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1.
Behav Pharmacol ; 34(7): 424-436, 2023 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-37578419

RESUMEN

Cognitive rigidity (CR) refers to inadequate executive adaptation in the face of changing circumstances. Increased CR is associated with a number of psychiatric disorders, for example, obsessive-compulsive disorder, and improving cognitive functioning by targeting CR in these conditions, may be fruitful. Levetiracetam (LEV), clinically used to treat epilepsy, may have pro-cognitive effects by restoring balance to neuronal signalling. To explore this possibility, we applied apomorphine (APO) exposure in an attempt to induce rigid cue-directed responses following a cue (visual pattern)-reward (social conspecifics) contingency learning phase and to assess the effects of LEV on such behaviours. Briefly, zebrafish were divided into four different 39-day-long exposure groups ( n  = 9-10) as follows: control (CTRL), APO (100 µg/L), LEV (750 µg/L) and APO + LEV (100 µg/L + 750 µg/L). The main findings of this experiment were that 1) all four exposure groups performed similarly with respect to reward- and cue-directed learning over the first two study phases, 2) compared to the CTRL group, all drug interventions, but notably the APO + LEV combination, lowered the degree of reward-directed behaviour during a dissociated presentation of the cue and reward, and 3) temporal and spatial factors influenced the manner in which zebrafish responded to the presentation of the reward. Future studies are needed to explore the relevance of these findings for our understanding of the potential cognitive effects of LEV.


Asunto(s)
Epilepsia , Piracetam , Animales , Levetiracetam/farmacología , Levetiracetam/uso terapéutico , Pez Cebra , Anticonvulsivantes/uso terapéutico , Apomorfina/farmacología , Epilepsia/tratamiento farmacológico
2.
BMC Cancer ; 17(1): 118, 2017 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-28183282

RESUMEN

BACKGROUND: We previously identified three short single peptides (P4, P6 and P7) representing different B-cell epitopes on the extracellular domain of Her-2/neu for a vaccine that was tested in a phase-I clinical trial. Here we describe the improvement of the multi peptide vaccine by fusing the single peptides to a hybrid peptide P467. METHODS: After coupling to either virosomes or to diphtheria toxoid CRM197 (CRM), the hybrid peptide was tested in different concentrations in combination with either Montanide or Aluminium hydroxide (Alum) in preclinical studies. RESULTS: Already low amount (10 µg) of P467 conjugated to CRM led to faster onset of high antibody levels compared to the P467-virosome. The formulation P467-CRM-Montanide induced higher serum IgG antibody titers, compared with P467-CRM-Alum, as examined by ELISA using recombinant Her-2/neu or Her-2/neu natively expressed on the tumor cell line SK-BR-3. Compared to P467-CRM-Alum, higher in vitro production of IL-2 and IFNγ in the Montanide-immunized mice was induced after re-stimulation of splenocytes with CRM but also with P467, indicating a clear Th1-biased response. In contrast to the single B cell peptides, the hybrid peptide led to T cell proliferation and cytokine production as CD4 T cell epitopes were generated in the fusion region of the single peptides P4 and P6 or P6 and P7. Additionally, a significantly higher proportion IFNγ-producing CD8+ T cells was found in the P467-CRM-Montanide immunized mice, probably by Montanide-driven bystander activation. Importantly, anti-P467 IgG antibodies exhibited anti-tumor properties and the combination of anti-P467 specific IgG with Herceptin® was found to inhibit the proliferation of Her-2/neu-overexpressing cell line SK-BR-3 in a significantly higher capacity than Herceptin® alone. CONCLUSIONS: Fusion of the B cell peptides has led to additional generation of CD4 T cell epitopes, and this P467-multi epitope vaccine was found to induce polyclonal antibody responses with anti-proliferative capacity against Her-2/neu. The hybrid vaccine together with Montanide induced higher and long-lasting antibody levels, Th1-biased cellular responses being superior to vaccination with the single B cell peptides. This vaccine formulation is now planned to be evaluated in a phase Ib/II study in Her-2/neu overexpressing cancer patients.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Receptor ErbB-2/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos Antineoplásicos/sangre , Anticuerpos Antineoplásicos/farmacología , Antígenos de Neoplasias , Antineoplásicos/farmacología , Proteínas Bacterianas/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Mapeo Epitopo , Femenino , Humanos , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Proteínas Recombinantes de Fusión/inmunología , Vacunas de Subunidad/inmunología
4.
Lab Anim ; : 236772231198733, 2023 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-38051824

RESUMEN

Empirical evidence suggests fishes meet the criteria for experiencing pain beyond a reasonable doubt and zebrafish are being increasingly used in studies of pain and nociception. Zebrafish are adopted across a wide range of experimental fields and their use is growing particularly in biomedical studies. Many laboratory procedures in zebrafish involve tissue damage and this may give rise to pain. Therefore, this FELASA Working Group reviewed the evidence for pain in zebrafish, the indicators used to assess pain and the impact of a range of drugs with pain-relieving properties. We report that there are several behavioural indicators that can be used to determine pain, including reduced activity, space use and distance travelled. Pain-relieving drugs prevent these responses, and we highlight the dose and administration route. To minimise or avoid pain, several refinements are suggested for common laboratory procedures. Finally, practical suggestions are made for the management and alleviation of pain in laboratory zebrafish, including recommendations for analgesia. Pain management is an important refinement in experimental animal use and so our report has the potential to improve zebrafish welfare during and after invasive procedures in laboratories across the globe.

