Targeted ED-B fibronectin SPECT in vivo imaging in experimental atherosclerosis.

AIM: The extracellular matrix protein ED-B fibronectin (ED-B) is upregulated in inflammatory atherosclerotic lesions. However, functional in vivo imaging of ED-B-containing plaques has not been explored. This study evaluated whether [(99m)Tc]-conjugated AP39 ([(99m)Tc]-AP39), a single-chain antibody specific to ED-B, can be used for in vivo detection of atherosclerotic plaques in Western diet (WD)-fed, apolipoprotein E-deficient (apoE-/-) mice as compared to wildtype (WT) control mice. METHODS: Using SPECT, 12-month-old WD-fed apoE-/- and WT mice were studied 4 hours after injecting [(99m)Tc]-AP39 (148 MBq). Subsequently, mice were sacrificed, thoracic aortas measured in a g-counter, and plaques analyzed using histology, immuno-histochemistry, autoradiography, and morphometry. RESULTS: In vivo [(99m)Tc]-AP39-SPECT imaging of apoE-/- mice demonstrated a significant signal activity in the plaque-ridden thoracic aorta (52.236 ± 40.646 cpm/cm³) that co-localized with the aortic arch and the supra-aortic arteries in MRI scans. Low signal activity (9.468 ± 4.976 cpm/cm³) was observed in WT mice. In apoE-/- mice, the strongest signals were detected in the aortic root, aortic arch and along the abdominal aorta. Autoradiography analysis of aortas from apoE-/- mice confirmed the in vivo observation by demonstrating signal localization in atherosclerotic plaques. The size of autoradiography-positive plaque areas correlated significantly with the size of ED-B-positive (r=0.645, P=0.044) or macrophage-infiltrated (r=0.84, P<0.002) plaques. A significant correlation was found between the sizes of ED-B-positive and macrophage-infiltrated plaque areas (r=0.93, P<0.01). CONCLUSION: [(99m)Tc]-AP39-SPECT in vivo imaging detects inflammatory plaque lesions in WD-fed apoE-/- mice.

The proline TP53 variant stimulates likely lymphangiogenesis in an orthotopic mouse model of pancreatic cancer.

BACKGROUND: Pancreatic cancer is a deadly disease characterised by high incidence of TP53 mutations. Restoration of TP53 function is perceived as a highly attractive therapeutic strategy, whose effects are not well characterised. METHODS: The current work adapted an inducible strategy of stage-specific reexpression of wild-type (wt) TP53 in an in vivo orthotopic mouse model of pancreatic cancer. RESULTS: The reconstitution of wt TP53 function in TP53-mutant DanG and MiaPaCa-2 cells caused G1 cell cycle arrest but no evidence of apoptosis induction. Consistent with subcutaneous xenograft models, we found that wt TP53 reduced primary tumour growth. Wt TP53 reexpression during early tumour growth led to significant increase in vascularisation. This correlated with an unexpectedly high rate of micro-metastases in lymph nodes of animals with wt TP53 induction, despite the 90% decrease in median primary tumour weight. Reexpression of wt TP53 later in tumour development did not significantly affect the number of CD31-reactive vessels, but increased lymphatic vessel density. CONCLUSION: The increased number of lymphatic vessels and micro-metastases suggests that wt TP53 induction complexly affected the biology of different tumour constituents of pancreatic cancer. Our observation suggests that combination of the inducible system with an orthotopic model can yield important insights into in vivo pancreatic cancer biology.

[Molecular imaging in oncology using targeted ultrasound contrast agents]. / Molekulare Bildgebung in der Onkologie: Tumor-spezifische Ultraschallkontrastmittel.

