RESUMO
BACKGROUND: The most common chronic complication of preterm birth is bronchopulmonary dysplasia (BPD), widely referred to as chronic lung disease of prematurity. All current definitions rely on characterizing the disease based on respiratory support level and do not provide full understanding of the underlying cardiopulmonary pathophysiology. OBJECTIVE: To evaluate a rapid functional lung imaging technique in premature infants and to quantitate pulmonary ventilation using 1.5 Tesla magnetic resonance imaging (MRI). MATERIALS AND METHODS: We conducted a prospective MRI study of 12 premature infants in the neonatal intensive care unit (NICU) using the phase resolved functional lung MRI technique to calculate pulmonary ventilation parameters in preterm infants with and without BPD grade 0/1 (n = 6) and grade 2/3 (n = 6). RESULTS: The total ventilation defect percentage showed a significant difference between groups (16.0% IQR (11.0%,18%) BPD grade 2/3 vs. 8.0% IQR (4.5%,9.0%) BPD grade 0/1, p = 0.01). CONCLUSION: Phase-resolved functional lung MRI is feasible for assessment of ventilation defect percentages in preterm infants and shows regional variation in localized lung function in this population.
Assuntos
Displasia Broncopulmonar , Nascimento Prematuro , Lactente , Feminino , Recém-Nascido , Humanos , Recém-Nascido Prematuro , Displasia Broncopulmonar/diagnóstico por imagem , Estudos Prospectivos , Pulmão/patologia , Imageamento por Ressonância Magnética/métodosRESUMO
Chronic lung disease of prematurity (CLD) is a frequent sequela of premature birth and oxygen toxicity is a major associated risk factor. Impaired alveolarization, scarring, and inflammation are hallmarks of CLD. Mast cell hyperplasia is a feature of CLD but the role of mast cells in its pathogenesis is unknown. We hypothesized that mast cell hyperplasia is a consequence of neonatal hyperoxia and contributes to CLD. Additionally, mast cell products may have diagnostic and prognostic value in preterm infants predisposed to CLD. To model CLD, neonatal wild-type and mast cell-deficient mice were placed in an O2 chamber delivering hyperoxic gas mixture [inspired O2 fraction (FiO2 ) of 0.8] (HO) for 2 wk and then returned to room air (RA) for an additional 3 wk. Age-matched controls were kept in RA (FiO2 of 0.21). Lungs from HO mice had increased numbers of mast cells, alveolar simplification and enlargement, and increased lung compliance. Mast cell deficiency proved protective by preserving air space integrity and lung compliance. The mast cell mediators ß-hexosaminidase (ß-hex), histamine, and elastase increased in the bronchoalveolar lavage fluid of HO wild-type mice. Tracheal aspirate fluids (TAs) from oxygenated and mechanically ventilated preterm infants were analyzed for mast cell products. In TAs from infants with confirmed cases of CLD, ß-hex was elevated over time and correlated with FiO2 Mast cell exosomes were also present in the TAs. Collectively, these data show that mast cells play a significant role in hyperoxia-induced lung injury and their products could serve as potential biomarkers in evolving CLD.
