Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

Factors influencing work functioning after cancer diagnosis: a focus group study with cancer survivors and occupational health professionals.

PURPOSE: Cancer survivors (CSs) frequently return to work, but little is known about work functioning after return to work (RTW). We aimed to identify barriers and facilitators of work functioning among CSs. METHODS: Three focus groups were conducted with CSs (n = 6, n = 8 and n = 8) and one focus group with occupational health professionals (n = 7). Concepts were identified by thematic analysis, using the Cancer and Work model as theoretical framework to structure the results. RESULTS: Long-lasting symptoms (e.g. fatigue), poor adaptation, high work ethics, negative attitude to work, ambiguous communication, lack of support and changes in the work environment were mentioned as barriers of work functioning. In contrast, staying at work during treatment, open dialogue, high social support, appropriate work accommodations and high work autonomy facilitated work functioning. CONCLUSIONS: Not only cancer-related symptoms affect work functioning of CSs after RTW but also psychosocial and work-related factors. The barriers and facilitators of work functioning should be further investigated in studies with a longitudinal design to examine work functioning over time.

Tissue characterization of acute lesions during cardiac magnetic resonance-guided ablation of cavo-tricuspid isthmus-dependent atrial flutter: a feasibility study.

AIMS: To characterize acute lesions during cardiac magnetic resonance (CMR)-guided radiofrequency (RF) ablation of cavo-tricuspid isthmus (CTI)-dependent atrial flutter by combining T2-weighted imaging (T2WI), T1 mapping, first-pass perfusion, and late gadolinium enhancement (LGE) imaging. CMR-guided catheter ablation offers a unique opportunity to investigate acute ablation lesions. Until present, studies only used T2WI and LGE CMR to assess acute lesions. METHODS AND RESULTS: Fifteen patients with CTI-dependent atrial flutter scheduled for CMR-guided RF ablation were prospectively enrolled. Directly after achieving bidirectional block of the CTI line, CMR imaging was performed using: T2WI (n = 15), T1 mapping (n = 10), first-pass perfusion (n = 12), and LGE (n = 12) imaging. In case of acute reconnection, additional RF ablation was performed. In all patients, T2WI demonstrated oedema in the ablation region. Right atrial T1 mapping was feasible and could be analysed with a high inter-observer agreement (r = 0.931, ICC 0.921). The increase in T1 values post-ablation was significantly lower in regions showing acute reconnection compared with regions without reconnection [37 ± 90 ms vs. 115 ± 69 ms (P = 0.014), and 3.9 ± 9.0% vs. 11.1 ± 6.8% (P = 0.022)]. Perfusion defects were present in 12/12 patients. The LGE images demonstrated hyper-enhancement with a central area of hypo-enhancement in 12/12 patients. CONCLUSION: Tissue characterization of acute lesions during CMR-guided CTI-dependent atrial flutter ablation demonstrates oedema, perfusion defects, and necrosis with a core of microvascular damage. Right atrial T1 mapping is feasible, and may identify regions of acute reconnection that require additional RF ablation.

Looking at individual symptoms: the dynamic network structure of depressive symptoms in cancer survivors and their preferences for psychological care.

PURPOSE: The majority of depressed cancer survivors do not receive psychological care, possibly because offered care does not align with their experiences and preferences. We examined (1) which depressive symptoms cancer survivors would like to receive psychological care for; (2) how distinct depressive symptoms are related to each other in the contemporaneous and temporal network of depressive symptoms; and (3) whether survivors' care needs correspond to the interconnectedness of these specific symptoms. METHOD: Fifty-two cancer survivors suffering from at least mild depressive symptoms and were not receiving psychological care filled out a baseline questionnaire about their care needs for distinct depressive symptoms, followed by ecological momentary assessments (EMA) assessing depressive symptoms (14 days, five times a day). Multi-level vector autoregression analysis was used to estimate associations between distinct depressive symptoms as well as their centrality within the network. RESULTS: Cancer survivors most strongly preferred to receive care for fatigue, feeling down, little enjoyment, and sleep problems. Fatigue, together with worry and lack of concentration, most strongly predicted the onset of other symptoms. Little enjoyment and feeling down were two of the most central symptoms (i.e., strongly connected to other symptoms) in the contemporaneous network and were most strongly influenced by other symptoms in the temporal network. CONCLUSIONS: Clinicians can offer specific interventions that target fatigue, as these played an important role in the onset of symptoms and would align with survivors' needs. IMPLICATIONS FOR CANCER SURVIVORS: Offering such symptom-specific care may increase the uptake of psychological interventions in cancer survivors.

Feasibility of in-vivo estimation of the brachial artery area-pressure relation from CINE and real-time MRI during upper arm cuff inflations.