Molecular imaging enables to assess disease-associated processes at the cellular and molecular level. Nuclear medicine techniques are already available in the clinical routine. Besides these techniques, intensive research has been performed in the field of ultrasound. The development of target-specific ultrasound contrast agents in combination with modern imaging systems transformed ultrasound to a capable molecular imaging technique. It has been shown that the expression of disease-associated endothelial receptors can be assessed using targeted microbubbles, demonstrating its high value in the diagnosis of several diseases. The broad availability of suitable ultrasound systems promises a wide utilisation in the clinical routine, once clinically approved contrast agents are available. This review summarizes the basics and the current status of molecular ultrasound imaging.

Repeated anaesthesia with isoflurane and xylazine/levomethadone/fenpipramide premedication in female Beagle dogs: influence on general health and wellbeing.

Beagle dogs continue to be used in experimental studies and preclinical and clinical trials, many of which address the usage of anaesthesia. In order to reduce the number of animals, researchers tend to conduct several experiments on a single animal. The question arises, however, as to whether or not this frequent usage involves more than simply additional stress and discomfort for the individual animal. Within the framework of an existing study involving six female Beagle dogs, we investigated the effects of repeated (5) isoflurane anaesthesia with xylazine/levomethadone/fenpipramide premedication carried out at short intervals (2 weeks) and compared these with the effects of two treatments intermitted by a longer resting period (8 weeks). To verify our hypothesis that frequent anaesthesia affects the dog's wellbeing more than the occasional anaesthesia, the following parameters were measured at regular intervals: body weight, body temperature, respiratory rate, blood pressure, reflexes and heart rate, both at rest and during a treadmill exercise. In addition, recovery behaviour subsequent to anaesthesia was monitored for one hour. Our observations indicate that the anaesthetic effects are most prominent 24 h after the anaesthetic treatment. However, crossover analysis of our data cannot show that there is no statistical difference of whether dogs were anaesthetized occasionally or frequently. In our study, it appears that frequent anaesthesia within a two-week period did not affect the wellbeing and general health of Beagle dogs in a super-additive manner and that a minimum of two-week testing-free period is sufficient to ensure complete recovery from the unwanted effects induced by anaesthesia.

Inducible re-expression of p16 in an orthotopic mouse model of pancreatic cancer inhibits lymphangiogenesis and lymphatic metastasis.

Functional inactivation of the tumour suppressor protein p16(INK4a) constitutes a key event in the multistep process of pancreatic ductal cell transformation. However, the significance of p16 inactivation for complex and tissue-specific aspects of pancreatic cancer progression, such as angiogenesis and metastasis, is less understood. Here, we inducibly re-expressed p16 in vivo in an orthotopic model of pancreatic cancer and examined the impact on these clinically relevant aspects of pancreatic cancer tumour biology. Consistent with previous work in subcutaneous xenograft models, we found p16 capable of reducing primary tumour growth. In addition, p16 restitution resulted in a marked reduction of tumour angiogenesis, largely accounted for by a p16-dependent inhibition of lymphangiogenesis. In excellent agreement with the antilymphangiogenic effect, re-expression of p16 almost completely prevented lymph node metastases of MiaPaca-2 pancreatic tumours. To our knowledge, this is the first report that experimentally links the tumour suppressor p16 to the process of lymphangiogenesis.

Ultrasound derived imaging and quantification of cell adhesion molecules in experimental autoimmune encephalomyelitis (EAE) by Sensitive Particle Acoustic Quantification (SPAQ).

Molecular imaging requires, not only the identification of an appropriate marker, but also its quantitative analysis. We used the Sensitive Particle Acoustic Quantification (SPAQ) technology - a novel ultrasound technique - for detection and quantification of cell adhesion molecules in isolated tissue and in live animals. By conjugating gas-filled microparticles (MPs) with antibodies to intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), we were able to depict and quantify ICAM-1 and VCAM-1 in isolated brain and spinal cord from rats with autoimmune encephalomyelitis (EAE), an established inflammatory disease model of human multiple sclerosis (MS). Depiction and quantification of specific MPs were also feasible in living animals with AT-EAE with similar results. After treatment with methylprednisolone, the measured number of targeted anti-ICAM-1 and VCAM-1-MPs was significantly lower (P<0.01) compared to untreated animals demonstrating the high sensitivity of this imaging technique. Depending on the antibody linked to the surface of the MPs, the technique can be used to quantify the expression of any accessible antigen expressed on the luminal surface of endothelial cells and is therefore a promising tool for the non-invasive and dynamic assessment of disease-related molecules.

