Interleukin-1ß signaling in fenestrated capillaries is sufficient to trigger sickness responses in mice.

BACKGROUND: The physiological and behavioral symptoms of sickness, including fever, anorexia, behavioral depression, and weight loss can be both beneficial and detrimental. These sickness responses are triggered by pro-inflammatory cytokines acting on cells within the brain. Previous research demonstrates that the febrile response to peripheral insults depends upon prostaglandin production by vascular endothelial cells, but the mechanisms and specific cell type(s) responsible for other sickness responses remain unknown. The purpose of the present study was to identify which cells within the brain are required for sickness responses triggered by central nervous system inflammation. METHODS: Intracerebroventricular (ICV) administration of 10 ng of the potent pro-inflammatory cytokine interleukin-1ß (IL-1ß) was used as an experimental model of central nervous system cytokine production. We examined which cells respond to IL-1ß in vivo via fluorescent immunohistochemistry. Using multiple transgenic mouse lines expressing Cre recombinase under the control of cell-specific promoters, we eliminated IL-1ß signaling from different populations of cells. Food consumption, body weight, movement, and temperature were recorded in adult male mice and analyzed by two-factor ANOVA to determine where IL-1ß signaling is essential for sickness responses. RESULTS: Endothelial cells, microglia, ependymal cells, and astrocytes exhibit nuclear translocation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in response to IL-1ß. Interfering with IL-1ß signaling in microglia, endothelial cells within the parenchyma of the brain, or both did not affect sickness responses. Only mice that lacked IL-1ß signaling in all endothelium including fenestrated capillaries lacked sickness responses. CONCLUSIONS: These experiments show that IL-1ß-induced sickness responses depend on intact IL-1ß signaling in blood vessels and suggest that fenestrated capillaries act as a critical signaling relay between the immune and nervous systems. TRIAL REGISTRATION: Not applicable.

Amplification and propagation of interleukin-1ß signaling by murine brain endothelial and glial cells.

BACKGROUND: During acute infections and chronic illnesses, the pro-inflammatory cytokine interleukin-1ß (IL-1ß) acts within the brain to elicit metabolic derangements and sickness behaviors. It is unknown which cells in the brain are the proximal targets for IL-1ß with respect to the generation of these illness responses. We performed a series of in vitro experiments to (1) investigate which brain cell populations exhibit inflammatory responses to IL-1ß and (2) examine the interactions between different IL-1ß-responsive cell types in various co-culture combinations. METHODS: We treated primary cultures of murine brain microvessel endothelial cells (BMEC), astrocytes, and microglia with PBS or IL-1ß, and then performed qPCR to measure inflammatory gene expression or immunocytochemistry to evaluate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. To evaluate whether astrocytes and/or BMEC propagate inflammatory signals to microglia, we exposed microglia to astrocyte-conditioned media and co-cultured endothelial cells and glia in transwells. Treatment groups were compared by Student's t tests or by ANOVA followed by Bonferroni-corrected t tests. RESULTS: IL-1ß increased inflammatory gene expression and NF-κB activation in primary murine-mixed glia, enriched astrocyte, and BMEC cultures. Although IL-1ß elicited minimal changes in inflammatory gene expression and did not induce the nuclear translocation of NF-κB in isolated microglia, these cells were more robustly activated by IL-1ß when co-cultured with astrocytes and/or BMEC. We observed a polarized endothelial response to IL-1ß, because the application of IL-1ß to the abluminal endothelial surface produced a more complex microglial inflammatory response than that which occurred following luminal IL-1ß exposure. CONCLUSIONS: Inflammatory signals are detected, amplified, and propagated through the CNS via a sequential and reverberating signaling cascade involving communication between brain endothelial cells and glia. We propose that the brain's innate immune response differs depending upon which side of the blood-brain barrier the inflammatory stimulus arises, thus allowing the brain to respond differently to central vs. peripheral inflammatory insults.

The support needs of parents having a child with a chronic kidney disease: a focus group study.

