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1.
Eur J Clin Microbiol Infect Dis ; 41(3): 395-405, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-34816330

RESUMEN

Viral respiratory tract infections cause significant morbidity in bone marrow transplant (BMT) patients. Speed and sensitivity of the FilmArray™ Respiratory Panel (FA-RP) can improve care but may prompt inappropriate testing. Studies describing FA-RP use in pediatric BMT patients are limited; we investigated FA-RP use, results, and clinical management to evaluate clinical significance of testing in pediatric BMT patients. Retrospective analysis of 671 respiratory specimens from 204 unique BMT patients between 01/01/2016 and 01/01/2019 was performed. Age, underlying diagnoses, FA-RP result, reason for FA-RP, and symptoms were abstracted. FA-RP impact on antimicrobial management, scheduled procedures, infection control measures, and hospital admission/discharge were investigated. Impacts of repeat testing were evaluated. Two hundred sixty-nine out of 671 specimens (40%) tested positive; human rhinovirus/enterovirus (hRV/hEV) was the most common (161/269, 60%). The primary reason for FA-RP was URI symptoms (402/671, 60%) with 54% testing positive. One hundred twenty-two out of 671 (18.2%) specimens were from asymptomatic patients; 14 (11.4%) tested positive. FA-RP informed antiviral initiation in 7/19 (36.8%), 7/8 (87.5%), and 5/30 (16.7%) of RSV, influenza, and human parainfluenza cases, respectively. In 11 cases, FA-RP informed azithromycin and ceftriaxone initiation, continuation, or discontinuation. BMT was delayed for three positives (two RSV, one hRV/hEV). In 22 instances, negative FA-RP cleared patients for BMT. In 70% of cases, repeats offered no new clinical information; all negative-to-positive cases had new or worsening respiratory symptoms. FA-RP was ordered on symptomatic and asymptomatic patients, provided rapid diagnosis in > 50% of symptomatic patients, and informed infection control measures for all inpatients and antiviral initiation in > 80% of influenza cases.


Asunto(s)
Infecciones del Sistema Respiratorio , Virus , Trasplante de Médula Ósea/efectos adversos , Niño , Humanos , Lactante , Sistema Respiratorio , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/epidemiología , Estudios Retrospectivos
2.
Mol Ther ; 22(6): 1139-1150, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24609143

RESUMEN

A subset of patients with severe hemophilia B, the X-linked bleeding disorder resulting from absence of coagulation factor IX (FIX), develop pathogenic antibody responses during replacement therapy. These inhibitors block standard therapy and are often associated with anaphylactic reactions to FIX. Established clinical immune tolerance induction protocols often fail for FIX inhibitors. In a murine model of this immune complication, retrovirally transduced primary B cells expressing FIX antigen fused with immunoglobulin-G heavy chain prevented antibody formation to FIX and was also highly effective in desensitizing animals with preexisting response. In contrast, transplant of B cells that received the identical expression cassette via nucleofection of plasmid vector substantially heightened antibody formation against FIX, a response that could be blocked by toll-like receptor 9 (TLR9) inhibition. While innate responses to TLR4 activation or to retrovirus were minimal in B cells, plasmid DNA activated TLR9, resulting in CpG-dependent NF-κB activation/IL-6 expression and adaptor protein 3 dependent, CpG-independent induction of IFN-I. Neither response was seen in TLR9-deficient B cells. Therefore, TLR9 signaling in B cells, in particular in response to plasmid vector, is highly immunogenic and has to be avoided in design of tolerance protocols.


Asunto(s)
Linfocitos B/inmunología , Factor IX/metabolismo , Terapia Genética/métodos , Hemofilia B/terapia , Plásmidos/genética , Retroviridae/genética , Receptor Toll-Like 9/metabolismo , Traslado Adoptivo/métodos , Animales , Linfocitos B/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Factor IX/genética , Hemofilia B/inmunología , Humanos , Tolerancia Inmunológica , Inmunidad Innata/inmunología , Ratones , Ratones Endogámicos C57BL , Plásmidos/metabolismo , Retroviridae/metabolismo , Transducción de Señal , Bazo/citología , Transducción Genética/métodos , Transfección/métodos
3.
Blood ; 117(24): 6459-68, 2011 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-21474674

RESUMEN

Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/ß expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.