Assuntos
Displasia Broncopulmonar/patologia , Exossomos/metabolismo , Hiperóxia/patologia , Mastócitos/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/imunologia , Displasia Broncopulmonar/metabolismo , Células Cultivadas , Humanos , Hiperóxia/imunologia , Hiperóxia/metabolismo , Recém-Nascido , Pulmão/imunologia , Pulmão/patologia , Camundongos , Proteoma/metabolismo , Traqueia/metabolismoRESUMO
Replication-deficient adenoviral (Ad) vectors of non-human serotypes can serve as Ad vaccine platforms to circumvent pre-existing anti-human Ad immunity. We found previously that, in addition to that feature, a non-human primate-based AdC7 vector expressing outer membrane protein F of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In this study we analysed if genetic modification of the AdC7 fibre to display an integrin-binding arginine-glycine-aspartic acid (RGD) sequence can further enhance lung mucosal immunogenicity of AdC7OprF. Intratracheal immunization of mice with either AdC7OprF.RGD or AdC7OprF induced robust serum levels of anti-OprF immunoglobulin (Ig)G up to 12 weeks that were higher compared to immunization with the human vectors Ad5OprF or Ad5OprF.RGD. OprF-specific cellular responses in lung T cells isolated from mice immunized with AdC7OprF.RGD and AdC7OprF were similar for T helper type 1 (Th1) [interferon (IFN)-γ in CD8(+) and interleukin (IL)-12 in CD4(+)], Th2 (IL-4, IL-5 and IL-13 in CD4(+)) and Th17 (IL-17 in CD4(+)). Interestingly, AdC7OprF.RGD induced more robust protective immunity against pulmonary infection with P. aeruginosa compared to AdC7OprF or the control Ad5 vectors. The enhanced protective immunity induced by AdC7OprF.RGD was maintained in the absence of alveolar macrophages (AM) or CD1d natural killer T cells. Together, the data suggest that addition of RGD to the fibre of an AdC7-based vaccine is useful to enhance its mucosal protective immunogenicity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Pulmão/imunologia , Oligopeptídeos , Porinas/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Células Th1/imunologia , Adenoviridae/genética , Animais , Células Cultivadas , Citocinas/imunologia , Feminino , Vetores Genéticos/genética , Humanos , Imunidade nas Mucosas , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/genética , Porinas/genética , Primatas , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas , Pseudomonas aeruginosa/genéticaRESUMO
We have investigated whether dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can trigger B cells to produce antigen-specific antibodies without CD4+ T-cell help. Dendritic cells modified with a recombinant adenovirus vector to express CD40 ligand and pulsed with heat-killed Pseudomonas induced naive B cells to produce antibodies against Pseudomonas in the absence of CD4+ T cells in vitro, initiated Pseudomonas-specific humoral immune responses in vivo in wild-type and CD4-/- mice, and protected immunized wild-type and CD4-/-, but not B-cell -/- mice, from lethal intrapulmonary challenge with Pseudomonas. Thus, genetic modification of dendritic cells with CD40 ligand enables them to present a complex mixture of microbial antigens and establish CD4+ T cell-independent, B cell-mediated protective immunity against a specific microbe.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/genética , Células Dendríticas/fisiologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Ligante de CD40/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Engenharia Genética/métodos , Vetores Genéticos , Imunização/métodos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/imunologiaRESUMO
Fcgamma receptors on the surface of phagocytic cells bind the Fc region of IgG and mediate binding, phagocytosis, and destruction of particulate antigens opsonized by the antigen-specific IgG molecule. The present study evaluates the feasibility of converting lung epithelial cells into phagocytic cells using adenovirus (Ad) vector-mediated gene transfer of FcgammaRIIA cDNA to induce expression of the human FcgammaRIIA receptor. Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcgammaRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with (51)Cr-labeled SRBCs. When A549 cells were infected with an Ad vector expressing a FcgammaRIIA mutant in which 2 of 3 cytoplasmic tyrosines have been replaced with phenylalanine, only binding, but not phagocytosis, of opsonized SRBCs was observed. In vivo expression of FcgammaRIIA in the lung after intratracheal administration of the AdFcgammaRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcgamma receptor expression. Similar results were observed with a chimeric FcgammaRIIA construct containing the extracellular domain of FcgammaRIIIA. Together, these data demonstrate that Ad-mediated FcgammaRIIA receptor cDNA expression can mediate the binding and phagocytosis of opsonized particulate antigens by normally nonphagocytic cells, suggesting that gene-transfer strategies might be used to utilize nonphagocytic cells to clear bacteria or other opsonized particulate antigens from the respiratory tract.
