RESUMO
Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable treatment risks may differ considerably from the ones in case of less serious disease. This theme was the focus of the international conference "Taking immunogenicity assessment of therapeutic proteins to the next level", held at the Paul-Ehrlich-Institut in Langen, Germany, on the 10-11. June 2010. The objectives of the conference were to highlight how the field could move from that of a mere description of risk factors to a system of risk assessment and mitigation, as well as an understanding of the impact of unwanted immunogenicity on the overall benefit/risk consideration for a medicinal product. More than 150 experts from industry, academia and regulatory authorities worldwide discussed the phenomenon of undesired immunogenicity from different perspectives. The conference focussed on issues relevant to three areas: (1) new European guidelines that are currently the subject of discussion; (2) testing strategies for immunogenicity assessment; and (3) scientific progress on the product-related factors that may contribute to the development of pathogenesis of immunogenicity, in particular in the field of protein aggregation and post-translational modifications. This report provides an overview of issues, insights, and conclusions that were discussed and achieved during the meeting.
Assuntos
Produtos Biológicos/efeitos adversos , Produtos Biológicos/imunologia , Avaliação de Medicamentos/tendências , Hipersensibilidade a Drogas/diagnóstico , Proteínas/efeitos adversos , Proteínas/imunologia , Algoritmos , Animais , Formação de Anticorpos/fisiologia , Congressos como Assunto , Avaliação de Medicamentos/legislação & jurisprudência , Avaliação de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Guias como Assunto , Humanos , Imunidade Inata/efeitos dos fármacos , Legislação de Medicamentos , Modelos Biológicos , Processamento de Proteína Pós-TraducionalRESUMO
Recombinant-activating gene 2 (RAG-2-/-) T cell receptor-transgenic mice repeatedly injected with the superantigen staphylococcal enterotoxin A entered a tolerant state in which splenic CD4+ T cells produced little interleukin (IL)-2, interferon gamma, or IL-4. This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression. The anergy persisted for at least 3 wk and could be distinguished from the suppression by a decrease in IL-2 production per cell, a block in the activation of early response kinases, and a failure to be reversed with anti-transforming growth factor (TGF)-beta. Full suppression lasted for only 1 wk and involved both IL-10 and TGF-beta, but required additional unknown molecules for optimal effect. These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels.
Assuntos
Anergia Clonal/imunologia , Citocinas/biossíntese , Proteínas de Ligação a DNA/imunologia , Enterotoxinas/imunologia , Tolerância Imunológica , Superantígenos/imunologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citocinas/imunologia , Tolerância Imunológica/efeitos dos fármacos , Interferon gama/biossíntese , Interleucina-10/farmacologia , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
A retroviral vector was designed to express toxic proteins only in the presence of the HIV-1 Rev and/or Tat protein(s). The design of this vector incorporates an HIV-specific expression cassette that consists of three elements: the U3R region of the HIV-1 IIIB LTR provides the promoter and Tat-responsive element, a modified intron derived from the human c-src gene facilitates the splicing of inserted genes, and the HIV-1 RRE region enhances the transport of unspliced mRNAs. To further limit potential readthrough transcription, the expression cassette was inserted in the reverse transcriptional orientation relative to the retroviral vector LTR. Three different genes, interferon alpha2, diphtheria toxin (DT-A), and cytosine deaminase, were inserted into this vector. Tat and Rev inducibility was demonstrated directly by a >300-fold induction of interferon production and functionally by a decrease in colony-forming units when a Tat and Rev expression vector was titered on HeLa cells harboring the inducible DT-A cassette. The Tat-inducible cytosine deaminase gene was tested in the Sup-T1 T cell line and shown to inhibit HIV-1 production only when engineered cells were grown in the presence of 5-fluorocytosine. To test the ability of this system to inhibit HIV-1 infection in bulk PBL cultures, a series of transduction and challenge experiments was initiated with both the interferon and DT-A vectors. Protection against infection was documented against three HIV strains in PBLs. Last, the interferon and DT-A vectors were compared with a vector encoding a transdominant Rev protein and were shown to mediate equal or greater inhibition of HIV-1.
