RESUMEN
Mutations that modify the amino acid sequence of C1-INH (except Val458Met) are associated with HAE. More than 200 different mutations scattering the entire C1-INH gene have been reported. The main objective of this study was to report the mutational findings in a HAE cohort of 138 Portuguese patients followed in specialized consultation all over the country. DNA was extracted from peripheral blood with QiaSymphony BioRobot (QIAGEN Portugal). The sequence reactions were performed by using a DNA sequencing kit (Big Dye terminator cycle sequencing v1.1/v3.1 from Applied Biosystems) and sequencing products were immediately submitted to direct sequencing on an Applied Biosystem 3130 DNA Analyser. DNA sequences were analyzed at four different stages. Raw data and sequence alignments of all 8 exons and intron-exon boundaries were performed for each patient individually with SeqScape software and using SERPING1 gene NG_009625 of 24,300 bp (12-March-2011) as reference sequence. Sequence comparisons among patients and controls were performed with software CodonCode Aligner v.3.7 from CodonCode Corp and with Geneious 4.5 from Biomatters Lda. A total of 94 point mutations were observed among patients, and 67% of them were located on exon 8. In addition, we noticed one not described stop codon at position c.1459 C>T in three different patients. Translation termination was also found on exon 3 and 7, as a result of mutations at positions c.481A>7, c.1174C>T. In this population, the prevalence of the missense mutation p.Arg444Cys was 39 out of 42. Mutational analysis revealed 22 different pathogenic mutations, of which 64% were not described on HAE database. Although identification of disease causing mutations is not necessary to establish HAE diagnosis, studies on gene expression and characterization of rearrangements in SERPING1 gene are suggested in order to get new insights on function and genetic tests of C1 inhibitor.
Asunto(s)
Angioedemas Hereditarios/genética , Proteínas Inactivadoras del Complemento 1/genética , Mutación , Población Blanca , Adolescente , Adulto , Anciano , Sustitución de Aminoácidos , Estudios de Casos y Controles , Niño , Proteína Inhibidora del Complemento C1 , Análisis Mutacional de ADN , Exones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistemas de Lectura Abierta , PortugalRESUMEN
Objective: to compare Doppler measurements with real flow in a in-vitro model, with variable diameters and insonation angle. Methods: The silicone tubes were 3-8 mm width, and set with variable inclinations from 40° to 70°. Two pumps with constant flow were used for all combinations of diameters and angles. A Sonoace 8000 from Medison was used. Real flow was compared to measured flow. Results: The measured flows varied importantly in different conditions. In a 3 mm tube, estimated flow increased from 212 ml/min to 403 ml/min when the angle was increased from 40° to 70°, when real flow was 140 ml. As well, in 8 mm tubes, estimated flow increased from 651 ml/min to 1.080 ml/min when the angle was increased from 40° to 70°, when real flow was 360 ml/min. Measured flows were 1.6 times greater than real flow. Conclusion: Measured flows were greater than real, with greater increase in larger tubes and greater angles. This confirms that velocity measurements need lowest angles possible. Measured flows are only representations of real flow, but can be considered useful as they were reproducible.
Objetivos: Recientes aplicaciones del Doppler pulsado se han orientado hacia la medición del flujo (ml/s) de la vena umbilical fetal, sin embargo sus mediciones han tenido cuestionamientos desde el punto de vista de la validez, por el efecto del ángulo y el flujo laminar. El objetivo de este estudio es probar la validez de las mediciones ecográficas en un modelo in vitro. Métodos: Se construyó un sistema dentro de un reservorio de agua, en que se instalaron tubos de silicona variando el lumen (3-8 mm), inclinación del tubo (40°-70°) y dos velocidades de infusión de agua. Se instaló la sonda convexa del transductor transabdominal a 5 cm sobre el tubo, de modo que todo el trayecto del tubo sea visualizado en la pantalla del ecógrafo SONOACE 8000 de Medison. Se corrigió el ángulo con la función del ecógrafo. El flujo se estimó por ecografía al multiplicar la velocidad medida por Doppler pulsado por el área interna del tubo. Se comparó el flujo medido real, obtenido por el volumen de agua obtenida en un minuto de funcionamiento de la bomba, y el medido por ecografía Doppler. El flujo medido por Doppler se obtuvo 2 veces, para comparar la variación intrínseca del método y verificar su confiabilidad. Resultados: Los flujos obtenidos variaron según las condiciones mecánicas importantemente. En tubos de 3 mm de diámetro, con ángulos crecientes de 40° a 70°, los fl ujos estimados aumentaron de 212 ml/min a 403 ml/min cuando el flujo real era de 140 ml/min. A su vez, en tubos de 8 mm, con ángulos crecientes de 40° a 70°, los flujos estimados aumentaron de 651 ml/min a 1.080 ml/min, cuando el flujo real era de 360 ml/min. Se demostró una correlación lineal entre el flujo estimado y el real de: Qestimado=(Qreal x 1,63)+181, r= 0,84. La aplicación del test de Bland y Altman demostró que las mediciones son repetibles y consistentes. Conclusión: Los flujos medidos en ecografía fueron 1,6 veces más que los obtenidos en flujo real en las condiciones mecánicas...