Assuntos
Técnicas de Transferência de Genes , Pulmão/imunologia , Pulmão/microbiologia , Pseudomonas aeruginosa/imunologia , Receptores de IgG/genética , Animais , Linhagem Celular , DNA Complementar/genética , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Eritrócitos/imunologia , Humanos , Pulmão/citologia , Camundongos , Proteínas Opsonizantes , Fagócitos/imunologia , Fagocitose , Pseudomonas aeruginosa/patogenicidade , Ratos , Receptores de IgG/deficiência , OvinosRESUMO
BACKGROUND AND PURPOSE: Late infantile neuronal ceroid lipofuscinosis (LINCL), a form of Batten disease, is a fatal neurodegenerative genetic disorder, diagnosed via DNA testing, that affects approximately 200 children in the United States at any one time. This study was conducted to evaluate whether quantitative data derived by diffusion-weighted MR imaging (DWI) techniques can supplement clinical disability scale information to provide a quantitative estimate of neurodegeneration, as well as disease progression and severity. MATERIALS AND METHODS: This study prospectively analyzed 32 DWI examinations from 18 patients having confirmed LINCL at various stages of disease. A whole-brain apparent diffusion coefficient (ADC) histogram was fitted with a dual Gaussian function combined with a function designed to model voxels containing a partial volume fraction of brain parenchyma versus CSF. Previously published whole-brain ADC values of age-matched control subjects were compared with those of the LINCL patients. Correlations were tested between the peak ADC of the fitted histogram and patient age, disease severity, and a CNS disability scale adapted for LINCL. RESULTS: ADC values assigned to brain parenchyma were higher than published ADC values for age-matched control subjects. ADC values between patients and control subjects began to differ at 5 years of age based on 95% confidence intervals. ADC values had a nearly equal correlation with patient age (R2=0.71) and disease duration (R2=0.68), whereas the correlation with the central nervous system disability scale (R2=0.27) was much weaker. CONCLUSION: This study indicates that brain ADC values acquired using DWI may be used as an independent measure of disease severity and duration in LINCL.
Assuntos
Encéfalo/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Interpretação de Imagem Assistida por Computador/métodos , Lipofuscinoses Ceroides Neuronais/diagnóstico , Índice de Gravidade de Doença , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Lipofuscinoses Ceroides Neuronais/classificação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Intravascular introduction of replication-deficient adenoviral vectors (Advectors) provides an ideal model of delivery of transgenes for the treatment of various vascular abnormalities. On the basis of the knowledge that Advectors can induce inflammatory responses after intravascular administration, we speculated that cellular activation by Advector infection could directly modulate the endothelial cell (EC) adhesion molecule/chemokine expression repertoire. Infection of human umbilical vein ECs or bone marrow microvascular ECs with an E1(-)E4(+) Advector resulted in the upregulation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD34, but not E-selectin, P-selectin, CD36, CD13, CD44, HLA-DR or PECAM. Upregulation of ICAM-1, VCAM-1, and CD34 was apparent 12 hours after infection and persisted for weeks after infection. Selective induction of adhesion molecules was mediated by the presence of the E4 gene in the Advector, because infection of ECs with an E1(-)E4(-) Advector had no effect on adhesion molecule expression. ECs infected with E1(-)E4(+) Advector, but not those infected with E1(-)E4(-) Advector, supported the adhesion of leukocytes. Monoclonal antibodies to ICAM-1 and VCAM-1 inhibited adhesion of leukocytes to E1(-)E4(+)-infected ECS: Infection of the ECs with E1(-)E4(+) Advector, but not E1(-)E4(-) Advector, resulted in downregulation of expression of chemocytokines, including interleukin-8, MCP-1, RANTES, and GM-CSF. Nonetheless, a large number of leukocytes migrated through ECs infected with E1(-)E4(+), but not those infected with E1(-)E4(l-), in response to exogenous chemokines. These results demonstrate that infection of ECs with E1(-)E4(+) Advectors, but not E1(-)E4(-) Advectors, may directly augment inflammatory responses by upregulating expression of adhesion molecules and enhancing migration through Advector-infected ECs and suggest that E1(-)E4(-) Advectors may be a better choice for gene-transfer strategies directed to the ECS:
Assuntos
Proteínas E1 de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Endotélio Vascular/metabolismo , Leucócitos/metabolismo , Proteínas/metabolismo , Adenoviridae/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Regulação para Baixo , Endotélio Vascular/citologia , Regulação da Expressão Gênica , Vetores Genéticos/genética , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/citologia , Proteínas/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
To evaluate the contribution of the innate immune component of host defense in clearing the genome of adenovirus (Ad) vectors following in vivo administration, the Ad vectors AdCMV.beta gal (expressing beta-galactosidase) or AdCMV.Null (expressing no gene) were administered intravenously to immunocompetent or immunodeficient mice, and the amount of vector genome was quantified in the liver. Strikingly, 90% of vector DNA was eliminated within 24 hr. There was no increase in vector DNA in other tissues over this period, suggesting that rapid clearance of vector genome resulted from local degradation. After 24 hr, vector elimination was slow, with only 9% of the initial amount of vector genome cleared over the subsequent 3 weeks. Importantly, early phase (0-24 hr) elimination of vector DNA was independent of the transgene and similar in immunocompetent and nude animals. These observations suggest two phases of Ad vector elimination: a previously recognized late, immune-related elimination, and the early, innate immune elimination described in the present study. The early phase of vector loss is, by far, the dominant mechanism, an observation that has implications in developing strategies to maintain persistent expression of the newly transferred gene following in vivo gene therapy.