OBJECTIVE: We investigate the basic feasibility of estimating the brachial artery area-pressure relationship from MRI data obtained during pressure cuff inflations in-vivo. METHODS: We acquired cross-sectional real-time MR images and cardiac-gated CINE MR images from the upper arm of a single male subject at rest during supra-systolic pressure cuff inflations and deflations. We estimate from the MR images the lumen area changes of the brachial artery, and, simultaneously, from the cuff pressure the systemic blood pressure of the subject. We reconstruct the area-pressure curve from two real-time and three CINE independent measurements. RESULTS: The area-pressure curve can be reconstructed, and it is plausible and appears largely consistent with the literature using other methods. CONCLUSION: MR imaging during pressure cuff inflations is an easy to use, non-invasive candidate method to estimate the brachial artery pressure-area curve.

Insights into oscillometry: An Experimental Study for Improvement of Cuff-Based Blood Pressure Measurement Technology.

Non-invasive blood pressure (BP) measurements are usually performed by means of an empirical interpretation of arterial oscillations recorded via cuff based oscillometic methods. Extensive effort has been put into development of a theoretical treatment of oscillometry aiming at more accurate BP estimations and measurement of additional hemodynamic parameters. However, oscillometry is still basically a heuristic method for BP inference.This study introduces an experimental setup and discusses experimental results to improve understanding of cuff characteristics and the process by which oscillometric signals are produced, with the aim of improving cuff-based non-invasive BP measurement technology relevant in clinical practice. The work focuses on mechanical simulations of arm volume pulsations in cuff pressure signals. The effects of air compression within the cuff and the influence of viscoelastic properties of exterior cuff material are also investigated. Additionally, arm volume changes and compressibility of arm tissue due to external cuff pressure were studied with an MRI system. Our results reveal novel insights into oscillometry and enable understanding of transducer design for cuffs including the importance of viscoelastic material properties and effects of cuff inflation on arm tissue.

Insulin-like growth factor (IGF) II induced changes in expression of IGF binding proteins in lymphoid tissues of hIGF-II transgenic mice.

Overexpression of human insulin-like growth factor II (IGF-II) in transgenic mice does not result in increased overall body growth. The IGF-II overexpression, however, specifically causes growth of the thymus and not of the spleen. We address the question whether the observed differences in growth induction in lymphoid tissues by IGF-II can be related to differences in local IGF binding protein (IGFBP) production, using nonradioactive in situ hybridization and Northern blot analysis. IGFBP-2, -4, and -5 are expressed in both lymphoid tissues of normal mice. The spleen additionally expresses IGFBP-3 and IGFBP-6. IGFBP-1 expression was not detected. Although the expression pattern of the IGFBPs did not change upon IGF-II overexpression, the level of expression changed in a specific manner for each IGFBP. In both the thymus and the spleen of transgenic mice, IGFBP-2 and -5 gene expression was slightly increased, whereas the level of IGFBP-4 expression was not altered. In the spleen, IGFBP-6 expression was not altered by IGF-II overexpression, whereas IGFBP-3 expression was strongly increased. The differences in IGFBP expression, and the difference in response of these IGFBPs to IGF-II overexpression in thymus and spleen suggests an important role of these proteins in growth regulation of both lymphoid tissues. We speculate that an increase of IGFBP-3 expression together with changes in expression of other IGFBPs, inhibits IGF-II stimulated growth in the spleen by an autocrine-/paracrine pathway.

Dexamethasone-induced growth inhibition of porcine growth plate chondrocytes is accompanied by changes in levels of IGF axis components.

High (pharmacological) doses of glucocorticoids inhibit the proliferation of growth plate chondrocytes, which leads to one of the side-effects of these steroids, namely suppression of longitudinal growth. Growth inhibition by glucocorticoids is thought to be mediated in part by impaired action of components of the IGF axis, which are important for chondrocyte regulation and hence for longitudinal growth. The aim of the present study was to determine whether glucocorticoid-induced growth retardation involves changes in IGF axis components. Chondrocytes were isolated from epiphyseal growth plates of neonatal piglets and treated with pharmacological doses of dexamethasone (DXM) for 24 h to study glucocorticoid-induced growth retardation. Under IGF-I-supplemented (10 nM) culture conditions, IGF-binding proteins (IGFBPs)-2, -4 and -5 were secreted by the growth plate chondrocytes and IGFBP-2 protein and mRNA levels were decreased by the DXM treatment, whereas IGFBP-4 and -5 were not affected. Proliferation of the chondrocytes, as measured by [(3)H]thymidine incorporation, was 3.5-fold higher in serum-supplemented medium in contrast to IGF-I-supplemented (10 nM) medium. In the presence of serum, DNA synthesis was significantly inhibited by 50-63% when treated with 100 nM DXM, which was prevented by the glucocorticoid-receptor antagonist Org34116. mRNA levels of IGF axis components were determined using Northern blot analysis. IGFBP-2 to -6 were expressed in the chondrocytes, IGFBP-1 was absent and both IGF-I and IGF-II, and the type I and type II IGF receptors were expressed. Treatment with DXM (100 nM) resulted in a 2-fold increase in mRNA levels of both IGFBP-5 and the type I IGF receptor, whereas IGFBP-2 mRNA levels decreased by 55%, in concert with the decrease in protein level observed under IGF-I-supplemented culture conditions. The changes in mRNA levels due to the DXM treatment were prevented by the glucocorticoid receptor antagonist. Our data show that exposure to pharmacological doses of DXM results in inhibition of proliferation and changes in components of the IGF axis, IGFBP-2 and -5 and the type I IGF receptor, suggesting a role for these components in glucocorticoid-induced growth retardation at the local level of the growth plate.