Asunto(s)
Humanos , Femenino , Embarazo , Velocidad del Flujo Sanguíneo , Ultrasonografía Doppler , Ultrasonografía Prenatal , Algoritmos , Modelos BiológicosRESUMEN
Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine (CsA), play an essential role in graft survival, preventing rejection. Large interindividual differences in drug-metabolizing enzymes as well as in drug transporters make the task of reaching the optimal concentrations difficult. The bioavailability of CsA and FK506 seems to be associated with the cytocrhome P450 IIIA (CYP3A) gene. It has also been described that the Multi Drug Resistance 1 (MDR1) gene that encodes for polyglycoprotein-P (P-gp) may influence the metabolizing action of FK506 and CsA. Therefore, we sought, to correlate single nucleotide polymorphisms (SNPs) in the CYP3A and MDR1 genes with the concentrations of FK506 and CsA. For this purpose we analyzed 2 groups of renal transplant recipients by sequencing: one receiving a CsA immunosuppressive regime, and other, an FK506-immunosuppression. This study showed that subjects in the FK506 group who had encoded the 1236C>T substitution in the MDR1 gene displayed 44.4% higher drug concentrations compared with ("wild-type") individuals. Individuals carrying the 2677G>T,A mutation showed FK506 concentrations that were 44.7% higher than the wild-type individuals. Concerning the CsA group, individuals carrying the 22915A>C substitution displayed CsA concentrations 52.1% higher than wild-type individuals.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Ciclosporina/sangre , Citocromo P-450 CYP3A/genética , Trasplante de Riñón/fisiología , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Tacrolimus/sangre , Subfamilia B de Transportador de Casetes de Unión a ATP , Adenina , Ciclosporina/uso terapéutico , Cartilla de ADN , Femenino , Guanina , Humanos , Inmunosupresores/sangre , Inmunosupresores/uso terapéutico , Trasplante de Riñón/inmunología , Masculino , Tacrolimus/uso terapéutico , TiminaAsunto(s)
Infecciones por Haemophilus/inmunología , Vacunas contra Haemophilus/inmunología , Haemophilus influenzae tipo b/inmunología , Alaska , Femenino , Infecciones por Haemophilus/prevención & control , Vacunas contra Haemophilus/administración & dosificación , Humanos , Inmunoglobulina G/análisis , Indígenas Norteamericanos , Lactante , Masculino , VacunaciónRESUMEN
The pneumococcal polysaccharide vaccine is recommended as a means of preventing invasive disease in the elderly. We compared responses to the 23-valent polysaccharide vaccine in 46 previously unvaccinated, healthy, institutionalized elderly persons (mean age, 85.5 years) with those in 12 healthy younger adults (mean age, 37 years) by measuring prevaccination and postvaccination serum IgG antibody concentrations (by ELISA), functional antibody activity (by opsonophagocytosis), IgG antibody avidity, and passive protection in mice. Postvaccination IgG antibody concentrations for two serotypes (6B and 19F) of the five studied (4, 6B, 14, 19F, and 23F) were significantly lower in elderly than in younger adults; however, opsonophagocytic activity was significantly reduced for all serotypes in the elderly. Sera with reduced opsonophagocytic activity (titer, <64) correlated with low IgG antibody avidity and protected mice poorly against pneumococcal challenge. In elderly persons receiving polysaccharide vaccination, there was a significant reduction in the functionality of postvaccination antibodies, and this appeared to increase with advanced age.