Assuntos
Adenoviridae/metabolismo , Vetores Genéticos/genética , Imunidade , Animais , Southern Blotting , Sondas de DNA , DNA Viral/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Imunidade/imunologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
To evaluate the hypothesis that innate immune mechanisms play a major role in eliminating adenovirus (Ad) vectors from the lung, the fate of adenoviral genome of an Ad vector was quantified in the first 24 h after intratracheal administration of an Ad vector coding for beta-galactosidase (beta gal) to mice. Southern analysis with an Ad specific probe showed that 70% of the Ad genome was lost within 24 h, in both immunocompetent and immunodeficient animals. When alveolar macrophages were eliminated by administration of liposomes containing dichloromethylene-biphosphanate, subsequent administration of Ad vector was associated with a 100%+/-8% increase in lung Ad DNA and 96%+/-9% rise in beta gal expression at 24 h compared to control animals. In vitro infection of mouse, rat, and human alveolar macrophages with an Ad vector resulted in 65% loss of vector genome within 24 h, whereas the vector genome was stable in lung epithelial cell lines. PCR in situ hybridization demonstrated that the Ad vector genome persisted A549 lung epithelial cell in vitro but not in alveolar macrophages. Finally, alveolar macrophages recovered from the mouse lung 30 min following intratracheal administration of an Ad vector showed large amounts of vector genome, whereas much less was evident in alveolar macrophages recovered after 24 h. These observations demonstrate that alveolar macrophages play an important role in elimination of Ad vectors from the lung and suggest that strategies to transiently suppress this major innate immune defense system might be rewarding in enhancing the efficiency Ad vectors for lung gene therapy.
Assuntos
Adenoviridae/imunologia , Proteínas do Capsídeo , Vetores Genéticos/imunologia , Macrófagos Alveolares/imunologia , Sistema Respiratório/imunologia , Adenoviridae/genética , Animais , Capsídeo/análise , Linhagem Celular , Ácido Clodrônico/farmacologia , DNA Viral/análise , Portadores de Fármacos , Epitélio , Vetores Genéticos/genética , Genoma Viral , Humanos , Lipossomos , Macrófagos Alveolares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Ratos , TraqueiaRESUMO
In animals, Ad-mediated gene transfer initiates anti-Ad host immune responses that vary, depending on vector design, dose, host, and transgene. To begin to understand whether the anti-Ad vector responses in humans simulate those in animals, Ad(GV)CD.10, an E1-E3- Ad5 vector encoding the E. coli cytosine deaminase gene, was administered by the intradermal route to six normal individuals (8 x 10(7) to 8 x 10(9) particle units, each dose administered to two sites; n = 2 per group). No adverse events were observed. Polymerase chain reaction/Southern analysis demonstrated vector genome in the skin through 28 days in all individuals except one of two at the lowest dose. Local induration, independent of vector dose and baseline systemic anti-Ad5 neutralizing antibodies, developed in all subjects (6 to 17 mm, peak by day 3). Biopsies revealed a mild to moderate T cell (CD3+, CD4+, CD8+), B cell, and macrophage infiltrate at day 3, all decreased by day 28. Langerhans cells accumulated primarily in the papillary dermis. The day 3 cellular response was dose independent. On day 28, CD4+ and CD8+ T lymphocytes and macrophages showed dose dependency. There was minimal systemic Ad5-specific lymphocyte proliferation induced by Ad vector administration in three individuals studied, and no Ad5-specific cytotoxic T lymphocytes (evaluated in two subjects) could be detected. Thus, intradermal administration of an E1-E3- Ad vector to normal subjects induces mild/moderate local cellular responses, even in Ad-immunized individuals. These observations provide a baseline to determine if these human anti-Ad vector host responses can be circumvented by using "stealth" vectors and/or immunosuppression.
