Normal radiological lymph node appearance in the thorax.

Modern treatment of esophageal cancer is multimodal and highly dependent on a detailed diagnostic assessment of clinical stage, which includes nodal stage. Clinical appraisal of nodal stage is highly dependent on knowledge of normal radiological appearance, information of which is scarce. We aimed to describe lymph node appearance on computed tomography (CT) investigations in a randomly selected cohort of healthy subjects. In a sample of the Swedish Cardiopulmonary bioimage study, which investigates a sample of the Swedish population aged 50-64 years, the CT scans of 426 subjects were studied in detail concerning intrathoracic node stations relevant in clinical staging of esophageal cancer. With stratification for sex, the short axis of visible lymph nodes was measured and the distribution of lymph node sizes was calculated as well as proportion of patients with visible nodes above 5 and 10 millimeters for each station. Probability of having any lymph node station above 5 and 10 millimeters was calculated with a logistic regression model adjusted for age and sex. In the 214 men (aged: 57.3 ± 4.1 years) and 212 women (aged: 57.8 ± 4.4 years) included in this study, a total of 309 (72.5%) had a lymph node with a short axis of 5 mm or above was seen in at least one of the node stations investigated. When using 10 mm as a cutoff, nodes were visible in 29 (6.81%) of the subjects. Men had higher odds of having any lymph node with short axis 5 mm or above (OR 3.03 95% CI 1.89-4.85, P < 0.001) as well as 10 mm or above (OR 2.31 95% CI 1.02-5.23, P = 0.044) compared to women. Higher age was not associated with propensity for lymph nodes above 5 or 10 millimeters in this sample. We conclude that, in a randomly selected cohort of patients between 50 and 64 years, almost 10% of the men and 4% of the women had lymph nodes above 10 millimeters, most frequently in the subcarinal station (station 107). More than half of the patients had nodes above 5 millimeters on CT and men were much more prone to have this finding. The probability of finding lymph nodes in specific stations relevant of esophageal cancer is now described.

Beneficial behavioral effects of chronic cerebral dopamine neurotrophic factor (CDNF) infusion in the N171-82Q transgenic model of Huntington's disease.

Huntington's disease (HD) is a progressive inherited neurological disease characterized by the degeneration of basal ganglia and the accumulation of mutant huntingtin (mHtt) aggregates in specific brain areas. Currently, there is no treatment for halting the progression of HD. Cerebral dopamine neurotrophic factor (CDNF) is a novel endoplasmic reticulum located protein with neurotrophic factor properties that protects and restores dopamine neurons in rodent and non-human primate models of Parkinson's disease. Our recent study showed that CDNF improves motor coordination and protects NeuN positive cells in a Quinolinic acid toxin rat model of HD. Here we have investigated the effect of chronic intrastriatal CDNF administration on behavior and mHtt aggregates in the N171-82Q mouse model of HD. Data showed that CDNF did not significantly decrease the number of mHtt aggregates in most brain regions studied. Notably, CDNF significantly delayed the onset of symptoms and improved motor coordination in N171-82Q mice. Furthermore, CDNF increased BDNF mRNA level in hippocampus in vivo in the N171-82Q model and BDNF protein level in cultured striatal neurons. Collectively our results indicate that CDNF might be a potential drug candidate for the treatment of HD.

Biomarkers and molecular imaging in gastroenteropancreatic neuroendocrine tumors.