Asunto(s)
Envejecimiento/inmunología , Anticuerpos Antibacterianos/inmunología , Vacunas Bacterianas/inmunología , Streptococcus pneumoniae/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Afinidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunización Pasiva , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratones , Vacunas Neumococicas , VacunaciónRESUMEN
Forty-two Gambian children randomised to receive two doses of meningococcal A/C polysaccharide vaccine (MPS) in infancy and either MPS (n = 15), meningococcal A/C conjugate (n = 13) or inactivated polio vaccine (IPV n = 14) at 2 years, were revaccinated with MPS at 5 years of age along with 39 matched control children. Meningococcal A and C polysaccharide antibodies were analysed by ELISA and bactericidal assay (SBA) in sera taken before and 10 days after revaccination. The geometric mean group SBA titre in the MPS group following revaccination was about half that of the unvaccinated controls (0.51 95%CI: 0.28, 0.90) for group A and less than half that of the controls for group C (0.41, 95%CI: 0.16, 1.03 P = 0.06). The group C SBA response in the conjugate group was 14-fold higher than in the MPS group (P < 0.001). Multiple doses of meningococcal polysaccharide in childhood may therefore attenuate the SBA response to both group A and group C polysaccharides. In contrast, vaccination with meningococcal A/C conjugate after MPS in infancy gives immunological memory to N. meningitidis group C.
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Vacunas Bacterianas/efectos adversos , Vacunas Bacterianas/inmunología , Esquemas de Inmunización , Polisacáridos Bacterianos/efectos adversos , Polisacáridos Bacterianos/inmunología , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/administración & dosificación , Preescolar , Humanos , Lactante , Meningitis Bacterianas/prevención & control , Vacunas Meningococicas , Polisacáridos Bacterianos/administración & dosificación , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/efectos adversos , Vacunas Conjugadas/inmunologíaRESUMEN
An infant mouse assay system for assessment of protective concentrations of human serum pneumococcal anticapsular antibodies is described. Passive immunization of anticapsular antibodies was evaluated for protection of infant mice challenged with Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 18C, and 23A, with bacteremia as an end point. Protection was defined as no detectable bacteremia in 70% of mice 48 h after challenge. Type-specific anticapsular concentrations required for protection varied with serotype (=0.05 to >0.4 microg/mL). Across serotypes, there was no significant correlation between human IgG concentration in mouse serum and protection from bacteremia or between IgG concentration and opsonophagocytic titer. Significant correlation (r=.84, P<.001) was observed between opsonophagocytic titer of human IgG antibody in mouse sera and protection from bacteremia. Thus, protective concentrations of anticapsular antibodies against bacteremia are serotype dependent. Opsonophagocytosis is a better predictor of in vivo protective capacity of pneumococcal anticapsular antibodies than are ELISA IgG antibody concentrations.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Cápsulas Bacterianas/inmunología , Bioensayo/métodos , Inmunización Pasiva , Streptococcus pneumoniae/inmunología , Animales , Animales Recién Nacidos , Bacteriemia , Humanos , Ratones , Proteínas Opsoninas , Fagocitosis , Streptococcus pneumoniae/patogenicidadRESUMEN
The simulated moving bed (SMB) technology, first conceived for large bulk-scale separations in the petrochemical industry, has found increasingly new applications in the pharmaceutical industry. Among these, the separation of fine chemicals has been the subject of considerable study and research. This work presents the modeling, simulation and design of the operation of a SMB plant in order to separate a binary chiral mixture. The usual assumption of instantaneous equilibrium at the solid-fluid interface is questioned and a first-order kinetics of adsorption is taken into account. The cases of linear, Langmuir and modified Langmuir equilibria are studied. The equivalent true moving bed (TMB) model was used assuming axial dispersion for the fluid flow and plug flow for the solid-phase flow. Intraparticle diffusion was described by a linear driving force (LDF) approximation. Simulation results indicate that, under certain conditions, equilibrium is not actually reached at the adsorbent surface. This leads to different unit performances, in terms of product purities and recoveries, as compared to those predicted assuming instantaneous equilibrium. Moreover, SMB units may be improperly designed by the usual methods (flow-rate ratio separation regions) if non-equilibrium effects are overlooked.