Asunto(s)
Dependovirus/genética , Vectores Genéticos/farmacología , Genoma Viral/fisiología , Inmunidad Innata/efectos de los fármacos , Hígado/inmunología , Receptor Toll-Like 9/fisiología , Animales , Dependovirus/inmunología , Dependovirus/fisiología , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Vectores Genéticos/fisiología , Genoma Viral/inmunología , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Hígado/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Transducción Genética , Transgenes/inmunología , Transgenes/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología
4.
Proc Natl Acad Sci U S A ; 107(15): 7101-6, 2010 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-20351275

RESUMEN

To address complications of pathogenic antibody or life-threatening anaphylactic reactions in protein replacement therapy for patients with hemophilia or other inherited protein deficiencies, we have developed a prophylactic protocol using a murine hemophilia B model. Oral delivery of coagulation factor IX fused with cholera toxin beta-subunit (with or without a furin cleavage site; CTB-FFIX or CTB-FIX), expressed in chloroplasts (up to 3.8% soluble protein or 0.4 mg/g leaf tissue), bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies (undetectable or up to 100-fold less than controls). Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after four to six exposures to intravenous F.IX. Whereas only 20-25% of control animals survived after six to eight F.IX doses, 90-93% of F.IX-fed mice survived 12 injections without signs of allergy or anaphylaxis. Immunostaining confirmed delivery of F.IX to Peyer's patches in the ileum. Within 2-5 h, feeding of CTB-FFIX additionally resulted in systemic delivery of F.IX antigen. This high-responder strain of hemophilia B mice represents a new animal model to study anaphylactic reactions. The protocol was effective over a range of oral antigen doses (equivalent to 5-80 microg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months (approximately 40% life span of this mouse strain). Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach of antigen delivery to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.


Asunto(s)
Administración Oral , Anafilaxia/prevención & control , Factor IX/administración & dosificación , Hemofilia B/sangre , Anafilaxia/mortalidad , Animales , Cloroplastos/metabolismo , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Factor IX/antagonistas & inhibidores , Factor IX/química , Vectores Genéticos , Hemofilia B/complicaciones , Inmunoglobulina E/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Modelos Genéticos , Nicotiana/genética
5.
Nat Commun ; 12(1): 511, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33479234

RESUMEN

The ability to utilize preclinical models to predict the clinical toxicity of chimeric antigen receptor (CAR) T cells in solid tumors is tenuous, thereby necessitating the development and evaluation of gated systems. Here we found that murine GD2 CAR-T cells, specific for the tumor-associated antigen GD2, induce fatal neurotoxicity in a costimulatory domain-dependent manner. Meanwhile, human B7H3 CAR-T cells exhibit efficacy in preclinical models of neuroblastoma. Seeking a better CAR, we generated a SynNotch gated CAR-T, GD2-B7H3, recognizing GD2 as the gate and B7H3 as the target. GD2-B7H3 CAR-T cells control the growth of neuroblastoma in vitro and in metastatic xenograft mouse models, with high specificity and efficacy. These improvements come partly from the better metabolic fitness of GD2-B7H3 CAR-T cells, as evidenced by their naïve T-like post-cytotoxicity oxidative metabolism and lower exhaustion profile.


Asunto(s)
Gangliósidos/inmunología , Inmunoterapia Adoptiva/métodos , Neuroblastoma/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Línea Celular Tumoral , Supervivencia Celular/inmunología , Citotoxicidad Inmunológica/inmunología , Gangliósidos/metabolismo , Humanos , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neuroblastoma/inmunología , Neuroblastoma/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Carga Tumoral/inmunología
6.
Oncoimmunology ; 10(1): 1838140, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33489468