5.
Transl Oncol ; 19: 101378, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35259675

RESUMEN

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4+ and CD8+ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer.

6.
Front Immunol ; 11: 895, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32528470

RESUMEN

Therapeutic monoclonal antibodies (mAbs), targeting tumor antigens, or immune checkpoints, have demonstrated a remarkable anti-tumor effect against various malignancies. However, high costs for mono- or combination therapies, associated with adverse effects or possible development of resistance in some patients, warrant further development and modification to gain more flexibility for this immunotherapy approach. An attractive alternative to passive immunization with therapeutic antibodies might be active immunization with mimotopes (B-cell peptides) representing the mAbs' binding epitopes, to activate the patient's own anti-tumor immune response following immunization. Here, we identified and examined the feasibility of inducing anti-tumor effects in vivo following active immunization with a mimotope of the immune checkpoint programmed cell death 1 (PD1), alone or in combination with a Her-2/neu B-cell peptide vaccine. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotopes, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effects were evaluated in a syngeneic tumor mouse model stably expressing human Her-2/neu. The identified PD1-derived mimotopes were shown to significantly block the mAbs' capacity in inhibiting the respective PD1/PD-L1 interactions. A significant reduction in tumor growth in vivo was observed following active immunization with the mPD1-derived mimotope, associated with a significant reduction in proliferation and increased apoptotic rates in the tumors. Particularly, combined vaccination with the mPD1-derived mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the vaccine's anti-tumor effect. Our results suggest active immunization with mimotopes of immune checkpoint inhibitors either as monotherapy or as combination therapy with tumor-specific vaccines, as a new strategy for cancer treatment.


Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Linfocitos B/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Vacunas contra el Cáncer/farmacología , Epítopos , Inhibidores de Puntos de Control Inmunológico/farmacología , Nivolumab/farmacología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor ErbB-2/antagonistas & inhibidores , Animales , Linfocitos B/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Estudios de Factibilidad , Femenino , Humanos , Inmunización , Células Jurkat , Células K562 , Ratones Endogámicos BALB C , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Prueba de Estudio Conceptual , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Carga Tumoral/efectos de los fármacos , Vacunas de Subunidad/farmacología
7.
PLoS Genet ; 1(5): e66, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-16311625

RESUMEN

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.


Asunto(s)
Conducta Animal , Visión Ocular , Animales , Axones , Etilnitrosourea/farmacología , Regulación de la Expresión Génica , Ligamiento Genético , Técnicas Genéticas , Mutagénesis , Fenómenos Fisiológicos Oculares , Fenotipo , Células Fotorreceptoras , Pez Cebra
8.
Nat Neurosci ; 7(12): 1329-36, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15516923

RESUMEN

The visual system adjusts its sensitivity to a wide range of light intensities. We report here that mutation of the zebrafish sdy gene, which encodes tyrosinase, slows down the onset of adaptation to bright light. When fish larvae were challenged with periods of darkness during the day, the sdy mutants required nearly an hour to recover optokinetic behavior after return to bright light, whereas wild types recovered within minutes. This behavioral deficit was phenocopied in fully pigmented fish by inhibiting tyrosinase and thus does not depend on the absence of melanin pigment in sdy. Electroretinograms showed that the dark-adapted retinal network recovers sensitivity to a pulse of light more slowly in sdy mutants than in wild types. This failure is localized in the retinal neural network, postsynaptic to photoreceptors. We propose that retinal pigment epithelium (which normally expresses tyrosinase) secretes a modulatory factor, possibly L-DOPA, which regulates light adaptation in the retinal circuitry.


Asunto(s)
Adaptación Ocular , Monofenol Monooxigenasa/fisiología , Red Nerviosa/enzimología , Estimulación Luminosa/métodos , Epitelio Pigmentado Ocular/enzimología , Adaptación Ocular/genética , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Monofenol Monooxigenasa/biosíntesis , Monofenol Monooxigenasa/genética , Mutación Missense , Pez Cebra
9.
Zebrafish ; 14(6): 547-551, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28968196

RESUMEN

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.


