RESUMO
The objective of the present work was to study peculiarities of the analgesic action of therapeutic electrical stimulation (TES therapy) in the early postoperative period in the patients who underwent tonsillectomy. A total of 60 patients admitted for this surgery to the specialized otorhinolaryngological department were available for observation. They were divided into two groups depending on the pain relief strategy. The patients of the study group (n=30) underwent courses of transcranial electrical stimulation on a daily basis (from the onset of hospitalization) in addition to the administration of a standard analgetic. The standard dose of tramadol (2.0 ml) was given to the patients of the control group (n=30) who complained of strong pain. The results of the objective and subjective estimations indicate that the degree of pharyngeal pain in the patients treated with TES therapy and the standard analgetic was significantly different. The patients receiving TES therapy could sooner resume their habitual diet and required smaller amounts of the analgetic which makes this modality a cost-effective supplement to the standard postoperative treatment.
Assuntos
Terapia por Estimulação Elétrica/métodos , Dor Pós-Operatória/terapia , Tonsilectomia/efeitos adversos , Tramadol/uso terapêutico , Adulto , Terapia Combinada , Terapia por Estimulação Elétrica/instrumentação , Humanos , Medição da Dor/métodos , Dor Pós-Operatória/tratamento farmacológico , Período Pós-Operatório , Fatores de Tempo , Resultado do TratamentoRESUMO
The objective of the present study was to estimate the incidence of vestibular symptoms among the patients presenting with otosclerosis and their relationship with the form of this pathology. A total of 90 patients had the confirmed diagnosis of otosclerosis in the absence of concomitant diseases known to cause vestibular disorders. The patients were interviewed and underwent thorough examination. It turned out that 16.7% of them exhibited vestibular asymmetry; in other words, the frequency of this condition was higher than the incidence of vestibular disbalance in the general population. Most patients with vestibular symptoms and complaints were referred to the group with unilateral sensorineural hearing impairment that, however, can not be a marker of unilateral vestibular deficiency.
Assuntos
Otosclerose/fisiopatologia , Doenças Vestibulares/fisiopatologia , Humanos , Incidência , Otosclerose/classificação , Otosclerose/complicações , Otosclerose/epidemiologia , Índice de Gravidade de Doença , Doenças Vestibulares/epidemiologia , Doenças Vestibulares/etiologiaRESUMO
The objective of the present investigation was to reveal clinical symptoms significantly associated with the cysts in the maxillary sinuses (MS). The study included 67 consecutive patients with the roentgenologically confirmed diagnosis of maxillary cysts in the absence of concomitant pathological changes in the nasal cavity and paranasal sinuses. Each patient was interviewed twice: first time before surgery when they were asked to fill a questionnaire and specify the disturbing symptoms, the second time postoperatively, i.e. during the follow-up examination within 4-6 months after the treatment. The statistical analysis demonstrated that MS cysts may be a cause of such symptoms as headache in the frontal region, feeling of pressure in the affected sinus, mucous rhinorrhoea, nasal obstruction, and probably coughing in the morning hours as a manifestation of postnasal drip syndrome.
