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BACKGROUND: The World Health Organization (WHO) surgical checklist is associated with reduced morbidity and mortality. Efficacy correlates with compliance. OBJECTIVE: This study aims to (1) establish completion rate and (2) identify and address barriers to use. METHODS: Records of patients undergoing dermatological surgery were studied. Staff completed attitude and barriers questionnaires. Checklist process was modified, and use was reassessed twice. RESULTS: Cycle 1 involved 217 subjects; 72% had excisions. Thirteen percent had surgery to multiple sites. Five percent of checklists were fully completed, with an average of 76% of available points per checklist marked as checked. The lowest single field use included "patient identity" (76%) and "surgical site" (72%). Questionnaire responses from 25 staff showed the checklist to be "important" and "relevant" in dermatology; key barrier to completion was lack of time. Checklist modifications and educational sessions were undertaken; checklist use was reassessed twice more with 103 and 134 patients. Average use increased to 96% and 98%; full completion increased to 71% and 70%; "surgical site" and "identity" completion increased to 100%. CONCLUSION: The WHO checklist is relevant and important in dermatology. Introduction must be supported by repeated training sessions. Adequate time and training can significantly improve checklist completion and patient safety.
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Lista de Checagem/normas , Dermatologia/normas , Segurança do Paciente/normas , Procedimentos Cirúrgicos Operatórios/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Atitude do Pessoal de Saúde , Feminino , Fidelidade a Diretrizes/normas , Acessibilidade aos Serviços de Saúde/normas , Humanos , Masculino , Pessoa de Meia-Idade , Salas Cirúrgicas/normas , Organização Mundial da Saúde , Adulto JovemRESUMO
This corrects the article DOI: 10.1038/leu.2016.388.
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B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3É (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.
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Anticorpos Biespecíficos/uso terapêutico , Antígeno de Maturação de Linfócitos B/imunologia , Complexo CD3/imunologia , Mieloma Múltiplo/terapia , Linfócitos T/imunologia , Animais , Apoptose , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Ativação Linfocitária , Macaca fascicularis , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The role of vaccination in the control and prevention of endemic and emerging diseases cannot be overemphasized. Induction of host protective immunity may be the most powerful tool and effective strategy in preventing the spread of potentially fatal disease and emerging illnesses, in particular in susceptible immunologically naive hosts. The strategy for vaccination programs is engrained in population studies recognizing benefit for the health and economic welfare of at-risk indigenous populations. Worldwide collaboration is a necessary aspect of vaccine-preventable diseases recognizing that even a small number of wild-type cases of an eradicated disease in one region presents opportunities for re-emergence of the disease in geographically remote areas.
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Erradicação de Doenças/métodos , Saúde Pública/métodos , Vacinação/métodos , Criança , Saúde Global , Humanos , Vacinas/imunologiaRESUMO
The oil sands of northern Alberta, Canada contain an estimated 170 billion barrels of crude oil. Extraction processes produce large amounts of liquid tailings known as oil sand process affected water (OSPW) that are toxic to aquatic organisms. Naphthenic acids (NAs), and their sodium salts, represent a significant contributor to the toxicity of these waters. Due to the recalcitrant nature of these compounds, an effective mode of remediation has yet to be established. This study investigates the suitability of the use of phytoplankton for remediation efforts based on two criteria: the ability of phytoplankton strains to withstand the toxic effects of NAs, and their rate of biomass accumulation. A total of 21 phytoplankton strains were isolated from waters containing NAs, cultured, and maintained under unialgal conditions. These strains were then exposed to NAs in concentrations ranging from 0mg L(-1) to 1000mg L(-1) over a 14 day period. Inhibition of growth was observed at 30mg L(-1) NA (one strain), 100mg L(-1) NA (one strain), 300mg L(-1) NA (six strains), and 1000mg L(-1) NA (six strains). Five strains failed to show any growth inhibition at any test concentration and two strains could not be analysed due to poor growth during the test period. Strains were then ranked based on their suitability for use in remediation efforts.
