Increased ozone-induced airway neutrophilic inflammation in extracellular-superoxide dismutase null mice.

Extracellular-superoxide dismutase (EC-SOD) exists primarily in the tissue interstitium and the lung contains particularly large amounts of the enzyme. To determine the roles of EC-SOD and extracellularly formed superoxide radicals in the pulmonary response to the common air pollutant ozone, wild-type mice and mice lacking EC-SOD were exposed to 1.5 ppm ozone for 48 h. The exposure resulted in a marked neutrophilic inflammatory reaction observed both in the bronchoalveolar lavage fluid (BALF) and by histopathology of the lungs, which was much stronger in the mice lacking EC-SOD. Unlike the wild-type mice, the null mutants also showed increased levels of interleukin-6 in the BALF. The ozone exposure also resulted in increased airway mucosal permeability and cell damage as indicated by increased protein and lactate dehydrogenase in the BALF. There was, however, no difference between the two groups of mice.The results suggest that extracellular superoxide radicals are important inflammatory mediators in the pulmonary response to ozone, but in the present model, the radical and the infiltrating neutrophils contributed little to the pulmonary injury The data, together with previous findings, support a role for EC-SOD as a modulator of inflammatory reactions.

Neural induction: toward a unifying mechanism.

Neural induction constitutes the initial step in the generation of the vertebrate nervous system. In attempting to understand the principles that underlie this process, two key issues need to be resolved. When is neural induction initiated, and what is the cellular source and molecular nature of the neural inducing signal(s)? Currently, these aspects of neural induction seem to be very different in amphibian and amniote embryos. Here we highlight the similarities and the differences, and we propose a possible unifying mechanism.

The status of Wnt signalling regulates neural and epidermal fates in the chick embryo.

The acquisition of neural fate by embryonic ectodermal cells is a fundamental step in the formation of the vertebrate nervous system. Neural induction seems to involve signalling by fibroblast growth factors (FGFs) and attenuation of the activity of bone morphogenetic protein (BMP). But FGFs, either alone or in combination with BMP antagonists, are not sufficient to induce neural fate in prospective epidermal ectoderm of amniote embryos. These findings suggest that additional signals are involved in the specification of neural fate. Here we show that the state of Wnt signalling is a critical determinant of neural and epidermal fates in the chick embryo. Continual Wnt signalling blocks the response of epiblast cells to FGF signals, permitting the expression and signalling of BMP to direct an epidermal fate. Conversely, a lack of exposure of epiblast cells to Wnt signals permits FGFs to induce a neural fate.

Fibroblast growth factor signaling is required for the proliferation and patterning of progenitor cells in the developing anterior pituitary.

The proliferation and patterning of progenitor cells in the anterior pituitary require signals derived from the neuroepithelium of the juxtaposed infundibulum. The infundibulum expresses Fibroblast growth factor (Fgf) 8 and Fgf 18, and FGFs can mimic some of the activities of the infundibulum. The requirement for FGF signaling during growth and patterning of the anterior pituitary has not, however, been established. By blocking FGF receptor signaling in explants of the anterior pituitary cultured in vitro we provide evidence that FGF signaling derived from the infundibulum is required for the proliferation and patterning of progenitor cells in the anterior pituitary.

Sonic hedgehog signaling at gastrula stages specifies ventral telencephalic cells in the chick embryo.

A secreted signaling factor, Sonic hedgehog (Shh), has a crucial role in the generation of ventral cell types along the entire rostrocaudal axis of the neural tube. At caudal levels of the neuraxis, Shh is secreted by the notochord and floor plate during the period that ventral cell fates are specified. At anterior prosencephalic levels that give rise to the telencephalon, however, neither the prechordal mesoderm nor the ventral neural tube expresses Shh at the time that the overt ventral character of the telencephalon becomes evident. Thus, the precise role and timing of Shh signaling relevant to the specification of ventral telencephalic identity remains unclear. By analysing neural cell differentiation in chick neural plate explants we provide evidence that neural cells acquire molecular properties characteristic of the ventral telencephalon in response to Shh signals derived from the anterior primitive streak/Hensen's node region at gastrula stages. Exposure of prospective anterior prosencephalic cells to Shh at this early stage is sufficient to initiate a temporal program of differentiation that parallels that of neurons generated normally in the medial ganglionic eminence subdivision of the ventral telencephalon.