Assuntos
Cistos/diagnóstico , Cistos/patologia , Seio Maxilar/fisiopatologia , Cistos/cirurgia , Seguimentos , Cefaleia/etiologia , Cefaleia/fisiopatologia , Humanos , Seio Maxilar/patologia , Seio Maxilar/cirurgia , Índice de Gravidade de Doença , Inquéritos e Questionários , Resultado do TratamentoRESUMO
mRNA-based technologies and preclinical research in a variety of animal models have shown that guanylyl cyclase C (GCC) is a highly sensitive and specific molecular marker for the diagnosis of colorectal cancer (CRC). GCC is also a receptor for Escherichia coli (E. coli) heat-stable enterotoxin (STa) and can be used for STa-directed delivery of small-sized imaging agents to human CRC tumours. In this study, we have evaluated GCC as a new immunohistochemical (IHC) marker for CRC tissues and STa as a suitable vector for delivering high-sized protein molecules to CRC cells. Firstly, we have developed a highly sensitive EnVision(+)-based IHC staining method for detecting GCC in serial paraffin-embedded sections of primary and metastatic CRC (38 cases) or non-CRC (14 cases) adenocarcinomas. Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) were chosen as controls. Our results indicate that GCC staining was positive in 100% of CRC tumours and was comparable to CEA (95%) or CK20 (92%). In contrast to CEA and CK20, GCC was negative in all of the extra-intestinal non-CRC tumours examined. GCC appears to display higher specificity than either CEA or CK20 while retaining high sensitivity, suggesting that it is a better CRC marker than CEA or CK20. Secondly, STa was genetically coupled to green fluorescent protein (GFP) and the resulting GFP-tagged STa was characterized for expression in E. coli and enterotoxicity in mouse. The binding characteristics of GFP-STa in CRC Caco-2 cells were followed by immunofluorescence microscopy. In this work we show that GFP-tagged STa is biologically active and has retained its ability to internalise into Caco-2 cells making it a potential vehicle for the delivery of anticancer therapeutic protein agents.
Assuntos
Toxinas Bacterianas/metabolismo , Neoplasias Colorretais/enzimologia , Enterotoxinas/metabolismo , Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Adulto , Idoso , Biomarcadores Tumorais , Células CACO-2 , Proteínas de Escherichia coli , Feminino , Imunofluorescência , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Veículos Farmacêuticos , Receptores de Enterotoxina , Receptores Acoplados a Guanilato CiclaseRESUMO
Two alternatively spliced forms of human PPAR alpha mRNA, PPAR alpha1 and PPAR alpha2, have been identified. PPAR alpha1 mRNA gives rise to an active PPAR alpha protein while PPAR alpha2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPAR alpha2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPAR alpha1 and PPAR alpha2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPAR alpha2 mRNA abundance is approximately half that of PPAR alpha1 mRNA; a correlation analysis of PPAR alpha1 and PPAR alpha2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPAR alpha2/PPAR alpha1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPAR alpha2/PPAR alpha1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPAR alpha activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPAR alpha2/PPAR alpha1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPAR alpha2/PPAR alpha1 mRNA. These data suggest that selective PPAR alpha2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators.
Assuntos
Fígado/metabolismo , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Células Cultivadas , DNA Complementar , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
We investigated whether the toxicity-associated receptor-binding domain of the non-immunogenic Escherichia coli heat-stable enterotoxin (STh) as a fusion with a carrier protein and the inclusion of an appropriate spacer are critical factors for eliciting antibody responses against the native toxin. The immunological properties of three toxic and one non-toxic fusion proteins, consisting of STh N-terminally joined to the C-terminus of the major subunit ClpG of E. coli CS31A fimbriae, were compared. In contrast to the non-toxic hybrid STh with glycine and leucine simultaneously substituted for the receptor-interacting Pro(13) and Ala(14) amino acids, the toxic chimeras responded by producing high serum levels of anti-STh antibodies in immunized animals. On the other hand, only the toxic ClpG-STh construct with the natural peptide 47KSGPESM(53) of Pro-STh as spacer stimulated STh-neutralizing responses against both native toxin and enterotoxigenic live E. coli cells. Altogether, these findings suggest a close relationship between conformational similarity to the native structure of STh and the ability to elicit specific antibody responses against STh.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli , Escherichia coli/imunologia , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Antibacterianos/análise , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Enterotoxinas/genética , Fímbrias Bacterianas/química , Fímbrias Bacterianas/genética , Glicina , Imunogenética , Leucina , Camundongos , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaAssuntos
Analgésicos não Narcóticos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Ácidos Pentanoicos/farmacologia , Administração Oral , Analgésicos não Narcóticos/química , Animais , Bloqueadores dos Canais de Cálcio/química , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo N/efeitos dos fármacos , Linhagem Celular , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos DBA , Medição da Dor , Ácidos Pentanoicos/química , RatosRESUMO
We have successfully used the major subunit ClpG of Escherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH(2) or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of E. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.
