Biotinylation and characterization of Cryptococcus neoformans cell surface proteins.

AIMS: To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation. METHODS AND RESULTS: Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls. CONCLUSIONS: A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation.

AIMS: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. METHODS AND RESULTS: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K(d) and relative B(max) values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K(d) and relative B(max), which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. CONCLUSIONS: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. SIGNIFICANCE AND IMPACT OF THE STUDY: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Production of passive immunity in neonatal ferrets following maternal vaccination with killed influenza A virus vaccines.

Neonatal ferrets may be passively immunized following maternal vaccination with formalin-inactivated influenza A virus vaccine, but the level of protection from partial to complete depends upon the number of doses used to vaccinate the mother, the presence or absence of aluminum hydroxide adjuvant, whether or not the mothers were 'primed' by prior infection with a serologically heterologous type A virus, and the age of the neonate at challenge. Neonates were completely protected up to 2 weeks of age, but susceptibility returned to nasal epithelium at 5 weeks and to lung at 7 weeks. Mothers immunized up to 9 months previously also partially or completely protected their offspring, this correlating with the maternal serum haemagglutination-inhibition (HI) antibody titre at the time of neonatal challenge, not the duration of immunity.

Further studies of the reasons for the lack of alveolar infection during influenza in ferrets.

Intratracheal inoculation of influenza virus in the ferret was followed by a more severe airway infection than that produced by nasal infection and was mainly bronchiolar rather than bronchial. Also, virus isolation from the alveolar zone of the lung together with immunofluorescence and immunoperoxidase techniques showed that some virus reached the alveoli. Nevertheless, there was no subsequent alveolitis suggesting the existence of a clearance phenomenon. Alveolar macrophages were shown to have phagocytosed virus in vivo and phagocytosis studies in vitro showed that two mechanisms could operate to eradicate the virus. First, a rapid destruction of virus and second an abortive cycle of replication which produced virus antigen but not infectious virus. Experiments with large doses of virus indicated that after intranasal inoculation little virus reached the alveoli so it would probably be quickly cleared by the macrophages.

Recent H1N1 viruses (A/USSR/90/77, A/Fiji/15899/83, A/Firenze/13/83) replicate poorly in ferret bronchial epithelium. Brief report.

Three recent wild-type H1N1 influenza virus isolates (A/USSR/90/77, A/Fiji/15899/83 and A/Firenze/13/83) replicated poorly in organ cultures of ferret bronchial tissue compared with the replication of an H3N2 wild-type virus (A/England/939/69). All four viruses replicated well in nasal turbinate tissue. Examination of one H1N1 virus (A/USSR/90/77) in vivo showed heavy infection in the upper respiratory tract of ferrets but little in the lower respiratory tract. These results raise the possibility that the mildness of human influenza arising from the H1N1 strains may be due to lack of capacity to attack the lower respiratory tract as well as the presence of antibody in previously exposed persons.

Differential replication of attenuated and virulent influenza viruses in organ cultures of ferret bronchial epithelium. Brief report.

In contrast to its abundant replication in ferret nasal epithelium in vivo and in vitro, comparable to that of the virulent strains, the attenuated influenza virus A/PR/8/34 produced much lower yields than the virulent strains in organ cultures of bronchial epithelium agreeing with its relative inability to infect the lower respiratory tract of ferrets. The replication of another attenuated strain showed different temperature characteristics in bronchial epithelium to that in nasal turbinate epithelium.

The similar interaction of ferret alveolar macrophages with influenza virus strains of differing virulence at normal and pyrexial temperatures.

The possibility that ferret lung macrophages may be one factor operating in vivo to prevent infection of susceptible alveolar cells (as demonstrated by organ cultures) by both virulent and attenuated strains of influenza virus has been investigated. Phagocytosis of four strains of influenza virus [A/PR/8/34 (H1N1) and clone 64d (attenuated for ferrets) and clones 64c and 7a (virulent for ferrets) of the recombinant virus A/PR/8/34-A/England/939/69 (H3N2)] by ferret alveolar macrophages in vitro showed that all strains, whether virulent or attenuated, attached equally well (72 to 93%). Recoveries of virus were similar (17 to 44%) whether phagocytosis occurred at the normal temperature of the ferret (39 degrees C) or at pyrexial temperatures induced during infection (40 degrees C for A/PR/8/34 and clone 64d; 41 degrees C for clones 7a and 64c). Thus, alveolar macrophages probably contribute to the lack of alveolar infection observed in vivo.