RESUMEN

Immune checkpoint therapy has resulted in minimal clinical response in many pediatric cancers. We sought to understand the influence of immune checkpoint inhibition using anti-PD-1 and anti-CTLA-4 antibodies individually, in combination, and after chemotherapy on immune responses in minimal and established murine neuroblastoma models. We also sought to understand the role of the tumor microenvironment (TME) and PD-L1 expression and their alteration post-chemotherapy in our models and human tissues. PD-L1 expression was enriched in human tumor-associated macrophages and up-regulated after chemotherapy. In a murine minimal disease model, single and dual immune checkpoint blockade promoted tumor rejection, improved survival, and established immune memory with long-term anti-tumor immunity against re-challenge. In an established tumor model, only dual immune checkpoint blockade showed efficacy. Interestingly, dual immune checkpoint therapy distinctly influenced adaptive and innate immune responses, with significant increase in CD8+CD28+PD-1+ T cells and inflammatory macrophages (CD11bhiCD11c-F4/80+Ly6Chi) in tumor-draining lymph nodes. Adding chemotherapy before immunotherapy provided significant survival benefit for mice with established tumors receiving anti-PD-1 or dual immune checkpoint blockade. Our findings demonstrate anti-PD-1 and anti-CTLA-4 therapy induces a novel subset of effector T cells, and support administration of induction chemotherapy immediately prior to immune checkpoint blockade in children with high-risk neuroblastoma.


Asunto(s)
Neuroblastoma , Receptor de Muerte Celular Programada 1 , Animales , Antígenos CD28 , Linfocitos T CD8-positivos , Humanos , Inhibidores de Puntos de Control Inmunológico , Ratones , Neuroblastoma/tratamiento farmacológico , Linfocitos T , Microambiente Tumoral
7.
Mol Ther ; 17(10): 1733-42, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19603001

RESUMEN

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.


Asunto(s)
Factor IX/inmunología , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Hemofilia B/terapia , Adenoviridae/genética , Animales , Linfocitos T CD8-positivos/inmunología , Ensayo de Inmunoadsorción Enzimática , Factor IX/genética , Terapia Genética/efectos adversos , Humanos , Inmunidad Humoral/genética , Inmunidad Humoral/inmunología , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Mutación , Mutación Missense
8.
J Cell Biochem ; 105(1): 9-16, 2008 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-18500726

RESUMEN

Many genes residing in gene clusters and expressed in a differentiation or developmental-stage specific manner are regulated by locus control regions (LCRs). These complex genetic regulatory elements are often composed of several DNAse I hypersensitive sites (HS sites) that function together to regulate the expression of several cis-linked genes. Particularly well characterized is the LCR associated with the beta-globin gene locus. The beta-globin LCR consists of five HS sites that are located upstream of the beta-like globin genes. Recent data demonstrate that the LCR is required for the association of the beta-globin gene locus with transcription foci or factories. The observation that RNA polymerase II associates with the LCR in erythroid progenitor or hematopoietic stem cells which do not express the globin genes suggests that the LCR is always in an accessible chromatin configuration during differentiation of erythroid cells. We propose that erythroid specific factors together with ubiquitous proteins mediate a change in chromatin configuration that juxtaposes the globin genes and the LCR. The proximity then facilitates the transfer of activities from the LCR to the globin genes. In this article we will discuss recent observations regarding beta-globin locus activation with a particular emphasis on LCR mediated activation of adult beta-globin gene expression.


Asunto(s)
Envejecimiento/fisiología , Regulación de la Expresión Génica/genética , Globinas/genética , Globinas/metabolismo , Región de Control de Posición , Animales , Humanos , Modelos Genéticos , Transactivadores/genética , Transactivadores/metabolismo
9.
Cancer Immunol Res ; 6(1): 36-46, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29180536

RESUMEN

The GD2 ganglioside, which is abundant on the surface of neuroblastoma cells, is targeted by an FDA-approved therapeutic monoclonal antibody and is an attractive tumor-associated antigen for cellular immunotherapy. Chimeric antigen receptor (CAR)-modified T cells can have potent antitumor activity in B-cell malignancies, and trials to harness this cytolytic activity toward GD2 in neuroblastoma are under way. In an effort to enhance the antitumor activity of CAR T cells that target GD2, we generated variant CAR constructs predicted to improve the stability and the affinity of the GD2-binding, 14G2a-based, single-chain variable fragment (scFv) of the CAR and compared their properties in vivo We included the E101K mutation of GD2 scFv (GD2-E101K) that has enhanced antitumor activity against a GD2+ human neuroblastoma xenograft in vivo However, this enhanced antitumor efficacy in vivo was concomitantly associated with lethal central nervous system (CNS) toxicity comprised of extensive CAR T-cell infiltration and proliferation within the brain and neuronal destruction. The encephalitis was localized to the cerebellum and basal regions of the brain that display low amounts of GD2. Our results highlight the challenges associated with target antigens that exhibit shared expression on critical normal tissues. Despite the success of GD2-specific antibody therapies in the treatment of neuroblastoma, the fatal neurotoxicity of GD2-specific CAR T-cell therapy observed in our studies suggests that GD2 may be a difficult target antigen for CAR T-cell therapy without additional strategies that can control CAR T-cell function within the CNS. Cancer Immunol Res; 6(1); 36-46. ©2017 AACR.


