Neutralizing activity and secretory IgA antibodies reactive with rotavirus SA-11 (serotype G3) in colostrum and milk from Brazilian women.

BACKGROUND: Rotavirus is an important aetiological agent for severe diarrhoea in infants and young children worldwide. Anti-rotavirus antibodies in human colostrum and milk may interfere with rotavirus vaccination seroconversion. AIMS: To verify the presence of anti-rotavirus secretory IgA antibodies (SIgA) and the neutralizing capacity of 30 colostrum and 30 milk samples from Brazilian women in two different centres and analyze their persistence throughout lactation. METHODS: Colostrum and milk samples from healthy nursing mothers were tested for the presence of anti-rotavirus SIgA using conventional ELISA and their capacity to neutralize rotavirus using MA-104 cell cultures. Total IgA concentrations and anti-rotavirus SIgA levels were measured in samples collected from three mothers during 90 or 240 days of the lactation period. RESULTS: Colostrum samples showed higher levels of anti-rotavirus SIgA and higher neutralizing ability than in milk. However, these antibodies levels were not statistically different. In addition, there was no correlation between antibody levels and the neutralizing activity observed in colostrum and milk samples. Follow-up of three mothers demonstrated the persistence of anti-rotavirus and total IgA levels throughout lactation. CONCLUSIONS: These results support the encouragement of breastfeeding as a mechanism of protection against rotavirus infection in lactating infants. Components other than SIgA antibodies might play an important role in virus neutralization.

Systemic antibody response to diarrheagenic Escherichia coli and LPS O111, O157 and O55 in healthy Brazilian adults.

Enterohaemorrhagic Escherichia coli (EHEC) can cause a variety of human illnesses ranging from uncomplicated diarrhoea to haemorrhagic colitis and haemolytic uremic syndrome. The serotype O157:H7 has been associated with numerous outbreaks worldwide, but in Brazil the infection is rare. Brazilian adults present antibodies reactive with the principal virulence factors of enteropathogenic E. coli (EPEC) that have many genetic and antigenic similarities with EHEC. Lipopolysaccharides (LPS) are components of outer membranes and important virulence factors of Gram-negative bacteria. LPS O111 is present in EPEC and EHEC strains. LPS O157 is found only in EHEC strains, but it has some structural similarities with LPS O55 present in EPEC strains. This study investigates the levels of IgG and IgM seric antibodies reactive with EHEC O157:H7, EHEC O111:H-, EPEC O111:H- and the levels of anti-LPS O111, LPS O157 and LPS O55 antibodies in healthy adults living in São Paulo, Brazil. The antibody levels were determined by an enzyme-linked immunosorbent assay for 100 individual serum samples, and the presence of anti-bacterial and anti-LPS seric antibodies was confirmed. Positive correlations were found among the three kinds of antibodies. The concentrations of IgM anti-LPS were significantly higher than those of IgG, and surprisingly, the concentrations of anti-LPS O157 were high in view of the infrequent isolation of O157 bacteria in Brazil. Our results suggest that there is a cross-reacting immunity to EHEC in the Brazilian population, which may be a result of the immunity to EPEC antigens. Alternatively, Brazilians may be exposed to EHEC more frequently than has previously been thought.

IgA secretora total e específica no colostro e leite de mães da cidade do Natal-Rio Grande do Norte, Brasil / Total and specific IgA in colostrum and milk of mothers of Natal-Rio Grande do Norte, Brasil

PURPOSE: To determine the concentration of total secretory IgA and evaluate the repertoire of IgA antibodies to enteropathogenic Escherichia coli and Shigella flexneri antigens in colostrums and milk from mothers in Natal, RN. METHODS: The sample was constituted by 22 healthy clinically women whose babies were born at public hospital in Natal, RN. To determine total secretory IgA a radial immunedifusion tecnique (Mancini et al, 1965), was employed and to detect specific antibodies, immuneenzimatic assays, ELISA was used. RESULTS: The median values of total secretory IgA concentration presented individual variations with high levels in colostrums samples, decreasing during lactation, it was observed a p < 0.001 among the samples from the first day of lactation, to the thirtieth for total IgA concentration. All the donators present in colostrum and milk specific antibodies to Escherichia coli enteropathogenic (EPEC) and Shigella flexneri with titles higer in colostrum. There was parallel and directional pattern between total IgA and IgA anti-EPEC and Shegella flexneri, during period. CONCLUSION: The concentrations of total SIgA and specific antibodies to enteropathogenic Escherichia coli and Shigella flexneri in colostrums and milk in our study do not differ from others accomplished among populations with the same social and econimic features, stressing the importance of human milk as a protector agent against pathogens.

