The CNS-penetrant soluble guanylate cyclase stimulator CYR119 attenuates markers of inflammation in the central nervous system.

BACKGROUND: Inflammation in the central nervous system (CNS) is observed in many neurological disorders. Nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling plays an essential role in modulating neuroinflammation. CYR119 is a CNS-penetrant sGC stimulator that amplifies endogenous NO-sGC-cGMP signaling. We evaluated target engagement and the effects of CYR119 on markers of neuroinflammation in vitro in mouse microglial cells and in vivo in quinolinic acid (QA)-induced and high-fat diet-induced rodent neuroinflammation models. METHODS: Target engagement was verified in human embryonic kidney (HEK) cells, rat primary neurons, mouse SIM-A9 cells, and in rats by measuring changes in cGMP and downstream targets of sGC signaling [phosphorylated vasodilator-stimulated phosphoprotein (pVASP), phosphorylated cAMP-response element binding (pCREB)]. In SIM-A9 cells stimulated with lipopolysaccharides (LPS), markers of inflammation were measured when cells were treated with or without CYR119. In rats, microinjections of QA and vehicle were administered into the right and left hemispheres of striatum, respectively, and then rats were dosed daily with either CYR119 (10 mg/kg) or vehicle for 7 days. The activation of microglia [ionized calcium binding adaptor molecule 1 (Iba1)] and astrocytes [glial fibrillary acidic protein (GFAP)] was measured by immunohistochemistry. Diet-induced obese (DIO) mice were treated daily with CYR119 (10 mg/kg) for 6 weeks, after which inflammatory genetic markers were analyzed in the prefrontal cortex. RESULTS: In vitro, CYR119 synergized with exogenous NO to increase the production of cGMP in HEK cells and in primary rat neuronal cell cultures. In primary neurons, CYR119 stimulated sGC, resulting in accumulation of cGMP and phosphorylation of CREB, likely through the activation of protein kinase G (PKG). CYR119 attenuated LPS-induced elevation of interleukin 6 (IL-6) and tumor necrosis factor (TNF) in mouse microglial cells. Following oral dosing in rats, CYR119 crossed the blood-brain barrier (BBB) and stimulated an increase in cGMP levels in the cerebral spinal fluid (CSF). In addition, levels of proinflammatory markers associated with QA administration or high-fat diet feeding were lower in rodents treated with CYR119 than in those treated with vehicle. CONCLUSIONS: These data suggest that sGC stimulation could provide neuroprotective effects by attenuating inflammatory responses in nonclinical models of neuroinflammation.

Validation of Poly(Propylene Imine) Glycodendrimers Towards Their Anti-prion Conversion Efficiency.

Prion diseases, such as the sporadic Creutzfeldt-Jakob disease (sCJD), are a class of fatal neurodegenerative disorders. Currently, there is no efficient treatment or therapy available. Hence, the search for molecules that may inhibit the conversion of the cellular prion protein (PrPC) into its pathological counterpart PrPScrapie (PrPSc) is of great urgency. Here, we report the generation- and dose-dependent biological action of dense-shell poly(propylene imine) (PPI) glycodendrimers by using scrapie-infected neuroblastoma (ScN2a) cells and the real-time quaking-induced conversion assay (RT-QuIC) for validation of anti-prion efficiencies. Whereas the 2nd and 3rd generation of PPI glycodendrimers exhibited anti-prion conversion efficiency in ScN2a cells validated by RT-QuIC analysis, we observed that the 4th generation of glycodendrimers had shown no significant effect. Translational RT-QuIC studies conducted with human prions derived from sCJD patients indicated an anti-prion conversion effect (not on PrPRes degradation) of PPI glycodendrimers against human prions with the highest inhibitory activity of the 4th generation of PPI glycodendrimers towards prion aggregation compared to the 2nd and 3rd generation. In conclusion, our study highlights the potential of PPI glycodendrimers as therapeutic compounds due to their anti-conversion activity on human prions in a PrPSc strain depending manner.

