RESUMO
The ocular involvement in psoriasis is not a completely well-known problem. The ophthalmologic involvement occurs in about 10 % of patients, particularly in case of arthropathic or pustular psoriasis. Ocular lesions are more common in males, and they often occur during psoriasis exacerbations. Our study aimed to assess the prevalence and type of ocular involvement in psoriasis, by a comparison between psoriasis and healthy subjects, and if/how a 12-week long systemic immunosuppressive therapy is able to modify them. This study involved thirty-two psoriatic patients and thirty-two healthy subjects. Dermatological evaluation was done using Psoriasis Area and Severity Index, Physician Global Assessment, and Dermatology Life Quality Index (PASI, PGA, and DLQI score). Ophthalmological evaluation included ocular surface involvement (Schirmer, Jones, break-up time--BUT, DR-1 camera), retinal pathologies, and ocular surface disease index. Laboratory investigations including the C-reactive protein (CRP) of all the patients were performed. At baseline, the values of Schirmer, Jones, and BUT tests in the patient group were significantly lower compared to controls; moreover, conjunctival hyperemia was more frequent in psoriatic patients than in healthy subjects. Ocular involvement was more prominent in the subset of psoriatic patients with sebo-psoriasis than in general psoriatic population. A statistically significant correlation was found in sebo-psoriasis between PASI and Schirmer, between PASI and Jones, and between PASI and BUT. On the other hand, the results obtained from DR1 camera showed statistically significant difference between psoriatic and sebo-psoriatic patients at the end of the follow-up. After 12 weeks of treatment, the mean values of PASI, PGA, DLQI, CRP, and BUT showed significant changes in psoriatic patients. Our findings suggest a high rate of ocular involvement in psoriatic patients, emphasizing the need of performing periodic ophthalmological examinations in order to avoid underestimating eye diseases and to allow early diagnosis and treatment of patients.
Assuntos
Oftalmopatias/etiologia , Psoríase/complicações , Adulto , Idoso , Proteína C-Reativa/análise , Estudos de Casos e Controles , Oftalmopatias/tratamento farmacológico , Oftalmopatias/epidemiologia , Feminino , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Prevalência , Estudos Prospectivos , Psoríase/tratamento farmacológico , Psoríase/metabolismo , Psoríase/patologia , Qualidade de Vida , Índice de Gravidade de DoençaRESUMO
Purpose: To compare immediate versus delayed introduction of immunosuppressives for naive noninfectious inflammatory choroidal neovascularization (iCNV).Methods: a retrospective, consecutive, comparative, interventional case series of patients with a diagnosis of inflammatory CNV and a minimum follow of 36 months. Patients were divided into two groups: Group A received Immunosuppressives if needed, while Group B since baseline. Both groups received systemic steroids and intravitreal ranibizumab since baseline. Primary end point was to compare the BCVA outcome till 36-month follow-up.Results: Twenty-nine eyes with iCNV were enrolled. In the long term, best-corrected visual acuity (BCVA) was significantly better in group B. At 3-month follow-up, Group B reduced steroids <10 mg/day significantly (p = .0001, Fisher's Exact Test). At 36 months of follow up, injections given were 2.9 (0.9 SD) in group A and 1.25 (0.4 SD) in group B.Conclusion: early immunosuppressive therapy exerts a positive action on the long-term control of uveitic CNV.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Corioidite/tratamento farmacológico , Glucocorticoides/uso terapêutico , Imunossupressores/uso terapêutico , Ranibizumab/uso terapêutico , Uveíte/tratamento farmacológico , Adulto , Neovascularização de Coroide/diagnóstico , Neovascularização de Coroide/fisiopatologia , Corioidite/diagnóstico , Corioidite/fisiopatologia , Feminino , Angiofluoresceinografia , Seguimentos , Humanos , Injeções Intravítreas , Masculino , Estudos Retrospectivos , Tomografia de Coerência Óptica , Uveíte/diagnóstico , Uveíte/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Acuidade Visual/fisiologiaRESUMO
Nowadays many authors suggest the use of intravitreal triamcinolone acetonide (TA) for the treatment of vitreoretinal diseases, although it can be associated with a high risk of local toxicity. In order to develop a safer injection for clinical use, the purpose of our study is to evaluate the in situ safety of two different triamcinolone preparations, a commercially available TA and a micronized triamcinolone. The experiments were performed on 18 adult male age-matched New Zealand rabbits. The clinical examination included funduscopy with an indirect ophthalmoscope and intraocular pressure (IOP) measurement. At the end of the clinical observations, the animals were sacrificed and the eyes enucleated and processed for the morphological evaluation. In our study the main side effect observed was the IOP elevation in the group injected with triamcinolone acetonide. In addition, in the TA-injected group, one eye was enucleated following an endophthalmitis. Our study highlights that doses as low as 4 mg of triamcinolone acetonide injected into the rabbit vitreous may have a local toxic effect in terms of IOP elevation, endophthalmitis occurrence and changes in the retinal morphology. In contrast, the micronized triamcinolone injection shows a less toxic effect in situ, thus suggesting the alternative use of this more reliable preparation which seems to be safer for a clinical use.