Neuroendocrine gastrointestinal and pancreatic tumors (GEP-NETs) are a heterogenous group of cancers with various clinical expressions. All tumors produce and secret various amines and peptides, which can be used as tissue and circulating markers. Chromogranin A (CgA) is a general tumor marker stored in secretory granules within the tumor cell and released upon stimulation. CgA is the best general tumor marker at the moment, expressed in 80-90% in all patients with GEP-NETs. CgA and NSE are used as tissue markers for the delineation of the neuroendocrine features of the tumors, but recently also the proliferation marker Ki-67 has been included in the standard procedure for evaluation of the proliferation. GEP-NETs are classified into well differentiated neuroendocrine tumors (Ki-67<2%), well-differentiated neuroendocrine carcinoma (Ki-67 2-20%), poorly differentiated neuroendocrine carcinoma (Ki-67>20%). The molecular imaging of NETs is based on the ability of these tumor cells to express somatostatin receptors as well as the APUD features. Octreoscan has been applied for imaging and staging of the disease for more than 2 decades and will nowadays be replaced by 68Ga-DOTA-Octreotate, with higher specificity and sensitivity. 18Fluoro-DOPA and 11C-5HTP are specific tracers for NETs with high specificity and selectivity. A new potential biomarker is auto-antibodies to paraneoplastic antigen MA2, which might indicate early recurrence of carcinoids after surgery with a curative intent. Circulating tumor cells (CTC) have been applied in GEP-NETs quite recently. There is still an unmet need for new markers.

Cerebral dopamine neurotrophic factor (CDNF) protects against quinolinic acid-induced toxicity in in vitro and in vivo models of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder with a progressive loss of medium spiny neurons in the striatum and aggregation of mutant huntingtin in the striatal and cortical neurons. Currently, there are no rational therapies for the treatment of the disease. Cerebral dopamine neurotrophic factor (CDNF) is an endoplasmic reticulum (ER) located protein with neurotrophic factor (NTF) properties, protecting and restoring the function of dopaminergic neurons in animal models of PD more effectively than other NTFs. CDNF is currently in phase I-II clinical trials on PD patients. Here we have studied whether CDNF has beneficial effects on striatal neurons in in vitro and in vivo models of HD. CDNF was able to protect striatal neurons from quinolinic acid (QA)-induced cell death in vitro via increasing the IRE1α/XBP1 signalling pathway in the ER. A single intrastriatal CDNF injection protected against the deleterious effects of QA in a rat model of HD. CDNF improved motor coordination and decreased ataxia in QA-toxin treated rats, and stimulated the neurogenesis by increasing doublecortin (DCX)-positive and NeuN-positive cells in the striatum. These results show that CDNF positively affects striatal neuron viability reduced by QA and signifies CDNF as a promising drug candidate for the treatment of HD.

Transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation.

Transforming growth factor-beta 1 (TGF-beta 1) has been shown to up-regulate the synthesis of nerve growth factor (NGF) in cultured rat astrocytes and in neonatal brain in vivo (Lindholm, D., B. Hengerer, F. Zafra, and H. Thoenen. 1990. NeuroReport. 1:9-12). Here we show that mRNA encoding TGF-beta 1 increased in rat cerebral cortex after a penetrating brain injury. The level of NGF mRNA is also transiently increased after the brain trauma, whereas that of brain-derived neurotrophic factor remained unchanged. In situ hybridization experiments showed a strong expression of TGF-beta 1 4 d after the lesion in cells within and in the vicinity of the wound. Staining of adjacent sections with OX-42 antibodies, specific for macrophages and microglia/brain macrophages, revealed a similar pattern of positive cells, suggesting that invading macrophages, and perhaps reactive microglia, are the source of TGF-beta 1 in injured brain. Both astrocytes and microglia express TGF-beta 1 in culture, and TGF-beta 1 mRNA levels in astrocytes are increased by various growth factors, including FGF, EGF, and TGF-beta itself. TGF-beta 1 is a strong inhibitor of astrocyte proliferation and suppresses the mitotic effects of FGF and EGF on astrocytes. The present results indicate that TGF-beta 1 expressed in the lesioned brain plays a role in nerve regeneration by stimulating NGF production and by controlling the extent of astrocyte proliferation and scar formation.

Regional and cellular codistribution of interleukin 1 beta and nerve growth factor mRNA in the adult rat brain: possible relationship to the regulation of nerve growth factor synthesis.