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Biomassa , Ácidos Carboxílicos/toxicidade , Petróleo/toxicidade , Fitoplâncton/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Alberta , Biodegradação Ambiental , Cianobactérias/efeitos dos fármacos , Cianobactérias/crescimento & desenvolvimento , Euglenozoários/efeitos dos fármacos , Euglenozoários/crescimento & desenvolvimento , Campos de Petróleo e Gás , Fitoplâncton/crescimento & desenvolvimento , Poluentes Químicos da Água/análiseRESUMO
In order to identify potential markers of renal cancer, the plasma membrane protein content of renal cell carcinoma (RCC)-derived cell lines was annotated using a proteomics process. One unusual protein identified at high levels in A498 and 786-O cells was CD70 (TNFSF7), a type II transmembrane receptor normally expressed on a subset of B, T and NK cells, where it plays a costimulatory role in immune cell activation. Immunohistochemical analysis of CD70 expression in multiple carcinoma types demonstrated strong CD70 staining in RCC tissues. Metastatic tissues from eight of 11 patients with clear cell RCC were positive for CD70 expression. Immunocytochemical analysis demonstrated that binding of an anti-CD70 antibody to CD70 endogenously expressed on the surface of A498 and 786-O cell lines resulted in the rapid internalisation of the antibody-receptor complex. Coincubation of the internalising anti-CD70 antibody with a saporin-conjugated secondary antibody before addition to A498 cells resulted in 50% cell killing. These data indicate that CD70 represents a potential target antigen for toxin-conjugated therapeutic antibody treatment of RCC.
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Ligante CD27/genética , Ligante CD27/imunologia , Carcinoma de Células Renais/imunologia , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Renais/imunologia , Anticorpos/farmacologia , Reações Antígeno-Anticorpo , Ligante CD27/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Ligação Proteica , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
STUDY DESIGN: A case report of blunt cervical spine trauma associated with cord oedema at the C3/C4 level with temporary Frankel/American Spinal Injury Association Grade A quadriparesis and motion segment instability without evidence of associated bony lesions (spinal cord injury without radiological abnormality, SCIWORA lesion). OBJECTIVES: By means of a rare and illustrative case, the reader's attention is focused on eventual marked cervical motion segment instability in SCIWORA patients. SETTING: A department of Neurology in Quito, Ecuador and a department of Neurosurgery in Bern, Switzerland. METHOD: A 73-year-old man sustained blunt cervical spine trauma. After resolution of paraparesis, dynamic studies of the cervical spine revealed translational instability of C3 over C4. The patient underwent segment fusion by intervertebral cage insertion and plate fixation. RESULTS: The patient had recovered almost completely from tetraparesis under conservative treatment. The postoperative course was uneventful. Solid bony fusion of the C3/C4 motion segment was obtained. CONCLUSION: Despite normal cervical alignment, the lack of bony lesions and neurological recovery, magnetic resonance imaging and dynamic studies may reveal marked translational cervical motion segment instability requiring segment fusion in order to prevent ongoing damage of the spinal cord.
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Vértebras Cervicais/lesões , Edema/complicações , Quadriplegia/complicações , Traumatismos da Medula Espinal/complicações , Idoso , Terapia Combinada , Humanos , Imageamento por Ressonância Magnética , Masculino , Quadriplegia/patologia , Quadriplegia/terapia , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Fusão Vertebral , Resultado do TratamentoRESUMO
B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.
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Linfócitos B/química , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Membrana/isolamento & purificação , Proteínas de Neoplasias/isolamento & purificação , Proteômica , Proteínas Reguladoras de Apoptose , Linfócitos B/patologia , Sequência de Bases , Western Blotting , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fases de Leitura Aberta , Isoformas de Proteínas , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
hAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT - PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P=0.0002; hAG-3, P=0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P=0.003). Yeast two-hybrid cloning identified metastasis-associated GPI-anchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.