Asunto(s)
Cromatografía/métodos , Adsorción , Industria Farmacéutica , Fructosa/aislamiento & purificación , Glucosa/aislamiento & purificación , Cinética , Matemática , Estereoisomerismo , TermodinámicaRESUMEN
OBJECTIVES: Pneumococcal polysaccharide vaccine is given after emergency splenectomy for trauma to lessen the risk of overwhelming postsplenectomy sepsis. This study was undertaken to determine optimal timing of vaccine administration as determined by serum type-specific polysaccharide antibody concentration titer and functional activity of the resulting antibodies. METHODS: Fifty-nine consecutive patients undergoing splenectomy after trauma were randomized to receive pneumococcal vaccine postoperatively at 1, 7, or 14 days. Immunoglobulin G serum antibody concentrations against serogroup 4 and serotypes 6B, 19F, and 23F were measured before vaccination and 4 weeks postvaccination. Antibody concentrations were determined by enzyme-linked immunosorbent assay, and functional antibody by opsonophagocytosis. Results were compared with a normal adult control group (n = 12). RESULTS: Postvaccination enzyme-linked immunosorbent assay immunoglobulin G antibody concentrations for all serogroups and serotypes studied were not significantly different in splenectomized patients and control subjects. Postvaccination functional antibody activity was significantly reduced in early vaccination groups (serotype 6B excepted). However, with the exception of 19F, all titers for the 14-day group approached those of the control subjects (p > 0.05). Fold-increases of opsonophagocytic titers for serogroup 4 and serotypes 6B and 19F showed progressive increases with delay in vaccination. Except for serotype 23F, the number of postsplenectomy patients with opsonophagocytic titers <64 significantly decreased with a delay in vaccination (14 days). CONCLUSIONS: Postvaccination immunoglobulin G serum antibody concentrations were not significantly different from normal control subjects regardless of the time of vaccination (1, 7, or 14 days). Although concentrations approach normal, functional antibody activity was significantly lower. Better functional antibody responses against the serogroup and serotypes studied seemed to occur with delayed (14-day) vaccination.
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Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Inmunoglobulina G/sangre , Esplenectomía , Streptococcus pneumoniae/inmunología , Adolescente , Adulto , Anciano , Esquema de Medicación , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Opsoninas/inmunología , Fagocitosis , Vacunas Neumococicas , Periodo Posoperatorio , Valores de Referencia , Heridas y Lesiones/cirugíaRESUMEN
Host protection against pneumococcal disease i primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes = 0.87: P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were 0.61, 0.60, 0.67 0.90, 0.61, 0.39, and 0.57, respectively, when HL-60 cells were used as effector cells and 0.56, 0.47, 0.61, 0.90, 0.71, 0.31, and 0.62, respectively, when PBLs were used. The assay requires small amounts of serum (40 microliters per serotype), making this test suitable for assaying infant sera. Culturable cells aid in assay standardization and likely reduce donor-to-donor variability. This standardized assay, in combination with the standardized ELISA, can be used to evaluate current and developing pneumococcal vaccines, in which functional opsonophagocytic antibody activity may correlate with protection against pneumococcal disease.
Asunto(s)
Anticuerpos Antibacterianos , Proteínas Opsoninas/inmunología , Infecciones Neumocócicas/diagnóstico , Infecciones Neumocócicas/inmunología , Adulto , Animales , Animales Recién Nacidos , Unión Competitiva/inmunología , Proteínas del Sistema Complemento/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Células HL-60/química , Células HL-60/inmunología , Células HL-60/microbiología , Humanos , Inmunoglobulina G , Inmunoglobulinas Intravenosas/administración & dosificación , Lactante , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Infecciones Neumocócicas/terapia , Conejos , Receptores de Superficie Celular/inmunologíaRESUMEN
Lack of primary immune response in severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL) has limited the applicability of this model. Use of human cytokines, in particular interleukin (IL)-12, was studied in the hu-PBL-SCID model. SCID mice were treated with IL-12 and reconstituted with hu-PBL in T replacement factor. The hu-PBL-SCID mice were immunized with serogroup C meningococcal polysaccharide (MCPS). The MCPS-specific antibody response was determined by ELISA. Thirteen of the 15 immunized, IL-12-treated hu-PBL-SCID mice demonstrated a primary human antibody response to MCPS ranging from 0.25 to 3.3 microg/mL, while no MCPS-specific antibody response was detectable in the 18 controls. Expression of cross-reactive idiotypic markers found on human anti-MCPS antibodies in the immunized hu-PBL-SCID mice was similar to that observed in immunized volunteers.
