sectional distribution of CA was consistent with that reported in a previous µCT study on bovine meniscus. The time required to reach diffusion equilibrium was found impractical for clinical applications. However, based on the present results, shorter delay between injection and imaging (e.g., 40 min) could be feasible in clinical diagnostics of meniscal pathologies.

Characterization of site-specific biomechanical properties of human meniscus-Importance of collagen and fluid on mechanical nonlinearities.

Meniscus adapts to joint loads by depth- and site-specific variations in its composition and structure. However, site-specific mechanical characteristics of intact meniscus under compression are poorly known. In particular, mechanical nonlinearities caused by different meniscal constituents (collagen and fluid) are not known. In the current study, in situ indentation testing was conducted to determine site-specific elastic, viscoelastic and poroelastic properties of intact human menisci. Lateral and medial menisci (n=26) were harvested from the left knee joint of 13 human cadavers. Indentation tests, using stress-relaxation and dynamic (sinusoidal) loading protocols, were conducted for menisci at different sites (anterior, middle, posterior, n=78). Sample- and site-specific axisymmetric finite element models with fibril-reinforced poroelastic properties were fitted to the corresponding stress-relaxation curves to determine the mechanical parameters. Elastic moduli, especially the instantaneous and dynamic moduli, showed site-specific variation only in the medial meniscus (p<0.05 between the sites). The instantaneous and dynamic elastic moduli of the anterior horn were significantly (p<0.05) greater in the medial than lateral meniscus. The phase angle showed no statistically significant variation between the sites (p>0.05). The values for the strain-dependent fibril network modulus (nonlinear behaviour of collagen) were significantly different (p<0.05) between all sites in the medial menisci. Additionally, there was a significant difference (p<0.01) in the strain-dependent fibril network modulus between the lateral and medial anterior horns. The initial permeability was significantly different (p<0.05) in the medial meniscus only between the middle and posterior sites. For the strain-dependent permeability coefficient, only anterior and middle sites showed a significant difference (p<0.05) in the medial meniscus. This parameter demonstrated a significant difference (p<0.05) between lateral and medial menisci at the anterior horns. Our results reveal that under in situ indentation loading, medial meniscus shows more site-dependent variation in the mechanical properties as compared to lateral meniscus. In particular, anterior horn of medial meniscus was the stiffest and showed the most nonlinear mechanical behaviour. The nonlinearity was related to both collagen fibrils and fluid.

No association between vitamin D and ß-cell autoimmunity in Finnish and Estonian children.

BACKGROUND: Vitamin D has immunomodulatory properties, such as regulation of FOXP3 expression and regulatory T-cell activity. Our aim was to investigate whether plasma 25-hydroxyvitamin D [25(OH)D] concentrations associate with the development of ß-cell autoimmunity and the transcriptional activity of FOXP3 or vitamin D3 convertase gene (CYP27B1) in CD4+ memory T cells. METHODS: We studied 83 Finnish and 32 Estonian children participating in the DIABIMMUNE and DIPP studies. Twenty-nine Finnish and six Estonian children tested positive for at least one diabetes-associated autoantibody. The plasma concentrations of 25(OH)D and 1,25(OH)2D were analysed with an enzyme immunoassay. Gene expression of FOXP3 and CYP27B1 in the isolated CD4+ memory T cells was studied with reverse transcription quantitative polymerase chain reaction. RESULTS: Vitamin D status did not differ between subjects positive and negative for ß-cell autoantibodies. Finnish children had higher vitamin D status than Estonian children (p < 0.001). FOXP3 expression was higher in Estonian CD4+ memory T-cell samples than in Finnish samples (p < 0.01) even when including in both groups only children with serum 25(OH)D concentrations in the range of 50-80 nmol/L (p < 0.001). CONCLUSIONS: These findings do not support a crucial role of circulating 25(OH)D as a regulator of ß-cell autoimmunity or FOXP3 expression.

Comparison of nonlinear mechanical properties of bovine articular cartilage and meniscus.