Asunto(s)
Encefalitis/etiología , Gangliósidos/inmunología , Inmunoterapia Adoptiva , Neuroblastoma/complicaciones , Neuroblastoma/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Animales , Complejo CD3/genética , Línea Celular Tumoral , Proliferación Celular , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Encefalitis/diagnóstico , Gangliósidos/metabolismo , Orden Génico , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Ratones , Neuroblastoma/terapia , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
10.
FEBS J ; 274(23): 6065-73, 2007 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17970752

RESUMEN

Upstream stimulatory factor and TFII-I are ubiquitously expressed helix-loop-helix transcription factors that interact with E-box sequences and or initiator elements. We previously demonstrated that upstream stimulatory factor is an activator of beta-globin gene expression whereas TFII-I is a repressor. In the present study, we demonstrate that upstream stimulatory factor interacts with the coactivator p300 and that this interaction is restricted to erythroid cells expressing the adult beta-globin gene. Furthermore, we demonstrate that Suz12, a component of the polycomb repressor complex 2, is recruited to the beta-globin gene. Reducing expression of Suz12 significantly activates beta-globin gene expression in an erythroid cell line with an embryonic phenotype. Suz12 also interacts with the adult beta-globin gene during early stages of erythroid differentiation of mouse embryonic stem cells. Our data suggest that TFII-I contributes to the recruitment of the polycomb repressor complex 2 complex to the beta-globin gene. Together, these data demonstrate that the antagonistic activities of upstream stimulatory factor and TFII-I on beta-globin gene expression are mediated at least in part by protein complexes that render the promoter associated chromatin accessible or inaccessible for the transcription complex.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Globinas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción TFII/metabolismo , Factores Estimuladores hacia 5'/metabolismo , Animales , Proteínas Portadoras/metabolismo , Diferenciación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Células Eritroides/citología , Globinas/genética , Histona Desacetilasas/metabolismo , Humanos , Células K562 , Ratones , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Células Madre/citología , Factores de Transcripción , Factores de Transcripción p300-CBP/metabolismo
11.
Hum Mutat ; 27(6): 598-9, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16705699

RESUMEN

The National Mutation Frequency Databases are continuously updated mutation depositories, which contain extensive information over the described genetic heterogeneity of an ethnic group or population. Here, we report the construction of the Cypriot (http://www.goldenhelix.org/cypriot) and Iranian National Mutation Frequency Databases (http://www.goldenhelix.org/iranian), both derived from an academic effort to provide high quality and up-to-date information on the underlying genetic heterogeneity of inherited disorders in the Cypriot and Iranian populations, respectively. Both databases have been built and maintained online using ETHNOS platform, a specialized software, which provides the means for national mutation database construction and curation. Each database contains brief summaries of the various genetic disorders studied for each population, and an easy-to-use query interface provides, both to specialist as well as to non-specialist users (i.e. patients and their families), instant access to the list and frequencies of the different mutations responsible for the inherited disorders in these populations. Furthermore, numerous links to the respective Online Mendelian Inheritance in Man (OMIM) entries and, when available, to the locus-specific databases fruitfully integrate the databases content into a single Web site. Both databases can serve as valuable online tools for molecular genetic testing of inherited disorders in these populations and could potentially motivate further investigations of yet unknown genetic diseases in the Cypriot and Iranian populations.