We have found a regional distribution of IL 1 beta mRNA and IL 1 activity in the normal adult rat brain, which reveals at least partially a colocalization with nerve growth factor (NGF). The predominantly neuronal signal patterns were found over the granule cells of the dentate gyrus, the pyramidal cells of the hippocampus, the granule cells of the cerebellum, the granule and periglomerular cells of the olfactory bulb, and over dispersed cells of the ventromedial hypothalamus and of the frontal cortex. In these areas also the highest levels of IL 1 activity were observed. In the striatum and septum much lower levels of IL 1 beta mRNA and IL 1 activity (shown for the striatum), most likely synthesized by glial cells, could be determined. IL 1 beta-expressing cells were mainly found in brain regions that also synthesize NGF mRNA as shown by in situ hybridization. NGF mRNA could be demonstrated over pyramidal cells of the hippocampus, granule cells of the dentate gyrus, periglomerular cells of the olfactory bulb and over prefrontal cortex neurons. These data indicate that IL 1 beta, among other factors, might also play a regulatory role in the synthesis of NGF in the CNS, as has been demonstrated in the peripheral nervous system (Lindholm, D., R. Heumann, M. Meyer, and H. Thoenen. 1987. Nature (Lond.). 330:658-659).

Expression and functional interaction of hepatocyte growth factor-scatter factor and its receptor c-met in mammalian brain.

Hepatocyte growth factor-scatter factor (HGF-SF) is a pleiotropic cytokine with mito-, morpho-, and motogenic effects on a variety of epithelial and endothelial cells. HGF-SF activity is mediated by the c-met protooncogene, a membrane-bound tyrosine kinase. Here, we demonstrate that both genes are expressed in developing and adult mammalian brains. HGF-SF mRNA is localized in neurons, primarily in the hippocampus, the cortex, and the granule cell layer of the cerebellum, and it is also present at high levels in ependymal cells, the chorioid plexus, and the pineal body. c-met is expressed in neurons, preferentially in the CA-1 area of the hippocampus, the cortex, and the septum, as well as in the pons. In the embryonic mouse, brain HGF-SF and c-met are expressed as early as days 12 and 13, respectively. Neuronal expression of HGF-SF is evolutionary highly conserved and detectable beyond the mammalian class. Incubation of septal neurons in culture with HGF-SF leads to a rapid increase of c-fos mRNA levels. The results demonstrate the presence of a novel growth factor-tyrosine kinase signaling system in the brain, and they suggest that HGF-SF induces a functional response in a neuronal subpopulation of developing and adult CNS.

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation.

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.

Evidence that fibroblast growth factor 5 is a major muscle-derived survival factor for cultured spinal motoneurons.

We examined the potential role of fibroblast growth factor 5 (FGF-5) as a target-derived trophic factor for spinal motoneurons. Northern analysis of total RNA from rat skeletal muscle revealed an FGF-5 mRNA transcript both during the period of embryonic motoneuron death and in the adult. Recombinant human FGF-5 supported the survival of highly enriched cultures of embryonic chick motoneurons. Significant proportions of the motoneuron survival activity of rat skeletal muscle extracts could be immunoprecipitated using an antiserum to FGF-5. The immunoprecipitable activity was present in soluble and matrix-bound forms in embryonic muscle, but bound exclusively to the extracellular matrix in adult muscle. These results, along with the secretory nature of FGF-5, suggest that FGF-5 may act as a target-derived trophic factor for motoneurons.

Expression of X-chromosome linked inhibitor of apoptosis protein in mature Purkinje cells and in retinal bipolar cells in transgenic mice induces neurodegeneration.