An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo.

BACKGROUND: In Xenopus embryos, fibroblast growth factors (FGFs) and secreted inhibitors of bone morphogenetic protein (BMP)-mediated signalling have been implicated in neural induction. The precise roles, if any, that these factors play in neural induction in amniotes remains to be established. RESULTS: To monitor the initial steps of neural induction in the chick embryo, we developed an in vitro assay of neural differentiation in epiblast cells. Using this assay, we found evidence that neural cell fate is specified in utero, before the generation of the primitive streak or Hensen's node. Early epiblast cells expressed both Bmp4 and Bmp7, but the expression of both genes was downregulated as cells acquired neural fate. During prestreak and gastrula stages, exposure of epiblast cells to BMP4 activity in vitro was sufficient to block the acquisition of neural fate and to promote the generation of epidermal cells. Fgf3 was also found to be expressed in the early epiblast, and ongoing FGF signalling in epiblast cells was required for acquisition of neural fate and for the suppression of Bmp4 and Bmp7 expression. CONCLUSIONS: The onset of neural differentiation in the chick embryo occurs in utero, before the generation of Hensen's node. Fgf3, Bmp4 and Bmp7 are each expressed in prospective neural cells, and FGF signalling appears to be required for the repression of Bmp expression and for the acquisition of neural fate. Subsequent exposure of epiblast cells to BMPs, however, can prevent the generation of neural tissue and induce cells of epidermal character.

Convergent inductive signals specify midbrain, hindbrain, and spinal cord identity in gastrula stage chick embryos.

In the chick embryo, neural cells acquire midbrain, hindbrain, and spinal cord character over a approximately 6 hr period during gastrulation. The convergent actions of four signals appear to specify caudal neural character. Fibroblast growth factors (FGFs) and a paraxial mesoderm-caudalizing (PMC) activity are involved, but neither signal is sufficient to induce any single region. FGFs act indirectly by inducing mesoderm that expresses PMC and retinoid activity and also directly on prospective neural cells, in combination with PMC activity and a rostralizing signal, to induce midbrain character. Hindbrain character emerges from cells that possess the potential to acquire midbrain character upon exposure to higher levels of PMC activity. Induction of spinal cord character appears to involve PMC and retinoid activities.

Dosimetric advantages of enhanced dynamic wedge in small field irradiation for the treatment of macular degeneration.

Exudative macular degeneration is a process that affects the central retina and is the chief cause of blindness in people over 55 years old. Radiation appears to improve or stabilize visual acuity in some of these patients, although definitive clinical studies are ongoing. The delivery of radiation in patients with macular degeneration is unique and challenging because of the location of the surrounding structures, such as the lens and the opposite retina. The "standard" treatment technique has been an anterior oblique beam 20 degrees above lateral to limit dose to these structures. In preference to the undesirable hot spot caused by this single-beam technique, we have employed a superior and inferior oblique pair utilizing the Enhanced Dynamic Wedge (EDW). Couch rotation was used to establish the desired treatment angles. The anterior half of the fields were blocked with the asymmetric jaw (AJ). Studies were performed to determine the value of these methods in reducing lens dose. Although the peripheral dose measurements at Dmax using a 60 degrees metal wedge for > or = 10 cm square fields were higher than for an open field or the EDW, for the small retinal fields there was no lens-dose reduction attributable to the EDW. However, the steep wedge angles achievable with the EDW were useful for optimizing the dose distribution for the most desirable field angles. As expected, the independent collimator was more effective in reducing transmitted dose to the lens than a typical Cerrobend block. Measurements required to predict dose in and out of the field are discussed along with the dosimeters employed in evaluating small fields (less than 5 cm square) with the EDW. The technical challenges of positioning, immobilization, treatment planning and treatment delivery are presented.