Assuntos
Anti-Inflamatórios/toxicidade , Retina/efeitos dos fármacos , Triancinolona Acetonida/toxicidade , Triancinolona/toxicidade , Animais , Endoftalmite/induzido quimicamente , Pressão Intraocular/efeitos dos fármacos , Masculino , Coelhos , Retina/patologia , Retina/ultraestrutura , Triancinolona/administração & dosagem , Triancinolona Acetonida/administração & dosagem , Corpo Vítreo/efeitos dos fármacosRESUMO
We have reported previously that R-enantiomer of etodolac (R-etodolac), which is under investigation in phase 2 clinical trials in chronic lymphocytic leukemia, induces potent cytotoxicity at clinically relevant concentrations in multiple myeloma (MM) cells. In this study, we demonstrated that SDX-308 (CEP-18082), a novel analog of etodolac, has more potent cytotoxicity than R-etodolac against both MM cell lines and patient MM cells, including tumor cells resistant to conventional (dexamethasone, doxorubicine, melphalan) and novel (bortezomib) therapies. SDX-308-induced cytotoxicity is triggered by caspase-8/9/3 activation and poly (ADP-ribose) polymerase cleavage, followed by apoptosis. SDX-308 significantly inhibits beta-catenin/T-cell factor pathway by inhibiting nuclear translocation of beta-catenin, thereby downregulating transcription and expression of downstream target proteins including myc and survivin. Neither interleukin-6 nor insulin-like growth factor-1 protect against growth inhibition triggered by SDX-308. Importantly, growth of MM cells adherent to bone marrow (BM) stromal cells is also significantly inhibited by SDX-308. Our data therefore indicate that the novel etodolac analog SDX-308 can target MM cells in the BM milieu.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mieloma Múltiplo/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição TCF/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Etodolac/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
BACKGROUND: Despite the undeniable impact of systemic sclerosis (SS) on quality of life, only a few studies so far have focused on its psychiatric or psychological aspects. We aimed at assessing psychiatric symptoms and self-image in inpatients with SS and comparing them with patients with either a very mild skin condition or a serious skin condition. METHODS: Three groups were recruited: (i) 38 consecutive female inpatients with SS; (ii) 38 age-matched female outpatients with melanocytic naevi; (iii) 35 age-matched female inpatients with melanoma. All participants completed the Zung Anxiety Scale, the Zung Depression Scale and a self-report questionnaire measuring self-perceived personal qualities. Patients with SS were also clinically interviewed by a psychologist. RESULTS: The clinical interview revealed the presence of a psychiatric disorder in most (81%) patients with SS. The Zung scales corroborated the presence of mild to moderate anxiety and depression among patients with SS, who scored significantly higher than patients with either naevi or melanoma on both scales. Scores on the questionnaire assessing self-perceived personal qualities were very similar in the three groups and indicated a fairly high level of self-esteem. CONCLUSIONS: This study suggested that psychosocial issues are quite relevant in patients with SS and underscored the need for a biopsychosocial approach to the clinical management of these patients. Timely detection of psychosocial difficulties and appropriate psychological or psychiatric intervention may represent important steps toward better adherence to medical treatment and improved psychological well-being and quality of life.
