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Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302–332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function., Graphical Abstract, The heterozygous in-frame GAG deletion in the TOR1A gene leads to the movement disorder DYT1 dystonia. Cruz et al. established a CRISPR strategy to selectively disrupt the mutant allele, aiming to restore torsinA function. They used the variant Cas9-VRQR, which recognizes a PAM site created by the GAG deletion.

Given the well-known antioxidant and neuroprotective properties of quercetin, the aim of this work was to evaluate the effects of quercetin stabilized by microencapsulation at two doses (10 mg kg−1 and 100 mg kg−1) on the oxidative/antioxidant status, number and morphological features of ICC, nitrergic neurons and M2-like macrophages in jejunum of diabetic rats. The rats were randomly distributed into six groups: normoglycemic control (N), diabetic control (D) and either normoglycemic or diabetic groups treated with quercetin-loaded microcapsules at a dose of 10 mg kg−1 (NQ10 and DQ10, respectively) or 100 mg kg−1 (NQ100 and DQ100, respectively). After 60 days, the jejunum was collected. Whole mounts were immunostained for Ano1, nNOS and CD206, and oxidative stress levels and total antioxidant capacity of the jejunum were measured. Diabetes led to a loss of ICC and nitrergic neurons, but increased numbers of M2-like macrophages and elevated levels of oxidative stress were seen in diabetic animals. High-dose administration of quercetin (100 mg kg−1) further aggravated the diabetic condition (DQ100) but this treatment resulted in harmful effects on healthy rats (NQ100), pointing to a pro-oxidant activity. However, low-dose administration of quercetin (10 mg kg−1) gave rise to antioxidant and protective effects on ICC, nNOS, macrophages and oxidative/antioxidant status in DQ100, but NQ100 displayed infrequent negative outcomes in normoglycemic animals. Microencapsulation of the quercetin may become promising alternatives to reduce diabetes-induced oxidative stress but antioxidant therapies should be careful used under healthy status to avoid toxic effects.

Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications.

Made available in DSpace on 2021-06-25T10:26:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) A new biomimetic sensor selective to folic acid based on a carbon paste modified with graphene oxide and Fe3O4 nanoparticles coated with molecularly imprinted polymer in the core@shell format (Fe3O4@MIPs) was obtained using the polyol method. The sensing phase was synthesized in a simple way and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Brunauer-Emmett-Teller (BET) surface area. In the binding experiments the selective material showed a high adsorption capacity (Q) of 30.4 mg g−1 for MMIP, while the Q value for MNIP (magnetic non-imprinted polymer) was 15.3 mg g−1 both of them following the Langmuir model to the adsorption procedure. After their efficiency was proven, these materials were used as modifiers in the electrochemical sensor (Fe3O4@MIP-GO/CPE). Under optimized conditions using the square-wave adsorptive voltammetry, the proposed sensor exhibited excellent response with a concentration linear range between 2.5 and 48 μmol L−1 and limit of detection of 0.65 μmol L−1 (S/σ = 3). The advantages obtained with the proposed method were high robustness, selectivity, and low cost being these characteristics due to the MIP; highly sensibility due to the high superficial area being the contribution of the magnetite in the core of material; and experimentally versatile since was possible carry out numerous reproductible analysis only make the renovation of the paste electrode surface by simple polishing. The sensor was applied successfully in pharmaceutical formulation and river water samples with recoveries percentages near 100%. Chemistry Institute São Paulo State University (UNESP) National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM) Chemistry Institute São Paulo State University (UNESP) CAPES: 0674/2018 FAPESP: 2018/09432–4 FAPESP: 2019/00677–7 CNPq: 301728/2019–4 CNPq: 408050/2018–7 CNPq: 465571/2014–0