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Peptídeos Catiônicos Antimicrobianos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Estrogênio/análise , Proteínas de Xenopus , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Diferenciação Celular , Cromossomos Humanos Par 7/genética , Distroglicanas , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica , Humanos , Magaininas , Dados de Sequência Molecular , Metástase Neoplásica , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Mapeamento Físico do Cromossomo , Proteínas de Plantas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-HíbridoRESUMO
While it is well established that phenotypic modulation of vascular smooth muscle cells (VSMCs) contributes to the development and progression of vascular lesions, little is known regarding the molecular mechanisms of phenotypic modulation in vivo. Here we show that vascular injury reduces transcription of VSMC differentiation marker genes, and we identify cis regulatory elements that may mediate this decrease. Using a carotid wire-injury model in mice carrying transgenes for smooth muscle alpha-actin, smooth muscle myosin heavy chain, or a SM22alpha promoter-beta-gal reporter, we collected arteries 7 and 14 days after injury and assessed changes in endogenous protein and mRNA levels and in beta-gal activity. Endogenous levels for all markers were decreased 7 days after injury and returned to nearly control levels by 14 days. beta-gal staining in all lines followed a similar pattern, suggesting that transcriptional downregulation contributed to the injury-induced decreases. To begin to dissect this response, we mutated a putative G/C-rich repressor in the SM22alpha promoter transgene and found that this mutation significantly attenuated injury-induced downregulation. Hence, transcriptional downregulation contributes to injury-induced decreases in VSMC differentiation markers, an effect that may be partially mediated through a G/C-rich repressor element.
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Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Sequência de Bases , Biomarcadores , Diferenciação Celular , Primers do DNA/genética , Expressão Gênica , Genes Reporter , Hibridização In Situ , Óperon Lac , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , beta-Galactosidase/genéticaRESUMO
Transforming growth factor beta (TGF-beta) is implicated in the regulation of smooth muscle cell (SMC) differentiation. We previously identified a novel TGF-beta control element (TCE) in the promoters of SMC differentiation marker genes, including alpha-smooth muscle actin and SM22alpha. In this study, the importance of the TCE in regulation of SM22alpha gene expression in vivo was investigated by mutating it within the context of a mouse SM22alpha promoter-lacZ transgenic construct. Mutation of the TCE completely abolished SM22alpha promoter activity in arterial SMCs as well as in developing heart and skeletal muscle. To identify the transcription factor(s) binding to the TCE, we performed yeast one-hybrid cloning analysis and identified gut-enriched Krüppel-like factor (GKLF). However, cotransfection studies in cultured cells showed that GKLF repressed the TGF-beta-dependent increases in SM22alpha and alpha-smooth muscle actin promoter activities. Furthermore, GKLF was not highly expressed in differentiated SMCs in vivo, and TGF-beta down-regulated GKLF expression in dedifferentiated cultured SMCs. In contrast, overexpression of a related factor (BTEB2) transactivated SM22alpha promoter activity. Thus, our findings suggest a reciprocal role for related Krüppel-like transcription factors in the regulation of SMC differentiation through a TCE-dependent mechanism.
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Biomarcadores , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso/citologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Aorta Torácica/citologia , Sequência de Bases , DNA , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Camundongos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Transforming growth factor beta (TGFbeta) is thought to play an important role in the development and/or progression of a number of vascular disorders through its numerous effects on vascular smooth muscle cells (VSMCs). In this study we sought to identify and characterize TGFbeta-regulated VSMC genes using differential mRNA display (DD-RT-PCR) analysis of RNA isolated from TGFbeta-stimulated cultured rat aortic VSMCs. Northern blot analysis was used to demonstrate that five of 19 differentially displayed bands identified represented VSMC transcripts differentially expressed by TGFbeta. DNA sequencing revealed that three of these TGFbeta regulated genes were novel whilst the remaining two were identified through homologies to known genes. One TGFbeta upregulated transcript represented the protease cathepsin B. Since cathepsins may play a role in TGFbeta activation, an enzyme-linked immunosorbent assay (ELISA) for active TGFbeta1 was used to demonstrate an effect of cathepsin B on TGFbeta1 activation in vitro using both recombinant and human serum platelet-derived latent TGFbeta1 as substrate. These results suggest that induction of cathepsin B by TGFbeta, and its ability to activate TGFbeta1, may represent a mechanism whereby the autocrine action of TGFbeta is facilitated through expression of a protein which can process its latent form.