Assuntos
Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/imunologia , Escherichia coli/metabolismo , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/imunologia , Adesinas de Escherichia coli/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Dissulfetos/metabolismo , Portadores de Fármacos , Enterotoxinas/genética , Escherichia coli/genética , Humanos , Camundongos , Oxirredução , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In 1998 we implemented a BCSH recommendation that addressograph labels should not be used on blood transfusion specimen tubes. Over a 12-month period before the ban was introduced our laboratory received 5964 red cell transfusion requests, 182 (3.1%) of which contained an error in the identification details (ID) supplied on the request form and/or specimen. Three of these errors were of the 'wrong patient' type, i.e. the sample belonged to a different patient from the one whose ID appeared on the specimen tube and request form. Over the 12 months after the ban was introduced 511 (8. 1%) of 6326 requests contained a labelling error, an increase in error rate of 165%; no wrong-patient errors were identified, however. In a survey, seven (29.2%) of 24 transfusion laboratories in the UK accepted specimens labelled with addressograph stickers; in four of these cases a local blood transfusion committee had agreed that the BCSH guideline should not be followed. We believe the BCSH guideline is valid; its implementation, however, has major financial and workload implications, which probably explains why many hospitals apparently do not comply with it.
Assuntos
Bancos de Sangue/organização & administração , Coleta de Amostras Sanguíneas/normas , Transfusão de Eritrócitos/normas , Registros Hospitalares/normas , Programas Nacionais de Saúde/normas , Sistemas de Identificação de Pacientes/normas , Guias de Prática Clínica como Assunto , Manejo de Espécimes/instrumentação , Controle de Formulários e Registros , Pesquisas sobre Atenção à Saúde , Humanos , Auditoria Médica , Flebotomia , Telefone , Reino UnidoRESUMO
To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity. These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo. In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity. This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted. Periplasmic and lysate fractions of dsbA(+) and dsbA(-) cells did not have any STh activity. In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background. Subsequently, a time course experiment in regard to the secretion of STh from both dsbA(+) and dsbA(-) cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time. Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Clonagem Molecular , Dissulfetos , Enterotoxinas/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Genótipo , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fenótipo , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The dissolution of the Soviet Union and the declaration of Independence by the Republic of Armenia created the need for significant changes in the healthcare delivery system in Armenia. The desire to raise the level of health care presented challenges and opportunities for nurses within the Republic. Members of the departments of nursing at Boston City Hospital/Boston Medical Center in Boston, Massachusetts, University of Massachusetts Medical Center, Worcester, Massachusetts, and the Emergency Scientific Medical Center of Yerevan, Armenia, joined forces through a grant written by Boston University School of Medicine and sponsored by the American International Health Alliance under a cooperative agreement with the United States Agency for International Development to expand the role of nursing. This article describes the assessment, planning, implementation, and evaluation of changes to the role of nursing and the development of new roles for nurses within a hospital in the capital city of Yerevan.
