Ryk-deficient mice exhibit craniofacial defects associated with perturbed Eph receptor crosstalk.

Secondary palate formation is a complex process that is frequently disturbed in mammals, resulting in the birth defect cleft palate. Gene targeting has identified components of cytokine/growth factor signalling systems such as Tgf-alpha/Egfr, Eph receptors B2 and B3 (Ephb2 and Ephb3, respectively), Tgf-beta2, Tgf-beta3 and activin-betaA (ref. 3) as regulators of secondary palate development. Here we demonstrate that the mouse orphan receptor 'related to tyrosine kinases' (Ryk) is essential for normal development and morphogenesis of craniofacial structures including the secondary palate. Ryk belongs to a subclass of catalytically inactive, but otherwise distantly related, receptor protein tyrosine kinases (RTKs). Mice homozygous for a null allele of Ryk have a distinctive craniofacial appearance, shortened limbs and postnatal mortality due to feeding and respiratory complications associated with a complete cleft of the secondary palate. Consistent with cleft palate phenocopy in Ephb2/Ephb3-deficient mice and the role of a Drosophila melanogaster Ryk orthologue, Derailed, in the transduction of repulsive axon pathfinding cues, our biochemical data implicate Ryk in signalling mediated by Eph receptors and the cell-junction-associated Af-6 (also known as Afadin). Our findings highlight the importance of signal crosstalk between members of different RTK subfamilies.

Regorafenib (BAY 73-4506) in advanced colorectal cancer: a phase I study.

BACKGROUND: In a phase I dose-escalation study, regorafenib demonstrated tolerability and antitumour activity in solid tumour patients. The study was expanded to focus on patients with metastatic colorectal cancer (CRC). METHODS: Patients received oral regorafenib 60-220 mg daily (160 mg daily in the extension cohort) in cycles of 21 days on, 7 days off treatment. Assessments included toxicity, response, pharmacokinetics and pharmacodynamics. RESULTS: Thirty-eight patients with heavily pretreated CRC (median 4 prior lines of therapy, range 0-7) were enrolled in the dose-escalation and extension phases; 26 patients received regorafenib 160 mg daily. Median treatment duration was 53 days (range 7-280 days). The most common treatment-related toxicities included hand-foot skin reaction, fatigue, voice change and rash. Twenty-seven patients were evaluable for response: 1 achieved partial response and 19 had stable disease. Median progression-free survival was 107 days (95% CI, 66-161). At steady state, regorafenib and its active metabolites had similar systemic exposure. Pharmacodynamic assessment indicated decreased tumour perfusion in most patients. CONCLUSION: Regorafenib showed tolerability and antitumour activity in patients with metastatic CRC. This expanded-cohort phase I study provided the foundation for further clinical trials of regorafenib in this patient population.

Vascular and pharmacokinetic effects of EndoTAG-1 in patients with advanced cancer and liver metastasis.

BACKGROUND: EndoTAG-1 (ET), a novel formulation of cationic liposomes carrying embedded paclitaxel (Taxol), shows antitumoral activity, targeting tumor endothelial cells in solid tumors. Patients with advanced metastatic cancer were evaluated investigating effects on pharmacokinetics and tumor vasculature using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and contrast-enhanced ultrasound (CEUS). PATIENTS AND METHODS: The pharmacokinetic (PK) profile of ET (22 mg/m(2) i.v.) was evaluated after single and repeated doses. DCE-MRI and CEUS explored hepatic metastases before, during and after the 4-week treatment cycle. Angiogenic biomarkers were assessed. Tumor response was evaluated by modified RECIST. RESULTS: The PK profile demonstrated slight accumulation of paclitaxel after repeated doses. DCE-MRI parameters K(trans) and/or iAUC(60) showed a trend to decrease. Changes of blood flow-dependent parameters of DCE-MRI and CEUS were well correlated. Angiogenic biomarkers revealed no clear trend. ET was generally well tolerated; common toxic effects were fatigue and hypersensitivity reactions. Nine (9 of 18) patients had stable disease after the first treatment cycle. Four patients without disease progression continued treatment. CONCLUSIONS: This study including multiple pretreated patients with different metastatic cancer revealed individually distinctive hemodynamic alterations by DCE-MRI. The PK profiles of ET were similar as observed previously.