BACKGROUND: Parents of children with a chronic kidney disease (CKD) have a crucial role in the management of their child's disease. The burden on parents is high: they are often exhausted, depressed and experience high levels of stress and a low quality of life, which could have a negative impact on their child's health outcomes. Support aiming at preventing and reducing parental stress is essential. Therefore, it is necessary to have insight in the problems and support needs among these parents. OBJECTIVE: Our aim is to describe parents' support needs regarding the problems they experience in having a child with CKD. METHODS: Five focus group interviews were conducted with parents of children: (i) with hereditary kidney disease, (ii) with nephrotic syndrome, (iii) with chronic kidney failure, (iv) using dialysis and (v) after renal transplantation. The children were treated at a paediatric nephrology unit in one university hospital in the Netherlands. The data were thematically analysed. RESULTS: Twenty-one parents participated in the focus groups. Parents need more information about their child's CKD and treatment options, and managing their own hobbies and work. Furthermore, parents need emotional support from their partner, family, friends, peers and healthcare professionals to help them cope with the disease of their child. Additionally, parents need practical support to hand over their care and support in transport, financial management and regarding their child at school. CONCLUSION: Needs regarding balancing their personal life are seldom prioritized by parents as the child's needs are considered more important. Therefore, it is important that healthcare professionals should not only attend to the abilities of parents concerning their child's disease management, but also focus on the parents' abilities in balancing their responsibilities as a caregiver with their own personal life.

Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling in skeletal muscle.

Cachexia is a wasting condition defined by skeletal muscle atrophy in the setting of systemic inflammation. To explore the site at which inflammatory mediators act to produce atrophy in vivo, we utilized mice with a conditional deletion of the inflammatory adaptor protein myeloid differentiation factor 88 (MyD88). Although whole-body MyD88-knockout (wbMyD88KO) mice resist skeletal muscle atrophy in response to LPS, muscle-specific deletion of MyD88 is not protective. Furthermore, selective reexpression of MyD88 in the muscle of wbMyD88KO mice via electroporation fails to restore atrophy gene induction by LPS. To evaluate the role of glucocorticoids as the inflammation-induced mediator of atrophy in vivo, we generated mice with targeted deletion of the glucocorticoid receptor in muscle (mGRKO mice). Muscle-specific deletion of the glucocorticoid receptor affords a 71% protection against LPS-induced atrophy compared to control animals. Furthermore, mGRKO mice exhibit 77% less skeletal muscle atrophy than control animals in response to tumor growth. These data demonstrate that glucocorticoids are a major determinant of inflammation-induced atrophy in vivo and play a critical role in the pathogenesis of endotoxemic and cancer cachexia.

Relating centromeric topography in fixed human chromosomes to α-satellite DNA and CENP-B distribution.

Despite extensive analyses on the centromere and its associated proteins, detailed studies of centromeric DNA structure have provided limited information about its topography in condensed chromatin. We have developed a method with correlative fluorescence light microscopy and atomic force microscopy that investigates the physical and structural organization of α-satellite DNA sequences in the context of its associated protein, CENP-B, on human metaphase chromosome topography. Comparison of centromeric DNA and protein distribution patterns in fixed homologous chromosomes indicates that CENP-B and α-satellite DNA are distributed distinctly from one another and relative to observed centromeric ridge topography. Our approach facilitates correlated studies of multiple chromatin components comprising higher-order structures of human metaphase chromosomes.

Allele specificity of DNA replication timing in the Angelman/Prader-Willi syndrome imprinted chromosomal region.

DNA replication within chromosome 15q11-q13, a region subject to genomic imprinting, was examined by fluorescence in situ hybridization. Asynchronous replication between homologues was observed in cells from normal individuals and in Prader-Willi (PWS) and Angelman syndrome (AS) patients with chromosome 15 deletions but not in PWS patients with maternal uniparental disomy. Opposite patterns of allele-specific replication timing between homologous loci were observed; paternal early/maternal late at D15S63, D15S10 and the gamma-aminobutyric acid receptor beta 3 subunit gene (GABRB3); and maternal early/paternal late at the more distal gamma-aminobutyric acid receptor alpha 5 subunit gene (GABRA5). At the most distal locus examined, D15S12, both patterns of allele-specific replication timing were detected.

Maternal but not paternal transmission of 15q11-13-linked nondeletion Angelman syndrome leads to phenotypic expression.

Angelman syndrome (AS) may result from either maternally inherited deletions of chromosome 15q11-13 or from paternal uniparental disomy for chromosome 15. This is in contrast to Prader-Willi syndrome (PWS), which is caused by either paternal deletion of this region or maternal disomy for chromosome 15. However, 40% of AS patients inherit an apparently intact copy of chromosome 15 from each parent. We now describe a family in which three sisters have given birth to four AS offspring who have no evidence of deletion or paternal disomy. We show that AS in this family is caused by a mutation in 15q11-13 that results in AS when transmitted from mother to child, but no phenotype when transmitted paternally. These results suggest that the loci responsible for AS and PWS, although closely linked, are distinct.

Multifunctional nanoparticles for dual imaging.