Assuntos
Melanoma/psicologia , Nevo Pigmentado/psicologia , Escleroderma Sistêmico/psicologia , Autoimagem , Neoplasias Cutâneas/psicologia , Adulto , Ansiedade/psicologia , Estudos de Casos e Controles , Depressão/psicologia , Feminino , Inquéritos Epidemiológicos , Humanos , Saúde Mental , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Qualidade de VidaRESUMO
Pulmonary arteriovenous fistulas (PAVFs) are rare vascular malformations (PAVMs) of the lung that could lead to severe hypoxiemia due to right-to-left intrapulmonary shunts. They may occur as isolated entities or associated with Osler-Rendu-Weber syndrome or hereditary haemorrhagic telangiectasia (HHT). We report a case of a 70 years old woman with Rendu-Osler-Weber disease and a large arteriovenous malformation involving the left pulmonary artery. We describe the successful transcatheter occlusion of the PAVF using an Amplatzer vascular plug. This work is an attempt to focus the attention on pulmonary arteriovenous malformations and on percutaneous treatment as an alternative to surgery, that consists of a conservative lung resection.
Assuntos
Malformações Arteriovenosas/terapia , Embolização Terapêutica/instrumentação , Artéria Pulmonar/anormalidades , Veias Pulmonares/anormalidades , Telangiectasia Hemorrágica Hereditária/complicações , Idoso , Feminino , HumanosRESUMO
14-3-3 proteins are a family of master regulators of intracellular signaling, yet their impact on proteasome function is unknown. We demonstrate that 14-3-3ζ binds the 11S proteasome activator, limiting proteasome assembly and cellular capacity for protein degradation. To define the functional impact of 14-3-3ζ proteasomal binding in myeloma cells, silencing and overexpression experiments are performed. We find that downregulation of 14-3-3ζ impairs myeloma cell growth and confers resistance to clinically used proteasome inhibitors. In a large cohort of newly diagnosed myeloma patients, elevated expression of 14-3-3ζ is associated with high risk myeloma genetic subtypes and worse prognosis overall. Our work demonstrates the important role of 14-3-3ζ in regulating proteasome function, myeloma cell growth and sensitivity to therapeutics, and suggests regulation of 14-3-3ζ as a new approach in myeloma therapy.
Assuntos
Proteínas 14-3-3/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas 14-3-3/genética , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Ligação Proteica , Proteólise , Transdução de Sinais/efeitos dos fármacosRESUMO
The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.
Assuntos
Fibronectinas , Fragmentos de Imunoglobulinas , Neovascularização Patológica/diagnóstico , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Fibronectinas/metabolismo , Humanos , Fragmentos de Imunoglobulinas/metabolismo , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Teratocarcinoma/irrigação sanguínea , Teratocarcinoma/diagnósticoRESUMO
Ketorolac tromethamine is a recent injectable non-steroidal anti-inflammatory drug (NSAID) with analgesic properties approved for short-term pain management. In spite of its increasing use both in adults and children, relatively few allergic-like reactions have been reported. Reactions are often severe, and a death occurred following an intramuscular injection of ketorolac.
Assuntos
Anafilaxia/fisiopatologia , Anti-Inflamatórios não Esteroides/efeitos adversos , Hipersensibilidade a Drogas/fisiopatologia , Cetorolaco de Trometamina/efeitos adversos , Adolescente , Anafilaxia/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Clorfeniramina/uso terapêutico , Hipersensibilidade a Drogas/tratamento farmacológico , Feminino , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Humanos , Metilprednisolona/uso terapêutico , Testes Cutâneos , Urticária/etiologiaRESUMO
BACKGROUND: The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. RESULTS: The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface. CONCLUSIONS: The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.
