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2.
Protein Expr Purif ; 23(2): 226-32, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11676596

RESUMO

The p55 tumor necrosis factor receptor (TNF-RI) is the main receptor by which TNF exerts its effects. The signaling capacity largely depends on the presence of an intact C-terminal protein-protein interaction domain, a so-called death domain (DD). Here we report the expression and purification of the human TNF-RI DD as a fusion with the Escherichia coli thioredoxin A (TRX) protein. When expressed under control of the bacteriophage T7 promoter, TRX-DD accumulates as a soluble protein in the cytoplasm of E. coli. The TRX-DD protein was released from the cells into the periplasmic fraction after osmotic shock. Due to self-association of the DD, a large part of the material appeared as multimers; it could be removed by selective precipitation and a combination of ion-exchange and size-exclusion chromatography. This purification protocol yielded 30 mg of purified, monomeric protein from 1 liter of shake-flask culture. The purified TRX-DD was found to be functional as it still bound to the TNF-RI-associated DD protein and the intracellular part of TNF-RI. We conclude that TRX-DD is correctly folded and can be used for further structure/function analysis.


Assuntos
Antígenos CD/genética , Receptores do Fator de Necrose Tumoral/genética , Antígenos CD/química , Antígenos CD/isolamento & purificação , Clonagem Molecular , Escherichia coli , Humanos , Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/isolamento & purificação , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/genética
3.
Eur J Biochem ; 268(5): 1382-91, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11231290

RESUMO

Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/química , Antígenos CD/metabolismo , Mutação/genética , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Proteínas de Transporte/química , Dicroísmo Circular , Escherichia coli , Proteína de Domínio de Morte Associada a Fas , Guanidina/farmacologia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fenótipo , Desnaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor de Fator de Crescimento Neural/química , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Células Tumorais Cultivadas , Receptor fas/química
4.
Biochem Pharmacol ; 60(8): 1185-95, 2000 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11007957

RESUMO

Interleukin (IL)-6 is a multifunctional cytokine that can be induced by a plethora of chemical or physiological compounds, including the inflammatory cytokines tumor necrosis factor (TNF) and IL-1. The molecule TNF has a trimeric configuration and thus binds to membrane-bound, cellular receptors to initiate cell death mechanisms and signaling pathways leading to gene induction. Previously, we showed that induced clustering of the intracellular domains of the p55 TNF receptor, or of their respective 'death domains' only, is sufficient to activate the nuclear factor kappa B (NF-kappa B) and several mitogen-activated protein kinase (MAPK) pathways. NF-kappa B is the exclusive transcription factor for induction of the IL-6 gene in response to TNF and functions as the final trigger to activate a multiprotein complex, a so-called 'enhanceosome', at the level of the IL-6 promoter. Furthermore, the enhanceosome displays histone acetylation activity, which turned out to be essential for IL-6 gene activation via NF-kappa B. However, activation of NF-kappa B alone is not sufficient for IL-6 gene induction in response to TNF, as inhibition of the coactivated extracellular signal-regulated kinase and p38 MAPK pathways blocks TNF-mediated gene expression. Nevertheless, the transactivating NF-kappa B subunit p65 is not a direct target of MAPK phosphorylation. Thus, we postulated that other components of the enhanceosome complex are sensitive to MAPK cascades and found that MAPK activity is unequivocally linked to the histone acetylation capacity of the enhanceosome to stimulate gene expression in response to TNF. In contrast, glucocorticoid repression of TNF-driven IL-6 gene expression does not depend on abrogation of histone acetyltransferase activity, but originates from interference of the liganded glucocorticoid receptor with the contacts between NF-kappa B p65 and the promoter configuration around the TATA box.


Assuntos
Regulação da Expressão Gênica , Interleucina-6/genética , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Elementos Facilitadores Genéticos/fisiologia , Humanos , NF-kappa B/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Ativação Transcricional
5.
J Biol Chem ; 275(48): 37596-603, 2000 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-10988295

RESUMO

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/química , Antígenos CD/metabolismo , Proteínas de Transporte/metabolismo , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Virais , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Proteína de Domínio de Morte Associada a Fas , Citometria de Fluxo , Humanos , Camundongos , Necrose , Receptores Tipo I de Fatores de Necrose Tumoral , Serpinas/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo
6.
FEBS Lett ; 441(2): 275-80, 1998 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-9883899

