Replacing the Draize eye test: Impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation.

Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test.

Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides.

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of ß-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

A multilayered electrospun graft as vascular access for hemodialysis.

Despite medical achievements, the number of patients with end-stage kidney disease keeps steadily raising, thereby entailing a high number of surgical and interventional procedures to establish and maintain arteriovenous vascular access for hemodialysis. Due to vascular disease, aneurysms or infection, the preferred access-an autogenous arteriovenous fistula-is not always available and appropriate. Moreover, when replacing small diameter blood vessels, synthetic vascular grafts possess well-known disadvantages. A continuous multilayered gradient electrospinning was used to produce vascular grafts made of collagen type I nanofibers on luminal and adventitial graft side, and poly-ɛ-caprolactone as medial layer. Therefore, a custom-made electrospinner with robust environmental control was developed. The morphology of electrospun grafts was characterized by scanning electron microscopy and measurement of mechanical properties. Human microvascular endothelial cells were cultured in the graft under static culture conditions and compared to cultures obtained from dynamic continuous flow bioreactors. Immunofluorescent analysis showed that endothelial cells form a continuous luminal layer and functional characteristics were confirmed by uptake of acetylated low-density-lipoprotein. Incorporation of vancomycin and gentamicin to the medial graft layer allowed antimicrobial inhibition without exhibiting an adverse impact on cell viability. Most striking a physiological hemocompatibility was achieved for the multilayered grafts.

Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion.

Investigation of percutaneous helminth infection is generally based on animal models or excised skin. As desirable replacement of animal experiments, tissue-engineered skin equivalents have recently been applied in microbial and viral in vitro infection models. In the present study, the applicability of tissue-engineered skin equivalents for the investigation of percutaneous helminth invasion was evaluated. Epidermal and a full-thickness skin equivalents that suit the requirements for helminth invasion studies were developed. Quantitative invasion assays were performed with the skin-invading larvae of the helminths Strongyloides ratti and Schistosoma mansoni. Both skin equivalents provided a physical barrier to larval invasion of the nematode S. ratti, while these larvae could invade and permeate a cell-free collagen scaffold and ex vivo epidermis. In contrast, the epidermal and full-thickness skin equivalents exhibited a human host-specific susceptibility to larvae of trematode S. mansoni, which could well penetrate. Invasion of S. mansoni in cell-free collagen scaffold was lowest for all experimental conditions. Thus, reconstructed epidermis and full-thickness skin equivalents confirmed a high degree of accordance to native tissue. Additionally, not only tailless schistosomula but also cercariae could permeate the skin equivalents, and thus, delayed tail loss hypothesis was supported. The present study indicates that the limitations in predictive infection test systems for human-pathogenic invading helminths can be overcome by tissue-engineered in vitro skin equivalents allowing a substitution of the human skin for analysis of the interaction between parasites and their hosts' tissues. This novel tissue-engineered technology accomplishes the endeavor to save animal lives.

Impedance spectroscopy for the non-destructive evaluation of in vitro epidermal models.

PURPOSE: Reconstructed human epidermis (RHE) is standardly used for the risk assessment of chemical compounds. However, analysis is dependent on invasive methods such as histological processing or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. METHODS: As an alternative, we have developed a non-destructive technology to analyze the integrity of epidermal equivalents based on impedance spectroscopy. RHEs were generated and impedance spectra were recorded. from these spectra, we extrapolated electrical characteristics such as the capacitance and the ohmic resistance. Furthermore, the measurable electrical parameters were used to quantify the effects of mechanical and chemical disruption of the epidermal integrity. RESULTS: A fully matured RHE exhibits typical impedance spectra in a frequency ranging between 1 Hz and 100 kHz, which is comparable to the spectra of freshly isolated human epidermal biopsies. We could show that, during RHE maturation, these characteristics change significantly. Thus, capacitance and ohmic resistance can be employed as a criterion for the quality control of skin equivalents. Additionally, our application of impedance spectroscopy reveals sufficient sensitivity to detect a transient decreased ohmic resistance caused by 2-propanol, which is classified as a non-irritant by MTT assays. CONCLUSION: These results indicate that impedance spectroscopy can be employed as a non-destructive complementary method to assess mild irritative effects, which is currently not possible.

