The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Red yeasts from leaf surfaces and other habitats: three new species and a new combination of Symmetrospora (Pucciniomycotina, Cystobasidiomycetes).

Our understanding of the systematics of red yeasts has greatly improved with the availability of sequence data and it is now clear that the majority of these fungi belong to three different classes of Pucciniomycotina (Basidiomycota): Agaricostilbomycetes, Cystobasidiomycetes, and Microbotryomycetes. Despite improvements in phylogenetic placement, the taxonomy of these fungi has long been in need of revision and still has not been entirely resolved, partly due to missing taxa. In the present study, we present data of culture-based environmental yeast isolation, revealing several undescribed species of Symmetrospora, which was recently introduced to accommodate six species previously placed in the asexual genera Sporobolomyces and Rhodotorula in the gracilis/marina clade of Cystobasidiomycetes. Based on molecular phylogenetic analyses of three rDNA loci, morphology, and biochemical studies, we formally describe the following new species: Symmetrospora clarorosea sp. nov. from leaf surfaces in Portugal and the USA; S. pseudomarina sp. nov. from leaf surfaces in Brazil, and the USA and decaying wood in the USA; and S. suhii sp. nov. from a beetle gut in the USA, leaf surfaces in Brazil and marine water in the Taiwan and Thailand. Finally, we propose a new combination for Sporobolomyces oryzicola based on our molecular phylogenetic data, Symmetrospora oryzicola comb. nov.

Electrokinetic device design and constraints for use in high conductance solutions.

The quest for new cell-free DNA and exosome biomarker-based molecular diagnostics require fast and efficient sample preparation techniques. Conventional methods for isolating these biomarkers from blood are both time-consuming and laborious. New electrokinetic microarray devices using dielectrophoresis (DEP) to isolate cell-free DNA and exosome biomarkers have now greatly improved the sample preparation process. Nevertheless, these devices still have some limitations when used with high conductance biological fluids, e.g. blood, plasma, and serum. This study demonstrates that electrochemical damage may occur on the platinum electrodes of DEP microarray devices. It further examines two model device designs that include a parallel wire arrangement and a planar array. Effective isolation of fluorescent beads with parallel wires is shown under low-conductance conditions (10-4  S/m), but electrothermal flow overcomes DEP forces under high conductance conditions (>0.1 S/m). Planar devices are shown to be effective under high conductance conditions (∼1 S/m) without the deleterious effects of electrothermal flow. This study provides new insights into design compromises and limitations for producing future electrokinetic devices for better performance with high conductance solutions.

Oesophageal involvement in 26 consecutive patients with mucous membrane pemphigoid.

BACKGROUND: Oesophageal involvement of mucous membrane pemphigoid (MMP) has not yet been thoroughly described. OBJECTIVES: To characterize systematically the endoscopic lesions of a series of patients with oesophageal symptoms seen at a referral centre for autoimmune bullous diseases. METHODS: Clinical, endoscopic and immunological findings of consecutively referred patients with MMP with oesophageal involvement, systemic and endoscopic treatments, and follow-up are described. RESULTS: Of 477 consecutive patients with MMP consulting between 2002 and 2012, 26 (5·4%) had symptomatic oesophageal involvement. Dysphagia, observed in 23 (88%) patients, was the most frequent symptom. Oesophageal symptoms could be the first sign of MMP. Patients with oesophageal involvement had a mean of three other involved sites. At initial oesophageal endoscopy, 17 of 26 patients had active lesions (intact bullae, erosions and/or erythema), 15 had stricture(s) and 12 had other cicatricial lesions. Systemic therapy alone achieved oesophageal symptom relief for five patients. Dilatation was combined with systemic therapy for 12 patients and was successful in nine; one perforation occurred. CONCLUSIONS: Symptomatic oesophageal involvement affected 5·4% of patients with MMP. Dermatologists and gastroenterologists should be aware of these mucocutaneous diseases and their oesophageal involvement, as it could lead to earlier diagnosis and better care. Oesophageal dilatation could be a therapeutic option for symptomatic stricture not relieved by optimized systemic therapy alone.