Assuntos
Adenoviridae/imunologia , Vetores Genéticos/imunologia , Imunidade Celular , Adenoviridae/genética , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dermatite/etiologia , Dermatite/imunologia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/efeitos adversos , Humanos , Injeções Intradérmicas , Masculino , Pessoa de Meia-Idade , Pele/imunologia , Pele/patologiaRESUMO
The pathogenic agent, adenovirus (Ad), has taken on a new role as a vector for gene transfer in both laboratory and clinical settings. To help understand the intracellular pathways and fate of Ad gene transfer vectors, we covalently conjugated fluorophores to E1-, E3- Ad vectors and used quantitative fluorescence microscopy to assess essential steps of Ad vector gene transfer to the A549 human epithelial lung cell line including binding, internalization, escape from endosomes, translocation to the nucleus, dissociation of capsids and gene expression. The data demonstrate that Ad internalizes with a t1/2 2.5 min, breaks out of endosomes early, likely prior to endosome-endosome fusion, exhibits sustained, intracellular velocities averaging 0.58 microm/sec, and translocates to the nucleus with >80% of internalized fluorophore demonstrating nuclear localization within 60 min of infection. Interestingly, 24 hr after infection, half of the initially internalized fluorescence was detected but lacked nuclear localization, suggesting that the capsid is released from the nucleus and is likely degraded. Fluorescent labeling of virions provides a novel quantitative, morphological strategy to characterize the interaction of gene transfer vectors with the intracellular environment.
Assuntos
Adenoviridae/fisiologia , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Adenoviridae/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Vetores Genéticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Fatores de Tempo , Células Tumorais Cultivadas , VírionRESUMO
OBJECTIVE: To characterize ouabain-like immunoreactivity in human urine. METHODS: Sensitive radioimmunoassay for ouabain characterized by high-performance liquid chromatography. RESULTS: Serial dilution of urinary immunoreactive ouabain paralleled the standard curve, but not so plasma immunoreactive ouabain. Intravenous administration of 86 nmol (62.5 micrograms) ouabain caused a rapid rise in ouabain immunoreactivity in plasma of healthy volunteers with a maximum of 1.7 nmol/l 8 min after injection and returned to basal levels after 6 h. Ouabain immunoreactivity rose to 36 nmol/l in urine, suggesting that exogenously administered ouabain can be measured reliably in plasma and urine. Analytical reversed-phase high-performance liquid chromatography (isopropanol-propanol biphasic gradient; linear acetonitrile gradient) of sample extracts before assay demonstrated measurable amounts of ouabain-related material only in native urine, but not in plasma. When plasma and urine were spiked with ouabain standard or normal volunteers were injected with ouabain, the assay reliably measured ouabain. CONCLUSION: A substance closely related to ouabain can be detected in urine, but circulates, if at all, in small amounts in human plasma.
Assuntos
Ouabaína/urina , Adulto , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Ouabaína/imunologia , CoelhosRESUMO
The expression of neuropeptide Y (NPY) and NPY-Y1 receptor (NPY-Y1R) in relation to that of tyrosine kinase A (trkA), nerve-growth-factor low-affinity-receptor (LNGFR) and the transcription factor N-myc was studied in 26 neuroblastomas and one ganglioneuroma by quantitative Northern-blot analysis. A correlation of NPY-Y1R with LNGFR (r = 0.85, p < 0.01) and trkA (r = 0.38, p < 0.05), respectively, could be shown, while no correlation between NPY and its receptor NPY-Y1R was observed. Comparison of a high and a low level NPY expressing group revealed that the high NPY expressing group also had high LNGFR and high trkA levels while the low NPY expressing group had low LNGFR and low trkA levels which were significantly different (NPY: p = 0.035, trkA: p = 0.008, LNGFR p = 0.004). Dividing the tumors in a high and a low N-myc expressing group showed that the low N-myc expressing group contained both high and low trkA expressing tumors while the high N-myc expressing group exclusively were low level trkA expressing tumors. The frequency distribution in both groups concerning trkA expression showed a significant difference (p < 0.01). In conclusion the coexpression of NPY-Y1R and LNGFR or trkA may indicate a similar gene regulation during ontogenesis of the peripheral nervous system.