Transgenic mice with overexpression of the caspase-inhibitor, X-chromosome-linked inhibitor of apoptosis protein (XIAP) in Purkinje cell (PC) and in retinal bipolar cells (RBCs) were produced to study the regulation of cell death. Unexpectedly, an increased neurodegeneration was observed in the PCs in these L7-XIAP mice after the third postnatal week with the mice exhibiting severe ataxia. The loss of PCs was independent of Bax as shown by crossing the L7-XIAP mice with Bax gene-deleted mice. Electron microscopy revealed intact organelles in PCs but with the stacking of ER cisterns indicative of cell stress. Immunostaining for cell death proteins showed an increased phosphorylation of c-Jun in the PCs, suggesting an involvement in cell degeneration. Apart from PCs, the number of RBCs was decreased in adult retina in line with the expression pattern for the L7 promoter. The data show that overexpression of the anti-apoptotic protein XIAP in vulnerable neurons leads to enhanced cell death. The mechanisms underlying this neurodegeneration can be related to the effects of XIAP on cell stress and altered cell signaling.

ER stress and neurodegenerative diseases.

Endoplasmic reticulum (ER) stress is caused by disturbances in the structure and function of the ER with the accumulation of misfolded proteins and alterations in the calcium homeostasis. The ER response is characterized by changes in specific proteins, causing translational attenuation, induction of ER chaperones and degradation of misfolded proteins. In case of prolonged or aggravated ER stress, cellular signals leading to cell death are activated. ER stress has been suggested to be involved in some human neuronal diseases, such as Parkinson's disease, Alzheimer's and prion disease, as well as other disorders. The exact contributions to and casual effects of ER stress in the various disease processes, however, are not known. Here we will discuss the possible role of ER stress in neurodegenerative diseases, and highlight current knowledge in this field that may reveal novel insight into disease mechanisms and help to design better therapies for these disorders.

Altered distribution and levels of cathepsinD and cystatins in amyotrophic lateral sclerosis transgenic mice: possible roles in motor neuron survival.

In amyotrophic lateral sclerosis (ALS) there is a selective degeneration of motor neurons leading to muscle paralysis and death. The mechanism underlying cell demise in ALS is not fully understood, but involves the activation of different proteolytic enzymes, including the caspase family of cysteine proteases. We have here studied whether other proteases, such as the cathepsins, residing in lysosomes, and the cathepsin inhibitors, cystatinB and -C are changed in ALS. The expression and protein levels of the cathepsinB, -L and -D all increased in the spinal cord in ALS mice, carrying the mutant copper/zinc superoxide dismutase (SOD1) gene. At the cellular level, cathepsinB and -L were present in ventral motor neurons in controls, but in the ALS mice cathepsinB was also expressed by glial fibrillary acidic protein (GFAP) positive astrocytes. The distribution of the aspartic protease, cathepsinD also changed in ALS with a loss of the lysosomal staining in motor neurons. Inhibition of caspases by means of X-chromosome-linked inhibitor of apoptosis protein (XIAP) overexpression did not inhibit cleavage of cathepsinD in ALS mice, suggesting a caspase-independent pathway. Expression of cystatinB and -C increased slightly in the ALS spinal cords. Immunostaining showed that in ALS, cystatinC was present in motor neurons and in GFAP positive astrocytes. CystatinB that is a neuroprotective factor decreased in motor neurons in ALS but was expressed by activated microglial cells. The observed changes in the levels and distributions of cathepsinD and cystatinB and-C indicate a role of these proteins in the degeneration of motor neurons in ALS.

Irradiation-induced progenitor cell death in the developing brain is resistant to erythropoietin treatment and caspase inhibition.