Short-term glucocorticoid treatment of prepubertal mice decreases growth and IGF-I expression in the growth plate.

The insulin-like growth factor (IGF) system is an important mediator of postnatal longitudinal growth, and the growth inhibiting effects of glucocorticoid (GC) treatment are suggested to be due to impaired action of the IGF system. However, the precise changes of the IGFs and the IGF-binding proteins (IGFBPs) in the growth plate, occurring upon short-term GC treatment have not been characterized. Prepubertal mice treated daily with dexamethasone (DXM) for 7 days, showed significant growth inhibition of total body length and weight and weight of the liver, thymus and spleen, whereas the weight of the kidneys was not affected. Analysis of the tibial growth plate showed that the total growth plate width significantly decreased to 84.5% of control values, caused by a significant decrease in the proliferative zone. The number of proliferating cell nuclear antigen (PCNA)-positive chondrocytes in the proliferative zone decreased significantly (to 40%) and TUNEL staining showed a significant 1.6-fold increase in apoptotic hypertrophic chondrocytes. In the growth plates, both IGF-I and IGF-II, as well as IGFBP-2 mRNAs were detected, mainly in the proliferative and prehypertrophic zones. DXM treatment significantly decreased the number of chondrocytes expressing IGF-I, whereas the number of chondrocytes expressing IGF-II and IGFBP-2 were not affected. The decrease in IGF-I expression in the growth plate indicates that GC treatment affects IGF-I at the local level of the growth plate, which could contribute to the GC-induced growth retardation.

IGF and IGF-binding protein expression in the growth plate of normal, dexamethasone-treated and human IGF-II transgenic mice.

Glucocorticoid (GC) treatment in childhood can lead to suppression of longitudinal growth as a side effect. The actions of GCs are thought to be mediated in part by impaired action of the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins (IGFBP-1 to -6). We have studied the effects of GCs on IGF and IGFBP expression at the local level of the growth plate, using non-radioactive in situ hybridization. We treated 3-week-old normal mice for 4 weeks with dexamethasone (DXM). We also treated human IGF-II (hIGF-II) transgenic mice in order to investigate whether IGF-II could protect against the growth retarding effect of this GC. DXM treatment resulted in general growth retardation in both mice strains, however, only in normal mice was tibial length decreased. In both normal and hIGF-II trangenic mice, the total width of the growth plate was not affected, whereas the width of the proliferative zone decreased as a result of the DXM treatment. Additionally, only in normal mice, the width of the hypertrophic zone thickened. Only expression of IGF-I, IGF-II and IGFBP-2 could be detected in the growth plates of 7-week-old normal mice. IGFBP-1, -3, -4, -5 and -6 mRNAs were not detected. DXM treatment of normal mice induced a significant 2.4-fold increase in the number of cells expressing IGF-I mRNA, whereas IGF-II and IGFBP-2 mRNA levels were not affected. In hIGF-II transgenic mice, IGF-I mRNA levels were significantly increased, while endogenous IGF-II and IGFBP-2 mRNAs were unaffected, compared to normal animals. DXM treatment of the hIGF-II transgenic mice induced a further increase of IGF-I mRNA expression, to a similar extent as in DXM-treated normal mice. The increase of IGF-I due to DXM treatment in normal mice might be a reaction in order to minimize the GC-induced growth retardation. Another possibility could be that the increase of IGF-I would contribute to the GC-induced growth retardation by accelerating the differentiation of chondrocytes, resulting in accelerated ossification. In the growth plates of hIGF-II transgenic mice, the higher basal level of IGF-I, might be responsible for the observed partial protection against the adverse effects of GCs on bone.