Single-voxel MRS with prospective motion correction and retrospective frequency correction.

Subject motion during MRS investigations is a factor limiting the quality and the diagnostic value of the spectra. The possibility of using external motion tracking data to correct for artefacts in MR imaging has been demonstrated previously. In this paper the utility of prospective motion correction for single-voxel proton MRS is investigated. The object motion data are used in real time to update the position of the spectroscopy voxel during the acquisition prior to every sequence repetition cycle. It is not, however, sufficient to update the voxel position alone due to shim changes accompanying subject motion. Adverse effects of frequency shifts induced by subject motion are effectively suppressed by the interleaved reference scan method.

The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.

The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

Evidence of disturbed amygdalar energy metabolism in patients with borderline personality disorder.

In order to detect possible links between structural and neurochemical brain abnormalities we applied high resolution morphometric imaging and short-echo time absolute-quantification magnetic resonance spectroscopy (MRS) at the left hand side to the amygdala in 12 patients with borderline personality disorder (BPD) and 10 group-matched healthy controls. Confirming earlier reports we found a significant 11-17% reduction of amygdalar volumes in patients with BPD. In addition there was a significant 17% increase of left amygdalar creatine concentrations in BPD patients. Left amygdalar creatine concentration correlated positively with measures of anxiety and negatively with amygdalar volume. This pilot study of simultaneous amygdalar morphometry and spectroscopy in BPD reveals a possible link between amygdalar volume loss, psychopathology and neurochemical abnormalities in terms of creatine signals.

Mutagenesis and selection of PDZ domains that bind new protein targets.

PDZ domains are a recently characterized protein-recognition module. In most cases, PDZ domains bind to the C-terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C-terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C-terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full-length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.

Targeting JAK kinase in solid tumors: emerging opportunities and challenges.

Various human malignancies are characterized by excessive activation of the Janus family of cytoplasmic tyrosine kinases (JAK) and their associated transcription factors STAT3 and STAT5. In the majority of solid tumors, this occurs in response to increased abundance of inflammatory cytokines in the tumor microenvironment prominently produced by infiltrating innate immune cells. Many of these cytokines share common receptor subunits and belong to the interleukin (IL)-6/IL-11, IL-10/IL-22 and IL-12/IL-23 families. Therapeutic inhibition of the JAK/STAT3 pathway potentially offers considerable benefit owing to the capacity of JAK/STAT3 signaling to promote cancer hallmarks in the tumor and its environment, including proliferation, survival, angiogenesis, tumor metabolism while suppressing antitumor immunity. This is further emphasized by the current successful clinical applications of JAK-specific small molecule inhibitors for the treatment of inflammatory disorders and hematopoietic malignancies. Here we review current preclinical applications for JAK inhibitors for the treatment of solid cancers in mice, with a focus on the most common malignancies emanating from oncogenic transformation of the epithelial mucosa in the stomach and colon. Emerging data with small molecule JAK-specific adenosine triphosphate-binding analogs corroborate genetic findings and suggest that interference with the JAK/STAT3 pathway may suppress the growth of the most common forms of sporadic colon cancers that arise from mutations of the APC tumor suppressor gene. Likewise inhibition of cytokine-dependent activation of the JAK/STAT3 pathway may also afford orthogonal treatment opportunities for other oncogene-addicted cancer cells that have gained drug resistance.

Endovascular Cooling Catheter for Selective Brain Hypothermia: An Animal Feasibility Study of Cooling Performance.