For imaging with different modalities, labels, which provide contrast for all modalities, are required. Colloidal nanoparticles composed out of an inorganic core and a polymer shell offer progress in this direction. Both, the core and the polymer shell, can be synthesized to be fluorescent, magnetic, or radioactive. When different cores are combined with different polymer shells, different types of particles for dual imaging can be obtained, as for example, fluorescent cores with radioactive polymer shells. Properties and perspectives of such nanoparticles for multimodal imaging are discussed.

Inhibition of telomerase limits the growth of human cancer cells.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator.

Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA.

Periventricular heterotopia: an X-linked dominant epilepsy locus causing aberrant cerebral cortical development.

Periventricular heterotopia (PH) involves dramatic malformations of the human cerebral cortex. Here we show that PH is closely linked to markers in distal Xq28 (maximal two-point lod score = 4.77 for F8C at theta = 0; maximal multipoint lod score = 5.37), so that affected females are obligatory mosaics for the mutation; that PH is lethal to at least some affected males; that PH malformations consist of well-differentiated cortical neurons filling the adult subependymal zone; and that individuals with PH are at high risk for epilepsy, though they have no other neurological or external stigmata. The PH gene may represent an important epilepsy susceptibility locus in addition to playing a key role in normal cortical development.

Neuroendocrine plasticity in the anterior pituitary: gonadotropin-releasing hormone-mediated movement in vitro and in vivo.

The secretion of LH is cued by the hypothalamic neuropeptide, GnRH. After delivery to the anterior pituitary gland via the hypothalamic-pituitary portal vasculature, GnRH binds to specific high-affinity receptors on the surface of gonadotrope cells and stimulates synthesis and secretion of the gonadotropins, FSH, and LH. In the current study, GnRH caused acute and dramatic changes in cellular morphology in the gonadotrope-derived alphaT3-1 cell line, which appeared to be mediated by engagement of the actin cytoskeleton; disruption of actin with jasplakinolide abrogated cell movement and GnRH-induced activation of ERK. In live murine pituitary slices infected with an adenovirus-containing Rous sarcoma virus-green fluorescent protein, selected cells responded to GnRH by altering their cellular movements characterized by both formation and extension of cell processes and, surprisingly, spatial repositioning. Consistent with the latter observation, GnRH stimulation increased the migration of dissociated pituitary cells in transwell chambers. Our data using live pituitary slices are a striking example of neuropeptide-evoked movements of cells outside the central nervous system and in a mature peripheral endocrine organ. These findings call for a fundamental change in the current dogma of simple passive diffusion of LH from gonadotropes to capillaries in the pituitary gland.

Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome.

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.

Activation of the Lbc Rho exchange factor proto-oncogene by truncation of an extended C terminus that regulates transformation and targeting.

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted alpha-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the alpha-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential.

A longevity study with enhancer substances (selegiline, BPAP) detected an unknown tumor-manifestation-suppressing regulation in rat brain.

AIMS: First proof to show that (-)-deprenyl/selegiline (DEP), the first selective inhibitor of MAO-B, later identified as the first ß-phenylethylamine (PEA)-derived synthetic catecholaminergic activity enhancer (CAE) substance and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP), the tryptamine-derived presently known most potent, selective, synthetic enhancer substance, are specific markers of unknown enhancer-sensitive brain regulations. MAIN METHODS: Longevity study disclosing the operation of tumor-manifestation-suppressing (TMS) regulation in rat brain. Immonohistochemical identification of a fibromyxosarcoma in rats. Experiments with human medulloblastoma cell lines. Analysis of the mechanism of action of enhancer substances. KEY FINDINGS: Whereas 20/40 saline-treated rats manifested a fibromyxosarcoma, in groups of rats treated with 0.001mg/kg DEP: 15/40 rats; with 0.1mg/kg DEP: 11/40 rats (P<0.01); with 0.0001mg/kg BPAP: 8/40 rats (P<0.001); with 0.05mg/kg BPAP: 7/40 rats (P<0.01) manifested the tumor. Experiments with human medulloblastoma cell lines, HTB-186 (Daoy); UW-228-2, showed that BPAP was devoid of direct cytotoxic effect on tumor cells, and did not alter the direct cytotoxic effectiveness of temozolomide, cisplatin, etoposide, or vincristine. Interaction with distinct sites on vesicular monoamine-transporter-2 (VMAT2) is the main mechanism of action of the enhancer substances which clarifies the highly characteristic bi-modal, bell-shaped concentration-effect curves of DEP and BPAP. SIGNIFICANCE: Considering of the safeness of the enhancer substances and the finding that DEP and BPAP, specific markers of unknown enhancer sensitive brain regulations, detected the operation of an enhancer-sensitive TMS-regulation in rat brain, it seems reasonable to test in humans low dose DEP or BPAP treatment against the spreading of a malignant tumor.