Assuntos
Toxina Diftérica/biossíntese , Produtos do Gene rev/metabolismo , Produtos do Gene tat/metabolismo , HIV-1/genética , Interferon-alfa/biossíntese , Nucleosídeo Desaminases/biossíntese , Northern Blotting , Linhagem Celular , Citosina Desaminase , Terapia Genética , Vetores Genéticos , Proteína do Núcleo p24 do HIV/química , HIV-1/fisiologia , Humanos , Imuno-Histoquímica , Plasmídeos , Retroviridae/genética , Fatores de Tempo , Transdução Genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We previously reported on the construction of retroviral vectors that produce a secreted form of the HIV-1 receptor, T cell antigen CD4 (Morgan et al., AIDS Res Hum Retroviruses 1990;6:183-191). In this article we test the ability of these sCD4-expressing retroviral vectors to protect human T-cell lines or primary T cells from HIV-1 infection. To demonstrate that protection from HIV-1 infection is mediated by the soluble nature of this protein, two coculture protection experiments were conducted. In these experiments, sCD4-expressing retroviral vectors were used to engineer mouse NIH 3T3 cells. In one coculture experiment the human SupT1 cell line was added directly to the culture of sCD4-producing NIH 3T3 cells, and in another experiment the two cell types were separated physically by a semipermeable membrane. In both coculture configurations, the T cell line was protected from HIV-1 challenge as measured by syncytium formation and indirect immunofluorescent assays. In addition, the SupT1 line was directly engineered with sCD4-expressing retroviral vectors and shown to be protected from HIV-1 challenge. As a prelude to further preclinical studies, we tested the ability of retroviral vectors to transduce primary human peripheral blood lymphocytes (PBLs). Conditions used to stimulate T cell growth resulted in significant shifts in the CD4/CD8 cell in favor of CD8 cells. Retroviral-mediated gene transfer under these conditions resulted in low levels of gene transfer (< 5%).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antígenos CD4/genética , Terapia Genética/métodos , Infecções por HIV/prevenção & controle , HIV-1 , Células 3T3 , Animais , Antígenos CD4/biossíntese , Antígenos CD4/uso terapêutico , Linhagem Celular , Células Cultivadas , Engenharia Genética , Vetores Genéticos , Infecções por HIV/terapia , Humanos , Linfócitos/virologia , Camundongos , Solubilidade , Transdução GenéticaRESUMO
The HIV-1 rev gene product facilitates the transport of singly spliced and unspliced HIV-1 transcripts and is necessary for productive HIV-1 infection. On the basis of the previously described trans-dominant Rev mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral expression vectors and analyzed for their ability to inhibit Rev-mediated gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-dependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expression was tested in the HeLa-T4 cell line and was also shown to be inhibited by the trans-dominant Rev mutants. Retroviral vector producer cell lines were constructed and used to transduce Rev trans-dominant genes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitored by Northern blot analysis and in situ hybridization. SupT1 cells expressing either a Rev point mutant or the frameshift mutant showed greatly reduced HIV-1 mRNA accumulation and the Rev-dependent singly spliced and unspliced HIV-1 mRNAs were reduced. The kinetics of viral replication following challenge of Rev trans-dominant-engineered SupT1 cells with both HIV-1 IIIB and MN strains was significantly reduced and cells were protected from viral lysis. Viruses that emerge late in infection from Rev trans-dominant-engineered cultures are not resistant to Rev-mediated inhibition. Last, trans-dominant Rev-mediated protection of human CD4+ lymphocytes from challenge with primary HIV-1 patient isolates confirms the potential utility of this system as an anti-HIV-1 gene therapy approach.
Assuntos
Produtos do Gene rev/genética , Terapia Genética , Infecções por HIV/terapia , HIV-1/genética , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Mutação da Fase de Leitura , Produtos do Gene env/genética , Produtos do Gene rev/uso terapêutico , Genes Dominantes , Genes Reporter , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação Puntual , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Transfecção , Replicação Viral/fisiologia , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
We describe two cases of coil malpositioning that occurred during endovascular occlusion of saccular basilar tip aneurysms with fibered platinum microcoils. The technique of endovascular coil extraction, accomplished successfully and without complication in both cases, is described and may be applicable to recently available controlled-detachment coil systems.