YKL-40 in the brain and cerebrospinal fluid of neurodegenerative dementias.

BACKGROUND: YKL-40 (also known as Chitinase 3-like 1) is a glycoprotein produced by inflammatory, cancer and stem cells. Its physiological role is not completely understood but YKL-40 is elevated in the brain and cerebrospinal fluid (CSF) in several neurological and neurodegenerative diseases associated with inflammatory processes. Yet the precise characterization of YKL-40 in dementia cases is missing. METHODS: In the present study, we comparatively analysed YKL-40 levels in the brain and CSF samples from neurodegenerative dementias of different aetiologies characterized by the presence of cortical pathology and disease-specific neuroinflammatory signatures. RESULTS: YKL-40 was normally expressed in fibrillar astrocytes in the white matter. Additionally YKL-40 was highly and widely expressed in reactive protoplasmic cortical and perivascular astrocytes, and fibrillar astrocytes in sporadic Creutzfeldt-Jakob disease (sCJD). Elevated YKL-40 levels were also detected in Alzheimer's disease (AD) but not in dementia with Lewy bodies (DLB). In AD, YKL-40-positive astrocytes were commonly found in clusters, often around ß-amyloid plaques, and surrounding vessels with ß-amyloid angiopathy; they were also distributed randomly in the cerebral cortex and white matter. YKL-40 overexpression appeared as a pre-clinical event as demonstrated in experimental models of prion diseases and AD pathology. CSF YKL-40 levels were measured in a cohort of 288 individuals, including neurological controls (NC) and patients diagnosed with different types of dementia. Compared to NC, increased YKL-40 levels were detected in sCJD (p < 0.001, AUC = 0.92) and AD (p < 0.001, AUC = 0.77) but not in vascular dementia (VaD) (p > 0.05, AUC = 0.71) or in DLB/Parkinson's disease dementia (PDD) (p > 0.05, AUC = 0.70). Further, two independent patient cohorts were used to validate the increased CSF YKL-40 levels in sCJD. Additionally, increased YKL-40 levels were found in genetic prion diseases associated with the PRNP-D178N (Fatal Familial Insomnia) and PRNP-E200K mutations. CONCLUSIONS: Our results unequivocally demonstrate that in neurodegenerative dementias, YKL-40 is a disease-specific marker of neuroinflammation showing its highest levels in prion diseases. Therefore, YKL-40 quantification might have a potential for application in the evaluation of therapeutic intervention in dementias with a neuroinflammatory component.

Applications of the real-time quaking-induced conversion assay in diagnosis, prion strain-typing, drug pre-screening and other amyloidopathies.

INTRODUCTION: The development of in vitro protein misfolding amplification assays for the detection and analysis of abnormally folded proteins, such as proteinase K resistant prion protein (PrPres) was a major innovation in the prion field. In prion diseases, these types of assays imitate the pathological conversion of the cellular PrP (PrPC) into a proteinase resistant associated conformer or amyloid, called PrPres. Areas covered: The most prominent protein misfolding amplification assays are the protein misfolding cyclic amplification (PMCA), which is based on sonication and the real-time quaking-induced conversion (RT-QuIC) technique based on shaking. The more recently established RT-QuIC is fully automatic and enables the monitoring of misfolded protein aggregates in real-time by using a fluorescent dye. Expert commentary: RT-QuIC is a very robust and highly reproducible test system which is applicable in diagnosis, prion strain-typing, drug pre-screening and other amyloidopathies.