Tissue engineered pre-vascularized buccal mucosa equivalents utilizing a primary triculture of epithelial cells, endothelial cells and fibroblasts.

Artificial generated buccal mucosa equivalents are a promising approach for the reconstruction of urethral defects. Limiting in this approach is a poor blood vessel supply after transplantation, resulting in increased morbidity and necrosis. We generated a pre-vascularized buccal mucosa equivalent in a tri-culture of primary buccal epithelial cells, fibroblasts and microvascular endothelial cells, using a native collagen membrane as a scaffold. A successful pre-vascularization and dense formation of capillary-like structures at superficial areas was demonstrated. The lumen size of pre-formed blood vessels corresponded to the capillary size in vivo (10-30 µm). Comparing native with a highly cross-linked collagen membrane we found a distinct higher formation of capillary-like structures on the native membrane, apparently caused by higher secretion of angiogenic factors such as PDGF, IL-8 and angiopoietin by the cells. These capillary-like structures became functional blood vessels through anastomosis with the host vasculature after implantation in nude mice. This in vitro method should result in an accelerated blood supply to the biomaterial with cells after transplantation and increase the succes rates of the implant material.

Association of osteolytic lesions, bone mineral loss and trabecular sclerosis with prevalent vertebral fractures in patients with multiple myeloma.

PURPOSE: In patients with multiple myeloma (MM), computed tomography is widely used for staging and to detect fractures. Detecting patients at severe fracture risk is of utmost importance. However the criteria for impaired stability of vertebral bodies are not yet clearly defined. We investigated the performance of parameters that can be detected by the radiologist for discrimination of patients with and without fractures. METHODS AND MATERIALS: We analyzed 128 whole body low-dose CT of MM patients. In all scans a QCT calibration phantom was integrated into the positioning mat (Image Analysis Phantom(®)). A QCT-software (Structural Insight) performed the volumetric bone mineral density (vBMD) measurements. Description of fracture risk was provided from the clinical radiological report. Suspected progressive disease (PD) was reported by the referring clinicians. Two radiologists that were blinded to study outcome reported on the following parameters based on predefined criteria: reduced radiodensity in the massa lateralis of the os sacrum (RDS), trabecular thickening and sclerosis of three or more vertebrae (TTS), extraosseous MM manifestations (EOM), visible small osteolytic lesions up to a length of 8mm (SO) and osteolytic lesions larger than 8mm (LO). Prevalent vertebral fractures (PVF) were defined by Genant criteria. Age-adjusted standardized odds ratios (sOR) per standard deviation change were derived from logistic regression analysis and area under the curve (AUC) from receiver operating characteristics (ROC) analyses were calculated. ROC curves were compared using the DeLong method. RESULTS: 45% of the 128 patients showed PVF (29 of 75 men, 24 of 53 women). Patients with PVF were not significantly older than patients without fractures (64.6 ± 9.2 vs. 63.3 ± 12.3 years: mean ± SD, p=0.5). The prevalence of each parameter did not differ significantly by sex. Significant fracture discrimination for age adjusted single models was provided by the parameters vBMD (OR 3.5 [1.4-8.8], AUC=0.64 ± 0.14), SO (sOR 1.6[1.1-2.2], AUC=0.63 ± 0.05), LO (sOR 2.1[1.1-4.2] AUC=0.69 ± 0.05) and RDS (sOR 2.6[1.6-4.7], AUC=0.69 ± 0.05). Multivariate models of these four parameters showed a significantly stronger association with the development of PVF (AUC=0.80 ± 0.04) than single variables. TTS showed a significant association with PVF in men(sOR 1.5 [0.8-3.0], AUC=0.63 ± 0.08), but not in women (sOR 2.3[1.4-3.7], AUC=0.70 ± 0.07). PD was significantly associated with PVF in women (sOR 1.9[1.1-3.6], AUC=0.67 ± 0.08) but not in men (sOR 1.4[0.9-2.3], AUC=0.57 ± .07). EOM were not associated with PVF (sOR 1.0[0.4-2.6], AUC=0.51 ± .05). CONCLUSION: In multiple myeloma, focal skeletal changes in low dose CT scans show a significant association with prevalent vertebral fractures. The combination of large osteolytic lesions and loss in radiodensity as can be detected with simple CT Hounsfield measurements of the os sacrum or BMD measurements showed the strongest association to fractures and may be of value for prospective studies.