Microtubule (MT) dynamics are central to eukaryotic cell organization, intracellular transport, division, adhesion, polarization, and migration. Unbalanced MT stability is associated with metastasis and neurodegenerative diseases. We show that altered expression of death-inducer obliterator isoform 3 (DIDO3) restricts cell adhesion and motility. Fibroblasts with a DIDO3 C-terminal delection (DIDO3ΔCT) show i) MT bundling and looping, ii) failure to localize the tubulin plus-end binding protein CLASP2 to the membrane, iii) deregulated tubulin-actin-binding proteins EB1, GAS2L1, and DNM1 downstream of GSK3β, and iv) transcriptionally deregulated cytoskeletal- and non-cytoskeletal-coding genes (Gas2l1, Dnm1, Tubb4a, Obsl1 and Sema7a, Lgals3bp, Cct3, respectively) associated to cell migration, adhesion, immune responses, and tubulin-actin folding. DIDO3ΔCT deletion delocalizes the GSK3β-interacting protein ninein and prevents MT growth from the centrosome. DIDO3 expression in DIDO3ΔCT fibroblasts largely reestablishes the normal phenotype. Furthermore, DIDO3ΔCT B cells do not polarize the centrosome to the immunological synapse, a tubulin-dependent process. DIDO3CT overexpression induces tubulin stabilization and CLASP2 accumulation at the cell membrane. We propose a mechanism by which DIDO3 links transcriptional and posttranslational regulation of MT stability.

Fluoride (F) is largely employed in dentistry, in therapeutic doses, to control caries. However, excessive intake may lead to adverse effects in the body. Since F is absorbed mostly from the gastrointestinal tract (GIT), gastrointestinal symptoms are the first signs following acute F exposure. Nevertheless, little is known about the mechanistic events that lead to these symptoms. Therefore, the present study evaluated changes in the proteomic profile as well as morphological changes in the jejunum and ileum of rats upon acute exposure to F. Male rats received, by gastric gavage, a single dose of F containing 0 (control) or 25 mg/Kg for 30 days. Upon exposure to F, there was a decrease in the thickness of the tunic muscularis for both segments and a decrease in the thickness of the wall only for the ileum. In addition, a decrease in the density of HuC/D-IR neurons and nNOS-IR neurons was found for the jejunum, but for the ileum only nNOS-IR neurons were decreased upon F exposure. Moreover, SP-IR varicosities were increased in both segments, while VIP-IR varicosities were increased in the jejunum and decreased in the ileum. As for the proteomic analysis, the proteins with altered expression were mostly negatively regulated and associated mainly with protein synthesis and energy metabolism. Proteomics also revealed alterations in proteins involved in oxidative/antioxidant defense, apoptosis and as well as in cytoskeletal proteins. Our results, when analyzed together, suggest that the gastrointestinal symptoms found in cases of acute F exposure might be related to the morphological alterations in the gut (decrease in the thickness of the tunica muscularis) that, at the molecular level, can be explained by alterations in the gut vipergic innervation and in proteins that regulate the cytoskeleton.

Considering the antioxidant, neuroprotective, inflammatory and nitric oxide modulatory actions of quercetin, the aim of this study was to test the effect of quercetin administration in drinking water (40 mg/day/rat) on neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), overall population of myenteric neurons (HuC/D) and nitric oxide (NO) levels in the jejunal samples from diabetic rats. Male Wistar rats were distributed into four groups (8 rats per group): euglycemic (E), euglycemic administered with quercetin (E+Q), diabetic (D) and diabetic administered with quercetin (D+Q). Rats were induced to diabetes with streptozotocin (35mg/kg/iv) and, after 120 days, the proximal jejunum were collected and processed for immunohistochemical (VIP, nNOS and HuC/D) and chemiluminescence (quantification of tissue NO levels) techniques. Diabetes mellitus reduced the number of nNOS-IR (immunoreactive) (p 0.05) and HuC/D-IR (p 0.001) neurons, however, promoted an increased morphometric area of nNOS-IR neurons (p 0.001) and VIP-IR varicosities (p0.05). In D+Q group, neuroplasticity effects were observed on HuC/D-IR neurons, accompanied by a reduction of cell body area of neurons nNOS- and VIP-IR varicosities (p 0.05). The NO levels were increased in the E+Q (p 0.05) and D+Q group (p 0.001) compared to the control group. In conclusion, the results showed that quercetin supplementation increased the bioavailability of NO in the jejunum in euglycemic and mitigate the effects of diabetes on nNOS-IR neurons and VIP-IR varicosities in the myenteric plexus of diabetic rats.