RESUMO
The suppression of neuropeptide Y Y1 receptor gene expression by antisense oligonucleotides targeted to different gene regions was monitored on mRNA and protein level in the human neuroblastoma SK-N-MC cell line. The antisense oligonucleotide targeted to the junction of the first intron and second exon suppressed specifically Y1 receptor subtype number by more than 50%, but only if oligonucleotides were administered by electroporation. Also, the formation of Y1 receptor mRNA as shown by reverse transcription-polymerase chain reaction was markedly blocked in this case. Using the antisense oligonucleotide targeted to the start of translation, no effect, neither on the Y1 receptor number nor on Y1 receptor mRNA, could be observed. This finding suggests that besides sequence-specific effects of antisense oligonucleotides gene site-specific effects play a major role in the efficacy of suppression.
Assuntos
Oligonucleotídeos Antissenso/farmacologia , Receptores de Neuropeptídeo Y/genética , Northern Blotting , Humanos , Técnicas In Vitro , Neuroblastoma/genética , Neuropeptídeo Y/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica/efeitos dos fármacos , Ensaio Radioligante , Células Tumorais CultivadasRESUMO
Endothelin 1-21 belongs to a family of locally produced regulatory peptides with potent vasoconstrictor activity and profound renal effects. To study the biological significance of endothelin in children with renal diseases, we measured urinary endothelin-like immunoreactivity (ETir) excretion in children and adolescents (60 normal controls and 57 patients with renal disease). ETir excretion was constant during childhood and adolescence (4-18 years). Compared to these normal controls elevated urinary excretions of ETir were found in children with chronic renal failure, following renal transplantation and with idiopathic hypercalciuria (all groups p < 0.001). However, ETir excretion was unchanged in children with idiopathic steroid sensitive nephrotic syndrome, with stable chronic glomerulonephritis and in 4 patients with hemolytic uremic syndrome. Urinary ETir concentrations were similar in controls and in various patient groups. ETir excretion correlated positively with urine flow rate in normal controls (r = 0.71) and in all patients studied (r = 0.91). Fractional excretion of ETir correlated negatively with glomerular filtration rate. Eight healthy volunteers (23-27 years old, 4 female, 4 male) were studied before and after oral water load (20 ml/kg) to investigated the effect of ETir excretion on urine flow rate. Urine flow rose tenfold in response to water load and urine concentration of ETir fell only by factor 3 and urinary ETir excretion rose fivefold. These results indicate that urinary ETir excretion is related to and depends at least in part on urine flow rate. ETir excretion may so reflect a role of ETir in renal disease, especially in the diuretic state.
