RESUMO
Antibody- and complement-mediated phagocytosis is the main defense mechanism against Streptococcus pneumoniae. A standardized, easy to perform phagocytosis assay for pneumococci would be a great asset for the evaluation of the potential efficacy of (experimental) pneumococcal vaccines. Such an assay could replace the laborious phagocytosis assay of viable pneumococci (classical killing assay). Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was compared with the conventional killing assay and enzyme-linked immunosorbent assay (ELISA), using sera obtained from adults pre- and postvaccination with either a bivalent conjugate, a tetravalent conjugate, or the 23-valent polysaccharide vaccine. Highly significant correlations were observed between flow assay phagocytosis titers, killing assay phagocytosis titers, and ELISA antibody titers for serotype 6B and 23F as well. For serotype 19F, strong correlations were only observed between killing assay and ELISA titers. A potential drawback of the flow assay might be the low sensitivity compared with that of the killing assay. The choice of what assay to use, however, will depend on the objectives of the assay. When speed, easy performance, sample throughput, improved worker safety, absence of influence of antibiotics, and absence of false positives are the major criteria, the flow assay is the method of choice. When higher sensitivity is the major requirement, the classical killing assay should be used.
Assuntos
Citometria de Fluxo/métodos , Fagocitose/imunologia , Vacinas Pneumocócicas/imunologia , Adulto , Anticorpos Antibacterianos/sangue , Testes Imunológicos de Citotoxicidade/métodos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/normas , Sensibilidade e Especificidade , Streptococcus pneumoniaeRESUMO
A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 microliter per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.
Assuntos
Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Cápsulas Bacterianas/imunologia , Citometria de Fluxo/métodos , Fagocitose/imunologia , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/imunologia , Proteínas do Sistema Complemento/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Neutrófilos/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Coelhos , Sorotipagem , Streptococcus pneumoniae/crescimento & desenvolvimento , VacinaçãoRESUMO
In perceptual systems, a stimulus parameter can be extracted by determining the center-of-gravity of the response profile of a population of neural sensors. Likewise at the motor end of a neural system, center-of-gravity decoding, also known as vector decoding, generates a movement direction from the neural activation profile. We evaluate these schemes from a statistical perspective, by comparing their statistical variance with the minimum variance possible for an unbiased parameter extraction from the noisy neuronal ensemble activation profile. Center-of-gravity decoding can be statistically optimal. This is the case for regular arrays of sensors with gaussian tuning profiles that have an output described by Poisson statistics, and for arrays of sensors with a sinusoidal tuning profile for the (angular) parameter estimated. However, there are also many cases in which center-of-gravity decoding is highly inefficient. This includes the important case where sensor positions are very irregular. Finally, we study the robustness of center-of-gravity decoding against response nonlinearities at different stages of an information processing hierarchy. We conclude that, in neural systems, instead of representing a parameter explicitly, it is safer to leave the parameter coded implicitly in a neuronal ensemble activation profile.