Characteristics of the genome of goose parvovirus.

The nucleic acid of goose parvovirus showed sensitivity to DNase and Mung Bean nuclease treatment and resistance to digestion with RNase. Viral DNA readily served as a template for self-primed conversion in vitro into a double-stranded form of about 5000 base pairs. There was evidence for encapsidation of strands of opposite polarities in equal amounts. The restriction enzyme cleavage patterns of goose and muscovy duck parvovirus DNAs differed significantly. However, they showed a high degree of homology by a hybridization test.

Lactoferrin binds to porins OmpF and OmpC in Escherichia coli.

Lactoferrin (Lf) is an iron-binding antimicrobial protein present in milk and on mucosal surfaces, with a suggested role in preimmune host defense. Certain strains of Escherichia coli (bacterial whole cells) demonstrate specific interaction with 125I-labeled Lf. A band with a mass of approximately 37 kDa, which was reactive with horseradish peroxidase-labeled Lf, was identified in the boiled cell envelope and outer membrane preparations of an Lf-binding E. coli strain, E34663, and a non-Lf-binding strain, HH45, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting). Such a band was not detected in the unboiled native cell envelope and outer membrane preparations. The molecular mass and the property of heat modifiability suggested that the Lf-binding proteins were porins. The native trimeric form of porin OmpF isolated from strain B6 and its dissociated monomeric form both reacted with horseradish peroxidase-labeled Lf and with monoclonal antibodies specific for OmpF. Furthermore, by using E. coli constructs with defined porin phenotypes, OmpF and OmpC were identified as the Lf-binding proteins by urea-SDS-PAGE and Western blotting and by 125I-Lf binding studies with intact bacteria. These data establish that Lf binds to porins, a class of well-conserved molecules common in E. coli and many other gram-negative bacteria. However, in certain strains of E. coli these pore-forming proteins are shielded from Lf interaction.

Elastin peptide concentration in human serum: variation with antibodies and elastin peptides used for the enzyme-linked immunosorbent assay.

Discrepancies exist between the reported values for the mean elastin peptide (EP) concentration in human sera. In order to understand these discrepancies, several EP preparations were obtained in vitro and monoclonal and polyclonal antibodies were produced against them. These different EP preparations and antibodies were used in an enzyme-linked immunosorbent assay (ELISA) to study cross-reactivity between EP preparations and to quantitate EP concentration in human sera. The method of purification of elastin, the method of hydrolysis of elastin and the molecular weight of EP influence their reactivity with antibodies and the results of EP measurements in human sera. However, there is a good correlation between EP measurements carried out in several human sera with the different EP preparations and different antibodies. Although absolute values of the EP concentrations varied with the EP preparation and antibodies used for the ELISA, the variations of this EP concentration measured from one human serum to another are significant.

Detection of bacterial interaction with lactoferrin by an enzyme-linked ligand binding assay (ELBA).

An enzyme-linked ligand binding assay (ELBA) was devised to measure the interaction between bacteria and human (H) or bovine (B) lactoferrin (Lf) linked to horseradish peroxidase. Reagents were calibrated for optimum colour development with o-phenylenediamine as chromophore and organisms that were either positive or negative in a radioisotope-labelled ligand binding assay (RLBA) with 125I-Lf. Good correlation of Lf binding (r = 0.89) was found between ELBA and RLBA with 169 randomly selected strains of Escherichia coli. A semi-quantitative scoring system for ELBA, corresponding to a similar system for RLBA, was established and shown to be valid for 517 strains from seven species of bacterial pathogens. ELBA was used to measure bacterial Lf binding-saturation and displacement kinetics and shown to be comparable with RLBA. ELBA may be a suitable method for examining the binding of Lf to bacteria without the need for radioactive isotopes.

Immunoenhancement and suppression induced by adenovirus in chicken.

Chickens injected intravenously (i.v.) with human adenovirus type 6 (Ad6) reveal a 2-17-fold increase in the number of plaque-forming cells producing antibody (Ab) against sheep red blood cells (SRBC) 2-6 days after virus infection. Further, polyclonal B-cell activation has been demonstrated by the quantitation of immunoglobulin-producing cells (IgPC) and cells producing immunoglobulin (Ig) of IgM isotype (IgPC mu) in the spleen of chicken inoculated with Ad6. Ad6 infection in chicken results in immunosuppression against SRBC when this unrelated antigen is given after virus infection. It seems that coincidence occurs between the B-cell mitogenic activation and the immunosuppression caused by Ad6, as the most pronounced change in both activities appears on the fourth day following virus infection. These findings suggest that the B-cell mitogenicity of the virus contributes to the impairment of the humoral immune response to SRBC.

Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections.

The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binding EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subepithelial matrix proteins and carbohydrates tested (in 10(4)-fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (Ka) of 1.4 x 10(-7) M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli.

Correlation between human lactoferrin binding and colicin susceptibility in Escherichia coli.

Escherichia coli H10407 demonstrated low 125I-human lactoferrin (HLf) binding (7%) and was insusceptible to group A (A, E1, E2, E3, E6, and K) and group B (B, D, Ia, Ib, and V) colicins. Conversely, a spontaneous HLf high-binding (44%) variant, H10407(Lf), demonstrated an increase susceptibility to both colicin groups. Colicin-insusceptible E. coli wild-type strains 75ColT, 84ColT, and 981ColT showed a low degree of HLf binding, i.e., 4, 8, and 10%, respectively. The HLf binding capacity was high in the corresponding colicin-susceptible mutants 75ColS (43%), 84ColS (32%), and 981ColS (43%). Furthermore, HLf low- (less than 5%) and high- (greater than 35%) binding E. coli clinical isolates (10 in each category) were tested for susceptibility against 11 colicins. Colicin V susceptibility did not correlate with HLf binding in either categories. However, with the remaining colicins, three distinct HLf-binding, colicin susceptibility patterns were observed; (i) 10 of 10 HLf low-binding strains were colicin insusceptible, (ii) 6 of 10 HLf high-binding strains were also colicin insusceptible, and (iii) the remaining HLf high binders were highly colicin susceptible. Certain proteins in the cell envelope and outer membrane of wild-type H10407 (HLf low binder, colicin insusceptible) showed a lower mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared to the corresponding proteins of mutant H10407(Lf) (HLf high binder, colicin susceptible). These mobility differences were also associated with HLf-binding proteins in Western blot (ligand blot) analysis. The wild type showed a smooth form of lipopolysaccharide (LPS) with a distinct ladder of O-chains, compared to the rough LPS of the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific binding of lactoferrin to Aeromonas hydrophila.

The interaction of lactoferrin (Lf) with Aeromonas hydrophila (n = 28) was tested in a 125I-labeled protein-binding assay. The mean per cent binding values for human Lf (HLf) and bovine Lf (BLf) were 13.4 +/- 2.0 (SEM), and 17.5 +/- 2.7 (SEM), respectively. The Lf binding was characterized in type strain A. hydrophila subsp. hydrophila CCUG 14551. The HLf and BLf binding reached a complete saturation within 2 h. Unlabeled HLf and BLf displaced 125I-HLf binding in a dose-dependent manner, and more effectively by the heterologous (1 microgram for 50% inhibition) than the homologous (10 micrograms for 50% inhibition) ligand. Apo- and holo-forms of HLf and BLf both inhibited more than 80%, while mucin caused approx. 50% inhibition of the HLf binding. Various other proteins (including transferrin) or carbohydrates did not block the binding. Two HLf-binding proteins with an estimated molecular masses of 40 kDa and 30 kDa were identified in a boiled-cell-envelope preparation, while the unboiled cell envelope demonstrated a short-ladder pattern at the top of the separating gel and a second band at approx. 60 kDa position. These data establish a specific interaction of Lf and the Lf-binding proteins seem to be porins in A. hydrophila.

Fibronectin in bronchoalveolar lavage fluid and plasma of dogs with acute inflammation of the lungs.

Bronchoalveolar inflammation, which was generated in dogs by Broncho-Vaxom instilled into the right lower lobe, was characterized first of all by an increased influx of macrophages. In this non-purulent acute-subacute inflammatory reaction, the lavage fibronectin decreased rapidly three hours after the incubation and then a marked gradual elevation was observed, which persisted throughout the whole two-week process, while plasma fibronectin concentrations were not altered significantly. Changes in the levels of lavage fibronectin may be an important sign for the control of the inflammatory reaction activity in the lungs.

Fibronectin in bronchoalveolar lavage fluid and plasma from children with chronic inflammation of lungs.