The organisation of paediatric renal care in different European countries: results of the PAC project.

The Paediatric Access Care (PAC) project, organised by the Research Board of EDTNA/ERCA, aimed to study the organisation of paediatric renal care in Europe and to investigate the practice of access care for both haemodialysis (HD) and peritoneal dialysis (PD) paediatric patients. This paper reports on the organisation of paediatric renal care. The majority of paediatric renal care units were located in specific paediatric units of university hospitals. Most of the centres had offered HD, PD and transplantation (Tx) for more than 20 years. Half of nursing staff had qualifications in paediatric and renal nursing. Most of the centres offered an extended multidisciplinary team approach with the family actively involved in the care of the patient. PD and HD were equally used. Automatic Peritoneal Dialysis (APD) was offered as the standard PD treatment in 2 out of 3 centres. The HD schedule mostly utilised was 3 x 4 hours a week. Half of the patients were on the Tx waiting list and one third of registered patients were transplanted in 2004.

Paediatric access care in Europe: results of the Paediatric Access Care (PAC) project.

Part two of the Paediatric Access Care (PAC) project, a research project of EDTNA/ERCA, investigated PAC in HD and PD patients including the policy for the creation and maintenance of access, and the registration of access related complications that occurred during the registration year of 2004. Data were collected from 39 centres of 13 European countries and included 379 paediatric patients. Fatal complications, resulting in terminating the use of the access, were noted in 59 HD and 22 PD patients. Paediatric access care varied considerably between European centres and in many areas consensus or best practice evidence is still lacking. There is a need for recommendations for the paediatric renal nurse, handling access care in the paediatric renal population.

Longevity study with low doses of selegiline/(-)-deprenyl and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP).

AIMS: The first longevity study demonstrating that rats treated with the MAO-B inhibitory dose of (-)-deprenyl (0.25mg/kg) lived significantly longer than their saline-treated peers was published in 1988, and corroborated in many papers. The recent findings that (-)-deprenyl is primarily a PEA-derived synthetic catecholaminergic activity enhancer substance; (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP) is a tryptamine-derived synthetic enhancer substance, initiated our first longevity study on rats with low enhancer doses of (-)-deprenyl and BPAP to test the enhancer effect's role in life extension. MAIN METHODS: We used the shuttle box technique for selecting the optimum doses of (-)-deprenyl and BPAP. (-)-Deprenyl exerts in rats in 0.001mg/kg its 'specific' enhancer effect and in 0.1mg/kg its 'non-specific' enhancer effect. BPAP exerts its 'specific' enhancer effect in 0.0001mg/kg and its 'non-specific' enhancer effect in 0.05mg/kg. Groups of male Wistar rats (N=40) were treated subcutaneously from their 10th week until death, three times weekly, with saline (0.5ml/kg), and the selected doses of (-)-deprenyl or BPAP, respectively. As an indicator of aging we tested the age-related changes in their learning ability. KEY FINDINGS: Rats treated with 0.0001 or 0.05mg/kg BPAP lived significantly longer than their saline treated peers (P<0.02) and BPAP was more potent in extending rats' lifespan than (-)-deprenyl. 18-month-old rats treated with 0.0001mg/kg BPAP were as good learners as 3-month-old saline treated rats. SIGNIFICANCE: The study revealed that the enhancer effect is responsible for life extension.

Live view of gonadotropin-releasing hormone containing neuron migration.

Neurons that synthesize GnRH control the reproductive axis and migrate over long distances and through different environments during development. Prior studies provided strong clues for the types of molecules encountered and movements expected along the migratory route. However, our studies provide the first real-time views of the behavior of GnRH neurons in the context of an in vitro preparation that maintains conditions comparable to those in vivo. The live views provide direct evidence of the changing behavior of GnRH neurons in their different environments, showing that GnRH neurons move with greater frequency and with more changes in direction after they enter the brain. Perturbations of guiding fibers distal to moving GnRH neurons in the nasal compartment influenced movement without detectable changes in the fibers in the immediate vicinity of moving GnRH neurons. This suggests that the use of fibers by GnRH neurons for guidance may entail selective signaling in addition to mechanical guidance. These studies establish a model to evaluate the influences of specific molecules that are important for their migration.

Excited state investigation of a new Ru(II) complex for dual reactivity with low energy light.

The new complex [Ru(tpy)(Me2dppn)(py)](2+) efficiently photodissociates py in CH3CN with Φ500 = 0.053(1) induced by steric bulk from methyl substituents and produces (1)O2 with ΦΔ = 0.69(9) from its long-lived (3)ππ* excited state. The unique excited state processes that result in dual reactivity were investigated using ultrafast transient absorption spectroscopy.