[Radiological diagnostics of the small bowel]. / Radiologische Diagnostik des Dünndarms.

The barium meal with plain X-ray films of the small intestine has for decades been the undisputed gold standard in imaging of the small intestine. More recently, X-rays and fluoroscopy with an overall accuracy of 73% have been replaced by multislice computed tomography (MSCT) or modern magnetic resonance imaging (MRI). Ultrasound is suitable for the orienting investigation of the small intestine in the context of general abdominal sonography as well as for dedicated examinations with a sensitivity of 67-96% and a specificity up to 97%. The endoscopic examinations of the small bowel, such as video capsule endoscopy and double-balloon enteroscopy are expensive and time-consuming techniques, which provide valuable information in special indications. Other than with the stomach or colon, the diagnostics of primary small intestine tumors plays a relatively subordinate role due to the low incidence of 3-5% of all gastrointestinal (GI) neoplasms but with a high sensitivity of 84% and a specificity of up to 97% for computed tomography (CT) and MRI. Predominant questions are those concerning ileus or the diagnostics of passage disturbances after preceding operations, to depict bowel obstructions, adhesions or the involvement of the small bowel in peritoneal carcinomatosis. The sensitivity per lesion in the initial evaluation of Crohn's disease (CD) is 47-68% for capsule endoscopy, 43% for MRI and 21% for CT enterography. In cases of known CD, the sensitivity is 70% for capsule endoscopy and 79% for MRI. A further indication is the evaluation of acute or occult gastrointestinal bleeding.

Bioreactor technology in cardiovascular tissue engineering.

Cardiovascular tissue engineering is a fast evolving field of biomedical science and technology to manufacture viable blood vessels, heart valves, myocardial substitutes and vascularised complex tissues. In consideration of the specific role of the haemodynamics of human circulation, bioreactors are a fundamental of this field. The development of perfusion bioreactor technology is a consequence of successes in extracorporeal circulation techniques, to provide an in vitro environment mimicking in vivo conditions. The bioreactor system should enable an automatic hydrodynamic regime control. Furthermore, the systematic studies regarding the cellular responses to various mechanical and biochemical cues guarantee the viability, bio-monitoring, testing, storage and transportation of the growing tissue.The basic principles of a bioreactor used for cardiovascular tissue engineering are summarised in this chapter.

Surgical therapy of extracranial carotid artery aneurysms: long-term results over a 24-year period.

BACKGROUND: To evaluate long-term results of surgical therapy of extracranial carotid artery aneurysms (ECCA) and to provide a morphologic classification for individual surgical reconstruction techniques. PATIENT AND METHODS: This retrospective analysis includes 57 patients (43 male, mean age 61.9 years.) with 64 carotid reconstructions for ECCA between 1980 and 2004. In 29 (50.9%) of the patients there was found a cerebral ischemic event as an initial symptom (18 transient ischemic attacks, 11 strokes). In patients without cerebral events, the presenting symptom was pulsatile cervical mass in 19 and cranial nerve dysfunction in 3 cases. ECCA was morphologically stratified in Type I=isolated aneurysms of the internal carotid artery (n=25), Type II=aneurysms of the complete internal carotid artery with involvement of the bifurcation (n=8), Type III=aneurysms of the carotid bifurcation (n=20), Type IV=combined aneurysm of the internal and common carotid artery (n=5) and Type V=isolated aneurysm of the common carotid artery (n=6). RESULTS: Perioperative stroke rate was 1.6%. 4 patients suffered from transient ischemic attacks (6.3%). Permanent and transient cranial nerve injury rate was 6.3% and 20.3% respectively. After 5, 10, 15 and 20 years the actuarial survival was 90%, 77%, 65% and 57%. The ipsilateral stroke-free time was 96%, 96%, 93% and 87%, respectively. CONCLUSIONS: Surgical reconstruction of extracranial carotid aneurysms is a safe procedure with good long-term results. The risk of a permanent, perioperative cerebral neurological deficit is low, but there is a considerable risk of cranial nerve injury.

[Radiological imaging of the upper gastrointestinal tract. Part II. The stomach]. / Radiologische Bildgebung des oberen Gastrointestinaltraktes : Teil II: Magen.