Edema hemorrágico agudo da infância: uma vasculite com bom prognóstico / Acute hemorrhagic edema of infancy: a vasculitis with good prognosis

OBJETIVOS: Relatar dois casos de edema hemorrágico agudo da infância, que consiste numa vasculite rara, caracterizada por lesões cutâneas purpúricas e edema periférico, sem envolvimento sistêmico (excetuando- se a febre), que tem início súbito, curso benigno e evolui espontaneamente para a cura. DESCRIÇÃO DOS CASOS: Os autores apresentam dois casos de crianças do sexo masculino, com 11 e 12 meses de idade, que foram atendidos no serviço de urgência por febre e lesões purpúricas exuberantes de agravamento progressivo. O diagnóstico de edema hemorrágico agudo da infância foi feito por exclusão e sustentado pelo fato de que, apesar da impressionante apresentação cutânea, ambos os lactentes apresentavam um ótimo estado geral e a tríade clássica desta entidade: febre, edema e lesões purpúricas da face, orelhas e extremidades. Houve regressão total das lesões cutâneas em aproximadamente uma semana, sem sequelas em ambos os casos. CONCLUSÕES: Existem pouco mais de 100 casos publicados mundialmente de edema hemorrágico agudo da infância. A raridade dessa vasculite pode dever-se a um subdiagnóstico ou diagnóstico equivocado de outras vasculites leucocitoclásticas, principalmente a Púrpura de HenochSchönlein. Além desta, existem outras doenças a considerar no diagnóstico diferencial, como meningococemia, doença de Kawasaki e eritema multiforme, que apresentam similaridades, mas ao mesmo tempo características distintas que permitem excluí-las. É fundamental o diagnóstico oportuno do edema hemorrágico agudo da infância, de modo a evitar exames complementares e terapêuticas desnecessárias, além de tranquilizar a família quanto ao bom prognóstico da doença.

AIMS: To report two cases of acute hemorrhagic edema of infancy, a rare vasculitis characterized by purpuric skin lesions and peripheral edema without systemic involvement (excluding fever), which has a sudden onset and an usually benign course, with spontaneous resolution. CASES DESCRIPTION: The authors describe two cases of male infants, 11 and 12 months old, who were admitted to the emergency department with fever and progressively worsening purpuric lesions. The diagnosis of acute hemorrhagic edema of infancy was made by exclusion and sustained by the fact that, despite the impressive skin presentation, both infants had a good general state and presented the classic triad of this entity: fever, peripheral edema, and purpuric lesions on the face, ears and extremities. Both boys presented total regression of the lesions in about one week, without sequelae. CONCLUSIONS: There are just over 100 cases of hemorrhagic edema of infancy reported worldwide. The rarity of this vasculitis may be due to underdiagnosis or mistaken diagnosis of other leukocytoclastic vasculitis, mostly Henoch-Schönlein purpura. In addition to this, there are other conditions to consider in the differential diagnosis, such as meningococcemia, Kawasaki disease and erythema multiforme, which have similarities, but at the same time distinctive features that allow to exclude them. Timely diagnosis of acute hemorrhagic edema of childhood is crucial to avoid unnecessary therapies and supplementary tests, as well as to reassure the family about the good prognosis of the disease.

Palhaços de hospital como estratégia de amenização da experiência de hospitalização infantil / Hospital clowns as a strategy for the mitigation of childhood hospitalization experience / Payasos del hospital como estrategia de mitigación de la experiencia de hospitalización infantil

A hospitalização pediátrica pode representar um acontecimento marcante para a criança e sua família, dado o desenraizamento de seus contextos e rotinas, e a imersão num ambiente estranho e ameaçador. Buscando minimizar o impacto dessa experiência, várias abordagens lúdicas têm surgido em pediatria, nomeadamente a intervenção de palhaços. Assumindo que, mesmo hospitalizada, existe, na criança, uma essência que deseja brincar, a intervenção do palhaço tem como propósito o seu resgate. Neste artigo, apresentam-se as expetativas de profissionais pediátricos (n=34) a respeito das (des)vantagens da presença dos palhaços junto dessas crianças/adolescentes, antes mesmo da sua intervenção. Os dados revelaram uma ampla abertura à presença desses artistas, apontando-os como potenciais amenizadores do impacto emocional da internação e dos tratamentos, e seu contributo para a humanização dos cuidados e desmistificação dos profissionais de saúde. Como desvantagens, referiram o medo do palhaço ou a percepção, entre adolescentes, da sua presença como uma infantilidade...