[Possible target specific molecular imaging with ultrasound contrast agents]. / Möglichkeiten Target-spezifischer molekularer Bildgebung mit Ultraschallkontrastmitteln.

Non-invasive molecular imaging technologies provide researchers with the opportunity to study cellular and molecular processes. Among different imaging technologies, ultrasound based molecular imaging methods are also of interest, since the use of ultrasound contrast agents allows specific and sensitive depiction of molecular targets. Recent studies are encouraging and have demonstrated the feasibility of ultrasound based molecular imaging. This review summarizes current experiences and recent preclinical studies with target-specific ultrasound contrast agents.

Tumor vascularization in experimental melanomas: correlation between unenhanced and contrast enhanced power Doppler imaging and histological grading.

The purpose of this study was to evaluate the reliability of unenhanced and enhanced power Doppler sonography in visualization of intratumoral angioneogenesis. Thirty-seven malignant melanomas, which had been implanted intra- or subcutaneously in 22 mice, were examined. Various B-mode criteria, power Doppler criteria and spectral Doppler parameters were evaluated before and after IV-application of the d-galactose-based contrast agent Levovist. After sonographic examination, all tumors were analyzed histologically with semiquantitative grading of tumoral vascularization. Unenhanced, in 70% of the tumors, no intratumoral vessels were visible using power Doppler, but only in 11% of the intracutaneous and in 0% of the subcutaneous after injection of the contrast agent. The enhanced mode was definitely superior to unenhanced Doppler in showing the intratumoral vascularity. The intratumoral vascular structure could be sufficiently analyzed in 30% of all tumors by unenhanced Doppler, but in 92% enhanced. The mean percentage vessel area increased about 433% after application of Levovist (intracutaneous: 485%, subcutaneous: 280%). Despite the missing direct correlation between the sonographically and histologically determined grade of tumor vascularization (Pearson's correlation unenhanced 0,356, p <.05/enhanced 0.395, p <.05), the correlation between the percentage vessel area and the histologic grade of vascularization was improved after application of the contrast agent (Pearson's correlation unenhanced 0.347, p <.05/enhanced 0.686, p <.01). We did not find a significant direct correlation between histologically and sonographically determined degree of vascularization. However, the correlation was improved using a d-galactose-based signal-enhancing agent in power Doppler sonography.

First experiences in using a new ultrasound mode and ultrasound contrast agent in the diagnosis of blunt renal trauma: a feasibility study in an animal model.

RATIONALE AND OBJECTIVES: To evaluate the diagnostic value of the new ultrasound mode "wide-band harmonic" (WBH) using an ultrasound contrast agent in blunt renal trauma in an animal model. METHODS: A defined blunt renal trauma was induced in 10 rabbits according to published standards. Ultrasound (B-mode, color and power Doppler, WBH) was performed before and after trauma, with and without using a contrast agent (Levovist). Ultrasound features were compared with histologic findings. RESULTS: In 2 of the 10 rabbits, three focal renal intraparenchymal lesions with diameters ranging from 1.0 to 1.8 mm were found that could be identified only using WBH with contrast. Six of the 10 rabbits developed a subcapsular hematoma with a thickness of up to 1.5 mm, which was identified by conventional B-mode as well as WBH. Histologic workup confirmed these findings of intraparenchymal hematomas and did not reveal further lesions. CONCLUSIONS: Only 20% of the experimental subjects developed parenchymal lesions with diameters of 1.0 mm or larger. All these lesions were identified only using WBH. These results indicate the potential to use WBH plus contrast for the diagnosis of blunt renal trauma.