One hemisphere of postnatal day 8 (P8) rats or P10 mice was irradiated with a single dose of 4-12 Gy, and animals were killed from 2 h to 8 weeks after irradiation (IR). In the subventricular zone (SVZ) and the granular cell layer (GCL) of the dentate gyrus, harboring neural and other progenitor cells, nitrosylation and p53 peaked 2-12 h after IR, followed by markers for active caspase-3, apoptosis-inducing factor and TUNEL (6-24 h). Ki67-positive (proliferating) cells had disappeared by 12 h and partly reappeared by 7 days post-IR. The SVZ and GCL areas decreased approximately 50% 7 days after IR. The development of white matter was hampered, resulting in 50-70% less myelin basic protein staining. Pretreatment with erythropoietin did not confer protection against IR. Caspase inhibition by overexpression of XIAP prevented caspase-9 and caspase-3 activation but not cell death, presumably because of increased caspase-independent cell death.

Brain-derived neurotrophic factor protects against ischemic cell damage in rat hippocampus.

The neuroprotective action of brain-derived neurotrophic factor (BDNF) was evaluated in a rat model of transient forebrain ischemia. A continuous intraventricular infusion of BDNF for 7 days starting immediately before the onset of ischemia significantly increased the number of pyramidal cells in the vulnerable CA1 sector of the hippocampus. In situ hybridization experiments suggest the neuroprotection to be mediated via trkB-receptors in the hippocampus. The data indicate a therapeutic potential for the treatment of cerebral ischemia.

Paradoxical increase in neuronal DNA fragmentation after neuroprotective free radical scavenger treatment in experimental traumatic brain injury.

The mechanisms and role of nerve cell death after traumatic brain injury (TBI) are not fully understood. The authors investigated the effect of pretreatment with the oxygen free radical spin trap alpha-phenyl-N-tert-butyl-nitrone (PBN) on the number of neurons undergoing apoptosis after TBI in rats. Apoptotic cells were identified by the TUNEL method combined with the nuclear stain, Hoechst 33258, and immunohistochemistry for the active form of caspase-3. Numerous neurons became positive for activated caspase 3 and TUNEL in the cortex at 24 hours after injury, suggesting ongoing biochemical apoptosis. In PBN-treated rats, a significantly greater number of cells were found to be TUNEL positive at 24 hours compared with controls. However, PBN treatment resulted in a reduced cortical lesion volume and improved behavioral outcome two weeks after injury. The authors conclude that a treatment producing an increase in DNA fragmentation in the early phase may be compatible with an overall beneficial effect on outcome after TBI. This should be considered in the screening process for future neuroprotective remedies.

Thyroxine preferentially stimulates transcription by isolated neuronal nuclei in the developing rat brain cortex.

The administration of thyroxine to neonatal rats stimulates RNA synthesis by neuronal nuclei isolated from the developing rat brain cortex. Glial nuclei are relatively resistant to thyroxine treatment. The activity of neuronal RNA polymerase II is particularly stimulated by the hormone. Thyroxine also affects neuronal chromatin structure as shown by changes in the relative proportion of different subnuclear fractions obtained by gentle micrococcal nuclease digestion of nuclei from hormone-treated rats.

Nerve Growth Factor Regulates Expression of the Nerve Growth Factor Receptor Gene in Adult Sensory Neurons.

Sensory neurons of the adult rat dorsal root ganglion (DRG) can be maintained in culture in the absence of nerve growth factor (NGF). We have thus used dissociated cultures of these neurons to study effects of NGF on the regulation of expression of mRNA encoding the nerve growth factor receptor (NGF-R). In the absence of NGF, levels of NGF-R mRNA remained constant for 7 days in cultures of adult rat DRG neurons. In the presence of NGF, NGF-R mRNA levels rose two - three-fold after 2 days, reaching plateau levels (five - six-fold elevation) after 5 days. This NGF-induced up-regulation could be demonstrated even after prior NGF-deprivation for 3 - 4 days. NGF had no effect upon NGF-R mRNA levels in DRG non-neuronal cells. Epidermal growth factor (EGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) were without effect on NGF-R mRNA levels, but 8-bromo-cAMP decreased NGF-R mRNA levels by 65% after 2 days. NGF also induced a rapid (30 min) rise in expression of c-fos in DRG neurons, but not in non-neuronal cells. Our results suggest that endogenous levels of NGF may regulate the expression of NGF-R in vivo.

