RESUMO
Mouse macrophage Fc receptors specific for IgG1/IgG2b mediate the binding and pinocytic uptake of soluble IgG-containing antibody-antigen complexes. Internalization of these multivalent IgG complexes is accompanied not only by the intracellular degradation of the ligand, but also by a net decrease in the number of plasma membrane Fc receptors and an accelerated rate of receptor turnover. In contrast, internalized receptors bound to a monovalent ligand, the high affinity Fab fragment of the antireceptor mAb 2.4G2, escape degradation by rapidly recycling to the cell surface. In this paper, we have characterized the intracellular pathway involved in the endocytosis and transport of Fc receptors in the J774 macrophage cell line. The results show that the uptake of multivalent ligands follows the normal pathway of receptor-mediated endocytosis: internalization in clathrin-coated pits and coated vesicles, delivery to endosomes, and finally to acid hydrolase-rich lysosomes. Immunoprecipitation of radiolabeled receptor from Percoll density gradients showed that endocytosis of the IgG complexes also results in the concomitant transport of the receptor to lysosomes. Although uptake of the monovalent Fab fragment had no detectable effect on intracellular receptor distribution, preparations of 2.4G2 Fab rendered multivalent by adsorption to colloidal gold were as effective as the IgG complexes at causing lysosomal accumulation of internalized receptors. Thus, it is likely that the down-regulation and degradation of Fc receptors which occurs during the endocytosis of antibody-antigen complexes is due to the transport of internalized receptors to lysosomes. Moreover, the ability of certain Fc receptor-bound ligands to interfere with receptor recycling and trigger lysosomal transport seems to depend on ligand valency rather than on the presence or absence of Fc domains on intact IgG molecules.
Assuntos
Lisossomos/metabolismo , Macrófagos/imunologia , Receptores Fc/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Fracionamento Celular , Linhagem Celular , Células Cultivadas , Endocitose , Ligantes/metabolismo , CamundongosRESUMO
Binding and pinocytosis of polyvalent IgG-containing immune complexes by mouse macrophages leads to the selective removal of Fc receptors (FcR) from the cell surface and to the rapid delivery of receptor and ligand to lysosomes, where both are degraded (I. Mellman and H. Plutner, 1984, Journal of Cell Biology, 98:1170-1177). In this paper, we have studied the internalization of FcR tagged with a monovalent probe that, unlike IgG-complexes, cannot cross-link adjacent receptors. We have used an Fab fragment of high affinity anti-FcR monoclonal antibody whose binding was completely sensitive to low pH (4.0) at 4 degrees C. Thus, surface-bound (acid-releasable) and intracellular (acid-resistant) 125I-Fab could be readily distinguished. Incubation of J774 macrophages with 125I-Fab at 37 degrees C did not lead to the accumulation of large amounts of the antibody in the acid-resistant compartment. After 3 h, only 20% of the total cell-associated radiolabel was intracellular. The internalized 125I-Fab was also shown by Percoll gradient centrifugation to be associated primarily with low density endosomes, as opposed to lysosomes. Significantly, most of the labeled antibody returned rapidly to the plasma membrane, still bound to FcR. This recycling was complete within 10 min, was unaffected by NH4Cl, and was only slightly inhibited by the Na+-H+ ionophore monensin. These results indicate that monovalent Fab-FcR complexes are internalized, delivered to endosomes, and rapidly returned to the cell surface. Since the internalization of polyvalent IgG-complexes removed the FcR from this recycling pathway and caused its transport to lysosomes, we suggest that the state of receptor aggregation in the endosome membrane helps determine its intracellular fate.
Assuntos
Complexo Antígeno-Anticorpo , Lisossomos/fisiologia , Macrófagos/imunologia , Receptores Fc/fisiologia , Animais , Linhagem Celular , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas/análise , Imunoglobulina G , Cinética , Macrófagos/fisiologia , CamundongosRESUMO
A new enzyme-immunoassay (EIA, Hepanostika Microelisa System) for the detection of hepatitis B surface antigen was evaluated against other methods, namely complement fixation, Hepanosticon, AusRIA II and Finnish Red Cross Radioimmunoassay (FRC-RIA). EIA detected the greatest number of positive samples in a serum panel consisting of 142 sera from clinical hepatitis patients. FRC-RIA was the most sensitive method for subtype ad, while EIA detected the ay specimen at the highest dilution. None of the test systems gave the 'optimal' result in the screening test, and it is proposed that a separate procedure for each antigen subtype should be carried out to detect the greatest number of positive samples.
