Referral Patterns and Outcome of Patients With Synchronous Brain Metastases From Non-small Cell Lung Cancer Treated With Gamma Knife Radiosurgery in a Third-Line Treatment Centre in The Netherlands - A Retrospective Analysis.

AIMS: Little understanding exists of referral patterns for patients with brain metastasis from non-small cell lung cancer (NSCLC) towards treatment with Gamma Knife radiosurgery (GKRS). Therefore, we explored current clinical daily practice and prognosis. MATERIAL AND METHODS: In total, 1129 patients with synchronously diagnosed brain metastasis from NSCLC diagnosed between 2008 and 2014 were selected from the population-based Netherlands Cancer Registry; 242 patients were treated with GKRS. RESULTS: Patients receiving GKRS were younger (62 years versus 64 years) and had lower tumour burden: the presence of T2 was higher and T4 was lower (43% versus 33%; P = 0.0158, 19% versus 28%; P = 0.0044, respectively). They more frequently had cN0 (32% versus 19%; P ≤ 0.0001), less frequently had N3 disease (18% versus 29%; P = 0.0004) and there were fewer metastatic sites. In multivariable logistic regression analysis, only age ≤60 years (odds ratio 1.4; 95% confidence interval 1.0-2.0) and patients with N0 stage, compared with those with N2, N3 and NX (odds ratio 0.6 [0.4-0.9], 0.3 [0.2-0.6], 0.3 [0.1-0.6], respectively), were more likely to receive GKRS. Gender, T-stage, histology, number of comorbidities, country of birth as proxy for ethnicity and socioeconomic status were not associated. The median survival was 9.6 months after GKRS versus 4.0 months in the noGKRS group (Log-rank: P ≤ 0.0001). Multivariably, GKRS, female, lower T-/N-stage, <2 comorbidities, adenocarcinoma and higher socioeconomic status were associated with a significantly reduced hazard of death. For the patients with at least one follow-up magnetic resonance image (80%), local intracranial tumour control was achieved in 93% at the last follow-up. CONCLUSION: Patients presenting with synchronic brain metastasis from NSCLC who are referred to a third-line treatment centre for GKRS are younger and have a lower tumour load. Due to a high level of local control, GKRS is able to provide a significant window of opportunity for additional treatment of the primary tumour.

Recellularization of auricular cartilage via elastase-generated channels.

Decellularized tissue matrices are promising substrates for tissue generation by stem cells to replace poorly regenerating tissues such as cartilage. However, the dense matrix of decellularized cartilage impedes colonisation by stem cells. Here, we show that digestion of elastin fibre bundles traversing auricular cartilage creates channels through which cells can migrate into the matrix. Human chondrocytes and bone marrow-derived mesenchymal stromal cells efficiently colonise elastin-treated scaffolds through these channels, restoring a glycosaminoglycan-rich matrix and improving mechanical properties while maintaining size and shape of the restored tissue. The scaffolds are also rapidly colonised by endogenous cartilage-forming cells in a subcutaneously implanted osteochondral biopsy model. Creating channels for cells in tissue matrices may be a broadly applicable strategy for recellularization and restoration of tissue function.

Evaluation of the impact of a clinical pathway on the organization of a multidisciplinary dental sleep clinic.

PURPOSE: Clinical pathways are used to organize complex care processes by providing structure and standardization. The multidisciplinary approach of oral appliance (OA) therapy for sleep-disordered breathing (SDB) is a complex and dynamic process suitable for such a structured pathway approach. METHODS: A clinical pathway for patients referred for OA therapy was developed and implemented. The aim of this study was to evaluate the impact of this clinical pathway on the time to delivery of the OA and the organization of the multidisciplinary dental sleep clinic (MDSC). The latter was achieved using the care process self-evaluation tool (CPSET). RESULTS: First, development and implementation of the clinical pathway gave structure and shortened the mean time to delivery by 102 days (240 ± 70 vs. 138 ± 33 days) (Mann-Whitney U: P < 0.001). Second, the CPSET scores were obtained in a cohort of 49 healthcare professionals involved in the pathway. Overall, patient-focused organization received the highest scores (80.5 ± 9.0%), whereas cooperation with primary care received the lowest score (66.7 ± 12.4%). CONCLUSIONS: This is the first project on clinical pathways in OA therapy for SDB. The implementation of the pathway in our MDSC has created a significant shortening of the time to delivery. A first evaluation of the clinical pathway using the CPSET scores indicates that all disciplines involved should be thoroughly informed in an ongoing approach.