CT, and multi-detector row computed tomography in particular, play a very important role in staging malignant tumors of the stomach. The optimized technique of so called "Hydro-CT", including distension to the gastric wall with 1-1.5 l oral contrast media, mainly water, has fostered the diagnostic value of CT in the diagnosis of diseases of the stomach. By using the "Hydro-CT" technique, the detection rate for gastric carcinoma is now between 89% and 94%, and for liver metastasis between 85% and 92%. For overall T staging, the sensitivity is rather low at between 43% and 65%, and for the lymph nodes between 64 and 88%. Depending on tumor type, multislice computed tomography (MSCT) supplies morphological details for defining the type of lesion (polyps, gastritis, lymphoma, gastrointestinal stromal tumours, carcinoma). Nevertheless, a definite differential diagnosis remains difficult. In addition, multiplanar reconstruction (MPR), derived from multi-detector row computed tomography data sets, is very helpful in localizing these pathologies and demonstrating their anatomic relationship to adjacent organs and vascular structures. MRI plays no major part in the diagnostic evaluation of the upper GI-tract.

[Radiological imaging of the upper gastrointestinal tract. Part 1. The esophagus]. / Radiologische Bildgebung des oberen Gastrointestinaltrakts. Teil I: Osophagus.

In the diagnosis of diseases of the esophagus, conventional x-ray evaluation still plays a more important role than endoscopy in the visualization of stenoses. CT plays a major role in the staging of malignancies of the esophagus, while MRI plays does not play a major part in the diagnostic evaluation of the upper GI-tract but is equal to CT for the staging and evaluation of the extent of local infiltration. The main indication for the radiological examination of the esophagus by barium studies is dysphagia. The use of barium allows a functional examination of esophageal motility. Swallow motility disorders can be diagnosed by videofluorography using high frame rate imaging. Zenker's diverticulum and other pulsion diverticula should also be investigated by functional esophageal imaging. Candida esophagitis can be identified by its characteristic ulcerations using barium swallow. The extension of gastroesophageal hernias are more accurately evaluated with barium studies than with endoscopy. The diagnosis of gastroesophageal reflux disease should be made by barium studies, but discrete inflammation as well as epithelial dysplasia are best investigated by classic endoscopy and modern endoscopic techniques. In cases of esophageal carcinoma, radiology adds to the findings of endoscopy and endosonography.

Comparison of CT during arterial portography and MR during arterial portography in the detection of liver metastases.

OBJECTIVE: This study compared MR during arterial portography (MRAP) with CT during arterial portography (CTAP) with regard to the detection and differentiation of liver metastases before surgery. MATERIALS AND METHODS: Fifteen patients with liver metastases were enrolled before surgery according to the guidelines of our institutional review board and good clinical practice. After mesentericography, unenhanced scans (Volume Zoom) were performed initially. For CTAP, the contrast medium was injected through the superior mesenteric artery. Images were acquired in portal and delayed enhancement. The MR protocol (1.5 T; Magnetom Symphony) started with T1-weighted fast low-angle shot (FLASH) T2-weighted turbo spin echo (TSE). MRAP followed with gadolinium-enhanced dynamic T1-weighted 3D FLASH. Delayed-phase T1-weighted 2D FLASH axial images were performed 2 min after IV injection of the contrast medium. Qualitative and quantitative evaluation of CTAP and MRAP was performed by three blinded radiologists regarding the number of lesions and their size, localization, and differential diagnosis. RESULTS: The overall sensitivity in detecting liver metastases was 97% with MRAP and 93% with CTAP (p > 0.05, not significant [n.s.]). The specificity was calculated to be 97% for MRAP and 82% for CTAP (p < 0.0001, statistically significant [s.s.]). The differences in sensitivity were more accentuated if only lesions 10 mm or smaller were considered (95% vs 88%, p > 0.05, n.s.), for which the respective specificities were 95% and 80% (p < 0.0014, s.s.). Improvements in sensitivity and specificity were associated with a higher lesion-to-liver contrast-to-noise ratio (59.4 +/- 51.0 for MRAP vs 10.4 +/- 7.3 for CTAP) and resulted in higher diagnostic confidence in the differential diagnosis of liver lesions (p < 0.001, s.s.) and better interobserver agreement (median kappa value, 0.88 vs 0.63). CONCLUSION: MRAP proved to be a reliable method in the preoperative detection of small liver metastases in particular, with a higher sensitivity and specificity than CTAP. If organizational difficulties of MRAP can be overcome, MRAP could be considered instead of CTAP in the preoperative invasive evaluation of metastatic liver disease.