A single isocenter technique using CT-based planning in the treatment of breast cancer.

Breast cancer will afflict 1 of every 9 women. With advances in mammography and public awareness, breast cancer is being detected at an earlier stage and is therefore more curable. The combination of lumpectomy and radiotherapy is a standard treatment option for most women with stage I and II invasive breast cancer. Radiotherapy treatment fields are usually tangential to encompass the breast, and in some cases, matched to a supraclavicular field. Due to variation in the size and shape of the breast, dosimetry and treatment planning can be challenging, yet many radiation oncology centers still achieve a treatment plan of the breast by using a single plane hand-generated contour 2-dimensional (2D) through the center of the breast. This type of planning neglects the variations in contours and chest-wall separation superior and inferior to that plane, which significantly impacts dose homogeneity and dosimetry. Computed tomography (CT) images are being utilized more often for planning in radiotherapy and should be considered for breast planning. CT-based treatment planning 3-dimensional (3D) allows the planner and physician to evaluate the dosimetry across the entire breast. Consequently, the plan can be optimized to limit lung volume with selective blocking, and minimize hot spots by using a higher energy and less wedge angle, thus improving dose homogeneity. For a larger breast, when the dosimetry using 6 MV is undesirable, a mix of 6 MV and higher energy may be used. This paper describes a single isocenter treatment technique using CT-based planning for tangents and a supraclavicular field and the advantages our department has found using 3D treatment planning vs. 2D. In addition, the creative planning done for larger breasts and the use of 18 MV combined with 6 MV to optimize a treatment plan will American Association of Medical Dosimetrists.

Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituitary.

The mechanisms by which inductive signals control the identity, proliferation and timing of differentiation of progenitor cells in establishing spatial pattern in developing vertebrate tissues remain poorly understood. We have addressed this issue in the embryonic anterior pituitary, an organ in which distinct hormone cell types are generated in a precise temporal and spatial order from an apparently homogenous ectodermal primordium. We provide evidence that in this tissue the coordinate control of progenitor cell identity, proliferation and differentiation is imposed by spatial and temporal restrictions in FGF- and BMP-mediated signals. These signals derive from adjacent neural and mesenchymal signaling centers: the infundibulum and ventral juxtapituitary mesenchyme. The infundibulum appears to have a dual signaling function, serving initially as a source of BMP4 and subsequently of FGF8. The ventral juxtapituitary mesenchyme appears to serve as a later source of BMP2 and BMP7. In vitro, FGFs promote the proliferation of progenitor cells, prevent their exit from the cell cycle and contribute to the specification of progenitor cell identity. BMPs, in contrast, have no apparent effect on cell proliferation but instead appear to act with FGFs to control the initial selection of thyrotroph and corticotroph progenitor identity.

Treatment planning of oblique wedge fields comparing enhanced dynamic wedge and standard 60 degree wedge for parotid type treatments.

The "standard" treatment planning for a parotid or similar treatment volume utilizes wedge field technique. This technique can be accomplished by using two beams on the same side of the patient with the heels of the wedges adjacent. This technique can also be accomplished by using standard or enhanced dynamic wedges. Advantages vs. disadvantages of enhanced dynamic wedged fields will be discussed in this paper.

Assignment of early caudal identity to neural plate cells by a signal from caudal paraxial mesoderm.