RESUMO

In the mouse fibrosarcoma cell line L929sA, tumor necrosis factor (TNF) stimulates activation of the stress-responsive p38 mitogen-activated protein kinase (MAPK), as well as the classical p42 and p44 MAPK. TNF signaling can be mediated by p55 or p75 TNF receptors. Here, we demonstrate that TNF-R55 is sufficient to activate p42/p44 MAPK and p38 MAPK. Moreover, by expressing different membrane-bound or purely cytoplasmic truncations of TNF-R55, we show that the intracellular death domain of TNF-R55 is the crucial domain involved. The cytoplasmic membrane-proximal region of TNF-R55, known to induce neutral sphingomyelinase activation, is not required for activation of p38 MAPK or p421p44 MAPK.


Assuntos
Antígenos CD/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD/química , Sequência de Bases , Primers do DNA , Ativação Enzimática , Fibrossarcoma/enzimologia , Fibrossarcoma/patologia , Camundongos , Dados de Sequência Molecular , Receptores do Fator de Necrose Tumoral/química , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno
7.
Artigo em Inglês | MEDLINE | ID: mdl-12167995

RESUMO

By designing four primers for an overlapping PCR, we created a fusion gene Ddlcat encoding human TNF receptor I death domain and chloramphenicol acetyltransferase (CAT). By DNA sequencing, the whole sequence of the fusion gene is confirmed to be correct. Two hours after induction with IPTG, we could see a 39 kD extra protein band on SDS-PAGE pattern. We proved that this 39 kD protein band is DdLcat protein by Western blotting. Then we purified this protein to the purity of 95% through Q-Sepharose chromatography.

8.
J Inflamm ; 47(1-2): 67-75, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-8913931

RESUMO

TNF-induced apoptosis, e.g. in murine PC60 cells, requires the TNF receptor p55 (TNF-R55) and the TNF receptor p75 (TNF-R75); the latter even does not have to be triggered. The intracellular domain of TNF-R55 can be activated in the cytosol by linking it to the trimeric CAT protein; induction of this fusion protein leads to a full TNF response. A new MAP kinase, p38, has been shown to be also activated by TNF. This activation is essential for gene induction, but not for cytotoxicity in L929 cells. TNF treatment of L929 leads to reactive oxygen formation in the mitochondria, resulting in cell death by necrosis. TNF treatment of many other cell types results in apoptosis, and this process involves activation of one or more ICE homologs (IHO). In the mouse, seven cysteine proteases of the IHO family have been cloned and partially characterized. One or more of these IHOs is involved in cell killing by proteolysis of critical substrate(s). One substrate, which may be a key effector molecule in the apoptotic process, is PITSLRE kinase.


Assuntos
Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Necrose , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/genética , Gatos , Linhagem Celular , Camundongos , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética
9.
Antimicrob Agents Chemother ; 38(9): 2180-2, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7811041

RESUMO

The inhibitory effects of the 9-(2-phosphonylmethoxyethyl)adenine-related compounds (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine, (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine, and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine on human hepatitis B virus replication in the human hepatoma cell line HepG2 2.2.15 and duck hepatitis B virus infection in primary duck hepatocytes were investigated. (R)-9-(2-phosphonylmethoxypropyl-2,6-diaminopurine had the lowest 50% inhibitory concentrations against hepatitis B virus and duck hepatitis B virus, 0.22 and 0.06 microM, respectively, i.e., two- to fivefold lower concentrations than required for (R)-9-(2-phosphonylmethoxypropyl)adenine and 9-(2-phosphonylmethoxyethyl)adenine. All compounds were not toxic in vitro at a concentration of 100 microM.


Assuntos
Antivirais/farmacologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Nucleosídeos/farmacologia , Organofosfonatos/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Carcinoma Hepatocelular/virologia , Citosina/análogos & derivados , Patos , Humanos , Fígado/citologia , Fígado/virologia , Neoplasias Hepáticas/virologia , Compostos Organofosforados/farmacologia , Purinas/farmacologia , Células Tumorais Cultivadas
10.
J Virol Methods ; 43(1): 41-51, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8360315