Asunto(s)
Bases de Datos Genéticas , Enfermedades Genéticas Congénitas/genética , Mutación , Chipre/epidemiología , Frecuencia de los Genes , Pruebas Genéticas , Genética de Población , Irán/epidemiología
12.
World J Gastroenterol ; 11(11): 1712-4, 2005 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-15786557

RESUMEN

AIM: Urea breath test (UBT) is a non-invasive diagnostic test for detecting the presence of Helicobacter pylori (H pylori). In this study we evaluated the effect of anti-tuberculosis therapy on the results of 14C-UBT. METHODS: Patients, with the diagnosis of tuberculosis (TB) who had a positive UBT at the point of starting anti-TB therapy, were included. None had a history of peptic ulcer disease or had taken antibiotics, bismuth compounds and/or PPI in the previous month. 14C-UBT was repeated at the end of the second month and the end of treatment period and one month after completion of treatment course. RESULTS: Thirty-five patients (23 males) were enrolled. 14C-UBT was negative in all 35 patients (100%) at the end of the second month and remained negative in 30 cases (85.7%) at the end of the treatment course. One month after completion of treatment course, UBT remained negative in 13 patients (37.1%). CONCLUSION: Our report underscores the need for caution while interpreting urea breath test results in patients undergoing anti-TB therapy. Furthermore, the combination of drugs used in this study resulted in H pylori eradication in a minority of patients.


Asunto(s)
Antituberculosos/uso terapéutico , Pruebas Respiratorias/métodos , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/aislamiento & purificación , Tuberculosis Pulmonar/tratamiento farmacológico , Adolescente , Adulto , Radioisótopos de Carbono , Femenino , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/tratamiento farmacológico , Humanos , Masculino , Persona de Mediana Edad , Tuberculosis Pulmonar/complicaciones , Urea
13.
Hum Mutat ; 21(2): 146-50, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12552562

RESUMEN

As Human Genome Project exploration continues, the necessity of having a broader spectrum of genomic DNA material from different nationalities to study various aspects of hereditary disease becomes more obvious. The existence of high genetic polymorphism within and between different communities in the world makes it necessary for the gene hunters to investigate many different populations. Iran, a large country with close to 66 million people, is a land of different nationalities, tribes, and religions that offers a highly heterogeneous gene pool to the genetics researcher. The purity of many different races in this country has been highly conserved by geographical borders and by an ancient culture that has always encouraged intrafamilial marriages. All these have created a population that is remarkably heterogeneous yet high in consanguinity rate. During the last five years of investigation we have established a DNA bank, the Iranian Human Mutation Gene Bank (www.IHMGB.com), which contains all genetic diseases studied in Iran that have the Mendelian mode of inheritance. Some of the samples are assigned to common or novel mutations and others belong to patients with clinical profiles associated with particular genetic diseases but undefined mutation. This bank stores samples of DNA from the patient and his/her first-degree relatives together with a comprehensive pedigree and clinical profile for each sample. To facilitate collaboration with other scientists around the world with the same interests, we decided to present our experimental projects online. This DNA bank provides opportunities for us to collaborate with scientists outside Iran. It offers a sample resource to research scientists around the world, at no charge, for the purpose of investigating the various aspects of genetic disorders from prenatal diagnosis to gene structure and function. It is strongly stressed that no commercial benefit is involved in the establishment of this DNA bank and the DNA samples are free of charge. However, to meet our goals and to respect ethical values, DNA samples can only be used under certain conditions stated in the User Consent Form.


Asunto(s)
Conducta Cooperativa , Bases de Datos Genéticas , Investigación Genética , Cooperación Internacional , Mutación/genética , Consanguinidad , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Heterogeneidad Genética , Humanos , Irán , Polimorfismo Genético/genética , Proyectos de Investigación , Enfermedades Cutáneas Genéticas/genética
14.
BMC Neurol ; 4(1): 22, 2004 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-15579201

RESUMEN

BACKGROUND: Cerebral venous sinus thrombosis is an infrequent disease with a variety of causes. Pregnancy, puerperium, contraceptive pills and intracranial infections are the most common causes. The patient may present with headache, focal neurological deficits and seizures.The clinical outcome is highly variable and treatment with heparin is advised. CASE PRESENTATION: The patient is a 22 year old male who presented with headache, repeated vomiting and papilledema.He was a bodybuilder doing exercise since 5 years ago, who had used nandrolone decaonoate 25 milligrams intramuscularly during the previous 5 months. Brain MRI and MRV showed superior sagital and transverse sinus thrombosis and extensive investigations did not reveal any known cause. CONCLUSIONS: We suggested that androgen was the predisposing factor in our patient. Androgens may increase coagulation factors or platelet activity and cause arterial or venous thrombosis. As athletes may hide using androgens it should be considered as a predisposing factor for thrombotic events in such patients.