Assuntos
Antígenos de Superfície da Hepatite B/análise , Técnicas Imunoenzimáticas , Testes de Fixação de Complemento , Humanos , Imunodifusão , RadioimunoensaioRESUMO
OBJECTIVE: Quinolones are used ever increasingly in pediatrics, although officially they are still contraindicated. Lack of evidence of arthropathic effects in human offspring favors their use, but little is known about the pharmacokinetics of oral or parenteral ciprofloxacin in children, especially those without cystic fibrosis. DESIGN: We studied 16 non-cystic fibrosis patients ranging in age from 0.3 to 7.1 years to whom the new suspension formulation of ciprofloxacin (10 mg/kg body weight) was given orally three times daily. Single-dose and steady-state pharmacokinetic parameters were elucidated. RESULTS: Ciprofloxacin was rapidly absorbed. The maximum plasma concentrations, with the means varying from 1.7 to 3.6 mg/L, were reached within 1 hour, almost regardless of whether single-dose administration or steady state. The mean oral clearance was lower in children <6 years of age than in those >/=6 years. Terminal half-life values, with the means varying only between 4.2 and 5.1, suggest that dosing recommendations based on body weight are pertinent, although caution should be exercised in small infants. No arthropathic or other adverse events attributable to ciprofloxacin suspension were observed. CONCLUSION: A dose of the suspension form of ciprofloxacin of 10 mg/kg body weight given orally three times daily seems appropriate in children, provided the drug is clearly indicated.
Assuntos
Ciprofloxacina/farmacocinética , Administração Oral , Criança , Pré-Escolar , Ciprofloxacina/administração & dosagem , Ciprofloxacina/efeitos adversos , Ciprofloxacina/metabolismo , Esquema de Medicação , Feminino , Humanos , Lactente , Masculino , SuspensõesRESUMO
Specific animal antisera to cytomegalovirus (CMV) and varicella-zoster virus (VZV), devoid of antibodies to host cell components, have been difficult to prepare because of the cell-associated nature of these viruses. A simple method for the purification of CMV and VZV antigens is described in this paper. The viral antigens were purified from commercially available crude antigens by affinity chromatography with human antibodies against CMV and VZV. Rabbits were immunized with the purified antigens. The produced antisera were specific for the respective viruses and did not contain antibodies to host cell components, as determined by immunofluorescence microscopy and enzyme-immunoassay. The antisera have been used for direct detection of viral antigens in clinical specimens and for identification of cytopathic effect in cell cultures by immunofluorescence microscopy. The CMV antiserum could also be used for the early detection of CMV in cell cultures inoculated with patient specimens, but it was less sensitive than a monoclonal antibody to the early antigen of CMV.
Assuntos
Antígenos Virais/análise , Citomegalovirus/imunologia , Herpesvirus Humano 3/imunologia , Soros Imunes/imunologia , Animais , Anticorpos Anti-Idiotípicos/análise , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , CoelhosRESUMO
Conventional virus isolation and detection of cytomegalovirus (CMV) early antigen by immunofluorescence staining of cultured cells were compared in the diagnosis of CMV infection from urine specimens. By virus isolation, 33 specimens out of 333 studied were positive, and the mean length of culturing time for a positive result was 23 days (range from 6 to 45 days). By early antigen detection, 35 specimens were positive after 20 hours in culture, but the number of positive findings increased to as high as 49 after 7 days in culture. It is recommended that, in addition to the early antigen staining after one day in culture, cells should also be stained after one week in culture, because the sensitivity is essentially improved by extended culturing time.
Assuntos
Antígenos Virais/análise , Infecções por Citomegalovirus/diagnóstico , Células Cultivadas , Citomegalovirus/imunologia , Imunofluorescência , Humanos , Testes SorológicosRESUMO
A simple method was developed for separation of cells from nasopharyngeal secretion for the diagnosis of respiratory virus infections by immunofluorescence microscopy. The diluted specimen, containing dithiothreitol to break up the mucus, was centrifuged once through a cushion of 20% Percoll (colloidal silica, a density gradient medium), which permitted sedimentation of cells through the cushion, but retained mucus on top of it. The pelleted cells were resuspended, and microscope slides were then prepared by standard techniques. The Percoll centrifugation method was also applicable for sputum and bronchoaveolar lavation specimens. Immunofluorescent antibody staining of nasopharyngeal secretions prepared by the described method was more sensitive than enzyme immunoassay for the detection of respiratory syncytial virus.
