Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

BACKGROUND: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. METHODS: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. RESULTS: The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. CONCLUSION: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

Minor changes in gene expression in the mouse preoptic hypothalamic region by inflammation-induced prostaglandin E2.

We investigated to what extent inflammation-induced prostaglandin E2 (PGE2 ) regulates gene expression in the central nervous system. Wild-type mice and mice with deletion of the gene encoding microsomal prostaglandin E synthase-1 (mPGES-1), which cannot produce inflammation-induced PGE2 , were subjected to peripheral injection of bacterial wall lipopolysaccharide (LPS) and killed after 5 h. The median and medial preoptic nuclei, which are rich in prostaglandin E receptors, were isolated by laser capture microdissection (LCM), and subjected to whole genome microarray analysis. Although the immune stimulus induced robust transcriptional changes in the brain, as seen by a quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on selected genes, only small PGE2 -dependent gene expression changes were observed in the gene array analysis and, for only two genes, a pronounced differential expression between LPS-treated wild-type and mPGES-1 knockout mice could be verified by qRT-PCR. These were Hspa1a and Hspa1b, encoding heat shock proteins, which showed a two- to three-fold higher expression in wild-type mice than in knockout mice after immune challenge. However, the induced expression of these genes was found to be secondary to increased body temperature because they were induced also by cage exchange stress, which did not elicit PGE2 synthesis, and thus were not induced per se by PGE2 -elicited transcriptional events. Our findings suggest that inflammation-induced PGE2 has little effect on gene expression in the preoptic region, and that centrally elicited disease symptoms, although PGE2 -dependent, occur as a result of regulation of neuronal excitability that is a consequence of intracellular, transcriptional-independent signalling cascades. Our findings also imply that the profound changes in gene expression in the brain that are elicited by peripheral inflammation occur independently of PGE2 via a yet unidentified mechanism.

Immune-induced expression of lipocalin-2 in brain endothelial cells: relationship with interleukin-6, cyclooxygenase-2 and the febrile response.

Interleukin (IL)-6 is critical for the febrile response to peripheral immune challenge. However, the mechanism by which IL-6 enables fever is still unknown. To characterise the IL-6-dependent fever generating pathway, we used microarray analysis to identify differentially expressed genes in the brain of lipopolysaccharide (LPS)-treated IL-6 wild-type and knockout mice. Mice lacking IL-6 displayed a two-fold lower expression of the lipocalin-2 gene (lcn2), and this difference was confirmed by real-time reverse transcriptase-polymerase chain reaction. Conversely, the induction of lipocalin-2 protein was observed in brain vascular cells following i.p. administration of recombinant IL-6, suggesting a direct relationship between IL-6 and lipocalin-2. Immunohistochemical analysis also revealed that LPS-induced lipocalin-2 is expressed by brain endothelial cells and is partly co-localised with cyclooxygenase-2 (Cox-2), the rate-limiting enzyme for the production of inflammatory induced prostaglandin E(2) (PGE(2) ), which is the key mediator of fever. The direct role of lipocalin-2 in fever was examined in LPS-challenged lipocalin-2 knockout mice. In both male and female mice, normal fever responses were observed at near-thermoneutral conditions (29-30 °C) but when recorded at normal room temperature (19-20 °C), the body temperature of lipocalin-2 knockout female mice displayed an attenuated fever response compared to their wild-type littermates. This difference was reflected in significantly attenuated mRNA expression of Cox-2 in the brain of lipocalin-2 knockout female mice, but not of male mice, following challenge with peripheral LPS. Our findings suggest that IL-6 influences the expression of lipocalin-2, which in turn may be involved in the control of the formation of Cox-2, and hence central PGE(2) -production. We have thus identified lipocalin-2 as a new factor in the pathway of inflammatory IL-6 signalling. However, the effect of lipocalin-2 on fever is small, being sex-dependent and ambient temperature-specific, and thus lipocalin-2 cannot be considered as a major mediator of the IL-6-dependent fever generating pathway.

Peripheral lipopolysaccharide administration induces cytokine mRNA expression in the viscera and brain of fever-refractory mice lacking microsomal prostaglandin E synthase-1.

We examined the expression of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF) alpha in mice lacking microsomal prostaglandin E synthase-1 (mPGES-1), which neither produce prostaglandin E(2), nor mount a febrile response upon immune challenge. Intraperitoneal lipopolysaccharide (LPS) injection resulted in a strongly induced expression of all three cytokines in the brain and viscera, similar to wild-type animals. Several brain regions additionally showed modest induction of receptors for these cytokines in both genotypes. Telemetric recordings of body temperature showed that the mPGES-1 deficient mice remained afebrile upon LPS challenge, in contrast to the prominent fever displayed by the wild-type mice. These data demonstrate that LPS-induced cytokine expression occurs independently of prostaglandin E(2), and imply that endogenously expressed IL-1beta, IL-6, and TNFalpha are not pyrogenic per se, supporting the role of prostaglandin E(2) as the final and obligatory mediator of LPS-induced fever.