Assuntos
Educação Continuada em Enfermagem/organização & administração , Enfermagem em Emergência/educação , Enfermagem em Emergência/organização & administração , Intercâmbio Educacional Internacional , Descrição de Cargo , Papel do Profissional de Enfermagem , Recursos Humanos de Enfermagem Hospitalar/educação , Recursos Humanos de Enfermagem Hospitalar/organização & administração , Autonomia Profissional , Desenvolvimento de Pessoal/organização & administração , Armênia , Boston , Currículo , Atenção à Saúde/organização & administração , Docentes de Enfermagem/organização & administração , Humanos , Liderança , Licenciamento em Enfermagem , Recursos Humanos de Enfermagem Hospitalar/psicologia , Inovação Organizacional , Sociedades de Enfermagem/organização & administração , Telemedicina/organização & administraçãoRESUMO
Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Dipeptídeos/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/efeitos dos fármacos , Dipeptídeos/química , Modelos Animais de Doenças , Humanos , Camundongos , Convulsões/tratamento farmacológico , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
PNQX (1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3, 4-f]quinoxaline-2,3-dione) is a potent AMPA (IC50 = 0.063 microM) and GlyN (IC50 = 0.37 microM) receptor antagonist that was developed in our laboratories. While possessing a desirable in vitro and in vivo activity profile, this compound suffers from low aqueous solubility. In an effort to improve its potency and physical properties, we have designed and synthesized novel ring-opened analogues 4, 6, 9, and 11. Modeling analyses demonstrated that, while the 5-substituent in these analogues was forced to adopt an out-of-plane conformation due to steric contacts with neighboring substituents, the overall structure retained a good fit to a previously described AMPA pharmacophore model. This nonplanar orientation may lessen efficient packing in the solid state, compared to PNQX, leading to increased water solubility. Indeed, several nonplanar analogues containing appropriate functionalities, for example, the sarcosine analogue 9, were found to retain AMPA (IC50 = 0.14 microM) and GlyN (IC50 = 0.47 microM) receptor affinity and possess improved aqueous solubility compared to PNQX. The synthesis and the SAR of these compounds are discussed.
Assuntos
Antagonistas de Aminoácidos Excitatórios/síntese química , Glicina/análogos & derivados , Quinoxalinas/síntese química , Receptores de AMPA/antagonistas & inibidores , Receptores de Glicina/antagonistas & inibidores , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Anticonvulsivantes/farmacologia , Ligação Competitiva , Córtex Cerebral/metabolismo , Desenho de Fármacos , Antagonistas de Aminoácidos Excitatórios/química , Antagonistas de Aminoácidos Excitatórios/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/síntese química , Glicina/química , Glicina/metabolismo , Glicina/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Modelos Moleculares , Quinoxalinas/química , Quinoxalinas/metabolismo , Quinoxalinas/farmacologia , Ratos , Receptores de AMPA/metabolismo , Receptores de Glicina/metabolismo , Solubilidade , Sinaptossomos/metabolismoRESUMO
Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown utility in several models of stroke and pain. We are especially interested in small molecule N-type calcium channel blockers for therapeutic use. Herein, we report a series of N,N-dialkyl-dipeptidylamines with potent functional activity at N-type VSCCs and in vivo efficacy. The synthesis, SAR, and pharmacological evaluation of this series are discussed.
Assuntos
Bloqueadores dos Canais de Cálcio/síntese química , Diaminas/síntese química , Diaminas/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos DBA , Modelos Químicos , Convulsões/tratamento farmacológicoAssuntos
Transtornos Psicóticos/etiologia , Estresse Fisiológico/complicações , Animais , Autoanticorpos/imunologia , Transtorno Bipolar/etiologia , Encéfalo/imunologia , Células Cultivadas , DNA/genética , Feminino , Humanos , Linfócitos/imunologia , Masculino , Gravidez , Transtornos Psicóticos/genética , Transtornos Psicóticos/imunologia , Coelhos , Ratos , Fatores de Risco , Transtorno da Personalidade Esquizoide/etiologia , Esquizofrenia/etiologia , Esquizofrenia/genética , Comportamento Estereotipado , Estresse Fisiológico/imunologiaRESUMO
The ICE family of cysteine proteases mediates necrotic or apoptotic events in the nervous system as well as in other tissues. This suggests that inhibitors may be of therapeutic value in acute and, perhaps, chronic neurodegenerative disease. In addition, some members of this family may respond to intercellular signals controlling proliferation or differentiation. This possibility should be kept in mind as therapeutics are pursued.