[Prevascularization of an in vitro buccal mucosa equivalent for regeneration of the urethra]. / Prävaskularisierung eines In-vitro-Mundschleimhautäquivalents zur Regeneration der Urethra.

For reconstructive interventions on the urethra the use of autologous buccal mucosa has a proven value. The aim of this study was to generate an in vitro preparation which is already infiltrated by capillary-like structures and is more rapidly incorporated after implantation. Commercially available collagen matrices which have been approved for use in humans were used as the substrate. Application possibilities of such artificial tissue in addition to reconstruction of the urethra include coverage of large defects in the oral, neck, nasal and aural areas, in gynecology and in ophthalmology.

Ultrasound-based computer navigation: an accurate measurement tool for determining combined anteversion?

INTRODUCTION: The present feasibility study examined the use of an ultrasound-based navigation system (UNS) for reliability of measurement the positions of both the femoral and acetabular components, a prerequisite to adjust the combined anteversion with sufficient accuracy when using a femur-first approach in total hip arthroplasty. METHOD: Using a UNS, five investigators performed five measurements of the posterior femoral condyles and the anterior pelvic planes (APP) of two cadavers with different body mass index. Deviations in stem and acetabular anteversion resulting from varying acquisition of the respective landmarks were determined relative to the reference measures of anteversion determined in the same cadavers from computed tomography (CT) scans. Here, both a freehand and guided ultrasound measurement methods were used to acquire the posterior femoral condyles. Femoral and acetabular anteversion values were added in order to estimate the combined anteversion of the reconstructed hip. RESULTS: Using an UNS, variations in the freehand technique for the acquisition of the posterior femoral condyles resulted in a mean error in the anteversion of the femoral component of -1.5° (SD 3.4°; -10.8° to 7.0°) while the mean error was -0.9° (SD 3.1°; -7.3° to 10.2°) when the UNS provided additional support to standardize the orientation of the UNS probe. In all cases, UNS navigation enabled to achieve combined anteversion values that fell within a clinically acceptable error range of less than ± 12.5° compared to the CT measures. CONCLUSION: Our investigations suggest that the anteversion of stem and cup can be measured with accuracy sufficient enough to utilize the concept of combined anteversion using UNS. Hence, the advantage of utilizing UNS's in a femur-first approach is the ability to intraoperatively compensate for deviations from the targeted anteversion of the stem (which is often difficult to control) by adjusting the acetabular anteversion in the final step of the implantation. In doing so, the placement of the components follows the concept of combined anteversion. Avoiding extreme anteversion values of combined anteversion could be an important step towards reducing post-operative complications following total hip arthroplasty (THA).

Lung, liver and lymph node metastases in follow-up MSCT: comprehensive volumetric assessment of lesion size changes.

PURPOSE: To investigate measurement accuracy in terms of precision and inter-rater variability in the simultaneous volumetric assessment of lung, liver and lymph node metastasis size change over time in comparison to RECIST 1.1. MATERIALS AND METHODS: Three independent readers evaluated multislice CT data from clinical follow-up studies (chest/abdomen) in 50 patients with metastases. A total of 117 lung, 77 liver and 97 lymph node metastases were assessed manually (RECIST 1.1) and by volumetry with semi-automated software. The quality of segmentation and need for manual adjustments were recorded. Volumes were converted to effective diameters to allow comparison to RECIST. For statistical assessment of precision and interobserver agreement, the Wilcoxon-signed rank test and Bland-Altman plots were utilized. RESULTS: The quality of segmentation after manual correction was acceptable to excellent in 95 % of lesions and manual corrections were applied in 21 - 36 % of all lesions, most predominantly in lymph nodes. Mean precision was 2.6 - 6.3 % (manual) with 0.2 - 1.5 % (effective) relative measurement deviation (p <.001). Inter-reader median variation coefficients ranged from 9.4 - 12.8 % (manual) and 2.9 - 8.2 % (volumetric) for different lesion types (p < .001). The limits of agreement were ± 9.8 to ± 11.2 % for volumetric assessment. CONCLUSION: Superior precision and inter-rater variability of volumetric over manual measurement of lesion change over time was demonstrated in a whole body setting.