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Anticuerpos Antibacterianos/biosíntesis , Antígenos T-Independientes/inmunología , Linfocitos B/inmunología , Interleucina-12/farmacología , Transfusión de Linfocitos , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Adulto , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antibacterianos/sangre , Linfocitos B/trasplante , Ensayo de Inmunoadsorción Enzimática , Hemocianinas/inmunología , Humanos , Inmunización , Cinética , Ratones , Ratones Endogámicos ICR , Ratones SCID , Trasplante HeterólogoRESUMEN
Two hundred twenty-one Gambian children vaccinated previously with one, two, or three doses of a meningococcal conjugate vaccine or two doses of polysaccharide vaccine before the age of 6 months were revaccinated at the age of 18-24 months with either meningococcal polysaccharide, conjugate, or inactivated polio vaccines. Children who had previously received one, two, or three doses of conjugate vaccine had significantly (P < .001) higher anti-group C meningococcal antibody levels following revaccination than did children vaccinated with a polysaccharide vaccine for the first time. Children vaccinated previously with two doses of polysaccharide vaccine had a lower group C antibody response than did control children. Group A antibody responses following revaccination of children who had previously received polysaccharide or conjugate vaccine were not significantly higher than those in control children. Thus, immunologic memory was probably induced by the group C but not by the group A component of the conjugate vaccine.
Asunto(s)
Vacunas Bacterianas/inmunología , Memoria Inmunológica , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Anticuerpos Antibacterianos/sangre , Cápsulas Bacterianas , Vacunas Bacterianas/administración & dosificación , Ensayo de Inmunoadsorción Enzimática , Gambia , Humanos , Esquemas de Inmunización , Inmunización Secundaria , Lactante , Vacunas Meningococicas , Vacunación , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunologíaRESUMEN
A new standard meningococcal reference serum designated CDC1992 was prepared to replace meningococcal reference sera ECG and PB-2, which are not available in sufficient quantities for continued use as primary reference sera. CDC1992 was prepared from 14 healthy adult volunteers who underwent plasmapheresis 4 to 12 weeks postvaccination with a single dose of a Neisseria meningitidis quadrivalent polysaccharide vaccine. Total and/or class-specific meningococcal serogroup A and C anticapsular antibody concentrations (in micrograms per milliliter) were assigned to CDC1992 by using homologous and heterologous enzyme-linked immunosorbent assay (ELISA) formats. The reference serum ECG was used as a reference standard to assign total anticapsular antibody concentrations to CDC1992 by a homologous ELISA format. A heterologous ELISA format, with the Haemophilus influenzae type b standard reference serum FDA 1983, was used to assign total and class-specific antibody concentrations to CDC1992. Alkaline phosphatase-labeled mouse anti-human monoclonal antibody conjugates were used as secondary antibodies in both ELISA formats. The total, immunoglobulin G (IgG), IgA, and IgM antibody concentrations, assigned to CDC1992 for serogroup A were 135.8, 91.8, 20.1, and 23.9 micrograms/ml, respectively, and those for serogroup C were 32.0, 24.1, 5.9, and 2.0 micrograms/ml, respectively. Meningococcal serogroup A and C antibody concentrations were in good agreement when homologous and heterologous ELISA format results were compared. Total and class-specific serogroup A and C antibody concentrations were determined in six adult quality control serum samples from the Centers for Disease Control and Prevention by using the homologous ELISA and our assigned antibody concentrations for CDC1992. Antibody concentrations in reference sera ECG and PB-2 were measured in order to provide a historical link to previous studies. The general acceptance of CDC1992 as the standard reference serum and the assigned antibody concentrations will allow investigators to compare antibody levels in serum to those in a single reference preparation.
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Anticuerpos Antibacterianos/análisis , Cápsulas Bacterianas/inmunología , Neisseria meningitidis/clasificación , Neisseria meningitidis/inmunología , Serología/normas , Adulto , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Humanos , Estándares de Referencia , VacunaciónRESUMEN
The safety and immunogenicity of a group A plus group C meningococcal polysaccharide-CRM197 conjugate vaccine was evaluated in 304 8- to 10-week-old Gambian infants. Infants were immunized with one, two, or three doses of conjugate vaccine or with two doses of a meningococcal A plus C polysaccharide vaccine. The conjugate vaccine produced few systemic side effects, and local reactions were similar to those produced by the polysaccharide vaccine. Postvaccination group A meningococcal polysaccharide antibody levels, measured by ELISA, increased progressively after one, two, or three doses of conjugate vaccine. However, one dose of conjugate vaccine given at the age of 6 months induced a higher group C meningococcal antibody response than did two doses of conjugate vaccine given at 2 and 6 months. Two doses of conjugate vaccine induced higher levels of antibody than did two doses of polysaccharide vaccine. Thus, this new meningococcal conjugate vaccine proved to be safe and immunogenic.