Li-decorated metal-organic framework 5: a route to achieving a suitable hydrogen storage medium.

A significant improvement in molecular hydrogen uptake properties is revealed by our ab initio calculations for Li-decorated metal-organic framework 5. We have found that two Li atoms are strongly adsorbed on the surfaces of the six-carbon rings, one on each side, carrying a charge of +0.9e per Li atom. Each Li can cluster three H(2) molecules around itself with a binding energy of 12 kJ (mol H(2))(-1). Furthermore, we show from ab initio molecular dynamics simulations with a hydrogen loading of 18 H(2) per formula unit that a hydrogen uptake of 2.9 wt % at 200 K and 2.0 wt % at 300 K is achievable. To our knowledge, this is the highest hydrogen storage capacity reported for metal-organic framework 5 under such thermodynamic conditions.

Lipopolysaccharide induces preproenkephalin transcription in hypophysiotropic neurons of the rat paraventricular hypothalamic nucleus suggesting a neuroendocrine role for enkephalins during immune stress.

Opioids have impact on stress responses and possess immune modulatory functions. We have previously shown that immune stress elevates the levels of preproenkephalin transcript in a variety of autonomic structures in the rat brain, including the paraventricular hypothalamic nucleus. By using in situ hybridization with an intronic probe recognizing the preproenkephalin heteronuclear RNA combined with retrograde tract tracing, we examined the efferent target of the enkephalinergic neurons in the paraventricular hypothalamic nucleus that display induced transcriptional activity during immune challenge. Rats were first given i.p. injections of the tracer substance Fluoro-Gold, which following this route of administration is taken up only by nerve terminals residing outside the blood-brain barrier, and were then given an i.v. injection of lipopolysaccharide. Neuronal cell bodies retrogradely labeled with Fluoro-Gold were detected by immunohistochemistry, and-using a dual-labeling approach-the same cells were then examined for their expression of preproenkephalin heteronuclear RNA. We found that over 90% of the neurons that expressed preproenkephalin heteronuclear RNA also contained Fluoro-Gold. In addition, approximately 40% of the neurons expressing preproenkephalin heteronuclear RNA co-expressed mRNA for corticotropin-releasing hormone, the main adrenocorticotropic hormone secretagogue. These data show that the paraventricular hypothalamic neurons that display induced preproenkephalin transcription following immune challenge are almost exclusively hypophysiotropic neurons, indicating a role for enkephalin in the hypothalamic control of hormone release during infectious and inflammatory conditions.

Expression of melanocortin-4 receptor by rat parabrachial neurons responsive to immune and aversive stimuli.

The pontine parabrachial nucleus is a major relay area for visceral and other interoceptive information, and has been implicated in mechanisms underlying anorexia and food aversion during disease. Thus, physiological studies have shown that peripheral immune stimuli, as well as the administration of aversive substances such as lithium chloride, evoke a prominent Fos-expression in the lateral parabrachial nucleus and behavioral experiments have demonstrated that this structure is critical for the acquisition of conditioned taste aversion. The present study examined in rats the relationship between parabrachial neurons activated by systemic administration of bacterial cell-wall lipopolysaccharide or lithium chloride and the melanocortin system, a major regulator of feeding and energy homeostasis that also has been implicated in aversive behavior. Dual-labeling in situ hybridization showed melanocortin-4 receptor expression on neurons in the external lateral parabrachial subnucleus that displayed lipopolysaccharide- or lithium chloride-induced expression of c-fos mRNA. Melanocortin-4 receptor mRNA was also co-expressed with mRNA for calcitonin gene-related peptide in this subnucleus. Taken together with previous observations showing that calcitonin gene-related peptide expressing neurons in the external lateral parabrachial subnucleus are activated by peripheral immune challenge, that lipopolysaccharide-activated external lateral parabrachial subnucleus neurons project to the amygdala, and that the amygdala-projecting neurons in the external lateral parabrachial subnucleus are calcitonin gene-related peptide-positive, the present findings suggest the presence of a melanocortin-regulated calcitonin gene-related peptide-positive pathway from the external lateral parabrachial subnucleus to the amygdala that relays information of importance to forebrain responses to certain aspects of sickness behavior. These observations may thus help explain how melanocortins can reduce feeding and influence conditioned taste aversion during inflammation and other disease conditions.