Asunto(s)
Anabolizantes/efectos adversos , Nandrolona/análogos & derivados , Trombosis del Seno Sagital/inducido químicamente , Levantamiento de Peso , Adulto , Cefalea/etiología , Humanos , Inyecciones Intramusculares , Masculino , Nandrolona/efectos adversos , Nandrolona Decanoato , Papiledema/etiología , Trombosis del Seno Sagital/diagnóstico , Vómitos/etiología
15.
J Genet Syndr Gene Ther ; 2(103)2011 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-23565344

RESUMEN

Primary autologous B-lymphocytes, following ex vivo gene transfer and re-implantation, have been successfully utilized to prevent autoimmune disease and adaptive responses to therapeutic proteins in several animal models. However, efficient gene transfer to primary B cells requires use of retroviral vectors, which increase the risk of insertional mutagenesis. Here, we evaluated several alternative gene transfer approaches. Resting splenic B cells were purified and activated with LPS, and ex vivo GFP gene transfer was performed by means of nucleofection, lipofectamine, adenoviral infection, or murine retroviral infection. The Adenoviral (Ad) vectors were added to B cell cultures with or without calcium phosphate precipitation. For transfection and nucleofection, naked plasmid DNA was utilized. Nucleofection technology represents a modified electroporation technique for effective transfer of nucleic acids to the nucleus and thus enhances the efficiency of transfer particularly for primary cells. Efficiency of ex vivo gene transfer was determined by flow cytometry using GFP, CD19, and a vital dye as markers. Nucleofection yielded the highest level of gene transfer with 60-65% of B cells being GFP+. Efficiencies were 30-35% for retrovirus, 20% for Ad5/11, 15% for Ad5/35, and 5% for lipofectamine-mediated transfection. Calcium phosphate precipitation increased efficiencies for Ad vectors to 30% (Ad5/11) and 25% (Ad5/35). Lipofectamin caused the greatest cell death at 80%, followed by nucleofection (35%), and viral vector (10-15% in each case). For all methods, gene transfer efficiencies were nearly identical for B cells from C57BL/6 or C3H/HeOuJ mice. In conclusion, recent advances in gene transfer technologies provide alternatives to retroviral vectors for primary B cells. If stable gene transfer is desired, non-integrating vector systems may be combined with transposon- or phage integrase-based systems or future site-specific systems to achieve integration into the host B cell genome.

16.
Front Microbiol ; 2: 244, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22279442

RESUMEN

Intramuscular (IM) administration of an adeno-associated viral (AAV) vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency). However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4(+) T cell epitope) caused an induction of regulatory T cells (Treg) with a concomitant apoptosis of antigen-specific effector T cells (Nayak et al., 2009). This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX) in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion). Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10 BU within 1 month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2 BU, which lasted for >4.5 months. Systemic hF.IX expression increased from undetectable to >200 ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.

17.
Mol Cell Biol ; 29(21): 5900-10, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19704006

RESUMEN

Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and beta-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all beta-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the beta-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expressing transgenic mice exhibit a defect in the formation of CD71(+) progenitor and Ter-119(+) mature erythroid cells. In summary, the data demonstrate that USF regulates globin gene expression indirectly by enhancing the expression of erythroid transcription factors and directly by mediating the recruitment of transcription complexes to the globin gene locus.