Assuntos
Separação Celular/métodos , Centrifugação/métodos , Muco/patologia , Nasofaringe/patologia , Infecções Respiratórias/patologia , Antígenos Virais/análise , Ditiotreitol , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Microscopia de Fluorescência , Povidona , Vírus Sinciciais Respiratórios/imunologia , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/patologia , Dióxido de Silício , Manejo de EspécimesRESUMO
Rabbits and guinea pigs were immunized with purified bovine rotavirus. Immunoglobulin G fractions of the resulting antisera were used in a standard four-layer solid-phase enzyme immunoassay (EIA) for rotavirus antigen in human faecal specimens. Samples negative for rotavirus in electron microscopy, when diluted in standard EIA buffers, regularly gave absorbance values lower than those obtained with buffer blank only. By further diluting of the samples the resulting absorbance values were found to increase to the blank levels. When all dilution buffers were supplemented with 1-5% of bovine serum, negative samples at any dilution gave absorbance values close to those of the buffer blanks. Similar results were obtained if the serum was replaced by 1-5 mM of phenylmethylsulphonyl fluoride, a synthetic broad spectrum serine-type protease inhibitor. Aprotinin, another protease inhibitor, was without effect. A similar inhibition pattern was obtained when faecal specimens were tested in a caseinolytic quantitative protease assay in the presence of the above inhibitors. These observations suggest that protease activity present in human faecal samples may cause false-negative results in solid-phase immunoassay for viral antigens, unless appropriate means are used to avoid this interference.
Assuntos
Antígenos Virais/análise , Fezes/imunologia , Peptídeo Hidrolases/metabolismo , Rotavirus/imunologia , Reações Falso-Negativas , Fezes/enzimologia , Humanos , Técnicas Imunoenzimáticas , Fluoreto de Fenilmetilsulfonil/farmacologiaAssuntos
Portador Sadio , Difteria/diagnóstico , Viagem , Pré-Escolar , Difteria/etiologia , Feminino , Finlândia , HumanosRESUMO
The persistence of hepatitis B surface antigen (HBsAg), e antigen (HGeAg) and antibodies to e antigen (anti-HBe) was studied retrospectively in 197 Finnish voluntary blood donors, who had been found positive for HBsAg by routine screening in 1970 or 1971. Two samples were available from each donor: the first was taken in 1970 or 1971 and the second in 1978; the average interval between the specimens was 7.6 years. All except one (99.5%) of the donors remained HGsAg carriers during the whole study period. HBeAg was detected initially in 3 (2%) cases, all of whom lost HBeAg after the follow-up, and 2 converted to anti-HBe-positive. Anti-HBe was found in the first samples of 158 (80%) HGsAg carriers, 154 (97%) of whom were still positive for anti-HBe in the second samples. These results suggest that the HBsAg carrier stage is stable as for the presence of HBsAg and anti-HBe.
Assuntos
Anticorpos Antivirais/análise , Doadores de Sangue , Anticorpos Anti-Hepatite B/análise , Antígenos de Superfície da Hepatite B/análise , Adulto , Fatores Etários , Seguimentos , Humanos , Fatores de TempoRESUMO
Serum samples from 94 Finnish hospital patients with hepatitis B were investigated for subtypes of HBsAg. Subtype ay predominated in patients with acute hepatitis (77%). All drug abusers had the ay subtype. Of 11 patients with chronic liver disease, 10 (91%) had the ad subtype. Five of 7 hepatitis cases associated with blood transfusion had subtype ay, although this determinant has not been detected among Finnish non-paid blood donors. These results suggest that blood donors who incubate hepatitis B (HBsAg not yet detectable) are an important source of transfusion hepatitis.
Assuntos
Antígenos de Superfície da Hepatite B , Hepatite B/imunologia , Doença Aguda , Adulto , Doadores de Sangue , Transfusão de Sangue , Doença Crônica , Epitopos , Hepatite B/transmissão , Humanos , Hepatopatias/imunologia , Pessoa de Meia-Idade , Transtornos Relacionados ao Uso de SubstânciasRESUMO
As the previous vaccination of 11- to 13-year-old girls proved ineffective, nationwide vaccination of preschool children with 2 doses of combined vaccine against measles, mumps, and rubella was started in Finland in 1982. To study the impact of vaccination, age-stratified rubella immunity and the occurrence of serologically verified rubella cases were determined using the computerized data of our diagnostic virus laboratory. The analysis covered the period 1979-1992, included all ages, and was based on the test results from 94,000 sera. By 1992, the seropositivity rate was 92-100% in 2- to 15-year-old children, remained high in females of all ages, but showed a gap in 16- to 19-year-old males. The number of verified rubella cases decreased to about 1/100, but outbreaks still occurred until 1991, when most cases were among adolescent males. The better protection of women was due to the vaccination of prepubertal girls since 1975. No congenital rubella infections were diagnosed after 1986. The 2-dose immunization of preschool children, complemented by selective vaccination of certain other groups, has resulted in excellent immunity in children and young adults, and practically eliminated rubella from the country.