Assuntos
Aneurisma/terapia , Artéria Basilar , Artérias Cerebrais , Embolização Terapêutica/instrumentação , Migração de Corpo Estranho/terapia , Platina , Adulto , Aneurisma Roto/terapia , Cateterismo/instrumentação , Angiografia Cerebral , Embolização Terapêutica/efeitos adversos , Embolização Terapêutica/métodos , Desenho de Equipamento , Migração de Corpo Estranho/etiologia , Humanos , Masculino , Micromanipulação , Pessoa de Meia-Idade , Radiografia Intervencionista/instrumentação , Radiografia Intervencionista/métodos , Hemorragia Subaracnóidea/etiologia , Propriedades de SuperfícieRESUMO
OBJECTIVE AND IMPORTANCE: Holocord intradural lipoma extending from the cervical to the lumbar spine is an exceedingly rare condition. An extensive review of the literature revealed that only eight other cases have been reported, and none occurred during the contemporary magnetic resonance imaging era. CLINICAL PRESENTATION: A case of holocord intradural lipoma that was partially resected with the aid of carbon dioxide laser and intraoperative ultrasound is reported. TECHNIQUE: The intimate relationship of the lipoma to the nerve roots and the absence of a distinct plane between tumor and spinal cord precluded a complete resection of this tumor. At 7 months postoperatively, the patient's dysesthetic pain had resolved almost completely. Proprioception in the lower extremities had improved significantly to the point that the patient was able to walk without a cane and his preoperative Romberg's sign had disappeared. CONCLUSION: Extensive intradural intramedullary spinal lipomas can present in adulthood with symptoms of myelopathy as well as nonradicular pain. We encourage early surgical debulking of the tumor to prevent further progression of symptoms and to offer the possibility of neurological improvement.
Assuntos
Lipoma/diagnóstico , Bulbo , Neoplasias da Medula Espinal/diagnóstico , Adulto , Feminino , Humanos , Lipoma/cirurgia , Região Lombossacral , Imageamento por Ressonância Magnética , Bulbo/diagnóstico por imagem , Bulbo/patologia , Bulbo/cirurgia , Pescoço , Radiografia , Futebol , Medula Espinal/diagnóstico por imagem , Medula Espinal/patologia , Neoplasias da Medula Espinal/cirurgia , TóraxRESUMO
To clarify the ideal timing of anterior decompression and stabilization for all patients with cervical spine trauma as well as its efficacy for patients with complete deficits, we reviewed the records of 103 consecutive patients with cervical spine trauma (50 incomplete deficits, Group A; 53 complete deficits, Group B) who underwent this procedure during a 5-year period at the Shock Trauma Center. We subdivided each group according to time of surgery: early and delayed (less than 24 and greater than 24 hours past injury, respectively). In Group A, 10 patients underwent early surgery and 40 patients underwent delayed surgery (range, 2 to 77 days past injury; mean, 13 days). One patient (2.5%) in the delayed group died. The following data refer to the early and delayed subgroups, respectively: average acute hospitalization, 20 and 22 days; patient motor score improvement (at discharge), 37.2 and 45.0%; functional grade improvement (at discharge), 5 (50.0%) and 9 (22.5%) patients. At 1-year follow-up, every patient who had had a deficit had progressed to a higher functional grade. In Group B, 35 patients underwent early surgery and 18 underwent delayed surgery (range, 2 to 45 days past injury; mean, 13 days). One patient (2.9%) in the early group died. The following data refer to the early and delayed subgroups, respectively: average acute hospitalization, 38.7 and 45.2 days (P less than 0.05); respiratory care (number of daily suction procedures), 6.0 and 9.86 (P less than 0.05); patient motor score improvement (at discharge), 3.9 and 4.5%; functional grade improvement (at discharge), 4 (11.4%) and 1 (5.6%) patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Vértebras Cervicais/lesões , Vértebras Cervicais/cirurgia , Adolescente , Adulto , Idoso , Placas Ósseas , Feminino , Seguimentos , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Prognóstico , Traumatismos da Medula Espinal/diagnóstico , Traumatismos da Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