The early patterning of the vertebrate central nervous system involves the generation of progenitor cells with distinct fates at rostral and caudal levels of the neuraxis. We provide evidence that the assignment of early rostrocaudal differences in progenitor cell properties is established by spatial restrictions in the signaling properties of the paraxial mesoderm and epidermal ectoderm. Caudal level paraxial mesoderm secretes a factor, distinct from retinoic acid or fibroblast growth factors (FGFs), that can impose caudal fates on prospective anterior proencephalic progenitors. The caudalizing activity of the paraxial mesoderm can, however, be induced by FGF signaling. The distinct properties of cells at rostral and caudal levels of the neural plate appear to depend, in addition, on the early exclusion of bone morphogenetic proteins (BMPs) from rostral level epidermal ectoderm. Thus, differences in the signaling properties of cell groups that flank the neural plate appear to contribute to the early rostrocaudal identity of neural cells, distinguishing progenitor cells at prospective anterior proencephalic regions from those at more caudal levels of the neuraxis.

Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells.

The mammalian pancreas is a specialized derivative of the primitive gut endoderm and controls many homeostatic functions through the activity of its component exocrine acinar and endocrine islet cells. The LIM homeodomain protein ISL1 is expressed in all classes of islet cells in the adult and its expression in the embryo is initiated soon after the islet cells have left the cell cycle. ISL1 is also expressed in mesenchymal cells that surround the dorsal but not ventral evagination of the gut endoderm, which together comprise the pancreatic anlagen. To define the role of ISL1 in the development of the pancreas, we have now analysed acinar and islet cell differentiation in mice deficient in ISL1 function. Dorsal pancreatic mesenchyme does not form in ISL1-mutant embryos and there is an associated failure of exocrine cell differentiation in the dorsal but not the ventral pancreas. There is also a complete loss of differentiated islet cells. Exocrine, but not endocrine, cell differentiation in the dorsal pancreas can be rescued in vitro by provision of mesenchyme derived from wild-type embryos. These results indicate that ISL1, by virtue of its requirement for the formation of dorsal mesenchyme, is necessary for the development of the dorsal exocrine pancreas, and also that ISL1 function in pancreatic endodermal cells is required for the generation of all endocrine islet cells.

The effect of antimicrobial therapy on periimplantitis lesions. An experimental study in the dog.

The objective of the present study was to evaluate the effect of systemic antibiotics and local debridement in the treatment of experimentally induced periimplantitis lesions. 5 Labrador dogs, and about 1-year old, were included in the study. In order to establish bilateral recipient sites for implants the mandibular right and left 1st molars, 4th and 3rd premolars were removed. 6 titanium fixtures (Brånemark System Nobelpharma AB, Göteborg, Sweden) were installed and standard abutments were connected 3 months after fixture installation. Cotton floss ligatures were placed in a submarginal position around the neck of the abutments and the animals were placed on a diet which allowed plaque accumulation. After 6-8 weeks, when the tissue destruction amounted to about 20% of the fixture length, the ligatures were removed. 1 month after ligature removal, an antibiotic regimen (amoxicillin and metronidazole) was initiated and maintained for 3 weeks. In the left side of the mandible, buccal and lingual mucoperiosteal flaps were elevated, the granulation tissue within the bone craters adjacent to the implants was curetted, and the abutments were removed. The exposed outer surface, the internal part of the fixtures, as well as the abutments were treated with a detergent, delmopinol. The cleaned abutments were autoclaved, and connected to the clean fixtures. The mucoperiosteal flaps were replaced to their original position, adapted to the abutments and sutured. A careful plaque control program was initiated for the left jaw quadrants. In the right side of the mandible no local treatment was given to the fixtures and the abutments following ligature removal. Furthermore, no plaque control was provided to the implant segments in the right jaws. After 4 months of healing block biopsies including one implant with adjacent hard and soft tissue were harvested and prepared for light microscopy. It was observed that systemic antimicrobial therapy, combined with implant cleaning, curettage of the bone defect and regular plaque control resulted in (i) resolution of the periimplantitis lesion, (ii) a significant recession of the marginal periimplant mucosa, and (iii) a minor additional apical shift of the base of the bone defect. In the untreated sites the plaque associated infiltrate remained and was in several sites examined in contact with the adjacent bone tissue.