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Regulação da Expressão Gênica , Fator de Crescimento Transformador beta/metabolismo , Animais , Northern Blotting , Catepsina B/genética , Catepsina B/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Músculo Liso Vascular/citologia , RNA Mensageiro , Ratos , Ratos Wistar , Análise de Sequência de DNA , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Transforming growth factor-beta (TGF-beta) has been shown to stimulate smooth muscle (SM) alpha-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-beta control element (TCE) located within the first 125 bp of the SM alpha-actin promoter were required for TGF-beta inducibility of SM alpha-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-beta induction of SM alpha-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-beta can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22alpha, h(1) calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-beta inducibility of SM alpha-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from (-155) completely repressed TGF-beta responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-beta enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-beta also stimulated expression of the SMC differentiation marker SM22alpha in non-SMCs. However, in contrast to SMCs, TGF-beta did not induce expression of h(1) calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-beta-induced expression of SM alpha-actin in SMCs and non-SMCs that is modified by a complex interplay of positive- and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-beta stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-beta alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.
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Actinas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Actinas/genética , Sequência de Aminoácidos/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Bovinos , Células Cultivadas , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/citologia , Cadeias Pesadas de Miosina/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Ratos , Fator de Resposta Sérica , CalponinasRESUMO
In 1992 a new approach for identifying differentially expressed genes was described by Liang and Pardee (1). Their method allowed the simultaneous differential display of mRNA from two or more cell types by means of the polymerase chain reaction (PCR). In a subsequent study demonstrating the technique Bauer, et al. (2) named it differential display reverse transcription PCR (DDRTPCR) in accordance with the series of steps required to identify differences in gene expression. Differential mRNA display offered a number of advantages over existing cDNA library hybridization based methods used to identify differentially expressed genes. Firstly, screening is fast, reproducible, and technically easier than existing protocols, especially since cDNA libraries do not need to be constructed. Secondly, PCR amplification not only detects low abundance mRNA species, but also allows the comparison of gene expression from very small amounts of starting material such as vascular biopsies.
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Endoglin, the gene linked to the autosomal dominant vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1), encodes a 95-kDa membrane-bound proteoglycan which binds TGF beta 1 and regulates signaling via the type I and II TGF beta receptors on the surface of vascular endothelial cells. Using reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analysis we have shown that endoglin mRNA is expressed in both cultured human VSMCs and VSMCs freshly isolated from human aortas. Northern blot analysis was also used to demonstrate that endoglin expression decreased in serum-stimulated cultured human VSMCs but could be maintained by exogenous TGF beta 1. Endoglin protein expression in human VSMCs was shown by immunocytochemistry. These data, the first describing the existence of endoglin in VSMCs, suggest that through regulating TGF beta 1 signaling endoglin may mediate the effects of TGF beta 1 on VSMC behavior in vitro and in vivo.