Assuntos
Cisteína Endopeptidases/fisiologia , Degeneração Neural/fisiopatologia , Animais , Apoptose/fisiologia , Caspase 1 , Humanos , NecroseRESUMO
CS31A fibrillae are thin, flexible, heteropolymeric proteinaceous appendages exposed as a capsule-like material around the cell surface of certain Escherichia coli strains. Two antigenic peptides of the S spike glycoprotein (TGEV-S) amino acids (aa) 363-371 and 521-531 of the transmissible gastroenteritis virus (TGEV) were tandemly introduced in the loop-structured, variable region aa 202-218 of the major ClpG subunit protein composing the bulk of CS31A. The resulting hybrid fibrillae with a 25 aa heterologous peptide were produced at the cell surface. Using a monoclonal antibody (Mab) specific for the TGEV epitopes, purified hybrid fibrillae were analysed in Western blotting under native conditions, which showed that the two viral epitopes were recognized immunologically as an integral part of the hybrid fibrillae, and therefore that they were antigenically active. The immunogenicity of the fusion construct was evaluated with live recombinant bacteria, purified hybrid ClpG monomers, and purified chimeric CS31A polymers. Whatever the form of hybrid used as antigen, intraperitoneally immunized outbred mice elicited serum anti-TGEV peptides antibodies (Abs) with significant titres and capable of recognizing native TGEV particles, indicating that the epitopes are exposed in an immunogenic conformation in all cases. However, virus neutralization titres were only obtained after immunization with either purified polymers or monomers. Furthermore, 4 months after an ultimate immunization with 20 micrograms of hybrid fibrillae mice developed a strong anamnestic Ab response against the two TGEV peptides following booster inoculation with virions. We conclude that CS31A fibrillae carrying a combination of TGEV epitopes as insert can induce an immunological memory in outbred animals infected with TGEV, and therefore that hybrid CS31A fibrillae may prove efficient as components of a subunit vaccine.
Assuntos
Adesinas de Escherichia coli/genética , Anticorpos Antivirais/biossíntese , Antígenos de Bactérias , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Fímbrias Bacterianas/genética , Imunização Secundária , Memória Imunológica/genética , Proteínas Recombinantes de Fusão/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Adesinas de Escherichia coli/imunologia , Adesinas de Escherichia coli/ultraestrutura , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Fímbrias Bacterianas/imunologia , Fímbrias Bacterianas/ultraestrutura , Haplótipos/imunologia , Memória Imunológica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Proteínas de Escherichia coli , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/química , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Sequência de Bases , Linhagem Celular , Genes Bacterianos , Humanos , Imunoglobulina G/farmacologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossínteseRESUMO
Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363-371 and the A epitope (TGEV-A) aa 522-531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG. Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process. Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding CIpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context. The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202-204 and 202-218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Vetores Genéticos , Vírus da Gastroenterite Transmissível/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Recombinante , Epitopos/genética , Escherichia coli , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Vírus da Gastroenterite Transmissível/imunologiaRESUMO
This study was performed to investigate whether the anticonvulsant phenytoin has neuroprotective effect in a model of hypoxia-ischemia with neonatal rats. The left carotid artery of each rat was ligated, followed by 3 h of hypoxic exposure (8% O2) in a temperature-regulated environment (36 degrees C). Two weeks later, brain damage was assessed by measuring loss of brain hemisphere weight. Phenytoin had no effect on body temperature or plasma glucose, but attenuated brain damage in a dose-dependent manner (3, 10, and 30 mg/kg i.p.) when administered before the hypoxic episode. Phenytoin administered during or after hypoxia did not alter hypoxic brain damage significantly. A parallel experiment using histological examination of frozen brain sections demonstrated less brain infarction after phenytoin treatment (30 mg/kg i.p.). In an additional experiment measuring breakdown of an endogenous brain calpain substrate, spectrin, phenytoin treatment reduced this measure of early cellular damage. Our results indicate that pretreatment with phenytoin is neuroprotective at a plasma phenytoin concentration of approximately 12 micrograms/ml. These results are consistent with the hypothesis that blockade of voltage-dependent sodium channels reduces brain damage following ischemia.