Anti-enteropathogenic Escherichia coli immunoglobulin Y isolated from eggs laid by immunised Leghorn chickens.

IgY, the egg yolk immunoglobulin, equivalent to the IgG from mammals, has been used in veterinary practice for passive immunisation against bacterial or viral infectious diseases. Enteropathogenic Escherichia coli (EPEC) is the main etiological agent of infantile diarrhoea in Brazil and other developing countries. Our aims were to isolate immunoglobulin IgY from egg yolk laid by EPEC -immunised Leghorn chickens and to study its reactivity to the antigens from this pathogen, including some virulence factors. Leghorn chickens were immunised with a bacterial suspension intramuscularly (three hens) or intravenously (three hens) or with PBS (two hens). Eggs were collected over a period of 17 weeks. IgY isolation procedures were carried out by salt precipitation (ammonium sulphate, in solid form) followed by centrifugations and dialysis. Final preparations were submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS - PAGE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting. All immunised animals developed good levels of antibodies reactive to whole bacteria or lipopolysaccharide (LPS), in contrast to the control ones. Immunoblottings allowed the recognition of several antigenic fractions of bacterial antigens, some of which had a molecular weight compatible with bacterial virulence factors, confirming the efficacy of the immunisation and the adequacy of the method.

Inhibition of enteroaggregative Escherichia coli adhesion to HEp-2 cells by secretory immunoglobulin A from human colostrum.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important agent of the persistent diarrhea among low socioeconomic level children in developing countries that may be associated with chronic undernourishment. Breast-feeding is effective in protecting infants against diarrhea and other infectious diseases. The aim of the study is to verify the ability of human colostrum to inhibit aggregative adhesion of EAEC to HEp-2 cells and the presence of antibodies reactive to antigenic fractions of EAEC in colostrum samples. METHODS: Enzyme-linked immunosorbent assay, immunoblotting and adhesion assays of EAEC to HEp-2 cells were done with pooled or individual colostrum samples (n = 35). Assays were performed with a well-known EAEC strain, 044:H18 E. coli (strain 042). Colostral IgA was isolated by affinity chromatography in Sepharose anti-human alpha chain column. RESULTS: Total colostrum and isolated IgA inhibited EAEC adhesion, and this ability was associated with the presence of IgA antibodies against a 15-kDa band, compatible with the subunits of aggregative adherence fimbrial adhesin II, characteristic of the 042 strain, absent in its plasmid-cured isogenic strain, that was used as control. Individual colostrum samples also inhibited adhesion, showed variable antibody titles against EAEC antigens in enzyme-linked immunosorbent assay and recognized many antigenic fractions in immunoblotting assays, including the 15-kDa band. CONCLUSIONS: These results confirm that IgA from human colostrum inhibits adhesion of EAEC to HEp-2 cells and suggest that colostrum IgA antibodies reactive to EAEC antigens may play a role in protection of infants against diarrhea caused by these bacteria.

Inhibition of enteropathogenic Escherichia coli (EPEC) adherence to HEp-2 cells by bovine colostrum and milk.

BACKGROUND: enteropathogenic Escherichia coli (EPEC) is the main etiological agent of infantile diarrhea in Brazil and other developing countries. Human milk IgA protects newborn intestinal mucosa by inhibiting bacterial adhesion to epithelial cells and this effect is shown by in vitro assays of EPEC adhesion to HEp-2 cultured cells. Bovine milk, if effective in promoting this protection, could be an useful tool in the absence of the natural breastfeeding, in high-risk nurseries or in hospital infections. METHODS: the effect of colostrum, milk, and serum from dairy cows on the adherence to EPEC to HEp-2 cells was investigated. Colostrum from immunized and control animals and industrialized milk formulas were fractionated through a membrane device with a molecular weight cut off 10 kDa. The high molecular weight fraction (HMWF) of bovine colostrum was depleted of IgG through an affinity column and absorbed with an EPEC adherent strain. Antibodies were searched by ELISA and immunoblotting (IB). RESULTS: colostrum and milk from EPEC-immunized animals showed and inhibitory activity on adherence similar to that of control non-immunized animals. The inhibitory effect on adhesion was related to the HMWF. IgG-depleted colostrum partially retained the inhibitory effect, whereas IgG-rich eluate lost this property. The EPEC-absorbed fraction retained the inhibitory property. Industrialized milk formulas and respective HMWF also inhibited bacterial adherence. In IB assays, colostrum and milk samples from immunized animals recognized proteins of 30-40 kDa and 94 kDa, a molecular weight consistent with the adhesin intimin, in EPEC extracts. CONCLUSIONS: the inhibitory effect of EPEC adherence may be mediated by HMWF components, and IgG was not the only component responsible for this phenomenon.