Nonlinear, linear and failure properties of articular cartilage and meniscus in opposing contact surfaces are poorly known in tension. Relationships between the tensile properties of articular cartilage and meniscus in contact with each other within knee joints are also not known. In the present study, rectangular samples were prepared from the superficial lateral femoral condyle cartilage and lateral meniscus of bovine knee joints. Tensile tests were carried out with a loading rate of 5mm/min until the tissue rupture. Nonlinear properties of the toe region, linear properties in larger strains, and failure properties of both tissues were analysed. The strain-dependent tensile modulus of the toe region, Young's modulus of the linear region, ultimate tensile stress and toughness were on average 98.2, 8.3, 4.0 and 1.9 times greater (p<0.05) for meniscus than for articular cartilage. In contrast, the toe region strain, yield strain and failure strain were on average 9.4, 3.1 and 2.3 times greater (p<0.05) for cartilage than for meniscus. There was a significant negative correlation between the strain-dependent tensile moduli of meniscus and articular cartilage samples within the same joints (r=-0.690, p=0.014). In conclusion, the meniscus possesses higher nonlinear and linear elastic stiffness and energy absorption capability before rupture than contacting articular cartilage, while cartilage has longer nonlinear region and can withstand greater strains before failure. These findings point out different load carrying demands that both articular cartilage and meniscus have to fulfil during normal physiological loading activities of knee joints.

Trophic transfer of pyrene metabolites and nonextractable fraction from Oligochaete (Lumbriculus variegatus) to juvenile brown trout (Salmo trutta).

The trophic transfer of pyrene metabolites was evaluated by a 2-month exposure of the freshwater annelid Lumbriculus variegatus (Oligochaeta) to pyrene, followed by feeding to juvenile brown trout (Salmo trutta). The results obtained by scintillation counting (SC) proved that the pyrene metabolites produced by L. variegatus were transferred to juvenile S. trutta through diet. More detailed analyses by LC-FLD (liquid chromatography with fluorescence detection) showed that an unknown pyrene metabolite originating from L. variegatus was present in fish liver. This metabolite, although yet not properly identified, may be the glucose conjugate of 1-hydroxy-pyrene. This metabolite was not present in chromatograms of fish that were fed pyrene-spiked food pellets. In addition, the strongly bound tissue residue of L. variegatus, which was nonextractable neither by organic solvents nor by the proteolytic enzyme Proteinase K, was most likely not available for the fish through diet. Altogether, the present study shows that the metabolites of pyrene produced at low levels of the food chain may be potentially available for upper levels through diet, raising a concern about their potential toxicity to predators and supporting their inclusion in the risk assessment of PAHs.

Humic substances modify accumulation but not biotransformation of pyrene in salmon yolk-sac fry.

Humic substances may influence the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in aquatic environment. Relatively little is known how humic substances affect the biotransformation of PAHs in aquatic animals. Here we report how two different types of humic substances affected the accumulation of pyrene, a four-ringed PAH, to yolk-sac fry of landlocked salmon (Salmo salar m. sebago). The accumulation of pyrene to yolk-sac fry tissues was inversely related to humic substance concentration in a short term (72h) exposure. However, the biotransformation of pyrene was not affected by humic substances. Pyrene or humic substances did not induce CYP1A activity in yolk-sac fry tissues contrasting to beta-naphthoflavone, which was used as a positive control. Yolk-sac fry were capable to biotransform pyrene to phase I (1-hydroxypyrene) and phase II (pyrene-1-sulphate) products. Interestingly, glucuronide conjugate (i.e. pyrene-1-glucuronide) was not present in yolk-sac fry tissues. The concentration of parent pyrene and 1-hydroxypyrene remained the same throughout the experiment but the concentration of pyrene-1-sulphate more than doubled from 24 to 72h. This finding suggests that salmon yolk-sac fry are not capable to excrete phase II biotransformation products or the excretion is very slow. Further, this could indicate that early life stage toxicity of many CYP1A inducing compounds is related to accumulation of phase II conjugates in fry tissues.

Toxicity of a phytosterol mixture to grayling (Thymallus thymallus) during early developmental stages.