Fifty-two patients with acute traumatic bilateral locked facets were treated at one trauma center during a 3 1/2-year period (July, 1987, to December, 1990). The patients presented with complete motor quadriplegia (34 cases), incomplete myelopathy (13 cases), or intact long-tract function (five cases). The injuries occurred at C2-3 (one patient, with intact function), C4-5 (12 patients), C5-6 (16 patients), C6-7 (19 patients), and C7-T1 (four patients). Immediate traction (with increasing weight and serial x-ray studies) and/or induction of general anesthesia and muscle relaxation reduced the dislocation in 40 patients, but 12 needed prompt operative reduction as their injuries failed to reduce within 4 hours. Stabilization was indicated for all patients, but three did not undergo surgery: two elderly patients with complete injuries (one refused surgery and one died), and one patient with multiple injuries (fusion was achieved by halo-vest immobilization for 3 months). Of the 49 patients treated operatively, 23 (44.2%) underwent surgery on the day of injury and 26 on a delayed basis (mean 8.7 days postinjury). Surgical treatment included fusion of the posterior facet to a spinous process (44 cases), an anterior Caspar plate technique (three cases), and both procedures (two cases). Of these 49 patients, three (6.1%) with complete injuries died due to an adult respiratory distress syndrome. Improvement of cord function, judged by functional grade change, was observed at discharge in 15 patients (31.9%) and in 15 (71.4%) of the 21 patients with a 1-year follow-up period. Of the 34 patients with complete myelopathy on admission, three are ambulatory after 1 year, and 13 others have gained function in at least one nerve root. It is concluded that prompt reduction (nonoperative or surgical) and internal stabilization facilitate recovery even in neurologically compromised patients, and that early operative intervention is a wiser option than conservative management. This report also documents a higher incidence of this injury without deficit (five of the 52 cases) than reported in other series.
Assuntos
Vértebras Cervicais/lesões , Vértebras Cervicais/cirurgia , Luxações Articulares/cirurgia , Traumatismos da Medula Espinal/etiologia , Adulto , Feminino , Humanos , Imobilização , Luxações Articulares/complicações , Luxações Articulares/mortalidade , Luxações Articulares/terapia , Masculino , Traumatismos da Medula Espinal/fisiopatologia , Fusão Vertebral/métodos , Fatores de Tempo , TraçãoRESUMO
OBJECTIVE AND IMPORTANCE: Adrenocortical carcinoma (ACC) is rare in the pediatric population, and brain metastasis seldom occurs. CLINICAL PRESENTATION: The authors report a case of metastatic ACC to the brain in a 9-year-old patient who had an adrenal cortex neoplasm removed at 4 years of age, and was free of symptoms for 5 years. Two weeks before admission she complained of blurred vision in both eyes. INTERVENTION: Examination revealed bilateral papilledema, and a Magnetic Resonance Imaging (MRI) of the brain revealed a mass in the left lateral ventricle with extensive vasogenic edema and hydrocephalus. The tumor was removed, and histopathologic examination demonstrated metastatic ACC. CONCLUSION: Although ACC is a rare neoplasm it must be considered in the differential diagnosis of cerebral lesions in patients with a history of this tumor. Periodic long-term brain imaging is suggested as part of the follow up in patients with adrenocortical neoplasms.