Defective reproductive organ morphology and function in domestic rooster embryonically exposed to o,p'-DDT or ethynylestradiol.

Environmental pollutants with estrogenic activity have a potential to disrupt estrogen-dependent developmental processes. The objective of this study was to investigate if embryonic exposure to the environmental estrogens o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane; 37, 75, 150 or 300 microg/g egg) and EE2 (17alpha-ethynyl estradiol; 60 ng/g egg) affects the reproductive system in domestic roosters. Following egg injection on Embryonic Day 4, the newly hatched chicks were sexed by cloacal inspection. A skewed phenotypic sex ratio with overrepresentation of chicks deemed as females was observed in the groups exposed to the three highest doses of o,p'-DDT but not in the EE2-exposed group. Normal sex ratios were observed in all groups at adulthood. However, a cloacal deformation seemed to remain in the adult roosters, causing an abnormal semen flow upon semen collection. Semen yield was significantly reduced in both o,p'-DDT-exposed and EE2- exposed birds, whereas semen quality was unaffected. When killed, deformations of the left testis were found in all treatment groups. Image analysis revealed a reduced seminiferous tubular area in the roosters exposed to the two highest doses of o,p'-DDT. Embryonic exposure to o,p'-DDT caused decreased comb weight and right-spur diameter, while EE2 only affected right-spur diameter. In conclusion, this study shows that embryonic exposure to estrogenic compounds can induce permanent effects in male birds. The effects of the two studied compounds were partly similar but o,p'-DDT also induced alterations not seen in the EE2-treated birds.

Distribution of growth hormone receptor mRNA in the brain stem and spinal cord of the rat.

By using in situ hybridization histochemistry the distribution of growth hormone (GH) receptor mRNA was examined in the rat brain stem and spinal cord. Dense labeling was seen in the arcuate nucleus of the hypothalamus, as reported previously, but also in several other areas, including the locus coeruleus, the area postrema, and the commissural part of the nucleus of the solitary tract. Other labeled structures included the superior lateral parabrachial nucleus, the facial, hypoglossal and trigeminal motor nuclei, the nucleus incertus, the dorsal tegmental nucleus, the dorsal raphe nucleus, the nucleus of the trapezoid body, and the superficial layers of the dorsal horn of the spinal cord. These findings provide support for a direct action of GH on brain regions involved in various aspects of homeostatic control. Thus, the distribution of GH receptor mRNA to visceral sensory and motor structures is consonant with a role of GH in the regulation of food intake and energy homeostasis. Its presence in the superficial dorsal horn of the spinal cord indicates a role for GH in the initial processing of fine afferent input, and may help explain the beneficial effects of GH replacement in certain unclear pain conditions.

Embryonic exposure to oestrogen causes eggshell thinning and altered shell gland carbonic anhydrase expression in the domestic hen.

Eggshell thinning among wild birds has been an environmental concern for almost half a century. Although the mechanisms for contaminant-induced eggshell thinning are not fully understood, it is generally conceived to originate from exposure of the laying adult female. Here we show that eggshell thinning in the domestic hen is induced by embryonic exposure to the synthetic oestrogen ethynyloestradiol. Previously we reported that exposure of quail embryos to ethynyloestradiol caused histological changes and disrupted localization of carbonic anhydrase in the shell gland in the adult birds, implying a functional disturbance in the shell gland. The objective of this study was to examine whether in ovo exposure to ethynyloestradiol can affect eggshell formation and quality in the domestic hen. When examined at 32 weeks of age, hens exposed to ethynyloestradiol in ovo (20 ng/g egg) produced eggs with thinner eggshells and reduced strength (measured as resistance to deformation) compared with the controls. These changes remained 14 weeks later, confirming a persistent lesion. Ethynyloestradiol also caused a decrease in the number of shell gland capillaries and in the frequency of shell gland capillaries with carbonic anhydrase activity. These data suggested that a disrupted carbonic anhydrase expression was involved in the mechanism for the oestrogen-induced eggshell thinning found in this study. The results support our hypothesis that eggshell thinning in avian wildlife can result from a structural and functional malformation in the shell gland, induced by xeno-oestrogen exposure during embryonic development.

Workshop 8 (synthesis): managing urban development and industrial growth from a drainage basin perspective.

Urban development and industrial growth give rise to the two problems of insidious and growing pollution, and an over-exploitation of water resources. To solve these problems, various solutions have been promoted, such as multi-stakeholder dialogues, establishment of river basin committees, strong regulation, compensation for water-exporting basins and convincing politicians and the public of the need for change.