RESUMO

An immuno disc assay (IDA) for semi-quantitative analysis of the surface antigen (DHBsAg) of duck hepatitis B virus (DHBV) is described. Unpurified antigen preparations were adsorbed onto punched-out nitrocellulose membrane discs. Rabbit antiserum raised against serum-derived gradient-purified DHBsAg was used for detecting the antigen. Cross-reacting antibodies in the rabbit antiserum were removed using normal duck serum and normal duck hepatocytes. The sensitivity of the IDA was compared with that of the Western blot analysis and was observed to be of the same order, but differed slightly for DHBsAg in liver and sera. In contrast to Western blot analysis, antigen specificity for the IDA included the S-protein. Immunodetection was carried out in microtitre plates, but the procedure was accelerated by attaching the antigen-adsorbed discs to an adhesive plate sealer. The IDA was exemplified for measuring DHBsAg in duck serum, duck liver homogenates and viral protein synthesis in cultures of DHBV-infected hepatocytes.


Assuntos
Antígenos Virais/análise , Patos/microbiologia , Vírus da Hepatite B do Pato/isolamento & purificação , Hepatite Viral Animal/imunologia , Imunoensaio/métodos , Fígado/microbiologia , Doenças das Aves Domésticas/imunologia , Viremia/veterinária , Animais , Southern Blotting , Western Blotting , Células Cultivadas , DNA Viral/análise , Patos/sangue , Patos/imunologia , Vírus da Hepatite B do Pato/imunologia , Hepatite Viral Animal/sangue , Hepatite Viral Animal/microbiologia , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/microbiologia , Coelhos , Sensibilidade e Especificidade , Viremia/sangue , Viremia/imunologia , Viremia/microbiologia
11.
Antiviral Res ; 21(2): 141-53, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8338351

RESUMO

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.


Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , Organofosfonatos , Replicação Viral/efeitos dos fármacos , Adenina/uso terapêutico , Animais , Células Cultivadas , DNA Viral/análise , Patos , Estudos de Avaliação como Assunto , Antígenos da Hepatite B/biossíntese , Humanos , Fígado/citologia
12.
J Pharm Sci ; 81(3): 245-8, 1992 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1640362

RESUMO

Two experimental formulations of theophylline with a hydrophilic starch matrix were evaluated for their sustained-release characteristics after single administration in healthy human volunteers. Theo-dur was chosen as a reference sustained-release formulation. In a first study, the extent of absorption was similar for a syrup, for Theo-dur, and for the experimental formulation of theophylline with 70% drum-dried corn starch as the sustained-release agent (DDCS-70). The maximal plasma concentration (Cmax) was significantly lower, and the time to reach Cmax as well as the time span during which the plasma concentration was at least 75% of the Cmax were significantly higher for Theo-dur than for the DDCS-70 formulation. A sustained-release profile, as for Theo-dur, was not reached for DDCS-70. In a second study the influence of the starch:drug ratio on the bioavailability was investigated. The decrease in starch content from 70 to 50% of the formulation did not improve the plasma concentration-time profile towards a sustained-release profile.


Assuntos
Amido/farmacocinética , Teofilina/farmacocinética , Adulto , Disponibilidade Biológica , Fenômenos Químicos , Química Farmacêutica , Físico-Química , Preparações de Ação Retardada , Portadores de Fármacos , Feminino , Humanos , Masculino , Teofilina/efeitos adversos , Teofilina/sangue
13.
Bull Soc Belge Ophtalmol ; 243: 129-38, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1302142

RESUMO

The Walker-Warburg syndrome is characterized by lissencephaly type II, cerebellar and retinal anomalies and congenital muscular dystrophy. A clinical and histopathological study of a case is presented and the differential diagnosis discussed.


Assuntos
Encéfalo/anormalidades , Hidrocefalia/diagnóstico , Distrofias Musculares/congênito , Retina/anormalidades , Cerebelo/anormalidades , Olho/patologia , Feminino , Humanos , Lactente , Síndrome
14.
Eur J Clin Pharmacol ; 42(5): 549-52, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1607003

RESUMO

The possibility of a pharmacokinetic interaction between isosorbide-5-mononitrate (5-ISMN) and epinine, the active metabolite of ibopamine, has been investigated in 8 healthy male subjects given single doses of 200 mg ibopamine and 20 mg 5-ISMN, separately and together. The plasma 5-ISMN concentration-time profile was the same whether 5-ISMN was administered concomitantly with ibopamine or alone [AUC(o-t): 2.24 micrograms.ml-1.h after 5-ISMN alone, 2.16 micrograms.ml-1.h after 5-ISMN+ibopamine]. The plasma concentrations of total and free epinine and the urinary recovery of total epinine, homovanillic acid and dihydroxyphenylacetic acid, too, were not different when ibopamine was administered alone or concomitantly with 5-ISMN. The intake of ibopamine did not change the blood pressure and heart rate. The decrease in diastolic blood pressure induced by 5-ISMN was not influenced by concomitant intake of ibopamine. The observations suggest that in healthy volunteers there is no pharmacokinetic interaction between 5-ISMN and ibopamine.