Assuntos
Neoplasias do Córtex Suprarrenal/diagnóstico , Neoplasias do Córtex Suprarrenal/cirurgia , Carcinoma Adrenocortical/diagnóstico , Carcinoma Adrenocortical/cirurgia , Neoplasias do Ventrículo Cerebral/diagnóstico , Neoplasias do Ventrículo Cerebral/cirurgia , Craniotomia/métodos , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/secundário , Neoplasias do Ventrículo Cerebral/patologia , Neoplasias do Ventrículo Cerebral/secundário , Criança , Pré-Escolar , Feminino , Humanos , Hidrocefalia/etiologia , Imageamento por Ressonância Magnética , Recidiva , Reoperação , Resultado do TratamentoRESUMO
OBJECTIVE: Incomplete resection of the epileptogenic zone (EZ) is the most important predictor of poor outcome after resective surgery for intractable epilepsy. We analyzed the contribution of preoperative and perioperative variables including MRI and EEG data as predictors of seizure-free (SF) outcome after incomplete resection. METHODS: We retrospectively reviewed patients <18 years of age with incomplete resection for epilepsy with 2 years of follow-up. Fourteen preoperative and perioperative variables were compared in SF and non-SF (NSF) patients. We compared lesional patients, categorized by reason for incompleteness, to lesional patients with complete resection. We analyzed for effect of complete EEG resection on SF outcome in patients with incompletely resected MRI lesions and vice versa. RESULTS: Eighty-three patients with incomplete resection were included with 41% becoming SF. Forty-eight lesional patients with complete resection were included. Thirty-eight percent (57/151) of patients with incomplete resection and 34% (47/138) with complete resection were excluded secondary to lack of follow-up or incomplete records. Contiguous MRI lesions were predictive of seizure freedom after incomplete resection. Fifty-seven percent of patients incomplete by MRI alone, 52% incomplete by EEG alone, and 24% incomplete by both became SF compared to 77% of patients with complete resection (p = 0.0005). CONCLUSIONS: Complete resection of the MRI- and EEG-defined EZ is the best predictor of seizure freedom, though patients incomplete by EEG or MRI alone have better outcome compared to patients incomplete by both. More than one-third of patients with incomplete resection become SF, with contiguous MRI lesions a predictor of SF outcome.
Assuntos
Epilepsia/cirurgia , Neurocirurgia/métodos , Convulsões/patologia , Adolescente , Criança , Pré-Escolar , Intervalo Livre de Doença , Eletroencefalografia/métodos , Epilepsia/patologia , Feminino , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética/métodos , Masculino , Exame Neurológico/métodos , Valor Preditivo dos Testes , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Focal cortical dysplasia (FCD) is recognized as the major cause of focal intractable epilepsy in childhood. Various factors influencing postsurgical seizure outcome in pediatric patients with FCD have been reported. OBJECTIVE: To analyze different variables in relation to seizure outcome in order to identify prognostic factors for selection of pediatric patients with FCD for epilepsy surgery. METHODS: A cohort of 149 patients with histologically confirmed mild malformations of cortical development or FCD with at least 2 years of postoperative follow-up was retrospectively studied; 113 subjects had at least 5 years of postoperative follow-up. Twenty-eight clinical, EEG, MRI, neuropsychological, surgical, and histopathologic parameters were evaluated. RESULTS: The only significant predictor of surgical success was completeness of surgical resection, defined as complete removal of the structural MRI lesion (if present) and the cortical region exhibiting prominent ictal and interictal abnormalities on intracranial EEG. Unfavorable surgical outcomes are mostly caused by overlap of dysplastic and eloquent cortical regions. There were nonsignificant trends toward better outcomes in patients with normal intelligence, after hemispherectomy and with FCD type II. Other factors such as age at seizure onset, duration of epilepsy, seizure frequency, associated pathologies including hippocampal sclerosis, extent of EEG and MRI abnormalities, as well as extent and localization of resections did not influence outcome. Twenty-five percent of patients changed Engel's class of seizure outcome after the second postoperative year. CONCLUSIONS: The ability to define and fully excise the entire region of dysplastic cortex is the most powerful variable influencing outcome in pediatric patients with focal cortical dysplasia.
Assuntos
Hemisferectomia/normas , Malformações do Desenvolvimento Cortical/complicações , Malformações do Desenvolvimento Cortical/cirurgia , Convulsões/etiologia , Convulsões/fisiopatologia , Adolescente , Adulto , Criança , Estudos de Coortes , Eletroencefalografia , Feminino , Seguimentos , Hipocampo/patologia , Humanos , Inteligência , Imageamento por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical/diagnóstico , Malformações do Desenvolvimento Cortical/psicologia , Estudos Retrospectivos , Esclerose , Convulsões/diagnóstico , Resultado do Tratamento , Adulto JovemRESUMO
Moloney murine leukemia virus ecotropic envelope expression plasmids were used to demonstrate that the synthesis of the retroviral envelope SU and TM polypeptides can be uncoupled with retention of biologic activity. By substituting a glycosyl-phosphatidylinositol (GPI) membrane anchor for part or all of the retroviral envelope transmembrane protein and creating several deletion variants of the TM subunit, we have begun to dissect the role of the TM protein in envelope function. We show that a GPI-anchored envelope can be incorporated into virions and binds receptor. We found that the envelope cytoplasmic tail, while not required, influences the efficiency of retroviral transduction at some step after membrane fusion (possibly by interacting with core). The membrane-spanning domain of TM is involved in membrane fusion, and this function is distinct from its role as a membrane anchor. As few as eight amino acids of the putative membrane-spanning domain are sufficient to achieve membrane anchoring of envelope but not to mediate membrane fusion. In addition, though not required, the membrane-spanning domain may have some direct role in the incorporation of envelope into virions. Finally, the extracellular domain of TM, besides containing the putative fusion domain and interacting with SU, may influence the synthesis or stability and the glycosylation of envelope, possibly by affecting oligomerization of the complex and proper intracellular transit.