Aims The aim of our study was to study the pathological mechanisms induced by the rheumatoid arthritis (RA) on the Enteric Nervous System (ENS). Main methods We evaluated the effect of the chronic arthritis and its treatment with 50 mg/kg quercetin alone (AQ) and combined with 17.5 mg/kg ibuprofen (AIQ) for 60 days on neurons, glial cells and intestinal wall. Other groups were used: control (C), arthritic (A) and arthritic treated with 17.5 mg/kg ibuprofen (AI). After 60 days, the jejunum was removed and processed for immunohistochemical techniques. Immunostainings were performed for HuC/D and S100 (myenteric and submucosal plexuses), and GFAP (only myenteric plexus), while immunolabeling for CD45 and CD20 lymphocytes was performed using cryosections. Western blot was performed for GDNF, S100 and GFAP. Key findings A group yielded a remarkable density decrease of the neurons and glial cells with morphometric changes in the myenteric and submucosal plexuses, reduction of the GDNF expression and GFAP-related parameters (GFAP expression, occupancy area and GFAP-expressing glial cells) and intestinal inflammation and atrophy of the mucosa and intestinal wall. AQ group substantially reversed most of these effects, except for intestinal atrophy of the jejunum. The AI and AIQ groups displayed lower beneficial results than AQ for parameters related to the neurons and glial cells, although AIQ did not prevent the inflammation of the mucosa. Significance The severe chronic rheumatoid arthritis induced severe effects on ENS and mucosa, and quercetin treatment continues to be an important antioxidant supplement preventing the progression of the RA severity.

Background Triple negative breast cancers (TNBC) are enriched with cells bearing stem-like features (CSC), which underlie cancer progression, thus targeting stemness could be an interesting treatment approach. In turn, the epigenetic machinery is crucial for the maintenance of the stemness phenotype. The BET family of epigenetic readers are emerging as novel targets for cancer therapy and have shown preclinical effect in breast cancer. In this work we evaluate the effect of the BET inhibitor (BETi) JQ1 on stemness in TNBC. Methods Transcriptomic, functional annotation and qPCR studies were performed on JQ1-exposed TNBC cells. Results were confirmed on spheroids and spheroid-derived tumours. Limiting dilution assays, matrigel invasion experiments, immunofluorescence staining, and flow cytometry studies were also performed to evaluate the effect of JQ1 on CSC features. For the outcome analysis, the online tool Kaplan-Meier Plotter and an integrated response database were used. Results JQ1 can modify the expression of stemness-related genes incultured TNBC cells. Among these changes, the CD44/CD24 and ALDH1A1 expression, both classical stemness markers, were diminished by JQ1. Using a validated spheroid model, to mimic the intrinsic characteristics of CSCs, we show that JQ1 decreased surface expression of CD44, inhibited self-renewal and invasion, and induced cells arrest in G0/G1, therefore altering the stemness phenotype in TNBC. Four of the identified stemness genes, GJA1, CD24, EPCAM, and SOX9, were found to be associated with worse patient outcome in TNBC. Furthermore, ABCG2 and RUNX2 predicted low response to chemotherapy in TNBC patients. Conclusions In this work we show how BETi modify the stemness landscape in TNBC. Thus, we propose a novel role for JQ1 as a stemness-targeting drug. Loss of the stem cell phenotype via JQ1 treatment could lead to less aggressive and more chemo-sensitive tumours, which would reflect in better patient prognosis. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background Antigen recognition by MHC class I molecules is a key step for the initiation of the immune response. We hypothesized that expression of these molecules could be a marker of immune activated breast cancers. Methods We used data from KM Plotter to develop an exploratory cohort and then utilized information from Cancer Genome Atlas (TCGA) and METABRIC to create two validation cohorts. Raw data were re-processed and analyzed using plyr R and Bioconductor. Definition of molecular subtypes as well as identification of mutations was performed using RNAseq from TCGA. We used NetMHC 4.0 epitope-HLA to predict binding to MHC I molecules. Cox proportional hazards regression was computed to correlate gene expression and survival outcome. Multiple genes were combined into a signature by using their mean expression. Results There was a weak but positive correlation between mutational burden and the expression of most MHC class I molecules. In the exploratory cohort, expression of HLA-A and HLA-B was associated with favorable relapse free survival (RFS) and overall survival (OS) in the basal-like subgroup. This was confirmed in the METABRIC dataset (OS HLA-A, HR: 0.59, CI 0.42-0.82; p = 0.002; HLA-B, HR: 0.60, CI 0.44-0.82; p = 0.001); and in the TCGA dataset (OS HLA-A, HR: 0.34, CI 0.16-0.75; p = 0.005; HLA-B, HR: 0.21, CI 0.01-0.71; p = 0.005). Expression of HLA-A and HLA-B was associated with biomarkers of T cell activation (GZMA, GZMB and PRF1) and improved the predictive capacity of known immunologic signatures (IFN gamma signature, expanded immune gene signature and Cytotoxic T lymphocyte). Several neopeptides expressed in breast cancer were also identified including FUK, SNAPC3, GC, ANO8, DOT1L, HIST1H3F, MYBPH, STX2, FRMD6, CPSF1, or SMTN, among others. Conclusions Expression of HLA-A and B is associated with T cell activation and identifies immune activated, basal-like breast cancers with favorable prognosis. Legal entity responsible for the study The authors. Funding Instituto de Salud Carlos III (PI16/01121), ACEPAIN; Diputacion de Albacete, CIBERONC, CRIS Cancer Foundation, scientific foundation of the AECC, implementation research program of the UCLM Spanish System of Science, Technology and Innovation-Secti (co-funded by the European Commission/FSE funds). Disclosure A. Ocana: Research grant / Funding (institution): Entrechem; Travel / Accommodation / Expenses: Merck. All other authors have declared no conflicts of interest.