Assuntos
Endotelinas/urina , Nefropatias/urina , Urodinâmica/fisiologia , Adolescente , Criança , Pré-Escolar , Feminino , Taxa de Filtração Glomerular , Humanos , Nefropatias/fisiopatologia , Transplante de Rim/fisiologia , Masculino , RadioimunoensaioRESUMO
The study was carried out to determine the urinary excretion of endothelin-1 (ET-1) in normal pregnancy and to define its possible role in mediating the renal response to aldosterone and arginine vasopressin (AVP). Measurements were performed in 12 healthy pregnant women serially in the 20th, 24th, 28th, 32nd and 36th weeks of pregnancy. Urinary ET-1, plasma and urinary aldosterone and AVP levels (RIA methods) as well as plasma and urine sodium, potassium, creatinine and osmolality were measured; creatinine clearance (Ccr), osmolar clearance (Cosm) and free water clearance (CH2O) calculated. Fractional sodium excretion (FENa), urine sodium/potassium ratio (Na/K) and transtubular potassium concentration gradient (TTKG) were also determined. It was demonstrated that urinary ET-1 excretion was higher in pregnant than in non-pregnant women and it increased further as the pregnancy progressed from 34.8 +/- 4.0 pmol/day in week 20 to 44.1 +/- 3.2 pmol/day in week 36 (P < 0.01). Daily ET-1 excretion significantly correlated with AVP (r = 0.39, P < 0.005) and aldosterone excretion (r = 0.62, P < 0.0001). Furthermore, there was a significant positive relationship between ET-1 excretion and urine flow rate (r = 0.67, P < 0.0001), CCR (r = 0.40, P < 0.0025), Cosm (r = 0.58, P < 0.001), sodium (r = 0.56, P < 0.001) and potassium excretion (r = 0.42, P < 0.001). However, such a relationship could not be established between ET-1 excretion and FENa, TTKG and Na/K.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aldosterona/urina , Arginina Vasopressina/urina , Endotelinas/fisiologia , Rim/metabolismo , Gravidez/urina , Equilíbrio Hidroeletrolítico/fisiologia , Adolescente , Adulto , Endotelinas/urina , Feminino , Humanos , Valores de ReferênciaRESUMO
BACKGROUND AND PURPOSE: LINCL is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene that encodes for tripeptidyl peptidase 1, a lysosomal enzyme necessary for the degradation of products of cellular metabolism. With the goal of developing quantitative noninvasive imaging biomarkers sensitive to disease progression, we evaluated a 5-component MR imaging metric and tested its correlation with a clinically derived disease-severity score. MATERIALS AND METHODS: MR imaging parameters were measured across the brain, including quantitative measures of the ADC, FA, nuclear spin-spin relaxation times (T2), volume percentage of CSF (%CSF), and NAA/Cr ratios. Thirty MR imaging datasets were prospectively acquired from 23 subjects with LINCL (2.5-8.4 years of age; 8 male/15 female). Whole-brain histograms were created, and the mode and mean values of the histograms were used to characterize disease severity. RESULTS: Correlation of single MR imaging parameters against the clinical disease-severity scale yielded linear regressions with R2 ranging from 0.25 to 0.70. Combinations of the 5 biomarkers were evaluated by using PCA. The best combination included ADC, %CSF, and NAA/Cr (R2=0.76, P<.001). CONCLUSIONS: The multiparametric disease-severity score obtained from the combination of ADC, %CSF, and NAA/Cr whole-brain MR imaging techniques provided a robust measure of disease severity, which may be useful in clinical therapeutic trials of LINCL in which an objective assessment of therapeutic response is desired.
Assuntos
Encéfalo/patologia , Imageamento por Ressonância Magnética/métodos , Lipofuscinoses Ceroides Neuronais/patologia , Índice de Gravidade de Doença , Fatores Etários , Aminopeptidases/genética , Artefatos , Biomarcadores/metabolismo , Encéfalo/metabolismo , Criança , Pré-Escolar , Bases de Dados Factuais , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Progressão da Doença , Feminino , Humanos , Masculino , Lipofuscinoses Ceroides Neuronais/genética , Serina Proteases/genética , Tripeptidil-Peptidase 1RESUMO
BACKGROUND: Late infantile neuronal ceroid lipofuscinosis (LINCL) is associated with progressive degeneration of the brain and retina starting in early childhood. METHODS: Thirty-two individual neurologic, ophthalmologic, and CNS imaging (MRI and MRS) assessments of 18 children with LINCL were analyzed. Disease severity was followed by two rating scales, one previously established but modified to solely assess the brain and exclude the retinal disease (modified Hamburg LINCL scale), and a newly developed scale, with expanded evaluation of the CNS impairment (Weill Cornell LINCL scale). RESULTS: For the 18 children, the Weill Cornell scale yielded a closer correlation with both age and time since initial clinical manifestation of the disease than did the modified Hamburg scale. There were no significant differences as a function of age or time since initial manifestation of the disease in the rating scales among the most frequent CLN2 mutations (G3556C, 56% of all alleles or C3670T, 22% of all alleles). Measurements of cortical MRS N-acetyl-aspartate content, MRI ventricular, gray matter and white matter volume, and cortical apparent diffusion coefficient correlated to a variable degree with the age of the children and the time since initial clinical manifestation of the disease. All imaging measurements correlated better with the Weill Cornell CNS scale compared to the modified Hamburg LINCL scale. CONCLUSION: The data suggest that the Weill Cornell late infantile neuronal ceroid lipofuscinosis (LINCL) scale, together with several of the MRI measurements, may be useful in the assessment of severity and progression of LINCL and for the evaluation of novel therapeutic strategies.