On the performance and accuracy of 2D navigator pulses.

The purpose of this study was to investigate and to optimize the performance of two-dimensional spatially selective excitation pulses used for navigator applications on a clinical scanner. The influence of gradient imperfections, off-resonance effects, and incomplete k-space covering on the pencil beam-shaped spatial excitation profile of the 2D RF pulse was studied. The studies involved experiments performed on phantoms and in vivo. In addition, simulations were carried out by numerical integration of the Bloch equations. The accuracy of positioning of the pencil beam was increased by a factor of three by employing a simple correction scheme for the compensation of gradient distortions. The spatial selectivity of the 2D RF pulse was improved by taking sampling density corrections into account. The 2D RF pulse performance was found to be sufficient to monitor the diaphragm motion even at moderate gradient strength. For applications, where a high spatial resolution is required or a less characteristic contrast is present a strong gradient system is recommended.

Short-term glucocorticoid treatment of piglets causes changes in growth plate morphology and angiogenesis.

OBJECTIVE: Glucocorticoid treatment of children often leads to growth retardation, and the precise target(s) in the growth plate responsible for this effect are unknown. Angiogenesis is an important part of the endochondral ossification process, and VEGF expressed in the growth plate is essential for proper angiogenesis to occur. Since glucocorticoid treatment down-regulates VEGF expression in cultured chondrocytes, we hypothesized that in vivo glucocorticoid treatment could result in VEGF down-regulation in the growth plate and disturbed angiogenesis, thus contributing to the growth retardation. DESIGN: We treated 6-week-old prepubertal piglets (10 kg) for 5 days with prednisolone (50 mg/day). Tibial growth plate sections were studied for apoptosis and the expression of VEGF protein and mRNA and MMP-9 protein. Capillaries in the metaphysis were visualized by CD31 immunostaining. Growth plate morphology (width of various zones) was determined by interactive measurements on hematoxylin/eosin stained sections and apoptotic cells were detected by TUNEL assay. RESULTS: In the prednisolone-treated animals, the total width of the growth plate decreased to 81% of controls (P<0.02), which was explained by a decrease of the width of the proliferative zone to 73% (P<0.05). The treatment had no effect on the orderly organization of the chondrocyte columns. In the growth plates of control animals, apoptosis was shown in 5.8% of the hypertrophic chondrocytes and was limited to the terminal hypertrophic chondrocytes. In prednisolone-treated animals, 40.5% of the hypertrophic chondrocytes was apoptotic (P<0.02), with apoptotic chondrocytes also appearing higher in the hypertrophic zone. We observed fewer capillaries and loss of their parallel organization in the metaphysis in the prednisolone-treated animals. The capillaries were shorter and chaotic in appearance. In contrast to controls, in prednisolone-treated animals VEGF mRNA and protein could not be detected in the hypertrophic zone of the growth plate. Trabecular bone length in the primary spongiosa was also diminished by the treatment. No changes were observed in the expression pattern of MMP-9, a matrix metalloproteinase, which is also important for angiogenesis and bone formation. CONCLUSIONS: These results indicate that short-term glucocorticoid treatment of growing piglets severely disturbs the width of the growth plate, apoptosis of chondrocytes, VEGF expression by hypertrophic chondrocytes, the normal invasion of blood vessels from the metaphysis to the growth plate and bone formation at the chondro-osseous junction. These effects could alter the dynamics of endochondral ossification and thus contribute to glucocorticoid-induced growth retardation.

Pharmacokinetic analysis of Gd-DTPA enhancement in dynamic three-dimensional MRI of breast lesions.

The purpose of this study was to demonstrate that dynamic MRI covering both breasts can provide sensitivity for tumor detection as well as specificity and sensitivity for differentiation of tumor malignancy. Three-dimensional gradient echo scans were used covering both breasts. Before Gd-DTPA bolus injection, two scans were obtained with different flip angles, and after injection, a dynamic series followed. Thirty-two patients were scanned according to this protocol. From these scans. In addition to enhancement, the value of T1 before injection was obtained. This was used to estimate the concentration of Gd-DTPA as well as the pharmacokinetic parameters governing its time course. Signal enhancement in three-dimensional dynamic scanning was shown to be a sensitive basis for detection of tumors. In our series, all but two mammographically suspicious lesions did enhance, and in three cases, additional enhancing lesions were found, two of which were in the contralateral breast. The parameter most suited for classification of breast lesions into benign or malignant was shown to be the pharmacokinetically defined permeability k31, which, for that test, gave a sensitivity of 92% and a specificity of 70%. Our three-dimensional dynamic MRI data are sensitive for detection of mammographically occult breast tumors and specific for classification of these as benign or malignant.