Assuntos
Proteínas de Fase Aguda/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Vírus da Leucemia Murina de Moloney/crescimento & desenvolvimento , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/metabolismo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Transporte Biológico , Análise Mutacional de DNA , Fusão de Membrana , Camundongos , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/metabolismo , Ligação Proteica , Receptores Virais/metabolismo , Proteínas Recombinantes/biossíntese , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
Cell-cell contact and exogenous cAMP regulate the expression of uridine diphosphoglucose pyrophosphorylase (UDPGP) of Dictyostelium discoideum (B. Haribabu, A. Rajkovic and R. P. Dottin, 1986, Dev. Biol., Vol. 113, 436-442). cAMP appears to regulate gene expression in Dictyostelium by transmembrane signal transduction (B. Haribabu and R. Dottin, 1986, Mol. Cell. Biol. 6, 2402-2408). To further characterize the mechanism of action of cAMP on the expression of this gene and the nature of the defects in UDPGP mutants that abort development, we sequenced the cDNA and the genomic DNA, including intervening and flanking sequences. The deduced amino acid sequence predicts a polypeptide of 57,893 d. molecular weight. Three short (100-200 nucleotides) A+T rich introns occur within the coding sequences but only one of them contains a sequence TAACTAAC, similar to the yeast lariat acceptor site. The 5' flanking sequences are also A+T rich and contain an oligo A tract (-14 to -24), a TATA box (-25 to -32), and a short G+C rich region (-63 to -101) which may be a control region. From -196 to -209 is a sequence AAAGTAGTATTCAA which matches in 11 of its 14 nucleotides, a sequence found upstream from the hormonally regulated P-enolypyruvate carboxykinase gene of rat.
Assuntos
Dictyostelium/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Nucleotidiltransferases/genética , UTP-Glucose-1-Fosfato Uridililtransferase/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , DNA Fúngico/genética , Dictyostelium/enzimologia , GenesRESUMO
Murine leukemia virus ecotropic and amphotropic envelope expression vectors were genetically engineered to generate truncations of the p15E TM cytoplasmic tail. The ecotropic construct CEET has the entire cytoplasmic tail of TM deleted, while the CEETR construct has only the R peptide portion of the tail deleted, thereby producing a TM subunit (p12E) that is identical to the one found in mature virions. The analogous amphotropic constructs were called CAET and CAETR. These envelopes, as opposed to their p15E TM counterparts, mediate cell-to-cell fusion at neutral pH in both transformed and nontransformed cell lines. Though the TM cytoplasmic domain is not required, its presence appears to augment such cell-to-cell fusion. This envelope-dependent fusion requires the presence of the viral receptor on the cell surface. Ecotropic virions bearing the p12E TM contain wild-type levels of the envelope complex and have near-normal titers. In contrast, virions which lack the cytoplasmic domain of TM (e.g., CEET) have 10- to 100-fold-lower titers but contain normal amounts of envelope. Both of the corresponding amphotropic virions contain normal amounts of envelope but have 10- to 100-fold-lower titers. Using immunofluorescent detection of envelope to monitor the fate of receptor-bound virions, we found that ecotropic murine leukemia virus envelope disappears from the cell surface while amphotropic envelope persists on the cell surface after virus binding. This pattern of immunofluorescence is consistent with the proposed routes of cell entry for these viruses, i.e., by endocytosis and direct fusion, respectively. In this assay, ecotropic virions bearing the genetically engineered p12E TM also appear to be internalized despite the ability of their envelope to mediate fusion at neutral pH in the same target cells. Our results show that direct fusion at neutral pH is a natural consequence of the surface expression of the mature ecotropic envelope and its receptor. We propose that the processing of the R peptide from the envelope TM (p15E) to yield p12E, at the time of virus budding or within virions, renders the envelope competent to fuse.