Asunto(s)
Bienestar del Animal , Eutanasia Animal , Pez Cebra/fisiología , Anestesia/veterinaria , Animales , Ciencia de los Animales de Laboratorio/educación
10.
Sci Rep ; 7(1): 5230, 2017 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-28701772

RESUMEN

Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression. We used image registration to characterize, compare, and digitally superimpose the labeling patterns from our newly generated transgenic lines. This analysis revealed highly restricted and mutually exclusive tissue distributions, with striking resolution of layered brain regions such as the tectum or the rhombencephalon. We further show that a combination of Gal4 and Cre transgenes allows intersectional expression of a fluorescent reporter in regions where the expression of the two drivers overlaps. Taken together, our study offers new tools for functional studies of specific neural circuits in zebrafish.


Asunto(s)
Encéfalo/fisiología , Cromosomas Artificiales Bacterianos , Marcación de Gen , Neuronas/fisiología , Transgenes , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/metabolismo
11.
J Immunol Methods ; 287(1-2): 203-15, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15099768

RESUMEN

We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.


Asunto(s)
Adyuvantes Inmunológicos , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli , Nicotiana/virología , Virus del Mosaico del Tabaco/fisiología , Vacunas Sintéticas , Animales , Toxinas Bacterianas/genética , Clonación Molecular , Enterotoxinas/genética , Ensayo de Inmunoadsorción Enzimática , Reacción en Cadena de la Polimerasa , ARN
12.
FEMS Immunol Med Microbiol ; 37(2-3): 179-83, 2003 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-12832123

RESUMEN

The protozoan parasite Entamoeba histolytica, which is responsible for intestinal amebiasis and amebic liver abscess, is causing significant morbidity and mortality worldwide. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) are major surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against the PPGs protected severe combined immune-deficient mice from amebic liver abscess. The structure of the PPGs is very complex and only known in part. To find peptide mimics of E. histolytica PPG antigens, we had screened phage-displayed random peptide libraries with the antibody EH5. We identified various peptide mimics of E. histolytica PPGs, all sharing a consensus sequence Gly-Thr-His-Pro-X-Leu. Several of the phage clones induced a significant, specific IgG response against membrane antigens of E. histolytica after immunization of mice with whole phage particles. In the present work, in order to avoid the use of phage particles for immunization, we coupled two selected chemically synthesized peptides to keyhole limpet hemocyanin (KLH). The two KLH-conjugated peptides were immunogenic in mice and induced the production of high titers of anti-peptide antibodies, and one of the two peptides was also able to induce significant titers of antibodies against E. histolytica PPGs. Our results demonstrate that the KLH-conjugated peptides are able to mimic the EH5 epitope without the M13 phage sequences flanking the peptide inserts and independent of the structural framework of the phage.


Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Superficie/inmunología , Entamoeba histolytica/inmunología , Péptidos/inmunología , Proteoglicanos/inmunología , Vacunas Antiprotozoos/inmunología , Adyuvantes Inmunológicos , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/inmunología , Epítopos , Hemocianinas/química , Inmunización , Ratones , Ratones Endogámicos BALB C , Imitación Molecular , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química
13.
PLoS One ; 8(4): e60866, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23593334

RESUMEN

The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1) gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na(+)/K(+)-ATPase pump (NKA) was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na(+) levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells.


Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Mutación/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Conducta Animal , Compartimento Celular , Clonación Molecular , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/patología , Espacio Intracelular/metabolismo , Mecanotransducción Celular , Datos de Secuencia Molecular , Transporte de Proteínas , Sodio/metabolismo , Estereocilios/metabolismo , Estereocilios/patología , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo
14.
J Allergy Clin Immunol ; 116(3): 683-9, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16159643

RESUMEN

BACKGROUND: 1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25[OH](2)D(3)) exerts its effects on the immune system, particularly through suppression of T helper/cytotoxic cell 1 (T(H)/T(C)1)-mediated reactions, although direct actions of 1 alpha,25(OH)(2)D(3) on human T lymphocytes have not yet been studied in detail. OBJECTIVE: We evaluated the effect of 1 alpha,25(OH)(2)D(3) on basal and cytokine-driven T-cell functions at the single-cell level. METHODS: We used 4-color flow cytometry for simultaneous detection of intracellular cytokines in CD4(+) and CD8(+) human PBMCs that had been cultured in the presence of 1 alpha,25(OH)(2)D(3) singly or in combination with either IL-12 or IL-4. According to the exploratory nature of these investigations, the Bonferroni correction was not applied for data analysis and presentation. RESULTS: 1 alpha,25(OH)(2)D(3) had little effect on T(H)/T(C)1 cytokines but significantly inhibited IL-12-induced IFN-gamma production. Constitutive synthesis of T(H)/T(C)2-related cytokines was also only modestly affected by 1 alpha,25(OH)(2)D(3) alone. When T(H)/T(C)2 differentiation was induced by IL-4, 1 alpha,25(OH)(2)D(3) significantly expanded the percentages of IL-4(+) and IL-13(+) cells. However, the predominant effect of 1 alpha,25(OH)(2)D(3) on T lymphocytes, particularly in the presence of IL-4, was the induction of separate CD4(+) and CD8(+) subpopulations with almost exclusive expression of IL-6. This might be an important facet of the immunomodulatory action of 1 alpha,25(OH)(2)D(3) because IL-6 might act in parallel with 1 alpha,25(OH)(2)D(3) in modulation of T(H)/T(C) effector cell functions. CONCLUSIONS: Our data imply that the specific actions of 1 alpha,25(OH)(2)D(3) on cytokine-stimulated T-cell functions could play a role in the prevention of T(H)/T(C)1-related autoimmune diseases but also predispose toward T(H)/T(C)2-mediated allergic reactions.