Rapid eye movement sleep behavior disorder (RBD) is a parasomnia characterized by the occurrence of intense movements during rapid eye movement (REM) sleep, also named paradoxical sleep. The neuronal dysfunctions at the origin of the loss of atonia in RBD patients are not known. One possibility is that RBD is due to the degeneration of neurons inducing the muscle atonia of REM sleep. Therefore, in our paper we review data on the populations of neurons responsible for the atonia of REM sleep before discussing their potential role in RBD. We first review evidence that motoneurons are tonically hyperpolarized by gamma-aminobutyric acid (GABA) and glycine and phasically excited by glutamate during REM sleep. Then, we review data indicating that the atonia of REM sleep is induced by glycinergic/GABAergic REM-on premotoneurons contained within the raphe magnus and the ventral and alpha gigantocellular reticular nuclei localized in the ventral medullary reticular formation. These neurons are excited during REM sleep by a direct projection from glutamatergic REM-on neurons localized in the pontine sublaterodorsal tegmental nucleus (SLD). From these results, we discuss the possibility that RBD is due to a specific degeneration of descending REM-on glutamatergic neurons localized in the caudal SLD or that of the REM-on GABA/glycinergic premotoneurons localized in the ventral medullary reticular formation. We then propose that movements of RBD are induced by descending projections of cortical motor neurons before discussing possible modes of action of clonazepam and melatonin.

DNA may be represented by sequences of four symbols, but it is often useful to convert those symbols into real or complex numbers for further analysis. Several mapping schemes have been used in the past, but most of them seem to be unrelated to any intrinsic characteristic of DNA. The objective of this work was to study a mapping scheme that is directly related to DNA characteristics, and that could be useful in discriminating between different species. Recently, we have proposed a methodology based on the inter-nucleotide distance, which proved to contribute to the discrimination among species. In this paper, we introduce a new distance, the distance to the nearest dissimilar nucleotide, which is the distance of a nucleotide to first occurrence of a different nucleotide. This distance is related to the repetition structure of single nucleotides. Using the information resulting from the concatenation of the distance to the nearest dissimilar and the inter-nucleotide distance, we found that this new distance brings additional discriminative capabilities. This suggests that the distance to the nearest dissimilar nucleotide might contribute with useful information about the evolution of the species.