Inhibition of enteropathogenic Escherichia coli adhesion to HEp-2 cells by colostrum and milk from mothers delivering low-birth-weight neonates.

UNLABELLED: Breast milk samples from three groups of Brazilian women were evaluated for their inhibitory effect on enteropathogenic Escherichia coli (EPEC) adhesion to HEp-2 cells: G1, mothers delivering preterm babies of appropriate birth weight (n = 12); G2, mothers delivering term babies of low birth weight (n = 11); G3, the control group, mothers delivering term babies of appropriate birth weight (n = 39). Colostrum samples were obtained at 48-72 h and milk samples on the 7th, 30th and 60th days after delivery. All samples showed strong inhibitory activity (66%-100%), without significant differences among the three groups and four periods. Total IgA and anti-EPEC IgA concentrations were significantly higher in colostrum than in milk samples in the three groups studied. The levels of colostral IgA and anti-EPEC IgA observed in G1 and G2 were significantly higher compared to the control group. Western blotting assays showed that individual samples as well as pools of colostrum or milk samples contain IgA antibodies to many EPEC outer membrane proteins. A 94 kDa band with molecular weight consistent with the EPEC adhesin named intimin; was recognized by all samples analysed. Bands of different molecular weight were also recognized by some samples of colostrum and milk, such as a band of approximately 18.4 kDa, with molecular weight equivalent to bundle-forming pilus subunits. CONCLUSION: Our results suggest that colostrum and milk from mothers of premature and small-for-date term neonates are as effective in protecting the newborn against EPEC infections as those from mothers of term babies of appropriate birth weight.

Human colostrum IgA antibodies reacting to enteropathogenic Escherichia coli antigens and their persistence in the faeces of a breastfed infant.

IgA antibodies reacting to enteropathogenic Escherichia coli (EPEC) antigens in human colostrum and their role in the inhibition of EPEC adherence to HEp-2 cells were studied. Colostrum IgA was isolated with a Sepharose anti-IgA column. IgA-depleted colostrum lost its inhibitory effect on EPEC adhesion, while the IgA-enriched eluate was a potent adherence inhibitor. The same eluate showed a significant loss of inhibitory activity after absorption with an EPEC strain showing localised adherence (LA+), but no alteration after absorption with an LA- strain. No bands were observed in Western blot analysis with LA+ absorbed eluate and with a crude extract of the EPEC strain, but the eluate absorbed with LA- showed a strong recognition of a 94-kDa band, a molecular weight equivalent to that of intimin. Colostrum antibodies reacting to non-protein antigens were not detected by Western blot analysis. The persistence of anti-EPEC IgA in the gastrointestinal tract was shown by the strong reactivity to the 94-kDa band in Western blot analysis of one mother's colostrum and her infant's faeces. These data confirm the role of colostrum antibodies in protecting the neonate against infections due to EPEC.

Monoclonal antibodies against conserved epitopes of a 46-kDa protein from Neisseria meningitidis.

The purpose of the present study was to generate monoclonal antibodies (mAbs) against conserved epitopes of B meningococcus which could be applicable to the immunoscreening of bacterial meningitis. Three mAbs reactive to a 46-kDa protein conserved in eight sero-groups and several sero(sub)types of Neisseria meningitidis were selected for the present study. No reaction was detected with whole-cell lysates of Staphylococcus aureus. Streptococcus pneumoniae, Haemophilus influenzae type b or Escherichia coli. Two of these mAbs recognized 46-kDa epitopes in four other Neisseria spp, and the third, MC3.13, cross-reacted only with N. lactamica. All mAbs reacted with whole-cell lysates from a N. meningitidis mutant strain lacking the class 1 outer membrane protein (43-47 kDa). Immunoelectron microscopy revealed a cytoplasmic location for the 46-kDa protein. The MC3.13 monoclonal antibody is potentially applicable to a rapid screening of bacterial meningitis.

Monoclonal antibodies against conserved epitopes of a 46-kDa protein from Neisseria meningitidis

The purpose of the present study was to generate monoclonal antibodies (mAbs) against conserved epitopes of B meningococcus which could be applicable to the immunoscreening of bacterial meningitis. Three mAbs reactive to a 46-kDa protein conserved in eight serogroups and several sero(sub)types of Neisseria meningitidis were selected for the present study. No reaction was detected wlth wholecell lysates of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae type b or Escherichia coli. Two of these mAbs recognized 46-kDa epitopes in four other Neisseria spp, and the third, MC3.13, cross-reacted only with N. lactamica. All mAbs reacted with whole-cell lysates from a N. meningitidis mutant strain lacking the class 1 outer membrane protein (43 -47 kDa). Immunoelectron microscopy revealed a cytoplasmic location for the 46-kDa protein. The MC3.13 monoclonal antibody is potentially applicable to a rapid screening of bacterial meningitis.