[Intraoperative endosonographic guided resection of tongue carcinoma]. / Intraoperative endosonographisch gesteuerte Resektion von Zungenkarzinomen.

BACKGROUND: Exact estimation of a tumor's size and the definition of adequate resection margins in carcinomas of the tongue are often difficult because of the tumor's extension and deep infiltration. METHODS: We have developed a method that allows intraoperative visualisation and marking of tumor margins. Intra-operative endosonography was performed on nine patients with carcinomas of the tongue using a 8-12 MHz linear array transducer. The oral cavity was flooded with normal saline solution and the transducer was immersed therein. This allowed scanning in a non-contact mode. The tumor margins were marked with a surgical suture under endosonographic monitoring. RESULTS: In the nine patients studied, the histological margins corresponded to the sonographic margins. The sonographic marking proved to be useful during the resection of the tumor and histological safety margins were respected in each case. CONCLUSIONS: This non-invasive procedure provides a quick and reliable orientation during the resection of tongue carcinoma, and a more precise and individual definition of resection margins is possible. Intraoperative non-contact use of endosonography is a promising method.

[Follow up of Crohn's disease under therapy with hydro-MRI]. / Hydro-MRT in der Verlaufsbeobachtung des Morbus Crohn unter Therapie.

PURPOSE: Evaluation of typical MRI-findings in patients with Crohn's disease receiving therapy. Correlation with the course of disease. PATIENTS AND METHODS: 81 follow-up MRI-studies in 25 patients conducted within a period of 3 weeks to 4 years were evaluated retrospectively. Therapy consisted in various combinations of antibiotics and immunosuppressive agents and if necessary operation. The findings of the MRI-studies were correlated with clinical data (e.g.operation of Crohn's complications) and the subjective perception during therapy. RESULTS: The morphological substrate of Crohn's disease in the Hydro-MRI images is reliably detected. Especially in a delineation of extraluminal changes MRI is superior to endoscopy and enteroclysis. Independent from clinical symptoms short- and mid-term follow-up showed inflammatory changes of the intestinal wall in all 25 patients. In 24/81 studies there was persistence or even progression of Crohn's disease in the MRI-studies, although patients were free of symptoms by the time of image acquisition. CONCLUSION: Hydro-MRI is a modality for the evaluation of inflammatory changes in patients with Crohn's disease. Independent from clinical symptoms persistence of Crohn's disease is detectable.

Radiation dose reduction in chest radiography using a flat-panel amorphous silicon detector.

AIM: The aim of this study was to evaluate the image quality and the potential for radiation dose reduction with a digital flat-panel amorphous silicon detector radiography system. MATERIAL AND METHODS: Using flat-panel technology, radiographs of an anthropomorphic thorax phantom were taken with a range of technical parameters (125kV, 200mA and 5, 4, 3.2, 2, 1, 0.5, and 0.25mAs) which were equivalent to a radiation dose of 332, 263, 209, 127, 58.7, 29, and 14 microGy, respectively. These images were compared to radiographs obtained by a conventional film-screen radiography system at 125kV, 200mA and 5mAs (equivalent to 252 microGy) which served as reference. Three observers evaluated independently the visibility of simulated rounded lesions and anatomical structures, comparing printed films from the flat-panel amorphous silicon detector and conventional x-ray system films. RESULTS: With flat-panel technology, the visibility of rounded lesions and normal anatomical structures at 5, 4, and 3.2mAs was superior compared to the conventional film-screen radiography system. (P< or =0.0001). At 2mAs, improvement was only marginal (P=0.19). At 1.0, 0.5 and 0.25mAs, the visibility of simulated rounded lesions was worse (P< or =0.004). Comparing fine lung parenchymal structures, the flat-panel amorphous silicon detector showed improvement for all exposure levels down to 2mAs and equality at 1mAs. CONCLUSION: Compared to a conventional x-ray film system, the flat-panel amorphous silicon detector demonstrated improved image quality and the possibility for a reduction of the radiation dose by 50% without loss in image quality.

Endoscopic variceal ligation plus propranolol vs. transjugular intrahepatic portosystemic stent shunt: a long-term randomized trial.