Assuntos
Desoxiepinefrina/farmacocinética , Dinitrato de Isossorbida/análogos & derivados , Ácido 3,4-Di-Hidroxifenilacético/sangue , Ácido 3,4-Di-Hidroxifenilacético/urina , Adulto , Pressão Sanguínea/efeitos dos fármacos , Desoxiepinefrina/administração & dosagem , Interações Medicamentosas , Feminino , Ácido Homovanílico/sangue , Ácido Homovanílico/urina , Humanos , Dinitrato de Isossorbida/administração & dosagem , Dinitrato de Isossorbida/farmacocinética , Masculino
15.
Bull Soc Belge Ophtalmol ; 241: 71-5, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1840999

RESUMO

The Hallermann-Streiff syndrome is characterised by systemic and ocular anomalies, including congenital cataract and microphthalmia. A case is presented and the differential diagnosis discussed.


Assuntos
Catarata/complicações , Síndrome de Hallermann/complicações , Microftalmia/complicações , Catarata/congênito , Catarata/diagnóstico , Criança , Opacidade da Córnea/diagnóstico , Humanos , Masculino , Microftalmia/diagnóstico , Acuidade Visual
16.
Eur J Clin Pharmacol ; 40(6): 629-30, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1884747

RESUMO

The bioavailability of two slow release preparations of disopyramide has been compared in a randomized cross-over trial of Rythmodan L.A. 250 mg b.d. and Dirytmin Durettes 300 mg b.d., given to 10 healthy volunteers. The plasma concentrations of disopyramide were measured on the 5th day of each treatment period. With both preparations, plasma concentrations were well sustained. The amount absorbed was slightly lower after Rythmodan L.A. than after Dirytmin Durettes, but the fluctuations over a dosing interval were significantly more pronounced for Dirytmin Durettes than for Rythmodan L.A.


Assuntos
Disopiramida/farmacocinética , Adulto , Disponibilidade Biológica , Preparações de Ação Retardada , Disopiramida/administração & dosagem , Disopiramida/sangue , Feminino , Humanos , Masculino
17.
Bull Soc Belge Ophtalmol ; 238: 137-43, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2131116

RESUMO

Fourty patients with a malignant melanoma of the choroid were treated with ruthenium applicators. Depending on the height of the tumour, the patients were divided into 3 groups. In the first group with a tumour height superior to 6 mm the results were poor as well from a functional point of view as considering tumour regression. In a second group with a tumour height between 5 and 6 mm the results were favourable concerning tumour regression, but only in 50% of the cases did the eye remain functional. The results in the third group with a tumour height between 2 and 5 mm were satisfactory. Tumour regression was obtained in 96% of the eyes; 86% of the eyes remained functional. Tumour related death (7.5%) was not correlated with initial height of the tumour at the time of diagnosis.


Assuntos
Braquiterapia/métodos , Neoplasias da Coroide/radioterapia , Melanoma/radioterapia , Radioisótopos de Rutênio/uso terapêutico , Adulto , Idoso , Humanos , Pessoa de Meia-Idade , Prognóstico , Radioisótopos de Rutênio/administração & dosagem
18.
Antiviral Res ; 12(2): 75-86, 1989 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2531990