EP3 and EP4 receptor mRNA expression in peptidergic cell groups of the rat parabrachial nucleus.

This study examines the distribution of prostaglandin E2 receptors of subtype EP3 and EP4 among brain stem parabrachial neurons that were characterized with respect to their neuropeptide expression. By using a dual-labeling in situ hybridization method, we show that preprodynorphin mRNA expressing neurons in the dorsal and central lateral subnuclei express EP3 receptor mRNA. Such receptors are also expressed in preproenkephalin, calcitonin gene related peptide and preprotachykinin mRNA positive neurons in the external lateral subnucleus, whereas preprodynorphin mRNA expressing neurons in this subnucleus are EP receptor negative. In addition, EP3 receptor expression is seen among some enkephalinergic neurons in the Kölliker-Fuse nucleus. Neurons in the central part of the cholecystokininergic population in the regions of the superior lateral subnucleus express EP4 receptor mRNA, whereas those located more peripherally express EP3 receptors. Taken together with previous findings showing that discrete peptidergic cell groups mediate nociceptive and/or visceral afferent information to distinct brain stem and forebrain regions, the present results suggest that the processing of this information in the parabrachial nucleus is influenced by prostaglandin E2. Recent work has shown that prostaglandin E2 is released into the brain following peripheral immune challenge; hence, the parabrachial nucleus may be a region where humoral signaling of peripheral inflammatory events may interact with neuronal signaling elicited by the same peripheral processes.

Management of acute cholecystitis in the laparoscopic era: results of a prospective, randomized clinical trial.

The aim of this prospective, randomized study was to determine whether laparoscopic cholecystectomy should be performed as an early or a delayed operation in patients with acute cholecystitis. After diagnostic workup, patients were randomized to one of two groups: (1) early laparoscopic cholecystectomy (i.e., within 7 days after onset of symptoms) or (2) initial conservative treatment followed by delayed laparoscopic cholecystectomy 6 to 8 weeks later. Seventy-four patients were placed in the early-operation group, and 71 patients were assigned to the delayed-operation strategy. There was no significant difference in conversion rates (early 31% vs. delayed 29%), operating times (early 98 [range 30 to 355] minutes vs. delayed 100 [45 to 280] minutes), or complications. Failure with the conservative treatment strategy was noted in 26% of these patients. The total hospital stay was significantly shorter in the early group (5 [range 3 to 63] days) vs. the delayed group (8 [range 4 to 50] days; P<0.05). Despite a high conversion rate, early laparoscopic cholecystectomy offered significant advantages in the management of acute cholecystitis compared to a conservative strategy. The greatest advantage was a reduced total hospital stay.

Workshop 2 (synthesis): water pollution abatement within the industrial sector.

This workshop aimed at demonstrating and discussing how effective abatement of water pollution can be achieved through introducing cleaner technologies, recycling and reuse of water, and implementing new public policy measures.

Synaptology of trigemino- and spinothalamic lamina I terminations in the posterior ventral medial nucleus of the macaque.

We used the electron microscope to examine lamina I trigemino- and spinothalamic (TSTT) terminations in the posterior part of the ventral medial nucleus (VMpo) of the macaque thalamus. Lamina I terminations were identified by anterograde labeling with biotinylated dextran, and 109 boutons on 38 terminal fibers were closely studied in series of ultrathin sections. Five unlabeled terminal boutons of similar appearance were also examined in detail. Three-dimensional, volume-rendered computer models were reconstructed from complete series of serial sections for 29 boutons on 10 labeled terminal fibers and one unlabeled terminal fiber. In addition, postembedding immunogold staining for GABA was obtained in alternate sections through 23 boutons. Lamina I TSTT terminations in VMpo generally have several large boutons (mean length = 2.16 microm, mean width = 1.29 microm) that are densely packed with vesicles and make asymmetric synaptic contacts on low-order dendrites of VMpo neurons (mean diameter 1.45 microm). They are closely associated with GABAergic presynaptic dendrites (PSDs), and nearly all form classic triadic arrangements (28 of 29 reconstructed boutons). Consecutive boutons on individual terminal fibers make multiple contacts with a single postsynaptic dendrite and can show evidence of progressive complexity. Dendritic appendages that enwrap and invaginate the terminal bouton constitute additional anatomic evidence for secure, high-fidelity synaptic transfer. These observations provide direct ultrastructural evidence supporting the hypothesis that VMpo is a lamina I TSTT thalamocortical relay nucleus in primates that subserves pain, temperature, itch, and other sensations related to the physiological condition of the body.