Effect of microwave radiation, pasteurization and lyophilization on the ability of human milk to inhibit Escherichia coli adherence to HEp-2 cells.

The effect of different physical treatments on the ability of colostrum and human milk to inhibit the adherence of enteropathogenic Escherichia coli (EPEC) to human epithelial cells was studied. Pools of colostrum and milk were submitted to microwave radiation, pasteurization or lyophilization, and then tested for the ability to inhibit the adherence of EPEC O111:H- to HEp-2 cells. The inhibitory effect of untreated colostrum and human milk on localized adherence was not significantly modified after exposure to any treatment. The total protein values of colostrum and milk were maintained, but IgA concentration and colostral anti-EPEC IgA were reduced after pasteurization. Nevertheless, the remaining IgA was sufficient to be effective in adhesion inhibition assay. Western blotting assays carried out with EPEC antigens showed that the treated and untreated pools recognize a 94-kDa outer-membrane protein which molecular weight is compatible with intimin, an EPEC adhesin related to bacterial attachment to epithelial cells. These results suggest that the protection of colostrum and milk to infantile diarrhoea due to EPEC remains unalterable after the physical treatments studied.

An in vitro model to study the effect of antibodies and cell receptor analogues on meningococcal adherence to human oroepithelial cells.

We have investigated different experimental schedules to achieve adherence of Neisseria meningitidis group B to cultured and buccal epithelial cells (BEC) and the effect of antibodies and receptor analogues on bacterial adherence. No adherence of meningococcus was observed when HeLa, HEp-2 or KB cells were used, but high rates of adherence to BEC occurred. The effect of antibodies on bacterial adherence was studied in assays carried out in the presence of saliva and serum collected from convalescing children with meningococcal meningitis and children vaccinated with VAMENGOC B-C. Both saliva and serum from the convalescent patients inhibited the adherence of meningococci, but saliva and serum from vaccinated children did not, corroborating our previous data of a poor antibody response induced by this vaccine. Human colostrum did not affect meningococcal adherence despite the presence of antibodies to N. meningitidis detected by ELISA. Inhibition of adherence by sera from an immunized horse, rabbits and mice, as well as by cell receptor analogues (outer-membrane complex and purified polysaccharide C), was observed. Our results show that up to now BEC continue to be the best cells to study meningococcal adherence and the effect of adherence inhibitors.

An in vitro model to study the effect of antibodies and cell receptor analogues on meningococcal adhrence to human oroepithelial cells

We have investigated different experimental schedules to achieve adherence of Neisseria meningitidis group B to cultured and buccal epithelial cells (BEC) and the effect of antibodies and receptor analogues on bacterial adherence. No adherence of meningococcus was observed when HeLa, HEp-2 or KB cells were used, but high rates of adherence to BEC occurred. The effect of antibodies on bacterial adherence was studied in assays carried out in the presence of saliva and serum collected from convalescing children with meningococcal meningitis and children vaccinated with VAMENGOC B-C. Both saliva and serum from the convalescent patients inhibited the adherence of meningococci, but saliva and serum from vaccinated children did not, corroborating our previous data of a poor antibody response induced by this vaccine. Human colostrum did not affect meningococcal adherence despite the presence of antibodies to N. meningitidis detected by ELISA. Inhibition of adherence by sera from an immunized horse, rabbits and mice, as well as by cell receptor analogues (outer-membrane complex and purified polysaccharide C), was observed. Our results show that up to now BEC continue to be the best cells to study meningococcal adherence and the effect of adherence inhibitors.

Inhibition of HEp-2 cell invasion by enteroinvasive Escherichia coli by human colostrum IgA.

This paper demonstrates that human colostrum can inhibit the invasion of HEp-2 cells by enteroinvasive Escherichia coli (EIEC) of serotypes O28:H- and O29:H-, and that IgA antibodies mediate the inhibitory process. Seventy three of 77 (95.9%) colostrum samples prevented invasion of HEp-2 cells by E. coli O28:H-. Most of these samples contained high levels of IgA reactive to EIEC in immunoenzymatic assays. IgA eluted from an affinity chromatography column strongly inhibited HEp-2 invasion by EIEC, whereas IgA-depleted colostrum had no inhibitory effect on bacterial invasion. Immunoblots of colostrum samples with high (> 60%) invasion-inhibiting levels were performed with water extracts of invasive and noninvasive strains. Bacterial antigens from the invasive strain were recognized and the size of some was consistent with the invasion plasmid antigens (Ipas) A, B, C, and D, with stronger reactions with Ipas A and C. Colostrum samples with high inhibitory levels showed a strong reaction in Western blot assays, in contrast to the faint bands observed with poor-inhibitory samples. The results obtained in the present study suggest that colostrum IgA may protect infants against invasive E. coli infections.