Assuntos
Fusão Celular , Vírus da Leucemia Murina/fisiologia , Fragmentos de Peptídeos/metabolismo , Proteínas Oncogênicas de Retroviridae/fisiologia , Proteínas do Envelope Viral/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Células CHO , Cricetinae , Técnicas de Transferência de Genes , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Vírus da Leucemia Murina/classificação , Fusão de Membrana , Camundongos , Dados de Sequência Molecular , Morfogênese , Mutação , Fragmentos de Peptídeos/genética , Provírus , Receptores Virais/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Oncogênicas de Retroviridae/genética , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/crescimento & desenvolvimentoRESUMO
Two patients with complex aneurysms of the vertebrobasilar system were treated using platinum coils. During these sessions, 22 coils were placed in a basilar tip aneurysm in a 48-year-old woman. Almost total occlusion of the lumen was achieved, excepted in the area from which the right posterior cerebral artery arose. A large fusiform vertebral artery aneurysm in a 6-year-old boy was treated using 16 coils, which partially occluded the lumen at initial embolization. Follow-up angiography 2 weeks later revealed almost complete occlusion of the lumen with preservation of the vertebral artery. Our experience indicates that coil embolization of complex intracranial aneurysms is an acceptable treatment alternative to surgery or to a detachable balloon.
Assuntos
Aneurisma/terapia , Artéria Basilar , Embolização Terapêutica , Artéria Vertebral , Adulto , Aneurisma/diagnóstico , Aneurisma/diagnóstico por imagem , Artéria Basilar/diagnóstico por imagem , Artéria Basilar/patologia , Criança , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Radiografia , Artéria Vertebral/diagnóstico por imagem , Artéria Vertebral/patologiaRESUMO
To investigate the role that translation plays in the stabilization of the IL-2 mRNA, we inhibited protein synthesis in both cis and trans. To block translation in trans, we utilized the inhibitors puromycin (PUR) and cycloheximide (CHX), which differentially effect polysome structure. We found that CHX enhances the stability of IL-2 mRNA in cells stimulated with anti-TCR Ab alone, but it inhibits CD28-induced message stabilization in costimulated cells. In contrast, PUR had a minimal effect on IL-2 mRNA stability in either the presence or absence of costimulation. The differential effects of these two inhibitors suggest that: 1) CHX is unlikely to stabilize the IL-2 mRNA by inhibiting the expression of a labile RNase; 2) CD28-mediated IL-2 mRNA stabilization does not require translation; and 3) IL-2 mRNA decay is not coupled to translation. To block translation in cis, we generated sequence-tagged IL-2 genomic reporters that contain a premature termination codon (PTC). In both the presence and absence of costimulation, these PTC-containing mRNAs exhibit drastically diminished stability. Interestingly, the addition of CHX but not PUR completely restored CD28-mediated stabilization, suggesting that CHX can block the enhanced decay induced by a PTC. Finally, CHX was able to superinduce IL-2 mRNA levels in anti-TCR Ab-stimulated cells but not in CD28-costimulated cells, suggesting that CHX may also act by other mechanisms.
Assuntos
Códon de Terminação/metabolismo , Interleucina-2/genética , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Transativadores/farmacologia , Animais , Antígenos CD28/fisiologia , Células Cultivadas , Códon de Terminação/efeitos dos fármacos , Códon de Terminação/imunologia , Cicloeximida/farmacologia , Genes Reporter/imunologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Camundongos , Mimetismo Molecular , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Terminação Traducional da Cadeia Peptídica/genética , Terminação Traducional da Cadeia Peptídica/imunologia , Biossíntese de Proteínas/imunologia , Puromicina/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Sitios de Sequências RotuladasRESUMO
Using sequence-tagged genomic reporter constructs, we investigated the contribution of IL-2 sequences to CD28-mediated regulation of mRNA stability. We find that CD28 signaling acts transiently to stabilize the IL-2 mRNA following T cell activation. Such stabilization requires sequences within both exon 2 and the coding region of exon 4. Unexpectedly, CD28 signaling at later times enhances the decay of the IL-2 mRNA. This CD28-dependent decay of IL-2 mRNA requires sequences localized between exon 3 and the stop codon. Our findings demonstrate that the coding region of the IL-2 mRNA contains previously undefined CD28-responsive sequence elements that are critical for the regulation of mRNA stability.