RESUMO

We investigated the development of anti-pre-S(2) antibodies by enzyme immunoassay and by Western blot analysis in a vaccination study in haemodialysis patients; both the Pasteur plasma vaccine retaining the pre-S(2) epitopes and the Merck, Sharp and Dohme (MSD) plasma vaccine containing only HBsAg were used. By enzyme immunoassay (EIA), one anti-pre-S(2) response at month 7 was registered in 23 patients after 5 injections with the 5 micrograms dose of Pasteur vaccine (PS group), whereas 6 responses were seen in 20 patients after 4 injections with the 10 micrograms dose (PD group). None of the 22 vaccines injected with MSD vaccine (40 micrograms) (4 injections, MD group) showed an anti-pre-S(2) response at month 7. In the Western blot an anti-pre-S(2) response was seen in 12 PS patients, in 8 PD patients and in none of the MD patients. Anti-pre-S(2) responses were predominantly observed in patients with a high anti-HBs response but exceptions occurred. Prevaccination anti-pre-S(2) positivity, in the absence of anti-HBc and anti-HBs, was detected in dialysis patients with EIA as well as Western blot, in 10.8 and 21.6%, respectively; similar findings were made in health care personnel. The possible nature of this phenomenon is discussed. In this study the Western blot technique has been shown to be a suitable test system for qualitative and semi-quantitative analysis of anti-pre-S(2) antibodies after hepatitis B vaccination with a higher sensitivity, but probably also a different specificity than the EIA.


Assuntos
Anticorpos Anti-Hepatite B/análise , Antígenos de Superfície da Hepatite B/imunologia , Hepatite B/prevenção & controle , Precursores de Proteínas/imunologia , Diálise Renal , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Adulto , Idoso , Especificidade de Anticorpos , Western Blotting , Feminino , Vacinas contra Hepatite B , Humanos , Técnicas Imunoenzimáticas , Estudos Longitudinais , Pessoa de Meia-Idade , Vacinação
19.
J Med Virol ; 27(2): 95-9, 1989 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2646395

RESUMO

We investigated sera from 39 patients, taken 1-8 years after recovery from acute hepatitis B for anti-pre-S(2) by Western blotting and for anti-HBs by radioimmunoassay. Anti-pre-S(2) antibodies were found in 27 out of 39 sera (69%) with the highest frequency in sera with anti-HBs greater than 100 IU/I (92%). However, sometimes sera with low anti-HBs titres showed a strong response in Western blotting. Acute hepatitis sera were also investigated from a limited number of patients (n = 14). Anti-pre-S(2) antibodies were found during antigenaemia (four out of six patients) and within 3 months after the maximum of alanine amino transferase (ALAT) (seven out of ten patients). Anti-pre-S(2) is an early antibody. It remains in the circulation for many years similar to but independent of anti-HBs.


Assuntos
Anticorpos Anti-Hepatite B/análise , Antígenos de Superfície da Hepatite B/imunologia , Hepatite B/imunologia , Precursores de Proteínas/imunologia , Proteínas do Envelope Viral/imunologia , Doença Aguda , Western Blotting , Antígenos de Superfície da Hepatite B/análise , Humanos , Técnicas Imunoenzimáticas , Precursores de Proteínas/análise
20.
Tijdschr Diergeneeskd ; 112(6): 322-33, 1987 Mar 15.
Artigo em Holandês | MEDLINE | ID: mdl-3824354

RESUMO

Dry fermented sausage (dfs) was the food most suspected in a number of outbreaks of salmonellosis and staphylococcal enterotoxaemia. Data on formulation and processing showed that over 75 per cent of 76 producers still manufactured dfs in a traditional manner: fermentation and drying at ambient temperature for ten days on an average, green room facilities not present. 'Modern' processes were characterised by fermentation in green rooms at elevated temperatures, thus limiting production time to six days on an average. However, precautions to prevent luxurious growth of S. aureus under these conditions were not adopted to any appreciable extent. Consequently, high S. aureus levels (greater than 10(4) cfu/g) were detected precisely in dfs from five manufacturers using rapid processes. Colony counts of Enterobacteriaceae were low in dfs (81 per cent of 151 samples less than 10(3) cfu/g), associated with relatively low pH and aw levels and a high concentration of salt. However, Salmonella was detected in 16 (11%) of the samples, both from 'traditional' and 'modern' producers. Improvement of manufacturing practices in the manufacture of dfs should be stimulated to guarantee wholesome and safe products.


Assuntos
Inspeção de Alimentos/normas , Microbiologia de Alimentos , Produtos da Carne/normas , Carne/normas , Enterobacteriaceae/isolamento & purificação , Humanos , Países Baixos , Intoxicação Alimentar por Salmonella/prevenção & controle , Intoxicação Alimentar Estafilocócica/prevenção & controle , Staphylococcus aureus/isolamento & purificação
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