Pediatric hospitalization may represent a significant event in the life of children and their families due to the uprooting of their contexts and routines, and the immersion in a strange and threatening environment. Seeking to minimize the impact of this experience, various ludic approaches emerged in pediatrics, namely the hospital clowns. Assuming that, even in the hospital, the child carries an essence that wants to play, the clowns' mission is to rescue it. This manuscript presents the staff's expectations regarding the (dis)advantages of clowning in their unit (n=34), a month before starting intervention. Results reveal an expressive opening regarding the clowns' presence, pointing them as potential buffers of the emotional impact of hospitalization and treatments on children, and contributing to the humanization of care and demystification of health staff. Regarding setbacks, participants mentioned the children's fear of clowns and the perception - by adolescents - of their presence as childish...

La hospitalización pediátrica puede representar un evento clave para los niños y sus familias, dado el desarraigo de sus contextos y rutinas, y la inmersión en un ambiente extraño y amenazante. Intentando minimizar el impacto de esta experiencia, diversos enfoques lúdicos han surgido en pediatría, incluyendo los payasos. Estas suponen que, aún hospitalizado, existe una esencia en el niño que quiere jugar. La misión del payaso es rescatarla. En este artículo se presentan las expectativas de 34 profesionales pediátricos en relación con las (des)ventajas de la presencia de payasos, un mes antes de iniciar su intervención. Los datos revelaron su amplia apertura a la presencia de payasos, señalándolos como potencialmente disminuyendo el impacto emocional de la hospitalización, del tratamiento, contribuyendo además a la humanización de los cuidados y desmitificación de los profesionales. Como desventajas, han mencionado el temor del payaso o la percepción, entre adolescentes, de su presencia como infantil...

Motor protein-dependent transport of AMPA receptors into spines during long-term potentiation.

The regulated trafficking of neurotransmitter receptors at synapses is critical for synaptic function and plasticity. However, the molecular machinery that controls active transport of receptors into synapses is largely unknown. We found that, in rat hippocampus, the insertion of AMPA receptors (AMPARs) into spines during synaptic plasticity requires a specific motor protein, which we identified as myosin Va. We found that myosin Va associates with AMPARs through its cargo binding domain. This interaction was enhanced by active, GTP-bound Rab11, which is also transported by the motor protein. Myosin Va mediated the CaMKII-triggered translocation of GluR1 receptors from the dendritic shaft into spines, but it was not required for constitutive GluR2 trafficking. Accordingly, myosin Va was specifically required for long-term potentiation, but not for basal synaptic transmission. In summary, we identified the specific motor protein and organelle acceptor that catalyze the directional transport of AMPARs into spines during activity-dependent synaptic plasticity.

[The clinical course of Burkholderia cepacia complex bacteria respiratory infection in cystic fibrosis patients].

Bacteria of the Burkholderia cepacia complex (Bcc), a group of nine related species, are opportunistic pathogens in cystic fibrosis (CF) patients, associated with a poor prognosis and patient-to-patient transmissibility. The pulmonary deterioration in Bcc-colonised/ infected patients has a heterogeneous pattern leading, sometimes, to a fulminant development - the cepacia syndrome. To evaluate the relationship between colonisation/ infection by the different Bcc species and the clinical course, the authors carried out a retrospective study of 31 CF patients with Bcc bacteria isolations followed at Hospital de Santa Maria from January 1995 to March 2006. Patients were categorised into two groups: Group I, with intermittent isolations and Group II with chronic isolations. The prevalence of Bcc species was as follows: B. cepacia 57%, B. cenocepacia 43%, B. multivorans 7%, B. stabilis 13%. Three of the patients died of cepacia syndrome. The species B. cepacia and B. stabilis, usually less frequent in CF populations of Europe and America, were isolated in a considerable percentage of the patients examined. No correlation could be established between the species and the clinical outcome. Deteriorated but not stable patients from group II, whose lung function and pulmonary exacerbation caused hospitalisation could be retrospectively analysed, exhibited significant differences in the number of hospitalisations and pulmonary function (FEV1) in the year prior to and the years following Bcc isolation. Based on the available data, it is not currently possible to outline preventive measures through the molecular characterisation of Bcc isolates, reinforcing the notion that the recommended control measures must be followed.