The antimeningococcal vaccine VAMENGOC B-C induced poor serum and salivary antibody response in young Brazilian children.

In 1989 about 2.3 million Brazilian children received the antimeningococcal vaccine VAMENGOC B-C (Havana, Cuba). We evaluated the serum and secretory immune response of vaccinated children by enzyme-linked immunosorbent assay with outer membrane complex antigens. Western blotting and bacterial adherence inhibition assays with human buccal epithelial cells were performed with some of the samples. Serum and salivary antibody concentrations to Neisseria meningitidis Group B of vaccinated children < 4 years old were not significantly higher than those of nonvaccinated children, as observed in convalescing patients used as positive controls. Older children (4 to 6 years old) presented a slight increase in antibody OD indexes. Sera and saliva from vaccinated children showed a weak reaction with meningococcal antigen by Western blotting and were unable to inhibit significantly the adherence of N. meningitidis B to buccal epithelial cells. These data suggest that this vaccine induced a poor serum and salivary antibody response in the population studied.

Inhibition of enteropathogenic Escherichia coli (EPEC) adhesion to HeLa cells by human colostrum: detection of specific sIgA related to EPEC outer-membrane proteins.

Human colostrum and a high molecular weight colostrum fraction (HMWF; > 14,000 D) prevented the adhesion of localized adherent (LA+) O111:H-enteropathogenic Escherichia coli (EPEC) to HeLa cells. This effect was abolished after absorption with an O111:H-LA + EPEC strain, but absorption with a LA- strain of same serotype had no effect on the process. A low molecular weight fraction (< 14,000 D), absorbed or not with LA+ or LA- bacterial strains, did not inhibit the adherence of E. coli to HeLa cells. IgA-depleted colostrum had no inhibitory effect on bacterial adhesion, demonstrating the critical role of this protein in the phenomenon. Heat inactivation of whole colostrum did not significantly modify the inhibition adherence levels. Immunoblots of O111:H-LA+ strain outer-membrane complex reacted with colostrum and HMWF showing that IgA antibodies were predominantly reactive with a 94-kD protein. These data confirm and extend observations about colostrum sIgA participation in adhesion inhibition of EPEC to HeLa cells and its response to a 94-kD outer-membrane protein.

Maternally acquired immunity in newborns from women infected by the human immunodeficiency virus.

Maternally acquired immunity was studied in 16 pairs of human immunodeficiency virus (HIV)-seropositive women and their newborns, and was compared to 18 control mother-newborn pairs. The HIV-infected women had higher IgG levels than the control subjects, but no difference was observed between newborn samples, presumably due to the limited placental IgG transfer in the HIV group. A poor type 2 poliovirus antibody transfer was also noted in this group. The population of newborns lacking demonstrable measles antibodies was higher in the HIV group than in the control group, probably because many of the HIV-infected mothers lacked measles antibodies also. These results show that maternally acquired immunity may be affected to newborns from HIV-infected women, either because of low maternal serum antibody levels or deficient transplacental transfer. If so, the measles vaccine schedule should be revised for these children and the same should be done for future passive immunization regarding fetus protection in pregnant HIV-seropositive women.

Frequency of selective IgA deficiency among Brazilian blood donors and healthy pregnant women.

Selective IgA deficiency (SIgAD) is the most common primary immunodeficiency, with frequencies ranging from 1:300 to 1:3,000 in populations surveyed in Europe and the US. In the present study we tested 11,576 clinically healthy persons (blood donors and pregnant women) for SIgAD (serum IgA less than 5 mg%). Serum samples were screened by double immunodiffusion with a sheep anti-human alpha-chain (minimal detection level of 30 mg%). Samples showing negative or doubtful reactions were submitted to the radial immunodiffusion test (minimal detection level of 0.5 mg%). For the samples with low or undetectable IgA levels, IgG and IgM concentrations were also determined. We found 12 individuals with SIgAD and 2 with deficiency of the 3 immunoglobulin classes. The prevalence of SIgAD in this Brazilian population (1:965) is equivalent to values reported for other countries.