BACKGROUND AND STUDY AIMS: After a first variceal bleeding episode in patients with cirrhosis of the liver, treatment with transjugular intrahepatic portosystemic stent shunt (TIPS) and endoscopic variceal ligation (EVL) plus propranolol were compared, with regard to prevention of variceal rebleeding, complications, and mortality. PATIENTS AND METHODS: 85 patients were randomly allocated to receive TIPS (n = 43) or EVL (n = 42). The groups were comparable regarding age, sex, etiology of liver cirrhosis, and liver function. RESULTS: The mean observation times were 4.1 years in the TIPS group and 3.6 years in the EVL group. Although the probability of rebleeding was higher in the EVL group (29.9%) than in the TIPS group (19.4%), the difference was not statistically significant. Three of five patients of the EVL group successfully underwent TIPS placement after treatment failure. The probability of TIPS dysfunction requiring shunt revision was 89 %. Hepatic encephalopathy was observed more often in the TIPS group (40.5%) than in the EVL group (20.5%; P < 0.05). The probability of survival was similar in both groups (TIPS group 75.9%, EVL group 82.2%; n.s.). CONCLUSIONS: In view of its good efficacy and the lower cost of treatment, endoscopic ligation plus propranolol may be recommended as initial procedure for prevention of recurrent variceal hemorrhage, whereas TIPS seems to be the preferable procedure in patients with recurrent bleeding after adequate endoscopic and pharmacological treatment.

[Organ-sparing surgery of renal cell carcinoma - operative technique and findings in radiological follow-up]. / Organerhaltende Chirurgie des Nierenzellkarzinoms - Operationstechniken und Befunde in der radiologischen Nachsorge.

Nephron-sparing surgery of renal cell carcinoma in the 1970's and 1980's in patients with bilateral renal tumors or reduced renal function (imperative indication) has shown a very low risk of recurrent cancer. Today, nephron-sparing surgery in renal cell carcinoma is considered in an increasing number of patients with expected sufficient renal function after nephrectomy (elective indication). Resection technique, the use of Tabotamp(R) to reduce bleeding, and pseudotumors do complicate the interpretation of the images. It has been not yet defined which diagnostic modality is best suited for follow-up after renal cell carcinoma resection. Follow-up protocols in different institutions show a wide variety. The follow-up of patients after nephron-sparing surgery is performed by annual sonography or MRI every three months. Up to now, CT and ultrasound are the standard methods. MRI with its multiplanar imaging and improved soft tissue contrast seems to have an equal diagnostic value. Additionally, MRI seems to be suited for patients with reduced renal function. The aim of this paper is to give guidelines for the radiologist to understand the different surgical procedures and to evaluate the postoperative findings. Different imaging modalities in the follow-up of patients and special radiological phenomena are discussed.

Restoration of liver function and portosystemic pressure gradient after TIPSS and late TIPSS occlusion.

TIPSS (transjugular intrahepatic portosystemic shunt) may be indicated to control bleeding from esophageal and gastric varicose veins, to reduce ascites, and to treat patients with Budd-Chiari syndrome and veno-occlusive disease. Numerous measures to improve the safety and methodology of the procedure have helped to increase the technical and clinical success. Follow-up of TIPSS patients has revealed shunt stenosis to occur more often in patients with preserved liver function (Child A, Child B). In addition, the extent of liver cirrhosis is the main factor that determines prognosis in the long term. Little is known about the effects of TIPSS with respect to portosystemic hemodynamics. This report deals with a cirrhotic patient who stopped drinking 7 months prior to admission. He received TIPSS to control ascites and recurrent esophageal bleeding. Two years later remarkable hypertrophy of the left liver lobe and shunt occlusion was observed. The portosystemic pressure gradient dropped from 24 mmHg before TIPSS to 11 mmHg and remained stable after shunt occlusion. The Child's B cirrhosis prior to TIPSS turned into Child's A cirrhosis and remained stable during the follow-up period of 32 months. This indicates that liver function of TIPSS patients may recover due to hypertrophy of the remaining non-cirrhotic liver tissue. In addition the hepatic hemodynamics may return to normal. In conclusion, TIPSS cannot cure cirrhosis but its progress may be halted if the cause can be removed. This may result in a normal portosystemic gradient, leading consequently to shunt occlusion.