Neoplastic and proliferative disorders of the perinephric space.

The perinephric space is a well-marginated central compartment of the retroperitoneum, located between the anterior and posterior pararenal spaces. Various neoplastic and proliferative disorders can affect the perinephric space, and there is a wide array of imaging findings. Although many perinephric lesions may extend directly from the kidney and adrenal gland, other lesions occur in the perinephric space due to haematogenous spread, as part of a systemic disease, or by extension from an adjacent retroperitoneal compartment. Imaging plays a pivotal role in the diagnosis of perinephric diseases, as many of the disease processes affecting this space will not result in clinical signs or symptoms until the disease is at an advanced stage. Despite the often shared non-specific clinical and imaging findings among these disease processes, application of a categorical differential diagnosis based on the imaging characteristics will serve to narrow the differential diagnosis and direct further evaluation and treatment. In this article, the lesions have been categorized as soft-tissue rind [nephroblastomatosis, fibrosis, Erdheim-Chester disease (ECD), extramedullary haematopoiesis, lymphoma, infiltrating metastases], focal solid lesions (extension of renal or adrenal malignancies, melanoma metastases, treated lymphoma), fat-containing lesions (angiomyolipoma, liposarcoma, myelolipoma), and cystic lesions (lymphangiomas, abscesses). The aim of this article is to demonstrate and describe the key imaging features of several neoplastic and proliferative disorders that affect the perinephric space.

Clinical lymph node staging--influence of slice thickness and reconstruction kernel on volumetry and RECIST measurements.

PURPOSE: Therapy response evaluation in oncological patient care requires reproducible and accurate image evaluation. Today, common standard in measurement of tumour growth or shrinkage is one-dimensional RECIST 1.1. A proposed alternative method for therapy monitoring is computer aided volumetric analysis. In lung metastases volumetry proved high reliability and accuracy in experimental studies. High reliability and accuracy of volumetry in lung metastases has been proven. However, other metastatic lesions such as enlarged lymph nodes are far more challenging. The aim of this study was to investigate the reproducibility of semi-automated volumetric analysis of lymph node metastases as a function of both slice thickness and reconstruction kernel. In addition, manual long axis diameters (LAD) as well as short axis diameters (SAD) were compared to automated RECIST measurements. MATERIALS AND METHODS: Multislice-CT of the chest, abdomen and pelvis of 15 patients with lymph node metastases of malignant melanoma were included. Raw data were reconstructed using different slice thicknesses (1-5 mm) and varying reconstruction kernels (B20f, B40f, B60f). Volume and RECIST measurements were performed for 85 lymph nodes between 10 and 60 mm using Oncology Prototype Software (Fraunhofer MEVIS, Siemens, Germany) and were compared to a defined reference volume and diameter by calculating absolute percentage errors (APE). Variability of the lymph node sizes was computed as relative measurement differences, precision of measurements was computed as relative measurement deviation. RESULTS: Mean absolute percentage error (APE) for volumetric analysis varied between 3.95% and 13.8% and increased significantly with slice thickness. Differences between reconstruction kernels were not significant, however, a trend towards middle soft tissue kernel could be observed.. Between automated and manual short axis diameter (SAD, RECIST 1.1) and long axis diameter (LAD, RECIST 1.0) no significant differences were found. The most unsatisfactory segmentation results occurred in higher slice thickness (3 and 5 mm) and sharp tissue kernel. CONCLUSION: Volumetric analysis of lymph nodes works satisfying in a clinical setting. Thin slice reconstructions (≤3 mm) and a middle soft tissue reconstruction kernel are recommended. LAD and SAD did not show significant differences regarding APE. Automated RECIST measurement showed lower APE than manual measurement in trend.