The study concerns the toxicity of a phytosterol mixture, ultrasitosterol, consisting mainly of beta-sitosterol 75.7% and beta-sitostanol 13.0%, to grayling (Thymallus thymallus) embryos. Eyed eggs were exposed to three concentrations (1 microg/l, 10 microg/l, and 50 microg/l) of ultrasitosterol for 4 weeks. Embryos and later on hatched fry were taken for triiodothyronine (T3), thyroxine (T4), and histopathological analyses after 7, 14, 21, and 28 days of exposure. Most of the eggs (>95%) hatched during the first week of exposure, and ultrasitosterol treatment shortened hatching time significantly (p < or = 0.0001) at all exposure concentrations in comparison to the control. Ultrasitosterol did not have any significant effect on T3 or T4 levels in the embryo extracts. However, an interesting observation was that T3 levels increased in all treatments and in the control near the time of hatching. In conclusion, ultrasitosterol showed potential to affect the development of grayling embryos and fry, but further long-term exposure experiments are needed to verify these changes in more detail.

Frequency of allergic rhinitis to laboratory animals in university employees as confirmed by chamber challenges.

BACKGROUND: Occupational risk for laboratory animal allergy (LAA) is well known, but prevention is often insufficient and new cases keep appearing. METHODS: A questionnaire on work-related symptoms was sent to all laboratory animal workers in Kuopio University. Subjects with rhinitis were invited to an examination for LAA, which consisted of an otorhinolaryngological examination and skin prick testing (SPT) with 16 common environmental allergens and two to five relevant laboratory animals. In cases of occupational sensitization, a challenge test was performed. RESULTS: The questionnaire was completed by 156 of 245 (64%) subjects. Rhinitis was reported by 65 of the respondents (42%) of whom 47 were examined for LAA. The duration of exposure before the onset of rhinitis varied from 1 month to 18 years. Twenty-seven persons (57%) had a history of some previous atopic symptoms. Forty subjects (85%) showed positive reactions in SPT, 24 (51%) for laboratory animals. Fifteen of the 26 animal challenge tests performed were positive, confirming the diagnosis of occupational allergic rhinitis in three subjects, occupational dermatitis in five and both diseases in seven subjects. The frequency of occupational allergic rhinitis among all participants was 10 of 156 (6%). CONCLUSIONS: Atopic constitution and work-related sensitization were common in rhinitic laboratory animal workers. Occupational allergic disease was diagnosed in nearly every third case.

Bisphenol A accumulation in the freshwater clam Pisidium amnicum at low temperatures.

Toxicokinetics of (14)C-labeled bisphenol A (BPA) was studied in the small freshwater clam Pisidium amnicum at four ecologically relevant low temperatures (2, 6, 8, and 12 degrees C). The uptake clearance (k(u)) of BPA increased from 1.49 to 6.55 ml x g(-1) x h(-1) as temperature increased from 2 to 8 degrees C but decreased slightly again at the highest temperature. The depuration of BPA was very slow and statistically insignificant at 2 degrees C, but the depuration rate ( k(d)) increased linearly as temperature increased from 2 to 12 degrees C. The longest half-life of BPA in clam tissues, 221 h, was found at the lowest temperature, and the highest bioconcentration factor of 144 was measured at 8 degrees C. Data show that P. amnicum accumulates BPA so that the bioconcentration factor rises over 100 at each temperature tested. Further, the data demonstrate the importance of temperature on the processes that control both the uptake and elimination of this compound.

Toxicokinetics of waterborne bisphenol A in landlocked salmon (Salmo salar m. sebago) eggs at various temperatures.

Eye-pigmented eggs of landlocked salmon were exposed to waterborne [14C]-labeled bisphenol A at four temperatures (2, 6, 8, and 12 degrees C). Both in accumulation and depuration experiments, the eggs were exposed to a bisphenol A concentration of 1 microg/L for 196 h. In the depuration experiment, the exposed eggs were placed into clean water for 96 h. At each sampling time, the eggs were dissected into three parts (eggshell, embryo, and yolk sac), and all of these parts were weighed and analyzed separately in a liquid scintillation counter. The results show that waterborne bisphenol A accumulates in developing salmon eggs. Accumulation of bisphenol A showed a clear trend both in whole eggs and in dissected parts: the higher the temperature, the higher the uptake rate constant and the concentration of bisphenol A. Steady state was reached only in the embryos at the lowest temperature; in other cases, accumulation was linear. Depuration was significant only in the embryos at 2 and 6 degrees C. As the accumulation and the depuration results show, the elimination of bisphenol A was evidently more significant at lower temperatures.