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Músculo Liso Vascular/metabolismo , RNA Mensageiro/biossíntese , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/genética , Antígenos CD , Aorta Torácica , Sequência de Bases , Células Cultivadas , Endoglina , Feminino , Humanos , Dados de Sequência Molecular , Músculo Liso Vascular/química , Músculo Liso Vascular/efeitos dos fármacos , Proteoglicanas/biossíntese , Proteoglicanas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/isolamento & purificação , Receptores de Superfície Celular , Fator de Crescimento Transformador beta/farmacologia , Molécula 1 de Adesão de Célula Vascular/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/isolamento & purificaçãoRESUMO
Replication-deficient adenoviral vectors have been widely used for gene transfer with the aim of delivering genes of interest to investigate their function and potentially to treat human disease. The ability to critically evaluate the biological role of a gene of interest, using adenovirus-based vectors, has been hampered by the development of local inflammation at the site of delivery. We have demonstrated that high multiplicity infection of human VSMCs with a replication-deficient adenoviral vector expressing no transgene leads to activation of the transcription factor NF kappa B. Activation of NF kappa B by this mechanism was able to augment gene expression from the human cytomegalo-virus immediate-early promoter (CMV-IEP) and induce expression of the adhesion molecule ICAM-1 in human VSMCs. These effects were inhibited by pretreatment with N alpha-p-tosyl1-L-lysine chloromethyl ketone (TLCK), a serine protease inhibitor known to inhibit the activation of NF kappa B. This important effect of the vector itself may have profound implications when replication-deficient adenoviral vectors are used for experimental gene transfer at a high multiplicity of infection.
Assuntos
Adenoviridae , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Músculo Liso Vascular/metabolismo , NF-kappa B/genética , Células Cultivadas , Citomegalovirus/genética , Genes Precoces , Vetores Genéticos , Humanos , Molécula 1 de Adesão Intercelular/genética , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Ativação Transcricional , Carga Viral , Replicação ViralRESUMO
OBJECTIVE: Medial vascular smooth muscle cells (VSMCs) in healthy vessels are phenotypically distinct from their intimal counterparts in vascular disease. To compare the genes expressed in these phenotypes we have previously performed a differential cDNA library screen on cultured rat VSMCs. The aim of this study was to identify and characterise a 2.8 kb transcript, 2E10, which was highly expressed in freshly dispersed rat aortic VSMCs and downregulated in multiply passaged cultured VSMCs. METHODS: Sequence analysis was used to identify the 2.8 kb rat cDNA. After trypsinisation of proliferating cultured rat and human VSMCs, or enzymatic digestion of aortic tunica media, total cytoplasmic RNA was isolated from VSMCs by lysis in Nonidet P-40 and extraction in phenol; 15 micrograms of total cytoplasmic RNA was used in Northern blot analysis with a 32P-[dCTP]-labelled 2E10 cDNA probe. 35S-[dATP]-labelled 2E10 riboprobe was hybridised in situ to frozen sections of normal and diseased human coronary arteries. RESULTS: DNA sequencing identified 2E10 as a rat polyubiquitin which is homologous to the human polyubiquitin, UbC. Northern blot analysis showed that this polyubiquitin was more highly expressed in differentiated, freshly dispersed rat and human aortic VSMCs compared with their dedifferentiated proliferating counterparts. This also identified a 3.2 kb transcript cross-reacting with the polyubiquitin probe which is specific to differentiated rat VSMCs only. However, expression in growth arrested and proliferating VSMCs was identical, suggesting that UbC does not have a role in VSMC growth arrest. In situ hybridisation of the polyubiquitin riboprobe to sections of diseased human coronary arteries indicated much higher expression in medial than in intimal VSMCs. Northern blot analysis of RNA from the developing rat aorta showed that polyubiquitin expression increased substantially after week 2 of neonatal life, coincident with expression of VSMC-specific contractile proteins. CONCLUSIONS: The greater expression of a UbC polyubiquitin transcript in contractile, differentiated VSMCs compared with proliferating, synthetic VSMCs provides a new gene marker for the phenotypic characterisation of VSMCs in vivo. This, and the finding that the developmental induction of expression of polyubiquitin (UbC) mirrors that of VSMC contractile proteins, suggests that ubiquitin, a protein known to associate with and degrade contractile proteins in skeletal muscle, is involved in the function or maintenance of the contractile phenotype of VSMCs.