Assuntos
Antígenos de Neoplasias/química , Fibronectinas/química , Espectroscopia de Ressonância Magnética , Neovascularização Patológica/metabolismo , Conformação Proteica , Isoformas de Proteínas/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/fisiologia , Sítios de Ligação , Biomarcadores , Epitopos/imunologia , Fibronectinas/imunologia , Fibronectinas/fisiologia , Produtos Finais de Glicação Avançada , Glicosilação , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Transplante de Neoplasias , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/fisiologia , Teratocarcinoma/irrigação sanguínea , Teratocarcinoma/metabolismo , Teratocarcinoma/patologiaRESUMO
MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, believed to be the driving factor in the pathogenesis of this MM subtype. MMSET overexpression in MM leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3), as well as changes in proliferation, gene expression and chromatin accessibility. Prior work linked methylation of histones to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET may play a role in DNA damage repair and response. In U2OS cells, we found that MMSET is required for efficient non-homologous end joining as well as homologous recombination. Loss of MMSET led to loss of expression of several DNA repair proteins, as well as decreased recruitment of DNA repair proteins to sites of DNA double-strand breaks (DSBs). By using genetically matched MM cell lines that had either high (pathological) or low (physiological) expression of MMSET, we found that MMSET-high cells had increased damage at baseline. Upon addition of a DNA-damaging agent, MMSET-high cells repaired DNA damage at an enhanced rate and continued to proliferate, whereas MMSET-low cells accumulated DNA damage and entered cell cycle arrest. In a murine xenograft model using t(4;14)+ KMS11 MM cells harboring an inducible MMSET shRNA, depletion of MMSET enhanced the efficacy of chemotherapy, inhibiting tumor growth and extending survival. These findings help explain the poorer prognosis of t(4;14) MM and further validate MMSET as a potential therapeutic target in MM and other cancers.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The three major proteins, EM1, EM5 and EM6, from the mature sperm of the bivalve mollusc Ensis minor have been partially sequenced in order to establish which category they belong to and their potential for phosphorylation. Protein EM1 is protamine-like with about 50% basic amino acids, some of which are included in SK(R) repeats. Three SPXX potential phosphorylation sites were observed in the N-terminal domain. EM1 does not fold (Giancotti et al. (1983) Eur. J. Biochem. 136, 509-516). Protein EM6 (approx. 270 residues) is histone H1-like, having a globular domain homologous to other H1 family proteins. The N-domain of EM6 contains SK(R) repeats like EM1, but there are few, if any, SPXX sites in the chain. Proteins EM1 and EM6 are the two proteins specific for mature sperm. Protein EM5, of about 150 residues and present at lower levels than EM1 and EM6, is also an H1-family molecule. A sequence from its globular domain shows close homology to chicken H5 and to sea urchin somatic H1. Its presence may relate to the existence of a low level of nucleosomal structure.
Assuntos
Histonas/química , Moluscos/química , Protaminas/química , Espermatozoides/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Dados de Sequência Molecular , Mapeamento de Peptídeos , Conformação ProteicaRESUMO
We previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, beta-actin, tubulin-alpha-1-chain and enolase-alpha. Interestingly, all carbonylated proteins except calreticulin and enolase-alpha showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.
Assuntos
Apoptose/fisiologia , Estresse Oxidativo/fisiologia , Fotoquimioterapia , Porfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Actinas/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Calreticulina/metabolismo , Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Proteínas de Choque Térmico/metabolismo , Humanos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo , Proteômica , Proteínas Supressoras de Tumor/metabolismoRESUMO
Calmodulin is a calcium-binding protein that regulates a wide range of enzymes. It is also one of the few examples of a small protein capable of binding to peptides with very high affinity, and is therefore an interesting candidate for biotechnological applications and a good model system for studying how proteins associate. We have synthesized a complete series of peptides derived from the recognition sequence of skeletal muscle myosin light-chain kinase, corresponding to single-point amino acid mutations to alanine. These peptides bind to calmodulin with a biphasic kinetic: a fast association step followed by a slow intramolecular isomerisation. We have measured the isomerisation rate (k(isom)) of these peptides for calmodulin by stopped-flow analysis, and their association and dissociation kinetic constants (k(on) and k(off)) by real-time interaction analysis using surface plasmon resonance detection. In addition, k(off) constants were measured by competition experiments using a high-sensitivity luminescence analyser and native polyacrylamide gels. We have observed that all the alanine-scanning peptides bound to calmodulin with better affinity than the wild-type. In one case, a Asn-->Ala substitution resulted in a 1000-fold improvement in affinity, owing to a slower off-rate. Our results indicate that naturally occurring calmodulin binders may have evolved to have high affinities, but far from the maximum. Our affinity data are in contrast with recently published predictions of interactions responsible for high-affinity calmodulin binding based on modelling and energy calculations.