Radiosensitivity of dermal and tumor fibroblasts derived from the same patient.

The in vitro radiosensitivity of dermal fibroblasts has been found to vary between individuals, and a number of studies have also shown that this parameter correlates with radiation-induced late injuries in clinical radiotherapy. In addition, certain genetic disorders are known to effect radiosensitivity, e.g. normal tissues of patients homozygous or heterozygous for the ataxia teleangiectasia gene show unusual sensitivity to radiation both in vivo and in vitro. Thus, it has been assumed that there is a genetically determined component resulting in a certain intrinsic cellular radiation response in an individual. To study this possible relationship between different cells of a specific patient, we established eight pairs of dermal and tumor fibroblast cultures. The donor patients had either adenocarcinoma of the uterus or squamous cell carcinoma (SCC) of the head and neck. The radiosensitivity of these strains was determined by a 96-well plate clonogenic assay, previously used by us for radiosensitivity testing of cancer cells. From a paired comparison, the values for the cell fraction surviving 2.0 Gy (SF2), of both fibroblast strains, were found to be on the same level in five out of eight cases. In patient 6, the SF2 of tumor fibroblasts was significantly higher than that of dermal fibroblasts (P=0.0014). In two additional cases the tendency was the same, but not statistically significant. As groups, the two types of fibroblasts did not differ from each other, mean SF2 values of 0.24+/-0.07 and 0.21+/-0.05, respectively. The SF2 of tumor fibroblasts from SCC patients proved to be significantly higher than that of the adenocarcinoma patients (P=0.030). These preliminary results indicate that the in vitro radiosensitivity of tumor fibroblasts correlates with normal cell sensitivity in many cases, but not in all. The radiosensitivity of tumor fibroblasts also seems to follow the level of in vitro radiosensitivity determined for the corresponding histological type of tumor cells. Further studies are needed to determine more closely the relationship between the radiosensitivities of tumor cells and tumor fibroblasts, thus evaluating the possibility of testing radiosensitivity from tumor fibroblasts in order to estimate tumor response.

Movement protein-derived resistance to triple gene block-containing plant viruses.

Two mutant potato virus X (PVX) movement protein (MP) genes (m 12K-Sal and m 12K-Kpn) were obtained by inserting specific linkers at the boundary between the N-terminal hydrophobic and putative transmembrane segment, and the central invariant hydrophilic region of the respective 12 kDa, 12K, triple gene block (TGB) protein. Several transgenic potato lines which expressed m 12K-Sal or m 12K-Kpn to different degrees were resistant to infection by PVX, potato aucuba mosaic potexvirus and the carlaviruses potato virus M and S over a wide range of inoculum concentrations (3-300 micrograms/ml). However, they were not resistant to potato virus Y, which lacks a TGB protein. We suggest that the resistance of m 12K-Sal and m 12K-Kpn transgenic potato lines is MP-derived and not RNA-mediated.

Transgenic potato plants expressing mammalian 2'-5' oligoadenylate synthetase are protected from potato virus X infection under field conditions.

We cloned and sequenced a rat cDNA encoding the 2'-5' oligoadenylate synthetase, a component of the mammalian interferon-induced antiviral response, and used Agrobacterium-mediated transformation to generate transgenic potato clones expressing this mammalian enzyme. In transgenic plants infected with potato virus X and followed under field conditions, virus concentrations in leaves and in tubers were significantly lower than in nontransgenic controls. Additionally, virus concentration in the leaves of five transgenic clones and in tubers of one clone was also lower than in transgenic potatoes expressing potato virus X coat protein.

Somatic embryogenesis and regeneration of flowering plants in rose.

Somatic embryos of the cut rose cultivars 'Domingo' and 'Vickey Brown' were obtained from callus derived from leaf explants on half strength Murashige and Skoog medium with low concentrations of kinetin and 1-naphthyl acetic acid or 2-naphthyloxyacetic acid. Somatic embryos were first observed after 6 to 12 weeks of culture on callus formed at the basis or midrib of the leaf. Embryos could be grown to phenotypically true to type greenhouse plants.