Fibronectin/albumin ratios in plasma and in bronchoalveolar lavage fluid were evaluated in patients (1-6 years of age) with recurrent obstructive bronchitis and different interstitial lung diseases. These inflammatory reactions were characterized by increased influx of macrophages on the bronchoalveolar surface, but an increase in the proportion of lymphocytes or neutrophils was also detected in the group of patients with lymphocyte-macrophage or neutrophil-macrophage alveolitis. There was no considerable difference in plasma fibronectin concentrations obtained from healthy children and patients with moderate obstructive bronchitis and slight inflammation of the bronchial mucosa observed bronchoscopically. Levels of plasma fibronectin were elevated in patients with serious bronchial inflammation and different alveolitis, but they were within the normal range. A comparison of lavage fibronectin/albumin ratios with plasma fibronectin/albumin ratios with plasma fibronectin/albumin ratios indicated significant local production of fibronectin in subjects with serious bronchial inflammation and interstitial lung disorders. Fibronectin detected on the bronchoalveolar surface seems to be an important factor in mediating cell-to-cell interactions in the repair of the bronchoalveolar structures, and in tracing the activity of the inflammatory reactions not only in patients with interstitial lung diseases, but also in patients with serious chronic bronchial inflammation.

Characterization of monoclonal antibodies to adenovirus type 35 hexon.

Twenty three monoclonal antibody-rich ascitic fluids (MIAFs) to human adenovirus (AV) type 35 hexon were studied by indirect ELISA using various tracer systems, passive haemagglutination (HA) as well as gel diffusion techniques. Eleven different human heterologous hexon types in addition to the homologous one, and two animal adenovirus (AV) hexons were used to determine the reactivity patterns (RPs) of the monoclonal antibodies (MoAbs). Based on the cross-reactivity with the different hexon types, the MoAbs exhibited genus, subgenus and type specificities; furthermore, a variety of intersubgenus and intertype specificities could be found. Fifteen of the MoAbs reacted in ELISA, but not in passive HA, suggesting that certain epitopes on the hexons bound to red blood cells were not available for the MoAbs in question. Four MoAbs were able to form a precipitin line with the hexon antigen in gel diffusion. Two of the four (MoAbs 35H10 and 35H51) formed with the homologous AV35 hexon a single confluent precipitin line only. In spite of the origin of these MoAbs from different hybrid cells (clones) their specificity was probably identical when recognizing the type-specific epitope of the AV35 hexon. The other two MoAbs (35H15 and 35H26) with a broad RPs were able to precipitate not only the homologous but also different heterologous hexon types.

Fibronectin on the bronchoalveolar surface in children with recurrent obstructive bronchitis.

Fibronectin is normally present in the lower respiratory tract. Significantly increased levels of it were detected in the lavage fluid in patients with interstitial lung diseases. Because this molecule appears to mediate a number of components of the inflammatory process, we evaluated the status of fibronectin in plasma and bronchoalveolar lavage in patients with recurrent obstructive bronchitis when signs of severe chronic mucosal inflammation were observed bronchoscopically. There was no considerable difference in plasma concentrations of fibronectin obtained from healthy children and patients. A comparison of lavage fibronectin/albumin ratios with plasma fibronectin/albumin ratios suggested significant local production, especially when the lavage and plasma ratios were measured in the same patients. Phagocytic activity of alveolar macrophages and blood granulocytes from the same patients was enhanced at both concentrations of fibronectin used. This concentrations referred to values quantified in the lavage fluid. The metabolism of fibronectin seems to be an important factor in tracing the inflammation process not only in adults with chronic interstitial lung diseases, but also in children with recurrent obstructive bronchitis.

Newcastle disease vaccine (La Sota) strain specific monoclonal antibody. Brief report.

Newcastle disease virus vaccine strain (La Sota) specific monoclonal antibody (La-1) was produced by immunizing mice with isolated glycoproteins of strain La Sota. This antibody was recognized only in the ELISA test in which it bound exclusively to La Sota strain out of a range of over 300 lentogenic, mesogenic and velogenic strains examined.

Monoclonal antibodies to adenovirus type 35 hexon.

A panel of 37 monoclonal antibodies (MAbs) directed against adenovirus type 35 (AV35) hexon was studied by indirect enzyme-linked immunosorbent assay (ELISA) and passive hemagglutination (HA) methods. Nine heterologous hexon types and the homologous type were used to determine the reactivity pattern (RP) of the MAbs and to study the antigenic relationship among the different hexon types. Eleven types of RPs were shown using ELISA and seven types were shown using the HA test. In the case of six MAbs, the RPs were identical in both assay systems; 31 MAbs showed some differences when the results of the two methods were compared. The common epitopes of the different hexon types studied seem to be characterized as genus, subgenus, intersubgenus, and intertype specificities. The type-specific determinant of AV35 hexon could be detected by several MAbs. The antigenic relationship seems to be closer between the two oncogenic subgenera (A and B) of adenoviruses, whereas the antigenic relationship to AV35 hexon is somewhat looser for subgenera D, and E. Hexon types of subgenus C showed the greatest differences in antigenic structure compared with the AV35 hexon.