The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid.

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.

Multi-fractionated stereotactic radiotherapy.

Precision and accuracy of a patient's treatment are key advantages of single-fraction stereotactic radiosurgery (SRS) for arteriovenous malformations (AVMS) and some small brain metastases. These advantages are equally valuable in fractionated treatment of the pituitary, brain metastases and brain boost fields. The need to implement the preciseness from stereotactic radiosurgery to fractionated treatments was recognized. Using our experience with single-fraction stereotactic radiosurgery as a model, we developed a multi-fractionated stereotactic radiotherapy technique that allows us to immobilize a patient daily and implement important existing devices such as the Brown-Roberts-Wells (BRW) angiographic localizer, CT scan localizer, and non-coplanar shaped field treatment planning. Development of this technique also allows us to achieve reproducible patient positioning based on immobilization techniques using polyurethane foam immobilization and heat moldable plastic technology without the necessity of the invasive technique of skull fixation. The development, implementation and dosimetry of this technique will be discussed in this paper.

Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation.

Motor neuron differentiation is accompanied by the expression of a LIM homeodomain transcription factor, Islet1 (ISL1). To assess the involvement of ISL1 in the generation of motor neurons, we analyzed cell differentiation in the neural tube of embryos in which ISL1 expression has been eliminated by gene targeting. Motor neurons are not generated without ISL1, although many other aspects of cell differentiation in the neural tube occur normally. A population of interneurons that express Engrailed1 (EN1), however, also fails to differentiate in Isl1 mutant embryos. The differentiation of EN1+ interneurons can be induced in both wild-type and mutant neural tissue by regions of the neural tube that contain motor neurons. These results show that ISL1 is required for the generation of motor neurons and suggest that motor neuron generation is required for the subsequent differentiation of certain interneurons.

Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish.

In zebrafish, individual primary motoneurons can be uniquely identified by their characteristic cell body positions and axonal projection patterns. The fate of individual primary motoneurons remains plastic until just prior to axogenesis when they become committed to particular identities. We find that distinct primary motoneurons express particular combinations of LIM homeobox genes. Expression precedes axogenesis as well as commitment, suggesting that LIM homeobox genes may contribute to the specification of motoneuronal fates. By transplanting them to new spinal cord positions, we demonstrate that primary motoneurons can initiate a new program of LIM homeobox gene expression, as well as the morphological features appropriate for the new position. We conclude that the patterned distribution of different primary motoneuronal types within the zebrafish spinal cord follows the patterned expression of LIM homeobox genes, and that this reflects a highly resolved system of positional information controlling gene transcription.

IPF1, a homeodomain protein with a dual function in pancreas development.

Insulin promoter factor 1 (IPF1), is a homeodomain protein which, in the adult mouse pancreas, is selectively expressed in beta-cells, and which binds to, and transactivates, the insulin promoter via the P1 element. In mouse embryos, IPF1 expression is initiated when the foregut endoderm commits to a pancreatic fate, i.e. prior to both morphogenesis and hormone specific gene expression. At later stages of development the expression is restricted to the dorsal and ventral walls of the primitive foregut at the positions where the pancreases will form. Mice homozygous for a targeted mutation in the Ipf1 gene selectively lack the pancreas. The mutant pups develop to term and are born alive, but die after a few days. The gastrointestinal tract with its associated organs show no obvious malformations. No pancreatic tissue and no ectopic expression of insulin or pancreatic amylase could be detected in this region in mutant neonates or embryos. These findings demonstrate that IPF1 is needed for the formation of the pancreas, and suggest that IPF1 acts to determine the fate of common pancreatic precursor cells and/or to regulate their propagation. The lack of a pancreas in the Ipf1-deficient mutants, the pattern of IPF1 expression and its ability to stimulate insulin gene transcription, strongly suggest that IPF1 functions both in the early specification of the primitive gut to a pancreatic fate and in the maturation of the pancreatic beta-cell.