Wideband harmonic imaging: a novel contrast ultrasound imaging technique.

A novel ultrasonic imaging method, wideband harmonic imaging, for nonlinear imaging of microbubble contrast agents is evaluated. In wideband harmonic mode, two pulses of alternate phase are send out. The image is then processed from the sum of both pulses, resulting in an image of nonlinear scatterers such as microbubbles. A prototype ultrasound system, Siemens Elegra, was evaluated with in vitro investigations and animal trials, using conventional, harmonic and wideband harmonic settings with the galactose based ultrasound contrast agent Levovist. Wideband harmonic imaging offers superior sensitivity for ultrasound contrast agents compared to conventional imaging and harmonic imaging. At low transmit power settings (MI 0. 1-0.5) the nonlinear response is already sufficient to generate a image of the blood pool distribution of Levovist in the rabbit kidney including the microvasculature, with clear delineation of vessels and perfused parenchyma. At high transmit amplitudes, nonlinear tissue response reduced the apparent image contrast between contrast agent and tissue. The results suggest that wideband harmonic imaging is currently the most sensitive contrast imaging technique, maintaining highest spatial resolution. This may add to image quality and offer new clinical potential for the use of ultrasound contrast agents such as Levovist.

Delineation of experimental liver tumors in rabbits by a new ultrasound contrast agent and stimulated acoustic emission.

RATIONALE AND OBJECTIVES: A new ultrasound contrast agent (SH U 563 A), consisting of hollow biodegradable polymeric microparticles, and a new imaging technique (stimulated acoustic emission) were used for delineation of experimental liver tumors. After intravenous injection, these microparticles are phagocytosed by cells of the reticuloendothelial system (RES) and create a color-coded signal using color Doppler. Because of the different distribution of phagocytic cells in healthy liver tissue and tumors, the delineation of focal lesions was to be tested. METHODS: Sixteen rabbits with VX2 liver tumors received doses of 0.15-mL SH U 563 A per kilogram of body weight intravenously. Liver investigations (UM9, HD1, L10.5, ATL, Bothell, USA) were performed in vivo before and after SH U 563 A application in B and color Doppler modes. Additionally, the liver and spleen of these rabbits were examined ex vivo in color Doppler. The sonographic diagnosis was confirmed by pathology. RESULTS: After application of SH U 563 A, the healthy liver tissue of all rabbits was characterized by a typical mosaic color pattern in vivo and ex vivo, using color Doppler. Entire VX2 liver tumors were detectable exclusively in color Doppler after SH U 563 A application. This was possible in 14 of 16 rabbits in vivo and in all 16 livers ex vivo. Furthermore, all ex vivo investigated spleens were color enhanced homogeneously. Sonographic diagnoses were in accordance with pathologic findings. CONCLUSIONS: SH U 563 A, combined with stimulated acoustic emission, provides potential for delineation of small isoechogenic liver lesions by sonography.

[Contrast medium supported imaging of tumorous vessels by color Doppler and power Doppler mode in animal experiments]. / Kontrastmittelgestützte Darstellung von Tumorgefässen in Farb-Doppler- und Power-Doppler-Mode im Tierexperiment.

PURPOSE: The purpose was to determine tumor neovascularisation via colour-coded Doppler (duplex) sonography and the "power mode", both visually and quantitatively, by means of videodensitometry. MATERIAL AND METHODS: 6 VX2 tumours of 4 to 11 mm size were implanted in 4 rabbits at various sites. The colour-coded duplex sonography and the new sonographic power technique were tested before and after having injected a new contrast medium (SH U 616A). RESULTS: If no contrast medium was injected, tumour neovascularisation was identified in only 50% of the cases. Injection of contrast medium increased signal intensity three to fourfold with all examined tumors. Combined use of the sonographic method by the power technique with injection of contrast medium is outstandingly suitable for tumor vessel imaging even of small tumors, as these initial results seem to show. CONCLUSION: If these results are corroborated by further studies, contrast-medium supported sonographic technique may possibly become established as an alternative method to other imaging procedures.