Assuntos
Alanina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/química , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/metabolismo , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , CoelhosRESUMO
We have aimed at developing a general methodology for the isolation of enzymatic activities from large repertoires of protein displayed on the surface of a filamentous phage. When selecting for protein binders by phage display, phage particles with suitable specificities are physically isolated by affinity capture and amplified by bacterial infection. Selection for catalysis mediated by enzymes displayed on filamentous phage is more difficult, as reaction products (which represent the biochemical memory of the reaction catalysed by the phage particle) diffuse away after the reaction is complete. We reasoned that if we were able to anchor the reaction products on the phage surface, the catalytically active phages could then be physically isolated using specific anti-product affinity reagents. We achieve the conditional anchoring of reaction substrates and products on phage by displaying enzyme-calmodulin chimeric proteins on filamentous phage as gene III fusions. Such phage particles can be targeted in a stable fashion (koff<10(-4) s(-1)) by chemical derivatives of a calmodulin-binding peptide. The peptide-phage complexes are stable in purification procedures such as capture with magnetic beads and polyethylene glycol precipitation, and can be conditionally dissociated by addition of calcium chelators. Glutathione-S-transferase and an endopeptidase were used in model selection experiments to demonstrate that it is possible to isolate catalytic activities from calmodulin-tagged enzymes displayed on filamentous phage, with enrichment factors >50 per round of selection.
Assuntos
Bacteriófagos/química , Catálise , Clonagem Molecular/métodos , Enzimas Imobilizadas/isolamento & purificação , Enzimas/metabolismo , Vetores Genéticos/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Biotinilação , Calmodulina/química , Proteínas de Ligação a Calmodulina/química , Capsídeo/genética , Precipitação Química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Enzimas/genética , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/metabolismo , Microesferas , Dados de Sequência Molecular , Polietilenoglicóis , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The results of competitive solid phase radioimmunoassay with labeled antigen, when analyzed by nonlinear regression, may reveal the presence of two different monoclonal antibodies, occasionally produced in a hybridoma culture. In fact, a hybridoma-derived antihuman chorionic somatomammotropin antibody prepared in our laboratories showed an unsatisfactory curve fitting when the experimental data were elaborated on the basis of a monoclonal model. The antibody was subjected to recloning, and gave two separate homogeneous monoclonal antibodies with different affinity constants.
Assuntos
Anticorpos Monoclonais/análise , Hibridomas/imunologia , Afinidade de Anticorpos , Hormônio do Crescimento/imunologia , Humanos , Lactogênio Placentário/imunologia , Radioimunoensaio , Análise de RegressãoRESUMO
The peptides corresponding to the fragments 135-140 and 166-174 of human chorionic somatomammotropin (hCS) were synthesized, and used to raise monoclonal antibodies to the native hCS molecule. The synthetic peptides were injected into BALB/c mice in the free form, i.e. not conjugated to a carrier, and the spleens were fused with Sp2/01Ag8 myeloma line to produce monoclonal antibodies. The antibodies produced belonged to the IgM and IgG classes and, once purified by affinity chromatography on hCS-Sepharose, they were covalently coupled to macroporous polystyrene beads and characterized by competitive radioimmunoassay. Their affinity constants were determined by elaborating the radioimmunoassay data by nonlinear regression analysis and they were found to range from 10(5) to 10(6) M-1. The evaluation of the affinity constant of the antibodies produced is always important as a measure of the immunogenicity of an antigen, particularly when synthetic peptides are used as immunogens.