Wnt signaling and stem cell control.

In many contexts, self-renewal and differentiation of stem cells are influenced by signals from their environment, constituting a niche. It is postulated that stem cells compete for local growth factors in the niche, thereby maintaining a balance between the numbers of self-renewing and differentiated cells. A critical aspect of the niche model for stem cell regulation is that the availability of self-renewing factors is limited and that stem cells compete for these factors (Fig. 1). Consequently, the range and concentrations of the niche factors are of critical importance. Now that some of the few self-renewing factors have become identified, aspects of the niche models can be tested experimentally. In this chapter, we address mechanisms of signal regulation that take place at the level of signal-producing cells, constituting a niche for stem cells. We emphasize the biochemical properties and posttranslational modifications of the signals, all in the context of Wnt signaling. We propose that these modifications control the range of Wnt signaling and have critical roles in establishing niches for stem cells in various tissues.

Translating insights from development into regenerative medicine: the function of Wnts in bone biology.

The Wnt pathway constitutes one of the most attractive candidates for modulating skeletal tissue regeneration based on its functions during skeletal development and homeostasis. Wnts participate in every stage of skeletogenesis, from the self-renewal and proliferation of skeletal stem cells to the specification of osteochondroprogenitor cells and the maturation of chondrocytes and osteoblasts. We propose that the function of Wnts depend upon a skeletogenic cell's state of differentiation. In this review we summarize recent data with a focus on the roles of Wnt signaling in mesenchymal stem cell fate, osteoprogenitor cell differentiation, chondrocyte maturation, bone remodeling, and bone regeneration.

Automated topographical cell proliferation analysis.

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.

Prx1 and Prx2 are upstream regulators of sonic hedgehog and control cell proliferation during mandibular arch morphogenesis.

The aristaless-related homeobox genes Prx1 and Prx2 are required for correct skeletogenesis in many structures. Mice that lack both Prx1 and Prx2 functions display reduction or absence of skeletal elements in the skull, face, limbs and vertebral column. A striking phenotype is found in the lower jaw, which shows loss of midline structures, and the presence of a single, medially located incisor. We investigated development of the mandibular arch of Prx1(-/-)Prx2(-/-) mutants to obtain insight into the molecular basis of the lower jaw abnormalities. We observed in mutant embryos a local decrease in proliferation of mandibular arch mesenchyme in a medial area. Interestingly, in the oral epithelium adjacent to this mesenchyme, sonic hedgehog (Shh) expression was strongly reduced, indicative of a function for Prx genes in indirect regulation of SHH: Wild-type embryos that were exposed to the hedgehog-pathway inhibitor, jervine, partially phenocopied the lower jaw defects of Prx1(-/-)Prx2(-/-) mutants. In addition, this treatment led to loss of the mandibular incisors. We present a model that describes how loss of Shh expression in Prx1(-/-)Prx2(-/-) mutants leads to abnormal morphogenesis of the mandibular arch.

Expression of retinoic acid 4-hydroxylase (CYP26) during mouse and Xenopus laevis embryogenesis.

Retinoids are important signal molecules during vertebrate embryonic development and their synthesis as well as catabolism should therefore be strictly regulated. The retinoic acid (RA) 4-hydroxylase, belonging to the cytochrome P450 family CYP26, is an enzyme catalyzing the 4-hydroxylation of RA, thereby regulating RA homeostasis. Here we describe the temporal and spatial expression patterns of mouse (mCYP26) and Xenopus laevis (xCYP26) homologues. In mouse, expression is detected in uterine crypt, around differentiating cartilage, several regions of the head, regions of the pharynx, the neural retina, and several regions of the trunk. In Xenopus, Northern blot analysis shows presence of xCYP26 transcripts before the MBT and an increased expression level during gastrulation. Whole-mount in situ hybridization shows a specific expression pattern arising at onset of gastrulation, with a ring around the blastopore. By mid gastrulation there is an anterior and a posterior expression domain, each of which gets more complex later in development. There are some important similarities and differences in expression pattern between Xenopus and mouse.

Prx1 and Prx2 in skeletogenesis: roles in the craniofacial region, inner ear and limbs.