Properties of two epithelial cell lines derived from HPV-associated cervical and vulvar lesions.

Two epithelial cell lines were established from human papilloma virus (HPV) 18 or 16 associated tumours, characterised as poorly and well differentiated squamous cell carcinomas of the cervix uteri (EC) and the vulva (GC), respectively. The cell lines are described by their morphology, biological parameters, and immunological markers. Both cell lines have undergone approximately 35 passages in vitro. HPV16 and 18 DNA are maintained integrated into the host cell DNA. Expression of epithelial cell markers--cytokeratins K1, K10, K13, K14 and involucrin, proliferation-specific proteins, proliferating cell nuclear antigen (PCNA) and Ki67 as well as the epidermal growth factor (EGF) receptor were monitored by indirect immunofluorescence studies. The cytoplasmic and membrane-associated locations of EGF receptor molecules in EC and GC cells, respectively, suggest a differently regulated expression. Studies of the HPV18 oncogene transcription revealed marked differences of amplimers between HeLa and EC cells, such as an additional fragment, probably corresponding to a E6**--E7 splice product, and a radical shift in transcription pattern observed in various sections of the tumour tissue. Injected subcutaneously into nu/nu mice both cell lines were non-tumorigenic.

Investigations of droloxifene and other hormone manipulations on N-nitrosomethylurea-induced rat mammary tumours. 1. Influence on tumour growth.

The effect of droloxifene, a new anti-oestrogenic drug, on N-nitrosomethylurea-induced mammary tumours of Sprague-Dawley rats was investigated and compared with that of tamoxifen. The response of tumour growth to ovariectomy or to treatment with aminoglutethimide or high doses of oestradiol was also studied. Ovariectomy was by far the most effective treatment for mammary-tumour-bearing animals. More than 75% of the tumours in ovariectomized rats did not grow progressively but remained in remission for up to 12 weeks after castration when the experiment was terminated. The inhibitory effects of droloxifene and tamoxifen on mammary tumour growth were similar, but body weight loss of animals treated with tamoxifen was more marked than that of animals treated with droloxifene at the same dose and schedule.

Investigations of droloxifene and other hormonal manipulations on N-nitrosomethylurea-induced rat mammary tumours. 2. Influence on oestrogen receptor.

In N-nitrosomethylurea-induced rat mammary tumours, tamoxifen is found to compete at the binding sites of the oestradiol receptor if a receptor determination is performed 1 day following the last drug application to animals. Despite a higher binding affinity of droloxifene (3-OH-tamoxifen) to oestradiol receptor, compared to tamoxifen, its influence on the measurable receptor quantity is only very weak or not demonstrable. Therefore, binding affinity is not a valid explanation for the different influences of the two anti-oestrogens on the receptor. These only can be attributed to different behaviour patterns of both substances in relation to their half-lives and metabolism and accumulation in the organism. Owing to the short half-life of droloxifene, even 1 day after the last application too little drug is available to compete for oestradiol binding sites. In the case of both anti-oestrogenic substances, cessation of drug application for 8 weeks abolished any influence on the oestradiol receptor. Furthermore, failure of aminoglutethimide to influence the oestradiol receptor could be observed because this substance does not act via this receptor. The experiments performed confirm literature data regarding the effect of aminoglutethimide therapy on oestradiol receptors in breast tumour tissue of human beings. In summary: receptor investigations of N-nitrosomethylurea-induced rat mammary tumours, used as a model to test therapy regimens with droloxifene or other drugs with a short half-life, may be of limited value only.

Spontaneous Hymenolepis nana infection in a breeding colony of nude mice.

The spontaneous occurrence of a parasitic infection with the dwarf tapeworm Hymenolepis nana is nude mice was observed under conventional conditions. Clinical, pathological and histological observations are described.