Asunto(s)
Eritropoyesis/genética , Genes Dominantes , Factores Estimuladores hacia 5'/genética , Animales , Antígenos CD/metabolismo , Pollos , Inmunoprecipitación de Cromatina , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Transgénicos , Modelos Genéticos , Especificidad de Órganos/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Polimerasa II/metabolismo , Receptores de Transferrina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Globinas beta/genética
18.
J Biol Chem ; 284(30): 20130-5, 2009 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-19491096

RESUMEN

Differentiation of erythroid cells is regulated by cell signaling pathways including those that change the intracellular concentration of calcium. Calcium-dependent proteases have been shown previously to process and regulate the activity of specific transcription factors. We show here that the protein levels of upstream stimulatory factor (USF) increase during differentiation of murine erythroleukemia (MEL) cells. USF was subject to degradation by the Ca(2+)-dependent protease m-calpain in undifferentiated but not in differentiated MEL cells. Treatment of MEL cells with the specific calpain inhibitor calpeptin increased the levels of USF and strongly induced expression of the adult alpha- and beta-globin genes. The induction of globin gene expression was associated with an increase in the association of USF and RNA po ly mer ase II with regulatory elements of the beta-globin gene locus. Calpeptin also induced high level alpha- and beta-globin gene expression in primary CD71-positive erythroid progenitor cells. The combined data suggest that inhibition of calpain activity is required for erythroid differentiation-associated increase in globin gene expression.


Asunto(s)
Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Células Eritroides/citología , Globinas/genética , Factores Estimuladores hacia 5'/metabolismo , Animales , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Diferenciación Celular , Células Eritroides/metabolismo , Femenino , Regulación de la Expresión Génica , Globinas/metabolismo , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Murinae , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Factores Estimuladores hacia 5'/genética
19.
Mol Cell Biol ; 28(5): 1584-95, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18086895

RESUMEN

CtBP is a transcriptional corepressor with tumorigenic potential that targets the promoter of the tumor suppressor gene E-cadherin. Pnn/DRS (Pnn) is a "nuclear speckle"-associated protein involved in mRNA processing as well as transcriptional regulation of E-cadherin via its binding to CtBP. Here, we show that CtBP can recruit Pnn to CtBP-associated complexes, resulting in Pnn-dependent chromatin remodeling at the E-cadherin promoter. In addition, CtBP and Pnn can differentially modulate E-cadherin mRNA splicing, with polymerase II serving as an interface in this event. Therefore, the Pnn/CtBP functional interplay represents a novel mechanism linking the corepressor CtBP and Pnn to the transcription-coupled mRNA splicing of a major tumor suppressor gene. Our findings implicate the existence of the molecular switches involved in tumorigenesis, which coordinate promoter-specific events and mRNA processing, by serving as bridging elements between the regulatory complexes both at gene promoters and within the mRNA splicing machineries.


Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Cadherinas/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Empalme del ARN , Transcripción Genética , Oxidorreductasas de Alcohol/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , ADN Complementario , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Hemaglutininas/metabolismo , Humanos , Riñón/citología , Luciferasas/metabolismo , Modelos Biológicos , Proteínas Nucleares/genética , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Transfección
20.
Hemoglobin ; 28(4): 353-6, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15658193

RESUMEN

Thalassemias are the most common hereditary diseases in Iran, resulting from synthesis defects in one or more hemoglobin (Hb) subunits. The majority of patients suffer from beta-thalassemia (thal), but cases with microcytic hypochromic anemia and normal electrophoretic patterns are suspected to have alpha- or silent beta-thal. A family from the northern part of Iran, an area highly prevalent for thalassemias, was referred to us for prenatal diagnosis. The hematological data of the father indicated a pattern of beta-thal minor. Reverse hybridization analysis for the most common beta-globin mutations identified IVS-II-1 (G-->A) in the heterozygous state. The maternal laboratory data indicated a case more compatible with alpha-thal. Iron deficient anemia was ruled out, and common alpha-thal point mutations and deletions were investigated. As no mutation was detected, chain synthesis was performed and showed an alpha/beta chain ratio of 2.1, that was in the range of beta-thal minor. DNA sequencing of the entire beta-globin gene identified a heterozygous GTG-->GGG (Val-->Gly) mutation at codon 126, also known as Hb Dhonburi (Neapolis). Prenatal diagnosis of the fetal DNA showed the absence of the IVS-II-1 and codon 126 anomalies. This result demonstrates the importance of screening of individuals with mild microcytic hypochromic anemia for both alpha- and silent beta-thal mutations.


Asunto(s)
Hemoglobinas Anormales/genética , Diagnóstico Prenatal , Talasemia beta/diagnóstico , Familia , Femenino , Humanos , Irán , Masculino , Embarazo
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