Assuntos
Anticorpos Monoclonais/biossíntese , Fragmentos de Peptídeos/imunologia , Lactogênio Placentário/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Cromatografia de Afinidade , Esquemas de Imunização , Camundongos , Camundongos Endogâmicos BALB C , RadioimunoensaioRESUMO
The synthetic peptide 166-174 of hCS sequence, corresponding to an antigenic determinant of the protein, was used to elicit MAbs to the native hCS molecule. The synthetic peptide was administered according to different immunization protocols to BALB/c mice, both in the free form or conjugated to carrier proteins. Spleens and popliteal lymph nodes from primed mice were fused with a myeloma cell line to produce MAbs, and selected clones were characterized for isotype and affinity. Spleen fusions gave rise to IgM MAbs, whereas lymph node fusions gave preferentially IgG MAbs. No correlation was found between antibody class and affinity since affinity is highly increased by carrier-conjugation, while it did not enhance IgG production. The free synthetic peptide showed a low immunogenicity: affinities of MAbs produced ranged from 10(5) to 10(7) l/mole, an average 1000-fold lower than the values obtained with carrier-conjugated peptide. In one case, however, carrier conjugation did not give rise to anti-hCS MAbs. Overall, these studies provide a rational approach to the production of anti-protein MAbs by synthetic peptide immunization and offer the opportunity to obtain MAbs of the desired isotype and affinity.
Assuntos
Afinidade de Anticorpos , Peptídeos/imunologia , Lactogênio Placentário/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Líquido Ascítico/imunologia , Imunoglobulinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Human chorionic somatomammotropin (hCS) and human growth hormone (hGH), despite their different biological activities, show a remarkable degree of homology in their primary structure, which explains their immunological cross-reactivity. On the basis of the hydrophilicity profiles, we predicted that the sequence 165-174 would correspond to an antigenic determinant common to both hormones. The sequence 165-174 was synthesized in the solid phase and the ability to bind antibodies to hCS was tested by a radioimmunoassay at each step of the synthesis, without detaching the peptide from the resin. We found that the immunological sequence able to bind antibodies to hCS is that corresponding to 167-174. In similar experiments, we showed that the sequence 166-174 is able to bind antibodies to hGH. In the plaque-forming cell test using the synthetic fragment 166-174 bound to sheep red blood cells, we also observed that the ratio between the number of cells forming antibodies to hCS and to the 166-174 peptide was always between 0.45 and 0.73, thus suggesting that the 166-174 peptide represents a major determinant of hCS.
Assuntos
Epitopos/análise , Hormônio do Crescimento/imunologia , Fragmentos de Peptídeos/imunologia , Lactogênio Placentário/imunologia , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Técnica de Placa Hemolítica , Humanos , Fragmentos de Peptídeos/síntese químicaRESUMO
OBJECTIVE: To understand emergency department (ED) physicians' use of electronic documentation in order to identify usability and workflow considerations for the design of future ED information system (EDIS) physician documentation modules. METHODS: We invited emergency medicine resident physicians to participate in a mixed methods study using task analysis and qualitative interviews. Participants completed a simulated, standardized patient encounter in a medical simulation center while documenting in the test environment of a currently used EDIS. We recorded the time on task, type and sequence of tasks performed by the participants (including tasks performed in parallel). We then conducted semi-structured interviews with each participant. We analyzed these qualitative data using the constant comparative method to generate themes. RESULTS: Eight resident physicians participated. The simulation session averaged 17 minutes and participants spent 11 minutes on average on tasks that included electronic documentation. Participants performed tasks in parallel, such as history taking and electronic documentation. Five of the 8 participants performed a similar workflow sequence during the first part of the session while the remaining three used different workflows. Three themes characterize electronic documentation: (1) physicians report that location and timing of documentation varies based on patient acuity and workload, (2) physicians report a need for features that support improved efficiency; and (3) physicians like viewing available patient data but struggle with integration of the EDIS with other information sources. CONCLUSION: We confirmed that physicians spend much of their time on documentation (65%) during an ED patient visit. Further, we found that resident physicians did not all use the same workflow and approach even when presented with an identical standardized patient scenario. Future EHR design should consider these varied workflows while trying to optimize efficiency, such as improving integration of clinical data. These findings should be tested quantitatively in a larger, representative study.