Assuntos
Antígenos CD28/fisiologia , Éxons/imunologia , Interleucina-2/genética , Interleucina-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Animais , Células Clonais , Éxons/genética , Genes Reporter/imunologia , Vetores Genéticos/síntese química , Vetores Genéticos/imunologia , Interleucina-2/química , Ativação Linfocitária/genética , Camundongos , RNA Mensageiro/química , Sitios de Sequências Rotuladas , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th1/imunologia , Transfecção/imunologiaRESUMO
The murine leukemia virus (MuLV) envelope protein was examined to determine which sequences are responsible for the differences in direct membrane fusion observed with the ecotropic and amphotropic MuLV subtypes. These determinants were studied by utilizing amphotropic-ecotropic chimeric envelope proteins that have switched their host range but retain their original fusion domain (TM subunit). Fusion was tested both in rodent cells and in 293 cells bearing the human homolog of the ecotropic MuLV receptor. The results demonstrate that the amphotropic TM is able to mediate cell-to-cell fusion to an extent equivalent to that mediated by the ecotropic TM, indicating that their fusion domains are equivalent. The "murinized" human homolog of the ecotropic receptor supports syncytium formation as well as the native murine receptor. These findings suggest that interactions between the ecotropic envelope protein and conserved sequences in the ecotropic receptor are the principal determinants of syncytium formation. The relationship of the fusion phenotype to pH-dependent infection and the route of viral entry was examined by studying virions bearing the chimeric envelope proteins. Such virions appear to enter cells via a pathway that is directed by the host range-determining region of their envelope rather than by sequences that confer pH dependence. Therefore, the pH dependence of infection may not reflect the initial steps in viral entry. Thus, it appears that both the syncytium phenotype and the route of viral entry are properties of the viral receptor, the amino-terminal half of the ecotropic envelope protein, or the interaction between the two.
Assuntos
Vírus da Leucemia Murina/fisiologia , Fusão de Membrana , Receptores Virais/fisiologia , Proteínas do Envelope Viral/fisiologia , Células 3T3 , Animais , Fusão Celular , Linhagem Celular , Células Gigantes , Humanos , Concentração de Íons de Hidrogênio , Cinética , Vírus da Leucemia Murina/classificação , Vírus da Leucemia Murina/patogenicidade , Substâncias Macromoleculares , Camundongos , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Fatores de Tempo , Transfecção , Proteínas do Envelope Viral/biossínteseRESUMO
The consequences of T-cell receptor engagement (signal 1) are profoundly affected by the presence or absence of co-stimulation (signal 2). T-cell receptor (TCR) stimulation in the absence of CD28-mediated co-stimulation not only results in little interleukin (IL)-2 production, but induces a long lasting hyporesponsive state known as T-cell clonal anergy. The addition of CD28 ligation to signal 1, on the other hand, results in the production of copious amounts of IL-2. Our laboratory has utilized CD4+ Th 1 clones in an effort to understand the molecular events resulting in enhanced IL-2 production by co-stimulation and the inhibition of IL-2 production in anergy. Our current studies have focused on defining the post-transcriptional effects of CD28-enhanced IL-2 production. The data suggest that a major component of CD28's ability to regulate IL-2 production occurs at the level of message stability and involves the 3'-untranslated region of the message. In terms of anergy, our recent studies support the notion that it is not the result of TCR engagement in the absence of co-stimulation, but rather signal 1 in the absence of IL-2 receptor signaling and proliferation. Furthermore, T-cell anergy appears to be an active negative state in which IL-2 production is inhibited both at the level of signal transduction and by cis-dominant repression at the level of the IL-2 promoter.