BDNF and NT-3 but not CNTF counteract the Ca2+ ionophore-induced apoptosis of cultured cortical neurons: involvement of dual pathways.

The effect of neurotrophic factors on apoptosis induced by ionomycin, a potent Ca2+ ionophore, was investigated using cultured cortical neurons from embryonic rats. Brain-derived neurotophic factor (BDNF) and neurotrophin-3 (NT-3) prevented the ionomycin-mediated cell death in a dose-dependent manner. In contrast to the neurotrophins, cilliary neurotrophic factor (CNTF) did not rescue neurons from cell death induced by ionomycin. The protective effect of BDNF was partially blocked by wortmannin, a phosphatidylinositol 3-kinase inhibitor, and by PD98059, a MAP kinase kinase inhibitor. However, the addition of both compounds together completely inhibited the survival promoting effect of BDNF. These results suggest that the neuroprotective effect of BDNF requires activation of both phosphatidylinositol-3 kinase and the Ras/MAP kinase cascade and that CNTF signaling through other pathways is without an effect in this system.

Distribution and protein synthetic activities of neuronal free and membrane-bound ribosomes during postnatal development of rat cerebral cortex.

The distribution and amino acid incorporative activities of free and membrane-bound ribosomes in neuronal perikarya of rat cerebral cortex at successive stages of postnatal development were investigated. The relative proportion of neuronal membrane-bound ribosomes increased significantly between 6 and 18 days of age, reaching 50% of the total ribosomal population around day 18. In contrast to the neuronal fraction, the membrane-bound ribosomes in unfractionated cerebral cortex decreased from 50% at birth to 35% in 18-day-old pups. When tested in a cell-free amino acid incorporation system the activities of both free and membrane-bound ribosomes increased up to day 10 post partum. However, whereas the activity of free ribosomes reached a constant level at this age, that of the membrane-bound fraction continued to rise until 18th day after birth. Crossover experiments employing homologous or heterologous cell sap fractions showed that the preparation from the 18-day-old animals was more efficient in supporting protein synthesis in vitro, if compared with similar preparation from the neonatal animals. This was attributed to an enhanced aminoacylation of transfer RNA in neuronal cell sap of older compared with younger rats. The observed developmental alterations in the distribution and activities of free and membrane-bound ribosomes are discussed in relation to changes that occur in morphology and function of brain during the early postnatal period.

Differential expression of bcl-w and bcl-x messenger RNA in the developing and adult rat nervous system.

The bcl-2 family of proteins comprises both anti-apoptotic and pro-apoptotic members, which play a pivotal role in regulating cell death. Bcl-w is a recently identified member of this family, which was shown to inhibit apoptosis in haemopoietic cell lines. However, the function and expression patterns of bcl-w in the nervous system have so far not been described. We have cloned complementary DNA corresponding to rat bcl-w and analysed the expression of bcl-w messenger RNA during rat brain development, using RNA blotting and in situ hybridization techniques. We also compared the expression patterns of bcl-w with those of bcl-xL. During brain development, the levels of bcl-w messenger RNA were found to increase, with highest expression located to specific regions of the mature brain, such as hippocampus, cerebellum, piriform cortex and locus coeruleus. Bcl-w messenger RNA was expressed by neurons, as shown with double labeling with neuronal markers. In contrast to bcl-w, bcl-xL messenger RNA expression levels were highest during early development, particularly in cortex, hippocampus, thalamus, spinal cord and dorsal root ganglia. During postnatal development the expression of bcl-xL messenger RNA decreased and were only detected at low levels in the adult nervous system. As shown earlier for bcl-2, the expression of bcl-w and bcl-x messenger RNA in cultured cerebellar granule cells was not altered by the deprivation of neurotrophic factors. The present results suggest that bcl-w may play an important role in the mature nervous system.