The role of genetic factors in the immunomodulating effect of thymosin.

The possible role of hypothetical genetic factors involved in the immunomodulating effect of thymosin fraction 7 (T7) was investigated. The model system was the in vitro immunization of murine spleen cell cultures with sheep red blood cells (SRBC), and the generation of antigen specific B cells in T7 treated cultures was compared to that of control values. It was found that T7 treatment enhanced the plaque forming cell (PFC) response of BALB/c spleen cells, while it proved to be suppressive in CBA cultures. Moreover, the T7 treatment of athymic BALB/c nude spleen cells resulted in a marked PFC response to SRBC, while a similar treatment of CBA nude cultures was ineffective in the same assay. The role of possible genetic factors was further confirmed using H-2 congenic and recombinant mouse strains on the C3H and B10 background. T7 elevated the PFC values in all B10 strains tested, and was suppressive in the case of C3H strains. It seems that the outcome of T7 treatment of murine target cells is determined by the genetic background and is independent of the H-2 haplotype.

Distribution pattern of adenovirus hexon epitopes in infected cells determined with monoclonal antibodies by immunofluorescence analysis.

Two monoclonal antibodies (MAbs) specific for two distinct epitopes on the human adenovirus type 1 (AV1) hexon were used to determine the subcellular localization of hexon epitopes in the infected HEp-2 cells by indirect immunofluorescence. On the basis of cross-reactivity pattern of MAbs, presumably one of the epitopes is genus specific and the other should be intertype specific. The epitopes, i.e. the adenovirus hexons could be detected throughout the cell and could display different accumulation forms. Fluorescence appeared either in the cytoplasm only or both in the nucleus and the cytoplasm. In the cytoplasm the hexons could be found in diffuse or perinuclear distribution or accumulated into discrete spots. In the nucleus they formed granules or clusters or were diffusely distributed causing a bright fluorescence of the whole nucleus. The different accumulation forms appeared at the same time in different cells of a culture, but in one given cell the fluorescence always appeared first in the cytoplasm.

Determination of different antigenic sites on the adenovirus hexon using monoclonal antibodies.

Eighteen mouse ascitic fluids containing monoclonal antibodies (MAbs) directed against crystallized hexon of adenovirus (AV) type 1 were used to map the antigenic structure of the capsomer in reciprocal competitive binding ELISA. With the help of peroxidase-labelled MAbs at least nine epitopes (epitope clusters) located on three distinct antigenic sites were identified on the hexon. Epitope on antigenic site I recognized by two MAbs could be the genus specific antigenic determinant based on the broad reactivity patterns of the MAbs. Epitopes on the antigenic site II recognized by fifteen MAbs could be divided into seven epitope clusters according to the competition patterns. Antigenic site III recognized by one MAb completely differs from the antigenic site I and on the basis of one-way blocking with all the MAbs specific for antigenic site II, should be also different from the latter one. The data suggest that the seven epitope clusters of antigenic site II contain partially overlapping epitopes and may be a part of a large single immunodominant antigenic region on AV 1 hexon as well as on hexons of heterologous types.

Multiple copies of identical epitopes on the adenovirus hexon.

A double monoclonal antibody (MAb) sandwich enzyme-linked immunosorbent assay (double MAb ELISA), which uses the same MAb as solid-phase immunosorbent (capture MAb) and as detector MAb (peroxidase-labeled), was developed to quantify the specific epitopes of adenovirus hexon. Four MAbs directed against crystallized adenovirus type 1 (Ad h 1) hexon were tested by this assay with homologous and different heterologous hexons. The lowest reacting concn with the homologous and heterologous hexon types both in direct and double MAb ELISA was determined and compared. At least two copies of four different epitopes were identified by the MAbs. Evidence is presented that more than one copy of identical or closely related epitopes exist on the homologous as well as on the heterologous hexon molecules. However, their presence could be detected only in higher concn of hexon preparations of subgenera A, B and D.