Interventional bleeding, hematoma and scar-formation after vacuum-biopsy under stereotactic guidance: Mammotome(®)-system 11 g/8 g vs. ATEC(®)-system 12 g/9 g.

PURPOSE: To evaluate prospectively the correlation of scar-formations after vacuum-assisted biopsy with different systems and needle-sizes and interventional bleeding/post-interventional hematoma. METHODS AND MATERIALS: Between 01/2008 and 12/2009, 479 patients underwent vacuum-assisted biopsy under stereotactic-guidance, using the Mammotome(®)-system with 11/8-gauge and ATEC(®)-system with 12/9-gauge, whereas in 178 cases with representative benign histology no surgical-biopsy after vacuum-assisted biopsy was performed and at least a 2-plane-follow-up-mammogram after 6 month post-vacuum-assisted biopsy was available. Bleeding during intervention, hematoma post-intervention and scar-tissue was scored as minimal and moderate/severe. Statistical analysis included Chi-Square-trend-test, p-value <0.05 was considered to be significant. RESULTS: Significantly more bleedings and post-interventional hematomas for 8-gauge-Mammotome(®)-system vs. 11-gauge-Mammotome(®)-system (41.9% vs. 8.4%, p<0.001/35.5% vs. 16.7%, p=0.029), no significant-differences for the ATEC(®)-systems 9-gauge vs. 12-gauge (26.9% vs. 29.7%, p=0.799/42.3% vs. 43.2%, p=0.596). 11-gauge-Mammotome(®)-system vs. ATEC(®)-12-gauge-system revealed significantly less bleedings/hematomas (8.4% vs. 29.7%, p=0.015/16.7% vs. 43.2%, p=0.001), no significant differences for the large-systems (p=0.135/p=0.352). Follow-up of Mammotome(®)-11/8-gauge-system system has shown 13.1/16.1% minimal scar-formation and 1.2/3.2% moderate/severe scars, whereas ATEC(®)-12/9-gauge-system has shown 10.8/3.8% minimal scar-formation and 0/11.5% moderate/severe scars, no significant differences. No significant difference was found when comparing Mammotome(®)-11/8-g-systems vs. ATEC(®)-12/9-g-systems (p=0.609/p=0.823). There was also no correlation between risk of scar-formation after occurrence of bleeding or hematoma with any examined VAB-system or any needle size in this study (p=0.800). CONCLUSION: Using larger needle-sizes significantly (Mammotome(®))/not significant for ATEC(®)) more interventional bleedings and post-interventional hematomas were detected, only a tendency concerning scar-formation.

Semi-automated volumetric analysis of lymph node metastases during follow-up--initial results.

OBJECTIVE: Quantification of tumour burden in oncology requires accurate and reproducible evaluation. The current standard is RECIST measurement with its inherent disadvantages. Volumetric analysis is an alternative for therapy monitoring. The aim of this study was to evaluate the feasibility of volumetric analysis of lymph node metastases using a software prototype in a follow-up setting. METHODS: MSCT was performed in 50 patients covering the chest, abdomen and pelvis. A total of 174 suspicious lymph nodes were evaluated by two radiologists regarding short axis diameters and volumetric analysis using semi-automated software. Quality of segmentation, time, maximum diameter and volume were documented. Variability of the derived change rates was computed as the standard deviation of the difference of the obtained respective change rates. RESULTS: The software performance provides robust volumetric analysis. Quality of segmentation was rated acceptable to excellent in 76-79% by each reader. Mean time spent per lesion was 38 s. The variability of change in effective diameters was 10.6%; for change rates of RECIST maximum diameter variability was 27.5%. CONCLUSION: Semi-automated volumetric analysis allows fast and convenient segmentation of most lymph node metastases. Compared with RECIST the inter-observer-variability in baseline and follow-up is reduced. This should principally allow subtle changes to be subclassified within the RECIST stable range as minor response [-15% to +10%].

Clinical-anthropometric and aesthetic analysis of nose and lip in unilateral cleft lip and palate patients.