Functional compartmentalization of endosomal trafficking for the synaptic delivery of AMPA receptors during long-term potentiation.

Endosomal membrane trafficking in dendritic spines is important for proper synaptic function and plasticity. However, little is known about the molecular identity and functional compartmentalization of the membrane trafficking machinery operating at the postsynaptic terminal. Here we report that the transport of AMPA-type glutamate receptors into synapses occurs in two discrete steps, and we identify the specific endosomal functions that control this process during long-term potentiation. We found that Rab11-dependent endosomes translocate AMPA receptors from the dendritic shaft into spines. Subsequently, an additional endosomal trafficking step, controlled by Rab8, drives receptor insertion into the synaptic membrane. Separate from this receptor delivery route, we show that Rab4 mediates a constitutive endosomal recycling within the spine. This Rab4-dependent cycling is critical for maintaining spine size but does not influence receptor transport. Therefore, our data reveal a highly compartmentalized endosomal network within the spine and identify the molecular components and functional organization of the membrane organelles that mediate AMPA receptor synaptic delivery during plasticity.

[Pregnancy and smoking: an opportunity to change behaviours]. / Gravidez e tabagismo: uma oportunidade para mudar comportamentos.

INTRODUCTION: Maternal smoking during pregnancy is related not only to perinatal adverse events but also to important postnatal problems. Smoking is very prevalent in women with several socio-demographic factors playing an important role. AIMS: To assess the frequency of smoking as well as mothers' change in behaviours during pregnancy. To identify socio-demographic factors associated to smoking and to assess women's knowledge and sources of information about adverse effects of smoking during pregnancy. METHODS: We started a cohort study between March and October 2003 at the Maternity, Hospital Santa Maria. After an informed consent the mothers in the post natal ward were asked to fill in a questionnaire. RESULTS: Four hundred and seventy five mothers responded to the questionnaire and among them 30% were smokers before pregnancy. Thirty five percent stopped smoking during pregnancy and those who continued significantly reduced the number of cigarettes per day. Smoking was less prevalent in married women, with higher educational level and with stable employment (statistically significant correlations). Forty five percent of the mothers were misinformed or had no information about the adverse effects of smoking during pregnancy. Their main source of information were the media. DISCUSSION: Smoking prevalence was high (30%) among the respondents but the pregnancy lowered the numbers of cigarettes per day. The percentage of misinformed mothers concerned about the adverse effects of smoking during pregnancy was high (45%), which also showed that doctors' role as a source of information was surpassed by the media. The study also brought to light the need for the health professionals to improve their competences in their role on prevention of smoking during pregnancy and the postnatal period.

Exceptionally high representation of Burkholderia cepacia among B. cepacia complex isolates recovered from the major Portuguese cystic fibrosis center.

Burkholderia cepacia, a species found infrequently in cystic fibrosis (CF), was isolated from 85% of patients infected with bacteria of the B. cepacia complex that visited the major Portuguese CF center, in Lisbon, during 2003 to 2005. A detailed molecular analysis revealed that this was mainly due to two B. cepacia clones. These clones were indistinguishable from two strains isolated from intrinsically contaminated nonsterile saline solutions for nasal application, detected during routine market surveillance by the Portuguese Medicines and Health Products Authority.