Prx1 and Prx2 are closely related paired-class homeobox genes that are expressed in very similar patterns predominantly in mesenchyme. Prx1 loss-of-function mutants show skeletal defects in skull, limbs and vertebral column (Martin, J. F., Bradley, A. and Olson, E. N. (1995) Genes Dev. 9, 1237-1249). We report here that mice in which Prx2 is inactivated by a lacZ insertion had no skeletal defects, whereas Prx1/Prx2 double mutants showed many novel abnormalities in addition to an aggravation of the Prx1 single mutant phenotype. We found defects in external, middle and inner ear, reduction or loss of skull bones, a reduced and sometimes cleft mandible, and limb abnormalities including postaxial polydactyly and bent zeugopods. A single, or no incisor was present in the lower jaw, and ectopic expression of Fgf8 and Pax9 was found medially in the mandibular arch. A novel method to detect &bgr ;-galactosidase activity in hydroxyethylmethacrylate sections allowed detailed analysis of Prx2 expression in affected structures. Our results suggest a role for Prx genes in mediating epitheliomesenchymal interactions in inner ear and lower jaw. In addition, Prx1 and Prx2 are involved in interactions between perichondrium and chondrocytes that regulate their proliferation or differentiation in the bones of the zeugopods.

Mouse Alx3: an aristaless-like homeobox gene expressed during embryogenesis in ectomesenchyme and lateral plate mesoderm.

Mouse Alx3 is a homeobox gene that is related to the Drosophila aristaless gene and to a group of vertebrate genes including Prx1, Prx2, Cart1, and Alx4. The protein encoded contains a diverged variant of a conserved peptide sequence present near the carboxyl terminus of at least 15 different paired-class-homeodomain proteins. Alx3 is expressed in mouse embryos from 8 days of gestation onward in a characteristic pattern, predominantly in neural crest-derived mesenchyme and in lateral plate mesoderm. We detected prominent expression in frontonasal head mesenchyme and in the first and second pharyngeal arches and some of their derivatives. High expression was also seen in the tail and in many derivatives of the lateral plate mesoderm including the limbs, the body wall, and the genital tubercle. aristaless-related genes like Alx3, Cart1, and Prx2 are expressed in overlapping proximodistal patterns in the pharyngeal arches. Similar, but more lateral patterns have been described for the Distal-less-related (Dlx) genes. Intriguingly, expression and to some extent function of aristaless and Distal-less in Drosophila also have overlapping as well as complementary aspects. Alx3 was localized to chromosome 3, near the droopy-ear (de) mutation.

Isolation of a bovine full length cytochrome P450 (CYP3A) cDNA sequence and its functional expression in V79 cells.

From a bovine liver cDNA library in λMaxl a 1870 bp cDNA was isolated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 507 amino acids and exhibited homologies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A1, respectively. Furthermore, a very high homology of 91.7% was observed with the deduced amino acid sequence of a partial CYP3A cDNA from dwarf goat. A striking observation was that both the bovine and the goat cDNA exhibit a 4 amino acid extension at the C-terminus, which is due to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was cloned in a retroviral vector, transfected to V79 cells and cells were selected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6ß-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6ß-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high levels of hydroxylase activity at the 2ß and 12ß position of testosterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazole.

Mode of insertion of the signal sequence of a bacterial precursor protein into phospholipid bilayers as revealed by cysteine-based site-directed spectroscopy.

The interactions between a bacterial precursor protein and phospholipids in bilayer-based model membrane systems is addressed in this study. The precursor-lipid interactions were assessed from the side of the lipid phase by fluorescence and electron spin resonance spectroscopy, using the precursor of the Escherichia coli outer membrane protein PhoE. The role of the signal sequence, as part of the precursor, in this interaction was investigated by using cysteine-based site-directed spectroscopy. For this purpose, purified cysteine-containing mutants of prePhoE, which were made by site-directed mutagenesis of the signal sequence part and of the mature part, and defined lipids were used. The location of the fluorescently labeled cysteine residues was established by resonance energy transfer and quenching experiments and those of the corresponding spin-labeled cysteine residues by paramagnetic relaxation enhancement. It was demonstrated that precursor-phospholipid interactions exist in model membrane systems and also that these interactions were dependent on the presence of anionic phospholipids and resulted in a deep insertion of (parts of) the precursor into the lipid bilayer. Furthermore, the results with the cysteine mutations in the signal sequence of the precursor indicate that both termini of the signal sequence are located near or at the membrane surface, with only the fluorescence of the labeled cysteines in the signal sequence part being protected against aqueous quenchers. The results demonstrate that, when part of the intact precursor, the signal sequence experiences similar lipid-protein interactions as do isolated signal peptides. They also indicate that the signal sequence inserts entirely as a looped structure into the membrane. In addition, the data also indicate that the mature part of the precursor has an affinity for the membrane.