OBJECTIVE: Presentation of a technique to determine objectively the degree of symmetry in the area of nose and lip in cleft patients based on analysis of photographs. To compare the objective measurements with the subjective impression. DESIGN: This was a retrospective study using a predefined photo documentation standard to capture images of the area of nose and lip. SETTING: Department of Oral and Maxillofacial Surgery, University of Jena, Germany. PATIENTS: Unilateral cleft patients following primary lip repair (group 1; n  =  36) or secondary correction (group 2; n  =  23). MAIN OUTCOME MEASURES: Measurements were taken on standardized photographs of three dimensions in the area of the nose and two dimensions in the upper lip region. Sign tests were used to ascertain differences between the cleft and unaffected sides separately for each group. Subjective impressions regarding symmetry were gathered and quantified by means of a visual analog scale (VAS). The Mann-Whitney U test was employed to compare the observers' impressions between the two groups. RESULTS: While significant side differences were found for all distances in group 1, only the side differences in the height of the nostril remained significant in group 2. Subjective evaluation of the nostril area improved significantly following corrective surgery. However, no such change in the area of the upper lip was recognized by the observers. CONCLUSIONS: We were able to demonstrate that the measurable symmetry of the nostril area, as well as the upper lip, was significantly enhanced by corrective surgery. However, only the subjective impression of the nostril was improved.

Breast ultrasound elastography--results of 193 breast lesions in a prospective study with histopathologic correlation.

PURPOSE: To evaluate the diagnostic performance of ultrasound elastography in breast masses. MATERIAL AND METHODS: 193 lesions (129 benign, 64 malignant) were analyzed with the EUB 8500 Logos-ultrasonic-unit (Hitachi Medical, Japan) and a linear-array-transducer of 7.5-13-MHz. Standard of reference was cytology (FNAfine needle aspiration) or histology (core biopsy). The elastic-score was classified according to a 6-point colour-scale (Ueno classification; 1-3 = benign, 4-5 = malignant). Conventional B-mode ultrasound (US) findings were classified according to the BI-RADS classification. Statistical analysis included sensitivity, specificity, ROC-analysis and kappa-values for intra-/interobserver reliability. RESULTS: The mean score for elasticity was 4.1 ± 0.9 for malignant lesions, and 2.1 ± 1.0 for benign lesions (p < 0.001). With a best cut-off point between elasticity scores 3 and 4, sensitivity was 96.9%, and specificity 76%. Setting a best cut-off point for conventional US between BI-RADS 4 and 5, sensitivity was 57.8%, and specificity 96.1%. Elastography provided higher sensitivity and lower specificity than conventional US, but two lesions with elasticity score 1 were false negative, whereas no lesion scored BI-RADS 1-3 were false negative. ROC-curve was 0.884 for elastography, and 0.820 for conventional US (p < 0.001). Weighted kappa-values for intra-/interobserver reliability were 0.784/0.634 for BI-RADS classification, and 0.720/0.561 for elasticity scores. CONCLUSION: In our study setting, elastography does not have the potential to replace conventional B-mode US for the detection of breast cancer, but may complement conventional US to improve the diagnostic performance.

Development and in vitro characterization of paclitaxel and docetaxel loaded into hydrophobically derivatized hyperbranched polyglycerols.

In this study we report the development and in vitro characterization of paclitaxel (PTX) and docetaxel (DTX) loaded into hydrophobically derivatized hyperbranched polyglycerols (HPGs). Several HPGs derivatized with hydrophobic groups (C(8/10) alkyl chains) (HPG-C(8/10)-OH) and/or methoxy polyethylene glycol (MePEG) chains (HPG-C(8/10)-MePEG) were synthesized. PTX or DTX were loaded into these polymers by a solvent evaporation method and the resulting nanoparticle formulations were characterized in terms of size, drug loading, stability, release profiles, cytotoxicity, and cellular uptake. PTX and DTX were found to be chemically unstable in unpurified HPGs and large fractions (∼80%) of the drugs were degraded during the preparation of the formulations. However, both PTX and DTX were found to be chemically stable in purified HPGs. HPGs possessed hydrodynamic radii of less than 10nm and incorporation of PTX or DTX did not affect their size. The release profiles for both PTX and DTX from HPG-C(8/10)-MePEG nanoparticles were characterized by a continuous controlled release with little or no burst phase of release. In vitro cytotoxicity evaluations of PTX and DTX formulations demonstrated a concentration-dependent inhibition of proliferation in KU7 cell line. Cellular uptake studies of rhodamine-labeled HPG (HPG-C(8/10)-MePEG(13)-TMRCA) showed that these nanoparticles were rapidly taken up into cells, and reside in the cytoplasm without entering the nuclear compartment and were highly biocompatible with the KU7 cells.