Asunto(s)
Citocinas/efectos de los fármacos , Interleucina-12/inmunología , Interleucina-4/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Vitamina D/análogos & derivados , Adulto , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/inmunología , Femenino , Citometría de Flujo , Humanos , Interferón gamma/efectos de los fármacos , Interferón gamma/inmunología , Interleucina-13/biosíntesis , Interleucina-13/inmunología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Masculino , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Vitamina D/farmacología
15.
Development ; 132(3): 615-23, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15634702

RESUMEN

In the sensory receptors of both the eye and the ear, specialized apical structures have evolved to detect environmental stimuli such as light and sound. Despite the morphological divergence of these specialized structures and differing transduction mechanisms, the receptors appear to rely in part on a shared group of genes for function. For example, mutations in Usher (USH) genes cause a syndrome of visual and acoustic-vestibular deficits in humans. Several of the affected genes have been identified, including the USH1F gene, which encodes protocadherin 15 (PCDH15). Pcdh15 mutant mice also have both auditory and vestibular defects, although visual defects are not evident. Here we show that zebrafish have two closely related pcdh15 genes that are required for receptor-cell function and morphology in the eye or ear. Mutations in pcdh15a cause deafness and vestibular dysfunction, presumably because hair bundles of inner-ear receptors are splayed. Vision, however, is not affected in pcdh15a mutants. By contrast, reduction of pcdh15b activity using antisense morpholino oligonucleotides causes a visual defect. Optokinetic and electroretinogram responses are reduced in pcdh15b morpholino-injected larvae. In electron micrographs, morphant photoreceptor outer segments are improperly arranged, positioned perpendicular to the retinal pigment epithelium and are clumped together. Our results suggest that both cadherins act within their respective transduction organelles: Pcdh15a is necessary for integrity of the stereociliary bundle, whereas Pcdh15b is required for alignment and interdigitation of photoreceptor outer segments with the pigment epithelium. We conclude that after a duplication of pcdh15, one gene retained an essential function in the ear and the other in the eye.


Asunto(s)
Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Peces/metabolismo , Genes Duplicados/genética , Audición/fisiología , Visión Ocular/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Animales , Proteínas Relacionadas con las Cadherinas , Electrofisiología , Proteínas de Peces/genética , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Audición/genética , Microscopía Electrónica , Mutación/genética , Fenotipo , Células Fotorreceptoras/citología , Células Fotorreceptoras/embriología , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestructura , Retina/embriología , Retina/metabolismo , Retina/ultraestructura , Sensibilidad y Especificidad , Visión Ocular/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética
16.
EMBO Rep ; 4(9): 894-9, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12947416

RESUMEN

The vertebrate eye forms by specification of the retina anlage and subsequent morphogenesis of the optic vesicles, from which the neural retina differentiates. chokh (chk) mutant zebrafish lack eyes from the earliest stages in development. Marker gene analysis indicates that retinal fate is specified normally, but optic vesicle evagination and neuronal differentiation are blocked. We show that the chk gene encodes the homeodomain-containing transcription factor, Rx3. Loss of Rx3 function in another teleost,medaka, has also been shown to result in an eyeless phenotype. The medaka rx3 locus can fully rescue the zebrafish mutant phenotype. We provide evidence that the regulation of rx3 is evolutionarily conserved, whereas the downstream cascade contains significant differences in gene regulation. Thus, these mutations in orthologous genes allow us to study the evolution of vertebrate eye development at the molecular level.


Asunto(s)
Anomalías del Ojo/genética , Proteínas de Peces/genética , Proteínas de Homeodominio/genética , Pez Cebra/genética , Animales , Proteínas del Ojo , Proteínas de Homeodominio/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Mutación Puntual , Proteínas Represoras , Retina/embriología , Retina/metabolismo , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética , Proteína Homeobox SIX3
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