Assuntos
Lipofuscinoses Ceroides Neuronais/fisiopatologia , Índice de Gravidade de Doença , Adolescente , Fatores Etários , Idade de Início , Aminopeptidases , Ácido Aspártico/análogos & derivados , Ácido Aspártico/análise , Córtex Cerebral/química , Córtex Cerebral/patologia , Criança , Pré-Escolar , Análise Mutacional de DNA , Dipeptidil Peptidases e Tripeptidil Peptidases , Progressão da Doença , Endopeptidases/genética , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Mutação de Sentido Incorreto , Exame Neurológico , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Ressonância Magnética Nuclear Biomolecular , Oftalmoscopia , Tamanho do Órgão , Mutação Puntual , Retina/patologia , Serina Proteases , Irmãos , Tripeptidil-Peptidase 1RESUMO
We investigated the role of endothelin-1 in the renal adaptation to alterations in sodium balance in premature infants. The postnatal course of urinary endothelin-1 excretion, an estimate of renal endothelin-1 production, was compared in premature infants receiving low or high sodium intake. Sodium supplementation was given in a dose of 3 to 5 mmol/kg per day and 1.5 to 2.5 mmol/kg per day at the postnatal ages of 8 to 21 and 22 to 35 days, respectively. Sodium balance and urinary endothelin-1 excretion were determined weekly up to the fifth week of life. Urinary endothelin-1 concentration (expressed in picomoles per liter) and urinary endothelin-1 excretion (expressed either in terms of picomoles per square meter per day or picomoles per millimole creatinine) were significantly lower in infants receiving a high sodium intake compared with those receiving low sodium intake (p < 0.001) in weeks 2 through 5. We conclude that in sodium-depleted premature infants with high urinary sodium excretion, an angiotensin II-mediated increase in renal endothelin-1 production occurs, which acts in concert with angiotensin II to restore sodium balance.
Assuntos
Endotelinas/urina , Alimentos Fortificados , Alimentos Infantis , Recém-Nascido Prematuro/fisiologia , Cloreto de Sódio na Dieta/farmacologia , Endotelinas/sangue , Endotelinas/efeitos dos fármacos , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro/urina , Masculino , Fatores de Tempo , Aumento de Peso/efeitos dos fármacosRESUMO
Infection of human endothelial cells with first-generation E1(-)E4(+) adenovirus (Ad) vectors leads to prolonged cell survival and changes in the cell phenotype to a more quiescent stage. Based on the concept that the CXCR4, the receptor for the endothelial chemoattractant stromal-derived factor-&alpha (SDF-alpha), is constitutively expressed by quiescent, resting endothelial cells, the present study analyzes the effect of Ad vector infection on CXCR4 expression and SDF-alpha responses of human umbilical vein endothelial cells (HUVEC). CXCR4 transcripts were markedly downregulated in E1(-)E4(+) Ad-infected cells 48 h following infection, but not in uninfected control cells or when the cells were infected with an E1(-)E4(-) Ad vector. Analysis of surface CXCR4 expression by flow cytometry demonstrated marked reduction of the CXCR4 receptor on cells infected with E1(-)E4(+) Ad compared to uninfected control cells or E1(-)E4(-) Ad-infected cells. Infection of other cell types which express CXCR4, such as dendritic cells and myeloma cells, did not exhibit CXCR4 receptor downregulation following infection with E1(-)E4(+) Ad. Consistent with the observed downregulation of CXCR4 mRNA and surface protein, infection of the endothelial cells with an E1(-)E4(+) Ad rendered the cells unresponsive to the chemoattractant SDF-alpha compared to naive or E1(-)E4(-) Ad-infected cells. Together, the data suggest that first-generation Ad vectors, likely the E4 region, modify the ability of endothelial cells to respond to at least one important chemoattractant.