Semi-automated volumetric analysis of artificial lymph nodes in a phantom study.

PURPOSE: Quantification of tumour burden in oncology requires accurate and reproducible image evaluation. The current standard is one-dimensional measurement (e.g. RECIST) with inherent disadvantages. Volumetric analysis is discussed as an alternative for therapy monitoring of lung and liver metastases. The aim of this study was to investigate the accuracy of semi-automated volumetric analysis of artificial lymph node metastases in a phantom study. MATERIALS AND METHODS: Fifty artificial lymph nodes were produced in a size range from 10 to 55mm; some of them enhanced using iodine contrast media. All nodules were placed in an artificial chest phantom (artiCHEST®) within different surrounding tissues. MDCT was performed using different collimations (1-5 mm) at varying reconstruction kernels (B20f, B40f, B60f). Volume and RECIST measurements were performed using Oncology Software (Siemens Healthcare, Forchheim, Germany) and were compared to reference volume and diameter by calculating absolute percentage errors. RESULTS: The software performance allowed a robust volumetric analysis in a phantom setting. Unsatisfying segmentation results were frequently found for native nodules within surrounding muscle. The absolute percentage error (APE) for volumetric analysis varied between 0.01 and 225%. No significant differences were seen between different reconstruction kernels. The most unsatisfactory segmentation results occurred in higher slice thickness (4 and 5 mm). Contrast enhanced lymph nodes showed better segmentation results by trend. CONCLUSION: The semi-automated 3D-volumetric analysis software tool allows a reliable and convenient segmentation of artificial lymph nodes in a phantom setting. Lymph nodes adjacent to tissue of similar density cause segmentation problems. For volumetric analysis of lymph node metastases in clinical routine a slice thickness of ≤3mm and a medium soft reconstruction kernel (e.g. B40f for Siemens scan systems) may be a suitable compromise for semi-automated volumetric analysis.

A potential role for indoleamine 2,3-dioxygenase (IDO) in Rhodococcus equi infection.

Rhodococcus equi is a facultative intracellular bacterial pathogen of foals and immunocompromised humans that infects and proliferates within host macrophages and dendritic cells (DC). Indoleamine 2,3-dioxygenase (IDO), the initial enzyme in the tryptophan catabolism pathway, is upregulated in R. equi infected equine monocyte-derived DC and alveolar macrophages. Tryptophan requirement of R. equi for extracellular and intracellular growth was assessed. Growth of R. equi in minimal media did not require tryptophan and pharmacologic inhibition of IDO had no effect on intracellular proliferation of R. equi in equine alveolar macrophages. To investigate an immune-regulatory role for INDO in R. equi infection, IDO(-/-) (B6.129-(Indotm1Alm)/J) (n=22) and strain matched control (C57BL/6J) (n=20) mice were infected with R. equi by intraperitoneal injection, for 3 and 6 days. There was no difference in bacterial counts in liver or spleen between the two groups. Histological sections of liver and spleen were assigned inflammation scores and RT-PCR for interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), IL-4, IL-6, IL-10, IL-12, IL-23, forkhead box P3 (FoxP3), and transforming growth factor-beta (TGFß) was performed on liver and spleen. Liver tissue of IDO(-/-) had higher inflammation scores at 6 days post-infection (PI) (P=0.05) and had decreased expression of TGFß at 3 days PI (P=0.01), and FOXP3 at 3 days (P=0.02) and 6 days (P=0.03) compared to control mice. Immunostaining for FOXP3 showed lower numbers of FOXP3+ regulatory T cells in liver of IDO(-/-) mice 6 days PI. Prolonged inflammation in the liver tissue of IDO(-/-) mice corresponded with lower expression of FOXP3 and TGFß in that tissue, and also with lower numbers of FOXP3+ regulatory T cells. We conclude that IDO expression by activated macrophages and DC plays a role in dampening the inflammatory response to R. equi infection in mice.