Asunto(s)
Proteínas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/efectos adversos , Femenino , Humanos , Lactante , Masculino , Vacunas Meningococicas , Vacunación , Vacunas Conjugadas/inmunologíaRESUMEN
Anti-hepatitis C virus antibody (anti-HCV) screening was performed in a sample of the adult population of the Coimbra District. 657 persons were included (267 male and 390 female, mean age of 42.7 + 13.1 years), aleatorily chosen from four characteristic regions. Anti-HCV was detected using an ELISA-2 test and all positive sera were also tested with RIBA-2. General prevalence of anti-HCV was 0.46%. All positive patients live in urban areas and presented risk factors for HCV infection. Anti-HCV was found in 33.3% of intravenous drug abusers, in 1.8% of transfused individuals, in 1.33% of alcoholics (higher than 80 g/d alcohol ingestion), in 1% of cases with history of surgical operations, and in 0.65% of persons who lived in risk regions for hepatitis B. We conclude that anti-HCV prevalence is low in our region. We think it is important to perform other studies on larger samples of general population and to study risk groups.
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Hepacivirus/inmunología , Anticuerpos Antihepatitis/sangre , Hepatitis C/epidemiología , Adulto , Anciano , Femenino , Hepatitis C/inmunología , Humanos , Masculino , Persona de Mediana Edad , Portugal/epidemiología , Prevalencia , Población Rural/estadística & datos numéricos , Estudios Seroepidemiológicos , Población Urbana/estadística & datos numéricosRESUMEN
There is a lack of consensus among investigators who use a variety of immunoassay techniques (e.g., enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay) regarding the protocols for describing and forming standard reference or calibration curves and interpolating serum antibody concentrations. This confounds the issue of detecting the presence or absence of parallelism between standard reference serum and serially diluted serum sample curves. These curves must be parallel to support the assumption that the antibody-binding characteristics are similar enough to allow the determination of antibody levels in the diluted serum sample. There is no universal and widely adopted strategy for assessing parallelism in bioassays, and without an assurance of parallelism, investigators are not able to calculate reliable estimates for antibody concentrations in serum samples. Furthermore, single-point (dilution) serum assays do not provide information related to parallelism and nonparallelism, and this, too, may lead to considerable error when calculating antibody concentrations. When assay methodology, technique, and precision improve to the extent that standard reference serum and serially diluted serum sample curves are fit with little error, standard analysis of variance techniques are overly sensitive to negligible departures from parallelism. We present a series of guidelines that compose a protocol for assessing parallelism between bioassay dilution curves that are applicable to data derived from ELISAs. These criteria should be applicable, with minor modifications, to most immunoassay experimental situations and, most importantly, are not dependent on the mathematical model used to form the standard reference curve. These guidelines have evolved in our laboratories over the past 4 years during the performance of thousands of ELISAs for antibodies to the capsular polysaccharides of Neisseria meningitidis groups A and C and Haemophilus influenzae type b.