Assuntos
Anticorpos Antivirais/sangue , Vacina contra Sarampo/imunologia , Vacina contra Caxumba/imunologia , Vacina contra Rubéola/imunologia , Rubéola (Sarampo Alemão)/prevenção & controle , Adolescente , Adulto , Idoso , Criança , Feminino , Finlândia , Humanos , Masculino , Vacina contra Sarampo-Caxumba-Rubéola , Pessoa de Meia-Idade , Rubéola (Sarampo Alemão)/epidemiologia , Fatores de Tempo , Vacinação , Vacinas Combinadas/imunologiaRESUMO
We determined the age-related prevalence of complement-fixing antibody to Mycoplasma pneumoniae from the computerized laboratory results of our routine diagnostic department. The material consisted of about 58,000 sera from an 8-year period, 1971 to 1978. Among children less than 1 month old, the frequency of complement-fixing antibody of titers greater than or equal to 8 was low (23%), and no decrease representing loss of maternal antibody was seen thereafter. An unexpectedly early increase in the antibody prevalence up to 62% was seen by 6 months of age. The frequency of antibody was high among young children from the age of 4 months, in whom symptomatic infection due to M. pneumoniae is rare. The frequency of higher titers (greater than or equal to 32) and the geometric mean titer increased more slowly, coinciding with the known age distribution of symptomatic M. pneumoniae disease; the frequency of high titers and the geometric mean titer peaked at the age of 7 to 10 years. Two explanations for the high frequency of complement-fixing antibody to M. pneumoniae in young children are discussed. It may be due to an asymptomatic infection caused by M. pneumoniae or to a nonspecific stimulus by lipids of other organisms, plants, or tissues leading to production of antibodies crossreacting with M. pneumoniae, or it may be due to both of the above. During the study, two extensive epidemics due to M. pneumoniae occurred in Finland, but the prevalence of complement-fixing antibody bore no correlation with these peaks of occurrence.
Assuntos
Anticorpos Antibacterianos/análise , Mycoplasma pneumoniae/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Criança , Pré-Escolar , Testes de Fixação de Complemento , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-IdadeRESUMO
A solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Rabbit and guinea pig antisera for the assay were raised against purified poliovirus type 3/Fin (strain 3/Fin/K) isolated from a fecal specimen from a meningitis patient during an outbreak of poliomyelitis in Finland in 1984. The EIA was highly specific for poliovirus type 3, and it was about 30 times more sensitive for strain 3/Fin/K than for strain 3/Saukett used in the inactivated poliovirus vaccine. The sensitivity of the EIA was 2 to 5 ng of purified strain 3/Fin/K per ml, whereas disrupted viruses and soluble viral proteins were almost undetectable by the assay. Only 5 of 51 (10%) stool specimens containing poliovirus type 3/Fin detected by virus isolation were positive by the EIA. Quantitation by the EIA, using purified poliovirus 3/Fin/K as a standard, revealed that concentrations of poliovirus type 3 in undiluted fecal specimens of patients with natural poliovirus infection were only 50 ng/ml or less. In conclusion, owing to the small amount of poliovirus in feces, the EIA is not suitable for the diagnosis of poliovirus infections directly from clinical specimens, but it can be used to detect, type, and quantitate poliovirus antigen in infected cells.
Assuntos
Antígenos Virais/análise , Poliomielite/diagnóstico , Poliovirus/imunologia , Animais , Especificidade de Anticorpos , Fezes/microbiologia , Cobaias , Humanos , Soros Imunes/imunologia , Técnicas Imunoenzimáticas , Poliovirus/isolamento & purificação , Valor Preditivo dos Testes , Coelhos , Especificidade da EspécieRESUMO
Effects of time, temperature, pH and stabilizers (i.e. medium) on inactivation of lipid-enveloped model viruses, Semliki Forest and vesicular stomatitis viruses in the production process of intravenous immunoglobulin were investigated on a laboratory scale. The lowering of pH, the raising of temperature and the increasing of incubation time improved the inactivation effect. However, small changes in pH and stabilizer concentrations did not influence the results. Inactivation was not linear and a clear tailing off could be seen. Therefore, for complete virus inactivation incubation times longer than 20 h are necessary. Inactivation took place much more rapidly in intravenous immunoglobulin solution than in intramuscular immunoglobulin solution. Processing steps such as freeze-dying in the presence of ethanol or storage of intramuscular immunoglobulin in the liquid state at pH7 only partially inactivated these viruses.