Detection of artificial air space opacities with digital radiography: ex vivo study on enhanced latitude post-processing.

PURPOSE: To evaluate in a.-p. digital chest radiograms of an ex vivo system if increased latitude and enhanced image detail contrast (EVP) improve the accuracy of detecting artificial air space opacities in parts of the lung that are superimposed by the diaphragm. MATERIALS AND METHODS: 19 porcine lungs were inflated inside a chest phantom, prepared with 20-50 ml gelatin-stabilized liquid to generate alveolar air space opacities, and examined with direct radiography (3.0 × 2.5 k detector/ 125 kVp/ 4 mAs). 276 a.-p. images with and without EVP of 1.0-3.0 were presented to 6 observers. 8 regions were read for opacities, the reference was defined by CT. Statistics included sensitivity/specificity, interobserver variability, and calculation of Az (area under ROC curve). RESULTS: Behind the diaphragm (opacities in 32/92 regions), the median sensitivity increased from 0.35 without EVP to 0.53-0.56 at EVP 1.5-3.0 (significant in 5/6 observers). The specificity decreased from 0.96 to 0.90 (significant in 6/6), and the Az value and interobserver correlation increased from 0.66 to 0.74 and 0.39 to 0.48, respectively. Above the diaphragm, the median sensitivity for artificial opacities (136/276 regions) increased from 0.71 to 0.77-0.82 with EVP (significant in 4/6 observers). The specificity and Az value decreased from 0.76 to 0.62 and 0.74 to 0.70, respectively, (significant in 3/6). CONCLUSION: In this ex vivo experiment, EVP improved the diagnostic accuracy for artificial air space opacities in the superimposed parts of the lung (area under the ROC curve). Above the diaphragm, the accuracy was not affected due to a tradeoff in sensitivity/specificity.

Development of a permanently controllable rotating biopsy device, part II: Competitive in-vitro testing of a cannula-like prototype compared to established end-cut and side-notch biopsy devices.

PURPOSE: To evaluate the quantitative and qualitative efficiency of a permanently controllable biopsy-cannula prototype with lancet-like helically bent cutting edge for safer biopsy compared to established end-cut and side-notch biopsy devices. MATERIALS AND METHODS: Each of the n = 100 specimens per organ and system were obtained by the prototype, an end-cut device and a side-notch device (18 gauge each) using a bovine liver, kidney and myocardium as the biopsy tissue. Quantitatively, the number of fragments, length in mm and weight in mg of the specimen were analyzed. Qualitatively, a histopathological analysis was performed with respect to tissue fragmentation, crush artifact and adequacy of tissue (score per category min. 1 and max 3). To identify significant differences (p < 0.025), chi-square and Kruskal-Wallis tests were calculated in the statistical analysis. RESULTS: For each of the n = 300 specimens, the one-piece fragment/mean length/mean weight were n = 232 / 10.34 mm/ 4.86 mg for the prototype, n = 210 / 12.16 mm/ 5.35 mg for the end-cut system and n = 248 / 11.63 mm/ 4.08 mg for the side-notch system. The differences reached a level of significance with p < 0.001. The mean histopathological score for the prototype/end-cut system/side-notch system was 5.60 / 5.60 / 5.25 for the liver, 5.65 / 4.65 / 4.60 for the kidney and 5.05 / 5.35 / 4.85 for the myocardium. The differences did not reach a level of significance for the liver/kidney/myocardium with p = 0.665 /p = 0.186 /p = 0.436. CONCLUSION: Compared to established core biopsy systems, the biopsy-cannula prototype offers diagnostically equivalent biopsy specimen quality in an in-vitro setting in bovine liver, kidney and myocardium.