Distribution of viral antigen with the lower respiratory tract of ferrets infected with a virulent influenza virus: production and release of virus from corresponding organ cultures.

Using fluorescent antibody techniques, a semi-quantitative survey has been made of the distribution of influenza virus antigen in the trachea, main bronchi, and three zones (hilar, intermediate and alveolar) of all four lung lobes of ferrets following intranasal inoculation of a virulent clone (7a) of the recombinant influenza virus A/PR/8/34-A/England/939/69 (H3N2). The results confirm the indications from our previous quantitative surveys of infectious virus and histological damage in these areas, namely that infection is confined largely to airway epithelium and is rare in the alveoli. Furthermore, in the lung zones, viral antigen resided mainly in the bronchial rather than bronchiolar epithelium. In attempts to identify the reasons for lack of alveolar involvement organ cultures of alveolar tissue, from which all major airways had been removed, produced levels of virus similar to cultures of bronchus and trachea and the hilar and intermediate lung zones which contain airway and alveolar tissue. Hence, the lack of alveolar infection in vivo must be due to factors which prevent virus attack of susceptible alveolar cells. However, these organ culture experiments showed that a contributing factor could be very poor release of virus from any alveolar cells that do become infected. In contrast, although cultures of bronchi produced less virus than those of nasal turbinates (the most susceptible tissue in vivo) they released a high proportion of their yield and this ease of release may contribute to spread of infection in vivo.

Differential distribution of virus and histological damage in the lower respiratory tract of ferrets infected with influenza viruses of differing virulence.

The distribution of four strains of influenza virus [A/PR/8/34 (H0N1) and clone 64d (attenuated for ferrets) and clones 64c and 7a (virulent for ferrets) of the recombinant virus A/PR/8/34--A/England/939/69 (H3N2)] in the lower respiratory tract (trachea, bronchi and the hilar, intermediate and outer alveolar zones of the lung) of ferrets was monitored daily for 4 days after intranasal inoculation. On day 1, some animals had high virus titres in all the tissues but in other animals virus was undetectable, irrespective of the virus strain. Two days after inoculation increase of virus contents of all tissues tended to be restricted. On days 3 and 4, the virulent clones (64c and 7a), in contrast to the attenuated strains (A/PR/8/34 and clone 64d), consistently infected the lower respiratory tissues. However, for all infected animals the virus contents of the hilar zones of the lungs were higher than those in the intermediate zones, while the alveolar zones were relatively free from virus. Quantitative estimations of the mild histological damage occurring in the lower respiratory tract 3 to 6 days after inoculation also indicated that bronchial and bronchiolar tissue were more susceptible to influenza virus than alveolar tissue and that clones 64c and 7a produced more damage than the other two strains. In agreement with the relative viral contents of clones 64c and 7a in the bronchi and in the hilar and intermediate zones of the lung, clone 64c produced more damage than clone 7a in the bronchi and less in the bronchioles of the lung parenchyma.

The behaviour in ferrets of two closely related clones of influenza virus of differing virulence for man.

Clones 7a and 64d of the recombinant influenza virus A/PR/8/34-A/England/939/69(H3N2) which are of different virulence for man as judged by clinical score (7a more virulent than 64d) showed similar differences in ferrets. With intranasal inoculation the approximate 50% minimal infectious doses of both clones were similar (between 10(0) and 10(2) EID(50)) as were their titres in nasal washes 24 h after inoculation and the histologically evident damage they caused in the nasal turbinates. However, clone 7a persisted in the nasal washes more than 64d and produced a more prolonged pyrexia. Furthermore, 7a consistently produced a lung infection which was produced only occasionally by 64d and then to a lesser extent than 7a. In contrast to nasal mucosa, histological damage in the lung was slight with both strains. Differences in replication of 7a and 64d in organ cultures of nasal turbinates appeared only after 24 h incubation. They were not sufficiently large to explain the markedly superior ability of 7a to persist in the nasal tract in vivo. This persistence, which coincides with the production of pyrexia, may be due to a greater ability of 7a to resist induced systemic host defences. Spasmodic isolations of infective virus of both clones were made from extra-respiratory tissues such as liver, spleen and kidney.