Assuntos
Transfusão de Sangue , Imunoglobulinas , Ativação Viral , Liofilização , Concentração de Íons de Hidrogênio , Imunoglobulinas/administração & dosagem , Imunoglobulinas/isolamento & purificação , Pepsina A , Vírus da Floresta de Semliki , Soluções , Temperatura , Fatores de Tempo , Vírus da Estomatite Vesicular IndianaRESUMO
The seroepidemiology of chlamydial infections in the Finnish population was studied by analysing the prevalence of chlamydial complement fixing (CF) antibodies in patients sera sent for virus serological screening tests over 17 years from 1971 to 1987. The total number of sera studied was over 160,000. In the early 1970s, the prevalence of chlamydial CF antibodies (CF titres greater than or equal to 8) was low (less than 2%), but later the proportion of seropositive cases rose, and in 1976, 18% of sera contained antibodies. In 1984, the seropositivity rate was over 31%. The prevalence of high chlamydial CF titres (titres greater than or equal to 64) also showed annual variation. In general, under 1% of sera contained chlamydial CF antibodies in high titre, but in 1979 and 1984, distinct peaks occurred when 1.3% and 1.4% of sera, respectively, had titres greater than or equal to 64. The age-related antibody positivity rate showed a decline during early infancy, an increase in childhood and adolescence, and a stable level in adulthood when approximately 20% of the sera contained antibodies. The chlamydial antigen used in this survey was genus-specific, i.e. it detects antibodies against all chlamydial species. Epidemiological data support the hypothesis that infections due to a novel chlamydial species, TWAR chlamydia, are the most likely explanation for the relatively frequent occurrence of chlamydial CF antibodies and for the variation in CF antibody prevalence.
Assuntos
Infecções por Chlamydia/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Anticorpos Antibacterianos/análise , Criança , Pré-Escolar , Chlamydia/imunologia , Testes de Fixação de Complemento , Finlândia , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Fatores de TempoRESUMO
The occurrence of rubella antibodies and frequency of virologically proven rubella infections in different age groups were analyzed in a large serum material (about 60,000 sera) collected both before and after the start of nationwide vaccination of children against measles, mumps and rubella in Finland in 1982. The combined live vaccine significantly raised the rubella immunity of children and shifted rubella infections to older ages. In 1986, 91-98% immunity was found in the 2-10-year-old children so far covered by the vaccination programme; no rubella cases were diagnosed in this age group. We also demonstrated that another rubella vaccine given to about 60% of 13-year-old girls since 1975 both raised the immunity and reduced the occurrence of rubella in the vaccinated population. It is concluded that the rubella vaccinations, especially the combined vaccine given to small children, are effective, although the total number of reported rubella cases in the whole population did not decrease significantly during the study period.
Assuntos
Vacina contra Rubéola/imunologia , Rubéola (Sarampo Alemão)/prevenção & controle , Vacinação , Adolescente , Adulto , Fatores Etários , Anticorpos Antivirais/análise , Criança , Pré-Escolar , Feminino , Finlândia , Humanos , Lactente , Masculino , Rubéola (Sarampo Alemão)/epidemiologia , Rubéola (Sarampo Alemão)/imunologia , Vacina contra Rubéola/administração & dosagem , Fatores SexuaisRESUMO
A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of mumps and parainfluenza type 1 antibodies (immunoglobulin G [IgG] and IgM classes) is described and compared with the conventional complement fixation (CF) test. A highly positive correlation was found between mumps IgG ELISA and the mumps CF test, whereas parainfluenza type 1 IgG ELISA had only a moderate positive correlation with the respective CF test. Mumps IgM antibodies could be demonstrated in all patients with serologically verified and clinically typical (parotitis, meningitis, or orchitis) mumps virus infection, but not in patients with rises in parainfluenza CF titers. Mumps IgM was already present in the acute-phase sera if they were not taken during the first 2 days after onset of disease. Mumps IgM was also found in some paired sera that were taken too late to demonstrate any significant increase in the antibody titers by CF. Therefore, mumps IgM ELISA provides an improvement over the conventional laboratory diagnosis of mumps infection, since the measurement of specific IgM antibodies in a single serum by ELISA is diagnostic, rather than the identification of a fourfold or greater rise in CF antibody titer. An unexpected finding was that parainfluenza type 1 IgM antibodies could not be demonstrated by ELISA in paired sera with rises in parainfluenza CF titers, suggesting a different antibody response from that occurring in mumps infection.