Resumen Fundamento El sindrome de Down (SD) es una alteracion genetica debida a la presencia de tres copias del cromosoma 21. Variaciones en los alelos del gen receptor de la vitamina D se han asociado a gran variedad de fenotipos y se han considerado factores de riesgo en determinadas poblaciones. En el presente trabajo se analiza si alguno de los genotipos del polimorfismo Bsm I del receptor de la vitamina D se presenta con mayor frecuencia en personas con SD con respecto a la poblacion general y la influencia que puede tener en diferentes fenotipos del SD. Pacientes y metodos Se estudiaron los polimorfismos del gen del receptor de la vitamina D en DNA de sangre periferica de 85 personas con SD y de 122 controles sin el sindrome. La determinacion de cada genotipo se realizo con amplificacion por tecnica de reaccion en cadena de la polimerasa (PCR) de los segmentos que contienen los polimorfismos situados en el intron 8 ( Bsm I ) . Se analizaron las diferencias de distribucion de genotipos entre los dos grupos, en el grupo con SD se relacionaron con diferentes parametros antroponometricos (edad, talla e indice de masa corporal), bioquimicos (calcio, vitamina D y paratohormona intacta) y con valores de masa osea por densitometria (DEXA). Resultados El analisis de distribucion del polimorfismo para Bsm I muestra una mayor frecuencia del alelo B en el grupo con SD y del b en los controles ( p = 0,015). En las personas con SD la combinacion de genotipos con respecto a los datos bioquimicos analizados y el resultado de la densitometria no muestra diferencias estadisticamente significativas. Sin embargo, el genotipo homocigoto bb es mas frecuente en los de talla alta ( p = 0,04) y el genotipo homocigoto BB en los de mayor edad ( p = 0,03). Conclusiones El alelo B del polimorfismo del intron 8 ( Bsm I ) del gen del receptor de la vitamina D es mas prevalente en personas con el SD. Los genotipos bb y BB son mas frecuentes en aquellas con el SD de mayor talla y mas longevas respectivamente, lo que hace pensar que estos podrian participar en estas dos caracteristicas fenotipicas del SD.

Background Down syndrome (DS) is a genetic alteration caused by having three copies of chromosome 21. Variations in the presence of alleles in the vitamin D receptor (VDR) gene have been linked to a variety in the phenotype and also considered a risk factor in some populations. In the present paper, we analyze whether a variation of the BsmI polymorphism in the VDR gene is overexpressed in patients with DS and whether it is related to any phenotype of the patients. Patients and methods We studied the BsmI polymorphism of the vitamin D receptor in DNA from peripheral blood of 85 patients with DS and 122 controls. The detection of each phenotype is performed by amplification of the DNA sequences of intron 8 of the VDR gene by polymerase chain reaction (PCR). We analyzed the differences in distribution of the alleles in patients with DS and the correlation of the genotype to different anthropometric (age, height, body mass index) and biochemical parameters (calcium, vitamin D,{PTH hormone, bone mass). Results The analysis of the distribution of the BsmI polymorphism showed a higher frequency of the B allele in the DS patients with respect to controls. In the same group of patients, regression analysis showed no link with any biochemical parameter. However, the homozygous genotype bb is more frequently found in taller individuals ( p = 0.04) and the BB in older individuals ( p = 0.03). Conclusions The B allele of the BsmI polymorphism of the VDR gene is more frequent in people with DS. The genotypes bb and BB are more frequent in taller and longer-living DS patients respectively. This result points out the possibility that VDR genotype could influence these two phenotypic characteristics of DS patients.

The phase behaviour of the binary mixture of carbon dioxide and the cobalt complex dicarbonyl(η 5 -cyclopentadienyl)-cobalt, CpCo(CO) 2 , has been investigated. This organometallic compound is one of the most effective catalysts of cyclotrimerization reactions of arylisocyanates and alkynes. Vapour–liquid equilibrium (VLE) measurements were undertaken in a static analytical apparatus at 313.15, 323.15 and 363.15 K at pressures up to 15 MPa. p , T isopleths were measured by a synthetic method in a Cailletet apparatus. Nine different compositions ranging from 17.56 to 94.23 mol% of CO 2 were measured up to 15 MPa. Modelling with the Peng–Robinson equation of state (PR EOS) gave reasonable results in the correlation of the experimental phase equilibrium compositions using two temperature-dependent interaction parameters.

Resumen La discinesia tardia es un efecto secundario grave de los antipsicoticos. A pesar de la introduccion de los nuevos antipsicoticos, siguen existiendo casos de discinesias tardias. Hay pocos estudios sobre el tratamiento de estos trastornos del movimiento, pero la clozapina sigue siendo el farmaco mas utilizado. A continuacion presentamos un caso de un paciente con esquizofrenia que sufrio discinesias tardias tras la supresion de un tratamiento con zuclopentixol. Posteriormente se introdujo clozapina, mejorando el cuadro hasta la remision clinica y consiguiendo una rehabilitacion psico-social del paciente.