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Ensayo de Inmunoadsorción Enzimática/normas , Análisis de Varianza , Anticuerpos Antibacterianos/sangre , Haemophilus influenzae/inmunología , Humanos , Neisseria meningitidis/inmunologíaRESUMEN
A meningococcal vaccine containing group A and C polysaccharides conjugated to CRM197 was evaluated in 50 adults. Vaccinees were entered into one of five groups: 30 adults received a single dose of either 22, 11, or 5.5 micrograms of the conjugated A-C vaccine; 10 received an approved meningococcal vaccine; and 10 received saline injections. Local and systemic reactions to vaccines were recorded, and immune responses were determined. The experimental meningococcal vaccine was well tolerated, with the most frequent reaction being pain at the injection site. Both A and C polysaccharide components of the experimental vaccine were highly immunogenic, and total antibody concentrations 1 month postvaccination were not significantly different from the mean antibody concentrations among adults given the approved meningococcal vaccine. In addition, significant rises in immunoglobulin G, A, and M antibodies to both A and C polysaccharides occurred. Antibody concentrations measured at 6 and 12 months postvaccination had declined but remained significantly higher than prevaccination concentrations. Postvaccination meningococcal group C functional antibody activity increased more than 600-fold for both the polysaccharide and the conjugate vaccines. Further studies of this conjugated meningococcal vaccine are indicated for young children and infants.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/inmunología , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Adolescente , Adulto , Vacunas Bacterianas/efectos adversos , Actividad Bactericida de la Sangre , Humanos , Vacunas Meningococicas , Persona de Mediana Edad , Vacunas Conjugadas/inmunologíaRESUMEN
A standardized enzyme-linked immunosorbent assay (ELISA) was used by 11 laboratories to measure levels of total serum antibody to Neisseria meningitidis serogroup C capsular polysaccharide in 16 unpaired pre- and postvaccination serum samples. Twelve serum samples were from adults, and four were from children aged 2, 3, 5, and 9. The between-laboratory coefficient of variation for pre- and postvaccination sera ranged from 16 to 59% and 11 to 21%, respectively. The average percent difference (absolute value) from the between-laboratory means for all prevaccination sera measured by each laboratory was 24%, whereas the average percent difference was 13% for all postvaccination sera. A postvaccination quality control serum was diluted three times to give optical densities on the high, middle, and low portions of the standard reference curve. The three dilutions were assayed by the 11 laboratories a total of 241 times and yielded an overall coefficient of variation of 20%. Antibody-binding inhibition curves showed that the standardized ELISA was specific for N. meningitidis serogroup C capsular polysaccharide antibody. Fifty percent inhibition of seven serum samples was obtained after reaction with an average concentration of 0.9 micrograms of meningococcal serogroup C polysaccharide per ml; an average of 93% inhibition was obtained with 50 micrograms of polysaccharide per ml. The acceptance and use of this standardized ELISA will reduce between-laboratory assay variability and ensure a more accurate and reproducible assessment of immunogenicity for vaccines under development.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Ensayo de Inmunoadsorción Enzimática , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Adulto , Anticuerpos Antibacterianos/inmunología , Niño , Preescolar , Humanos , Neisseria meningitidis/clasificación , Estándares de Referencia , Reproducibilidad de los Resultados , VacunaciónRESUMEN
The long-term kinetics of the immunologic response after vaccination of adults with Neisseria meningitidis polysaccharide vaccine is unknown. Total meningococcal anti-capsular antibody response (measured by ELISA) and serum bactericidal activity after routine vaccination with quadrivalent meningococcal vaccine were evaluated in US Air Force personnel. In a retrospective cross-sectional study, blood samples were obtained from approximately 40 personnel before vaccination, at 1 and 4-6 months, and at 2, 3, 4, 6, 8, and 10 years after vaccination. Total anti-group A and -group C capsular antibody levels and bactericidal activity peaked 1 month after vaccination and declined substantially by 2 years. At each interval, significantly higher levels of total antibody and bactericidal activity were detected than before vaccination. Anti-capsular antibodies and bactericidal activity persisted for up to 10 years after immunization. These and further studies on the serologic measure of protection against meningococcal disease are important for evaluation of candidate vaccines and development of recommendations for immunization.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/inmunología , Personal Militar , Neisseria meningitidis/inmunología , Adulto , Población Negra , Estudios Transversales , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Vacunas Meningococicas , Estudios Retrospectivos , Factores Sexuales , Factores de Tiempo , Vacunación , Población BlancaRESUMEN
Anti-hepatitis C virus antibody (anti-HCV) screening was performed in a sample of the adult population of the Coimbra District. 657 persons were included (267 male and 390 female, mean age of 42.7 +/- 13.1 years), aleatorily chosen from four characteristic councils. Anti-HCV was detected using an ELISA-2 test and all positive sera were also tested with RIBA-2. General prevalence of anti-HCV was 0.46%. All positive patients live in urban areas and presented risk factors for HCV infection. Anti-HCV was found in 33.3% of intravenous drug abusers, in 1.8% of transfused individuals, in 1.33% of alcoholics (higher than 80 g/d alcohol ingestion), in 1% of cases with history of surgical operations, and in 0.65% of persons who lived in risk regions for hepatitis B. We conclude that anti-HCV prevalence is low in our region. We think it is important to perform other studies in larger samples of general population and to study risk groups.