Association of spinothalamic lamina I neurons and their ascending axons with calbindin-immunoreactivity in monkey and human.

The calbindin-immunoreactivity of spinothalamic (STT) lamina I neurons and their ascending axons was examined in two experiments. In the first experiment, lamina I STT neurons in macaque monkeys were double-labeled for calbindin and for retrogradely transported WGA*HRP following large (n=2) or small (n=1) injections that included the posterior thalamus. Most, but not all (78%) of the contralateral retrogradely labeled lamina I STT cells were positive for calbindin. Calbindin-immunoreactivity was not selectively associated with any particular anatomical type of lamina I STT cell; 82% of the fusiform cells, 78% of the pyramidal cells and 67% of the multipolar cells were double-labeled. In the second experiment, oblique transverse sections from upper cervical spinal segments of three macaque monkeys, one squirrel monkey and five humans were stained for calbindin-immunoreactivity. In each case, a distinct bundle of fibers was densely stained in the middle of the lateral funiculus. This matches the location of anterogradely labeled ascending lamina I axons observed in prior work in cats and monkeys, and it matches the location of the classically described 'lateral spinothalamic tract' in humans. This bundle had variable shape across cases, an observation that might have clinical significance. These findings support the view that lamina I STT neurons are involved in spinal cordotomies that reduce pain, temperature and itch sensations.

Activation of prostanoid EP(3) and EP(4) receptor mRNA-expressing neurons in the rat parabrachial nucleus by intravenous injection of bacterial wall lipopolysaccharide.

Systemic inflammation activates central autonomic circuits, such as neurons in the pontine parabrachial nucleus. This activation may be the result of afferent signaling through the vagus nerve, but it may also depend on central prostaglandin-mediated mechanisms. Recently, we have shown that neurons in the parts of the parabrachial nucleus that are activated by immune challenge express prostaglandin receptors of the EP(3) and EP(4) subtypes, but it remains to be determined if the prostaglandin receptor-expressing neurons are identical to those that respond to immune stimuli. In the present study, bacterial wall lipopolysaccharide was injected intravenously in adult male rats and the expression of c-fos mRNA and of EP(3) and EP(4) receptor mRNA was examined with complementary RNA probes labeled with digoxigenin and radioisotopes, respectively. Large numbers of neurons in the external lateral parabrachial subnucleus, a major target of vagal-solitary tract efferents, expressed c-fos mRNA. Quantitative analysis showed that about 60% (range 40%-79%) of these neurons also expressed EP(3) receptor mRNA. Conversely, slightly more than 50% (range 48%-63%) of the EP(3) receptor-expressing neurons in the same subnucleus coexpressed c-fos mRNA. In contrast, few EP(4) receptor-expressing neurons were c-fos positive, with the exception of a small population located in the superior lateral and dorsal lateral subnuclei. These findings show that immune challenge activates central autonomic neurons that could be the target of centrally produced prostaglandin E(2), suggesting that synaptic signaling and paracrine mechanisms may interact on these neurons.

Preproenkephalin mRNA expression in rat parabrachial neurons: relation to cells activated by systemic immune challenge.

By using a dual-labeling immunohistochemical/in situ hybridization technique we examined if enkephalin-expressing neurons in the pontine parabrachial nucleus, a major brain stem relay for ascending visceral and homeostatic information, were activated by systemic immune challenge. While rats subjected to intravenous injection of bacterial wall lipopolysaccharide expressed dense labeling for the immediate-early gene product FOS in parts of the parabrachial nucleus that also demonstrated dense preproenkephalin expression, only a small proportion of the enkephalin-positive neurons were FOS-positive. These data indicate that enkephalins, although implicated in a variety of autonomic responses, are not primarily involved in the transmission of immune-related information from the parabrachial nucleus to its different forebrain and brain stem targets.

Feeding-related immune responsive brain stem neurons: association with CGRP.

Using dual-labeling in situ hybridization histochemistry, the neurotransmitter expression of immune-responsive neurons in the pontine parabrachial nucleus, a major relay for interoceptive information, was investigated. Intravenous injection of bacterial wall lipopolysaccharide resulted in dense c-fos mRNA expression in the external lateral parabrachial nucleus, and a majority of the c-fos expressing cells also expressed calcitonin gene-related peptide (CGRP) mRNA. In contrast CGRP-positive cells in the adjoining external medial subnucleus were c-fos negative. Taken together with previous hodological and behavioral studies, these data suggest that CGRPergic parabrachial neurons may mediate lipopolysaccharide-induced anorexia by means of their projection to central nucleus of the amygdala.