Introduction Substance P (SP) is a neuropeptide present in enteric neurons, involved not only in the control of intestinal motility, but also in the development of inflammation in the gastrointestinal tract (GIT). The increase in the SP production is associated with the progress of inflammatory process, since SP leads directly to the secretion of cytokines, such as IL-6, IL-8, and TNFα. The GIT is considered the main route of F exposure. Daily, high F concentrations are ingested through dental products, water and food consumption, and in cases of intoxication, important intestinal symptoms are described. Aim To evaluate SP-containing enteric neurons, in the myenteric plexus of the small intestine, since there is no information about F effects on enteric neurons, even with the report of intestinal symptomatology as result of excessive F intake. Thus, we analysed the effects of an acute F exposure by the morphology evaluation of the SP varicosities, which are the axonal portion that concentrates SP. Methods 12 male rats ( Rattus norvegicus – Wistar type) were divided into 2 groups: Control (C) and Acute Dose (AD). The animals received deionized water for 29 days, and at the 30th day they received 0 or 25 mgF/kg orally by gavage. The segments of the small intestine (duodenum, jejunum, and ileum) were collected, and processed for immunohistochemical technique for the SP identification. Morphometric analyses were carried out in 400 varicosities from each animal. The groups were compared by Student’s t -test ( p Results The 3 segments (duodenum, jejunum, and ileum) presented a statistically significant increase (44.6%, 57.1%, and 56.1%, respectively) in the mean value of the areas of the SP myenteric varicosities for DA in relation to C. Conclusion: we may infer that the dose of 25 mgF/kg could lead to an increase in the production of SP, which could impair the intestinal motility, and induce an intestinal inflammatory process.

Sodium absorption across the airway epithelium is increased in cystic fibrosis (CF) patients and may contribute to abnormal water transport and accummulation of sticky sputum, a hallmark of the CF lung disease. Down-regulation of epithelial sodium channels (ENaC) may attenuate the CF lung disease. One approach to decrease ENaC function is the specific down-regulation of its mRNA. Single-stranded DNA antisense oligonucleotides (ssDNA) bind to mRNA sequence-specifically and trigger RNase-H mediated degradation of the ssDNA/mRNA hybrid. However, careful selection of the ssDNA sequence is required to obtain optimal mRNA reduction. Here, we compared five murine ENaC antisense DNAs (20 nucleotides each) for their ability to decrease ENaC mRNA in M1 cells, a murine kidney cell line that expresses ENaC abundantly. It has previously been demonstrated that the alpha subunit of ENaC is the most crucial for channel function and all five ssDNAs were therefore directed against this subunit. First, fluorescently labelled ssDNA was complexed with Lipofectamine 2000 and transfection efficiency determined. Interestingly, the ssDNA rapidly (after 30 min) accumulated in the nucleus of transfected cells and after 6 hrs >90% cells were transfected. The nuclear localisation of the antisense ODNs was consistent with their proposed site of action. Forty-eight hrs after transfection cells were harvested and mRNA was prepared for quantitative RT-PCR using Taqman. The relative degree of aENaC mRNA reduction varied between antisense molecules and ranged from 5 to 60% (n=6, p

Top of pageAbstract Sendai virus (SeV) is currently the only viral vector that transfects airway epithelial cells efficiently in vivo. However, as for any other viral vector repeated administration, leads to a significant drop in gene expression. Induction of immunological tolerance may permit repeated administration of the SeV vector. To achieve this we investigated the induction of immunological tolerance to the CD4 and CD8 T cell immunodominant epitopes of SeV. These peptides (100 mg/mouse) or a control peptide were administered to the lungs of C57Bl/6 mice (female, 6–8 weeks) via “sniffing” 10 days before the first virus administration (dF/SeV-GFP 106 pfu/mouse). 21 days after the first virus administration, mice were repeatedly transfected with dF/SeV. To avoid interference from anti-GFP antibodies, the second transfection was carried out with dF/SeV-lacZ (107 pfu/mouse). Two days after dF/SeV-lacZ administration all animals were culled. The lungs were harvested for βgal quantification and T-cell proliferation in response to the immunodominant epitopes was assessed to determine immunological tolerance. In addition serum was collected to measure anti-SeV neutralising antibodies. Administration of peptide 1 or 2 and 1+2 reduced CD4 and CD8 T-cell proliferation ex vivo after re-challenge with the respective protein by >60%. βgal expression following a second administration of SeV in mice receiving no peptide or the control peptide was 40–50% lower compared to mice that only had a single administration. Administration of the peptides did not elevate expression levels over levels measured in these control groups. This result indicates either that T cell responses need to be further reduced or that immune mechanisms other than T cells play a major role in eliminating SeV.