BACKGROUND AND PURPOSE: Therapeutic hypothermia represents a promising neuroprotective treatment in acute ischemic stroke. Selective cerebral hypothermia applied early, prior to and during endovascular mechanical recanalization therapy, may be beneficial in the critical phase of reperfusion. We aimed to assess the feasibility of a new intracarotid cooling catheter in an animal model. MATERIALS AND METHODS: Nine adult sheep were included. Temperature probes were introduced into the frontal and temporal brain cortices bilaterally. The cooling catheter system was introduced into a common carotid artery. Selective blood cooling was applied for 180 minutes. Systemic and local brain temperatures were measured during cooling and rewarming. Common carotid artery diameters and flow were measured angiographically and by Doppler sonography. RESULTS: The common carotid artery diameter was between 6.7 and 7.3 mm. Common carotid artery blood flow velocities increased moderately during cooling and after catheter removal. Maximum cerebral cooling in the ipsilateral temporal cortex was -4.7°C (95% CI, -5.1 to -4.0°C). Ipsilateral brain temperatures dropped significantly faster and became lower compared with the contralateral cortex with maximum temperature difference of -1.3°C (95% CI, -1.5 to -1.0°C; P < .0001) and compared with systemic temperature (-1.4°C; 95% CI, -1.7 to -1.0°C; P < .0001). CONCLUSIONS: Sheep proved a feasible animal model for the intracarotid cooling catheter. Fast induction of selective mild hypothermia was achieved within the cooled cerebral hemisphere, with stable temperature gradients in the contralateral brain and systemic blood. Further studies are required to demonstrate any therapeutic benefit of selective cerebral cooling in a stroke model.

An epitope tagged mammalian/prokaryotic expression vector with positive selection of cloned inserts.

A dual eukaryotic/prokaryotic expression vector has been developed which combines the features of positive selection for cloned inserts along with the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells as well as high level induced expression in E. coli cells harbouring T7 RNA polymerase. This vector, pZilch, has two MCSs flanking a mutant E. coli phenylalanyl-tRNA synthetase gene, pheS, which when expressed in combination with the phenylalanine analog p-CI-Phe, results in termination of host cell protein synthesis. Cloning of inserts using unique sites in the flanking MCS regions results in loss of the pZilch pheS allele and hence permits growth of colonies harbouring recombinants on p-Cl-Phe plates. Additional features of the vector include an optimal Kozak consensus sequence for high level eukaryotic cell expression and an efficient prokaryotic translation initiation site in frame and downstream from the eukaryotic initiation site. Recombinant proteins can be produced with an N-terminal FLAG epitope which can be removed via a specific protease cleavage site. Flanking T7 and SP6 RNA polymerase promoter sites permit in vitro transcription and translation of cloned inserts. A derivative of the vector has also been constructed enabling nuclear accumulation of the tagged proteins via an SV40 nuclear localisation signal upstream of the 5' MCS.

Lhermitte-Duclos disease: assessment with MR imaging, positron emission tomography, single-photon emission CT, and MR spectroscopy.

SUMMARY: Lhermitte-Duclos disease (LDD) is a rare cerebellar lesion with features of both malformation and benign neoplasm. However, the fundamental nature of the entity, its pathogenesis, and the exact genetic alterations remain unknown. We describe MR findings (including perfusion- and diffusion-weighted images) in two patients with LDD, as well as findings from single-photon emission CT (SPECT), MR spectroscopy (MRS), and fluorodeoxyglucose (FDG) positron emission tomography (PET) that give additional information about tumor pathophysiology. MR imaging usually distinguishes the LDD by its characteristic "tiger-striped" appearance. The regions of increased regional cerebral blood volume (rCBV) within the lesion correlated closely to the regions of FDG-hypermetabolism and high thallium (201-Tl) uptake. Proton MRS revealed an increased level of lactate and decreased level of myo-inositiol and N-acetyl-aspartate, as observed in low-grade gliomas, but decreased levels of choline. Our cases indicate that the functional investigations give additional information about tumor pathophysiology and reflect the histopathologic controversial entity with both characteristics found in low-grade gliomas and characteristics not typical for tumors.

Breath-hold projection magnetic resonance-cholangio-pancreaticography (MRCP): a new method for the examination of the bile and pancreatic ducts.

Breath-hold MR cholangio-pancreaticography using the RARE method was evaluated as a noninvasive alternative to direct cholangio-pancreaticography in 30 healthy volunteers and in 30 patients with various diseases of the respective duct systems. In contrast to 3D MR cholangiography, RARE MRCP generates projection images requiring a 4-s acquisition time and no postprocessing. Diagnostic information gained from the cholangio-pancreaticograms was fully equivalent to that of conventional x-ray imaging by invasive procedures. Additional information was gained whenever endoscopic access was impossible and/or cystic malformations were present.

TSE-sequences with spin-echo contrast.

The article presents a discussion of the basic signal behavior of contrast-modified RARE(TSE,FSE...)-sequences which have been modified such that the echo train used for image encoding is preceded by a long echo interval in order to introduce the T(2)-contrast of conventional spin-echo sequences while maintaining the high imaging speed of TSE. Sequences aimed at breathhold abdominal imaging as well as for the detection of hemorrhages in the CNS have been implemented and optimized. The significant difference in image contrast at identical echo times compared to unmodified TSE is demonstrated for different tissues.

Transcription factor AP-2 essential for cranial closure and craniofacial development.

During closure of the neural tube in the mouse, transcription factor AP-2 is expressed in ectoderm and in neural-crest cells migrating from the cranial neural folds. Cranial neural crest cells provide patterning information for craniofacial morphogenesis, generate most of the skull bones, and together with placodal ectoderm, form the cranial ganglia. To study the role of AP-2 during embryogenesis, we undertook a targeted mutagenesis of the AP-2 gene in the mouse. Here we report that AP-2(-/-) mice died perinatally with cranio-abdominoschisis and severe dismorphogenesis of the face, skull, sensory organs and cranial ganglia. Failure of cranial closure between 9 and 9.5 days postcoitum coincided with increased apoptosis in the midbrain, anterior hindbrain and proximal mesenchyme of the first branchial arch, but did not involve loss of expression of twist or Pax-3, two other regulatory genes known to be required for cranial closure.

MRI-diffusion imaging of neuroblastomas: first results and correlation to histology.

The purpose of this study was to evaluate diffusion-weighted MR imaging in neuroblastomas. We prospectively examined seven children (age range 1-3 years) with seven solid body neuroblastomas. Diagnosis was established histologically. Diffusion-weighted echo-planar imaging (EPI) sequence was performed in all patients, with a repetition time of 5400 ms and an echo time of 103 ms, and with a b-value of 1000 s/mm(2). The contrast of tumour tissue depicted with T2-weighted images and diffusion-weighted images were evaluated by means of region-of-interest measurements and a calculation of the apparent diffusion coefficient (ADC) was done. The ADC calculation showed a mean ADC of 1.1x10(-3) (SD 0.14x10(-3), range 0.9-1.2x10(-3)) mm(2)/s of all tumours. Diffusion-weighted images showed an increased tumour signal. Water proton diffusion within the tumour matrix of neuroblastomas is especially restricted by the molecular and macromolecular barriers due to the very dense structure of this tumour tissue. We hypothesize that high nuclear-to-cytoplasm ratio of neuroblastoma cells limits intracellular motion. Furthermore, the very densely packed tumour cells inhibit effective motion of extracellular water protons. Restricted proton motion leads to a reduction in the rate of apparent diffusion and to a marked increase in signal on diffusion-weighted EPI MR images.

Functional magnetic resonance imaging evidence for binocular interactions in human visual cortex.

Using functional magnetic resonance imaging (fMRI), we explored the binocular interactions occurring when subjects viewed dichoptically presented checkerboard stimuli. A flickering radial checkerboard was presented to each eye of the subject, while T2*-weighted images were acquired over the visual cortex with gradient-echo, echoplanar sequences. We compared responses in striate and extrastriate visual cortex under four conditions: both eyes were stimulated at the same time (binocular condition), each eye was stimulated in alternation (monocular condition) or first the one eye then the other eye was stimulated (left eye first - right eye trailing, or vice versa). The results indicate that only the striate area, in and near the calcarine fissure, shows significant differences for these stimulation conditions. These differences are not evident in more remote extrastriate or associational visual areas, although the BOLD response in the stimulation-rest comparison was robust. These results suggest that the effect could be related to inhibitory interactions across ocular dominance columns in striate visual cortex.